1. J Am Chem Soc. 2010 Mar 3;132(8):2558-60.

Cyclobutanone Analogues of beta-Lactams Revisited: Insights into Conformational
Requirements for Inhibition of Serine- and Metallo-beta-Lactamases.

Johnson JW, Gretes M, Goodfellow VJ, Marrone L, Heynen ML, Strynadka NC,
Dmitrienko GI.

Department of Chemistry, University of Waterloo, 200 University Avenue West,
Waterloo, Ontario, Canada N2L 3G1, and Department of Biochemistry and Molecular
Biology and the Center for Blood Research, University of British Columbia, 2350
Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

The most important mode of bacterial resistance to beta-lactam antibiotics is the
expression of beta-lactamases. New cyclobutanone analogues of penams and penems
have been prepared and evaluated for inhibition of class A, B, C, and D
beta-lactamases. Inhibitors which favor conformations in which the C4 carboxylate
is equatorial were found to be more potent than those in which the carboxylate is
axial, and molecular modeling studies with enzyme-inhibitor complexes indicate
that an equatorial orientation of the carboxylate is required for binding to
beta-lactamases. An X-ray structure of OXA-10 complexed with a cyclobutanone
confirms that a serine hemiketal is formed in the active site and that the
inhibitor adopts the exo envelope. An unsaturated penem analogue was also found
to enhance the potency of meropenem against carbapenem-resistant MBL-producing
strains of Chryseobacterium meningosepticum and Stenotrophomonas maltophilia .
These cyclobutanones represent the first type of reversible inhibitors to show
moderate (low micromolar) inhibition of both serine- and metallo-beta-lactamases 
and should be considered for further development into practical inhibitors.

PMID: 20141132 [PubMed - in process]


2. J Microbiol Biotechnol. 2009 Dec;19(12):1547-56.

Recombinant expression and characterization of Thermoanaerobacter tengcongensis
thermostable alpha-glucosidase with regioselectivity for high yield
isomaltooligosaccharides synthesis.

Zhou C, Xue Y, Zhang Y, Zeng Y, Ma Y.

The Graduate School, Chinese Academy of Sciences, Beijing 100049, China, Tianjin 
Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin
300308, China.

A novel thermostable alpha-glucosidase (TtGluA) from Thermoanaerobacter
tengcongensis MB4 was successfully expressed in E. coli and characterized. The
TtgluA gene contained 2253bp which encodes 750 amino acids. The native TtGluA was
a trimer with monomer molecular weight of 89 kDa shown by SDS-PAGE. The purified 
recombinant enzyme showed hydrolytic activity on maltooligosaccharides,
pNP-alpha-D-glucopyranide and dextrin with an exo-type cleaving manner. TtGluA
showed preference for short-chain maltooligosaccharides and the highest specific 
activity for maltose of 3.26 units/mg. Maximal activity was observed at 60
degrees and pH 5.5. The half-life was 2 hours at 60 masculineC. The enzyme showed
good tolerance to urea and SDS but was inhibited by Tris. When maltose with the
concentration over 50 mM was used as substrate, TtGluA was also capable of
catalyzing transglycosylation to produce alpha-1, 4 linked maltotriose and
alpha-1, 6 linked isomaltooligosaccharides. More importantly, TtGluA showed
exclusive regiospecificity with high yield to produce alpha-1, 6 linkages
isomaltooligosaccharides when the reaction time extended to more than 10 hours.

PMID: 20075617 [PubMed - in process]


3. Mol Microbiol. 2010 Jan 5. [Epub ahead of print]

Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's
progression from liver infection to blood infection.

Pei Y, Tarun AS, Vaughan AM, Herman RW, Soliman JM, Erickson-Wayman A, Kappe SH.

Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500,
Seattle, WA 98109, USA.

Summary Plasmodium parasites possess a single pyruvate dehydrogenase (PDH) enzyme
complex that is localized to the plastid-like organelle known as the apicoplast. 
Unlike most eukaryotes, Plasmodium lacks a mitochondrial PDH. The PDH complex
catalyzes the conversion of pyruvate to acetyl-CoA, an important precursor for
the tricarboxylic acid cycle and type II fatty acid synthesis (FAS II). In this
study, using a rodent malaria model, we show that the PDH E1alpha and E3 subunits
co-localize with the FAS II enzyme FabI in the apicoplast of liver stages but are
not significantly expressed in blood stages. Deletion of the E1alpha or E3
subunit genes of P. yoelii PDH caused no defect in blood stage development,
mosquito stage development or early liver stage development. However, the gene
deletions completely blocked the ability of the e1alpha(-) and e3(-) parasites to
form exo-erythrocytic merozoites during late liver stage development, thus
preventing the initiation of a blood stage infection. This phenotype is similar
to that observed for deletions of genes involved in FAS II elongation. The data
strongly support the hypothesis that the sole role of PDH is to provide
acetyl-CoA for FAS II.

PMID: 20059687 [PubMed - as supplied by publisher]


4. Inorg Chem. 2010 Feb 1;49(3):1238-44.

Discrete mercury(II) complexes, one-dimensional and palladium(II)-mediated
two-dimensional silver(I) coordination polymers of NS2-macrocycle: synthesis and 
structural characterization.

Park S, Lee SY, Lee SS.

Department of Chemistry and Research Institute of Natural Science, Gyeongsang
National University, Jinju 660-701, S. Korea.

A range of thiaphilic mononuclear (Hg(2+) and Ag(+)) and heterobinuclear
(Ag(+)/Pd(2+)) complexes (1-6) of an NS(2)-donor macrocycle L with discrete and
networked forms were prepared and structurally characterized. Reaction of L with 
HgCl(2) afforded a unique 1:1 (cationic/anionic) complex [Hg(2)(L)Cl(5)][Hg(L)Cl]
(1). Reaction of L with Hg(ClO(4))(2) yielded the sandwich-type 1:2
(metal-to-ligand) complex [Hg(L)(2)](ClO(4))(2) (2). Contrasting with the
mercury(II) salts, the reactions of L with AgX (X = NO(3)(-) and CF(3)SO(3)(-))
yielded an isostructural one-dimensional (1D) zigzag networks [Ag(L)X](n) (3; X =
NO(3)(-) and 4; X = CF(3)SO(3)(-)) in which each ring of L is exo-coordinated via
two S atoms and one N atom to a silver ion which is also bound to one S atom in
the neighboring L such that the overall coordination geometry about each silver
is four-coordinate. Whereas, reaction of L with K(2)PdCl(4) gave a discrete
exocyclic complex 5, [cis-Cl(2)Pd(L)]. Through a successive reaction of the
complex 5 with AgNO(3), a heterobinuclear two-dimensional (2D) coordination
polymer 6, [Pd(L)Ag(2.5)(NO(3))(4.5)(H(2)O)(0.5)](n), utilizing exocyclic Pd(II) 
and Ag(I) was isolated and characterized.

PMID: 20050601 [PubMed - in process]


5. Acta Crystallogr C. 2009 Dec;65(Pt 12):o645-8. Epub 2009 Nov 21.

2'-Deoxy-5-propynyluridine: a nucleoside with two conformations in the asymmetric
unit.

Budow S, Eickmeier H, Reuter H, Seela F.

Laboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstrasse 11, 48149 Munster, Germany.

The title compound,
1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-(prop-1-ynyl)pyrimidin-2,4(1H,3H)-dio
ne, C(12)H(14)N(2)O(5), shows two conformations in the crystalline state:
conformer 1 adopts a C2'-endo (close to (2)E; S-type) sugar pucker and an anti
nucleobase orientation [chi = -134.04 (19) degrees], while conformer 2 shows an S
sugar pucker (twisted C2'-endo-C3'-exo), which is accompanied by a different anti
base orientation [chi = -162.79 (17) degrees]. Both molecules show a +sc (gauche,
gauche) conformation at the exocyclic C4'-C5' bond and a coplanar orientation of 
the propynyl group with respect to the pyrimidine ring. The extended structure is
a three-dimensional hydrogen-bond network involving intermolecular N-H...O and
O-H...O hydrogen bonds. Only O atoms function as H-atom acceptor sites.

PMID: 19966450 [PubMed - indexed for MEDLINE]


6. Can J Microbiol. 2009 Oct;55(10):1145-52.

Gene SMb21071 of plasmid pSymB is required for osmoadaptation of Sinorhizobium
meliloti 1021 and is implicated in modifications of cell surface polysaccharides 
structure in response to hyperosmotic stress.

Reguera M, Lloret J, Margaret I, Vinardell JM, Martin M, Buendia A, Rivilla R,
Ruiz-Sainz JE, Bonilla I, Bolanos L.

Departamento de Biologia, Facultad de Ciencias, Universidad Autonoma de Madrid,
Darwin 2, 28049-Madrid, Spain.

Megaplasmid pSymB of the nitrogen-fixing symbiont Sinorhizobium meliloti,
implicated in adaptation to hyperosmotic stress, contains 11 gene clusters that
apparently encode surface polysaccharides. However, only 2 of these clusters,
containing the exo and exp genes, have been associated with the synthesis of the 
acidic exopolysaccharides succinoglycan and galactoglucan, respectively. The
functions of the other 9 clusters remain unsolved. The involvement of one of
those regions, pSymB cluster 3, on surface polysaccharide synthesis and its
possible implication in osmoadaptation were investigated. In silico analysis of
cluster 3 showed that it putatively encodes for the synthesis and transport of a 
methylated surface polysaccharide. Mutants affected in this cluster were
symbiotically effective but showed defects in growth under saline and nonsaline
osmotic stress. The gene SMb21071, encoding a putative initiating
glycosyltransferase, is transcriptionally induced under hyperosmotic conditions. 
Sodium dodecyl sulfate - polyacrylamide gel electrophoresis and silver staining
showed that osmotic stresses changed the profiles of surface polysaccharides of
wild-type and mutants strains in different ways. The overall results suggest that
cluster 3 is important for growth under saline stress and essential for growth
under nonsaline hyperosmotic stress, and it appears to be implicated in
maintaining and (or) modifying surface polysaccharides in response to osmotic
stress.

PMID: 19935886 [PubMed - in process]


7. Biochemistry (Mosc). 2009 Sep;74(9):1002-8.

Isolation and properties of extracellular beta-xylosidases from fungi Aspergillus
japonicus and Trichoderma reesei.

Semenova MV, Drachevskaya MI, Sinitsyna OA, Gusakov AV, Sinitsyn AP.

Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071,
Russia. margs@mail.ru

Homogeneous beta-xylosidases with molecular mass values 120 and 80 kDa (as shown 
by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated
by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme
preparations based on the fungi Aspergillus japonicus and Trichoderma reesei,
respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5-4.0), 
and temperature activity optimum was 70 degrees C for the beta-xylosidase of A.
japonicus and 60 degrees C for the beta-xylosidase of T. reesei. Kinetic
parameters of p-nitrophenyl beta-xylopyranoside and xylooligosaccharide
hydrolysis by the purified enzymes were determined, which showed that
beta-xylosidase of A. japonicus was more specific towards low molecular weight
substrates, while beta-xylosidase of T. reesei preferred high molecular weight
substrates. The competitive type of inhibition by reaction product (xylose) was
found for both enzymes. The interaction of the enzymes of different specificity
upon hydrolysis of glucurono- and arabinoxylans was found. The beta-xylosidases
exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as 
with alpha-L-arabinofuranosidase and endoxylanase upon hydrolysis of
arabinoxylan. Addition of beta-xylosidases increased efficiency of hydrolysis of 
plant raw materials with high hemicellulose content (maize cobs) by the enzymic
preparation Celloviridine G20x depleted of its own beta-xylosidase.

PMID: 19916911 [PubMed - indexed for MEDLINE]


8. J Biosci Bioeng. 2009 Dec;108(6):455-9.

Characterization of glycosyl hydrolase family 3 beta-N-acetylglucosaminidases
from Thermotoga maritima and Thermotoga neapolitana.

Choi KH, Seo JY, Park KM, Park CS, Cha J.

Department of Microbiology, Pusan National University, Busan 609-735, Republic of
Korea.

The genes encoding beta-N-acetylglucosaminidase (nagA and cbsA) from Thermotoga
maritima and Thermotoga neapolitana were cloned and expressed in Escherichia coli
in order to investigate whether Thermotoga sp. is capable of utilizing chitin as 
a carbon source. NagA and CbsA were purified to homogeneity by HiTrap Q HP and
Sephacryl S-200 HR column chromatography. Both enzymes were homodimers containing
a family 3 glycoside hydrolase (GH3) catalytic domain, with a monomer molecular
mass of 54 kDa. The optimal temperatures and pHs for the activities of the
beta-N-acetylglucosaminidases were found to be 65-75 degrees C and 7.0-8.0,
respectively. Both enzymes hydrolyzed chitooligomers such as
di-N-acetylchitobiose and tri-N-acetylchitotriose, and synthetic substrates such 
as p-nitrophenyl-beta-D-glucose (pNPGlc), p-nitrophenyl N-acetyl
beta-D-glucosamine (pNPGlcNAc), p-nitrophenyl di-N-acetyl beta-D-chitobiose
(pNPGlcNAc(2)) and p-nitrophenyl tri-N-acetyl beta-D-chitotriose (pNPGlcNAc(3)). 
However, the enzymes had no activity against p-nitrophenyl-beta-D-galactose
(pNPGal) and p-nitrophenyl N-acetyl beta-D-galactosamine (pNPGalNAc) or highly
polymerized chitin. The k(cat) and K(m) values were determined for pNPGlcNAc,
pNPGlcNAc(2) and pNPGlcNAc(3). The k(cat)/K(m) value for pNPGlcNAc was the
highest among three synthetic substrates. NagA and CbsA initially hydrolyzed
p-nitrophenyl substrates to give GlcNAc, suggesting that the enzymes have
exo-activity with chitin oligosaccharides from the non-reducing ends, like other 
beta-N-acetylglucosaminidases. However, NagA and CbsA can be distinguished from
other GH3-type beta-N-acetylglucosaminidases in that they are highly active
against di-N-acetylchitobiose. Thus, the present results suggest that the
physiological role of both enzymes is to degrade the chitooligosaccharides
transported through membrane following hydrolysis of chitin into
beta-N-acetylglucosamine to be further metabolized in Thermotoga sp.

PMID: 19914575 [PubMed - in process]


9. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(9):391-408.

Chemistry and biosynthesis of isoprenylated flavonoids from Japanese mulberry
tree.

Nomura T, Hano Y, Fukai T.

Faculty of Pharmaceutical Sciences, Toho University, Chiba, Japan.
hano@phar.toho-u.ac.jp

Many isoprenylated flavonoids have been isolated from Japanese mulberry tree
(Moraceae). Among them, kuwanons G (1) and H (2) were the first isolated active
substances exhibiting a hypotensive effect. These compounds are considered to be 
formed through an enzymatic Diels-Alder type reaction between an isoprenyl
portion of an isoprenylphenol as the diene and an alpha, beta-double bond of
chalcone as the dienophile. The absolute configurations of these Diels-Alder type
adducts were confirmed by three different methods. The stereochemistries of the
adducts were consistent with those of ones in the Diels-Alder reaction involving 
exo- and endo-addition. Some strains of Morus alba callus tissues have a high
productivity of mulberry Diels-Alder type adducts, such as chalcomoracin (3) and 
kuwanon J (4). The biosynthetic studies of the mulberry Diels-Alder type adducts 
have been carried out with the aid of the cell strain. Chalcomoracin (3) and
kuwanon J (4) were proved to be enzymatic Diels-Alder type reaction products by
the administration experiments with O-methylchalcone derivatives. Furthermore,
for the isoprenoid biosynthesis of prenylflavonoids in Morus alba callus tissues 
by administration of [1,3-(13)C(2)]- and [2-(13)C]-glycerol, a novel way through 
the junction of glycolysis and pentose-phosphate cycle was proved. Two
independent isoprenoid biosynthetic pathways, that for sterols and that for
isoprenoid-phenols, operate in the Morus alba cell cultures. The former is
susceptible to compactin (ML-236) and the latter resists to compactin in the cell
cultures, respectively.

PMID: 19907125 [PubMed - in process]


10. J Biol Chem. 2010 Jan 22;285(4):2734-49. Epub 2009 Nov 10.

Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2.

Kahlfeldt N, Vahedi-Faridi A, Koo SJ, Schafer JG, Krainer G, Keller S, Saenger W,
Krauss M, Haucke V.

Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry,
Freie Universitat Berlin, 14195 Berlin, Germany.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant
in clathrin-mediated endocytosis (CME). In mammals three type I
phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the 
I gamma-p90 isoform being highly expressed in the brain where it regulates
synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely
PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain,
but recent data indicate that direct interactions between type I PIPK and
components of the endocytic machinery, in particular the AP-2 adaptor complex,
are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the 
mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that
a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds 
to a cognate recognition site on the sandwich subdomain of the beta2 appendage.
Partly overlapping aromatic and hydrophobic residues within the same peptide also
can engage the C-terminal sorting signal binding domain of AP-2mu, thereby
potentially competing with the sorting of conventional YXXO motif-containing
cargo. Biochemical and structure-based mutagenesis analysis revealed that
association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of
these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair 
endocytosis of SVs in primary neurons largely depends on its association with
AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and
the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and
enzymatic activity. We postulate a model according to which multiple interactions
between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) 
synthesis during clathrin-mediated SV endocytosis.

PMCID: PMC2807329 [Available on 2011/1/22]
PMID: 19903820 [PubMed - indexed for MEDLINE]


11. J Biol Chem. 2010 Jan 8;285(2):1000-7. Epub 2009 Nov 2.

Structural insights into vinyl reduction regiospecificity of
phycocyanobilin:ferredoxin oxidoreductase (PcyA).

Hagiwara Y, Sugishima M, Khawn H, Kinoshita H, Inomata K, Shang L, Lagarias JC,
Takahashi Y, Fukuyama K.

Department of Biological Sciences, Graduate School of Science, Osaka University, 
Toyonaka, Osaka 560-0043, Japan.

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member
of the ferredoxin-dependent bilin reductase family. Unlike other
ferredoxin-dependent bilin reductases that catalyze a two-electron reduction,
PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of
biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the 
light-harvesting antennae complexes of red algae, cyanobacteria, and
cryptophytes. To address the structural basis for the reduction regiospecificity 
of PcyA, we report new high resolution crystal structures of bilin substrate
complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl
reduction activity. These include the BV complex of the E76Q mutant as well as
substrate-bound complexes of wild-type PcyA with the reaction intermediate
18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha
(BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall
folds and the binding sites of the U-shaped substrates of all three complexes
were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain,
which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away 
from the bilin D-ring. The local structures around the A-rings in the three
complexes, which all retain the ability to reduce the A-ring of their bound
pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with
the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for
exo-vinyl reduction, these structures reveal new insight into the reduction
regiospecificity of PcyA.

PMCID: PMC2801226 [Available on 2011/1/8]
PMID: 19887371 [PubMed - indexed for MEDLINE]


12. Carbohydr Res. 2009 Dec 14;344(18):2444-53. Epub 2009 Sep 12.

Synthesis, regioselective hydrogenolysis, partial hydrogenation, and
conformational study of dioxane and dioxolane-type (9'-anthracenyl)methylene
acetals of sugars.

Jakab Z, Mandi A, Borbas A, Benyei A, Komaromi I, Lazar L, Antus S, Liptak A.

Research Group for Carbohydrates of the Hungarian Academy of Sciences, University
of Debrecen, PO Box 94, H-4010 Debrecen, Hungary.

Dioxane-type (9'-anthracenyl)methylene acetal of methyl
2,3-di-O-methyl-alpha-D-glucopyranoside was cleaved with LiAlH(4)/AlCl(3) (3:1)
or with Na(CN)BH(3)-HCl regioselectively to provide the 4- or
6-O-(9'-anthracenyl)methyl ether, respectively. Hydrogenolytic reaction of the
exo and endo isomers of dioxolane-type acetals proved to be directed by the
configuration of the acetalic carbon as well as by the intramolecular
participation of the adjacent-free hydroxyl; ring-opening reaction of the endo
isomer of the methyl 2,3-O-(9'-anthracenyl)methylene-alpha-L-rhamnopyranoside
took place with complete selectivity resulting in the axial
(9'-anthracenyl)methyl ether, whereas a 1:1 mixture of the axial and equatorial
ethers was formed upon the same reaction of the exo isomer. Catalytic
hydrogenation of the sugar acetals resulted in
(9',10'-dihydro-9'-anthracenyl)methylene derivatives without affecting the
acetalic center. High-temperature molecular dynamics simulations and DFT (Density
Functional Theory) geometry optimizations were carried out to study the
conformation of the dioxane-type (9',10'-dihydro-9'-anthracenyl)methylene acetal.

PMID: 19879559 [PubMed - indexed for MEDLINE]


13. Appl Microbiol Biotechnol. 2010 Mar;86(2):555-66. Epub 2009 Oct 16.

Characterization of an exo-acting intracellular alpha-amylase from the
hyperthermophilic bacterium Thermotoga neapolitana.

Park KM, Jun SY, Choi KH, Park KH, Park CS, Cha J.

Department of Microbiology, College of Natural Sciences, Pusan National
University, San 30, Jangjeon-dong, Geumjeong-gu, Busan, 609-735, Korea.

We cloned and expressed the gene for an intracellular alpha-amylase, designated
AmyB, from the hyperthermophilic bacterium Thermotoga neapolitana in Escherichia 
coli. The putative intracellular amylolytic enzyme contained four regions that
are highly conserved among glycoside hydrolase family (GH) 13 alpha-amylases.
AmyB exhibited maximum activity at pH 6.5 and 75 degrees C, and its
thermostability was slightly enhanced by Ca(2+). However, Ca(2+) was not required
for the activity of AmyB as EDTA had no effect on enzyme activity. AmyB
hydrolyzed the typical substrates for alpha-amylase, including soluble starch,
amylose, amylopectin, and glycogen, to liberate maltose and minor amount of
glucose. The hydrolytic pattern of AmyB is most similar to those of maltogenic
amylases (EC 3.2.1.133) among GH 13 alpha-amylases; however, it can be
distinguished by its inability to hydrolyze pullulan and beta-cyclodextrin. AmyB 
enzymatic activity was negligible when acarbose, a maltotetraose analog in which 
a maltose residue at the nonreducing end was replaced by acarviosine, was
present, indicating that AmyB cleaves maltose units from the nonreducing end of
maltooligosaccharides. These results indicate that AmyB is a new type exo-acting 
intracellular alpha-amylase possessing distinct characteristics that distinguish 
it from typical alpha-amylase and cyclodextrin-/pullulan-hydrolyzing enzymes.

PMID: 19834705 [PubMed - in process]


14. J Phys Chem A. 2010 Jan 28;114(3):1427-31.

Determination of the structure of cyclopentene oxide and the argon-cyclopentene
oxide van der Waals complex.

Minei AJ, van Wijngaarden J, Novick SE, Pringle WC.

Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, USA.

Rotational spectra of cyclopentene oxide and the argon-cyclopentene oxide van der
Waals complex were studied using pulsed-jet Fabry-Perot Fourier transform
microwave (FTMW) spectroscopy. Spectra of the parent along with those of the
(13)C and (18)O singly substituted isotopologues, in natural abundance, of the
monomer and of the complex were measured in the frequency region of 5-26.5 GHz.
The complete heavy atom substitution structure was determined for the monomer and
complex. The boat structure for cyclopentene oxide was confirmed with naturally
abundant (13)C and (18)O isotopes. For the argon cyclopentene oxide complex, both
a and b-type transitions were observed and the rotational constants for the
all-(12)C (16)O isotopologue were determined to be A = 3268.254(2), B =
993.345(1), and C = 950.430(9) MHz. The r(0) coordinates of the argon in the
principal axis system of cyclopentene oxide are a = 0.27, b = 0.42, and c = 3.91 
A, such that the argon is exo to the boat of the ring and on the opposite side of
the ring from the oxygen and is 0.42 A off to the side and 0.27 A from the center
of mass toward the back end of the ring (again away from the oxygen). Large
amplitude van der Waals bending vibrations require an averaging model to account 
for differences between the observed complex and monomer planar moments of
inertia.

PMID: 19831342 [PubMed - in process]


15. Nucleic Acids Res. 2009 Dec;37(22):7612-22.

Cleavage of adenine-modified functionalized DNA by type II restriction
endonucleases.

Macickova-Cahova H, Hocek M.

Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, Gilead & IOCB Research Center, Flemingovo nam. 2, CZ-16610, Prague 6,
Czech Republic.

A set of 6 base-modified 2'-deoxyadenosine derivatives was incorporated to
diverse DNA sequences by primer extension using Vent (exo-) polymerase and the
influence of the modification on cleavage by diverse restriction endonucleases
was studied. While 8-substituted (Br or methyl) adenine derivatives were well
tolerated by the restriction enzymes and the corresponding sequences were
cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence
resulted in blocking of cleavage by some enzymes depending on the nature and size
of the 7-substituent. All sequences with modifications outside of the recognition
sequence were perfectly cleaved by all the restriction enzymes. The results are
useful both for protection of some sequences from cleavage and for manipulation
of functionalized DNA by restriction cleavage.

PMCID: PMC2794189
PMID: 19820117 [PubMed - indexed for MEDLINE]


16. J Biochem. 2010 Feb;147(2):237-44. Epub 2009 Oct 9.

Structural explanation for the acquisition of glycosynthase activity.

Hidaka M, Fushinobu S, Honda Y, Wakagi T, Shoun H, Kitaoka M.

Department of Biotechnology, The University of Tokyo, Tokyo, Japan.

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the
synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We
have determined five crystal structures of the glycosynthase mutants reducing-end
xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels 
of glycosynthetic activities. At the active site of the Y198F mutant, the most
efficient glycosynthase, a water molecule is observed at the same position as
nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the
direction of the lone pair of water molecules in the mutant drastically decreases
hydrolytic activity. Water molecules were also observed at each active site of
the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild 
type. Their positions exhibited a strong correlation with the strength of
glycosynthase activity. Here, we propose that a structural prerequisite for the
sufficient glycosynthase reaction is the presence of a water molecule at the NW
position, and mutation at the NW holder provides a general strategy for inverting
GHs. The idea on the position of a water molecule may also be applicable to the
design of efficient glycosynthases from retaining GHs.

PMID: 19819900 [PubMed - in process]


17. J Neurochem. 2009 Dec;111(6):1434-46. Epub 2009 Oct 6.

Nitric oxide and cyclic nucleotide signal transduction modulates synaptic vesicle
turnover in human model neurons.

Tegenge MA, Stern M, Bicker G.

Division of Cell Biology, Institute of Physiology, University of Veterinary
Medicine Hannover, Hannover, Germany.

The human Ntera2 (NT2) teratocarcinoma cell line can be induced to differentiate 
into post-mitotic neurons. Here, we report that the human NT2 neurons generated
by a spherical aggregate cell culture method express increasing levels of typical
pre-synaptic proteins (synapsin and synaptotagmin I) along the neurite depending 
on the length of in vitro culture. By employing an antibody directed against the 
luminal domain of synaptotagmin I and the fluorescent dye
N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide, we
show that depolarized NT2 neurons display calcium-dependent exo-endocytotic
synaptic vesicle recycling. NT2 neurons express the neuronal isoform of neuronal 
nitric oxide synthase and soluble guanylyl cyclase (sGC), the major receptor for 
nitric oxide (NO). We tested whether NO signal transduction modulates synaptic
vesicle turnover in human NT2 neurons. NO donors and cylic
guanosine-monophosphate analogs enhanced synaptic vesicle recycling while a sGC
inhibitor blocked the effect of NO donors. Two NO donors, sodium nitroprusside,
and and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino) ethanamine evoked
vesicle exocytosis which was partially blocked by the sGC inhibitor. The
activator of adenylyl cyclase, forskolin, and a cAMP analog induced synaptic
vesicle recycling and exocytosis via a parallel acting protein kinase A pathway. 
Our data from NT2 neurons suggest that NO/cyclic nucleotide signaling pathways
may facilitate neurotransmitter release in human brain cells.

PMID: 19807845 [PubMed - indexed for MEDLINE]


18. Appl Microbiol Biotechnol. 2010 Mar;86(1):227-34. Epub 2009 Oct 3.

Overexpression and molecular characterization of Aga50D from Saccharophagus
degradans 2-40: an exo-type beta-agarase producing neoagarobiose.

Kim HT, Lee S, Lee D, Kim HS, Bang WG, Kim KH, Choi IG.

School of Life Sciences and Biotechnology, Korea University, Anam-Dong
Seongbuk-Gu, Seoul 136-713, Korea.

beta-Agarases are mostly categorized into three glycoside hydrolase (GH) families
16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2-40 revealed
the presence of five agarase genes belonging to these GH families. Among the five
agarases, Aga50D (a member of GH50) had neither been functionally characterized
nor overexpressed. In this report, we present soluble overexpression and
molecular characterization of Aga50D. Aga50D was expressed in an active form
resulting in a single major product from agarose without intermediates. While
known GH50 agarases have both endo-lytic and exo-lytic activities, which produce 
neoagarobiose as a final product through the intermediate,
neoagaro-oligosaccharides, identification and analysis of the reaction product by
mass spectrometry and 13C NMR showed that Aga50D had unique exo-lytic activity
and was able to produce neoagarobiose directly from agarose. The optimum pH and
temperature for the activity were 7.0 and 30 degrees C, respectively. The K (m)
and V (max) for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.

PMID: 19802606 [PubMed - in process]


19. Protoplasma. 2009 Oct;237(1-4):27-39. Epub 2009 Sep 8.

Oxidative defence reactions in sunflower roots induced by methyl-jasmonate and
methyl-salicylate and their relation with calcium signalling.

Garrido I, Espinosa F, Alvarez-Tinaut MC.

Area de Fisiologia Vegetal, Facultad de Ciencias, Universidad de Extremadura,
Badajoz, Spain. igarridoc@unex.es

Ca(2+) plays a critical role as second messenger in the signal-response coupling 
of plant defence responses, and methyl-jasmonate and methyl-salicylate are
important components of signal transduction cascades activating plant defences.
When intact axenic non-induced seedling roots of sunflower were treated with
different Ca(2+) concentrations up to 1 mM, there was no significant increase in 
O(2)(*-) generation or DMAB-MBTH peroxidase (extracellular, ECPOX) activities in 
the apoplast, probably because these roots had enough Ca(2+) in their exo- and
endocellular reservoirs. Both activities were strongly inhibited by the
RBOH-NADPH oxidase inhibitor DPI and by the Ca(2+) surrogate antagonist La(3+),
but the voltage-dependent Ca(2+) channel blocker verapamil was only inhibitory at
concentrations higher than those active on animal L-type Ca(2+) channels.
Concentrations >5 mM EGTA (chelating Ca(2+) in the apoplast) and Li(+)
(inhibiting PI cycle dependent endogenous Ca(2+) fluxes) also inhibited both
activities. W7, inhibitor of binding of Ca-CaM to its target protein, enhanced
both activities, but the inactive analogue W5 showed a similar effect. Our data
suggest that Ca(2+) from exocellular and, to a lesser extent, from endocellular
stores is involved in oxidative activities, and that RBOH-NADPH oxidase is the
main system supporting them. Ca(2+) activation of the PM cytosolic side of
RBOH-NADPH oxidase is probably the key to Ca(2+) involvement in these processes. 
Roots induced by MeJA or MeSA showed significant enhancement of both oxidative
activities, as corresponding to the oxidative burst evoked by the two
phytohormones in the root apoplast. But while ECPOX activity showed a response to
the effectors similar to that described above for non-induced roots, O(2)(*-)
generation activity in the apoplast of induced roots was insensitive to EGTA,
verapamil and Li(+), the inhibitors of exogenous and endogenous Ca(2+) fluxes;
only DPI and La(3+) were inhibitory. As exogenously added 0.1 mM Ca(2+) also
increased O (2) (.-) generation, we propose that, in these roots, activation of
RBOH-NADPH oxidase by Ca(2+) could be regulated by Ca(2+) sensors in the
apoplast.

PMID: 19763783 [PubMed - in process]


20. Chemistry. 2009 Oct 12;15(40):10423-31.

Alpha-O-linked glycopeptide mimetics: synthesis, conformation analysis, and
interactions with viscumin, a galactoside-binding model lectin.

Jimenez-Barbero J, Dragoni E, Venturi C, Nannucci F, Arda A, Fontanella M, Andre 
S, Canada FJ, Gabius HJ, Nativi C.

Chemical and Physical Biology, Centro de Investigaciones Biologicas, CSIC, Ramiro
de Maeztu 9, 28040 Madrid, Spain. jjbarbero@cib.csic.es

Efficient cycloaddition of a silylidene-protected galactal with a suitable
heterodiene yielded the basis for a facile diastereoselective route to a
glycopeptide-mimetic scaffold. Its carbohydrate part was further extended by
beta1-3-linked galactosylation. The pyranose rings retain their (4)C(1) chair
conformation, as shown by molecular modeling and NMR spectroscopy, and the
typical exo-anomeric geometry was observed for the disaccharide. The expected
bioactivity was ascertained by saturation-transfer-difference NMR spectroscopy by
using the galactoside-specific plant toxin viscumin as a model lectin. The
experimental part was complemented by molecular docking. The described synthetic 
route and the strategic combination of computational and experimental techniques 
to reveal conformational properties and bioactivity establish the prepared
alpha-O-linked glycopeptide mimetics as promising candidates for further
exploitation of this scaffold to give O-glycans for lectin blocking and
vaccination.

PMID: 19746469 [PubMed - indexed for MEDLINE]


21. FEBS J. 2009 Sep;276(18):5094-100. Epub 2009 Aug 4.

The crystal structure of a xyloglucan-specific endo-beta-1,4-glucanase from
Geotrichum sp. M128 xyloglucanase reveals a key amino acid residue for substrate 
specificity.

Yaoi K, Kondo H, Hiyoshi A, Noro N, Sugimoto H, Tsuda S, Miyazaki K.

Institute for Biological Resources and Functions, National Institute of Advanced 
Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
k-yaoi@aist.go.jp

Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases
belonging to family 74, xyloglucan-specific endo-beta-1,4-glucanase (XEG) and
oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their
similar amino acid sequences (48% identity), their modes of action and substrate 
specificities are distinct. XEG catalyzes the hydrolysis of xyloglucan
polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides 
at the reducing end in exo mode. Here, we determined the crystal structure of XEG
at 2.5 A resolution, and compared it to a previously determined structure of
OXG-RCBH. For the most part, the amino acid residues that interact with substrate
are conserved between the two enzymes. However, there are notable differences at 
subsite positions -1 and +2. OXG-RCBH has a loop around the +2 site that blocks
one end of the active site cleft, which accounts for its exo mode of action. In
contrast, XEG lacks a corresponding loop at this site, thereby allowing binding
to the middle of the main chain of the substrate. At the -1 site in OXG-RCBH,
Asn488 interacts with the xylose side chain of the substrate, whereas the -1 site
is occupied by Tyr457 in XEG. To confirm the contribution of this residue to
substrate specificity, Tyr457 was substituted by Gly in XEG. The wild-type XEG
cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the
same substrate, but at various sites. Together, the absence of a loop in the
cleft and the presence of bulky Tyr457 determine the substrate specificity of
XEG.

PMID: 19682300 [PubMed - indexed for MEDLINE]


22. Mov Disord. 2009 Oct 15;24(13):2007-11.

Analysis of SCA2 and SCA3/MJD repeats in Parkinson's disease in mainland China:
genetic, clinical, and positron emission tomography findings.

Wang JL, Xiao B, Cui XX, Guo JF, Lei LF, Song XW, Shen L, Jiang H, Yan XX, Pan Q,
Long ZG, Xia K, Tang BS.

Department of Neurology, Xiangya Hospital, Central South University, Changsha,
Hunan, People's Republic of China.

To investigate the prevalence and clinical feature(s) of Parkinson's disease (PD)
patients with expanded (ATXN2 and MJD1) genes of spinocerebellar ataxia type 2
and 3 (SCA2 and SCA3/MJD) in a mainland Chinese population, CAG triplet repeat
expansions of (SCA2 and SCA3/MJD) genes (ATXN2 and MJD1) were analyzed in a
cohort of 452 PD patients, including 386 sporadic and 66 familial forms. Striatal
dopamine transporter was evaluated in two SCA2 and two SCA3/MJD-positive family
members, an idiopathic PD patient and a healthy control using carbon (C11)
[(11)C]-radiolabeled-CFT positron emission tomography (PET). We found two
patients in one familial PD (FPD) family (1.5%) and two sporadic PD patients
(0.5%) with expanded CAG repeats in the ATXN2 locus, four patients in two FPD
families (3%) and another three sporadic PD patients (0.8%) in the MJD1 locus.
[(11)C]-CFT PET in detected members in SCA2 and SCA3/MJD families showed
decrements of (11)C-CFT uptake. These findings suggest that a mutation in SCA2 or
SCA3/MJD may be one of the genetic causes of PD.

PMID: 19672991 [PubMed - indexed for MEDLINE]


23. J Cell Mol Med. 2009 Jul 20. [Epub ahead of print]

Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more
efficient CD8(+) CTL- and NK-mediated antitumor immunity than exosomes released
from heat-shocked tumor cells expressing cytoplasmic HSP70.

Xie Y, Bai O, Zhang H, Yuan J, Zong S, Chibbar R, Slattery K, Qureshi M, Wei Y,
Deng Y, Xiang J.

Research Unit, Division of Health Research, Saskatchewan Cancer Agency,
Department of Oncology, University of Saskatchewan, Saskatoon, Saskatchewan S7N
4H4, Canada.

ABSTRACT Exosomes (EXO) derived from tumor cells have been used to stimulate
antitumor immune responses, but only resulting in prophylatic immunity.
Tumor-derived heat shock protein 70 (HSP70) molecules are molecular chaperones
with a broad repertoire of tumor antigen peptides capable of stimulating
dendritic cell (DC) maturation and T cell immune responses. To enhance EXO-based 
antitumor immunity, we generated an engineered myeloma cell line J558(HSP)
expressing endogenous P1A tumor antigen and transgenic form of membrane-bound
HSP70 and heat-shocked J558(HS) expressing cytoplasmic HSP70, and purified
EXO(HSP) and EXO(HS) from J558(HSP) and J558(HS) tumor cell culture supernatants 
by ultracentrifugation. We found that EXO(HSP) were able to more efficiently
stimulate maturation of DC with upregulation of Ia(b), CD40, CD80 and
inflammatory cytokines than EXO(HS) after overnight incubation of immature
bone-marrow-derived DC (5X10(6) cells) with EXO (100 mug), respectively. We also 
i.v. immunized BALB/c mice with EXO (30 mug/mouse) and assessed P1A-specific T
cell responses after immunization. We demonstrate that EXO(HSP) are able to
stimulate type 1 CD4(+) helper T (Th1) cell responses, and more efficient
P1A-specific CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumor immunity
than EXO(HS). In addition, we further elucidate that EXO(HSP)-stimulated
antitumor immunity is mediated by both P1A-specific CD8(+) CTL and
non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound
HSP70-expressing tumor cell-released EXO may represent a more effective EXO-based
vaccine in induction of antitumor immunity.

PMID: 19627400 [PubMed - as supplied by publisher]


24. Mol Biol Cell. 2009 Aug;20(16):3725-39. Epub 2009 Jun 24.

{alpha}-synuclein and its A30P mutant affect actin cytoskeletal structure and
dynamics.

Sousa VL, Bellani S, Giannandrea M, Yousuf M, Valtorta F, Meldolesi J,
Chieregatti E.

Department of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.

The function of alpha-synuclein, a soluble protein abundant in the brain and
concentrated at presynaptic terminals, is still undefined. Yet, alpha-synuclein
overexpression and the expression of its A30P mutant are associated with familial
Parkinson's disease. Working in cell-free conditions, in two cell lines as well
as in primary neurons we demonstrate that alpha-synuclein and its A30P mutant
have different effects on actin polymerization. Wild-type alpha-synuclein binds
actin, slows down its polymerization and accelerates its depolymerization,
probably by monomer sequestration; A30P mutant alpha-synuclein increases the rate
of actin polymerization and disrupts the cytoskeleton during reassembly of actin 
filaments. Consequently, in cells expressing mutant alpha-synuclein,
cytoskeleton-dependent processes, such as cell migration, are inhibited, while
exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying 
a deletion of the alpha-synuclein gene, electroporation of wild-type
alpha-synuclein increases actin instability during remodeling, with growth of
lamellipodia-like structures and apparent cell enlargement, whereas A30P
alpha-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. 
In conclusion, alpha-synuclein appears to play a major role in actin cytoskeletal
dynamics and various aspects of microfilament function. Actin cytoskeletal
disruption induced by the A30P mutant might alter various cellular processes and 
thereby play a role in the pathogenesis of neurodegeneration.

PMCID: PMC2777932
PMID: 19553474 [PubMed - indexed for MEDLINE]


25. Mol Plant Microbe Interact. 2009 Jul;22(7):783-9.

Gene disruption of an arabinofuranosidase/beta-xylosidase precursor decreases
Sclerotinia sclerotiorum virulence on canola tissue.

Yajima W, Liang Y, Kav NN.

Department of Agriculture, Food and Nutritional Science, University of Alberta,
Edmonton, Alberta, Canada.

Although Sclerotinia sclerotiorum (Lib.) de Bary has been studied extensively,
there are still aspects of this important phytopathogen's ability to cause
disease in susceptible plants that remain unclear. A recent comprehensive
proteome-level investigation of this fungus identified a number of proteins whose
functions in disease initiation and progression have not been clearly
established. Included among these proteins was an
arabinofuranosidase/beta-xylosidase precursor whose role as a potential virulence
factor had not been investigated previously. This article describes the
generation of gene-disrupted mutant S. sclerotiorum unable to produce this
arabinofuranosidase/beta-xylosidase precursor as well as the comparison of the
virulence of this mutant with that of wild-type mycelia on susceptible canola
leaves and stems. At all time points tested, the degree of necrosis was observed 
to be significantly greater on the plant tissue inoculated with wild-type
mycelia. To our knowledge, this is the first report that clearly demonstrates
that this arabinofuranosidase/beta-xylosidase precursor is a virulence factor for
S. sclerotiorum.

PMID: 19522560 [PubMed - indexed for MEDLINE]


26. Int J Syst Evol Microbiol. 2009 Jun;59(Pt 6):1342-7.

Paenibacillus pectinilyticus sp. nov., isolated from the gut of Diestrammena
apicalis.

Park DS, Jeong WJ, Lee KH, Oh HW, Kim BC, Bae KS, Park HY.

Biological Resources Center, KRIBB, Daejeon 305-806, Republic of Korea.

During a search for exo-enzyme-producing bacteria in the gut of an insect,
Diestrammena apicalis, a novel bacterium capable of degrading pectin was
isolated. The isolate, designated strain RCB-08(T), comprised Gram-positive,
endospore-forming, motile rods capable of growth at 15-30 degrees C and pH
6.0-8.7. The DNA G+C content of the isolate was 51.5 mol% and the predominant
cellular fatty acid was anteiso-C(15 : 0) (74.1 %). Phylogenetic analysis based
on 16S rRNA gene sequences showed that strain RCB-08(T) was affiliated with a
cluster within the Paenibacillaceae, and was related most closely to
Paenibacillus chondroitinus NBRC 15376(T), with a sequence similarity of 96.7 %. 
The DNA-DNA relatedness value for strain RCB-08(T) with P. chondroitinus NBRC
15376(T) was 15.0 %. Strain RCB-08(T) hydrolysed pectin, but not cellulose,
casein, starch or xylan. Strain RCB-08(T) could be clearly distinguished from
other Paenibacillus species on the basis of characteristics observed using a
polyphasic approach. Therefore strain RCB-08(T) is considered to represent a
novel species of the genus Paenibacillus, for which the name Paenibacillus
pectinilyticus sp. nov. is proposed. The type strain is RCB-08(T) (=KCTC
13222(T)=CECT 7358(T)).

PMID: 19502313 [PubMed - indexed for MEDLINE]


27. Acc Chem Res. 2009 Aug 18;42(8):1026-36.

Mechanistic insights on the cycloisomerization of polyunsaturated precursors
catalyzed by platinum and gold complexes.

Soriano E, Marco-Contelles J.

Laboratorio de Radicales Libres y Quimica Computacional, IQOG (CSIC), Juan de la 
Cierva 3; 28006 Madrid, Spain.

Organometallic chemistry provides powerful tools for the stereocontrolled
synthesis of heterocycles and carbocycles. The electrophilic transition metals
Pt(II) and Au(I, III) are efficient catalysts in these transitions and promote a 
variety of organic transformations of unsaturated precursors. These reactions
produce functionalized cyclic and acyclic scaffolds for the synthesis of natural 
and non-natural products efficiently, under mild conditions, and with excellent
chemoselectivity. Because these transformations are strongly substrate-dependent,
they are versatile and may yield diverse molecular scaffolds. Therefore,
synthetic chemists need a mechanistic interpretation to optimize this reaction
process and design a new generation of catalysts. However, so far, no
intermediate species has been isolated or characterized, so the formulated
mechanistic hypotheses have been primarily based on labeling studies or trapping 
reactions. Recently, theoretical DFT studies have become a useful tool in our
research, giving us insights into the key intermediates and into a variety of
plausible reaction pathways. In this Account, we present a comprehensive
mechanistic overview of transformations promoted by Pt and Au in a
non-nucleophilic medium based on quantum-mechanical studies. The calculations are
consistent with the experimental observations and provide fundamental insights
into the versatility of these reaction processes. The reactivity of these metals 
results from their peculiar Lewis acid properties: the alkynophilic character of 
these soft metals and the pi-acid activation of unsaturated groups promotes the
intra- or intermolecular attack of a nucleophile. 1,n-Enynes (n = 3-8) are
particularly important precursors, and their transformation may yield a variety
of cycloadducts depending on the molecular structure. However, the calculations
suggest that these different cyclizations would have closely related reaction
mechanisms, and we propose a unified mechanistic picture. The intramolecular
nucleophilic attack of the double bond on the activated alkyne takes place by an 
endo-dig or exo-dig pathway to afford a cyclopropyl-metallocarbenoid. Through
divergent routes, the cyclopropyl intermediate formed by exo-cyclopropanation
could yield the metathesis adduct or bicyclic compounds. The endo-cyclization may
be followed by a [1,2]-migration of the propargyl moiety to the internal
acetylenic position to afford bicyclic [n.1.0] derivatives. This reaction
mechanism is applicable for functional groups ranging from H to carboxylate
propargyl substituents (Rautenstrauch reaction). In intramolecular reactions in
which a shorter enyne bears a propargyl ester or in intermolecular reactions of
an ester with an alkene, the ester preferentially attacks the activated alkyne
because of enthalpic (ring strain) and entropic effects. Our calculations can
predict the correct stereochemical outcome, which may aid the rational design of 
further stereoselective syntheses. The alkynes activated by electrophilic species
can also react with other nucleophiles, such as aromatic rings. The calculations 
account for the high endo-selectivity observed and suggest that this
transformation takes place through a Friedel-Crafts-type alkenylation mechanism, 
where the endo-dig cyclization promoted by PtCl(2) may involve a
cyclopropylmetallacarbene as intermediate before the formation of the expected
Wheland-type intermediate. These comparisons of the computational approach with
experiment demonstrate the value of theory in the development of a solid
mechanistic understanding of these reaction processes.

PMID: 19480448 [PubMed]


28. Bioorg Med Chem. 2009 Jun 1;17(11):3782-8. Epub 2009 May 3.

Transcription and reverse transcription of artificial nucleic acids involving
backbone modification by template-directed DNA polymerase reactions.

Kuwahara M, Takeshima H, Nagashima J, Minezaki S, Ozaki H, Sawai H.

Department of Chemistry and Chemical Biology, Graduate School of Engineering,
Gunma University, Gunma 376-8515, Japan. kuwahara@chem-bio.gunma-u.ac.jp

Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been
replaced with an amide-type linker [-CH(2)C=ONH-] or an amine-type linker
[-CH(2)CH(2)NH-] were synthesized to investigate the effect of these backbone
modifications on polymerase reactions. In addition, a triphosphate analogue of
thymidine dinucleotide with the amide-type linker was synthesized and enzymatic
insertion of the amide linkage into ODN was attempted using this analogue for the
polymerase reaction. Primer extension reactions using three types of thermostable
DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays.
Analysis of these data indicate that (i) the polymerase reaction tends to be
affected much more by insertion of the cationic flexible amine-type linker than
by insertion of the neutral rigid amide-type linker; (ii) the backbone
modification has a greater effect on the polymerase reaction when it is adjacent 
to the 3'-end of a primer as the elongation terminus than when it is on the
template, as well as in base or sugar modification; (iii) although the modified
linker in the modified DNA template is passed beyond by the polymerase, it still 
affects the extension reaction several bases downstream from its location; (iv)
the modified linker in the template, in some cases, also affects the extension
reaction upstream from its location; (v) further improvement of the chemical
structure is required for dinucleotide-mimic incorporation.

PMID: 19427792 [PubMed - indexed for MEDLINE]


29. J Biol Chem. 2009 Jun 26;284(26):17404-10. Epub 2009 May 1.

A new sialidase mechanism: bacteriophage K1F endo-sialidase is an inverting
glycosidase.

Morley TJ, Willis LM, Whitfield C, Wakarchuk WW, Withers SG.

Department of Chemistry, University of British Columbia, Vancouver, British
Columbia V6T 1Z1.

Bacteriophages specific for Escherichia coli K1 express a tailspike protein that 
degrades the polysialic acid coat of E. coli K1 that is essential for
bacteriophage infection. This enzyme is specific for polysialic acid and is a
member of a family of endo-sialidases. This family is unusual because all other
previously reported sialidases outside of this family are exo- or
trans-sialidases. The recently determined structure of an endo-sialidase derived 
from bacteriophage K1F (endoNF) revealed an active site that lacks a number of
the residues that are conserved in other sialidases, implying a new,
endo-sialidase-specific catalytic mechanism. Using synthetic
trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for
hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a
series of pH values revealed a dependence on a single protonated group of pKa 5. 
Mutation of a putative active site acidic residue, E581A, resulted in complete
loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of
trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting
sialidase. All other wild type sialidases previously reported are retaining
glycosidases, implying a new mechanism of sialidase action specific to this
family of endo-sialidases.

PMCID: PMC2719380 [Available on 2010/6/26]
PMID: 19411257 [PubMed - indexed for MEDLINE]


30. J Leukoc Biol. 2009 Sep;86(3):491-504. Epub 2009 Apr 28.

Lethal pulmonary infection with Francisella novicida is associated with severe
sepsis.

Sharma J, Li Q, Mishra BB, Pena C, Teale JM.

South Texas Center for Emerging Diseases and Department of Biology, University of
Texas at San Antonio, San Antonio, Texas 78249-1644, USA.

Comment in:
    J Leukoc Biol. 2009 Sep;86(3):469-70.

The bacterial or host determinants of lethality associated with respiratory
Francisella infections are currently unknown. No exo- or endotoxins that
contribute to the severity of this disease have been identified. However, a
deregulated host immune response upon infection is characterized by an initial
36- to 48-h delay followed by a rapid and excessive inflammatory response prior
to death at 72-120 h. Here, we extend these findings by comparing host immune
responses between sublethal and lethal respiratory infections of mice with an
attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) 
versus the wild-type (WT) strain (lethal). Infection with WT bacteria, but not
the Mut, was characterized by sustained bacteremia and systemic dissemination of 
the pathogen with temporal increases in bacterial burdens in liver and spleen.
Severe pathology with large foci of infiltrates associated with extensive tissue 
damage was evident in WT-infected lungs, and Mut-infected mice displayed much
reduced pathology with intact lung architecture. Similar to other experimental
models of severe sepsis, WT- but not the Mut-infected mice exhibited a robust
increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant
depletion of alphabeta T cells. Further, a dramatic up-regulation of multiple
cytokines and chemokines was observed only in lethal WT infection. In addition,
an earlier and larger increased expression of S100A9, a known mediator of sepsis,
was observed in WT-infected mice. Taken together, these results show that a
hyperinflammatory host immune response, culminating in severe sepsis, is
responsible for the lethal outcome of respiratory tularemia.

PMCID: PMC2735285 [Available on 2010/9/1]
PMID: 19401387 [PubMed - indexed for MEDLINE]


31. Bioorg Med Chem Lett. 2009 May 1;19(9):2444-7. Epub 2009 Mar 18.

Molecular design, synthesis and docking study of benz[b]oxepines and
12-oxobenzo[c]phenanthridinones as topoisomerase 1 inhibitors.

Lee SH, Van HT, Yang SH, Lee KT, Kwon Y, Cho WJ.

College of Pharmacy and Research Institute of Drug Development, Chonnam National 
University, Gwangju 500-757, Republic of Korea.

Benz[b]oxepines 4a-g and 12-oxobenzo[c]phenanthridines 5a-d were designed and
synthesized as constrained forms of 3-arylisoquinolines through an intramolecular
radical cyclization reaction. Radical cyclization of O-vinyl compounds
preferentially led to the 7-endo-trig cyclization pathway to the benz[b]oxepines 
and 12-oxobenzo[c]phenanthridines through 6-exo-trig path as minor products.
Among the synthesized compounds, benz[b]oxepine derivative 4e exhibited potent in
vitro cytotoxicity against three different tumor cell lines, as well as
topoisomerase 1 inhibitory activity. A Surflex-Dock docking study was performed
to clarify the topoisomerase 1 activity of 4e.

PMID: 19345580 [PubMed - indexed for MEDLINE]


32. J Org Chem. 2009 May 1;74(9):3424-9.

Efficient diastereoselective synthesis of trifarane-type sesquiterpenes,
trifarienols A and B.

Takahashi K, Akao R, Honda T.

Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41, Shinagawa-ku,
Tokyo 142-8501, Japan.

Diastereoselective total synthesis of trifarienols A and B, trifarane-type
sesquiterpenes isolated from the Malaysian Cheilolejeunea trifaria, was achieved 
via an intramolecular Hosomi-Sakurai reaction of the aldehyde to construct a
substituted bicyclo[3.3.1]nonane skeleton having the exo-methylene moiety of the 
target compounds in one step.

PMID: 19334700 [PubMed - indexed for MEDLINE]


33. Strabismus. 2009 Jan-Mar;17(1):37-40.

Recurrence of intermittent exotropia: factors associated with surgical outcomes.

Koklanis K, Georgievski Z.

Department of Clinical Vision Sciences, La Trobe University, Department of
Ophthalmology, Royal Children's Hospital, Melbourne, Australia.
k.koklanis@latrobe.edu.au

INTRODUCTION: The surgery of choice for intermittent exotropia (XT) continues to 
be debated, with little evidence that one procedure provides a more successful
outcome than the other in the longer term. Many factors, however, may potentially
influence the outcome of strabismus surgery for intermittent XT. This study aimed
to investigate factors associated with the recurrence of an exo-deviation
following horizontal muscle surgery for intermittent XT of the divergence excess 
type. METHODS: We retrospectively reviewed the medical histories of patients who 
underwent surgery for the correction of intermittent XT between January 1998 and 
June 2005. The factors analyzed were as follows: sex, age of onset, age at
initial surgery, family history, size of preoperative deviation, near binocular
single vision, amblyopia, oblique dysfunction, refractive error, type of surgery,
and postoperative alignment. RESULTS: Eighty-nine patients were included in the
final analysis. Of these, 19 demonstrated recurrence of their deviation. The mean
follow-up period was 2 years. None of the factors analysed appeared to influence 
the outcome of intermittent XT surgery. CONCLUSION: We found that no single
factor influenced patients' responses to the surgical treatment of intermittent
XT. To address controversies and improve the evidence base regarding surgical
intervention of this condition, randomized controlled trials are needed and
justified because the results indicate that it would be relatively safe to
randomly allocate patients to groups who could receive differing treatments so as
to determine optimum management strategies.

PMID: 19301192 [PubMed - indexed for MEDLINE]


34. Inorg Chem. 2009 Apr 20;48(8):3581-90.

Influence of conformational flexibility on self-assembly and luminescence
properties of lanthanide coordination polymers with flexible exo-bidentate
biphenol derivatives.

Guo Y, Dou W, Zhou X, Liu W, Qin W, Zang Z, Zhang H, Wang D.

College of Chemistry and Chemical Engineering and State Key Laboratory of Applied
Organic Chemistry, Lanzhou University, Lanzhou 730000, China.

To explore how nonplanar conformational distortions affect supramolecular
self-assembly and properties of lanthanide complexes, we have designed and
synthesized two new flexible exo-bidentate ligands derived from biphenol
featuring two salicylamide pendant arms,
2,2'-bis{[(2'-benzylaminoformyl)phenoxyl]ethoxyl}-1,1'-biphenylene (L(I)) and
5,5'-dibromo-2,2'-bis{[(2'-benzylaminoformyl)phenoxyl]ethoxyl}-1,1'-biphenylene
(L(II)). These two structurally related ligands can have different conformations 
and are used for constructing diverse lanthanide polymers with interesting
luminescence properties. Among two series of lanthanide nitrate complexes which
have been characterized by elemental analysis, X-ray powder diffraction, and IR
spectroscopy, four new coordination polymers have been determined using X-ray
diffraction analysis. The coordination polymer type
{Ln(2)(NO(3))(6)(L(I))(3).3H(2)O}(infinity) (Ln = Nd, Sm, Eu, Gd, Tb or Dy)
displays a two-dimensional honeycomb-like framework in the ab plane, which can be
regarded as a (6,3) topological network with neodymium atoms acting as
"three-connected" centers. In contrast, the coordination polymer types
{[Nd(NO(3))(3)(L(II))(CH(3)OH)] x CH(3)OH}(infinity) and
[Ln(NO(3))(3)(L(II))(C(2)H(5)OH)](infinity) (Ln = Sm, Eu, Gd, Tb or Dy) possess
single-stranded helix chains which can be further connected through
intermolecular hydrogen bonds to form two-dimensional supramolecular sheets. The 
photophysical properties of trivalent Sm, Eu, Tb, and Dy complexes at room
temperature were investigated. The present work substantiates the claim that the 
supramolecular structure as well as the luminescence properties of the
coordination polymer can be tuned by controlling the conformational distortion of
a nonplanar flexible ligand in the supramolecular self-assembly.

PMID: 19290612 [PubMed - indexed for MEDLINE]


35. Inorg Chem. 2009 Mar 16;48(6):2526-33.

Eu(3)Co(2)In(15) and KM(2)In(9) (M = Co, Ni): 3D frameworks based on transition
metal centered In(9) clusters.

Lei XW, Zhong GH, Li LH, Hu CL, Li MJ, Mao JG.

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the
Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, P. R. China.

The title three compounds have been synthesized by solid state reactions at high 
temperatures, with excess indium as flux, and structurally characterized by
single-crystal X-ray diffraction studies. Eu(3)Co(2)In(15) forms a new structure 
type and crystallizes in the tetragonal space group P4/mbm (No. 127), whereas
KM(2)In(9) (M = Co, Ni) in the BaFe(2)Al(9) type crystallizes in the hexagonal
space group P6/mmm (No. 191). Their structures all feature a three-dimensional
anionic framework based on 1D [MIn(6)] single cluster chains composed of
face-sharing [MIn(9)] clusters. In Eu(3)Co(2)In(15), two adjacent [CoIn(6)]
single cluster chains form a [Co(2)In(11)] double cluster chain via
corner-sharing In atoms as well as In-In bonds; the latter chains are further
interconnected by additional indium atoms via In-In bonds into a complicated 3D
framework, forming two types of tunnels along the c-axis, which are filled by the
europium atoms. In KM(2)In(9), the [MIn(6)] single cluster chains are directly
interconnected via corner sharing and exo In-In bonds into a 3D framework with
the K(+) ions encapsulated in the 1D tunnels along the c-axis. Band structure
calculations of three compounds based on density functional theory methods
indicate that all three compounds are metallic.

PMID: 19267504 [PubMed - in process]


36. Prosthet Orthot Int. 2009 Mar;33(1):10-6.

Improvement of the soft socket after rotationplasty: a single case study.

Wessling M, Aach M, Hardes J, Janssen E, Rosenbaum D, Winkelmann W, Gebert C.

Department of Orthopaedics, University Hospital, University of Muenster,
Muenster, Germany. wessling@uni-muenster.de

Rotationplasty is established as a functionally improving and partially ablative 
method of tumour surgery, but good clinical and functional results do not only
depend on a successful surgery. Due to the changed biomechanical situation the
activity level is limited by the weight bearing capacity of the rotated foot.
Painful blisters and callosities may limit the use of the exo-prosthesis, because
the skin is overstressed in the soft socket. A 28-year-old patient with a
rotationplasty type A2 suffered from painful callosities of the rotated foot.
Capacitive pressure measurements were performed as well as a gait analysis for
kinematics and kinetic characteristics. Clinically a decrease of the callosities 
and a pain relieve was obvious and the patient learned skiing without prior
knowledge. Biomechanically a decrease of the peak pressure (from 240.6-135.0 kPa)
and the mean pressure (from 83.2-66.2 kPa), was observed with an increased weight
bearing area. The study has shown that a modification of the heel bench can
considerably improve pressure distribution. An increase of the load bearing area 
appears to enable the skin to compensate even intensive strain during athletic
activities.

PMID: 19235061 [PubMed - indexed for MEDLINE]


37. BMC Plant Biol. 2009 Feb 13;9:20.

The extracellular EXO protein mediates cell expansion in Arabidopsis leaves.

Schroder F, Lisso J, Lange P, Mussig C.

Max Planck Institute of Molecular Plant Physiology, Dept. Willmitzer, Am
Muhlenberg 1, 14476, Potsdam, Golm, Germany. schroeder@mpimp-golm.mpg.de

BACKGROUND: The EXO (EXORDIUM) gene was identified as a potential mediator of
brassinosteroid (BR)-promoted growth. It is part of a gene family with eight
members in Arabidopsis. EXO gene expression is under control of BR, and EXO
overexpression promotes shoot and root growth. In this study, the consequences of
loss of EXO function are described. RESULTS: The exo loss of function mutant
showed diminished leaf and root growth and reduced biomass production. Light and 
scanning electron microscopy analyses revealed that impaired leaf growth is due
to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were 
smaller in comparison to the wild-type. The exo mutant showed reduced
brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were 
significantly more sensitive to the inhibitory effect of synthetic brassinolide. 
Apart from reduced growth, exo did not show severe morphological abnormalities.
Gene expression analyses of leaf material identified genes that showed robust
EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and 
genes involved in primary and secondary metabolism showed weaker expression in
exo than in wild-type plants. However, the vast majority of BR-regulated genes
were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to 
the apoplast. CONCLUSION: The EXO gene is essential for cell expansion in leaves.
Gene expression patterns and growth assays suggest that EXO mediates BR-induced
leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the
shoot. EXO presumably is involved in a signalling process which coordinates
BR-responses with environmental or developmental signals. The hypersensitivity of
exo roots to BR suggests that EXO plays a diverse role in the control of BR
responses in the root.

PMCID: PMC2661892
PMID: 19216774 [PubMed - indexed for MEDLINE]


38. Appl Microbiol Biotechnol. 2009 Jun;83(4):659-68. Epub 2009 Feb 13.

An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8.

Papaneophytou CP, Pantazaki AA, Kyriakidis DA.

Department of Chemistry, Aristotle University of Thessaloniki, Greece.

The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a
polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and 
forms clear zones on agar plates containing PHB as sole carbon source. T.
thermophilus extracellular PHB depolymerase was purified to homogeneity using an 
affinity chromatography protocol. The purified enzyme was estimated to have an
apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was
identified as the TTHA0199 gene product of T. thermophilus HB8. The amino acid
sequence of the TTHA0199 gene product shared significant homologies to other
carboxylesterases. A catalytic triad was identified consisting of S(183), E(310),
and H(405). A pentapeptide sequence (GX(1)SX(2)G) exists within the molecule,
characteristic for PHB depolymerases (lipase box) and for other serine
hydrolases. Purified extracellular PHB depolymerase was stable at high
temperatures with an optimum activity at pH 8.0. The apparent Km value of the
purified enzyme for PHB was 53 microg/ml. As the main product of the enzymic
hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that 
the enzyme acts principally as an exo-type hydrolase.

PMID: 19214501 [PubMed - indexed for MEDLINE]


39. Chemistry. 2009;15(12):2861-73.

Synthesis and conformational analysis of (alpha-D-galactosyl)phenylmethane and
alpha-,beta-difluoromethane analogues: interactions with the plant lectin
viscumin.

Kolympadi M, Fontanella M, Venturi C, Andre S, Gabius HJ, Jimenez-Barbero J,
Vogel P.

Laboratoire de Glycochimie et Synthese Asymetrique, Swiss Federal Institute of
Technology (EPFL), Batochime, 1015 Lausanne, Switzerland.

(Alpha-D-galactosyl)phenylmethane (1), (alpha- and
beta-D-galactosyl)(difluoro)phenylmethane (2 and 3) have been prepared and their 
conformations in solution were described by using a combination of force-field
calculations and NMR spectroscopic studies. Galactoside 1 adopts a (4)C(1) chair 
conformation and an exo anomeric orientation, as is the case for natural
alpha-galactosides. The X-ray crystal structure of its difluoromethylene
derivative 2 similarly shows a (4)C(1) chair conformation. Surprisingly, compound
2 exhibits a different equilibrium between (1)C(4) chair and (1)S(3) skew boat
conformations and significant flexibility around the pseudoglycosidic linkage
when in solution. The beta-stereoisomer 3 adopts a major (4)C(1) chair
conformation. Interestingly, C-galactosides 1, 2, and 3 bind to viscumin (VAA), a
galactoside-specific lectin, which is confirmed by NMR experiments and docking
calculations.

PMID: 19185038 [PubMed - indexed for MEDLINE]


40. Biol Pharm Bull. 2009 Feb;32(2):186-9.

Oxidative stress induced by a dihydropyrazine derivative.

Takechi S, Nakahara K, Adachi M, Yamaguchi T.

Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan.
stakechi@ph.sojo-u.ac.jp

The Maillard reaction contributes to the complications of diabetes and normal
aging. Dihydropyrazines (DHPs), which are produced during the Maillard reaction, 
generate radicals and possess DNA strand-cleaving activities in vitro. In the
present study, we evaluated the genotoxic and cytotoxic potentials of a DHP
derivative, cyclohexyl-DHP, which is obtained as a mixture of two isomers,
2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline
(exo-type), fused with a cyclohexyl ring. Cyclohexyl-DHP caused DNA strand breaks
in plasmid pUC18, especially in the presence of Cu(2+). By using Escherichia coli
mutant strains, we observed that cyclohexyl-DHP exposure strongly reduced the
survival rate of a cytosolic sodium dodecyl sulfate (SOD)-deficient strain (sodA 
sodB), significantly reduced the survival rates of DNA repair-deficient strains
(recA and uvrB) and mildly reduced the survival rate of a catalase-deficient
strain (katE katG) compared with the survival rate of the wild-type strain.
Addition of Cu(2+) enhanced the cell killing ability of cyclohexyl-DHP. The
frequency of mutations induced by cyclohexyl-DHP increased dose-dependently in
the sodA sodB strain. Assays with the highly water-soluble tetrazolium salt WST-1
revealed that cyclohexyl-DHP strongly generated superoxide anions. Moreover,
cyclohexyl-DHP elevated the protein carbonyl levels in E. coli. These findings
indicate that cyclohexyl-DHP could potentially generate superoxide anions, and
cause not only breakage of chromosomal DNA leading to mutagenic lesions but also 
induce damage to cellular proteins.

PMID: 19182373 [PubMed - indexed for MEDLINE]


41. Blood. 2009 Mar 19;113(12):2673-83. Epub 2009 Jan 27.

Antigen-loaded exosomes alone induce Th1-type memory through a B-cell-dependent
mechanism.

Qazi KR, Gehrmann U, Domange Jordo E, Karlsson MC, Gabrielsson S.

Department of Medicine, Clinical Allergy Research Unit, Karolinska University
Hospital Solna, Stockholm, Sweden. khaleda.qazi@ki.se

Exosomes are nanovesicles harboring proteins important for antigen presentation. 
We compared the potency of differently loaded exosomes, directly loaded with
OVA(323-339) peptide (Pep-Exo) or exosomes from OVA-pulsed DCs (OVA-Exo), for
their ability to induce specific T-cell proliferation in vitro and in vivo. Both 
Pep-Exo and OVA-Exo elicited specific transgenic T-cell proliferation in vitro,
with the Pep-Exo being more efficient. In contrast, only OVA-Exo induced specific
T-cell responses in vivo highlighting the importance of indirect loading
strategies in clinical applications. Coadministration of whole OVA overcame the
unresponsiveness with Pep-Exo but still elicited a lower response compared with
OVA-Exo. In parallel, we found that OVA-Exo not only augmented the specific
T-cell response but also gave a Th1-type shift and an antibody response even in
the absence of whole OVA. We detected IgG2a and interferon-gamma production from 
splenocytes showing the capability of exosomes to provide antigen for B-cell
activation. Furthermore, we found that B cells are needed for exosomal T-cell
stimulation because Bruton tyrosine kinase-deficient mice showed abrogated B- and
T-cell responses after OVA-Exo immunization. These findings reveal that exosomes 
are potent immune regulators and are relevant for the design of vaccine adjuvants
and therapeutic intervention strategies to modulate immune responses.

PMID: 19176319 [PubMed - indexed for MEDLINE]


42. J Virol. 2009 Apr;83(7):2941-50. Epub 2009 Jan 7.

Reovirus FAST protein transmembrane domains function in a modular, primary
sequence-independent manner to mediate cell-cell membrane fusion.

Clancy EK, Duncan R.

Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova
Scotia, Canada B3H 1X5.

The FAST proteins are a unique family of virus-encoded cell-cell membrane fusion 
proteins. In the absence of a cleavable N-terminal signal peptide, a single-pass 
transmembrane domain (TMD) functions as a reverse signal-anchor to direct the
FAST proteins into the plasma membrane in an N(exo)/C(cyt) topology. There is
little information available on the role of the FAST protein TMD in the cell-cell
membrane fusion reaction. We show that in the absence of conservation in the
length or primary amino acid sequence, the p14 TMD can be functionally exchanged 
with the TMDs of the p10 and p15 FAST proteins. This is not the case for chimeric
p14 proteins containing the TMDs of two different enveloped viral fusion proteins
or a cellular membrane protein; such chimeric proteins were defective for both
pore formation and syncytiogenesis. TMD structural features that are conserved
within members of the FAST protein family presumably play direct roles in the
fusion reaction. Molecular modeling suggests that the funnel-shaped architecture 
of the FAST protein TMDs may represent such a conserved structural and functional
motif. Interestingly, although heterologous TMDs exert diverse influences on the 
trafficking of the p14 FAST protein, these TMDs are capable of functioning as
reverse signal-anchor sequences to direct p14 into lipid rafts in the correct
membrane topology. The FAST protein TMDs are therefore not primary determinants
of type III protein topology, but they do play a direct, sequence-independent
role in the membrane fusion reaction.

PMCID: PMC2655562
PMID: 19129451 [PubMed - indexed for MEDLINE]


43. AJNR Am J Neuroradiol. 2009 Mar;30(3):629-34. Epub 2008 Dec 26.

Abnormal basiocciput development in CHARGE syndrome.

Fujita K, Aida N, Asakura Y, Kurosawa K, Niwa T, Muroya K, Adachi M, Nishimura G,
Inoue T.

Department of Radiology, Endocrinology, Kanagawa Children's Medical Center,
Kanagawa, Japan. kazu_kcmc@yahoo.co.jp

BACKGROUND AND PURPOSE: The causative gene of the common congenital malformation 
referred to as CHARGE syndrome is CHD7. Affected individuals often undergo head
and neck imaging to assess abnormalities of the olfactory structures,
hypothalamus-pituitary axis, and inner ear. We encountered a few children with
severe hypoplasia of the basiocciput during a radiologic assessment of patients
with CHARGE syndrome. To our knowledge, this anomaly has not been reported. Our
purpose was to evaluate the incidence and severity of this anomaly in this
syndrome. MATERIALS AND METHODS: Sagittal MR images of 8 patients with CHARGE
syndrome were retrospectively reviewed by 2 radiologists who consensually
evaluated the status of the basiocciput of the patients with CHARGE syndrome, as 
either normal or hypoplastic; and associated anomalies, which include basilar
invagination, Chiari type I malformation, and syringomyelia, as either present or
absent. The length between the basion (Ba) and the endo-sphenobasion (Es) and
between the basion and the exo-sphenobasion (Xs) was measured on midsagittal MR
images of the 8 patients and 70 age-matched controls. We searched for trends
related to age in the length of Ba-Es and Ba-Xs of the control children by using 
a matched t test. RESULTS: Basioccipital hypoplasia was identified in 7 of the 8 
patients with CHARGE syndrome and was severe in 6. Of those, 5 had associated
basilar invagination and 1 had Chiari type I malformation with syringomyelia.
CONCLUSIONS: Basioccipital hypoplasia and basilar invagination are prevalent in
patients with CHARGE syndrome.

PMID: 19112063 [PubMed - indexed for MEDLINE]


44. J Biomol Struct Dyn. 2009 Feb;26(4):509-15.

Inactivation kinetics of beta-N-acetyl-D-glucosaminidase from green crab (Scylla 
serrata) in dioxane solution.

Xie JJ, Chen CQ, Yan YW, Zhang JP, Lin JC, Wang Q, Zhou HT, Chen QX.

Key Lab of Ministry of Education for Cell Biology and Tumor Cell Engineering,
School of Life Sciences, Xiamen University, Xiamen 361005, China.

Beta-N-acetyl-D-glucosaminidase (NAGase, EC.3.2.1.52), which catalyzes the
cleavage of N-acetylglucosamine polymers, is a composition of chitinase and
cooperates with endo-chitinase and exo-chitinase to disintegrate chitin into
N-acetylglucosamine (NAG). In this investigation, A NAGase from green crab
(Scylla serrata) was purified and the effects of dioxane on the enzyme activity
for the hydrolysis of p-Nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were 
studied. The results show that appropriate concentrations of dioxane can lead to 
reversible inactivation of the enzyme and the inactivation is classified as mixed
type. The value of IC50, the dioxane (inactivator) concentration leading to 50%
activity lost, is estimated to be 0.68%. The kinetics of inactivation of NAGase
in the appropriate concentrations of dioxane solution has been studied using the 
kinetic method of the substrate reaction. The rate constants of inactivation have
been determined. The results showed that k+0 is much larger than k'+0, indicating
the free enzyme molecule is more fragile than the enzyme-substrate complex in the
dioxane solution. It is suggested that the presence of the substrate offers
marked protection of this enzyme against inactivation by dioxane.

PMID: 19108590 [PubMed - in process]


45. Cell Microbiol. 2009 Mar;11(3):506-20. Epub 2008 Dec 3.

Type II fatty acid synthesis is essential only for malaria parasite late liver
stage development.

Vaughan AM, O'Neill MT, Tarun AS, Camargo N, Phuong TM, Aly AS, Cowman AF, Kappe 
SH.

Seattle Biomedical Research Institute, Seattle, WA 98109, USA.

Intracellular malaria parasites require lipids for growth and replication. They
possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that
localizes to the apicoplast plastid organelle and is assumed to be necessary for 
pathogenic blood stage replication. However, the importance of FAS II throughout 
the complex parasite life cycle remains unknown. We show in a rodent malaria
model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. 
Targeted deletion of FabB/F, a critical enzyme in fatty acid synthesis, did not
affect parasite blood stage replication, mosquito stage development and initial
infection in the liver. This was confirmed by knockout of FabZ, another critical 
FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to
form exo-erythrocytic merozoites, the invasive stage that first initiates blood
stage infection. Furthermore, deletion of FabI in the human malaria parasite
Plasmodium falciparum did not show a reduction in asexual blood stage replication
in vitro. Malaria parasites therefore depend on the intrinsic FAS II pathway only
at one specific life cycle transition point, from liver to blood.

PMCID: PMC2688669
PMID: 19068099 [PubMed - indexed for MEDLINE]


46. Beilstein J Org Chem. 2008;4:38. Epub 2008 Oct 24.

Radical cascades using enantioenriched 7-azabenzonorbornenes and their
applications in synthesis.

Hodgson DM, Winning LH.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford,
Mansfield Road, Oxford, OX1 3TA, UK;

Tandem deoxygenation-neophyl-type radical rearrangement-electrophile trapping
using xanthates from 7-azabenzonorbornadienes gives 3-exo-substituted
2-aza-5,6-benzonorbornenes, which in some cases undergo isomerisation to
(aminomethyl)indenes. The starting xanthates are accessible in good yields and
high enantiomeric ratios via asymmetric hydroboration of (aryne/pyrrole-derived) 
7-azabenzonorbornadienes. Oxidation (using RuO(4)) and Birch reduction of the
2-aza-5,6-benzonorbornenes provide access to substituted pyrrolidines and
tetrahydroindenes, respectively.

PMCID: PMC2587947
PMID: 19043626 [PubMed - in process]


47. Cancer Lett. 2009 Mar 18;275(2):256-65. Epub 2008 Nov 25.

MHC independent anti-tumor immune responses induced by Hsp70-enriched exosomes
generate tumor regression in murine models.

Cho JA, Lee YS, Kim SH, Ko JK, Kim CW.

Tumor Immunity Medical Research Center and Cancer Research Institute, Department 
of Pathology, Seoul National University College of Medicine, 28 Jong-ro gu,
Yun-gun dong, Seoul, Republic of Korea.

The ideal cancer vaccine should work regardless of MHC types but currently the
barrier generated by MHC specificity hampers the development of human cancer
vaccines, requesting to identify strong immunogenic molecules that can induce
anti-cancer immune responses without being affected by MHC polymorphism.
Tumor-derived exosomes are small membrane vesicles containing tumor antigens as
well as other immunologically important molecules such as MHC molecules and heat 
shock proteins (HSPs). Because of their potential immunogenicity, the plausible
utility of tumor-derived exosomes as an MHC independent cancer vaccine was
proposed. Here, we investigated whether Hsp70-enriched tumor exosomes can induce 
stronger immunogenicity as compared to normal tumor-derived exosomes in
autologous as well as allogeneic murine models in vitro and in vivo. Western
blotting showed that the exosomes of heat-treated tumor cells (HS Exo) contained 
higher amounts of Hsp70 than the exosomes of untreated cells (CNTL Exo). In both 
MHC type-identical and -irrelevant antigen-presenting cell models in vitro, HS
Exo triggered the increased expressions of MHC class II molecules. Crucially, HS 
Exo performed greater therapeutic capability in regressing pre-established MHC
type-identical and -irrelevant tumors than CNTL Exo in vivo. The analyses of
anti-tumor function in allogeneic mouse model demonstrated that HS Exo elicited
Th1-polarized immune responses defined by the increased productions of IgG2a and 
IFN-gamma. In summary, the Hsp70-enriched exosomes extracted from heat-treated
tumors induced strong Th1 immune responses, resulting in eliminating cancer cells
in allogeneic hosts in vivo. These results indicate that HS Exo is a potent MHC
independent cell-free cancer therapeutic agent that can be developed for clinical
trials.

PMID: 19036499 [PubMed - indexed for MEDLINE]


48. Neuropharmacology. 2009 Feb;56(2):531-40. Epub 2008 Oct 26.

Zinc regulates the dopamine transporter in a membrane potential and chloride
dependent manner.

Pifl C, Wolf A, Rebernik P, Reither H, Berger ML.

Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090
Vienna, Austria. christian.pifl@meduniwien.ac.at

The dopamine transporter (DAT), a membrane protein specifically expressed by
dopaminergic neurons and mediating the action of psychostimulants and
dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with
the protein. Herein, we report a host-cell-specific direction of the Zn(2+)
effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by 
DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC
cells. Inhibition or stimulation was lost in a DAT construct without the binding 
site for Zn(2+). Also reverse transport was differentially affected by Zn(2+),
dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells.
Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an
activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on
uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells.
Patch-clamp experiments under non-voltage-clamped conditions revealed a
significantly higher membrane potential of HEK293 than SK-N-MC cells and a
reduced membrane potential after phorbol ester treatment. Lowering chloride in
the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to
an inhibitory, whereas high potassium depolarization of HEK293 cells switched the
inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first
evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane
potential and chloride.

PMID: 19000913 [PubMed - indexed for MEDLINE]


49. Bioorg Med Chem. 2008 Dec 1;16(23):9891-7. Epub 2008 Oct 17.

Design and synthesis of novel 2',3'-dideoxy-4'-selenonucleosides as potential
antiviral agents.

Jeong LS, Choi YN, Tosh DK, Choi WJ, Kim HO, Choi J.

Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University,
Seoul 120-750, Republic of Korea. lakjeong@ewha.ac.kr

On the basis of potent anti-HIV activity of 2',3'-dideoxynucleosides (ddNs),
their bioisosteric analogues, 2',3'-dideoxy-4'-selenonucleosides (4'-seleno-ddNs)
were first synthesized from a chiral template, d-glutamic acid using
stereoselective ring-closure reaction of the dimesylate with Se(2-) and Pummerer 
type condensation of the selenoxide with nucleobases as key steps. X-ray
crystallographic analysis indicated that 4'-seleno-ddNs adopted the same
C2'-endo/C3'-exo (South) conformation as anti-HIV active ddNs, but did not show
anti-HIV activity, indicating that RT seems to prefer the C2'-exo/C3'-endo
(North) conformation on binding with their triphosphates.

PMID: 18977147 [PubMed - indexed for MEDLINE]


50. Biochim Biophys Acta. 2009 Jan;1794(1):144-58. Epub 2008 Oct 10.

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from 
Selenomonas ruminantium.

Jordan DB, Mertens JA, Braker JD.

National Center for Agricultural Utilization Research, U.S. Department of
Agriculture, Agricultural Research Service, Peoria, IL 61604, USA.
douglas.jordan@ars.usda.gov

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the
protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186,
pK(a) 7.2) which reside in subsite -1 of the two-subsite active site. Cationic
aminoalcohols are shown to bind exclusively to subsite -1 of the
catalytically-inactive, dianionic enzyme (D14(-)E186(-)). Enzyme (E) and
aminoalcohols (A) form E-A with the affinity progression:
triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the
K(i)(triethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols,
affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The
single access route for ligands into the active site dictates ordered formation
of E-A followed by E-A-I. E-A-I forms with the affinity progression:
ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is
seen in formation of E-A-substrate complexes with substrate
4-nitrophenyl-beta-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols 
versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on
the strength of E-A-4NPX. E-A-substrate complexes form weakly with substrate
4-nitrophenyl-alpha-l-arabinofuranoside (4NPA), and inhibition patterns appear
competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%)
contamination of E186A preparations (including a His-Tagged form) by
wild-type-like enzyme, likely originating from translational misreading.
Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts
when used unwarily as buffer components.

PMID: 18973836 [PubMed - indexed for MEDLINE]


51. J Ind Microbiol Biotechnol. 2009 Feb;36(2):195-203. Epub 2008 Oct 21.

Purification and characterization of chitinases from Paecilomyces variotii DG-3
parasitizing on Meloidogyne incognita eggs.

Nguyen VN, Oh IJ, Kim YJ, Kim KY, Kim YC, Park RD.

Division of Applied Bioscience and Biotechnology, Institute of Agricultural
Science and Technology, Chonnam National University, Gwangju, 500-757, South
Korea. ngdaonam963@yahoo.com

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a
chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE
Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a
monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46),
respectively, and showed chitinase activity bands with 0.01% glycol chitin as a
substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of 
Chi32 and Chi46 were determined to be
Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe
and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively.
Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60
degrees C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal
ions Ag(+) and Hg(2+) while Chi46 by Hg(2+) and Pb(2+) at a 10 mM concentration
but both enzymes were enhanced by 1 mM concentration of Co(2+). On analyzing the 
hydrolyzates of chitin oligomers [(GlcNAc)( n ), n = 2-6)], it was considered
that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an
endo-type chitinase.

PMID: 18936994 [PubMed - indexed for MEDLINE]


52. J Am Chem Soc. 2008 Nov 12;130(45):15105-15. Epub 2008 Oct 18.

The structure of a TNA-TNA complex in solution: NMR study of the octamer duplex
derived from alpha-(L)-threofuranosyl-(3'-2')-CGAATTCG.

Ebert MO, Mang C, Krishnamurthy R, Eschenmoser A, Jaun B.

Laboratory of Organic Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093
Zurich, Switzerland.

TNA (alpha-( l)-threofuranosyl-(3'-2') nucleic acid) is a nucleic acid in which
the ribofuranose building block of the natural nucleic acid RNA is replaced by
the tetrofuranose alpha-( l)-threose. This shortens the repetitive unit of the
backbone by one bond as compared to the natural systems. Among the alternative
nucleic acid structures studied so far in our laboratories in the etiological
context, TNA is the only one that exhibits Watson-Crick pairing not only with
itself but also with DNA and, even more strongly, with RNA. Using NMR
spectroscopy, we have determined the structure of a duplex consisting entirely of
TNA nucleotides. The TNA octamer (3'-2')-CGAATTCG forms a right-handed double
helix with antiparallel strands paired according to the Watson-Crick mode. The
dominant conformation of the sugar units has the 2'- and 3'-phosphodiester
substituents in quasi-diaxial position and corresponds to a 4'-exo puckering.
With 5.85 A, the average sequential P i -P i+1 distances of TNA are shorter than 
for A-type DNA (6.2 A). The helix parameters, in particular the slide and
x-displacement, as well as the shallow and wide minor groove, place the TNA
duplex in the structural vicinity of A-type DNA and RNA.

PMID: 18928287 [PubMed - indexed for MEDLINE]


53. Channels (Austin). 2008 Nov-Dec;2(6):419-28. Epub 2008 Nov 2.

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases.

Marin AN, Prica CM, Amuzescu BP, Neaga EI, Flonta ML.

Department of Biophysics and Physiology, Faculty of Biology, University of
Bucharest, Bucharest, Romania.

Analgesia induced by certain tricyclic antidepressants has been largely used for 
decades, yet the mechanisms involved are incompletely understood. Starting from
previously reported dual effects of amitriptyline on wild-type ENaC (Pena F, et
al. J Pharm Pharmacol 54:1393-8: 2002), we extended our study to ASIC1a by
performing a series of whole cell and single-channel recordings of
proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min
intervals, and amitriptyline (1-500 microM) was applied at a holding pH of 7.4 or
8.4 between pulses. Dose-response plots were fitted with dual Hill type
functions, yielding a half-activatory constant of 0.3 microM and a
half-inhibitory constant of 382 microM at pH 7.4. At pH 8.4 both constants were
shifted to higher values (0.5 and 444 microM, respectively). In whole-cell
experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 
microM and decreased it at 1 and 100 microM. Single-channel recordings were
idealized and fitted using an 8-state linear connectivity model comprising four
consecutive activation steps. Both amitriptyline (1 microM) and FMRF-amide (0.1
microM) increased the unitary current amplitude, and modified the opening and
closing rates of the first gating mode. They also increased the transition rate
from the second to the first gating mode, and the rate of final closure. The
activatory effect of both compounds vanished after a mild trypsin pretreatment,
suggesting the existence of activatory sites for FMRF-amide and amitriptyline in 
the outer vestibule of ASIC1a, which can be removed by exo- or endogenous
serine-proteases.

PMID: 18927513 [PubMed - indexed for MEDLINE]


54. J Neurochem. 2008 Nov;107(4):928-40. Epub 2008 Sep 11.

Interaction of cocaine-, benztropine-, and GBR12909-like compounds with wild-type
and mutant human dopamine transporters: molecular features that differentially
determine antagonist-binding properties.

Schmitt KC, Zhen J, Kharkar P, Mishra M, Chen N, Dutta AK, Reith ME.

Department of Psychiatry, New York University School of Medicine, New York, New
York, USA.

The widely abused psychostimulant cocaine is thought to elicit its reinforcing
effects primarily via inhibition of the neuronal dopamine transporter (DAT).
However, not all DAT inhibitors share cocaine's behavioral profile, despite
similar or greater affinity for the DAT. This may be due to differential
molecular interactions with the DAT. Our previous work using transporter mutants 
with altered conformational equilibrium (W84L and D313N) indicated that
benztropine and GBR12909 interact with the DAT in a different manner than
cocaine. Here, we expand upon these previous findings, studying a number of
structurally different DAT inhibitors for their ability to inhibit [(3)H]CFT
binding to wild-type, W84L and D313N transporters. We systematically tested
structural intermediates between cocaine and benztropine, structural hybrids of
benztropine and GBR12909 and a number of other structurally heterologous
inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine)
exhibited a cocaine-like binding profile with respect to mutation, whereas
compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were 
dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine
and tropane analogues, compounds with 3alpha stereochemistry tended to exhibit
benztropine-like binding, whereas those with 3beta stereochemistry were more
cocaine-like. Our results point to the importance of specific molecular
features--most notably the presence of a diphenylmethoxy moiety--in determining a
compound's binding profile. This study furthers the concept of using DAT mutants 
to differentiate cocaine-like inhibitors from atypical inhibitors in vitro.
Further studies of the molecular features that define inhibitor-transporter
interaction could lead to the development of DAT inhibitors with differential
clinical utility.

PMCID: PMC2728472
PMID: 18786172 [PubMed - indexed for MEDLINE]


55. J Org Chem. 2008 Oct 17;73(20):7921-7. Epub 2008 Sep 12.

Synthesis of tricyclic phosphonopyrrolidines via IMDAF: experimental and
theoretical investigation of the observed stereoselectivity.

Claeys DD, Moonen K, Roman BI, Nemykin VN, Zhdankin VV, Waroquier M, Van
Speybroeck V, Stevens CV.

Research Group SynBioC, Department of Organic Chemistry, Faculty of Bioscience
Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium.

During the synthesis of tricyclic phosphonopyrrolidines via intramolecular
Diels-Alder reactions of 1-acylamino(furan-2-yl)methyl phosphonates, two isomers 
are formed in most cases. The presence of a short three-atom tether together with
spectroscopic data, including difference NOE, revealed that the cycloaddition
occurred exo, but the phosphonate substituent on the tether had an exo or endo
orientation. This was confirmed via X-ray analysis. A thermodynamic preference
for the product with the phosphonate function in the endo position was observed
experimentally and was confirmed theoretically. Density functional theory methods
and several high-level post Hartree-Fock procedures were used to rationalize the 
observed isomer ratio of the IMDAF-reactions. This was done for two different
types of reagents: with the activating carbonyl group in the tether or as a
substituent on the tether. For the first type of molecules there is a large
steric hindrance of the bulky tether substituents that disfavors the exo-isomer. 
In the latter case, there was a very small energy difference between the
transition states causing a mixture of epimers being formed.

PMID: 18785707 [PubMed - indexed for MEDLINE]


56. J Neurosci. 2008 Sep 10;28(37):9122-32.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like 
microvesicles in astrocytes.

Marchaland J, Cali C, Voglmaier SM, Li H, Regazzi R, Edwards RH, Bezzi P.

Department of Cell Biology and Morphology, Faculty of Biology and Medicine,
University of Lausanne, 1005 Lausanne, Switzerland.

Astrocytes are the most abundant glial cell type in the brain. Although not
apposite for long-range rapid electrical communication, astrocytes share with
neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter
exocytosis. Despite this recent finding, little is known about the specific
properties of regulated secretion and vesicle recycling in astrocytes. Important 
differences may exist with the neuronal exocytosis, starting from the fact that
stimulus-secretion coupling in astrocytes is voltage independent, mediated by
G-protein-coupled receptors and the release of Ca(2+) from internal stores.
Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is,
therefore, of primary importance for understanding the mode of communication of
these cells and their role in brain signaling. We here take advantage of
fluorescent tools recently developed for studying recycling of glutamatergic
vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine 
epifluorescence and total internal reflection fluorescence imaging to investigate
with unprecedented temporal and spatial resolution, the stimulus-secretion
coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles 
(SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in
astrocytes proceeds with a time course on the millisecond time scale
(tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) 
exocytosis is controlled by local Ca(2+) microdomains. We identified
submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli
reaching beneath the plasma membrane and containing SLMVs at which fast
(time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise
spatial-temporal correlation with exocytic fusion events. Overall, the above
characteristics of transmitter exocytosis from astrocytes support a role of this 
process in fast synaptic modulation.

PMID: 18784293 [PubMed - indexed for MEDLINE]


57. Biotechnol Bioeng. 2008 Dec 1;101(5):1053-71.

Genome-scale model for Clostridium acetobutylicum: Part II. Development of
specific proton flux states and numerically determined sub-systems.

Senger RS, Papoutsakis ET.

Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way,
Newark, Delaware 19711, USA. senger@dbi.udel.edu

A regulated genome-scale model for Clostridium acetobutylicum ATCC 824 was
developed based on its metabolic network reconstruction. To aid model convergence
and limit the number of flux-vector possible solutions (the size of the
phenotypic solution space), modeling strategies were developed to impose a new
type of constraint at the endo-exo-metabolome interface. This constraint is
termed the specific proton flux state, and its use enabled accurate prediction of
the extracellular medium pH during vegetative growth of batch cultures. The
specific proton flux refers to the influx or efflux of free protons (per unit
biomass) across the cell membrane. A specific proton flux state encompasses a
defined range of specific proton fluxes and includes all metabolic flux
distributions resulting in a specific proton flux within this range. Effective
simulation of time-course batch fermentation required the use of independent flux
balance solutions from an optimum set of specific proton flux states. Using a
real-coded genetic algorithm to optimize temporal bounds of specific proton flux 
states, we show that six separate specific proton flux states are required to
model vegetative-growth metabolism and accurately predict the extracellular
medium pH. Further, we define the apparent proton flux stoichiometry per weak
acids efflux and show that this value decreases from approximately 3.5 mol of
protons secreted per mole of weak acids at the start of the culture to
approximately 0 at the end of vegetative growth. Calculations revealed that when 
specific weak acids production is maximized in vegetative growth, the net proton 
exchange between the cell and environment occurs primarily through weak acids
efflux (apparent proton flux stoichiometry is 1). However, proton efflux through 
cation channels during the early stages of acidogenesis was found to be
significant. We have also developed the concept of numerically determined
sub-systems of genome-scale metabolic networks here as a sub-network with a
one-dimensional null space basis set. A numerically determined sub-system was
constructed in the genome-scale metabolic network to study the flux magnitudes
and directions of acetylornithine transaminase, alanine racemase, and D-alanine
transaminase. These results were then used to establish additional constraints
for the genome-scale model.

PMCID: PMC2745297
PMID: 18767191 [PubMed - indexed for MEDLINE]


58. Chemistry. 2008;14(31):9772-8.

Recent advances in "formal" ruthenium-catalyzed [2+2+2] cycloaddition reactions
of diynes to alkenes.

Garcia-Rubin S, Varela JA, Castedo L, Saa C.

Departamento de Quimica Organica, Facultad de Quimica, Universidad de Santiago de
Compostela, 15782 Santiago de Compostela (Spain).

"Formal" and standard RuII-catalyzed [2+2+2] cycloaddition of 1,6-diynes to
alkenes gave bicyclic 1,3-cyclohexadienes in relatively good yields. When
terminal 1,6-diynes 1 were used, two isomeric bicyclic 1,3-cyclohexadienes 4 or 6
were obtained, depending on the acyclic or cyclic nature of the alkene partner.
When unsymmetrical substituted 1,6-diynes 7 were used, the reaction with acyclic 
alkenes took place regio- and stereoselectively to afford bicyclic
1,3-cyclohexadienes 8. A cascade process that behaves as a "formal"
RuII-catalyzed [2+2+2] cycloaddition explained these results. Initially, a
Ru-catalyzed linear coupling of 1,6-diynes 1 and 7 with acyclic alkenes occurs to
give open 1,3,5-trienes of type 3, which after a thermal disrotatory 6e(-)
pi-electrocyclization led to the final 1,3-cyclohexadienes 4 and 8. When
disubstituted 1,6-diyne 10 was used with electron-deficient alkenes, new
exo-methylene cyclohexadienes 12 arose from a competitive reaction pathway.

PMID: 18767111 [PubMed]


59. J Org Chem. 2008 Oct 3;73(19):7721-30. Epub 2008 Aug 30.

Gold(I)-catalyzed intermolecular addition of carbon nucleophiles to 1,5- and
1,6-enynes.

Amijs CH, Lopez-Carrillo V, Raducan M, Perez-Galan P, Ferrer C, Echavarren AM.

Institute of Chemical Research of Catalonia (ICIQ), Av. Paisos Catalans 16, 43007
Tarragona, Spain.

Gold(I)-catalyzed addition of carbon nucleophiles to 1,6-enynes gives two
different type of products by reaction at the cyclopropane or at the carbene
carbons of the intermediate cyclopropyl gold carbenes. The 5-exo-dig cyclization 
is followed by most 1,6-enynes, although those bearing internal alkynes and
alkenes react by the 6-endo-dig pathway. The cyclopropane versus carbene
site-selectivity can be controlled in some cases by the ligand on the gold
catalyst. In addition to electron-rich arenes and heteroarenes, allylsilanes and 
1,3-dicarbonyl compounds can be used as the nucleophiles. In the reaction of
1,5-enynes with carbon nucleophiles, the 5-endo-dig pathway is preferred.

PMID: 18759485 [PubMed]


60. Yakugaku Zasshi. 2008 Sep;128(9):1259-66.

Development of new palladium(0)-catalyzed reactions based on novel oxidative
addition mode.

Tsukamoto H.

Graduate School of Pharmaceutical Sciences, Tohoku University, 603 Aramaki-aza
aoba, Aoba-ku, Sendai, Japan. hirokazu@mail.pharm.tohoku.ac.jp

We have developed palladium(0)/monophosphine-catalyzed trans-selective arylative,
alkenylative, alkylative, and alkynylative cyclization reactions of
alkyne-aldehydes and -ketones with organoboron reagents. These reactions afford
six-membered allylic alcohols with endo tri- or tetra-substituted olefin groups
and/or five-membered counterparts with exo olefin groups. The ratios of these
products are dramatically affected by alkyne substituents as well as the
phosphine ligand. The remarkable trans selectivity of the process results from
the novel reaction mechanism involving 'anti-Wacker'-type oxidative addition.
Although the cyclization reactions are influenced by the length of the tether
between the alkyne and carbonyl group, they can be applied to a multi-component
synthesis of biologically important indenes bearing three substituent groups at
1, 2, 3-positions from available o-ethynylbenzaldehyde derivatives. A
two-component coupling reaction in methanol provides 1H-indenols, while a
three-component reaction involving secondary aliphatic amines as the third
component in DMF affords 1H-indenamines. This method allows combinatorial
preparation of unsymmetrically substituted 1H-indenes that cannot be prepared via
previous synthetic routes. The same catalytic system can also transform
allene-carbonyl compounds into 3-cyclohexenols and -cyclopentenols with alkyl,
aryl, alkenyl, alkynyl, and boryl groups at C-3. Microwave irradiation
efficiently increases not only the reaction rate but also the product yield by
suppressing formation of hydroarylation byproducts. Cyclization of optically
active 1,3-disubstituted allene-aldehyde reveals that the reaction proceeds
through not carbopalladation but 'anti-Wacker'-type oxidative addition.

PMID: 18758139 [PubMed - indexed for MEDLINE]


61. J Am Chem Soc. 2008 Aug 27;130(34):11535-45. Epub 2008 Aug 2.

Radical cascade transformations of tris(o-aryleneethynylenes) into substituted
benzo[a]indeno[2,1-c]fluorenes.

Alabugin IV, Gilmore K, Patil S, Manoharan M, Kovalenko SV, Clark RJ, Ghiviriga
I.

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, 
Florida 32306-4390, USA. alabugin@chem.fsu.edu

Oligomeric o-aryleneethynylenes with three triple bonds undergo cascade radical
transformations in reaction with a Bu 3SnH/AIBN system. These cascades involve
three consecutive cycle closures with the formation of substituted benzo[
a]indeno[2,1- c]fluorene or benzo[1,2]fluoreno[4,3- b]silole derivatives. The
success of this sequence depends on regioselectivity of the initial attack of the
Bu 3Sn radical at the central triple bond of the o-aryleneethynylene moiety. The 
cascade is propagated through the sequence of 5-exo-dig and 6-exo-dig
cyclizations which is followed by either a radical attack at the terminal Ar
substituent or radical transposition which involves H-abstraction from the
terminal TMS group and 5-endo-trig cyclization. Overall, the transformation has
potential to be developed into an approach to a new type of graphite ribbons.

PMID: 18680260 [PubMed - indexed for MEDLINE]


62. Fungal Genet Biol. 2008 Sep;45(9):1307-14. Epub 2008 Jul 8.

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis.

Spiering MJ, Faulkner JR, Zhang DX, Machado C, Grossman RB, Schardl CL.

Department of Plant Pathology, University of Kentucky, 1405 Veterans Drive,
Lexington, KY 40546-0312, USA.

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related
endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and
C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the
1-amine, which affect their biological activity. Enzymes for key loline
biosynthesis steps are probably encoded by genes in the LOL cluster, which is
duplicated in N. uncinatum, except for a large deletion in lolP2. The role of
lolP1 was investigated by its replacement with a hygromycin B phosphotransferase 
gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 
cultures had greatly elevated levels of N-methylloline (NML) and lacked
N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of
the Emericella nidulans trpC promoter restored NFL production. These results and 
the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing 
oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

PMID: 18655839 [PubMed - indexed for MEDLINE]


63. Neuropharmacology. 2008 Oct;55(5):771-9. Epub 2008 Jun 27.

Differential cocaine-induced modulation of glutamate and dopamine transporters
after contingent and non-contingent administration.

Miguens M, Crespo JA, Del Olmo N, Higuera-Matas A, Montoya GL, Garcia-Lecumberri 
C, Ambrosio E.

Departamento de Psicobiologia, Universidad Nacional de Educacion a Distancia, C/ 
Juan del Rosal n degrees 10, 28040 Madrid, Spain.

Although dopamine and glutamate transmission has been implicated in cocaine
dependence, the effects of the extinction of cocaine self-administration on
protein transporters in both of these neurotransmitter systems remain unknown. We
have used a yoked-box procedure to simultaneously test rats in triads, one rat
that actively self-administered cocaine (CONT), while the other two received
yoked injections of either cocaine (NON-CONT) or saline (SALINE). The brains in
each triad were removed and processed for quantitative autoradiography
immediately after the last session of cocaine self-administration (Day 0), or
after 1, 5, or 10 days of extinction, and excitatory amino acid transporters
(EAATs) and dopamine transporter (DAT) binding was examined. When compared to
NON-CONT and SALINE animals, binding of radioligand to EAATs was significantly
lower in the hippocampal CA1 field and the cerebellar cortex of CONT rats on Day 
0, although it was significantly higher after 1 day of extinction in the
infralimbic cortex. No differences in EAAT binding were observed after 5 or 10
days of extinction in any of the brain regions analyzed. In contrast and at all
the time points of extinction, binding to DAT was significantly enhanced in CONT 
animals when compared to SALINE and NON-CONT rats in different forebrain and
mesencephalic regions, including the nucleus accumbens, ventral tegmental area or
caudate putamen. These results suggest that changes in protein transporter
binding after cocaine self-administration and extinction are transient for EAAT
while they are more enduring for DAT, and that they depend on the type of access 
to cocaine.

PMID: 18634806 [PubMed - indexed for MEDLINE]


64. Biochem J. 2008 Aug 1;413(3):e11-2.

CD95 ligation and intracellular membrane flow.

Reinehr R, Haussinger D.

Clinic for Gastroenterology, Hepatology and Infectiology,
Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany.

Comment on:
    Biochem J. 2008 Aug 1;413(3):467-78.

Whereas ligation of the CD95 death receptor in the plasma membrane of so-called
type I cells leads to a direct caspase 8-dependent activation of downstream
effector caspases, mitochondrial amplification of caspase 8-derived signals is
required in so-called type II cells in order to execute apoptotic cell death. In 
type I cells CD95L (CD95 ligand) binding to CD95 results in a ceramide-dependent 
formation of the DISC (death-inducing signalling complex) and caspase 8-dependent
CD95 clustering in the plasma membrane, followed by an internalization of these
multimeric-receptor-DISC complexes. In contrast, in the hepatocyte, a type II
cell, the bulk of CD95 is stored intracellularly under resting conditions and
only a few 'sentinel' CD95 receptors are present in the plasma membrane. However,
their activation by CD95L is sufficient to trigger a caspase 8-dependent
endosomal acidification and a ceramide-dependent trafficking of intracellularly
stored CD95 to the plasma membrane, thereby amplifying CD95 activation. Thus, in 
both type I and type II cells, ceramide and CD95 receptor endo- and exo-cytosis
are involved in CD95-mediated apoptosis, but apparently in different ways. This, 
however, is not the only effect of CD95 ligation on intracellular membrane flow
in type II cells, and evidence has been presented that soon after CD95 ligation
Golgi elements intermix caspase-dependently with mitochondria. In this issue of
the Biochemical Journal, Matarrese et al. report another aspect on endocytosis in
response to CD95 ligation in type II cells, namely a caspase-independent
endocytosis with vesicle translocation to the mitochondrial compartment,
suggestive of an interplay between both organelles in the sense of an 'organelle 
scrambling'. Thus early effects of CD95 activation on intracellular membrane flow
may be much more complex than previously thought, but much has still to be
learned about signalling mechanisms and the role they play in apoptosis.

PMID: 18613813 [PubMed - indexed for MEDLINE]


65. Carbohydr Res. 2008 Jun 17. [Epub ahead of print]

The dual exo/endo-type mode and the effect of ionic strength on the mode of
catalysis of chitinase 60 (CHI60) from Serratia sp. TU09 and its mutants.

Kuttiyawong K, Nakapong S, Pichyangkura R.

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok
10330, Thailand.

Mutations of the tryptophan residues in the tryptophan-track of the N-terminal
domain (W33F/Y and W69F/Y) and in the catalytic domain (W245F/Y) of Serratia sp. 
TU09 Chitinase 60 (CHI60) were constructed, as single and double point
substitutions to either phenylalanine or tyrosine. The enzyme-substrate
interaction and mode of catalysis, exo/endo-type, of wild type CHI60 and mutant
enzymes on soluble (partially N-acetylated chitin), amorphous (colloidal chitin),
and crystalline (beta-chitin) substrates were studied. All CHI60 mutants
exhibited a reduced substrate binding activity on colloidal chitin. CHI60
possesses a dual mode of catalysis with both exo- and endo-type activities
allowing the enzyme to work efficiently on various substrate types. CHI60
preferentially uses the endo-type mode on soluble and amorphous substrates and
the exo-type mode on crystalline substrate. However, the prevalent mode of
hydrolysis mediated by CHI60 is regulated by ionic strength. Slightly elevated
ionic strength, 0.1-0.2M NaCl, which promotes enzyme-substrate interactions,
enhances CHI60 hydrolytic activity on amorphous substrate and, interestingly, on 
partially N-acetylated chitin. High ionic strength, 0.5-2.0M NaCl, prevents the
enzyme from dissociating from amorphous substrate, occupying the enzyme in an
enzyme-substrate non-productive complex. However, on crystalline substrates, the 
activity of CHI60 was only inhibited approximately 50% at high ionic strength,
suggesting that the enzyme hydrolyzes crystalline substrates with an exo-type
mode processively while remaining tightly bound to the substrate. Moreover,
substitution of Trp-33 to either phenylalanine or tyrosine reduced the activity
of the enzyme at high ionic strength, suggesting an important role of Trp-33 on
enzyme processivity.

PMID: 18565500 [PubMed - as supplied by publisher]


66. J Org Chem. 2008 Jul 18;73(14):5558-65. Epub 2008 Jun 13.

Microwave-assisted syntheses of N-heterocycles using alkenone-, alkynone- and
aryl-carbonyl O-phenyl oximes: formal synthesis of neocryptolepine.

Portela-Cubillo F, Scott JS, Walton JC.

School of Chemistry, EaStChem, University of St. Andrews, St. Andrews, Fife KY16 
9ST, UK.

This research aimed to provide a new and "clean" synthetic method that would
enable both known and novel N-heterocycles to be prepared efficiently. O-Phenyl
oximes were found to be excellent precursors for iminyl radicals with a variety
of acceptor side chains. Dihyropyrroles were made in good yields from O-phenyl
oximes containing pent-4-ene acceptors. The analogous process with a hex-5-enyl
acceptor did not yield a dihydropyridine, probably because the 6-exo-trig ring
closure of the iminyl radical was too slow to compete with H-atom abstraction.
The iminyl radical from a precursor with a pent-4-yne type side chain underwent
ring closure followed by rearrangement to afford a pyrrole derivative. Suitably
substituted iminyl radicals ring closed readily onto aromatic acceptors, thus
enabling several polycyclic systems to be accessed. Quinolines were made from
3-phenylpropanones via their O-phenyl oximes. Syntheses of phenanthridines
starting from 2-formylbiphenyls were particularly efficient, and this approach
enabled the natural product trisphaeridine to be made. Starting from
2-phenylnicotinaldehyde derivatives, ring closures of the derived iminyl radicals
onto the phenyl rings yielded benzo[h][1,6]naphthyridines. Similarly, ring
closure onto a phenyl ring from a benzothiophene-based iminyl yielded a
benzo[b]thieno[2,3-c]quinoline. By way of contrast, iminyl radical ring closure
onto pyridine rings was not observed. However, iminyl radicals did cyclize onto
indoles, enabling indolopyridines to be prepared. The latter route was exploited 
in a short formal synthesis of neocryptolepine starting from
2-((1H-indol-3-yl)methyl)cyclohexanone.

PMID: 18549288 [PubMed - indexed for MEDLINE]


67. Protein Eng Des Sel. 2008 Sep;21(9):561-6. Epub 2008 Jun 6.

Structural simulation and protein engineering to convert an endo-chitosanase to
an exo-chitosanase.

Yao YY, Shrestha KL, Wu YJ, Tasi HJ, Chen CC, Yang JM, Ando A, Cheng CY, Li YK.

Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh
Road, Hsinchu, Taiwan.

To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by
converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from
Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using
molecular modeling, two peptides with five amino acids (PCLGG) and six amino
acids (SRTCKP) were designed and inserted after the positions of D(115) and
T(222) of Csn, respectively. The inserted fragments are expected to form loops
that might protrude from opposite walls of the substrate-binding cleft, thus
forming a 'roof' over the catalytic site that might alter the product
specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase
(WT-Csn) were both over-expressed in Escherichia coli and purified nearly to
homogeneity. The products formed from chitosan were analyzed by ESI-MS
(electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and
GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller
catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product.
Measurements of viscosity showed that, with similar amounts of enzyme activity,
Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity
decrease than WT-Csn. The results indicate that, on inserting two surface loops, 
the endo-type chitosanase was converted into an exo-type chitosanase, which to
our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the
dominant product.

PMID: 18540010 [PubMed - indexed for MEDLINE]


68. Inorg Chem. 2008 Jul 7;47(13):5992-6000. Epub 2008 Jun 5.

Synthesis and characterization of aluminum- and gallium-bridged
[1.1]chromarenophanes and [1.1]molybdarenophanes.

Lund CL, Schachner JA, Burgess IJ, Quail JW, Schatte G, Muller J.

Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon, Saskatchewan S7N 5C9, Canada.

The synthesis and structural characterization of the first [1.1]chromarenophanes 
and the first [1.1]molybdarenophanes are described. A salt-metathesis reaction of
[2-(Me 2NCH 2)C 6H 4]AlCl 2 with freshly prepared [Cr(LiC 6H 5) 2].TMEDA (TMEDA =
N, N, N', N'-tetramethylethylenediamine) resulted in the
dialumina[1.1]chromarenophane [{2-(Me 2NCH 2)C 6H 4}Al(eta (6)-C 6H 5) 2Cr] 2 (
2a). The poor solubility of 2a in organic solvents prompted us to synthesize the 
new intramolecularly coordinated aluminum- and gallium dichlorides [5- tBu-2-(Me 
2NCH 2)C 6H 3]ECl 2 [E = Al ( 3a), Ga ( 3b)] in which the phenyl group was
equipped with a tert-butyl group. Salt-metathesis reactions of 3a and 3b,
respectively, with freshly prepared [M(LiC 6H 5) 2].TMEDA (M = Cr, Mo) resulted
in four new [1.1]metallarenophanes of the general type [{5- tBu-2-(Me 2NCH 2)C 6H
3}E(eta (6)-C 6H 5) 2M] 2 [E = Al, M = Cr ( 4a); E = Ga, M = Cr ( 4b); E = Al, M 
= Mo ( 5a); E = Ga, M = Mo ( 5b)]. 2a, 4a, b, and 5a, b have been structurally
characterized by single-crystal analysis [ 2a.1/2C 6H 12: C 48H 56Al 2Cr 2N 2,
monoclinic, P2 1/ c, a = 9.9117(9) A, b = 19.9361(16) A, c = 10.638(2) A, alpha =
90 degrees , beta = 112.322(5) degrees , gamma = 90 degrees , Z = 2; 4a.2C 6H 6: 
C 62H 72Al 2Cr 2N 2, monoclinic, P2 1/ c, a = 10.9626(9) A, b = 19.3350(18) A, c 
= 12.4626(9) A, alpha = 90 degrees , beta = 100.756(5) degrees , gamma = 90
degrees , Z = 2; 4b.2C 6H 6: C 62H 72Cr 2Ga 2N 2, monoclinic, P2 1/ c, a =
10.8428(2) A, b = 19.4844(4) A, c = 12.4958(2) A, alpha = 90 degrees , beta =
100.6187 degrees , gamma = 90 degrees , Z = 2; 5a.2C 6H 6: C 62H 72Al 2Mo 2N 2,
triclinic, P1, a = 10.4377(4) A, b = 11.6510(4) A, c = 11.6514(4) A, alpha =
73.545(3) degrees , beta = 89.318(2) degrees , gamma = 76.120(2) degrees , Z = 1;
5b.2C 6H 6: C 62H 72Ga 2Mo 2N 2, triclinic, P1, a = 10.3451(5) A, b = 11.6752(6) 
A, c = 11.6900(5) A, alpha = 73.917(3) degrees , beta = 89.550(3) degrees , gamma
= 76.774(2) degrees , Z = 1]. All five [1.1]metallarenophanes 2a, 4a, b, and 5a, 
b crystallize as anti isomers with both Me 2N donor groups in exo positions ( C i
point group symmetry). The new [1.1]metallarenophanes show NMR spectra that can
be interpreted as being caused by time-averaged C 2 h symmetrical species, which 
is consistent with the findings of their molecular structures in the solid state.
Variable-temperature (1)H NMR measurements for 4a, b and 5a, b (500 MHz; -90 to
90 degrees C) revealed only peak broadening in the lower temperature range of -70
to -90 degrees C. (1)H NMR saturation transfer difference experiments did not
show an expected anti-to-anti isomerization, rendering the new
[1.1]metallacyclophanes rigid on the NMR time scale. Electrochemical measurements
were performed for 4a, b and 5a, b. However, reproducible cyclic voltammograms
could only be obtained for the two gallium species 4b and 5b, revealing the
expected weak communication between the two transition-metal atoms in both
compounds (class II).

PMID: 18533628 [PubMed]


69. Protein Expr Purif. 2008 Aug;60(2):170-5. Epub 2008 Apr 8.

Cloning, heterologous gene expression and biochemical characterization of the
alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum.

Shalom G, Pratten J, Wilson M, Nair SP.

Division of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray's Inn
Road, London WC1X 8LD, UK.

There has been much recent interest in alpha-1,3-glucanases (mutanases) as they
have the potential to be used in the treatment of dental caries. Mutanases have
been reported in a number of bacteria, yeast and fungi but remain a relatively
uncharacterised family of enzymes. In this study we heterologously expressed the 
mutanase gene from the filamentous fungus Penicillium purpurogenum to enable
further characterization of its enzymatic activity. The mutanase cDNA was cloned 
and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of 
the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan
with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was
specific for mutan and could not cleave a variety of other polysaccharides
demonstrating a specificity for alpha-1,3-glucosidic linkages. The pH and
temperature optima were pH 4.6 and 45 degrees C, respectively. Synthetic
compounds were also tested as substrates to assess whether the P. purpurogenum
mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an
endo-hydrolytic mode of action. The type of mechanism was confirmed since
mutanase activity was not suppressed in the presence of inhibitors of exo-type
enzymes.

PMID: 18490176 [PubMed - indexed for MEDLINE]


70. Congenit Anom (Kyoto). 2008 Jun;48(2):81-6.

Adrenocorticotropic hormone affects nonapoptotic cell death of undifferentiated
germ cells in the fetal mouse testis: in vivo study by exo utero transplantation 
of corticotropic tumor cells into embryos.

Nimura M, Udagawa J, Otani H.

Research Project Promoting Institute, Shimane University, Izumo, Shimane, Japan.

Adrenocorticotropic hormone (ACTH) has been suggested to have possible roles in
the fetal testes, one of the organs that express its specific receptors,
melanocortin type 2 and 5 receptors (MC2R and MC5R), during the fetal period. We 
investigated the effect of ACTH on the cells in the testis cord of the fetal
mouse testis by inducing ACTH-secreting AtT20 tumor cells in mouse fetuses. We
first identified that mouse testicular germ cells at embryonic day (E) 16.5 and
E18.5 spermatogonia were entirely CDH1 (E-cadherin)-positive by
immunohistochemistry. We next performed AtT20-cell transplantation into the mouse
fetus at E12.5, and analyzed ACTH effects on the development of fetal male mouse 
germ cells that express MC2R and MC5R at E16.5 and E18.5. The spermatogonia in
the testis of AtT20-implanted embryos exhibited morphological changes, including 
pyknotic nuclei and swollen cytoplasm. In the AtT20-implanted embryos, the number
of spermatogonia per unit area of the testis cord was significantly lower, but
there were more pyknotic spermatogonia than in the controls. Single-stranded
DNA-positive (apoptotic) and histone H3-positive (mitotic) spermatogonia were
rarely observed and their numbers did not significantly differ in the two groups.
Anti-Mullerian hormone (AMH)-positive Sertoli cells, another cell type that
constitutes the fetal testis cord but does not express MC2R or MC5R, showed no
apparent morphological changes compared with controls, nor were their numbers in 
the two groups significantly different between the two groups. These results
suggest that ACTH, via MC2R and/or MC5R, may be involved in the nonapoptotic cell
death of fetal mouse spermatogonia that is observed during the normal perinatal
period.

PMID: 18452489 [PubMed - indexed for MEDLINE]


71. J Mol Recognit. 2008 May-Jun;21(3):154-62.

RPA repair recognition of DNA containing pyrimidines bearing bulky adducts.

Petruseva IO, Tikhanovich IS, Chelobanov BP, Lavrik OI.

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, pr. Lavrentieva, 8, Novosibirsk 630090, Russia.

Recognition of new DNA nucleotide excision repair (NER) substrate analogs, 48-mer
ddsDNA (damaged double-stranded DNA), by human replication protein A (hRPA) has
been analyzed using fluorescence spectroscopy and photoaffinity modification. The
aim of the present work was to find quantitative characteristics of RPA-ddsDNA
interaction and RPA subunits role in this process. The designed DNA structures
bear bulky substituted pyrimidine nitrogen bases at the inner positions of duplex
forming DNA chains. The photoreactive 4-azido-2,5-difluoro-3- pyridin-6-yl (FAP) 
and fluorescent antracenyl, pyrenyl (Antr, Pyr) groups were introduced via
different linker fragments into exo-4N of deoxycytidine or 5C of deoxyuridine.
J-dU-containing DNA was used as a photoactive model of undamaged DNA strands. The
reporter group was a fluorescein residue, introduced into the 5'-phosphate end of
one duplex-forming DNA strand. RPA-dsDNA association constants and the molar
RPA/dsDNA ratio have been calculated based on fluorescence anisotropy
measurements under conditions of a 1:1 RPA/dsDNA molar ratio in complexes. The
evident preference for RPA binding to ddsDNA over undamaged dsDNA distinctly
depends on the adduct type and varies in the following way: undamaged dsDNA <
Antr-dC-ddsDNA < mmdsDNA < FAPdU-, Pyr-dU-ddsDNA < FAP-dC-ddsDNA (K(D) = 68 +/-
1; 25 +/- 6; 13 +/- 1; 8 +/- 2, and 3.5 +/- 0.5 nM correspondingly) but weakly
depends on the chain integrity. Interestingly the bulkier lesions not in all
cases have a greater effect on RPA affinity to ddsDNA. The experiments on
photoaffinity modification demonstrated only p70 of compactly arranged RPA
directly interacting with dsDNA. The formation of RPA-ddsDNA covalent adducts was
drastically reduced when both strands of DNA duplex contained virtually opposite 
located FAP-dC and Antr-dC. Thus RPA requires undamaged DNA strand presence for
the effective interaction with dsDNA bearing bulky damages and demonstrates the
early NER factors characteristic features underlying strand discrimination
capacity and poor activity of the NER system toward double damaged DNA. 2008 John
Wiley & Sons, Ltd

PMID: 18438969 [PubMed - indexed for MEDLINE]


72. J Neurosci. 2008 Apr 2;28(14):3668-82.

Presynaptic calcium channel localization and calcium-dependent synaptic vesicle
exocytosis regulated by the Fuseless protein.

Long AA, Kim E, Leung HT, Woodruff E 3rd, An L, Doerge RW, Pak WL, Broadie K.

Department of Biological Sciences, Kennedy Center for Research on Human
Development, Vanderbilt University, Nashville, Tennessee 37235-1634, USA.

A systematic forward genetic Drosophila screen for electroretinogram mutants
lacking synaptic transients identified the fuseless (fusl) gene, which encodes a 
predicted eight-pass transmembrane protein in the presynaptic membrane. Null fusl
mutants display >75% reduction in evoked synaptic transmission but, conversely,
an approximately threefold increase in the frequency and amplitude of spontaneous
synaptic vesicle fusion events. These neurotransmission defects are rescued by a 
wild-type fusl transgene targeted only to the presynaptic cell, demonstrating a
strictly presynaptic requirement for Fusl function. Defects in FM dye turnover at
the synapse show a severely impaired exo-endo synaptic vesicle cycling pool.
Consistently, ultrastructural analyses reveal accumulated vesicles arrested in
clustered and docked pools at presynaptic active zones. In the absence of Fusl,
calcium-dependent neurotransmitter release is dramatically compromised and there 
is little enhancement of synaptic efficacy with elevated external Ca(2+)
concentrations. These defects are causally linked with severe loss of the
Cacophony voltage-gated Ca(2+) channels, which fail to localize normally at
presynaptic active zone domains in the absence of Fusl. These data indicate that 
Fusl regulates assembly of the presynaptic active zone Ca(2+) channel domains
required for efficient coupling of the Ca(2+) influx and synaptic vesicle
exocytosis during neurotransmission.

PMCID: PMC2769928
PMID: 18385325 [PubMed - indexed for MEDLINE]


73. Ann Ig. 2007 Nov-Dec;19(6):499-508.

[Epidemiology of HPV infections and cervical cancer in Apulia: a survey study and
current data analysis]

[Article in Italian]

Martinelli D, Chironna M, Tafuri S, Neve A, Caputi G, Prato R, Germinario C,
Quarto M.

Dipartimento di Scienze Mediche e del Lavoro, Sezione di Igiene, Universita degli
Studi di Foggia, Osservatorio Epidemiologico Regione Puglia, Bari.

HPV infection is common in sexually active women and is an important risk factor 
for cervical cancer. The aim of this article is to describe the prevalence of HPV
infection, the incidence and the mortality rates for cervical cancer and
adherence to screening programs in Apulia in the light of recent introduction of 
anti-HPV vaccines. The prevalence of HPV was evaluated testing biological samples
from 1082 women. The 33% resulted positive for HPV (80% for high-risk genotypes
and 20% for low-risk genotypes). The 59% of positive samples showed only a single
viral type while 37% multiple genotypes. In Apulia, from 1998 to 2005, a total of
1849 women were hospitalized for cervical cancer with a decreasing trend; the 46%
had exo cervical cancer; the 22% endocervical cancer, 29% cancer of cervix
without specification and 3% infiltrating cervical cancer. The mortality rate was
1,5 x 100.000. Data from PASSI study regarding cervical cancer screening showed
that 62% of Apulian women 25-64 years aged had a Pap smear and 54.7% get it every
three years. The viral genotypes circulating in Apulia region are present in
anti-HPV vaccines; this item could give information on their introduction
together with actions to implement the adherence to screening program that
results lower than international standard.

PMID: 18376570 [PubMed - indexed for MEDLINE]


74. Strabismus. 2008 Apr-Jun;16(2):57-63.

Changes in binocular alignment after surgery for concomitant and pattern
intermittent exotropia.

Pineles SL, Rosenbaum AL, Demer JL.

Jules Stein Eye Institute and Department of Ophthalmology, UCLA, Los Angeles, CA 
90095-7002, USA.

INTRODUCTION: Although early post-surgical over-correction for intermittent
exotropia is widely advised, post-operative drift has not been well quantified in
concomitant intermittent exotropia, and has not been described specifically with 
A and V patterns. While such patterns have been proposed to result from abnormal 
locations of the rectus muscle pulleys, others have suggested that A and V
patterns may result from the disruption of fusion arising from exotropia itself. 
METHODS: We prospectively performed Hess screen analysis in 20 exotropic patients
(mean age 42 +/- 16 yrs) before and two to six times after strabismus surgery,
with a post-operative follow-up of 2-108 weeks. Primary surgery cases included
medial rectus resection (2) and lateral rectus recession (10), combined
resection/recession (6), and superior oblique tenectomy (2). Alignment trends in 
primary and secondary gazes were analyzed for concomitant, pattern, and
re-operated subgroups. Results were also analyzed by type of surgery performed.
RESULTS: Mean pre-operative central gaze exotropia was 8.6 +/- 7.1 degrees .
Twelve cases were concomitant, while 8 exhibited A or V patterns. Twelve cases
were re-operations. In initial surgery for concomitant exotropia, there was a
well-defined exotropic drift approaching 5 degrees by 30 weeks post-operatively
(linear regression, r = 0.43, p = 0.01). There was similar exo drift in
re-operations. However, in pattern exotropia, post-operative drift was more
variable, with mean esotropic drift of approximately 5 degrees (r = 0.18, p =
0.43). For all patients, final post-operative central gaze exotropia was 1.9 +/- 
5.8 degrees , with significant pattern collapse (p < 0.01). DISCUSSION:
Post-operative exo-shift of about 5 degrees occurs in initial and re-operated
concomitant exotropia. However, in A and V patterns, there is no definitive
direction of post-operative drift, suggesting that pattern strabismus may be more
likely due to mechanical factors in the orbit than to neural factors associated
with fusion disruption. CONCLUSIONS: Alignment following strabismus surgery
differs in concomitant vs. pattern exotropia. Initial over-correction of about 5 
degrees is advisable for concomitant exotropia, but should be avoided in A and V 
patterns.

PMCID: PMC2796603
PMID: 19995177 [PubMed - indexed for MEDLINE]


75. J Biotechnol. 2008 Apr 30;134(3-4):253-60. Epub 2008 Feb 16.

Oligosaccharide hydrolysis by chitosanase enzymes monitored by real-time
electrospray ionization-mass spectrometry.

Dennhart N, Fukamizo T, Brzezinski R, Lacombe-Harvey ME, Letzel T.

Analytical Research Group, Department for Basic Life Sciences, Technische
Universitat Munchen, Weihenstephaner Berg 3, 85354 Freising-Weihenstephan,
Germany.

The chitosanase-catalyzed hydrolysis of chitosan oligosaccharides was
investigated for the first time by real-time electrospray ionization-mass
spectrometry (ESI-MS). As chitosan oligosaccharides (GlcNn, n=2-6) were
hydrolyzed by exochitosanase (exo-beta-glucosaminidase) from Amycolatopsis
orientalis, the reaction time-courses of substrate, intermediate and products
could be monitored simultaneously by direct infusion of the reaction solvent into
the mass spectrometer. Consequently, the analytical approach of real-time MS is
an enormous time-saving method. Furthermore, the high sensitivity of the mass
spectrometric detection allows the determination of the reaction time-courses
with very low quantities of substrate and therefore also a low amount of applied 
enzyme. Real-time mass spectrometric detection was also applicable in
investigating the reaction behaviour of Streptomyces sp. N174 endochitosanase
wild type and of two of its mutants. This technique establishes the fast and
efficient determination of in vitro enzymatic activities of various enzyme
systems.

PMID: 18359118 [PubMed - indexed for MEDLINE]


76. Appl Environ Microbiol. 2008 May;74(9):2759-65. Epub 2008 Mar 7.

Biochemical and molecular characterization of a novel type of Mutanase from
Paenibacillus sp. strain RM1: identification of its mutan-binding domain,
essential for degradation of Streptococcus mutans biofilms.

Shimotsuura I, Kigawa H, Ohdera M, Kuramitsu HK, Nakashima S.

Oral Care Research Laboratories, Lion Corporation, 3-7 Honjyo 1-chome, Sumida-ku,
Tokyo 130-8644, Japan. isaosimo@lion.co.jp

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus
sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-1,3-glucan
(mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major
etiologic agent in the progression of dental caries, even following brief
incubation. The nucleotide sequence of the gene for this protein contains a
3,873-bp open reading frame encoding 1,291 amino acids with a calculated
molecular mass of 135 kDa. The protein contains two major domains, the N-terminal
domain (277 residues) and the C-terminal domain (937 residues), separated by a
characteristic sequence composed of proline and threonine repeats. The
characterization of the recombinant proteins for each domain which were expressed
in Escherichia coli demonstrated that the N-terminal domain had strong
mutan-binding activity but no mutanase activity whereas the C-terminal domain was
responsible for mutanase activity but had mutan-binding activity significantly
lower than that of the intact protein. Importantly, the biofilm-degrading
activity observed with the intact protein was not exhibited by either domain
alone or in combination with the other. Therefore, these results indicate that
the structural integrity of mutanase RM1 containing the N-terminal mutan-binding 
domain is required for the biofilm-degrading activity.

PMCID: PMC2394904
PMID: 18326674 [PubMed - indexed for MEDLINE]


77. J Fr Ophtalmol. 2007 Dec;30(10):992-7.

[Postoperative pain in retinal detachment surgery]

[Article in French]

Marzak S, Miloudi Y, El Harrar N, Bensaid A, Zaghloul K, Amraoui A.

Service d'Anesthesie Reanimation, Hopital 20 aout, Casablanca, Maroc.

Postoperative pain in retinal detachment surgery is frequent but it is often
underestimated. The aim of this study was to determine the incidence of
postoperative pain after retinal detachment surgery and to identify its
predictive factors in a longitudinal study. We included 106 patients operated for
retinal detachment surgery using an endo-ocular or exo-ocular approach with
general anesthesia. Postoperative monitoring for 24 h evaluated the intensity of 
pain using a numerical scale. The possible predictive factors of this pain were
studied: ocular antecedents, premedication, total amount of morphine used, type
of surgery, duration of surgery, and vomiting. The incidence of postoperative
pain was 57.5%, 56% of which was intense pain. Postoperative pain was greatest
during the first 4 h. The predictive factors of this pain revealed by bivariate
analysis of the data were the type of surgery and vomiting. The incidence and
intensity of postoperative pain after retinal detachment surgery remain high.
Pain management requires postoperative treatment of vomiting as well as the
development of the endo-ocular surgery and locoregional anesthesia techniques.

PMID: 18268438 [PubMed - indexed for MEDLINE]


78. Org Biomol Chem. 2008 Feb 21;6(4):727-31. Epub 2008 Jan 10.

Transient kinetic studies of protein hydrolyses by endo- and exo-proteases on a
27 MHz quartz-crystal microbalance.

Furusawa H, Takano H, Okahata Y.

Department of Biomolecular Engineering and Frontier Collaborative Research
Centre, Tokyo Institute of Technology, B-53, 4259 Nagatsuta-cho, Midori-ku,
Yokohama 226-8501, Japan.

We have compared endo- and exo-type protease reactions and characterized the
enzymatic reaction mechanisms by determining all kinetic parameters (k(on),
k(off), k(cat), K(d) = k(off)/k(on), and K(m) = (k(off) + k(cat))/k(on)) by
following the mass change of the formation and the decay of the enzyme-substrate 
(ES) complex (k(on) and k(off)), and the formation of the product (k(cat)) on a
27 MHz quartz-crystal microbalance in aqueous solutions. The K(m) value was
nearly equal to the K(d) value for the endo-type protease (subtilisin and
alpha-chymotrypsin); however, in the case of exo-type protease (carboxypeptidase 
P), the K(m) value was quite different from the K(d) value, due to k(cat) >>
k(off).

PMID: 18264573 [PubMed - indexed for MEDLINE]


79. Biosci Biotechnol Biochem. 2008 Feb;72(2):514-22. Epub 2008 Feb 7.

Salt-adaptation of tobacco BY2 cells induces change in glycoform of N-glycans:
enhancement of exo- and endo-glycosidase activities by salt-adaptation.

Kimura Y, Watanabe T, Kimura M, Maeda M, Murata Y, Fujiyama K.

Department of Biofunctional Chemistry, Graduate School of Natural Science and
Technology, Okayama University, Japan. yosh8mar@cc.okayama-u.ac.jp

In this report, we describe that a salt adaptation of plant cells induces
glycoform changes in N-glycoproteins. Intracellular and cell-wall glycopeptides
were prepared from glycoproteins expressed in wild-type BY2 cells and
salt-adapted cells. N-Glycans were liberated from those glycopeptides by
hydrazinolysis, and the released oligosaccharides were N-acetylated and
pyridylaminated. The structures of pyridylaminated (PA-) N-glycans were analyzed 
by a combination of two-dimensional sugar-chain mapping, MS analysis, and
exoglycosidase digestion. In both wild-type cells and salt-adapted cells, the
plant complex type structure was predominant among N-glycans expressed on
glycoproteins, but we found that the Man2Xyl1Fuc1GlcNAc2 structure was
significantly expressed on intracellular and cell-wall glycoproteins of the
salt-adapted cells. Furthermore, enhancement of the specific activities of
alpha-mannosidase and beta-N-acetylglucosaminidase was observed in the
salt-adapted BY2 cells, suggesting that the glycoform changes are due to changes 
in glycosidase activities.

PMID: 18256500 [PubMed - indexed for MEDLINE]


80. J Chromatogr A. 2008 May 16;1191(1-2):274-81. Epub 2008 Jan 8.

Ring-opening metathesis polymerization-derived monolithic capillary columns for
high-performance liquid chromatography. Downscaling and application in medical
research.

Sinner FM, Gatschelhofer C, Mautner A, Magnes C, Buchmeiser MR, Pieber TR.

Institute of Medical Technologies and Health Management, Joanneum Research,
Auenbruggerplatz 20/3, A-8036 Graz, Austria. frank.sinner@joanneum.at

Monolithic capillary columns were prepared via ring-opening metathesis
polymerization (ROMP) using norborn-2-ene (NBE) and 1, 4, 4a, 5, 8,
8a-hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The
monolithic polymer was copolymerized with Grubbs-type initiator
RuCl(2)(PCy(3))(2)(CHPh) and a suitable porogenic system within the confines of
fused silica capillaries of different inner diameter (I.D.). The first part of
the study focused on batch-to-batch reproducibility of ROMP-derived capillary
monoliths. Capillary monoliths of 200 microm I.D. showed good reproducibility in 
terms of retention times, with relative standard deviations (RSD) of 1.9% for
proteins and 2.2% for peptides. However, the separately synthesized capillary
monoliths revealed pronounced variation in back pressure with RSD values of up to
31%. These variations were considerably reduced by cooling of the capillaries
during polymerization. Using this optimized preparation procedure capillary
monoliths of 100 and 50 microm I.D. were synthesized and the effects of scaling
down the column I.D. on the morphology and on the reproducibility of the
polymerization process were investigated. In the second part, the applicability
of ROMP-derived capillary monoliths to a separation problem common in medical
research was assessed. A 200 microm I.D. monolithic column demonstrated excellent
separation behavior for insulin and various insulin analogs, showing equivalent
separation performance to Vydac C4 and Zorbax C3-based stationary phases.
Moreover, the high permeability of monoliths enabled chromatographic separations 
at higher flow rates, which shortened analysis time to about one third. For the
analysis of insulin in human biofluid samples, enhanced sensitivity was achieved 
by using a 50 microm I.D. ROMP-derived monolith.

PMID: 18242625 [PubMed - indexed for MEDLINE]


81. Dev Dyn. 2008 Mar;237(3):630-9.

Cellular and molecular determinants targeting the Caenorhabditis elegans PHR
protein RPM-1 to perisynaptic regions.

Abrams B, Grill B, Huang X, Jin Y.

Department of Molecular, Cell and Developmental Biology, Sinsheimer Laboratories,
University of California Santa Cruz, Santa Cruz, California, USA.

Caenorhabditis elegans RPM-1 is a member of a conserved protein family, the PHR
proteins, that includes human Pam, mouse Phr1, zebrafish Esrom, and Drosophila
Highwire. PHR proteins play important roles in the development of the nervous
system. In particular, mutations in rpm-1 cause a disruption of synaptic
architecture, affecting the distribution of synaptic vesicles and the number of
presynaptic densities. Using antibodies against RPM-1, we determined the
localization of the endogenous RPM-1 protein in wild-type and in several mutants 
that affect synaptic development. Our analyses show that, in mature neurons,
RPM-1 resides in a distinct region that is close to, but does not overlap with,
the synaptic exo- and endocytosis domains. The localization of RPM-1 occurs
independently of several proteins that function in the transport or assembly of
synapse components, and its abundance is partially dependent on its binding
partner the F-box protein FSN-1. RPM-1 has been shown to target the MAPKKK DLK-1 
for degradation. We show that activated DLK-1 may be preferentially targeted for 
degradation. Furthermore, using transgene analysis, we identified a critical role
of the conserved PHR domain of RPM-1 in its subcellular localization. (c) 2008
Wiley-Liss, Inc.

PMCID: PMC2657606
PMID: 18224716 [PubMed - indexed for MEDLINE]


82. J Bioenerg Biomembr. 2008 Feb;40(1):53-7. Epub 2008 Jan 24.

Vacuolar-type proton ATPase as regulator of membrane dynamics in multicellular
organisms.

Wada Y, Sun-Wada GH, Tabata H, Kawamura N.

Division of Biological Science, Institute for Scientific and Industrial Research,
Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
yohwada@sanken.osaka-u.ac.jp

Acidification inside membrane compartments is a common feature of all eukaryotic 
cells. The acidic milieu is involved in many physiological processes including
secretion, protein processing, and others. However, its cellular relevance has
not been well established beyond the results of in vitro studies involving
cultured cell systems. In the last decade, human and mouse genetics have revealed
that the acidification machinery is implicated in multiple pathophysiological
disorders, and thus our understanding of physiological consequences of the
defective acidification in multicellular organisms has improved. In invertebrates
including Drosophila and nematodes, mutations of V-ATPase were found to lead the 
development of rather unexpected phenotypes. Studies have suggested that V-ATPase
may be involved in membrane fusion and vesicle formation, important processes for
membrane trafficking, and have further implied its involvement in cell-cell
fusion. This rather novel idea arose from the phenotypes associated with genetic 
disorders involving V-ATPase genes in various genetic model systems. In this
article, we focus and overview the non-classical, beyond proton-pumping function 
of the vacuolar-type ATPase in exo/endocytic systems.

PMID: 18214654 [PubMed - indexed for MEDLINE]


83. Anal Chem. 2008 Feb 15;80(4):1005-11. Epub 2008 Jan 23.

Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase
P on a 27-MHz quartz crystal microbalance.

Furusawa H, Takano H, Okahata Y.

Frontier Collaborative Research Center, Department of Biomolecular Engineering,
Tokyo Institute of Technology and CREST, JST, Nagatsuda, Midori-ku, Yokohama,
Japan.

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein
(myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were
obtained by using a protein-immobilized quartz crystal microbalance (QCM) in
acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex
(k(on)), the decay of the ES complex (k(off)), and the formation of the product
(k(cat)) could be analyzed by transient kinetics as mass changes on the QCM
plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km
calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The
rate-determining step was the binding step (k(on), and the catalytic rate k(cat) 
was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values
of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7,
values of k(off) were independent of pH, and k(cat) increased gradually with
decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a
maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at
3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH
terminus of the substrate with hydrogen bonds. The increase of k(cat) at the
lower pH correlated with the decrease of alpha-helix contents of the myoglobin
substrate on the QCM.

PMID: 18211097 [PubMed - indexed for MEDLINE]


84. J Am Chem Soc. 2008 Jan 30;130(4):1440-52. Epub 2008 Jan 4.

Metal-catalyzed 1,2-shift of diverse migrating groups in allenyl systems as a new
paradigm toward densely functionalized heterocycles.

Dudnik AS, Sromek AW, Rubina M, Kim JT, Kel'in AV, Gevorgyan V.

Department of Chemistry, University of Illinois at Chicago, 845 West Taylor
Street, 4500 SES, M/C 111, Chicago, Illinois 60607-7061, USA.

A general, mild, and efficient 1,2-migration/cycloisomerization methodology
toward multisubstituted 3-thio-, seleno-, halo-, aryl-, and alkyl-furans and
pyrroles, as well as fused heterocycles, valuable building blocks for synthetic
chemistry, has been developed. Moreover, regiodivergent conditions have been
identified for C-4 bromo- and thio-substituted allenones and alkynones for the
assembly of regioisomeric 2-hetero substituted furans selectively. It was
demonstrated that, depending on reaction conditions, ambident substrates can be
selectively transformed into furan products, as well as undergo selective
6-exo-dig or Nazarov cyclizations. Our mechanistic investigations have revealed
that the transformation proceeds via allenylcarbonyl or allenylimine
intermediates followed by 1,2-group migration to the allenyl sp carbon during
cycloisomerization. It was found that 1,2-migration of chalcogens and halogens
predominantly proceeds via formation of irenium intermediates. Analogous
intermediate can also be proposed for 1,2-aryl shift. Furthermore, it was shown
that the cycloisomerization cascade can be catalyzed by Bronsted acids, albeit
less efficiently, and commonly observed reactivity of Lewis acid catalysts cannot
be attributed to the eventual formation of proton. Undoubtedly, thermally induced
or Lewis acid-catalyzed transformations proceed via intramolecular Michael
addition or activation of the enone moiety pathways, whereas certain carbophilic 
metals trigger carbenoid/oxonium type pathway. However, a facile
cycloisomerization in the presence of cationic complexes, as well as observed
migratory aptitude in the cycloisomerization of unsymmetrically disubstituted
aryl- and alkylallenes, strongly supports electrophilic nature for this
transformation. Full mechanistic details, as well as the scope of this
transformation, are discussed.

PMID: 18173272 [PubMed - indexed for MEDLINE]


85. J Biol Chem. 2008 Mar 21;283(12):7568-79. Epub 2007 Dec 31.

The dystonia-associated protein torsinA modulates synaptic vesicle recycling.

Granata A, Watson R, Collinson LM, Schiavo G, Warner TT.

Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 
44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with
diverse cellular activities) torsinA is responsible for most cases of early onset
autosomal dominant primary dystonia. In this study, we found that snapin, which
binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the
association of the SNARE complex with synaptotagmin, is an interacting partner
for both wild type and mutant torsinA. Snapin co-localized with endogenous
torsinA on dense core granules in PC12 cells and was recruited to perinuclear
inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In
view of these observations, synaptic vesicle recycling was analyzed using the
lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope 
of synaptotagmin I. We found that overexpression of wild type torsinA negatively 
affects synaptic vesicle endocytosis. Conversely, overexpression of
DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of
snapin and/or torsinA using small interfering RNAs had a similar inhibitory
effect on the exo-endocytic process. In addition, down-regulation of torsinA
causes the persistence of synaptotagmin I on the plasma membrane, which closely
resembles the effect observed by the overexpression of the DeltaE-torsinA mutant.
Altogether, these findings suggest that torsinA plays a role together with snapin
in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects
through a loss of function mechanism. This may affect neuronal uptake of
neurotransmitters, such as dopamine, playing a role in the development of
dystonic movements.

PMID: 18167355 [PubMed - indexed for MEDLINE]


86. Magn Reson Chem. 2008 Feb;46(2):107-9.

The effect of carbonyl group in the asymmetry of 3,4JCH coupling constants in
norbornanones.

dos Santos FP, Tormena CF, Contreras RH, Rittner R, Magalhaes A.

Chemistry Institute, State University of Campinas, C.P. 6154, 13084-971,
Campinas-SP, Brazil.

A rationalization of the known difference between the 3,4JC4H1 and 3,4JC1H4
couplings transmitted mainly through the 7-bridge in norbornanone is presented in
terms of the effects of hyperconjugative interactions involving the carbonyl
group. Theoretical and experimental studies of 3,4JCH couplings were carried out 
in 3-endo- and 3-exo-X-2-norbornanone derivatives (X = Cl, Br) and in exo- and
endo-2-noborneol compounds. Hyperconjugative interactions were studied with the
natural bond orbital (NBO) method. Hyperconjugative interactions involving the
carbonyl pi*(C2=O) and sigma*(C2=O) antibonding orbitals produce a decrease of
three-bond contribution to both 3,4JC4H1 and 3,4JC1H4 couplings. However, the
latter antibonding orbital also undergoes a strong sigmaC3--C4 --> sigma*(C2=O)
interaction, which defines an additional coupling pathway for 3,4JC4H1 but not
for 3,4JC1H4. This pathway is similar to that known for homoallylic couplings,
the only difference being the nature of the intermediate antibonding orbital;
i.e. for 3,4JC4H1 it is of sigma*-type, while in homoallylic couplings it is of
pi*-type. Copyright (c) 2007 John Wiley & Sons, Ltd.

PMID: 18092306 [PubMed]


87. Org Lett. 2008 Jan 17;10(2):209-12. Epub 2007 Dec 19.

First synthesis of 4'-selenonucleosides showing unusual Southern conformation.

Jeong LS, Tosh DK, Kim HO, Wang T, Hou X, Yun HS, Kwon Y, Lee SK, Choi J, Zhao
LX.

Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University,
Seoul 120-750, Korea. lakjeong@ewha.ac.kr

The first synthesis of 4'-selenonucleosides was achieved using a Pummerer-type
condensation as a key step. All stereoelectronic effects shown in
4'-oxonucleosides were overwhelmed by the size of selenium and steric
interactions, driving the conformation to the C2'-endo/ C3'-exo twist (Southern) 
conformation.

PMID: 18088134 [PubMed - indexed for MEDLINE]


88. J Org Chem. 2008 Jan 18;73(2):502-10. Epub 2007 Dec 18.

Studies toward the synthesis of
4-(2-R-ethyl)amino-2,2,5,5-tetramethyl-3-imidazoline 1-oxyls. nucleophilic
substitution of bromide in the N-Alkyl chain of the 1,2,4-oxadiazol-2-one
precursor.

Polienko JF, Schanding T, Gatilov YV, Grigor'ev IA, Voinov MA.

Institute of Organic Chemistry, Ave. akad. Lavrent'eva 9, 630090, Novosibirsk,
Russia.

A synthetic approach to access the new nitroxides of the amidine type exhibiting 
pH-dependent EPR spectra through substitution of a halide in the
exo-N-halogenoalkyl chain of
1-(2-bromoethyl)-6-oxyl-5,5,7,7-tetramethyltetrahydroimidazo[1,5-b][1,2,4]oxadiaz
ol-2-one is reported. In this approach, an oxycarbonyl moiety of the oxadiazolone
heterocycle plays the role of a "protecting group" for the amidine functionality.
A nucleophilic cleavage of the oxadiazolone heterocycle under mild nonbasic
conditions, applicable to substrates bearing substituents vulnerable to attack by
strong basic nucleophiles, is elaborated. The approach allows for the new amidine
nitroxides bearing various functional groups (e.g., such as CN, N3, NH2, COOEt)
to be synthesized. A series of nitroxides obtained through the
Staudinger/intermolecular aza-Wittig reaction of the azido derivative is also
described. The nitroxides synthesized here were found to have pH-dependent
two-component EPR spectra indicative of a slow, on the EPR time scale, R* left
arrow over right arrow R*H+ chemical exchange, and pKa values ranging from 2.8 to
12.5 units. The guanidine derivatives synthesized in this work show the highest
pKa values (pKa = 10.2 and 12.5, respectively) ever reported for the nitroxide
pH-probes of a "basic type".

PMID: 18085792 [PubMed - indexed for MEDLINE]


89. J Am Chem Soc. 2008 Jan 9;130(1):269-79. Epub 2007 Dec 13.

Gold(I)-catalyzed intramolecular [4+2] cycloadditions of arylalkynes or
1,3-enynes with alkenes: scope and mechanism.

Nieto-Oberhuber C, Perez-Galan P, Herrero-Gomez E, Lauterbach T, Rodriguez C,
Lopez S, Bour C, Rosellon A, Cardenas DJ, Echavarren AM.

Institute of Chemical Research of Catalonia (ICIQ), Av. Paisos Catalans 16, 43007
Tarragona, Spain.

The cyclizations of enynes substituted at the alkyne gives products of formal
[4+2] cyclization with Au(I) catalysts. 1,8-Dien-3-ynes cyclize by a 5-exo-dig
pathway to form hydrindanes. 1,6-Enynes with an aryl ring at the alkyne give
2,3,9,9a-tetrahydro-1H-cyclopenta[b]naphthalenes by a 5-exo-dig cyclization
followed by a Friedel-Crafts-type ring expansion. A 6-endo-dig cyclization is
also observed in some cases as a minor process, although in a few cases, this is 
the major cyclization pathway. In addition to cationic gold complexes bearing
bulky biphenyl phosphines, a gold complex with
tris(2,6-di-tert-butylphenyl)phosphite is exceptionally reactive as a catalyst
for this reaction. This cyclization can also be carried out very efficiently with
heating under microwave irradiation. DFT calculations support a stepwise
mechanism for the cycloaddition by the initial formation of an anti-cyclopropyl
gold(I)-carbene, followed by its opening to form a carbocation stabilized by a pi
interaction with the aryl ring, which undergoes a Friedel-Crafts-type reaction.

PMID: 18076170 [PubMed]


90. Mol Biol Evol. 2008 Feb;25(2):287-300. Epub 2007 Dec 7.

Diversifying selection and concerted evolution of a type IV secretion system in
Bartonella.

Nystedt B, Frank AC, Thollesson M, Andersson SG.

Department of Molecular Evolution, Evolutionary Biology Centre, Uppsala
University, Uppsala, Sweden.

We have studied the evolution of a type IV secretion system (T4SS), in
Bartonella, which is thought to have changed function from conjugation to
erythrocyte adherence following a recent horizontal gene transfer event. The
system, called Trw, is unique among T4SSs in that genes encoding both exo- and
intracellular components are located within the same duplicated fragment. This
provides an opportunity to study the influence of selection on proteins involved 
in host-pathogen interactions. We sequenced the trw locus from several strains of
Bartonella henselae and investigated its evolutionary history by comparisons to
other Bartonella species. Several instances of recombination and gene conversion 
events where detected in the 2- to 5-fold duplicated gene fragments encompassing 
trwJIH, explaining the homogenization of the anchoring protein TrwI and the
divergence of the minor pilus protein TrwJ. A phylogenetic analysis of the 7- to 
8-fold duplicated gene coding for the major pilus protein TrwL displayed 2
distinct clades, likely representing a subfunctionalization event. The analyses
of the B. henselae strains also identified a recent horizontal transfer event of 
almost the complete trwL region. We suggest that the switch in function of the
T4SS was mediated by the duplication of the genes encoding pilus components and
their diversification by combinatorial sequence shuffling within and among
genomes. We suggest that the pilus proteins have evolved by diversifying
selection to match a divergent set of erythrocyte surface structures, consistent 
with the trench warfare coevolutionary model.

PMID: 18065487 [PubMed - indexed for MEDLINE]


91. Nucleosides Nucleotides Nucleic Acids. 2007;26(8-9):1079-82.

Synthesis of novel 2'-deoxy type trans-3',4'-bridged nucleic acid.

Osaki T, Obika S, Harada Y, Mitsuoka Y, Sugaya K, Sekiguchi M, Roongjang S,
Imanishi T.

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

We newly designed and synthesized a 2'-deoxy type trans-3',4'-bridged nucleic
acid (trans-3',4'-BNA) analogues bearing a 4,7-dioxabicyclo[4.3.0]nonane
structure. The synthesis of the trans-3',4'-BNA was carried out successfully from
thymidine over 21 steps. The structure of trans-3',4'-BNA was confirmed by x-ray 
crystallographic analysis, indicating that the furanose ring has a typical S-type
conformation with C(3')-exo puckering.

PMID: 18058540 [PubMed - indexed for MEDLINE]


92. J Org Chem. 2008 Jan 4;73(1):249-58. Epub 2007 Dec 4.

Carbocyclization reaction of omega-iodo- and 1,omega-diiodo-1-alkynes without the
loss of iodine atoms through a carbenoid-chain process.

Harada T, Muramatsu K, Mizunashi K, Kitano C, Imaoka D, Fujiwara T, Kataoka H.

Department of Chemistry and Materials Technology, Kyoto Institute of Technology, 
Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.

Atom-economical carbocyclization reactions of omega-iodo-1-alkynes and
1,omega-diiodo-1-alkynes to give products with incorporation of iodine atoms is
described. Cycloisomerization of 2-(2-propynyloxy)ethyl iodides is initiated by a
catalytic amount of LDA to give 3-(iodomethylene)tetrahydrofurans in high yields.
Upon treatment of with a catalytic amount of 1-hexynyllithium,
1,omega-diiodo-1-alkynes efficiently undergo cycloisomerization to give
(diiodomethylene)cycloalkanes. The diiodomethylene products are also obtained by 
iodine atom-transfer-type cyclization of omega-iodo-1-alkynes, using
1-iodo-1-hexyne as an external iodine atom source. Bromine atom-transfer and
proton-transfer cyclization proceed as well by employing 1-bromo-1-octyne and
1-octyne, respectively. These reactions are proposed to proceed through a
carbenoid-chain process involving exo-cyclization of the lithium acetylide
intermediates to give Li,I-alkylidene carbenoids. It is shown that the
exo-cyclization proceeded stereospecifically through inversion of the
stereochemistry at the electrophilic carbon.

PMID: 18052194 [PubMed - indexed for MEDLINE]


93. J Microbiol Biotechnol. 2007 Mar;17(3):481-9.

Rapid detection and isolation of known and putative alpha-L-arabinofuranosidase
genes using degenerate PCR primers.

Park JM, Han NS, Kim TJ.

Department of Food Science and Technology, School of Applied Life Science and
Environment, Chungbuk National University, Cheongju 361-763, Korea.

alpha-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which
hydrolyze terminal nonreducing arabinose residues from various polysaccharides
such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various
bacterial strains possess the putative AFase genes with well-conserved motif
sequences at the nucleotide and amino acid sequence levels. In this study, two
sets of degenerate PCR primers were designed and tested to detect putative AFase 
genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG,
and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to
have putative AFase genes in their genome and they share over 67% of amino acid
identities with each other. Based on the partial sequence obtained from an
isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli.
Enzymatic characterization has verified that the resulting enzyme corresponds to 
a typical AFase. Accordingly, degenerate PCR primers developed in this work can
be used for fast, easy, and specific detection and isolation of putative AFase
genes from bacterial cells.

PMID: 18050953 [PubMed - indexed for MEDLINE]


94. J Neurochem. 2008 Apr;105(1):78-90. Epub 2007 Nov 5.

VMAT2 and dopamine neuron loss in a primate model of Parkinson's disease.

Chen MK, Kuwabara H, Zhou Y, Adams RJ, Brasic JR, McGlothan JL, Verina T, Burton 
NC, Alexander M, Kumar A, Wong DF, Guilarte TR.

Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of
Public Health, Baltimore, Maryland 21205, USA.

We used positron emission tomography (PET) to measure the earliest change in
dopaminergic synapses and glial cell markers in a chronic, low-dose MPTP
non-human primate model of Parkinson's disease (PD). In vivo levels of dopamine
transporters (DAT), vesicular monoamine transporter-type 2 (VMAT2),
amphetamine-induced dopamine release (AMPH-DAR), D2-dopamine receptors (D2R) and 
translocator protein 18 kDa (TSPO) were measured longitudinally in the striatum
of MPTP-treated animals. We report an early (2 months) decrease (46%) of striatal
VMAT2 in asymptomatic MPTP animals that preceded changes in DAT, D2R, and
AMPH-DAR and was associated with increased TSPO levels indicative of a glial
response. Subsequent PET studies showed progressive loss of all pre-synaptic
dopamine markers in the striatum with expression of parkinsonism. However, glial 
cell activation did not track disease progression. These findings indicate that
decreased VMAT2 is a key pathogenic event that precedes nigrostriatal dopamine
neuron degeneration. The loss of VMAT2 may result from an association with
alpha-synuclein aggregation induced by oxidative stress. Disruption of dopamine
sequestration by reducing VMAT2 is an early pathogenic event in the dopamine
neuron degeneration that occurs in the MPTP non-human primate model of PD.
Genetic or environmental factors that decrease VMAT2 function may be important
determinants of PD.

PMID: 17988241 [PubMed - indexed for MEDLINE]


95. J Am Chem Soc. 2007 Nov 21;129(46):14281-95. Epub 2007 Oct 31.

Mechanistic studies of olefin and alkyne trimerization with chromium catalysts:
deuterium labeling and studies of regiochemistry using a model chromacyclopentane
complex.

Agapie T, Labinger JA, Bercaw JE.

Arnold and Mabel Beckman Laboratories of Chemical Synthesis, California Institute
of Technology, Pasadena, California 91125, USA.

A system for catalytic trimerization of ethylene utilizing chromium(III)
precursors supported by diphosphine ligand PNP(O4) =
(o-MeO-C6H4)2PN(Me)P(o-MeO-C6H4)2 has been investigated. The mechanism of the
olefin trimerization reaction was examined using deuterium labeling and studies
of reactions with alpha-olefins and internal olefins. A well-defined chromium
precursor utilized in this studies is Cr(PNP(O4))(o,o'-biphenyldiyl)Br. A
cationic species, obtained by halide abstraction with NaB[C6H3(CF3)2]4, is
required for catalytic turnover to generate 1-hexene from ethylene. The
initiation byproduct is vinylbiphenyl; this is formed even without activation by 
halide abstraction. Trimerization of 2-butyne is accomplished by the same
cationic system but not by the neutral species. Catalytic trimerization, with
various (PNP(O4))Cr precursors, of a 1:1 mixture of C2D4 and C2H4 gives
isotopologs of 1-hexene without H/D scrambling (C6D12, C6D8H4, C6D4H8, and C6H12 
in a 1:3:3:1 ratio). The lack of crossover supports a mechanism involving
metallacyclic intermediates. Using a SHOP catalyst to perform the oligomerization
of a 1:1 mixture of C2D4 and C2H4 leads to the generation of a broader
distribution of 1-hexene isotopologs, consistent with a Cossee-type mechanism for
1-hexene formation. The ethylene trimerization reaction was further studied by
the reaction of trans-, cis-, and gem-ethylene-d2 upon activation of
Cr(PNP(O4))(o,o'-biphenyldiyl)Br with NaB[C6H3(CF3)2]4. The trimerization of cis-
and trans-ethylene-d2 generates 1-hexene isotopomers having terminal CDH groups, 
with an isotope effect of 3.1(1) and 4.1(1), respectively. These results are
consistent with reductive elimination of 1-hexene from a putative
Cr(H)[(CH2)4CH=CH2] occurring much faster than a hydride 2,1-insertion or with
concerted 1-hexene formation from a chromacycloheptane via a 3,7-H shift. The
trimerization of gem-ethylene-d2 has an isotope effect of 1.3(1), consistent with
irreversible formation of a chromacycloheptane intermediate on route to 1-hexene 
formation. Reactions of olefins with a model of a chromacyclopentane were
investigated starting from Cr(PNP(O4))(o,o'-biphenyldiyl)Br. alpha-Olefins react 
with cationic biphenyldiyl chromium species to generate products from
1,2-insertion. A study of the reaction of 2-butenes indicated that beta-H
elimination occurs preferentially from the ring CH rather than exo-CH bond in the
metallacycloheptane intermediates. A study of cotrimerization of ethylene with
propylene correlates with these findings of regioselectivity. Competition
experiments with mixtures of two olefins indicate that the relative insertion
rates generally decrease with increasing size of the olefins.

PMID: 17973377 [PubMed]


96. Guang Pu Xue Yu Guang Pu Fen Xi. 2007 Jul;27(7):1393-7.

[Study on the molecular recognition of perhydroxycucurbit[6] uril with methyl
orange by spectroscopic methods]

[Article in Chinese]

Li LS, Ge XH, Huang ZB, Li YP.

The Center of Analysis and Test, Nanchang University, Nanchang 330047, China.
lilaishengcn@163.com

The inclusion interaction of perhydroxycucurbit[6] uril (HOCB6) with methyl
orange (MO) was studied by UV spectroscopic and fluorimetric methods. Several
effect factors, such as pH values, common organic solvents and surfactants on the
fluorescence intensity and the stability of the complex were investigated. The
results indicate that the fluorescence intensity of MO was enhanced with a blue
shift as host molecules were added, showing that MO was accommodated into the
hydrophobic cavities of HOCB6 and an endo-inclusion complex was formed. The
hydrophobic interaction between HOCB6 and MO mainly contributed to the formation 
of 1 : 1 type HOCB6-MO. Its complex constant was determined to be 1.41 x 10(2) L 
x mol(-1). The comparative study of HOCB6 with other supramolecules, such as
cucurbit[6] uril (CB6), p-(N,N-dimethyl-aminomethyl) calix [8]arene and
beta-cyclodextrin, was also carried out by using MO as a guest probe. The spectra
changes showed that cucurbit[6] uril (CB6) can also form 1 : 1 type
endo-inclusion complex with MO, which is similar to HOCB6, but its complex
constant (34.65 L x mol(-1)) is small. The spectra changes also showed that the
endo-inclusion complex with 2 : 1 type was formed between beta-cyclodextrin and
MO, While the exo-inclusion complex was formed between
p-(N,N-dimethyl-aminomethyl) calix[8] are-ne and MO, leading to fluorescence
quenching, and their complex constants were determined to be 6.14 x 10(6) L2
mol(-2) and 1.35 x 10(4) L x mol(-1), respectively.

PMID: 17944422 [PubMed - in process]


97. Brain Res. 2007 Oct 31;1178:52-64. Epub 2007 Aug 24.

Synapsin maintains the reserve vesicle pool and spatial segregation of the
recycling pool in Drosophila presynaptic boutons.

Akbergenova Y, Bykhovskaia M.

Department of Biological Sciences, Lehigh University, 111 Research Dr.,
Bethlehem, PA 18015, USA.

We employed optical detection of the lipophylic dye FM1-43 and focal recordings
of quantal release to investigate how synapsin affects vesicle cycling at the
neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye 
employing high K+ stimulation, which presumably involves the recycling pool of
vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons,
while in Syn KO the dye was redistributed towards the center of the bouton. When 
endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was
significantly enhanced in WT boutons, and the entire boutons were stained,
suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same
loading paradigm produced fainter staining and significantly faster destaining.
When the axon was stimulated electrically, a distinct difference in dye loading
patterns was observed in WT boutons at different stimulation frequencies: a low
stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a 
higher frequency (10 Hz) the dye was redistributed towards the center of the
bouton and the fluorescence intensity was significantly increased. This
difference in staining patterns was essentially disrupted in Syn KO boutons,
although synapsin did not affect the rate of quantal release. Stimulation of the 
nerve in the presence of bafilomycin, the blocker of the transmitter uptake,
produced significantly stronger depression in Syn KO boutons. These results,
taken together, suggest that synapsin maintains the reserve pool of vesicles and 
segregation between the recycling and reserve pools, and that it mediates
mobilization of the reserve pool during intense stimulation.

PMID: 17904536 [PubMed - indexed for MEDLINE]


98. DNA Repair (Amst). 2008 Jan 1;7(1):95-107. Epub 2007 Sep 27.

Mechanisms by which herpes simplex virus DNA polymerase limits translesion
synthesis through abasic sites.

Zhu Y, Song L, Stroud J, Parris DS.

Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State
University, Columbus, OH 43210, United States.

Results suggest a high probability that abasic (AP) sites occur at least once per
herpes simplex virus type 1 (HSV-1) genome. The parameters that control the
ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS)
were examined because AP lesions could influence the completion and fidelity of
viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that
wildtype (WT) and exonuclease-deficient (exo-) pol could incorporate opposite an 
AP lesion, but full TLS required absence of exo function. Virtually all of the WT
pol was bound at the exo site to AP-containing primer-templates (P/Ts) at
equilibrium, and the pre-steady-state rate of excision by WT pol was higher on
AP-containing than on matched DNA. However, several factors influencing
polymerization work synergistically with exo activity to prevent HSV-1 pol from
engaging in TLS. Although the pre-steady-state catalytic rate constant for
insertion of dATP opposite a T or AP site was similar, ground-state-binding
affinity of dATP for insertion opposite an AP site was reduced 3-9-fold.
Single-turnover running-start experiments demonstrated a reduced proportion of
P/Ts extended to the AP site compared to the preceding site during processive
synthesis by WT or exo- pol. Only the exo- pol engaged in TLS, though
inefficiently and without burst kinetics, suggesting a much slower rate-limiting 
step for extension beyond the AP site.

PMID: 17904428 [PubMed - indexed for MEDLINE]


99. Org Lett. 2007 Oct 25;9(22):4431-4. Epub 2007 Sep 27.

Functionalized cyclobutanes via Heck cyclization.

Innitzer A, Brecker L, Mulzer J.

Institut fur Organische Chemie der Universitat Wien, Wahringerstrasse 38, 1090
Wien, Austria.

Heck-type 4-exo-trig cyclization of linear 2-enol triflate-1,5-hexadienes
provides functionalized methylene cyclobutanes. Intramolecular palladium
coordination can initiate beta-hydride elimination leading to 1,2-dimethylene
cyclobutane derivatives, which are obtained with high selectivity if substrates
having a geminal diphenyl group at Calpha are used. In parallel, formal
5-endo-trig cyclization and beta-hydride elimination form 1-methylene
cyclopent-2-en derivatives.

PMID: 17900129 [PubMed]


100. J Mol Model. 2007 Dec;13(12):1215-20. Epub 2007 Sep 15.

Ab initio and DFT study of the inner mechanism and dynamic stereochemistry of
electrophilic addition reaction of bromine to bisbenzotetracyclo[6.2.2.2(3,6).0
(2,7)]tetradeca-4,9,11,13-tetraene.

Abbasoglu R.

Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey. 
rabbas@ktu.edu.tr

The inner mechanism and dynamic stereochemistry of electrophilic addition of
bromine to
bisbenzotetracyclo[6.2.2.2(3,6).0(2,7)]tetradeca-4,9,11,13-tetraene(BBTT)
molecule have been investigated by the methods of quantum chemistry. The
structure of the BBTT molecule has been studied by ab initio and DFT/B3LYP
methods using the 6-31G(d) and 6-311G(d) basis sets. The double bonds of BBTT
molecule are endo-pyramidalized. The structure and stability of the cationic
intermediates and products of the addition reaction have been investigated by
HF/6-311G(d), HF/6-311G(d,p), B3LYP/6-311G(d) and
B3LYP/6-311++G(2d,p)//B3LYP/6-311G(d) methods. The bridged bromonium cation
isomerized into the more stable nonclassical delocalized N- and U-type cations
and the difference between the stability of these cations is small. For the
determination of the direction of addition reaction and the stereochemistry of
the products, the stability of nonclassical delocalized N- and U-type ions and
the structure of their cationic centres play a vital role. Since the cationic
centre of the N-type ion is in interaction with the benzene ring from the exo
face, the nucleofilic attackof the bromide anion to this centre occurs from the
endo face and the exo,endo-isomer of the N-type product is obtained. The attack
of bromide anion, towards the cationic centre of U-type ion from the endo face is
sterically hindered by the hydrogen atom therefore the attack occurs from the exo
face, which interacts with the benzene ring and the more stable exo,exo-isomer of
U-type product is formed. Although, the U-type cation was 2.232 kcal mol(-1) more
stable than the N-type cation, the U-type product was 0.587 kcal mol(-1) less
stable than the N-type product.

PMID: 17874151 [PubMed - indexed for MEDLINE]


101. Arq Bras Oftalmol. 2007 May-Jun;70(3):429-32.

Intermittent exotropia: comparative surgical results of lateral recti-recession
and monocular recess-resect.

Fiorelli VM, Goldchmit M, Uesugui CF, Souza-Dias C.

Cornea and External Disease Sector, Ophthalmology Department, Santa Casa de
Misericordia de Sao Paulo, Sao Paulo, SP, Brazil. fiorelliliv@uol.com.br

PURPOSE: To compare the results between recession of the lateral recti and
monocular recess-resect procedure for the correction of the basic type of
intermittent exotropia. METHODS: 115 patients with intermittent exotropia were
submitted to surgery. The patients were divided into 4 groups, according to the
magnitude of preoperative deviation and the surgical procedure was subsequently
performed. Well compensated orthophoria or exo-or esophoria were considered
surgical success, with minimum of 1 year follow-up after the operation. RESULTS: 
Success was obtained in 69% of the patients submitted to recession of the lateral
recti, and in 77% submitted to monocular recess-resect. In the groups with
deviations between 12 PD and 25 PD, surgical success was observed in 74% of the
patients submitted to recession of the lateral recti and in 78% of the patients
submitted to monocular recess-resect. (p=0.564). In the group with deviations
between 26 PD and 35 PD, surgical success was observed in 65% out of the patients
submitted to recession of the lateral recti and in 75% of the patients submitted 
to monocular recess-resect. (p=0.266). CONCLUSION: Recession of lateral recti and
monocular recess-resect were equally effective in correcting basic type
intermittent exotropia according to its preoperative deviation in primary
position.

PMID: 17768548 [PubMed - indexed for MEDLINE]


102. J Org Chem. 2007 Aug 31;72(18):6982-91. Epub 2007 Aug 7.

Origin of the detrimental effect of lithium halides on an enantioselective
nucleophilic alkylation of aldehydes.

Pate F, Duguet N, Oulyadi H, Harrison-Marchand A, Fressigne C, Valnot JY, Lasne
MC, Maddaluno J.

Laboratoire de Chimie Organique et Biologique Structurale, Laboratoire des
Fonctions Azotees & Oxygenees Complexes de l'IRCOF, UMR CNRS 6014, Universite et 
INSA de Rouen, 76821 Mont St Aignan Cedex, France.

The effect of lithium halides on the enantioselectivity of the addition of
methyllithium on o-tolualdehyde, in the presence of chiral lithium amides derived
from chiral 3-aminopyrrolidines (3APLi), has been investigated. The enantiomeric 
excess of the resulting 1-o-tolylethanol was found to drop upon addition of
significant amounts of LiCl, introduced before the aldehyde. The competitive
affinity between the lithium amide, the methyllithium, and the lithium halides in
THF was examined by multinuclear NMR spectroscopy and DFT calculations. The
results showed that the original mixed aggregate of the chiral lithium amide and 
methyllithium is rapidly, totally, and irreversibly replaced by a similar 1:1
complex involving one lithium chloride or bromide and one lithium amide. While
the MeLi/LiX substitution occurs with some degree of epimerization at the
nitrogen for the endo-MeLi:3APLi complex, it is mostly stereospecific for the
exo-type arrangements of the aggregate. The thermodynamic preference for mixed
aggregates between 3APLi and LiX was confirmed by static DFT calculations: the
data show that the LiCl and LiBr aggregates are more stable than their MeLi
counterparts by more than 10 kcal.mol(-1) provided THF is explicitly taken into
account. These results suggest that a sequestration of the source of chirality by
the lithium halides is at the origin of the detrimental effect of these additives
on the ee of the model reaction.

PMID: 17683145 [PubMed]


103. J Leukoc Biol. 2007 Oct;82(4):829-38. Epub 2007 Jul 11.

Nonspecific CD4(+) T cells with uptake of antigen-specific dendritic
cell-released exosomes stimulate antigen-specific CD8(+) CTL responses and
long-term T cell memory.

Hao S, Yuan J, Xiang J.

Research Unit, Division of Health Research, Saskatchewan Cancer Agency,
Saskatoon, Saskatchewan, Canada.

Dendritic cell (DC) and DC-derived exosomes (EXO) have been used extensively for 
tumor vaccination. However, its therapeutic efficiency is limited to only
production of prophylactic immunity against tumors. T cells can uptake
DC-released EXO. However, the functional effect of transferred exosomal molecules
on T cells is unclear. In this study, we demonstrated that OVA protein-pulsed
DC-derived EXO (EXO(OVA)) can be taken up by Con A-stimulated, nonspecific CD4(+)
T cells derived from wild-type C57BL/6 mice. The active EXO-uptaken CD4(+) T
cells (aT(EXO)), expressing acquired exosomal MHC I/OVA I peptide (pMHC I)
complexes and costimulatory CD40 and CD80 molecules, can act as APCs capable of
stimulating OVA-specific CD8(+) T cell proliferation in vitro and in vivo and
inducing efficient CD4(+) Th cell-independent CD8(+) CTL responses in vivo. The
EXO(OVA)-uptaken CD4(+) aT(EXO) cell vaccine induces much more efficient CD8(+) T
cell responses and immunity against challenge of OVA-transfected BL6-10 melanoma 
cells expressing OVA in wild-type C57BL/6 mice than EXO(OVA). The in vivo
stimulatory effect of the CD4(+) aT(EXO) cell to CD8(+) T cell responses is
mediated and targeted by its CD40 ligand signaling/acquired exosomal CD80 and
pMHC I complexes, respectively. In addition, CD4(+) aT(EXO) vaccine stimulates a 
long-term, OVA-specific CD8(+) T cell memory. Therefore, the EXO(OVA)-uptaken
CD4(+) T cells may represent a new, effective, EXO-based vaccine strategy in
induction of immune responses against tumors and other infectious diseases.

PMID: 17626150 [PubMed - indexed for MEDLINE]


104. J Morphol. 2007 Sep;268(9):815-25.

Feeding, nutrient flow, and functional gut morphology in the cricket Gryllus
bimaculatus.

Woodring J, Lorenz MW.

Department of Animal Ecology I, University of Bayreuth, 95440 Bayreuth, Germany. 
joseph.woodring@uni-bayreuth.de

The flow of nutrients through the digestive tract of Gryllus bimaculatus is
regulated by the proventriculus, which effectively triturates the partially
digested food coming from the crop and shoves the mushy nutrient mass into the
space between the paired caeca. The many folds at the base of the caeca form a
sieve, and only fine food particles (4-10 microm) and fluids in the mush are
filtered under pressure (produced by proventricular peristalsis) into the caeca. 
Combined with the release of enzymes in the caeca and the influx of water, the
caeca are rapidly inflated on day 1 after the terminal molt. The remaining,
mostly undigested food is shoved into a tube formed by the peritrophic membrane, 
which is first formed at the anterior end of the ventriculus. A mucous membrane
(peritrophic gel) covers the caecal epithelium, and seems to merge with the true 
peritrophic membrane at the beginning of the ventriculus. The Type I peritrophic 
membrane is dragged posteriorly through the entire ventriculus and ileum by the
posterior movement of the food bolus, which is shoved posteriorly at a rate of 6 
mm/h by proventricular pressure. The growth rate of the peritrophic membrane is
about 3 mm/h. Peristalsis does not occur in the midgut or ileum; the muscles in
these regions function solely to counteract the internal pressure produced by the
proventriculus. The exo- and endoperitrophic space in newly molted animals is
open and fluids can flow in both directions. The endoperitrophic space becomes
filled on day 1, and leads to a great reduction of the exoperitrophic space. In
the ileal pouch (exoperitrophic space) the peritrophic membrane separates the
mass of bacteria from the waste bolus within the endoperitrophic space. Feathery 
bristles arising from the cuticular covering of the finger-like invaginations of 
the ileal wall hold most of the bacterial mass in place. The crop weight
decreases from day 1 to day 3 as the weight of caeca, ventriculus, and ileum
increases. After day 3, food uptake and the weight of the entire gut system
decrease in female crickets, partly in response to space restrictions in the
abdomen caused by rapid ovarial growth. (c) 2007 Wiley-Liss, Inc.

PMID: 17624929 [PubMed - indexed for MEDLINE]


105. Chem Commun (Camb). 2007 Jul 14;(26):2698-700. Epub 2007 Apr 11.

Nucleophilic 5-endo-trig cyclizations of N-homoallylic sulfonamides: a facile
method for the construction of pyrrolidine rings.

Ichikawa J, Lapointe G, Iwai Y.

Department of Chemistry, Graduate School of Science, The University of Tokyo,
Hongo, Bunkyo-ku, Tokyo, Japan. junji@chem.s.u-tokyo.ac.jp

Normally disfavored 5-endo-trig cyclizations proceed in N-homoallylsulfonamides
bearing a CF(3), CCl(3), CO(2)Et or CN group at the C-3 position, via an
intramolecular S(N)2'-type or addition reaction to construct pyrrolidine rings,
even though the system allows a more favorable 5-exo-trig pathway.

PMID: 17594024 [PubMed]


106. J Nutr. 2007 Jul;137(7):1725-33.

Contribution of mucosal maltase-glucoamylase activities to mouse small intestinal
starch alpha-glucogenesis.

Quezada-Calvillo R, Robayo-Torres CC, Opekun AR, Sen P, Ao Z, Hamaker BR, Quaroni
A, Brayer GD, Wattler S, Nehls MC, Sterchi EE, Nichols BL.

CIEP-Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi, Zona
Universitaria, San Luis Potosi, S.L.P., Mexico 78360.

Digestion of starch requires activities provided by 6 interactive small
intestinal enzymes. Two of these are luminal endo-glucosidases named
alpha-amylases. Four are exo-glucosidases bound to the luminal surface of
enterocytes. These mucosal activities were identified as 4 different maltases.
Two maltase activities were associated with sucrase-isomaltase. Two remaining
maltases, lacking other identifying activities, were named maltase-glucoamylase. 
These 4 activities are better described as alpha-glucosidases because they digest
all linear starch oligosaccharides to glucose. Because confusion persists about
the relative roles of these 6 enzymes, we ablated maltase-glucoamylase gene
expression by homologous recombination in Sv/129 mice. We assayed the
alpha-glucogenic activities of the jejunal mucosa with and without added
recombinant pancreatic alpha-amylase, using a range of food starch substrates.
Compared with wild-type mucosa, null mucosa or alpha-amylase alone had little
alpha-glucogenic activity. alpha-Amylase amplified wild-type and null mucosal
alpha-glucogenesis. alpha-Amylase amplification was most potent against amylose
and model resistant starches but was inactive against its final product
limit-dextrin and its constituent glucosides. Both sucrase-isomaltase and
maltase-glucoamylase were active with limit-dextrin substrate. These mucosal
assays were corroborated by a 13C-limit-dextrin breath test. In conclusion, the
global effect of maltase-glucoamylase ablation was a slowing of rates of mucosal 
alpha-glucogenesis. Maltase-glucoamylase determined rates of digestion of starch 
in normal mice and alpha-amylase served as an amplifier for mucosal starch
digestion. Acarbose inhibition was most potent against maltase-glucoamylase
activities of the wild-type mouse. The consortium of 6 interactive enzymes
appears to be a mechanism for adaptation of alpha-glucogenesis to a wide range of
food starches.

PMID: 17585022 [PubMed - indexed for MEDLINE]


107. Arch Virol. 2007;152(9):1655-64. Epub 2007 Jun 8.

Functional characterization of chitinase from Cydia pomonella granulovirus.

Daimon T, Katsuma S, Kang WK, Shimada T.

Graduate School of Agricultural and Life Sciences, Department of Agricultural and
Environmental Biology, The University of Tokyo, Tokyo, Japan.

Baculovirus chitinases (V-CHIAs) play a crucial role in the terminal liquefaction
of virus-infected larvae after death. Although v-chiAs from
nucleopolyhedroviruses (NPVs) have been well characterized, little is known about
v-chiAs from granuloviruses (GVs). We characterized the v-chiA of Cydia pomonella
GV (CpGV) by constructing a recombinant Bombyx mori NPV (BmNPV) in which BmNPV
v-chiA was replaced by CpGV v-chiA (103CpGV virus). CpGV v-chiA encoded an
approximately 70-kDa chitinase with an exo-type substrate preference. CpGV V-CHIA
lacked a C-terminal KDEL endoplasmic reticulum retention motif and was suggested 
to be a secretory protein. Terminal host liquefaction of B. mori larvae and
proper folding of BmNPV-encoded cysteine protease (BmNPV V-CATH) were observed
following infection with 103CpGV, indicating that CpGV v-chiA is able to
compensate for the absence of its BmNPV counterpart. Our data suggest that the
molecular interaction between V-CHIA and V-CATH may be conserved across a broad
range of lepidopteran GVs and NPVs.

PMID: 17557135 [PubMed - indexed for MEDLINE]


108. Anal Chim Acta. 2007 Jun 5;592(2):146-53. Epub 2007 Apr 20.

Glycosyl linkage characteristics and classifications of exo-polysaccharides of
some regionally different strains of Lentinula edodes by amplified fragment
length polymorphism assay and cluster analysis.

Lo TC, Kang MW, Wang BC, Chang CA.

Department of Biological Science and Technology, National Chiao Tung University, 
Hsinchu, Taiwan, ROC.

We report here the first combined amplified fragment length polymorphism (AFLP)
analysis of genomic DNA fingerprinting data and cluster analysis of the
exo-polysaccharide glycosyl linkage data of 10 regionally different strains of
Lentinula edodes to compare their genetic and structural similarities and
differences. In addition, the monosaccharide compositions, molecular weights,
glycosyl structural linkages were investigated for the exo-polysaccharides
extracted from these different phylogenetic groups of regionally different L.
edodes. All exo-polysaccharides had similar molecular weight distribution between
1x10(4) and 3x10(6) Da and the monosaccharide composition analysis revealed the
presence of heterogeneous materials containing glucose, mannose, xylose,
galactose, fucose, rhamnose and arabinose in different ratios. Among these
monosaccharides, the glucose contents are the highest for all but one strain,
indicating that glucose probably is the building block of the backbones of these 
exo-polysaccharides. The AFLP assay data helped to classify the 10 L. edodes
strains into three distinct genetic groups. Gas chromatographic and mass
spectrometric (GC-MS) data revealed five different glycosyl linkage types for
these exo-polysaccharides. Most of the exo-polysaccharide backbone structures
contain (1-->4)-linked-D-glucopyranosyl and (1-->6)-linked-D-glucopyranosyl
moieties. Arabinose 1-->4 linkages and mannose 1-->2 linkages also exist in all
strains. The only differences among these linkages are their monosaccharide
compositions leading to different degree of backbone and branch formations.
Cluster analyses of the GC-MS data of the exo-polysaccharides of the 10 strains
resulted in 10 dendrograms. However, four of the 10 dendrograms were identical
and were obtained using the average, Ward and weighted linkage type method of
Manhattan distance and using the Ward method of Euclidean distance. The results
of cluster analyses were not very much different from that of the AFLP assay and 
allowed the comparison of genetic and structural similarities and differences.

PMID: 17512819 [PubMed - indexed for MEDLINE]


109. Biophys J. 2007 Sep 1;93(5):1707-18. Epub 2007 May 11.

Effects of serine-to-cysteine mutations on beta-lactamase folding.

Santos J, Risso VA, Sica MP, Ermacora MR.

Departamento de Ciencia y Tecnologia, Universidad Nacional de Quilmes, Roque
Saenz Pena 352, B1876XD Bernal, Buenos Aires, Argentina.

B. licheniformis exo-small beta-lactamase (ESBL) has two nonsequential domains
and a complex architecture. We replaced ESBL serine residues 126 and 265 with
cysteine to probe the conformation of buried regions in each domain.
Spectroscopic, hydrodynamic, and chemical methods revealed that the mutations do 
not alter the native fold but distinctly change stability (S-126C > wild-type >
S-126/265C > S-265C ESBL) and the features of partially folded states. The
observed wild-type ESBL equilibrium intermediate has decreased fluorescence but
full secondary structure. S-126C ESBL intermediate has the fluorescence of the
unfolded state, no thiol reactivity, and partial secondary structure. S-265C and 
S-126/265C ESBL populate intermediate states unfolded by fluorescence and thiol
reactivity but with full secondary structure. Mass analysis of S-126/265C ESBL in
the partially folded state proved that both thiol groups become exposed
simultaneously. None of the intermediates is compatible with sequential domain
unfolding. Molecular dynamics simulation suggests that the stabilizing effect of 
the S-126C substitution is due to optimization of van der Waals interactions and 
packing. On the other hand, destabilization induced by the S-265C mutation
results from alteration of the hydrogen-bond network. The results illustrate the 
large impact that seemingly conservative serine-to-cysteine changes can have on
the energy landscape of proteins.

PMCID: PMC1948053
PMID: 17496026 [PubMed - indexed for MEDLINE]


110. Biochem Biophys Res Commun. 2007 Jun 15;357(4):834-9. Epub 2007 Apr 16.

L-type calcium channels are preferentially coupled to endocytosis in bovine
chromaffin cells.

Rosa JM, de Diego AM, Gandia L, Garcia AG.

Instituto Teofilo Hernando, Departamento de Farmacologia y Terapeutica, Facultad 
de Medicina, Universidad Autonoma de Madrid, C/ Arzobispo Morcillo 4, 28029
Madrid, Spain.

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of
high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly
studied in chromaffin cells. However, similar reports concerning endocytosis are 
unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N
(omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+)
currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane
capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin 
cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 
mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the
exo-endocytotic responses while omega-agatoxin IVA markedly blocked those
responses. However, nifedipine blocked little exocytosis but almost completely
inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be
selectively coupled to endocytosis.

PMID: 17451644 [PubMed - indexed for MEDLINE]


111. J Phys Chem A. 2007 May 10;111(18):3585-91. Epub 2007 Apr 13.

Resonance-assisted hydrogen bonds: a critical examination. Structure and
stability of the enols of beta-diketones and beta-enaminones.

Sanz P, Mo O, Yanez M, Elguero J.

Departamento de Quimica, C-9, Universidad Autonoma de Madrid, Cantoblanco, 28049 
Madrid, Spain.

The characteristics of the intramolecular hydrogen bond (IMHB) for a series of 40
different enols of beta-diketones and their nitrogen counterparts have been
systematically analyzed at the B3LYP/6-311+G(3df,2p)//B3LYP/6-311+G(d,p) level of
theory. In some cases, two tautomers may exist which are interconnected by a
hydrogen shift through the IMHB. In tautomer a the HB donor group (YH) is
attached to the six-membered ring, while in tautomer b the HB acceptor (X) is the
one that is attached to the six-membered ring. We found that changing an O to a N
favors the a tautomer when the atom is endo and the contrary when it is exo,
while the presence of a double bond favors the a tautomers. As expected, the OH
group behaves as a better HB donor than the NH2 group and the C=NH group as a
better HB acceptor than the C=O group, although the first effect clearly
dominates. Accordingly, the expected IMHB strength follows the [donor, acceptor] 
trend: [OH, C=NH] > [OH, C=O] > [NH2, C=NH] > [NH2, C=O]. For all those compounds
in which the functionality exhibiting the IMHB is unsaturated (I-type), the IMHB 
is much stronger than in their saturated counterparts (II-type). However, when
the systems of the II-type subset, which are saturated, are constrained to have
the HB donor and the HB acceptor lying in the same plane and at the same distance
as in the corresponding unsaturated analogue, the IMHB is of similar or even
larger strength. Hence, we conclude that, at least for this series of unsaturated
compounds, the resonance-assisted hydrogen bond effect is not the primary reason 
behind the strength of their IMHBs, which is simply a consequence of the
structure of the sigma-skeleton of the system that keeps the HB donor and the HB 
acceptor coplanar and closer to each other.

PMID: 17429952 [PubMed - indexed for MEDLINE]


112. J Org Chem. 2007 May 11;72(10):3667-71. Epub 2007 Apr 12.

Synthesis of dimethyl gloiosiphone a by way of palladium-catalyzed domino
cyclization.

Doi T, Iijima Y, Takasaki M, Takahashi T.

Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1 Ookayama, 
Meguro, Tokyo 152-8552, Japan.

The synthesis of a spiro[4.4]nonane skeleton by the palladium-catalyzed domino
cyclization of a linear 7-methylene-2,10-undecadienyl acetate is described. The
pi-allylpalladium intermediate underwent intramolecular alkene insertion with
high intraannular diastereoselectivity, followed by intramolecular Heck-type
cyclization, leading to a spiro[4.4]nonane system. Oxidation of the allylic ether
moiety and transformation of the vinyl group to an exo-methylene unit provided 3,
which is the known synthetic intermediate of dimethyl gloiosiphone A (2).

PMID: 17428095 [PubMed]


113. Physiology (Bethesda). 2007 Apr;22:113-21.

The ins and outs of secretion from pancreatic beta-cells: control of
single-vesicle exo- and endocytosis.

MacDonald PE, Rorsman P.

Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada.
pmacdonald@bcell.org

Exocytosis of insulin-containing secretory vesicles in pancreatic beta-cells is
crucial to maintenance of plasma glucose levels. They fuse with the plasma
membrane in a regulated manner to release their contents and are subsequently
recaptured either intact or through conventional clathrin-mediated endocytosis.
Here, we discuss these mechanisms in beta-cells at the single-vesicle level.

PMID: 17420302 [PubMed - indexed for MEDLINE]


114. Bioresour Technol. 2008 Mar;99(5):1417-24. Epub 2007 Apr 3.

Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in
relation to their activity against various cellulosic substrates.

Martins LF, Kolling D, Camassola M, Dillon AJ, Ramos LP.

Department of Chemistry, Federal University of Parana, PO Box 19081, Curitiba, PR
81531-990, Brazil.

Penicillium echinulatum has been identified as a potential cellulase producer for
bioconversion processes but its cellulase system has never been investigated in
detail. In this work, the volumetric activities of P. echinulatum cellulases were
determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), 
hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan
(3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and
p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in
relation to the amount of protein and compared those of Trichoderma reesei
cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to
have similar total cellulase and xylanase activities. Analysis of substrate
hydrolysates demonstrated that P. echinulatum enzymes have higher
beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to
have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P.
echinulatum cellulases had enough beta-glucosidase activity to remove most of the
cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became
more powerful than P. echinulatum cellulases when supplemented with exogenous
beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the
same influence over the degree of polymerization of cellulose, revealing that
hydrolyzes were carried out under the typical endo-exo synergism of fungal
enzymes.

PMID: 17408952 [PubMed - indexed for MEDLINE]


115. Toxicol Sci. 2007 Jul;98(1):57-62. Epub 2007 Mar 30.

Elevation of 8-hydroxydeoxyguanosine in DNA from isolated mouse lung cells
following in vivo treatment with aflatoxin B(1).

Guindon KA, Bedard LL, Massey TE.

Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario,
Canada, K7L 3N6.

Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by some strains of Aspergillus
and is a recognized pulmonary and hepatic carcinogen. The most widely accepted
mechanism of AFB(1) carcinogenicity involves bioactivation to
AFB(1)-8,9-exo-epoxide and binding to DNA to form AFB(1)-N(7)-guanine. Another
potential cause of DNA damage is AFB(1)-mediated stimulation of reactive oxygen
species formation, leading to oxidation of DNA bases. The objective of this study
was to determine the ability of AFB(1) to cause oxidative DNA damage in lung cell
types of the A/J mouse. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in 
freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and
nonciliated bronchial epithelial (Clara) cells was assessed by high-performance
liquid chromatography with electrochemical detection. An approximately 3-fold
increase in 8-OHdG formation occurred in both alveolar macrophage and Clara cell 
preparations isolated from A/J mice 2 h following treatment with a single
tumorigenic dose of 50 mg/kg AFB(1) ip (n = 3, p < 0.05). Prior treatment with
300 kU/kg polyethylene glycol-conjugated catalase prevented the AFB(1)-induced
increase in 8-OHdG levels in all mouse lung cell preparations (n = 3, p < 0.05). 
These results support the possibility that oxidative DNA damage in mouse lung
cells contributes to AFB(1) carcinogenicity.

PMID: 17400578 [PubMed - indexed for MEDLINE]


116. Inorg Chem. 2007 Apr 2;46(7):2691-9. Epub 2007 Mar 9.

Sulfur ligand substitution at the nickel(II) sites of cubane-type and cubanoid
NiFe3S4 clusters relevant to the C-clusters of carbon monoxide dehydrogenase.

Sun J, Tessier C, Holm RH.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, USA.

Substitution reactions at the nickel site of the cubane-type cluster
[(Ph3P)NiFe3S4(LS3)]2- (2) have been investigated in the course of a synthetic
approach to the C-clusters of CODH. Reaction of 2 with RS- or
toluene-3,4-dithiolate affords [(RS)NiFe3S4(LS3)]3- (R = Et (5), H (6)) or
[(tdt)NiFe3S4(LS3)]3- (7), demonstrating that anionic sulfur ligands can be bound
at the NiII site. Clusters 5 and 6 contain tetrahedral Ni(micro3-S)3(SR) sites.
Cluster 7 is of particular interest because it includes a cubanoid
NiFe3(micro2-S)(micro3-S)3 core and an approximately planar Ni(tdt)(micro3-S)2
unit. The cubanoid structure is found in all C-clusters, and an NiS4-type unit
has been reported in C. hydrogenoformans CODH. Clusters 5/6 are formulated to
contain the core [NiFe3S4]1+ identical with Ni2+ (S = 1) + [Fe3S4]1- (S = 5/2)
and 7 the core [NiFe3S4]2+ identical with Ni2+ (S = 0) + [Fe3S4]0 (S = 2) on the 
basis of structure, 57Fe isomer shifts, and 1H NMR isotropic shifts. Also
reported are [(EtS)CuFe3S4(LS3)]3- (9) and [Fe4S4(LS3)(tdt)]3- (11). The
structures of 5-7, 9, and 11 are presented. Cluster 11, with a five-coordinate
Fe(tdt)(micro3-S)3 site, provides a clear structural contrast with 7, which is
currently the closest approach to a C-cluster but lacks the exo iron atom found
in the NiFe4S4,5 cores of the native clusters. (CODH = carbon monoxide
dehydrogenase, LS3 =
1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-
), tdt = toluene-3,4-dithiolate).

PMID: 17346040 [PubMed - indexed for MEDLINE]


117. J Am Chem Soc. 2007 Mar 28;129(12):3745-53. Epub 2007 Mar 7.

Stable exo-nido-metallacarboranes (M = Os(IV)) that incorporate
meta-carborane-based dicarbollide ligands.

Balagurova EV, Cheredilin DN, Kolomnikova GD, Tok OL, Dolgushin FM, Yanovsky AI, 
Chizhevsky IT.

A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov Street, 119991
Moscow, Russia.

Reactions of the [K]+ salts of the [nido-7,9-C2B9H12]- anion (2) and its
C-phenylated derivative [7-Ph-nido-7,9-C2B9H11]- (4) with [OsCl2(PPh3)3] (3)
proceed in benzene at ambient temperature with the formation of 16-electron
chlorohydrido-Os(IV) exo-nido complexes,
[exo-nido-10,11-{(Ph3P)2OsHCl}-10,11-(mu-H)2-7-R-7,9-C2B9H8] (5: R = H; 6: R =
Ph), along with the small amounts of the charge-compensated nido-carboranes
[nido-7,9-C2B9H11PPh3] (7) and [7-Ph-nido-7,9-C2B9H10PPh3] (8) as byproducts.
However, when carried out under mild heating in ethanol, the reaction of 2 with 3
selectively afforded a 16-electron dihydrido-Os(IV) exo-nido complex
[exo-nido-10,11-{(Ph3P)2OsH2}-10,11-(mu-H)2-7,9-C2B9H9] (9). Structures of both
complexes 5 and 9 have been confirmed by single-crystal X-ray diffraction
studies, which revealed that nido-carboranes in these species function as a
bidentate dicarbollide ligands [7-R-nido-7,9-C2B9H10]2- linked to the Os(IV)
center via two B-H...Os bonds involving adjacent B-H vertices in the upper CBCBB 
belt of the carborane cage. Thus, compounds 5 and 9 represent the first
structurally characterized exo-nido-metallacarboranes based on
meta-dicarbollide-type ligands. Variable-temperature 1H and 31P{1H} NMR
experiments indicate that complex 9 is fluxional in solution and shows an unusual
exchange between terminal Os-(H)2 and bridging {B-H}2...Os hydrogen atoms. Upon
heating in d8-THF at 65 degrees C, complex 9 converts irreversibly to its closo
isomer [2,2-(PPh3)2-2,2-H2-closo-2,1,7-OsC2B9H11] (13), which could thus be
obtained as a pure crystalline solid. The structure of 13 has been established on
the basis of analytical and multinuclear NMR data and a single-crystal X-ray
diffraction study.

PMID: 17341073 [PubMed]


118. Infect Immun. 2007 May;75(5):2351-8. Epub 2007 Mar 5.

Role of Bacillus anthracis spore structures in macrophage cytokine responses.

Basu S, Kang TJ, Chen WH, Fenton MJ, Baillie L, Hibbs S, Cross AS.

Center for Vaccine Development, Department of Medicine, University of Maryland,
685 W. Baltimore Street, HSF I-480, Baltimore, MD 21201, USA.
sbasu1@medicine.umaryland.edu

The innate immune response of macrophages (Mphi) to spores, the environmentally
acquired form of Bacillus anthracis, is poorly characterized. We therefore
examined the early Mphi cytokine response to B. anthracis spores, before
germination. Mphi were exposed to bacilli and spores of Sterne strain 34F2 and
its congenic nongerminating mutant (DeltagerH), and cytokine expression was
measured by real-time PCR and an enzyme-linked immunosorbent assay. The
exosporium spore layer was retained (exo+) or removed by sonication (exo-).
Spores consistently induced a strong cytokine response, with the exo- spores
eliciting a two- to threefold-higher response than exo+ spores. The threshold for
interleukin-1beta (IL-1beta) production by wild-type Mphi was significantly lower
than that required for tumor necrosis factor alpha expression. Cytokine
production was largely dependent on MyD88, suggesting Toll-like receptor
involvement; however, the expression of beta interferon in MyD88-/- Mphi suggests
involvement of a MyD88-independent pathway. We conclude that (i) the B. anthracis
spore is not immunologically inert, (ii) the exosporium masks epitopes recognized
by the Mphi, (iii) the Mphi cytokine response to B. anthracis involves multiple
pattern recognition receptors and signaling pathways, and (iv) compared to other 
cytokines, IL-1beta is expressed at a lower spore concentration.

PMCID: PMC1865778
PMID: 17339355 [PubMed - indexed for MEDLINE]


119. Phytochemistry. 2007 Apr;68(7):1046-58. Epub 2007 Mar 6.

An immunomodulating pectic polymer from Glinus oppositifolius.

Inngjerdingen KT, Kiyohara H, Matsumoto T, Petersen D, Michaelsen TE, Diallo D,
Inngjerdingen M, Yamada H, Paulsen BS.

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo,
P.O. Box 1068 Blindern, N-0316 Oslo, Norway. k.t.inngjerdingen@farmasi.uio.no
<k.t.inngjerdingen@farmasi.uio.no>

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the
Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has
previously been reported to consist of arabinogalactans type I and II, probably
linked to a rhamnogalacturonan backbone. To further elucidate the structure of
the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were
performed. Five different glycosidases were used,
endo-alpha-D-(1-->4)-polygalacturonase, exo-alpha-L-arabinofuranosidase,
endo-alpha-L-(1-->5)-arabinanase, endo-beta-D-(1-->4)-galactanase and
exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety
consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked
Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp
side chain may be branched in position 3 with arabinofuranosyl (Araf) side
chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl
units attached to position 3 is also a structural element in the polymer. We
further show that GOA1 induce proliferation of B cells and the secretion of
IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma
in NK-cells. To elucidate structure-activity relations the native polymer and the
digested fractions were tested for complement fixing activity and intestinal
immune stimulating activity. The partial removal of Araf residues after enzymatic
degradations did not affect the bioactivities, while the acid hydrolysed fraction
showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked
Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a
reduction in molecular weight are factors that might have contributed to reduced 
bioactivity.

PMID: 17337024 [PubMed - indexed for MEDLINE]


120. Org Lett. 2007 Mar 29;9(7):1399-402. Epub 2007 Feb 28.

Hg(OAc)(2).0.1Sc(OTf)(3)-catalyzed cycloisomerization of 2-(4-pentynyl)furan.

Yamamoto H, Sasaki I, Imagawa H, Nishizawa M.

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.

[structure: see text]. Although the Hg(OTf)2.3TMU-catalyzed Friedel-Crafts-type
reaction of 3-(4-pentynyl)furan afforded the exo cyclization product, the
reaction of 2-(4-pentynyl)furan furnished a very low yield. We found a 10:1 mixed
reagent of Hg(OAc)2 and Sc(OTf)3 showed remarkable catalytic activity for the
latter transformation. The actual reacting species is presumed to be
Hg(OAc)(OTf), which is efficiently generated in situ by mixing the two reagents.

PMID: 17326651 [PubMed]


121. Org Biomol Chem. 2006 Dec 21;4(24):4453-9. Epub 2006 Oct 30.

Natural sialoside analogues for the determination of enzymatic rate constants.

Indurugalla D, Watson JN, Bennet AJ.

Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby,
British Columbia, Canada V5A 1S6.

Two isomeric 4-methylumbelliferyl-alpha-D-N-acetylneuraminylgalactopyranosides (1
and 2) were synthesised. These compounds contain either the natural alpha-2,3 or 
alpha-2,6 sialyl-galactosyl linkages, as well as an attached
4-methylumbelliferone for convenient detection of their hydrolyses. These
compounds were designed as natural sialoside analogues to be used in a continuous
assay of sialidase activity, where the sialidase-catalysed reaction is coupled
with an exo-beta-galactosidase-catalysed hydrolysis of the released galactoside
to give free 4-methylumbelliferone. The kinetic parameters for 1 and 2 were
measured using the wild-type and nucleophilic mutant Y370G recombinant sialidase 
from Micromonospora viridifaciens. Kinetic parameters for these analogues
measured using the new continuous assay were in good agreement with the
parameters for the natural substrate, 3'-sialyl lactose. Given the selection of
commercially available exo-beta-galactosidases that possess a variety of pH
optima, this new method was used to characterise the full pH profile of the
wild-type sialidase with the natural sialoside analogue 1. Thus, use of these new
substrates 1 and 2 in a continuous assay mode, which can be detected by UV/Vis or
fluorescence spectroscopy, makes characterisation of sialidase activity with
natural sialoside linkages much more facile.

PMID: 17268638 [PubMed - indexed for MEDLINE]


122. J Am Chem Soc. 2007 Feb 7;129(5):1076-88.

The mechanistically significant coordination chemistry of dinitrogen at FeMo-co, 
the catalytic site of nitrogenase.

Dance I.

School of Chemistry, University of New South Wales, Sydney 2052, Australia.
i.dance@unsw.edu.au

Reported here is a comprehensive theoretical investigation of the binding of N(2)
to the Fe(7)MoS(9)N(homocitrate)(cysteine)(histidine) active site (FeMo-co) of
the enzyme nitrogenase, as a prerequisite to elucidation of the chemical
mechanism of the catalyzed reduction to NH(3). The degree and type of
hydrogenation of FeMo-co, with H atoms and possibly an H(2) molecule, are key
variables, following the Thorneley-Lowe kinetic scheme. Ninety-four local energy 
minima were located for N(2) coordinated in eta(2) (side) and eta(1) (end) modes 
at the endo and exo coordination positions of Fe2 and Fe6. The stabilities of 57 
representative structures are assessed by calculation of the reaction profiles
and activation energies for the association and dissociation of N(2). Barriers to
association of N(2) depend mainly on the location of the hydrogenation and the
location of N(2) coordination, while dissociation barriers depend primarily on
whether N(2) is eta(2)- and eta(1)-coordinated, and secondarily on the location
of the hydrogenation. Increased negative charge on FeMo-co increases the
barriers, while C in place of N at the center of FeMo-co has little effect. The
interactions of the models of ligated FeMo-co with the surrounding protein,
including proteins with mutations of key amino acids, are assessed by in silico
cofactor transplantations and calculations of protein strain energies. From these
results, which identify models involving contacts and interactions with the
surrounding residues that have been shown by mutation to affect the N(2) activity
of nitrogenase, and from the N(2) coordination profiles, it is concluded that
endo-eta(1)-N(2) coordination at Fe6 is most probable. There is strong reason to 
believe that the mechanism of nitrogenase will involve one or more of the
preferred models presented here, and a detailed foundation of structures and
principles is now available for postulation and calculation of the profiles of
the steps in which H atoms bound to FeMo-co are transferred to bound N(2).

PMID: 17263388 [PubMed - indexed for MEDLINE]


123. Res Microbiol. 2007 Mar;158(2):150-8. Epub 2006 Dec 29.

Characterization of a luxI/luxR-type quorum sensing system and N-acyl-homoserine 
lactone-dependent regulation of exo-enzyme and antibacterial component production
in Serratia plymuthica RVH1.

Van Houdt R, Moons P, Aertsen A, Jansen A, Vanoirbeek K, Daykin M, Williams P,
Michiels CW.

Laboratory of Food Microbiology, Katholieke Universiteit Leuven, Kasteelpark
Arenberg 22, B-3001 Leuven, Belgium. rob.van.houdt@sckcen.be

Quorum sensing by means of N-acyl-l-homoserine lactones (AHLs) is widespread in
Gram-negative bacteria, where diverse AHLs influence a wide variety of functions,
even in a single genus such as Serratia. Here we report the identification and
characterization of the quorum sensing system of Serratia plymuthica strain RVH1.
This strain isolated from a raw vegetable processing line produces at least three
AHLs which were identified as N-butanoyl- (C4-HSL), N-hexanoyl- (C6-HSL) and
N-(3-oxo-hexanoyl)-homoserine lactone (3-oxo-C6-HSL). The identified LuxI homolog
SplI synthesizes 3-oxo-C6-HSL, and influences the production of C4-HSL and
C6-HSL, as splI gene inactivation resulted in loss of 3-oxo-C6-HSL production and
smaller amounts of C4-HSL and C6-HSL produced. SplI-dependent quorum sensing
controls 2,3-butanediol fermentation (previously reported) and the production of 
an extracellular chitinase, nuclease, protease and antibacterial compound. The
identity of the latter is not yet elucidated, but appears to be different from
the known antibacterial compounds produced by Serratia strains. SplR, the homolog
of the LuxR regulator, appears to act as a repressor of synthesis of
extracellular enzymes and antibacterial compound and to autorepress its own
expression, probably by binding to a 21bp lux box sequence.

PMID: 17258895 [PubMed - indexed for MEDLINE]


124. Biochemistry. 2007 Jan 23;46(3):792-8.

Kinetic and structural analysis of enzyme sliding on a substrate: multiple attack
in beta-amylase.

Ishikawa K, Nakatani H, Katsuya Y, Fukazawa C.

National Institute of Advanced Industrial Science and Technology, 1-8-31
Midorigaoka, Ikeda, Osaka 563-8577, Japan. kazu-ishikawa@aist.go.jp

Beta-amylase (EC 3.2.1.2) is starch-hydrolyzing exo-type enzyme that can catalyze
the successive liberation of beta-maltose from the nonreducing ends of
alpha-1,4-linked glucopyranosyl polymers. There is a well-known phenomenon called
multiple or repetitive attack where the enzyme releases several maltose molecules
in a single enzyme-substrate complex. In order to understand it further, we
examined the beta-amylase-catalyzed reaction using maltooligosaccharides. The
Monte Carlo method was applied for simulation of the beta-amylase-catalyzed
reaction including the multiple attack mechanism. Through site-directed
mutagenesis, we have successfully prepared a mutant enzyme which may be simulated
as a multiple attack action reduced one with retaining significant hydrolytic
activity. From the results of X-ray structure analysis of the mutant enzyme, it
was clarified that one carboxyl residue plays a very important role in the
multiple attack. The multiple attack action needs the force of enzyme sliding on 
the substrate. In addition, it is important for the multiple attack that the
enzyme and substrate have the characteristics of a stable productive
substrate-enzyme complex through a hydrogen bond between the nonreducing end of
the substrate and the carboxyl residue of the enzyme.

PMID: 17223700 [PubMed - indexed for MEDLINE]


125. Int J Dermatol. 2007 Jan;46(1):77-9.

Multiple keratoacanthomas in a young woman: report of a case emphasizing medical 
management and a review of the spectrum of multiple keratoacanthomas.

Feldman RJ, Maize JC.

Department of Dermatology, Medical University of South Carolina, Charleston,
South Carolina, USA.

Comment in:
    Int J Dermatol. 2007 Oct;46(10):1105.

A 27-year-old white woman was referred for consultation with regard to the
presence of extensive multiple keratotic lesions. She began to develop these
lesions at the age of 9 years, with healing of the lesions resulting in scar
formation. A biopsy was performed at the age of 16 years, but the patient was
unsure of the results. Since then, she had not had any treatment or biopsies, and
stated that she had not suffered from any health problems during the intervening 
period. She was most concerned about the tumors on her heels and soles, which
caused difficulty with ambulation. The family history was negative for skin
diseases, including melanoma, nonmelanoma skin cancer, psoriasis, and eczema, and
positive for Type II diabetes mellitus. A relative reported that the patient's
grandfather had similar lesions, but the patient's parents and siblings were
healthy. She was married and had one child, a 9-year-old daughter. Her child had 
no skin lesions. The patient's only medication was Ortho-Tricyclene birth control
pills. She had no known drug allergies. Physical examination revealed the
presence of multiple lesions on her body (Fig. 1). Her left superior helix
contained a well-demarcated, dome-shaped nodule with a rolled, mildly
erythematous border with a central hyperkeratotic plug. A similar lesion was
present in the scaphoid fossa of the left ear and smaller lesions were scattered 
on her face. Numerous lesions were present on the arms and legs bilaterally, with
the majority of lesions being located on the anterior lower legs. There were also
lesions present on the palms and soles. The lesions ranged in size from 5 mm to 3
cm, the largest being a verrucous exophytic nodule on the anterior aspect of her 
left leg. Overall, there appeared to be two distinct types of lesion. One type
appeared round, oval, and symmetric with a central keratotic plug, similar to
that on the ear. The other type was larger, more exophytic, and verrucous,
including the lesions on the volar surfaces. Also present were numerous,
irregularly shaped atrophic scars where previous lesions had healed
spontaneously. There were no oral lesions or lesions on her fingernails or
toenails, and her teeth and hair were normal. A biopsy was obtained from an early
lesion on the right dorsal forearm. Histology revealed an exo-/endophytic growth 
having a central crater containing keratinous material (Fig. 2). The crater was
surrounded by markedly hyperplastic squamous epithelium with large squamous
epithelial cells having abundant glassy cytoplasm. Some cells were dyskeratotic. 
Within the dermis was a dense, chiefly mononuclear inflammatory infiltrate. A
buttress of epidermis surrounded the crater. The clinical and pathologic data
were consistent with keratoacanthomas. Initial laboratory screenings revealed
elevated triglycerides and total cholesterol, 537 mg/dL (normal, < 150 mg/dL) and
225 mg/dL (normal, < 200 mg/dL), respectively, with all other laboratory results 
within normal limits. In anticipation of starting oral retinoid therapy for her
multiple keratoacanthomas, she was referred to her primary care physician for
control of hyperlipidemia. After her lipids had been controlled, she was placed
on isotretinoin (Accutane) 40 mg/day. There was some interval improvement with
regression of some lesions leaving atrophic scars. She was also started on
topical application of tazarotene (Tazorac) for all nonresolving lesions.
Possible side-effects from the isotretinoin occurred, including dry mouth and
eyes. After 8 months of isotretinoin, the patient was switched to acitretin
(Soriatane) 25 mg to determine whether it might have a more beneficial effect on 
the resistant lesions. Many of the larger lesions regressed leaving atrophic
scars. The dose of acitretin was subsequently increased to 35 mg because the
lesions on her heel and the ball of her foot persisted. Almost all of the lesions
resolved, except those on her feet, which are slowly regressing. Currently, the
patient is on a regimen of acitretin 25 mg once a day with tazarotene 0.1% gel
applied directly to the few residual keratoacanthomas on her feet, which are
slowly improving.

PMID: 17214727 [PubMed - indexed for MEDLINE]


126. J Bacteriol. 2007 Mar;189(6):2510-20. Epub 2007 Jan 5.

Sinorhizobium meliloti SyrA mediates the transcriptional regulation of genes
involved in lipopolysaccharide sulfation and exopolysaccharide biosynthesis.

Keating DH.

Department of Microbiology and Immunology, Loyola University Chicago, Building
105, 2160 S. First Avenue, Maywood, IL 60153, USA. dkeati1@lumc.edu

Sinorhizobium meliloti is a gram-negative soil bacterium found either in
free-living form or as a nitrogen-fixing endosymbiont of leguminous plants such
as Medicago sativa (alfalfa). S. meliloti synthesizes an unusual sulfate-modified
form of lipopolysaccharide (LPS). A recent study reported the identification of a
gene, lpsS, which encodes an LPS sulfotransferase activity in S. meliloti.
Mutants bearing a disrupted version of lpsS exhibit an altered symbiosis, in that
they elicit more nodules than wild type. However, under free-living conditions,
the lpsS mutant displayed no change in LPS sulfation. These data suggest that the
expression of lpsS is differentially regulated, such that it is transcriptionally
repressed during free-living conditions but upregulated during symbiosis. Here, I
show that the expression of lpsS is upregulated in strains that constitutively
express the symbiotic regulator SyrA. SyrA is a small protein that lacks an
apparent DNA binding domain and is predicted to be located in the cytoplasmic
membrane yet is sufficient to upregulate lpsS transcription. Furthermore, SyrA
can mediate the transcriptional upregulation of exo genes involved in the
biosynthesis of the symbiotic exopolysaccharide succinoglycan. The SyrA-mediated 
transcriptional upregulation of lpsS and exo transcription is blocked in mutants 
harboring a mutation in chvI, which encodes the response regulator of a conserved
two-component system. Thus, SyrA likely acts indirectly to promote
transcriptional upregulation of lpsS and exo genes through a mechanism that
requires the ExoS/ChvI two-component system.

PMCID: PMC1899389
PMID: 17209018 [PubMed - indexed for MEDLINE]


127. Cell Mol Life Sci. 2007 Jan;64(2):230-43.

Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation
during development and after neurotrauma.

Savaskan NE, Rocha L, Kotter MR, Baer A, Lubec G, van Meeteren LA, Kishi Y, Aoki 
J, Moolenaar WH, Nitsch R, Brauer AU.

Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam,
The Netherlands. n.savaskan@nki.nl

Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with
lysophospholipase D activity that generates bioactive lysophosphatidic acid.
Lysophosphatidic acid has been implicated in various neural cell functions such
as neurite remodeling, demyelination, survival and inhibition of axon growth.
Here, we report on the in vivo expression of autotaxin in the brain during
development and following neurotrauma. We found that autotaxin is expressed in
the proliferating subventricular and choroid plexus epithelium during embryonic
development. After birth, autotaxin is mainly found in white matter areas in the 
central nervous system. In the adult brain, autotaxin is solely expressed in
leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, 
autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion.
The present study revealed the cellular distribution of autotaxin in the
developing and lesioned brain and implies a function of autotaxin in
oligodendrocyte precursor cells and brain injuries.

PMID: 17192809 [PubMed - indexed for MEDLINE]


128. J Neurol Sci. 2007 Jan 31;252(2):181-4. Epub 2006 Dec 19.

Dopaminergic neuronal dysfunction associated with parkinsonism in both a Gaucher 
disease patient and a carrier.

Kono S, Shirakawa K, Ouchi Y, Sakamoto M, Ida H, Sugiura T, Tomiyama H, Suzuki H,
Takahashi Y, Miyajima H, Hattori N, Mizuno Y.

First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1
Handayama, Hamamatsu 431-3192, Japan. satokono@hama-med.ac.jp

A clinical association between Gaucher disease and parkinsonism has been
demonstrated. We herein report a Japanese patient with type 3 Gaucher disease who
was compound heterozygous for F213I and L444P mutations in the glucocerebrosidase
gene while his father was heterozygous for the L444P mutation. They both
presented with parkinsonism characterized by a predominance of akinetic-rigid
signs and a favorable response to anti-Parkinson therapies. We investigated the
dopaminergic neuronal function using positron emission tomography (PET) with
radioligands, [(11)C] CFT and [(11)C] raclopride. PET studies of both patients
demonstrated the [(11)C] CFT uptake to be severely decreased in the putamen and
the caudate nucleus, however, the [(11)C] raclopride uptake was normal in the
basal ganglia. Although the majority of Gaucher disease patients with
parkinsonism tend to be refractory to anti-Parkinson therapies. The clinical
features and the findings of the PET studies suggest that patients with
parkinsonism associated with the mutation in the glucocerebrosidase gene, even in
heterozygosis, may be related to the presynaptic dopaminergic neuronal
dysfunction reported in Parkinson's disease. A PET study to evaluate the
dopaminergic neuronal function in Gaucher disease would provide both a better
understanding of the effects of anti-Parkinson therapies and a help to improve
our ability to make an early diagnosis of parkinsonism associated with Gaucher
disease.

PMID: 17182061 [PubMed - indexed for MEDLINE]


129. J Biol Chem. 2007 Feb 16;282(7):4951-62. Epub 2006 Dec 14.

Biochemical evidence that phaZ gene encodes a specific intracellular medium chain
length polyhydroxyalkanoate depolymerase in Pseudomonas putida KT2442:
characterization of a paradigmatic enzyme.

de Eugenio LI, Garcia P, Luengo JM, Sanz JM, Roman JS, Garcia JL, Prieto MA.

Departamento de Microbiologia Molecular, Centro de Investigaciones Biologicas,
Consejo Superior de Investigaciones Cientificas (CSIC), C. Ramiro de Maeztu, 9,
28040 Madrid, Spain.

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using
intra- or extracellular PHA depolymerases. Most of our current knowledge of these
intracellular enzyme-coding genes comes from the analysis of short chain length
PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular
depolymerization systems still remained to be characterized. The phaZ gene of
some Pseudomonas putida strains has been identified only by mutagenesis and
complementation techniques as putative intracellular mcl-PHA depolymerase.
However, none of their corresponding encoded PhaZ enzymes have been characterized
in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been
purified and biochemically characterized after its overexpression in Escherichia 
coli. To facilitate these studies we have developed a new and very sensitive
radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated
that PhaZ is an intracellular depolymerase that is located in PHA granules and
that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers.
The enzyme behaves as a serine hydrolase that is inhibited by
phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of
PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that
the enzyme appears to be built up from a corealpha/beta hydrolase-type domain
capped with a lid structure with an active site containing a catalytic triad
buried near the connection between domains. All these data constitute the first
biochemical characterization of PhaZ and allow us to propose this enzyme as the
paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.

PMID: 17170116 [PubMed - indexed for MEDLINE]


130. J Physiol. 2007 Mar 1;579(Pt 2):413-29. Epub 2006 Dec 14.

Cholesterol-dependent balance between evoked and spontaneous synaptic vesicle
recycling.

Wasser CR, Ertunc M, Liu X, Kavalali ET.

Center for Basic Neuroscience, The University of Texas Southwestern Medical
Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA.

Cholesterol is a prominent component of nerve terminals. To examine cholesterol's
role in central neurotransmission, we treated hippocampal cultures with
methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an
inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used
hippocampal cultures from Niemann-Pick type C1-deficient mice defective in
intracellular cholesterol trafficking. These conditions revealed an augmentation 
in spontaneous neurotransmission detected electrically and an increase in
spontaneous vesicle endocytosis judged by horseradish peroxidase uptake after
cholesterol depletion by methyl-beta-cyclodextrin. In contrast, responses evoked 
by action potentials and hypertonicity were severely impaired after the same
treatments. The increase in spontaneous vesicle recycling and the decrease in
evoked neurotransmission were reversible upon cholesterol addition. Cholesterol
removal did not impact on the low level of evoked neurotransmission seen in the
absence of synaptic vesicle SNARE protein synaptobrevin-2 whereas the increase in
spontaneous fusion remained. These results suggest that synaptic cholesterol
balances evoked and spontaneous neurotransmission by hindering spontaneous
synaptic vesicle turnover and sustaining evoked exo-endocytosis.

PMCID: PMC2075401
PMID: 17170046 [PubMed - indexed for MEDLINE]


131. J Org Chem. 2006 Dec 22;71(26):9751-64.

Asymmetric platinum group metal-catalyzed carbonyl-ene reactions: carbon-carbon
bond formation versus isomerization.

Doherty S, Knight JG, Smyth CH, Harrington RW, Clegg W.

School of Natural Sciences, Chemistry, Bedson Building, Newcastle University,
Newcastle upon Tyne, NE1 7RU, United Kingdom. simon.doherty@ncl.ac.uk

A comparative study of the carbonyl-ene reaction between a range of
1,1'-disubstituted or trisubstituted alkenes and ethyl trifluoropyruvate
catalyzed by Lewis acid-platinum group metal complexes of the type
[M{(R)-BINAP}]2+ (M = Pt, Pd, Ni; BINAP is
2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) revealed subtle but significant
differences in their reactivity. For instance, the palladium-based Lewis acid
[Pd{(R)-BINAP}]2+ catalyzes the ene reaction between methylene cycloalkane to
afford the expected alpha-hydroxy ester in good yield and excellent diastereo-
and enantioselectivity. In contrast, under the same conditions, the corresponding
[M{(R)-BINAP}]2+ (M = Pt, Ni) catalyzes isomerization of methylene cycloalkane
and the ene reaction of the resulting mixture of methylene cycloalkane and
1-methylcycloalkene at similar rates to afford a range of -hydroxy esters in high
regioselectivity, good diastereoselectivity, and good to excellent
enantioselectivity. In addition, [Pt{(R)-BINAP}]2+ also catalyzes postreaction
isomerization of the ene product as well as consecutive ene reactions to afford a
double carbonyl-ene product. The sense of asymmetric induction has been
established by single-crystal X-ray crystallography, and a stereochemical model
consistent with the formation of (S)-configured -hydroxy ester has been proposed;
the same model also accounts for the observed exo-diastereoselectivity as well as
the level of diastereoselectivity.

PMID: 17168594 [PubMed - indexed for MEDLINE]


132. J Am Chem Soc. 2006 Dec 20;128(50):16169-77.

Iridium-iron-monocarborane clusters from oxidative insertion reactions of
[IrCl(CO)(PPh3)2] with ferracarborane anions.

Franken A, McGrath TD, Stone FG.

Department of Chemistry & Biochemistry, Baylor University, Waco, Texas
76798-7348, USA.

The nine-vertex ferracarborane salt [N(PPh3)2][7,7,7-(CO)3-closo-7,1-FeCB7H8] (1)
reacts with an excess of [IrCl(CO)(PPh3)2] in the presence of Tl[PF6] to form,
successively, the bimetallic species
[7,7,9,9,9-(CO)5-7-PPh3-closo-7,9,1-IrFeCB6H7] (3), in which one {BH}- vertex has
formally been subrogated by an {Ir(CO)2(PPh3)} unit, and the trimetallic complex 
[6,7,9-{Ir(CO)(PPh3)2}-7,9-(mu-H)2-7,9,9-(CO)3-7-PPh3-closo-7,9,1-IrFeCB6H6] (5),
which contains an {FeIr2} triangle. The {FeIrCB6} core in 5 resembles that in 3
with, in addition, the Fe...Ir connectivity being spanned by an {Ir(CO)(PPh3)2}
fragment and the consequent Fe-Ir and Ir-Ir bonds bridged by hydrido ligands. In 
contrast to the above, treatment of the 10-vertex diferracarborane salt
[N(PPh3)2][6,6,6,10,10,10-(CO)6-closo-6,10, 1-Fe2CB7H8] (2) with the same
reagents yields two very different, trimetallic complexes, namely
[8,10-{Ir(mu-PPh2)(Ph)(CO)(PPh3)}-8-(mu-H)-6,6,6,10,10-(
CO)5-closo-6,10,1-Fe2CB7H7] (6) and
[6,7,10-{Fe(CO)3}-6-(mu-H)-6,10,10,10-(CO)4-6-PPh3-closo-6,10,1-IrFeCB7H7] (7).
In 6, an exo-polyhedral {IrPh(CO)(PPh3)} moiety is attached to a
{closo-6,10,1-Fe2CB7} framework via a PPh2-bridged Fe-Ir bond and a B-HIr
agostic-type linkage, the iridium center formally having inserted into one P-Ph
bond of a PPh3 unit. Complex 7 contains an {IrFeCB7} cluster core, with an
exo-polyhedral {Fe(CO)3} moiety bridging a {BIrFe} triangular face and with an
additional Ir-H-Fe bridge. However, this metal atom arrangement reveals that
iridium and iron moieties have exchanged exo- and endo-polyhedral sites with
respect to the 10-vertex metallacarborane. X-ray diffraction studies upon 3, 5,
6, and 7 confirmed their novel structural features; some preliminary reactivity
studies upon these compounds are also reported.

PMID: 17165770 [PubMed]


133. J Gynecol Obstet Biol Reprod (Paris). 2006 Dec;35(8 Pt 1):785-9.

[Treatment of squamous intraepithelial lesion of type CIN2 et CIN3 with laser CO2
vaporization: retrospective study of 52 cases]

[Article in French]

Saah-Briffaut E, Collinet P, Saah R, Boman F, Leroy JL.

Clinique de Gynecologie, Hopital Jeanne-de-Flandre, CHRU de Lille, 2, avenue
Oscar-Lambret, 59037 Lille Cedex.

OBJECTIVES: This study was carried out over an 8-year period in order to evaluate
the long-term effectiveness of laser CO2 vaporization in the treatment of
squamous intraepithelial lesion of type CIN2 and CIN3. MATERIALS AND METHODS: A
retrospective study of 52 cases of cervical lesions of type CIN2 and CIN3 treated
in first intention by laser CO2 vaporization was carried out at the hospital
Jeanne-de-Flandre in CHRU of Lille from 1996 to 2003. This treatment was
performed on only high-grade exo-cervical lesions, of small size (<2cm2), after a
complete colposcopic examination. RESULTS: Fifty-two patients were treated by
first-intention laser vaporization only. Mean age was 29.4 years and 51.9% were
nulliparous. At the first cyto-colposcopic control, there were 17 persistent
lesions (32.7%). Among the 35 patients without persistent lesion, 29 achieved
cure (absence of recurrence), 4 presented a recurrence and 2 were lost to
follow-up. CONCLUSION: The current data of the literature concerning the
treatment by laser CO2 vaporization authorize application of this method for
certain high-grade exocervical lesions after a complete colposcopic examination. 
This type of treatment remains less aggressive than a surgical treatment. The
high rate of residual lesions in particular in the event of CIN3 can be due to an
incomplete destruction of the lesion. Patients should thus be advised that
monitoring is an integral part of the treatment. Laser vaporization could be
limited to CIN1 and CIN2 lesions.

PMID: 17151534 [PubMed - indexed for MEDLINE]


134. J Mol Model. 2007 Mar;13(3):425-30. Epub 2006 Nov 28.

Ab initio and DFT investigation of electrophilic addition reaction of bromine to 
endo,endo-tetracyclo[4.2.1.1(3,6).0 (2,7)]dodeca-4,9-diene.

Abbasoglu R.

Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey. 
rabbas@ktu.edu.tr

Full geometric optimization of
endo,endo-tetracyclo[4.2.1.1(3,6).0(2,7)]dodeca-4,9-diene (TTDD) has been carried
out by ab initio and DFT/B3LYP methods and the structure of the molecule
investigated. The double bonds of TTDD molecule are endo pyramidalized. The
structure of pi-orbitals and their mutual interactions for TTDD molecule were
investigated. The cationic intermediates and products obtained as a result of the
addition reaction have been studied using the HF/6-311G(d), HF/6-311G(d,p) and
B3LYP/6-311G(d) methods. The bridged bromonium cation isomerized into the more
stable N- and U-type cations and the difference between the stability of these
cations is small. The N- and U-type reaction products are obtained as a result of
the reaction, which takes place via the cations in question. The stability of
exo, exo and exo, endo isomers of N-type product are nearly the same and the
formation of both isomers is feasible. The U-type product basically formed from
the exo, exo-isomer. Although the U-type cation was 0.68 kcal mol(-1) more stable
than the N-type cation, the U-type product was 4.79 kcal mol(-1) less stable than
the N-type product.

PMID: 17131135 [PubMed - indexed for MEDLINE]


135. Orig Life Evol Biosph. 2006 Dec;36(5-6):541-7.

Characterizing extrasolar terrestrial planets with reflected, emitted and
transmitted spectra.

Tinetti G.

European Space Agency, Institut d'Astrophysique de Paris, IAP-Section Planetes
extra solaires, 98 bis boulevard Arago, 75014 Paris, France. tinetti@iap.fr

NASA and ESA are planning missions to directly detect and characterize
terrestrial planets outside our solar system (nominally NASA-Terrestrial Planet
Finder and ESA-DARWIN missions). These missions will provide our first
opportunity to spectroscopically study the global characteristics of those
planets, and search for signs of habitability and life. We have used spatially
and spectrally-resolved models to explore the observational sensitivity to
changes in atmospheric and surface properties, and the detectability of surface
biosignatures, in the globally averaged spectra and light-curves of the Earth.
Atmospheric signatures of Earth-size exoplanets might be detected, in a near
future, by stellar occultation as well. Detectability depends on planet's size,
atmospheric composition, cloud cover and stellar type. According to our
simulations, Earth's land vegetation signature (red-edge) is potentially visible 
in the disk-averaged spectra, even with cloud cover, and when the signal is
averaged over the daily time scale. Marine vegetation is far more difficult to
detect. We explored also the detectability of an exo-vegetation responsible for
producing a signature that is red-shifted with respect to the Earth vegetation's 
one.

PMID: 17120124 [PubMed - indexed for MEDLINE]


136. Chem Res Toxicol. 2006 Nov;19(11):1506-17.

Translesion synthesis past the C8- and N2-deoxyguanosine adducts of the dietary
mutagen 2-Amino-3-methylimidazo[4,5-f]quinoline in the NarI recognition sequence 
by prokaryotic DNA polymerases.

Stover JS, Chowdhury G, Zang H, Guengerich FP, Rizzo CJ.

Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt Institute
of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235-1822, USA.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is found in cooked meats and forms
DNA adducts at the C8- and N2-positions of dGuo after appropriate activation. IQ 
is a potent inducer of frameshift mutations in bacteria and is carcinogenic in
laboratory animals. We have incorporated both IQ-adducts into the G1- and
G3-positions of the NarI recognition sequence (5'-G1G2CG3CC-3'), which is a
hotspot for arylamine modification. The in vitro replication of the
oligonucleotides was examined with Escherichia coli pol I Klenow fragment exo-,
E. coli pol II exo-, and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and
the extension products were sequenced by tandem mass spectrometry. Replication of
the C8-adduct at the G3-position resulted in two-base deletions with all three
polymerases, whereas error-free bypass and extension was observed at the
G1-position. The N2-adduct was bypassed and extended by all three polymerases
when positioned at the G1-position, and the error-free product was observed. The 
N2-adduct at the G3-position was more blocking and was bypassed and extended only
by Dpo4 to produce an error-free product. These results indicate that the
replication of the IQ-adducts of dGuo is strongly influenced by the local
sequence and the regioisomer of the adduct. These results also suggest a possible
role for pol II and IV in the error-prone bypass of the C8-IQ-adduct leading to
frameshift mutations in reiterated sequences, whereas noniterated sequences
result in error-free bypass.

PMID: 17112239 [PubMed - indexed for MEDLINE]


137. J Bacteriol. 2007 Jan;189(2):422-9. Epub 2006 Nov 3.

Characterization of Helicobacter pylori lytic transglycosylases Slt and MltD.

Chaput C, Labigne A, Boneca IG.

Unite de Pathogenie Bacterienne des Muqueuses, Department of Microbiology,
Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris cedex 15, France.

Peptidoglycan (PG) is a cell wall heteropolymer that is essential for cell
integrity. PG hydrolases participate in correct assembly of the PG layer and have
been shown to be required for cell division, cell daughter separation, and
maintenance of bacterial morphology. In silico analysis of the Helicobacter
pylori genome resulted in identification of three potential hydrolases, Slt,
MltD, and AmiA. This study was aimed at determining the roles of the putative
lytic transglycosylases, Slt and MltD, in H. pylori morphology, growth, and PG
metabolism. Strain 26695 single mutants were constructed using a nonpolar
kanamycin cassette. The slt and mltD mutants formed normal bacillary and coccoid 
bacteria in the exponential and stationary phases, respectively. The slt and mltD
mutants had growth rates comparable to the growth rate of the parental strain.
However, the mltD mutant exhibited enhanced survival in the stationary phase
compared to the wild type or the slt mutant. PG was purified from exponentially
growing bacteria and from bacteria in the stationary phase, and its muropeptide
composition was analyzed by high-pressure liquid chromatography. This analysis
revealed changes in the muropeptide composition indicating that MltD and Slt have
lytic transglycosylase activities. Glycan strand analysis suggested that Slt and 
MltD have exo and endo types of lytic transglycosylase activity, indicating that 
Slt is involved mainly in PG turnover and MltD is involved mainly in
rearrangement of the PG layer. In this study, we determined the distinct roles of
the lytic transglycosylases Slt and MltD in PG metabolism.

PMCID: PMC1797392
PMID: 17085576 [PubMed - indexed for MEDLINE]


138. Dalton Trans. 2006 Nov 21;(43):5167-75. Epub 2006 Sep 28.

Insertion reaction of carbon dioxide into Sn-OR bond. Synthesis, structure and
DFT calculations of di- and tetranuclear isopropylcarbonato tin(IV) complexes.

Ballivet-Tkatchenko D, Chermette H, Plasseraud L, Walter O.

LSEO, UMR 5188 CNRS-Universite de Bourgogne, UFR Sciences et Techniques, BP
47870, 21078, Dijon Cedex, France. ballivet@u-bourgogne.fr

The reaction of carbon dioxide with the stannane nBu2Sn(OiPr)2 and distannoxane
[nBu2(iPrO)Sn]2O leads to the selective insertion into one Sn-OiPr bond
generating the corresponding nBu2Sn(OiPr)(OCO2(i)Pr) and
nBu2(iPrO)SnOSn(OCO2(i)Pr)nBu2 species. Both compounds are characterised by
multinuclear NMR, FT-IR and single-crystal X-ray crystallography. In the solid
state, they adopt a dimeric arrangement with bridging isopropoxy and terminal
isopropylcarbonato ligands. The X-ray crystal structure of the dinuclear stannane
shows that the Sn2O2 ring and the two Sn-OCO2C fragments are nearby coplanar. The
same holds for the ladder-type tetranuclear distannoxane. The dimeric structures 
are also evidenced by solution NMR in non-coordinating solvents. Interestingly,
the assignment of the exo and endo tin resonances of the dimeric distannoxane is 
unambiguous using a labeled 13CO2 experiment. The stability of the dimeric
association has been probed in the stannane series on the basis of DFT
calculations.

PMID: 17077890 [PubMed - indexed for MEDLINE]


139. Immunology. 2007 Jan;120(1):90-102. Epub 2006 Oct 31.

Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic
T-lymphocyte responses and antitumour immunity.

Hao S, Bai O, Li F, Yuan J, Laferte S, Xiang J.

Research Unit, Division of Health Research, Saskatchewan Cancer Agency,
Department of Oncology, College of Medicine, University of Saskatchewan,
Sakatoon, Saskatchewan, Canada.

Exosomes (EXO) derived from dendritic cells (DC), which express major
histocompatibility complex (MHC) and costimulatory molecules, have been used for 
antitumour vaccines. However, they are still less effective by showing only
prophylatic immunity in animal models or very limited immune responses in
clinical trials. In this study, we showed that ovalbumin (OVA) protein-pulsed DC 
(DC(OVA))-derived EXO (EXO(OVA)) displayed MHC class I-OVA I peptide (pMHC I)
complexes, CD11c, CD40, CD80, CCR7, DEC205, Toll-like receptor 4 (TLR4), TLR9,
MyD88 and DC-SIGN molecules, but at a lower level than DC(OVA). EXO(OVA) can be
taken up by DC through LFA-1/CD54 and C-type lectin/mannose (glucosamine)-rich
C-type lectin receptor (CLR) interactions. Mature DC pulsed with EXO(OVA), which 
were referred to as mDC(EXO), expressed a higher level of pMHC I, MHC II, and
costimulatory CD40, CD54 and CD80 than DC(OVA). The mDC(EXO) could more strongly 
stimulate OVA-specific CD8(+) T-cell proliferation in vitro and in vivo, and more
efficiently induce OVA-specific cytotoxic T-lymphocyte responses, antitumour
immunity and CD8(+) T-cell memory in vivo than EXO(OVA) and DC(OVA). In addition,
mDC(EXO) could also more efficiently eradicate established tumours. Therefore,
mature DC pulsed with EXO may represent a new, highly effective DC-based vaccine 
for the induction of antitumour immunity.

PMCID: PMC2265880
PMID: 17073943 [PubMed - indexed for MEDLINE]


140. Biochem J. 2007 Mar 1;402(2):321-9.

Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase
in replicating past an abasic site.

Blanca G, Delagoutte E, Tanguy le Gac N, Johnson NP, Baldacci G, Villani G.

Institut de Pharmacologie et Biologie Structurale CNRS-UMR 5089, 205 route de
Narbonne, 31077 Toulouse cedex 4, France.

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and
3'-5' exonuclease proofreading catalytic activities. They arrest at the base
preceding DNA damage on the coding DNA strand and specialized DNA polymerases
have evolved to replicate across the lesion by a process known as TLS
(translesion DNA synthesis). TLS is considered to take place in two steps that
often require different enzymes, insertion of a nucleotide opposite the damaged
template base followed by extension from the inserted nucleotide. We and others
have observed that inactivation of the 3'-5' exonuclease function of T4
polymerase enables TLS across a single site-specific abasic [AP
(apurinic/apyrimidinic)] lesion. In the present study we report a role for
auxiliary replicative factors in this reaction. When replication is performed
with a large excess of DNA template over DNA polymerase in the absence of
auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' 
exonuclease activity) inserts one nucleotide opposite the AP site but does not
extend past the lesion. Addition of the clamp processivity factor and the clamp
loader complex restores primer extension across an AP lesion on a circular
AP-containing DNA substrate by the exo- polymerase, but has no effect on the
wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA 
damage. It can behave as a replicative polymerase or (in the absence of
proofreading activity) as a specialized DNA polymerase and carry out TLS. As a
specialized polymerase it can function either as an inserter or (with the help of
accessory proteins) as an extender. The capacity to separate these distinct
functions in a single DNA polymerase provides insight into the biochemical
requirements for translesion DNA synthesis.

PMCID: PMC1798438
PMID: 17064253 [PubMed - indexed for MEDLINE]


141. Acta Crystallogr C. 2006 Oct;62(Pt 10):o593-5. Epub 2006 Sep 12.

2-Amino-7-chloro-2'-deoxytubercidin.

Seela F, Peng X, Eickmeier H, Reuter H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.
frank.seela@uni-osnabrueck.de

In
4-chloro-7-(2-deoxy-beta-D-erythro-pentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-2,
4-diamine, C11H14ClN5O3, the conformation of the N-glycosylic bond is between
anti and high-anti [chi = -102.5 (6) degrees]. The 2'-deoxyribofuranosyl unit
adopts the C3'-endo-C4'-exo (3T4) sugar pucker (N-type) with P = 19.6 degrees and
taum = 32.9 degrees [terminology: Saenger (1989). Landolt-Bornstein New Series,
Vol. 1, Nucleic Acids, Subvol. a, edited by O. Madelung, pp. 1-21. Berlin:
Springer-Verlag]. The orientation of the exocyclic C4'-C5' bond is +ap (trans)
with a torsion angle gamma = 171.5 (4) degrees. The compound forms a
three-dimensional network that is stabilized by four intermolecular hydrogen
bonds (N-H...O and O-H...N) and one intramolecular hydrogen bond (N-H...Cl).

PMID: 17008745 [PubMed - indexed for MEDLINE]


142. Microbiology. 2006 Oct;152(Pt 10):3003-12.

Ecophysiology of different filamentous Alphaproteobacteria in industrial
wastewater treatment plants.

Kragelund C, Kong Y, van der Waarde J, Thelen K, Eikelboom D, Tandoi V, Thomsen
TR, Nielsen PH.

Section of Environmental Engineering, Department of Biotechnology, Chemistry and 
Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000
Aalborg, Denmark.

The ecophysiology of five filamentous species affiliated to the
Alphaproteobacteria was investigated in industrial activated sludge systems. The 
five species, 'Candidatus Alysiosphaera europaea', 'Candidatus Monilibacter
batavus', 'Candidatus Alysiomicrobium bavaricum', 'Candidatus Sphaeronema
italicum' and Meganema perideroedes, are very abundant in industrial wastewater
treatment plants and are often involved in bulking incidents. The morphology of
these filamentous bacterial species resembled Eikelboom's Nostocoida limicola, or
Type 021N, and could only be correctly identified by using fluorescence in situ
hybridization (FISH), applying species-specific gene probes. Two physiological
groupings of the five species were found using microautoradiography combined with
FISH. Group 1 ('Ca. Monilibacter batavus' and 'Ca. Sphaeronema italicum')
utilized many short-chained fatty acids (acetate, pyruvate and propionate),
whereas Group 2 ('Ca. Alysiosphaera europaea', 'Ca. Alysiomicrobium bavaricum'
and Meganema perideroedes) could also exploit several sugars, amino acids and
ethanol. All species had polyhydroxyalkanoate granules present and several of the
species had a very large storage capacity. No activity was found under strict
anaerobic conditions, while uptake of substrate was observed in the presence of
nitrate or nitrite as potential electron acceptor. However, for all species a
reduced number of substrates could be consumed under these conditions compared to
aerobic conditions. Only a little exo-enzymic activity was found and nearly all
species had a hydrophobic cell surface. Based on knowledge of the
ecophysiological potential, control strategies are suggested.

PMID: 17005981 [PubMed - indexed for MEDLINE]


143. J Phys Chem A. 2006 Sep 14;110(36):10633-42.

Structure of the boronic acid dimer and the relative stabilities of its
conformers.

Larkin JD, Bhat KL, Markham GD, Brooks BR, Schaefer HF 3rd, Bock CW.

Center for Computational Chemistry, University of Georgia, Athens, GA 30602, USA.
jlarkin@ccqc.uga.edu

Despite the widespread use of boronic acids in materials science and as
pharmaceutical agents, many aspects of their structure and reactivity are not
well understood. In this research the boronic acid dimer, [HB(OH)(2)](2), was
studied by second-order Moller-Plesset (MP2) perturbation theory and coupled
cluster methodology with single and double excitations (CCSD). Pople
split-valence 6-31+G*, 6-311G**, and 6-311++G** and Dunning-Woon
correlation-consistent cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, and aug-cc-pVTZ basis sets 
were employed for the calculations. A doubly hydrogen-bonded conformer (1) of the
dimer was consistently found to be lowest in energy; the structure of 1 was
planar (C(2h)) at most computational levels employed but was significantly
nonplanar (C(2)) at the MP2/6-311++G** and CCSD/6-311++G** levels, the result of 
an intrinsic problem with Pople-type sp-diffuse basis functions on heavy atoms.
The dimerization energy, enthalpy, and free energy for the formation of (1) from 
the exo-endo conformer of the monomer were -10.8, -9.2, and +1.2 kcal/mol,
respectively, at the MP2/aug-cc-pVTZ level. Several other hydrogen-bonded
conformers of the dimer were local minima on the potential energy surface (PES)
and ranged from 2 to 5 kcal/mol higher in energy than 1. Nine doubly OH-bridged
conformers, in which the boron atoms were tetracoordinated, were also local
minima on the PES, but they were all greater than 13 kcal/mol higher in energy
than 1; doubly H-bridged structures proved to be transition states. MP2 and CCSD 
results were compared to those from the BLYP, B3LYP, OLYP, O3LYP, PBE1PBE, and
TPSS functionals with the 6-311++G** and aug-cc-pVTZ basis sets; the PBE1PBE
functional performed best relative to the MP2 and CCSD results. Self-consistent
reaction field (SCRF) calculations predict that boronic acid dimerization is less
favorable in solution than in vacuo.

PMID: 16956246 [PubMed - indexed for MEDLINE]


144. Dalton Trans. 2006 Sep 21;(35):4277-85. Epub 2006 Jul 6.

Intramolecular hydroamination/cyclisation of aminoallenes mediated by a cationic 
zirconocene catalyst: a computational mechanistic study.

Tobisch S.

University of St. Andrews, School of Chemistry, Purdie Building, North Haugh, St.
Andrews, Fife, Scotland, UK KY16 9ST. st40@st-andrews.ac.uk

The complete sequence of steps of a tentative catalytic cycle for intramolecular 
hydroamination/cyclisation (IHC) of 4,5-hexadien-1-ylamine (1) by a prototypical 
cationic [Cp(2)ZrCH(3)](+) zirconocene precatalyst (2) has been examined by
employing a gradient-corrected DFT method. The predicted smooth overall reaction 
energy profile is consistent with the available experimental data, thereby
providing further confidence in the proposed mechanism. Following activation of
the precatalyst by protonolytic cleavage of the Zr-Me bond, the catalytically
active amidoallene-Zr complex undergoes addition of an allenic C[double bond,
length as m-dash]C linkage across the Zr-N sigma-bond. The alternative exo- and
endocyclic pathways show similar probabilities for the sterically less encumbered
reactants {1 + 2} investigated herein. However, steric factors are expected to
exert control on the regioselectivity of ring closure. On the other hand, the
metathesis-type transition states for subsequent protonolysis are indicated to be
less sensitive to steric demands. Formation of the six-membered azacycle-Zr
intermediate through intramolecular C[double bond, length as m-dash]C insertion
into the Zr-N sigma-bond is predicted to be turnover limiting. The factors that
govern the regioselectivity of the aminoallene IHC have been elucidated.

PMID: 16932821 [PubMed - indexed for MEDLINE]


145. Neuropharmacology. 2006 Sep;51(4):885-95. Epub 2006 Aug 8.

Protection against MDMA-induced dopaminergic neurotoxicity in mice by
methyllycaconitine: involvement of nicotinic receptors.

Chipana C, Camarasa J, Pubill D, Escubedo E.

Unitat de Farmacologia i Farmacognosia, Facultat de Farmacia, Universitat de
Barcelona, Av. Joan XXIII s/n., Zona Universitaria Pedralbes, 08028 Barcelona,
Spain.

Methylenedioxymethamphetamine (MDMA) is a relatively selective dopaminergic
neurotoxin in mice. Previous studies demonstrated the participation of alpha-7
nicotinic receptors (nAChR) in the neurotoxic effect of methamphetamine. The aim 
of this paper was to study the role of this receptor type in the acute effects
and neurotoxicity of MDMA in mice. In vivo, methyllycaconitine (MLA), a specific 
alpha-7 nAChR antagonist, significantly prevented MDMA-induced neurotoxicity at
dopaminergic but not at serotonergic level, without affecting MDMA-induced
hyperthermia. Glial activation was also fully prevented by MLA. In vitro, MDMA
induced intrasynaptosomal reactive oxygen species (ROS) generation, which was
calcium-, nitric-oxide synthase-, and protein kinase C-dependent. Also, the
increase in ROS was prevented by MLA and alpha-bungarotoxin. Experiments with
reserpine point to endogenous dopamine (DA) as the main source of MDMA-induced
ROS. MLA also brought the MDMA-induced inhibition of [3H]DA uptake down, from 73%
to 11%. We demonstrate that a coordinated activation of alpha-7 nAChR, blockade
of DA transporter function and displacement of DA from intracellular stores
induced by MDMA produces a neurotoxic effect that can be prevented by MLA,
suggesting that alpha-7 nAChR have a key role in the MDMA neurotoxicity in mice; 
however, the involvement of nicotinic receptors containing the beta2 subunit
cannot be conclusively ruled out.

PMID: 16901518 [PubMed - indexed for MEDLINE]


146. Glycobiology. 2006 Dec;16(12):1194-206. Epub 2006 Aug 9.

Molecular cloning and characterization of a novel human
beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and 
glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain.

Sato T, Sato M, Kiyohara K, Sogabe M, Shikanai T, Kikuchi N, Togayachi A, Ishida 
H, Ito H, Kameyama A, Gotoh M, Narimatsu H.

Glycogene Function Team of Research Center for Glycoscience (RCG), National
Institute of Advanced Industrial Science and Technology (AIST), Tukuba, Ibaraki, 
Japan.

Protein O-linked fucosylation is an unusual glycosylation associated with many
important biological functions such as Notch signaling. Two fucosylation pathways
synthesizing O-fucosylglycans have been reported on cystein-knotted proteins,
that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin
Type 1 repeat (TSR) domains. We report here the molecular cloning and
characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that
synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel
glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in
databases. The recombinant enzyme expressed in human embryonic kidney 293T
(HEK293T) cells exhibited glucosyltransferase activity toward
fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC)
analysis revealed that the product of the recombinant enzyme migrated to the same
position as did the product of endogenous beta3Glc-T of Chinese hamster ovary
(CHO) cells. The two products could be digested by beta-glucosidase from almond
and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly
suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we
named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged
beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention 
signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain 
expressed in Escherichia coli was first fucosylated by the recombinant protein
O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for 
the recombinant beta3Glc-T, which could apparently transfer Glc to the
fucosylated TSR domain. Our results suggest that a novel glycosyltransferase,
beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs
specifically on TSR domains.

PMID: 16899492 [PubMed - indexed for MEDLINE]


147. Biochemistry. 2006 Aug 8;45(31):9566-74.

Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show 
endo activity and different degrees of processivity during enzymatic hydrolysis
of chitosan.

Sikorski P, Sorbotten A, Horn SJ, Eijsink VG, Varum KM.

Department of Physics, Norwegian University of Science and Technology, 7491
Trondheim, Norway. pawel.sikorski@phys.ntnu.no

The modes of action of three family 18 chitinases (ChiA, ChiB, and ChiC) from
Serratia marcescens during the degradation of a water-soluble polymeric
substrate, chitosan, were investigated using a combination of viscosity
measurements, reducing end assays, and characterization of the size-distribution 
of the oligomeric products. All three enzymes yielded a fast reduction in
molecular weight of the chitosan substrate at a very early stage of hydrolysis,
which is typical for endo-acting enzymes. For ChiA and ChiB, this is inconsistent
with the previously proposed exo-attack mode of action. The main difference
between ChiA, ChiB, and ChiC is the degree of processivity. ChiC is an endo
enzyme with no apparent processivity. ChiA and ChiB are processive enzymes in
which the substrate remains bound to the active cleft after successful hydrolysis
and is moved along for the next hydrolysis to occur. ChiA and ChiB perform on
average 9.1 and 3.4 cleavages, respectively, for the formation of each
enzyme-substrate complex. ChiA and ChiB have deep, tunnel-like substrate-binding 
grooves. The demonstration of endo activity shows that substrate binding must
involve the temporary restructuring of the loops that make up the roofs of the
substrate-binding grooves, similar to what has been proposed for
cellobiohydrolase Cel6A. The data suggest that the exo-type of activity observed 
for ChiA and ChiB during the degradation of solid crystalline chitin is due to
the better accessibility of chain ends, rather than intrinsic enzyme properties.

PMID: 16878991 [PubMed - indexed for MEDLINE]


148. Glycobiology. 2006 Nov;16(11):1064-72. Epub 2006 Jul 28.

Exo-beta-D-glucosaminidase from Amycolatopsis orientalis: catalytic residues,
sugar recognition specificity, kinetics, and synergism.

Fukamizo T, Fleury A, Cote N, Mitsutomi M, Brzezinski R.

Department of Advanced Bioscience, Kinki University, 3327-204 Nakamichi, Nara
631-8505, Japan.

Catalytic residues and the mode of action of the exo-beta-D-glucosaminidase
(GlcNase) from Amycolatopsis orientalis were investigated using the wild-type and
mutated enzymes. Mutations were introduced into the putative catalytic residues
resulting in five mutated enzymes (D469A, D469E, E541D, E541Q, and S468N/D469E)
that were successfully produced. The four single mutants were devoid of enzymatic
activity, indicating that Asp469 and Glu541 are essential for catalysis as
predicted by sequence alignments of enzymes belonging to GH-2 family. When
mono-N-acetylated chitotetraose [(GlcN)3-GlcNAc] was hydrolyzed by the enzyme,
the nonreducing-end glucosamine unit was produced together with the
transglycosylation products. The rate of hydrolysis of the disaccharide,
2-amino-2-deoxy-D-glucopyranosyl 2-acetamido-2-deoxy-D-glucopyranose
(GlcN-GlcNAc), was slightly lower than that of (GlcN)2, suggesting that N-acetyl 
group of the sugar residue located at (+1) site partly interferes with the
catalytic reaction. The time-course of the enzymatic hydrolysis of the completely
deacetylated chitotetraose [(GlcN)4] was quantitatively determined by
high-performance liquid chromatography (HPLC) and used for in silico modeling of 
the enzymatic hydrolysis. The modeling study provided the values of binding free 
energy changes of +7.0, -2.9, -1.8, -0.9, -1.0, and -0.5 kcal/mol corresponding, 
respectively, to subsites (-2), (-1), (+1), (+2), (+3), and (+4). When chitosan
polysaccharide was hydrolyzed by a binary enzyme system consisting of A.
orientalis GlcNase and Streptomyces sp. N174 endochitosanase, the highest synergy
in the rate of product formation was observed at the molar ratio 2:1. Thus,
GlcNase would be an efficient tool for industrial production of glucosamine
monosaccharide.

PMID: 16877749 [PubMed - indexed for MEDLINE]


149. J Am Chem Soc. 2006 Aug 2;128(30):9730-40.

Enantioselective synthesis of vinylcyclopropanes and vinylepoxides mediated by
camphor-derived sulfur ylides: rationale of enantioselectivity, scope, and
limitation.

Deng XM, Cai P, Ye S, Sun XL, Liao WW, Li K, Tang Y, Wu YD, Dai LX.

State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China.

By a sidearm approach, camphor-derived sulfur ylides 1 were designed and
synthesized for the cyclopropanation of electron-deficient alkenes and
epoxidation of aldehydes. Under the optimal conditions, the exo-type sulfonium
salts 4a and 4b reacted with beta-aryl-alpha,beta-unsaturated esters, amides,
ketones, and nitriles to give 1,3-disubstituted-2-vinylcyclopropanes with high
diastereoselectivities and enantioselectivities. When the endo-type sulfonium
salts 5a and 5b were used, the diastereoselectivities were not changed, whereas
the absolute configurations of the products became the opposite to those of the
reactions of 4a and 4b. An ylide cyclopropanation of chalcone derivatives with
phenylvinyl bromide in the presence of catalytic amount of chiral sulfonium salts
4b and 5b has been developed. The sidearmed hydroxyl group was found to play a
key role in the control of enantioselectivity and diastereoselectivity. The
origins of the high diastereoselectivity and enantioselectivity were also studied
by density functional theory calculations, which reveal the importance of the
hydrogen-bonding between the sidearmed hydroxyl group and the substrate in
determining the diastereoselectivity and enantioselectivity. The ylides 1 were
also successfully applied for the epoxidation of aromatic aldehydes.

PMID: 16866528 [PubMed]


150. Chemistry. 2006 Oct 25;12(31):8084-9.

Azanonaboranes containing imidazole derivatives for boron neutron capture
therapy: synthesis, characterization, and in vitro toxicity evaluation.

El-Zaria ME, Genady AR, Gabel D.

Department of Chemistry, Faculty of Science, University of Tanta, 31527 Tanta,
Egypt. mohamedel-zaria@web.de

A number of azanonaboranes containing imidazole derivatives have been synthesized
by a ligand-exchange reaction. The exo-NH(2)R group of the azanonaborane of the
type [(RH(2)N)B(8)H(11)NHR] can be exchanged by one hetero-nitrogen atom of the
imidazole ring. In the case of histamine, the exchange takes place on the
aliphatic amino group, the hetero-nitrogen atom of the imidazole ring or both of 
them. The products were confirmed by NMR, IR spectroscopy, elemental analysis,
and mass spectrometry. The electron-withdrawing effect of the nitro group in
2-nitroimidazole is the main hindrance to achieve the exchange reaction. In vitro
experiments were performed with B16 melanoma cells. A comparison of the
biological properties of the products in which the B(8)N cluster is connected to 
the hetero-nitrogen atom of imidazole ring or the aliphatic NH(2) group showed
that incorporation of B(8)N cluster unit into primary amino group increases the
compound's toxicity. In contrast, this specificity for cytotoxicity effect was
not observed in the case of histamine containing two B(8)N clusters which was
relatively nontoxic and did not inhibit colony formation up to concentrations of 
2 mM.

PMID: 16865752 [PubMed - indexed for MEDLINE]


151. Dalton Trans. 2006 Aug 14;(30):3624-6. Epub 2006 Jun 15.

Macropolyhedral boron-containing cluster chemistry. Metallathiaboranes from
S2B17H17: isolation and characterisation of [(eta6-MeC6H4isoPr)RuS2B16H16] and
[(eta6-MeC6H4isoPr)RuS2B15H15].

Carr MJ, Londesborough MG, Hamilton Mcleod AR, Kennedy JD.

School of Chemistry of the University of Leeds, Leeds LS2 9JT, UK.

Deprotonation of [S2B17H17] with NaH and subsequent reaction with
[{RuCl2(eta6-MeC6H4(iso)Pr)}2] gives new nineteen-vertex
[(eta6-MeC6H4(iso)Pr)RuS2B16H16] , with a nido {SB9} unit and an arachno-type
{RuSB9} unit conjoined with a B-B edge in common, and new eighteen-vertex
[(eta6-MeC6H4(iso)Pr)RuS2B15H15] with a nido {RuSB9} subcluster fused via a
common B-B edge to a nido {B8} subcluster that is additionally linked exo to the 
{RuSB9} unit by a bridging sulfur atom that is held endo to the {B8} unit.

PMID: 16865172 [PubMed]


152. Biol Rev Camb Philos Soc. 2006 Nov;81(4):501-29. Epub 2006 Jul 24.

Intake rates and the functional response in shorebirds (Charadriiformes) eating
macro-invertebrates.

Goss-Custard JD, West AD, Yates MG, Caldow RW, Stillman RA, Bardsley L, Castilla 
J, Castro M, Dierschke V, Durell SE, Eichhorn G, Ens BJ, Exo KM,
Udayangani-Fernando PU, Ferns PN, Hockey PA, Gill JA, Johnstone I,
Kalejta-Summers B, Masero JA, Moreira F, Nagarajan RV, Owens IP, Pacheco C,
Perez-Hurtado A, Rogers D, Scheiffarth G, Sitters H, Sutherland WJ, Triplet P,
Worrall DH, Zharikov Y, Zwarts L, Pettifor RA.

Centre for Ecology and Hydrology, Winfrith Technology Centre, Dorchester DT2 8ZD,
UK. j.d.goss-custard@exeter.ac.uk

As field determinations take much effort, it would be useful to be able to
predict easily the coefficients describing the functional response of free-living
predators, the function relating food intake rate to the abundance of food
organisms in the environment. As a means easily to parameterise an
individual-based model of shorebird Charadriiformes populations, we attempted
this for shorebirds eating macro-invertebrates. Intake rate is measured as the
ash-free dry mass (AFDM) per second of active foraging; i.e. excluding time spent
on digestive pauses and other activities, such as preening. The present and
previous studies show that the general shape of the functional response in
shorebirds eating approximately the same size of prey across the full range of
prey density is a decelerating rise to a plateau, thus approximating the Holling 
type II ('disc equation') formulation. But field studies confirmed that the
asymptote was not set by handling time, as assumed by the disc equation, because 
only about half the foraging time was spent in successfully or unsuccessfully
attacking and handling prey, the rest being devoted to searching.A review of 30
functional responses showed that intake rate in free-living shorebirds varied
independently of prey density over a wide range, with the asymptote being reached
at very low prey densities (<150/m-2). Accordingly, most of the many studies of
shorebird intake rate have probably been conducted at or near the asymptote of
the functional response, suggesting that equations that predict intake rate
should also predict the asymptote.A multivariate analysis of 468 'spot' estimates
of intake rates from 26 shorebirds identified ten variables, representing prey
and shorebird characteristics, that accounted for 81% of the variance in
logarithm-transformed intake rate. But four-variables accounted for almost as
much (77.3%), these being bird size, prey size, whether the bird was an
oystercatcher Haematopus ostralegus eating mussels Mytilus edulis, or breeding.
The four variable equation under-predicted, on average, the observed 30 estimates
of the asymptote by 11.6%, but this discrepancy was reduced to 0.2% when two
suspect estimates from one early study in the 1960s were removed. The equation
therefore predicted the observed asymptote very successfully in 93% of cases. We 
conclude that the asymptote can be reliably predicted from just four easily
measured variables. Indeed, if the birds are not breeding and are not
oystercatchers eating mussels, reliable predictions can be obtained using just
two variables, bird and prey sizes. A multivariate analysis of 23 estimates of
the half-asymptote constant suggested they were smaller when prey were small but 
greater when the birds were large, especially in oystercatchers. The resulting
equation could be used to predict the half-asymptote constant, but its predictive
power has yet to be tested. As well as predicting the asymptote of the functional
response, the equations will enable research workers engaged in many areas of
shorebird ecology and behaviour to estimate intake rate without the need for
conventional time-consuming field studies, including species for which it has not
yet proved possible to measure intake rate in the field.

PMID: 16863594 [PubMed - indexed for MEDLINE]


153. J Phys Chem B. 2005 Oct 27;109(42):19936-45.

A conformational dynamics study of
alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution by NMR
experiments and molecular simulations.

Eklund R, Lycknert K, Soderman P, Widmalm G.

Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
S-10691 Stockholm, Sweden.

The conformational preference of
alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been 
studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments
and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular
dynamics (MD) simulations with a CHARMM22 type of force field modified for
carbohydrates were performed with water as the explicit solvent. The homonuclear 
cross-relaxation rates, interpreted as effective proton-proton distances, were
compared to those obtained from simulation. Via a Karplus torsional relationship,
(3)J(C,H) values were calculated from simulation and compared to experimental
data. Good agreement was observed between experimental data and the MD
simulation, except for one inter-residue T-ROE between protons in the terminal
sugar residues. The results show that the trisaccharide exhibits substantial
conformational flexibility, in particular along the psi glycosidic torsion
angles. Notably, for these torsions, a high degree of correlation (77%) was
observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or
psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric
conformations were present at the phi torsion angles, but to a limited extent,
with the phi(3) state populated to a larger extent than the phi(2) state. Further
NMR analysis of the trisaccharide by translational diffusion measurements and
(13)C T(1) relaxation experiments quantified global reorientation using an
anisotropic model together with interpretation of the internal dynamics via the
"model-free" approach. Fitting of the dynamically averaged states to experimental
data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%,
psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately
12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE
buildup curves were calculated using a full relaxation matrix approach and were
found to be in excellent agreement with experimental data, in particular for the 
above inter-residue proton-proton interaction between the terminal residues.

PMID: 16853578 [PubMed - indexed for MEDLINE]


154. J Org Chem. 2006 Jul 21;71(15):5662-73.

Photochemistry of 4-(2'-aminoethyl)quinolones: enantioselective synthesis of
tetracyclic tetrahydro-1aH-pyrido[4',3':2,3]- cyclobuta[1,2-c]
quinoline-2,11(3H,8H)-diones by intra- and intermolecular [2 +
2]-photocycloaddition reactions in solution.

Selig P, Bach T.

Lehrstuhl fur Organische Chemie I, Technische Universitat Munchen, D-85747
Garching, Germany.

Enantioselective [2 + 2]-photocycloaddition reactions on
4-(2'-aminoethyl)quinolones in solution were studied using the enantiomerically
pure complexing agent 1 as source of chirality. The intermolecular reactions of
fully N-protected substrates 5a-5c with different 2-alkyl-substituted acrylates
12-15 represent the first systematic study on the diastereoselectivity of their
intermolecular [2 + 2]-photocycloadditions to unsymmetrically 1,1-disubstituted
olefins (75-91% yield, d.r. = 58/42-95/5). N-Benzylic-protected photoproducts
exo-16a/b-19a/b could easily be converted into lactams 20a/b-23a/b by a sequence 
of Boc deprotection and thermal lactamization (74-98% yield). Identical products 
20a-22a were directly accessible by the intramolecular [2 + 2]-photocycloaddition
of acrylic acid amides 2-4 (41-61% yield). The suitability of both pathways for
an enantioselective reaction variant was proven (70-92% ee). Thus, tetracyclic
lactams possessing the carbon framework C were obtained with good yields and
enantioselectivities of up to 92% ee in intramolecular reactions. Comparative
investigation of both routes showed that quinolone dimerization was the single
most decisive factor preventing a complete chirality transfer. Functional group
manipulations were successfully conducted with the primary photoproduct exo-17a. 
Finally, a new and unexpected type of benzylic hydrogen abstraction-radical
cyclization reaction was discovered for substrate 5a, which explains the
photochemical instability of substrates 2-5 under short wavelength irradiation
(lambda = 300 nm).

PMID: 16839147 [PubMed - indexed for MEDLINE]


155. J Exp Bot. 2006;57(10):2353-62.

An alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish
(Raphanus sativus L.).

Kotake T, Tsuchiya K, Aohara T, Konishi T, Kaneko S, Igarashi K, Samejima M,
Tsumuraya Y.

Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama
University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
kotake@molbiol.saitama-u.ac.jp

The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for
their physiological functions and undergo rapid turnover in vivo. Degradation of 
the carbohydrate moieties of AGPs seems to occur by concerted action of several
glycosidases, among them alpha-L-arabinofuranosidase, beta-D-galactosidase, and
beta-D-glucuronidase. Here, a bifunctional
alpha-L-arabinofuranosidase/beta-D-xylosidase from immature seeds of radish
(Raphanus sativus L.), which hydrolyses alpha-L-arabinofuranosyl residues of the 
carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The 
gene, designated RsAraf1, contained an open reading frame of 2343 bp (780 amino
acids), including a putative signal sequence (33 amino acids) at the N-terminus. 
RsAraf1 is highly similar to barley
alpha-L-arabinofuranosidase/beta-D-xylosidases and belongs to family 3 of the
glycosyl hydrolases based on sequence homology. Southern blot analysis revealed
that several related genes exist in the radish genome. RsAraf1 is expressed
throughout seed development and weakly expressed in young seedlings. It was found
that alpha-L-arabinofuranosidase activity in a cell-wall protein fraction
prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1
was significantly higher than that in a wild-type protein fraction; the crude
enzyme preparation released L-arabinose from radish AGPs as well as
alpha-(1-->5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl 
residues in the cell walls of transgenic plants was significantly decreased.
These results indicate that RsAraf1 encodes a bifunctional
alpha-L-arabinofuranosidase/beta-D-xylosidase and suggest that RsAraf1 is
involved in the hydrolysis of the carbohydrate moieties of AGPs in immature
radish seeds.

PMID: 16831850 [PubMed - indexed for MEDLINE]


156. Acta Crystallogr C. 2006 Jul;62(Pt 7):o379-81. Epub 2006 Jun 15.

7-Deaza-2,8-diazaadenosine.

Lin WQ, Ming X, Eickmeier H, Seela F.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.

In the title compound [systematic name:
4-amino-7-(beta-D-ribofuranosyl)-7H-pyrazolo[3,4-d][1,2,3]triazine],
C(9)H(12)N(6)O(4), the torsion angle of the N-glycosylic bond is high anti [chi =
-83.2 (3) degrees ]. The ribofuranose moiety adopts the C2'-endo-C1'-exo
((2)T(1)) sugar conformation (S-type sugar pucker), with P = 152.4 degrees and
tau(m) = 35.0 degrees . The conformation at the C4'-C5' bond is +sc
(gauche,gauche), with the torsion angle gamma = 52.0 (3) degrees . The compound
forms a three-dimensional network that is stabilized by several hydrogen bonds
(N-H...O, O-H...N and O-H...O).

PMID: 16823207 [PubMed - indexed for MEDLINE]


157. J Org Chem. 2006 Jul 7;71(14):5208-20.

Total synthesis and biological evaluation of neodysiherbaine A and analogues.

Shoji M, Akiyama N, Tsubone K, Lash LL, Sanders JM, Swanson GT, Sakai R,
Shimamoto K, Oikawa M, Sasaki M.

Laboratory of Biostructural Chemistry, Graduate School of Life Sciences, Tohoku
University, Sendai 981-8555, Japan.

Dysiherbaine (1) and its congener neodysiherbaine A (2) are naturally occurring
excitatory amino acids with selective and potent agonistic activity for
ionotropic glutamate receptors. We describe herein the total synthesis of 2 and
its structural analogues 3-8. Advanced key intermediate 16 was employed as a
branching point to assemble a series of these analogues 3-8 with respect to the
C8 and C9 functionalities, which would not have been accessible through
manipulations of the natural product itself. The synthesis of key intermediate 16
features (i) stereocontrolled C-glycosylation to set the C6 stereocenter, (ii)
concise synthesis of the bicyclic ether skeleton through chemo- and
stereoselective dihydroxylation of the exo-olefin and stereoselective epoxidation
of the endo-olefin, followed by epoxide ring opening/5-exo ring closure, and
(iii) catalytic asymmetric hydrogenation of enamide ester to construct the amino 
acid appendage. A preliminary biological evaluation of analogues for their in
vivo toxicity against mice and binding affinity for glutamate receptors showed
that both the type and stereochemistry of the C8 and C9 functional groups
affected the subtype selectivity of dysiherbaine analogues for members of the
kainic acid receptor family.

PMID: 16808508 [PubMed - indexed for MEDLINE]


158. J Exp Bot. 2006;57(10):2339-51. Epub 2006 Jun 23.

Purification, functional characterization, cloning, and identification of mutants
of a seed-specific arabinan hydrolase in Arabidopsis.

Minic Z, Do CT, Rihouey C, Morin H, Lerouge P, Jouanin L.

Laboratoire de Biologie Cellulaire - Institut National de la Recherche
Agronomique, Route de St-Cyr, F-78026 Versailles Cedex, France.

This work describes the purification and characterization of an enzyme that
exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The
enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified
to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser
desorption ionization-time of flight) as a putative beta-D-xylosidase that
belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3
hydrolysed synthetic substrates such as p-nitrophenyl-alpha-L-arabinofuranoside
and p-nitrophenyl-beta-D-xyloside with similar catalytic efficiency. XYL3
released L-arabinose from (1-->5)-alpha-L-arabinofuranobiose, arabinoxylan, sugar
beet arabinan, and debranched arabinan. The enzyme hydrolysed both
arabinosyl-substituted side group residues and terminal arabinofuranosyl residues
(1-->5)-alpha-linked to the arabinan backbone. This indicates that XYL3 is able
to degrade all terminal arabinosyl residues and suggests that it participates in 
the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by
semi-quantitative RT-PCR, in-situ hybridization and a promoter-GUS fusion
demonstrated that AtBX3 was specifically expressed in the seed endosperm at the
globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera
found that arabinan, the XYL3 substrate, was also present in this seed tissue.
T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the
alpha-L-arabinofuranosidase and beta-D-xylosidase activities corresponding to
XYL3. Mutants showed reduced seed size and are delayed in seedling germination
compared with the wild type.

PMID: 16798843 [PubMed - indexed for MEDLINE]


159. J Prosthet Dent. 2006 Jun;95(6):421-9.

Influence of cavity preparation design on fracture resistance of posterior
Leucite-reinforced ceramic restorations.

Soares CJ, Martins LR, Fonseca RB, Correr-Sobrinho L, Fernandes Neto AJ.

School of Dentistry, Federal University of Uberlandia, Minas Gerais, Brazil.
carlosjsoares@umuarama.ufu.br

STATEMENT OF PROBLEM: Controversy exists concerning the preferred cavity design
for posterior ceramic restorations to improve their resistance to fracture under 
occlusal load. PURPOSE: The aim of this study was to assess the resistance to
fracture of leucite-reinforced ceramic restorations placed on molars with
different cavity preparation designs. MATERIAL AND METHODS: Ninety noncarious
molars were selected, stored in 0.2% thymol solution, and divided into 9 groups
(n = 10): IT, intact teeth; CsI, conservative inlay; ExI, extensive inlay;
CsO/mb, conservative onlay with mesio-buccal cusp coverage; ExO/mb, entensive
onlay with mesio-buccal cusp coverage; CsO/b, conservative onlay with buccal cusp
coverage; ExO/b, entensive onlay with buccal cusp coverage; CsO/t, conservative
onlay with total cusp coverage; ExO/t, extensive onlay with total cusp coverage. 
Teeth were restored with a Leucite-reinforced ceramic (Cergogold). The fracture
resistance (N) was assessed under compressive load in a universal testing
machine. The data were analyzed with 1-way and 2-way analyses of variance,
followed by the Tukey HSD test (alpha = .05). Fracture modes were recorded, based
on the degree of tooth structure and restoration damage. RESULTS: One-way
analysis showed that intact teeth had the highest fracture resistance values.
Two-way analyses showed no significant differences for the isthmus extention
factor, but showed a significant difference for the preparation design type of
fracture (P = .03), and also for the interaction between both factors (P = .013).
The fracture mode observed in all groups tended to involve only restorations.
CONCLUSION: Within the limitations of this study, it was observed that cuspal
coverage does not increase fracture resistance of the posterior tooth-restoration
complex restored with leucite-reinforced ceramics.

PMID: 16765154 [PubMed - indexed for MEDLINE]


160. Physiology (Bethesda). 2006 Jun;21:189-96.

Insulin vesicle release: walk, kiss, pause ... then run.

Rutter GA, Hill EV.

Department of Biochemistry, School of Medical Sciences, University Walk
University of Bristol, Bristol, United Kingdom. g.a.rutter@bris.ac.uk

The mechanisms by which insulin-containing dense core secretory vesicles approach
and finally fuse with the plasma membrane are of considerable current interest:
defects in these processes may be one of the contributing factors to Type 2
diabetes. In this review, we discuss the molecular mechanisms involved in vesicle
trafficking within the pancreatic beta-cell and the mechanisms whereby these may 
be regulated. We then go on to describe recent evidence that suggests that
vesicle fusion at the plasma membrane is a partly reversible process ("kiss and
run" or "cavity recapture"). We propose that vesicles may participate in a
exo-endocytotic cycle in which a proportion of those that have already undergone 
an interaction with the plasma membrane may exchange exocytotic machinery with
maturing vesicles.

PMID: 16714477 [PubMed - indexed for MEDLINE]


161. DNA Repair (Amst). 2006 Jul 13;5(7):799-809. Epub 2006 May 19.

Trapping of DNA topoisomerase I on nick-containing DNA in cell free extracts of
Saccharomyces cerevisiae.

Lebedeva N, Auffret Vander Kemp P, Bjornsti MA, Lavrik O, Boiteux S.

CEA, UMR217 CNRS Radiobiologie Moleculaire et Cellulaire, route du Panorama, BP6,
92265-Fontenay aux Roses, France.

The aim of the present study was to identify proteins that bind nicked DNA
intermediates formed in the course of base excision repair (BER) in cell free
extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are
targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One
of the most promising methodologies to trap proteins that interact with damaged
DNA lies in using a photocrosslinking technique with photoactivable dNTP
analogues such as
exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoe
thyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA 
probes with a photoreactive dNMP residue at the 3'-margin of a nick. Using this
approach, we identified a major covalent DNA-protein adduct between a
nick-containing 34-mer DNA duplex and a protein of a molecular mass of around
100-kDa. Unexpectedly, the formation of the 100-kDa adduct did not require the
incorporation of the photoreactive dNMP residue at the 3'-margin of the nick nor 
exposure to near UV-light. However, the formation of the 100-kDa adduct strictly 
required a nick or a short gap in the DNA probe. Furthermore, the 100-kDa adduct 
was not detected in yeast extracts lacking DNA topoisomerase I (Top1). To further
establish the nature of crosslinked protein, yeast Top1 was tagged with a
Myc-epitope. In this case, the mobility of the Top1-DNA adduct increased by 7-
kDa. Therefore, our data speak in favor of Top1 trapping by nicked DNA. In
support of this hypothesis, purified yeast Top1 was also crosslinked to nicked
DNA structures. Undamaged, uracil- and abasic (AP) site-containing DNAs were
unable to trap Top1 under the same assay conditions. Since nicked DNA structures 
are frequently formed in the course of BER, their covalent linkage to Top1 has
the potential to interfere with BER in vivo.

PMID: 16713756 [PubMed - indexed for MEDLINE]


162. J Org Chem. 2006 May 26;71(11):4066-77.

Cyclopropenes in the 1,3-dipolar cycloaddition with carbonyl ylides: experimental
and theoretical evidence for the enhancement of sigma-withdrawal in
3-substituted-cyclopropenes.

Diev VV, Kostikov RR, Gleiter R, Molchanov AP.

Department of Chemistry, Saint Petersburg State University, 198504 Universitetsky
pr. 26, Saint Petersburg, Russian Federation.

The carbonyl ylide dipoles generated by the dirhodium tetra-acetate-catalyzed
decomposition of diazocarbonyl precursors 1, 5, and 8 cycloadd to 3-substituted
1,2-diphenylcyclopropenes 3a-e and 3,3-disubstituted cyclopropenes 13, 14, 19,
and 20 to give polycyclic compounds with 8-oxatricyclo[3.2.1.0(2,4)]octane and
9-oxatricyclo[3.3.1.0(2,4)]nonane frameworks. Generally, reactions proceed
stereoselectively to give adducts of exo stereochemistry with the approach of the
carbonyl ylide dipoles from the less-hindered face of cyclopropenes. The
electronic properties of the substituent at the C3 position of cyclopropenes play
an important role in governing the reactivity of cyclopropenes: when the C3
position is substituted by electron-acceptors such as the methoxycarbonyl or
cyano groups, the yields of adducts are decreased significantly or no adducts can
be detected at all. Relative reactivities of cyclopropenes were quantified by
competition experiments to give the best correlation with sigmaF-Taft constants. 
Both measured photoelectron spectra and ground-state calculations of a series of 
1,2-diphenylcyclopropenes indicate considerable lowering of cyclopropene pi-HOMO 
energies by substitution with an acceptor group. Such changes in electronic
structures of cyclopropenes may cause the inversion of frontier molecular orbital
(FMO) interactions from HOMO(cyclopropene)-LUMO(ylide) to
LUMO(cyclopropene)-HOMO(ylide) type. In terms of philicity, nucleophilic
properties of acceptor-substituted cyclopropenes are diminished to such an extent
that these species are no longer good nucleophiles in the reaction with carbonyl 
ylides, and neither are they good electrophiles, being unreactive. This was shown
by the B3LYP calculations of addends.

PMID: 16709045 [PubMed]


163. J Mol Model. 2006 Sep;12(6):991-5. Epub 2006 May 6.

Ab initio and DFT study on the electrophilic addition reaction of bromine to
tetracyclo[5.3.0.0(2,6).0 (3,10)]deca-4,8-diene.

Abbasoglu R.

Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey. 
rabbas@ktu.edu.tr

The electronic and geometric structures of
tetracyclo[5.3.0.0(2,6).0(3,10)]deca-4,8-diene (hypostrophene) have been
investigated by ab initio and DFT/B3LYP methods using the 6-31G* and 6-311G*
basis sets. The double bonds of hypostrophene are endo-pyramidalized. The
cationic intermediates and products formed in the addition reaction have been
investigated using the HF/6-311G*, HF/6-311G**, and B3LYP/6-311G* methods. The
bridged bromonium cation was more stable than the U-type cation. Considering that
the bridged cation does not isomerize to the less stable U-type cation, it is not
possible for the U-type product to be obtained in the reaction. The bridged
bromonium cation transformed into the more stable N-type cation and the N-type
product was obtained via this cation. The thermodynamic stability of the exo, exo
and exo, endo isomers of the N-type dibromide molecule were almost identical. The
N-type product was 16.6 kcal mol(-1) more stable than the U-type product.

PMID: 16680412 [PubMed - indexed for MEDLINE]


164. Acta Crystallogr C. 2006 May;62(Pt 5):o246-8. Epub 2006 Apr 13.

2'-Deoxy-7-propynyl-7-deazaadenosine: a DNA duplex-stabilizing nucleoside.

Seela F, Shaikh KI, Budow S, Eickmeier H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.
frank.seela@uni-osnabrueck.de

In the title compound, 2'-deoxy-7-propynyl-7-deazaadenosine, C14H16N4O3, the
torsion angle of the N-glycosylic bond is anti [chi = -130.7 (2) degrees ]. The
sugar pucker of the 2'-deoxyribofuranosyl moiety is C2'-endo-C3'-exo, 2T3
(S-type), with P = 185.9 (2) degrees and tau(m) = 39.1 (1) degrees , and the
orientation of the exocyclic C4'-C5' bond is -ap (trans). The 7-substituted
propynyl group is nearly coplanar with the heterocyclic base moiety. Molecules of
the nucleoside form a layered network in which the heterocyclic bases are stacked
head-to-tail with a closest distance of 3.197 (1) A. The crystal structure of the
nucleoside is stabilized by three intermolecular hydrogen bonds of types N-H...
O, O-H... N and O-H... O.

PMID: 16679593 [PubMed - indexed for MEDLINE]


165. Eur J Pharmacol. 2006 May 24;538(1-3):43-7. Epub 2006 Mar 28.

Dopamine uptake and cocaine binding mechanisms: the involvement of charged amino 
acids from the transmembrane domains of the human dopamine transporter.

Dar DE, Metzger TG, Vandenbergh DJ, Uhl GR.

Molecular Neurobiology Branch, National Institute on Drug Abuse, Intramural
Research Program, National Institutes of Health, Baltimore, MD 21224, USA.
dalit.dar@weizmann.ac.il

The wild type human dopamine transporter (DAT) and five DAT mutants were
transfected into COS-7 cells and their ability to uptake dopamine or to bind
cocaine was examine three days later. In each mutant, a single charged amino
acid, located in areas that initial hydrophobic analysis had indicated were DAT
transmembrane domains was substituted by alanine. Mutants used in this study were
lysines 257 and 525 (termed K257A and K525A), arginines 283 and 521 (termed R283A
and R521A), and glutamate 491 (termed E491A). Dopamine affinity was significantly
enhanced in the K257A and R283A mutants, and the IC(50) for displacement of the
radioactive cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane
(CFT) by cocaine was significantly elevated in the E491A mutant. All mutants
displayed a reduction or complete loss of the maximal velocity (V(m)) of dopamine
transport.

PMID: 16674939 [PubMed - indexed for MEDLINE]


166. J Mol Biol. 2006 May 5;358(3):741-53. Epub 2006 Mar 7.

The autocrine motility factor (AMF) and AMF-receptor combination needs sugar
chain recognition ability and interaction using the C-terminal region of AMF.

Haga A, Tanaka N, Funasaka T, Hashimoto K, Nakamura KT, Watanabe H, Raz A, Nagase
H.

Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502-8585, Japan.
arayo@gifu-pu.ac.jp

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and
regulates tumor angiogenesis or ascites accumulation. These signals are triggered
by binding between AMF and its receptor (AMFR), a glycoprotein on the cell
surface. AMF has been identified as phosphohexose isomerase (PHI). Previous
reports have suggested that the substrate-recognition of exo-PHI is significant
for receptor binding. Crystallographic studies have shown that AMF consists of
three domains, and that the substrate or inhibitor of PHI is stored between the
large and small domains, corresponding to approximately residues 117-288. Here,
site-directed mutagenesis was used to investigate 18 recombinant human AMF point 
mutants involving critical amino acid residues for substrate or enzyme inhibitor 
recognition or binding. Mutation of residues that interact with the phosphate
group of the PHI substrate significantly reduced the cell motility-stimulating
activity. Their binding capacities for AMFR were also lower than wild-type human 
AMF. Mutants that retained the enzymic activity showed the motility-stimulating
effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR
lacking the N-sugar chain was expressed on the cell membrane but did not respond 
to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor 
binding of AMF. Furthermore, the AMF domains that contain the substrate storage
domain and C-terminal region stimulate cell locomotion. These results suggest
that the N-glyco side-chain of AMFR is a trigger and that interaction between the
117-C-terminal part of AMF and the extracellular core protein of AMFR is needed
during AMF-AMFR interactions.

PMID: 16563432 [PubMed - indexed for MEDLINE]


167. J Org Chem. 2006 Mar 31;71(7):2690-8.

Synthesis, properties, and NAD+-NADH-type redox ability of 14-substituted
1,3-dimethyl-5,10-methanocycloundeca[4,5]pyrrolo[2,3-d]pyrimidine-2,4(1,3h)-diony
lium tetrafluoroborates and their hydride adducts.

Igarashi K, Yamaguchi Y, Mitsumoto Y, Naya S, Nitta M.

Department of Chemistry, Faculty of Science and Engineering, Waseda University,
Shinjuku-ku, Tokyo 169-8555, Japan.

A synthesis of 14-substituted
1,3-dimethyl-5,10-methanocycloundeca[4,5]pyrrolo[2,3-d]pyrimidine-2,4(1,3H)-diony
lium tetrafluoroborates 11a,b(+).BF4- was accomplished by the methylation of
5,10-methanocycloundeca[4,5]pyrrolo[2,3-d]pyrimidine-2,4(1,3H)-dione derivatives 
with MeI and following anion-exchange reaction by treatment with 42% aq HBF(4).
Compound 11b(+).BF4- was synthesized alternatively by the reaction of
1,6-methano[11]annulenylium tetrafluoroborate with
6-phenylamino-1,3-dimethyluracil and following oxidative cyclization reaction.
Remarkable structural characteristics of 11a,b(+)were clarified on inspection of 
the UV-vis and NMR spectral data as well as X-ray crystal analyses. The stability
of cations 11a,b(+)() is expressed by the pK(R+) values which were determined
spectrophotometrically as 9.8 and 9.7, which are smaller by 1.4 and 1.2 pH units 
than those of the corresponding seven-membered ring cations, respectively;
however, the values are larger by 3.6 and 3.5 pH units than that of the parent
1,6-methano[11]annulenylium ion (pK(R+) = 6.2). The feature is rationalized on
the basis of the perturbation derived from the bond fixation of the parent cation
and the electron-donating ability of pyrrolopyrimidine. The electrochemical
reduction of 11a,b(+).BF4- exhibited reduction potential at -0.58 and -0.52 (V vs
Ag/AgNO3) upon cyclic voltammetry (CV). Reaction of 11a(+).BF4- with hydride
afforded mixures of the C13- and C11-adducts in a ratio with hydride afforded, on
the other hand, the C13-adduct as a single product. In both cations, the
methano-bridge seemed to control the nucleophilic attack to the C13 favorably
with exo-selectivity. The photoinduced autorecycling oxidation reactions of
11a,b(+).BF4- toward some amines under aerobic conditions were carried out to
give the corresponding imines (isolated by converting to the corresponding
2,4-dinitrophenylhydrazones) with the recycling number of 1.1 to 32.2.
Furthermore, as an example of the NAD+-NADH models, the reduction of a pyruvate
analogue and some carbonyl compounds with the hydride adducts of 11a,b+.BF4- was 
accomplished for the first time to give the corresponding alcohol derivatives.

PMID: 16555822 [PubMed - indexed for MEDLINE]


168. Gene Ther. 2006 Jun;13(12):974-85. Epub 2006 Mar 2.

Adenoviral gene transfer of a mutant surfactant enzyme ameliorates
pseudomonas-induced lung injury.

Zhou J, Wu Y, Henderson F, McCoy DM, Salome RG, McGowan SE, Mallampalli RK.

Department of Internal Medicine, Pulmonary and Critical Care Division, Roy J and 
Lucille A Carver College of Medicine, The University of Iowa, Iowa City, IA
52242, USA.

Surfactant deficiency is an important contributor to the acute respiratory
distress syndrome, a disorder that commonly occurs after bacterial sepsis.
CTP:phosphocholine cytidylyltransferase (CCTalpha) is the rate-limiting enzyme
required for the biosynthesis of dipalmitoylphosphatidylcholine (DPPC), the major
phospholipid of surfactant. In this study, a cDNA encoding a novel,
calpain-resistant mutant CCTalpha enzyme was delivered intratracheally in mice
using a replication-deficient adenovirus 5 CTP:phosphocholine
cytidylyltransferase construct (Ad5-CCT(Penta)) in models of bacterial sepsis.
Ad5-CCT(Penta) gene transfer produced high-level CCTalpha gene expression,
increased alveolar surfactant (DPPC) levels and improved lung surface tension and
pressure-volume relationships relative to control mice. Pseudomonas aeruginosa
(PA103) decreased DPPC synthesis, in part, via calpain-mediated degradation of
CCTalpha. Deleterious effects of Pseudomonas on surfactant were lessened after
infection with a mutant strain lacking the type III exotoxin, Exo U.
Replication-deficient adenovirus 5 CTP:phosphocholine cytidylyltransferase gene
delivery improved lung biophysical properties by optimizing surface activity in
this Pseudomonas model of proteinase-mediated lung injury. The studies are the
first demonstration of in vivo gene transfer of a lipogenic enzyme resulting in
improved lung mechanics. The studies suggest that augmentation of DPPC synthesis 
via gene delivery of CCTalpha can attenuate impaired lung function in
surfactant-deficient states such as bacterial sepsis.

PMID: 16511521 [PubMed - indexed for MEDLINE]


169. Mol Cells. 2006 Feb 28;21(1):104-11.

Intracellular localization and sustained prodrug cell killing activity of
TAT-HSVTK fusion protein in hepatocelullar carcinoma cells.

Cao L, Si J, Wang W, Zhao X, Yuan X, Zhu H, Wu X, Zhu J, Shen G.

Department of Immunology, Tongji Medical College, Huazhong University of Science 
and Technology, Wuhan 430030, China.

Gene therapy with nonviral vectors using the suicide gene/prodrug activating
system of herpes simplex vi-rus type-1 thymidine kinase (HSV1-TK)/ganciclovir
(GCV) is inefficient in killing malignant tumor cells due to two major factors:
(a) an unsatisfactory by-stander effect; (b) short-lived expression of the
pro-tein. To study the capacity of the protein transduction domain (PTD) of HIV-1
TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three
fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as
TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much 
of it is translocated to the nu-cleus. The transduced HepG2 cells are killed by
exo-genously added GCV and have bystander effects on untransduced HepG2 cells.
Most importantly, the in-troduced recombinant protein is stable and remains
functional for several days at least, probably because nuclear localization
protects it from the cytoplasmic degradation machinery and provides access to the
nu-clear transcription machinery. Our results indicate that TAT fusion proteins
traffic intercellularly and have enhanced stability and prodrug cell killing
activ-ity. We conclude that TAT has potential for enhancing enzyme prodrug
treatment of liver cancers.

PMID: 16511352 [PubMed - indexed for MEDLINE]


170. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Feb 1;61(Pt 2):255-7. Epub
2005 Feb 1.

Crystallization and preliminary X-ray crystallographic studies of XynX, a family 
10 xylanase from Aeromonas punctata ME-1.

Fujimoto Z, Usui K, Kondo Y, Yasui K, Kawai K, Suzuki T.

Department of Biochemistry, National Institute of Agrobiological Sciences,
Tsukuba 305-8602, Japan. zui@affrc.go.jp

Xylanases catalyze the hydrolysis of beta-1,4-glycosidic linkages within the
xylan backbone. XynX is a xylanase from Aeromonas punctata ME-1 and belongs to
glycoside hydrolase family 10. While most xylanases show endo-type catalytic
activities, XynX shows exo-like catalytic activities, selectively producing
xylobiose from birchwood xylan. In this study, XynX was crystallized by the
hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic
space group P2(1)2(1)2(1), with unit-cell parameters a = 79.0, b = 88.6, c = 93.2
A, and diffracted to beyond 1.8 A resolution.

PMCID: PMC1952265
PMID: 16511010 [PubMed - indexed for MEDLINE]


171. Mol Cell Neurosci. 2006 Apr;31(4):702-12. Epub 2006 Feb 28.

Postsynaptic protein mobility in dendritic spines: long-term regulation by
synaptic NMDA receptor activation.

Sharma K, Fong DK, Craig AM.

Department of Anatomy and Neurobiology, Washington University School of Medicine,
St. Louis, MO 63110, USA. ksharma5@jhem.jhmi.edu

Reorganization of molecular components represents a cellular mechanism for
synaptic plasticity. Dendritic spines, major sites for glutamatergic synapses,
compartmentalize dynamic changes in molecular composition. Here, we use
fluorescence recovery after photobleaching (FRAP) in cultured hippocampal neurons
to show that spine proteins undergo continual exchange with extra-spine pools.
Each spine component has a distinctive mobility: calcium/calmodulin activated
protein kinase CaMKIIalpha > GluR1 AMPA glutamate receptor > PSD-95 scaffolding
protein > NR1 NMDA glutamate receptor. Stimulation of synaptic NMDA receptors by 
a protocol that induces chemical LTP resulted in a long-lasting reduction in the 
mobility of spine CaMKIIalpha and an increased mobile fraction but slower
kinetics for spine GluR1. Stimulation also increased the resistance of
postsynaptic CaMKIIalpha to detergent extraction. These results suggest
long-lasting changes in affinity of protein-protein interactions and/or ongoing
alterations in exo/endocytosis. Such lasting changes in protein mobility may
contribute to maintaining alterations in synaptic efficacy.

PMID: 16504537 [PubMed - indexed for MEDLINE]


172. RNA. 2006 Mar;12(3):476-87.

T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion
cycle.

Zhelonkina AG, O'Hearn SF, Law JA, Cruz-Reyes J, Huang CE, Alatortsev VS,
Sollner-Webb B.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, 
725 N. Wolfe Street, Baltimore, MD 21205, USA.

Trypanosome RNA editing is massive post-transcriptional U-insertion and
U-deletion, which generates mature mRNA coding regions through cycles of
endonuclease, terminal U transferase (TUTase) or 3'-U-exo, and ligase action.
Both types of editing are thought to be catalyzed by distinct sets of proteins of
a multiprotein complex, and no enzymatic activity of wild-type editing complex
had been shown to function in both forms of editing. By examining the individual 
steps of the U-deletion cycle using purified editing complex, traditional
mitochondrial extract, and rapidly prepared cell lysate, we here demonstrate that
TbMP57 TUTase of U-insertion can act efficiently within a U-deletion cycle. When 
physiological UTP levels are provided, it adds U's to the upstream cleavage
fragment after U-deletional endonuclease and 3'-U-exo action, but before
rejoining by the U-deletional ligase, generating partial U-deletion products.
TUTase activity in U-deletion was not previously appreciated since its detection 
requires UTP, which is not normally added to in vitro U-deletion reactions.
Fractionation and RNAi analyses show this U-addition in U-deletion requires
TbMP57 TUTase be present and competent for U-insertion; such U-addition does not 
occur with another mitochondrial TUTase that is separate from the basic editing
complex. Efficient TbMP57 action in both U-insertion and U-deletion suggests
these two editing forms may be less separate than generally envisioned. Should
such promiscuous TUTase action also occur in vivo, it could explain why editing
utilizes substantially fewer U-deletional than U-insertional events and why
partial editing appears preferential in U-deletion.

PMCID: PMC1383585
PMID: 16495238 [PubMed - indexed for MEDLINE]


173. Microb Cell Fact. 2006 Feb 16;5:7.

Rhizobial exopolysaccharides: genetic control and symbiotic functions.

Skorupska A, Janczarek M, Marczak M, Mazur A, Krol J.

Department of General Microbiology, University of M, Curie-Sklodowska, Akademicka
19 st, 20-033 Lublin, Poland. genet@biotop.umcs.lublin.pl

Specific complex interactions between soil bacteria belonging to Rhizobium,
Sinorhizobium, Mesorhizobium, Phylorhizobium, Bradyrhizobium and Azorhizobium
commonly known as rhizobia, and their host leguminous plants result in
development of root nodules. Nodules are new organs that consist mainly of plant 
cells infected with bacteroids that provide the host plant with fixed nitrogen.
Proper nodule development requires the synthesis and perception of signal
molecules such as lipochitooligosaccharides, called Nod factors that are
important for induction of nodule development. Bacterial surface polysaccharides 
are also crucial for establishment of successful symbiosis with legumes. Sugar
polymers of rhizobia are composed of a number of different polysaccharides, such 
as lipopolysaccharides (LPS), capsular polysaccharides (CPS or K-antigens),
neutral beta-1, 2-glucans and acidic extracellular polysaccharides (EPS). Despite
extensive research, the molecular function of the surface polysaccharides in
symbiosis remains unclear. This review focuses on exopolysaccharides that are
especially important for the invasion that leads to formation of indetermined
(with persistent meristem) type of nodules on legumes such as clover, vetch, peas
or alfalfa. The significance of EPS synthesis in symbiotic interactions of
Rhizobium leguminosarum with clover is especially noticed. Accumulating data
suggest that exopolysaccharides may be involved in invasion and nodule
development, bacterial release from infection threads, bacteroid development,
suppression of plant defense response and protection against plant antimicrobial 
compounds. Rhizobial exopolysaccharides are species-specific heteropolysaccharide
polymers composed of common sugars that are substituted with non-carbohydrate
residues. Synthesis of repeating units of exopolysaccharide, their modification, 
polymerization and export to the cell surface is controlled by clusters of genes,
named exo/exs, exp or pss that are localized on rhizobial megaplasmids or
chromosome. The function of these genes was identified by isolation and
characterization of several mutants disabled in exopolysaccharide synthesis. The 
effect of exopolysaccharide deficiency on nodule development has been extensively
studied. Production of exopolysaccharides is influenced by a complex network of
environmental factors such as phosphate, nitrogen or sulphur. There is a strong
suggestion that production of a variety of symbiotically active polysaccharides
may allow rhizobial strains to adapt to changing environmental conditions and
interact efficiently with legumes.

PMCID: PMC1403797
PMID: 16483356 [PubMed]


174. Biochemistry. 2006 Feb 21;45(7):2211-20.

The L561A substitution in the nascent base-pair binding pocket of RB69 DNA
polymerase reduces base discrimination.

Zhang H, Rhee C, Bebenek A, Drake JW, Wang J, Konigsberg W.

Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar
Street, New Haven, Connecticut 06520, USA.

Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements 
in the nascent base-pair binding pocket are less discriminating with respect to
noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the
loss in base selectivity, we determined the transient-state kinetic parameters
for incorporation of correct and all combinations of incorrect dNMPs by the
exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid 
chemical quench assays. The L561A variant did not significantly alter the k(pol) 
and K(D) values for incorporation of correct dNMPs, but it showed increased
incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the
wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A
variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite
templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C.
These k(pol)/K(D) values are 3-60-fold greater than those observed with the
wild-type enzyme. The effect of the L561A replacement on the mutation frequency
in vivo was determined by infecting Escherichia coli harboring a plasmid encoding
the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the
rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol
L561A displayed a weak mutator phenotype. In contrast, no progeny phage were
produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol
L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype
was attributed to error catastrophe caused by the high rate of mutation expected 
from combining the pol L561A and exo(-) mutator activities.

PMID: 16475809 [PubMed - indexed for MEDLINE]


175. J Org Chem. 2006 Feb 17;71(4):1471-9.

Solid-supported copper catalysts for atom-transfer radical cyclizations:
assessment of support type and ligand structure on catalyst performance in the
synthesis of nitrogen heterocycles.

Clark AJ, Geden JV, Thom S.

Department of Chemistry, University of Warwick, Coventry, West Midlands CV4 7AL, 
UK. msrir@csv.warwick.ac.uk

A range of solid supported pyridinemethanimine 9-11 and polyamine 12-15 ligands
have been prepared on silica, polystyrene, and JandaJel supports. The CuCl and
CuBr complexes of these supported ligands have been used to assess both the
effect of the ligand type and the nature of the support upon a representative
range of copper-mediated atom transfer 5-exo-trig 6, 24-25, 5-exo-dig 26,
4-exo-trig 28, and 5-endo-trig 27, 38 radical cyclizations to give nitrogen
heterocycles. In addition, the effect of the nature of the support on the
stereochemical outcome of the 5-exo cyclization of 25 has been probed. Generally,
it was found that the type of support (e.g., polystyrene, silica, or JandaJel)
had very little effect upon the efficiency and selectivity of the processes but
that the nature of the ligand type immobilized was the important factor. Thus,
the 5-exo cyclization of 6 and 24-26 proceeded more rapidly with the PMI ligands 
9-11, whereas 4-exo cyclizations 28 and 5-endo radical polar crossover reactions 
27 and 38 proceeded more efficiently with the JJ-TEDETA ligand 15. The efficiency
of the supported ligands was also compared to their solution counterparts 4 and
5. The reusability of P-PMDETA ligand system 13 was assessed in the cyclization
of 6.

PMID: 16468796 [PubMed - indexed for MEDLINE]


176. Methods Enzymol. 2005;404:388-98.

Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2
coat recruitment to membranes.

Krauss M, Haucke V.

Department of Biochemistry II, Center for Biochemistry and Molecular Cell
Biology, University of Gottingen, Germany.

Arf proteins play pivotal roles in membrane traffic, cell signaling, and actin
cytoskeletal rearrangements. We describe here methods to functionally analyze
interacting partner proteins of recombinantly produced N-myristoylated Arf6.
Combined evidence from affinity purification and chemical crosslinking
experiments, in vitro recruitment assays, and the analysis of lipid kinase
activities indicates that Arf6-GTP facilitates clathrin/AP-2 recruitment to
synaptic membranes by direct binding and activation of the brain-specific
phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPK Igamma). These
methods shall help to mechanistically dissect the role of Arf6 in regulating
exo-endocytic vesicle cycling at synapses and in related membrane trafficking
events.

PMID: 16413285 [PubMed - indexed for MEDLINE]


177. J Org Chem. 2006 Jan 20;71(2):704-12.

Intramolecular additions of various pi-nucleophiles to chemoselectively activated
amides and application to the synthesis of (+/-)-tashiromine.

Belanger G, Larouche-Gauthier R, Menard F, Nantel M, Barabe F.

Laboratoire de synthese organique et de developpement de strategies de synthese
Departement de Chimie, Universite de Sherbrooke 2500 boulevard Universite,
Sherbrooke, Quebec, J1K 2R1, Canada.

[reaction: see text] Vilsmeier-Haack type cyclizations proved to be particularly 
efficient for generating parts of the polycyclic cores of many alkaloids,
although only monocyclizations have so far been reported. With the goal of
rapidly and efficiently constructing polycyclic alkaloids, we decided to exploit 
the Vilsmeier-Haack reaction by utilizing iminium ions successively generated and
trapped with tethered nucleophiles. To develop such a strategy, we had to set the
first cyclization. This constitutes a great challenge in itself because amide
activation conditions are usually not compatible with tethered nucleophiles,
except for indoles and aromatic rings which have already been reported. This
paper describes the comprehensive study of intramolecular addition of silyl enol 
ethers, allylsilanes, and enamines to chemoselectively activated formamides,
aliphatic amides, and lactams. Good to excellent yields were obtained for the
5-exo, 6-exo, and 6-endo modes of cyclization. Moreover, we demonstrated that the
species in solution after the cyclization are iminium ions. This is highly
encouraging for the development of bis-cyclization strategies. An expeditious
total synthesis of (+/-)-tashiromine is also reported.

PMID: 16408983 [PubMed - indexed for MEDLINE]


178. Proc Natl Acad Sci U S A. 2006 Jan 17;103(3):532-7. Epub 2006 Jan 9.

Manganese(II), iron(II), cobalt(II), and copper(II) complexes of an extended
inherently chiral tris-bipyridyl cage.

Perkins DF, Lindoy LF, McAuley A, Meehan GV, Turner P.

Centre for Heavy Metals Research, School of Chemistry, University of Sydney,
Sydney NSW 2006, Australia.

Manganese(II), iron(II), cobalt(II), and copper(II) derivatives of two inherently
chiral, Tris(bipyridyl) cages (L and L') of type [ML]-(PF(6))(2)(solvent)(n) and 
[FeL'](ClO(4))(2) are reported, where L is the hexa-tertiary butyl-substituted
derivative of L'. These products were obtained by using the free cage and metal
template procedures; the latter involved the reductive amination of the
respective Tris-dialdehyde precursor complexes of iron(II), cobalt(II), or
nickel(II). Electrochemical, EPR, and NMR studies have been used to probe the
nature of the individual complexes. X-ray structures of the manganese(II),
iron(II), and copper(II) complexes of L and the iron(II) complex of L' are
presented; these are compared with the previously reported structures of the
corresponding nickel(II) complex and metal-free cage (L). In each complex the
metal cation occupies the cage's central cavity and is coordinated to six
nitrogens from the three bipyridyl groups. The cations [MnL](2+) and [FeL](2+)
are isostructural but both exhibit a different arrangement of the bound cage to
that observed in the corresponding nickel(II) and copper(II) complexes. The
latter have an exo-exo arrangement of the bridgehead nitrogen lone pairs, with
the metal inducing a triple helical twist that extends approximately 22 A along
the axial length of each complex. In contrast, [MnL](2+) and [FeL](2+) have their
terminal nitrogen lone pairs directed endo, causing a significant change in the
configuration of the bound ligand. In [FeL'](2+), the cage has both bridgehead
nitrogen lone pairs orientated exo. Semiempirical calculations indicate that the 
observed endo-endo and exo-exo arrangements are of comparable energy.

PMCID: PMC1334659
PMID: 16407129 [PubMed]


179. Inorg Chem. 2005 Dec 26;44(26):9930-7.

Novel rhenium chelate system derived from dimercaptosuccinic acid for the
selective labeling of biomolecules.

Heinrich TK, Kraus W, Pietzsch HJ, Smuda C, Spies H.

Forschungszentrum Rossendorf, Institut fuer Bioanorganische und
Radiopharmazeutische Chemie, PF 510 119, D-01314 Dresden, Germany.

This work is part of an effort to develop chelating agents for stable binding and
easy conjugation of Re-188 to biologically interesting structures. Starting from 
the well-known in vivo stability of [(188)ReO(DMSA)(2)](-), we want to exploit
this coordination system for the design of (188)ReO(V) chelates, which are stable
toward reoxidation to perrhenate and toward ligand exchange under all conditions 
of radiopharmaceutical development. Therefore, a new type of tetradentate ligand 
has been synthesized by bridging two molecules of
N,N'-diisobutyl-2,3-dimercaptosuccinamide with
N-(3-aminopropyl)propane-1,3-diamine. The resulting stereoisomeric tetrathiolato 
S(4) ligand of composition
((i)()Bu)(2)N(O)C-C(SH)-C(SH)-C(O)NH-(CH(2))(3)-NH-(CH(2))(3)-NHC(O)-C(SH)-C(SH)-
C(O)N((i)Bu)(2) forms anionic five-coordinate oxorhenium(V) complexes by a
ligand-exchange reaction of NBu(4)[ReOCl(4)] in methanol. In the absence of a
base, the compounds were isolated as "betaine", [ReO(S(4))], with the protonated 
nitrogen of the bridge serving as an internal "counterion". Two representatives
have been fully characterized in both the solid and solution states and found to 
adopt the expected square-pyramidal coordination geometry. The equatorial plane
is formed by four thiolate sulfur atoms, whereas the oxygen occupies the apical
position. The orientation of the metal oxo group is exo in relation to the
carbamido groups in both isomers. Both complexes are stereoisomeric regarding the
junction of the triamine chain.

PMID: 16363864 [PubMed - indexed for MEDLINE]


180. Appl Environ Microbiol. 1998 Mar;64(3):890-895.

Purification and Characterization of Exo-beta-d-Glucosaminidase from a
Cellulolytic Fungus, Trichoderma reesei PC-3-7.

Nogawa M, Takahashi H, Kashiwagi A, Ohshima K, Okada H, Morikawa Y.

Department of Bioengineering, Nagaoka University of Technology, 1603-1
Kamitomioka, Nagaoka, Niigata 940-21, Japan.

Chitosan-degrading activities induced by glucosamine (GlcN) or
N-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma
reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity 
by precipitation with ammonium sulfate followed by anion-exchange and
hydrophobic-interaction chromatographies. The enzyme was monomeric, and its
molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 
and 50 degrees C, respectively. The activity was stable in the pH range 6.0 to
9.0 and at a temperature below 50 degrees C. Reaction product analysis from the
viscosimetric assay and thin-layer chromatography and H nuclear magnetic
resonance spectroscopy clearly indicated that the enzyme was an exo-type
chitosanase, exo-beta-d-glucosaminidase, that releases GlcN from the nonreducing 
end of the chitosan chain. H nuclear magnetic resonance spectroscopy also showed 
that the exo-beta-d-glucosaminidase produced a beta-form of GlcN, demonstrating
that the enzyme is a retaining glycanase. Time-dependent liberation of the
reducing sugar from partially acetylated chitosan with exo-beta-d-glucosaminidase
and the partially purified exo-beta-d-N-acetylglucosaminidase from T. reesei
PC-3-7 suggested that the exo-beta-d-glucosaminidase cleaves the glycosidic link 
of either GlcN-beta(1-->4)-GlcN or GlcN-beta(1-->4)-GlcNAc.

PMCID: PMC106342
PMID: 16349528 [PubMed - as supplied by publisher]


181. Appl Microbiol Biotechnol. 2006 Jul;71(3):294-303. Epub 2005 Dec 10.

Purification, characterization, and gene cloning of 46 kDa chitinase (Chi46) from
Trichoderma reesei PC-3-7 and its expression in Escherichia coli.

Ike M, Nagamatsu K, Shioya A, Nogawa M, Ogasawara W, Okada H, Morikawa Y.

Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.

We purified a chitinase (named Chi46), with a molecular mass of 46 kDa estimated 
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from the culture
filtrate of Trichoderma reesei PC-3-7 grown on N-acetylglucosamine (GlcNAc). The 
relative activity of this enzyme reduced when the degrees of acetylation (DA) of 
chitosan decreased. Furthermore, the enzyme was able to hydrolyze colloidal
chitin and ethylene glycol chitin. The gene chi46 was cloned and sequenced. chi46
encodes a protein of 424 amino acid residues containing a 35-amino acid
prepro-type secretion signal peptide. The molecular mass of mature Chi46
calculated from deduced amino acid sequence was 42,265 Da. The chi46 transcript
was biphasic when the mycelia were grown on GlcNAc, suggesting that the multiple 
regulatory proteins are involved in the chi46 expression. The chi46 cDNA was
expressed in Escherichia coli (ca. 0.23 mg/ml culture). To determine substrate
cleavage fashion of Chi46 in more detail, we carried out high-performance liquid 
chromatography analysis and viscosimetric assay using recombinant Chi46 (rChi46).
Chi46 was shown to release mainly (GlcNAc)(2) from colloidal chitin (insoluble
chitin) as an exo-type manner and to act on chitosan 7B (DA ca. 30%) and
N-acetylchitooligosaccharides (soluble chitins) in an endo-type one.

PMID: 16341861 [PubMed - indexed for MEDLINE]


182. Appl Environ Microbiol. 2005 Dec;71(12):7670-8.

Cloning and characterization of two xyloglucanases from Paenibacillus sp. strain 
KM21.

Yaoi K, Nakai T, Kameda Y, Hiyoshi A, Mitsuishi Y.

Institute for Biological Resources and Functions, National Institute of Advanced 
Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,
Ibaraki 305-8566, Japan. k-yaoi@aist.go.jp

Two xyloglucan-specific endo-beta-1,4-glucanases (xyloglucanases [XEGs]), XEG5
and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were
isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which
degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and 
sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and
XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively.
XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5
lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 
type carbohydrate-binding module at its C terminus. The two XEGs were expressed
in Escherichia coli, and recombinant forms of the enzymes were purified and
characterized. Both XEGs had endoglucanase active only toward xyloglucan and not 
toward Avicel, carboxymethylcellulose, barley beta-1,3/1,4-glucan, or xylan. XEG5
is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain,
while XEG74 has dual endo- and exo-mode activities or processive endo-mode
activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, 
whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting 
that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses
using various oligosaccharide structures revealed that unique structures of
xyloglucan oligosaccharides can be prepared with XEG74.

PMCID: PMC1317386
PMID: 16332739 [PubMed - indexed for MEDLINE]


183. Nucleic Acids Res. 2005 Nov 27;33(20):6515-21. Print 2005.

Synthesis of novel poly(dG)-poly(dG)-poly(dC) triplex structure by Klenow exo-
fragment of DNA polymerase I.

Kotlyar A, Borovok N, Molotsky T, Klinov D, Dwir B, Kapon E.

Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv
University, Ramat Aviv, 69978 Israel. s2shak@post.tau.ac.il

The extension of the G-strand of long (700 bp) poly(dG)-poly(dC) by the Klenow
exo(-) fragment of DNA polymerase I yields a complete triplex structure of the
H-DNA type. High-performance liquid chromatography analysis demonstrates that the
length of the G-strand is doubled during the polymerase synthesis. Fluorescence
resonance energy transfer analysis shows that the 5' ends of the G- and the
C-strands, labeled with fluorescein and TAMRA, respectively, are positioned close
to each other in the product of the synthesis. Atomic force microscopy morphology
imaging shows that the synthesized structures lack single-stranded fragments and 
have approximately the same length as the parent 700 bp poly(dG)-poly(dC). CD
spectrum of the polymer has a large negative peak at 278 nm, which is
characteristic of the poly(dG)-poly(dG)-poly(dC) triplex. The polymer is
resistant to DNase and interacts much more weakly with ethidium bromide as
compared with the double-stranded DNA.

PMCID: PMC1292991
PMID: 16314313 [PubMed - indexed for MEDLINE]


184. Biosci Biotechnol Biochem. 2005 Nov;69(11):2200-6.

Enzymic activity of the K5-type yeast killer toxin and its characterization.

Izgu F, Altinbay D, Sertkaya A.

Department of Biological Sciences, Middle East Technical University, Ankara,
Turkey. izgu@metu.edu.tr

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad
killing spectrum. Competitive inhibiton of killer activity showed that glucans,
mainly the beta-1,3 glucan, represent the primary toxin binding site within the
cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like
fashion revealed that the toxin exerts its killing effect by
exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg,
and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25
mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h
contact with the target cells. Production of the toxin by the cells was induced
only when they were grown in culture media rich in beta-glucan sources, and the
addition of glucose increased the specific production rate. The enzymic activity 
of the toxin was fully inhibited by Hg(+2), but increased with some other metal
ions, most of all by Pb(+2).

PMID: 16306703 [PubMed - indexed for MEDLINE]


185. J Biol Chem. 2006 Feb 17;281(7):4242-53. Epub 2005 Nov 17.

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations.

Liou B, Kazimierczuk A, Zhang M, Scott CR, Hegde RS, Grabowski GA.

Division and Program in Human Genetics, Children's Hospital Research Foundation, 
Cincinnati, OH 45229, USA.

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal 
exo-beta-glucosidase whose defective activity leads to the Gaucher disease
phenotypes. To move toward a structure/function map for disease mutations, 52
selected single amino acid substitutions were introduced into GCase, expressed in
an insect cell system, purified, and characterized for basic kinetic, stability, 
and activator response properties. The variant GCases from Gaucher disease
patients and selected variant GCases from the mouse had decreased relative k(cat)
and differential effects on active site binding and/or attachment of
mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin
derivatives) inhibitors. A defect in negatively charged phospholipid activation
was present in the majority of variant GCases but was increased in two, N370S and
V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases
involving residues 48-122, whereas approximately 2-fold increases were obtained
with the L264I GCase. About 50% of variant GCases each had wild-type or increased
sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto
the crystal structures of GCase indicated wide dispersion of functional
properties that can affect catalytic function and stability. Site-directed
mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) 
and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free
Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His
substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic
conservation indicate localized and general structural effects of Gaucher disease
mutations that were not obvious from the nature of the amino acid substitution,
including those predicted to be nondisruptive (e.g. Val --> Leu). These results
provide initial studies for the engineering of variant GCases and, potentially,
molecular chaperones for therapeutic use.

PMID: 16293621 [PubMed - indexed for MEDLINE]


186. Protein Expr Purif. 2006 Jan;45(1):125-31. Epub 2005 Jul 27.

Purification and characterization of exo-beta-D-glucosaminidase from Aspergillus 
fumigatus S-26.

Jung WJ, Kuk JH, Kim KY, Jung KC, Park RD.

Glucosamine Saccharide Materials National Research Laboratory, Department of
Agricultural Chemistry, Institute of Agricultural Science and Technology, Chonnam
National University, Gwangju 500-757, Republic of Korea.

An extracellular 104 kDa exo-beta-d-glucosaminidase was purified and
characterized from the culture supernatant of Aspergillus fumigatus S-26, which
showed exceptionally strong chitosanolytic enzyme activity. The purified enzyme
showed optimum pH of 3.0-6.0 and optimum temperature of 50-60 degrees C, and was 
stable between pH 2.0 and 10.0 and under 35 degrees C. The Km, Vmax, and kcat
were determined to be 1.0 mg chitosan/ml, 7.8x10(-8) mol/s/mg protein, and 28.3
s-1, respectively. The exo-beta-D-glucosaminidase was severely inactivated by
Cu2+ and Hg2+ at 10 mM. 2-Hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, and 
p-chloromercuribenzoic acid inhibited the enzyme. The enzyme did not degrade
chitin, cellulose, and starch. The exo-beta-D-glucosaminidase did not reduce the 
viscosity of chitosan solutions at early stage of reaction, suggesting the
exo-type of cleavage in polymeric chitosan chains. The exo-beta-D-glucosaminidase
liberated only GlcN from chitosan, and GlcN plus the one-residue shortened
oligomers from (GlcN)2-7. The exo-beta-D-glucosaminidase exhibited
transglycosylation activity, resulting in the one-residue elongated oligomers.

PMID: 16289917 [PubMed - indexed for MEDLINE]


187. J Org Chem. 2005 Sep 30;70(20):7985-95.

Free-radical-5-exo-trig cyclization of chiral 3-silylhepta-1,6-dienes: concise
approach to the A-B-C ring core of hexacyclinic acid.

James P, Felpin FX, Landais Y, Schenk K.

Universite Bordeaux-I, Laboratoire de Chimie Organique et Organometallique, 351, 
Cours de la Liberation, 33405 Talence Cedex, France.

[Chemical reaction: See text] Free-radical-mediated 5-exo-trig cyclization of
hepta-1,6-dienes 6a-c incorporating an allylsilane moiety was shown to provide at
low temperature exclusively the trans-cis cyclopentanes 7a-c in good yield. In
contrast, the same reaction with the alkyl analogue 9a led to the formation of
all four possible stereoisomers. Interestingly, extension of this sequence of
radical processes to alkoxy analogues 12-14 provided the complementary cis-cis
stereoisomers with modest to excellent stereoselectivity. It is noteworthy that
under such conditions allylsilanes were found to be much more reactive than their
alkyl and alkoxy analogues. Beckwith-Houk-type models were proposed that
rationalize the stereochemical course of these 5-exo-trig cyclizations. Finally, 
an illustration of the value of this methodology was proposed with the synthesis 
of the A-B-C tricyclic unit of polyketide hexacyclinic acid 3a.

PMID: 16277319 [PubMed]


188. Mol Ther. 2006 Feb;13(2):289-300. Epub 2005 Nov 7.

Exosomes derived from genetically modified DC expressing FasL are
anti-inflammatory and immunosuppressive.

Kim SH, Bianco N, Menon R, Lechman ER, Shufesky WJ, Morelli AE, Robbins PD.

Department of Molecular Genetics and Biochemistry, University of Pittsburgh
School of Medicine, PA 15261, USA.

We previously have demonstrated the ability of primary murine bone marrow-derived
DC (BM-DC), genetically modified by adenoviral infection to express FasL, to
inhibit progression of established collagen-induced arthritis (CIA) following
systemic delivery. Here we demonstrate that exosomes derived from genetically
modified BM-DC expressing FasL are able to inhibit inflammation in a murine
footpad model of delayed-type hypersensitivity (DTH). Local administration of
exosomes derived from DC expressing FasL (Exo/FasL) as well as the parental
DC/FasL resulted in a significant reduction in swelling in both the treated and
the untreated distal paw. However, both the DC/FasL and the Exo/FasL were unable 
to suppress the DTH response in lpr (Fas-deficient) mice. Gene transfer of FasL
to BM-DC from gld (FasL-deficient) mice resulted in restoration of the ability of
DC as well as DC-derived exosomes to suppress DTH. The ability of DC-derived
exosomes and DC to suppress DTH responses was antigen specific and MHC class II
dependent, but class I independent. The injected exosomes were found to be
internalized into CD11c(+) cells at the site of injection and in the draining
popliteal lymph node. Systemic injection of exosome/FasL into mice with
established CIA resulted in significant disease amelioration. These results
demonstrate that both systemic and local administration of exosomes derived from 
FasL-expressing DC are able to suppress antigen-specific immune responses through
an MHC class II-dependent pathway, resulting in effective and sustained treatment
of established collagen-induced arthritis and suppression of the DTH inflammatory
response. These results suggest that DC/FasL-derived exosomes could be used
clinically for the treatment of inflammatory and autoimmune diseases.

PMID: 16275099 [PubMed - indexed for MEDLINE]


189. J Am Chem Soc. 2005 Nov 2;127(43):15071-82.

A versatile toolbox for variable DNA functionalization at high density.

Jager S, Rasched G, Kornreich-Leshem H, Engeser M, Thum O, Famulok M.

Kekuke-Institut fur Organische Chemie und Biochemie, Universitat Bonn,
Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany.

To broaden the applicability of chemically modified DNAs in nano- and
biotechnology, material science, sensor development, and molecular recognition,
strategies are required for introducing a large variety of different
modifications into the same nucleic acid sequence at once. Here, we investigate
the scope and limits for obtaining functionalized dsDNA by primer extension and
PCR, using a broad variety of chemically modified deoxynucleotide triphosphates
(dNTPs), DNA polymerases, and templates. All natural nucleobases in each strand
were substituted with up to four different base-modified analogues. We studied
the sequence dependence of enzymatic amplification to yield high-density
functionalized DNA (fDNA) from modified dNTPs, and of fDNA templates, and found
that GC-rich sequences are amplified with decreased efficiency as compared to
AT-rich ones. There is also a strong dependence on the polymerase used. While
family A polymerases generally performed poorly on "demanding" templates
containing consecutive stretches of a particular base, family B polymerases were 
better suited for this purpose, in particular Pwo and Vent (exo-) DNA polymerase.
A systematic analysis of fDNAs modified at increasing densities by CD
spectroscopy revealed that single modified bases do not alter the overall B-type 
DNA structure, regardless of their chemical nature. A density of three modified
bases induces conformational changes in the double helix, reflected by an
inversion of the CD spectra. Our study provides a basis for establishing a
generally applicable toolbox of enzymes, templates, and monomers for generating
high-density functionalized DNAs for a broad range of applications.

PMID: 16248646 [PubMed - indexed for MEDLINE]


190. Clin Cancer Res. 2005 Oct 15;11(20):7554-63.

More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen
(CEA)-specific CTL response by immunization with exosomes prepared from
heat-stressed CEA-positive tumor cells.

Dai S, Wan T, Wang B, Zhou X, Xiu F, Chen T, Wu Y, Cao X.

Institute of Immunology, Zhejiang University, Hangzhou, People's Republic of
China.

PURPOSE: Tumor-derived exosomes are proposed as a new type of cancer vaccine.
Heat shock proteins are potent Th1 adjuvant, and heat stress can induce heat
shock protein and MHC-I expression in tumor cells, leading to the increased
immunogenicity of tumor cells. To improve the immunogenicity of exosomes as
cancer vaccine, we prepared exosomes from heat-stressed carcinoembryonic antigen 
(CEA)-positive tumor cells (CEA+/HS-Exo) and tested the efficacy of these
exosomes in the induction of CEA-specific antitumor immunity. EXPERIMENTAL
DESIGN: First, we identified the composition of CEA+/HS-Exo and observed their
effects on human dendritic cell maturation. Then, we evaluated their ability to
induce a CEA-specific immune response in vivo in HLA-A2.1/Kb transgenic mice and 
CEA-specific CTL response in vitro in HLA-A*0201+ healthy donors and
HLA-A*0201+CEA+ cancer patients. RESULTS: CEA+/HS-Exo contained CEA and more heat
shock protein 70 and MHC-I and significantly induced dendritic cell maturation.
Immunization of HLA-A2.1/Kb transgenic mice with CEA+/HS-Exo was more efficient
in priming a CEA-specific CTL, and the CTL showed antitumor effect when
adoptively transferred to SW480-bearing nude mice. Moreover, in vitro incubation 
of lymphocytes from HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer
patients with CEA+/HS-Exo-pulsed autologous dendritic cells induces
HLA-A*0201-restricted and CEA-specific CTL response. CONCLUSIONS: Our results
show that CEA+/HS-Exo has superior immunogenicity than CEA+/Exo in inducing
CEA-specific CTL response and suggest that exosomes derived from heat-stressed
tumor cells may be used as efficient vaccine for cancer immunotherapy.

PMID: 16243831 [PubMed - indexed for MEDLINE]


191. Org Biomol Chem. 2005 Nov 7;3(21):3926-36. Epub 2005 Sep 29.

Azetidinic amino acids: stereocontrolled synthesis and pharmacological
characterization as ligands for glutamate receptors and transporters.

Brauner-Osborne H, Bunch L, Chopin N, Couty F, Evano G, Jensen AA, Kusk M,
Nielsen B, Rabasso N.

Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, 
Universitetsparken 2, DK-2100, Copenhagen, Denmark.

A set of ten azetidinic amino acids, that can be envisioned as C-4 alkyl
substituted analogues of trans-2-carboxyazetidine-3-acetic acid (t-CAA) and/or
conformationally constrained analogues of (R)- or (S)-glutamic acid (Glu) have
been synthesized in a diastereo- and enantiomerically pure form from beta-amino
alcohols through a straightforward five step sequence. The key step of this
synthesis is an original anionic 4-exo-tet ring closure that forms the azetidine 
ring upon an intramolecular Michael addition. This reaction was proven to be
reversible and to lead to a thermodynamic distribution of two diastereoisomers
that were easily separated and converted in two steps into azetidinic amino
acids. Azetidines 35-44 were characterized in binding studies on native
ionotropic Glu receptors and in functional assays at cloned metabotropic
receptors mGluR1, 2 and 4, representing group I, II and III mGlu receptors,
respectively. Furthermore, azetidine analogues 35, 36, and 40 were also
characterized as potential ligands at the glutamate transporter subtypes EAAT1-3 
in the FLIPR Membrane Potential (FMP) assay. The (2R)-azetidines 35, 37, 39, 41
and 43 were inactive in iGlu, mGlu and EAAT assays, whereas a marked change in
the pharmacological profile at the iGlu receptors was observed when a methyl
group was introduced in the C-4 position, compound 36 versus t-CAA. At EAAT1-3,
compound 35 was inactive, whereas azetidines 36 and 40 were both identified as
inhibitors and showed selectivity for the EAAT2 subtype.

PMID: 16240010 [PubMed - indexed for MEDLINE]


192. FEMS Microbiol Lett. 2005 Dec 1;253(1):95-101. Epub 2005 Oct 5.

PscF is a major component of the Pseudomonas aeruginosa type III secretion
needle.

Pastor A, Chabert J, Louwagie M, Garin J, Attree I.

Laboratoire de Biochimie et Biophysique des Systemes Integres (UMR 5092
CNRS/CEA/UJF), DRDC/BBSI, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 
09, France.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, translocates
exoenzymes (Exo) directly into the eukaryotic cell cytoplasm. This is
accomplished by a type III secretion/translocation machinery. Here, we show that 
the P. aeruginosa type III secretory needle structure is composed essentially of 
PscF, a protein required for secretion and P. aeruginosa cytotoxicity. Partially 
purified needles, detached from the bacterial surface, are 60-80 nm in length and
7 nm in width, resembling needles from Yersinia spp.. YscF of Yersinia
enterocolitica was able to functionally complement the pscF deletion, but
required 11 P. aeruginosa-specific amino acids at the N-terminus for its
function.

PMID: 16239085 [PubMed - indexed for MEDLINE]


193. J Biosci Bioeng. 2003;96(6):529-36.

Cloning and characterization of the constitutively expressed chitinase C gene
from a marine bacterium, Salinivibrio costicola strain 5SM-1.

Aunpad R, Panbangred W.

Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI
Road, Bangkok 10400, Thailand.

The chitinase C gene (chiC) encoding chitinase C (ChiC) from Salinivibrio
costicola 5SM-1 was cloned and the nucleotide sequence was determined. S.
costicola ChiC was expressed constitutively and repressed by glucose. A single
operon composed of two complete open reading frames organized in the order of
chiB, chiC and one partial open reading frame of chiA was found in the same
transcriptional direction. chiC was composed of 2610 bp encoding for 870 amino
acids with a calculated molecular mass of 94 kDa including a signal peptide.
Analysis of the deduced amino acid sequence alignment revealed a domain structure
consisting of an N-terminal catalytic domain, followed by a putative
cadherin-like domain and two type 3 chitin-binding domains located at the C
terminus. Mutation of three highly conserved amino acid residues, two aspartic
acids (Asp-313 and Asp-315) and one glutamic acid (Glu-317) resulted in a
complete loss of chitinase activity against colloidal chitin substrate. This
suggests that these amino acid residues which reside in the putative catalytic
domain play an important role in catalysis. chiB classified as a chitin-binding
protein with C-terminal type 3 chitin-binding domain was composed of 390 amino
acids with the molecular mass of 43 kDa and does not have any detectable
chitinase activity. Chitinase C was identified as an exo-type chitinase releasing
chitobiose as a major product from colloidal chitin hydrolysis.

PMID: 16233569 [PubMed]


194. J Biosci Bioeng. 2000;89(1):107-9.

A universal assay for screening expression libraries for carbohydrases.

Meeuwsen PJ, Vincken JP, Beldman G, Voragen AG.

Food Chemistry Group, Department of Food Technology and Nutritional Sciences,
Wageningen Agricultural University, Bomenweg 2, NL-6703 HD Wageningen, The
Netherlands.

Although many assays are available for the screening of expression libraries for 
carbohydrases, some enzymes cannot be detected because their substrates are
incompatible with the existing assays. One thing that all carbohydrases have in
common is that they increase the number of reducing ends when degrading their
substrates. In this paper we explore the possibility of detecting this increase
with the highly sensitive bicinchoninic acid (BCA) reducing value assay. This
assay can be used for the detection of all carbohydrases degrading any
polysaccharide; enzymes with either an exo- or an endo-type of mechanism can be
detected at the same time. A cDNA library of Aspergillus tubigensis expressed in 
Kluyveromyces lactis clones, was screened with this assay for the presence of
xylogalacturonan degrading enzyme(s). High background absorbances caused by
culture medium, by proteins produced by the clones and by substrate could be
dealt with by using the precautions described in this note. Three
xylogalacturonase producing clones were found using this procedure.

PMID: 16232711 [PubMed]


195. J Biosci Bioeng. 2000;89(1):27-32.

Purification and characterization of a 49-kDa chitinase from Streptomyces griseus
HUT 6037.

Tanabe T, Kawase T, Watanabe T, Uchida Y, Mitsutomi M.

Department of Applied Biological Sciences, Faculty of Agriculture, Saga
University, Saga 840-8502, Japan.

A 49-kDa chitinase (pI7.3) was purified to homogeneity from the culture
supernatant of Streptomyces griseus HUT 6037 by ultrafiltration, DEAE-Sephadex
A-50 and Sephadex G-100 column chromatographies, and chromatofocusing. The
purified enzyme was stable up to 40 degrees C. The N-terminal amino acid sequence
of the enzyme was highly homologous to the N-terminal region of the fibronectin
type III-like domain of S. olivaceoviridis chitinase 01 belonging to family 18
glycosyl hydrolases. The 49-kDa chitinase hydrolyzed partially N-acetylated
chitosan more easily than colloidal chitin. The hydrolyzate of 54% deacetylated
chitosan by the enzyme was separated by CM-Sephadex C-25 column chromatography.
The structures of the oligosaccharides obtained were determined by MALDI-TOF MS
analysis combined with exo-glycosidase digestion. In addition to GlcNAc,
(GlcNAc)2, and (GlcNAc)3, hetero-chitooligosaccharides with GlcNAc at the
reducing end were detected. Thus, the specificity of the enzyme for the
hydrolysis of the beta-1,4-glycosidic linkages in partially N-acetylated chitosan
was similar to that of the family 18 chitinases.

PMID: 16232694 [PubMed]


196. J Biosci Bioeng. 1999;88(2):130-5.

Gene analysis and enzymatic properties of thermostable beta-glycosidase from
Pyrococcus kodakaraensis KOD1.

Ezaki S, Miyaoku K, Nishi K, Tanaka T, Fujiwara S, Takagi M, Atomi H, Imanaka T.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of
Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.

A beta-glycosidase with broad substrate specificity was identified from a
hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding
beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a
polypeptide of 483 amino acids. The protein showed similarity with other
beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed
high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from
Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%).
The cloned gene was expressed in Escherichia coli and the recombinant protein was
purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an
extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90
degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides,
with kcat K(m) values in the order of pNp-beta-glucopyranoside =
pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. 
pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value
[0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside.
This substrate specificity was distinct from previously reported
beta-glycosidases. We observed that the region in PK-Gly corresponding to the
fifth alpha-helix and beta-strand region of beta-glycosidase from S.
solfataricus, which constitutes a large portion of the channel for substrate
incorporation, displayed a chimeric structure, with the N-terminal region similar
to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An
exo-type hydrolytic activity and transglycosylation activity were also observed
towards cellooligomers.

PMID: 16232586 [PubMed]


197. J Biosci Bioeng. 1999;87(4):442-51.

Purification, characterization and gene analysis of exo-cellulase II (Ex-2) from 
the white rot basidiomycete Irpex lacteus.

Hamada N, Ishikawa K, Fuse N, Kodaira R, Shimosaka M, Amano Y, Kanda T, Okazaki
M.

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu
University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.

A new exo-type cellulase, named exo-cellulase II (Ex-2), was purified from the
crude enzyme preparation of Irpex lacteus. Ex-2 was very similar to the
previously characterized exo-cellulase I (Ex-1) with respect to enzymatic
features such as optimal pH, temperature, heat stability, and catalytic activity.
However, Ex-2 exhibited greater pH stability than Ex-1. The molecular mass and
carbohydrate content of Ex-2 (56,000, 4.0%) were different from those of Ex-1
(53,000, 2.0%). A cellulase gene (named cel2) encoding both Ex-2 and Ex-1 was
isolated from an I. lacteus genomic library. The cel2 gene was found to consist
of 1569 bp with an open reading frame encoding 523 amino acids, interrupted by
two introns. The deduced amino acid sequences revealed that cel2 ORF has a
modular structure consisting of a catalytic domain and a fungal-type
cellulose-binding domain (CBD) separated by a serine-rich linker region. The
catalytic domain was homologous to those of fungal cellobiohydrolases belonging
to family 7 of the glycosyl hydrolases. Northern blot analysis showed that
expression of the cel2 gene was induced by various cellulosic substrates and
repressed by glucose, fructose, and lactose.

PMID: 16232497 [PubMed]


198. J Bacteriol. 2005 Oct;187(20):7038-44.

Characterization of a novel glucosamine-6-phosphate deaminase from a
hyperthermophilic archaeon.

Tanaka T, Takahashi F, Fukui T, Fujiwara S, Atomi H, Imanaka T.

Department of Synthetic and Biological Chemistry, Graduate School of Engineering,
Kyoto University, Katsura, Kyoto, Japan.

A key step in amino sugar metabolism is the interconversion between
fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This
conversion is catalyzed in the catabolic and anabolic directions by GlcN6P
deaminase and GlcN6P synthase, respectively, two enzymes that show no
relationship with one another in terms of primary structure. In this study, we
examined the catalytic properties and regulatory features of the glmD gene
product (GlmD(Tk)) present within a chitin degradation gene cluster in the
hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein 
GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal
sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly
exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P.
This enzyme also catalyzed the reverse reaction, the ammonia-dependent
amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity
dependent on glutamine was observed. Kinetic analyses clarified the preference of
this enzyme for the deaminase reaction rather than the reverse one, consistent
with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was 
polycistronically transcribed together with upstream genes encoding an ABC
transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the 
gene cluster, and their expression was induced by the chitin degradation
intermediate, diacetylchitobiose. The results presented here indicate that
GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of
chitin-derived monosaccharides to glycolysis in this hyperthermophile. This
enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally
novel type distinct from any previously known GlcN6P deaminase.

PMCID: PMC1251617
PMID: 16199574 [PubMed - indexed for MEDLINE]


199. Biosci Biotechnol Biochem. 2005 Sep;69(9):1700-5.

Glycoform analysis of Japanese cedar pollen allergen, Cry j 1.

Maeda M, Kamamoto M, Hino K, Yamamoto S, Kimura M, Okano M, Kimura Y.

Division of Biomolecular Science, Graduate School of Natural Science and
Technology, Okayama University, Okayama 700-8530, Japan.

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69,
137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a
epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a
glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1.
Although it has been found that some plant complex type N-glycans are linked to
Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been
proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find
whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the 
cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and
the resulting sugar chains were N-acetylated and then coupled with
2-aminopyridine. Three pyridylaminated sugar chains were purified by
reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by 
a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and
electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly
indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of 
the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.

PMID: 16195587 [PubMed - indexed for MEDLINE]


200. Biochemistry. 2005 Sep 27;44(38):12737-45.

Functional roles of ATP-binding residues in the catalytic site of human
mitochondrial NAD(P)+-dependent malic enzyme.

Hung HC, Chien YC, Hsieh JY, Chang GG, Liu GY.

Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan.
hchung@dragon.nchu.edu.tw

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray
crystal structures have revealed that two ATP molecules occupy both the active
and exo site of the enzyme, suggesting that ATP might act as an allosteric
inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences
indicated that the catalytic activity of the enzyme is inhibited by ATP through a
competitive inhibition mechanism in the active site and not in the exo site.
Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded 
with NAD+ or ATP, are chosen to characterize their possible roles on the
inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate
that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme
activity, and it was only slightly inhibited by ATP and not activated by
fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were
elevated. Elimination of the guanidino side chain of R165 made the enzyme
defective on the binding of NAD+ and ATP, and it caused the charge imbalance in
the active site. These effects possibly caused the enzyme to malfunction on its
catalytic power. The N259A enzyme was less inhibited by ATP but could be fully
activated by fumarate at a similar extent compared with the wild-type enzyme. For
the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated,
indicating that the hydrogen bonding between ATP and the amide side chain of this
residue is important for the binding stability of ATP. Removal of this side chain
did not cause any harmful effect on the fumarate-induced activation of the
enzyme. The E314A enzyme, however, was severely inhibited by ATP and only
slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP)
to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with
those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala
eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and
thus the binding affinities of malate, NAD+, and ATP in the active site of the
enzyme were enhanced.

PMID: 16171388 [PubMed - indexed for MEDLINE]


201. Mutat Res. 2006 Jan 29;593(1-2):187-95. Epub 2005 Sep 8.

Epistatic participation of REV1 and REV3 in the formation of UV-induced
frameshift mutations in cell cycle-arrested yeast cells.

Heidenreich E, Eisler H, Steinboeck F.

Institute of Cancer Research, Department of Medicine I, Medical University of
Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
erich.heidenreich@meduniwein.ac.at

Mutations arising in times of cell cycle arrest may provide a selective advantage
for unicellular organisms adapting to environmental changes. For multicellular
organisms, however, they may pose a serious threat, in that such mutations in
somatic cells contribute to carcinogenesis and ageing. The budding yeast
Saccharomyces cerevisiae presents a convenient model system for studying the
incidence and the mechanisms of stationary-phase mutation in a eukaryotic
organism. Having studied the emergence of frameshift mutants after several days
of starvation-induced cell cycle arrest, we previously reported that all
(potentially error-prone) translesion synthesis (TLS) enzymes identified in S.
cerevisiae did not contribute to the basal level of spontaneous stationary-phase 
mutations. However, we observed that an increased frequency of stationary-phase
frameshift mutations, brought about by a defective nucleotide excision repair
(NER) pathway or by UV irradiation, was dependent on Rev3p, the catalytic subunit
of the TLS polymerase zeta (Pol zeta). Employing the same two conditions, we now 
examined the effect of deletions of the genes coding for polymerase eta (Pol eta)
(RAD30) and Rev1p (REV1). In a NER-deficient strain background, the increased
incidence of stationary-phase mutations was only moderately influenced by a lack 
of Pol eta but completely reduced to wild type level by a knockout of the REV1
gene. UV-induced stationary-phase mutations were abundant in wild type and
rad30Delta strains, but substantially reduced in a rev1Delta as well as a
rev3Delta strain. The similarity of the rev1Delta and the rev3Delta phenotype and
an epistatic relationship evident from experiments with a double-deficient strain
suggests a participation of Rev1p and Rev3p in the same mutagenic pathway. Based 
on these results, we propose that the response of cell cycle-arrested cells to an
excess of exo- or endogenously induced DNA damage includes a novel
replication-independent cooperative function of Rev1p and Pol zeta, which has the
potential to generate mutations.

PMID: 16154164 [PubMed - indexed for MEDLINE]


202. Acta Crystallogr C. 2005 Sep;61(Pt 9):o562-4. Epub 2005 Aug 20.

8-Aza-7-deaza-2'-deoxy-2-(methylsulfanyl)adenosine.

Seela F, Jawalekar AM, Budow S, Eickmeier H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.
frank.seela@uni-osnabrueck.de

In the title compound,
4-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-6-methylsulfanyl-1H-pyrazolo[3,
4-d]pyrimidine, C11H16N5O3S, the conformation of the glycosidic bond is between
anti and high anti. The 2'-deoxyribofuranosyl moiety adopts the C3'-exo-C4'-endo 
conformation (3T4, S-type sugar pucker), and the conformation at the exocyclic
C-C bond is +sc (+gauche). The exocyclic 6-amine group and the 2-methylsulfanyl
group lie on different sides of the heterocyclic ring system. The molecules form 
a three-dimensional hydrogen-bonded network that is stabilized by O-H...N,
N-H...O and C-H...O hydrogen bonds.

PMID: 16143781 [PubMed - indexed for MEDLINE]


203. Insect Biochem Mol Biol. 2005 Oct;35(10):1112-23.

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a
functional exochitinase that plays a role in the chitin degradation during the
molting process.

Daimon T, Katsuma S, Iwanaga M, Kang W, Shimada T.

Department of Agricultural and Environmental Biology, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, 
Tokyo 113-8657, Japan.

The silkworm, Bombyx mori, has been recently demonstrated to contain a
bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized
endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h
showed extensive structural similarities with chitinases from bacteria such as
Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase
genes have not been found from any eukaryotes and viruses except for lepidopteran
insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may
be derived from a bacterial or baculovirus chitinase gene via horizontal gene
transfer. In this report, we investigated the biological function of BmChi-h. Our
enzymological study indicated that a chitinase encoded by BmChi-h has exo-type
substrate preference, which is the same as S. marcescens chiA and v-CHIA, and
different from BmChitinase, which has endo-type substrate preference. An
immunohistochemical study revealed that BmChi-h localizes in the
chitin-containing tissues during the molting stages, indicating that it plays a
role in chitin degradation during molting. These results suggest that BmChi-h
(exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a 
concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that 
bacterial-type chitinase genes are highly conserved among lepidopteran insects,
suggesting that the utilization of a bacterial-type chitinase during the molting 
process may be a general feature of lepidopteran insects.

PMID: 16102417 [PubMed - indexed for MEDLINE]


204. J Am Chem Soc. 2005 Aug 10;127(31):11092-101.

Synthesis of MFe3S4 clusters containing a planar M(II) site (M = Ni, Pd, Pt), a
structural element in the C-cluster of carbon monoxide dehydrogenase.

Panda R, Berlinguette CP, Zhang Y, Holm RH.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
02138, USA.

Synthesis of an analogue of the C-cluster of C. hydrogenoformans carbon monoxide 
dehydrogenase requires formation of a planar Ni(II) site and attachment of an exo
iron atom in the core unit NiFe(4)S(5). The first objective has been achieved by 
two reactions: (i) displacement of Ph(3)P or Bu(t)()NC at tetrahedral Ni(II)
sites of cubane-type [NiFe(3)S(4)](+) clusters with chelating diphosphines, and
(ii) metal atom incorporation into a cuboidal [Fe(3)S(4)](0) cluster with a M(0) 
reactant in the presence of bis(1,2-dimethylphosphino)ethane (dmpe). The isolated
product clusters [(dmpe)MFe(3)S(4)(LS(3))](2-) (M = Ni(II) (9), Pd(II) (12),
Pt(II) (13); LS(3) =
1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-
)) contain the cores [MFe(3)(mu(2)-S)(mu(3)-S)(3)](+) having planar M(II)P(2)S(2)
sites and variable nonbonding M...S distances of 2.6-3.4 A. Reaction (i) involves
a tetrahedral --> planar Ni(II) structural change between isomeric cubane and
cubanoid [NiFe(3)S(4)](+) cores. Based on the magnetic properties of 12 and
earlier considerations, the S = (5)/(2) ground state of the cubanoid cluster
arises from the [Fe(3)S(4)](-) fragment, whereas the S = (3)/(2) ground state of 
the cubane cluster is a consequence of antiferromagnetic coupling between the
spins of Ni(2+) (S = 1) and [Fe(3)S(4)](-). Other substitution reactions of
[NiFe(3)S(4)](+) clusters and 1:3 site-differentiated [Fe(4)S(4)](2+) clusters
are described, as are the structures of 12, 13, [(Me(3)P)NiFe(3)S(4)(LS(3))](2-),
and [Fe(4)S(4)(LS(3))L'](2-) (L' = Me(2)NC(2)H(4)S(-), Ph(2)P(O)C(2)H(4)S(-)).
This work significantly expands our initial report of cluster 9 (Panda et al. J. 
Am. Chem. Soc. 2004, 126, 6448-6459) and further demonstrates that a planar M(II)
site can be stabilized within a cubanoid [NiFe(3)S(4)](+) core.

PMCID: PMC2631389
PMID: 16076217 [PubMed - indexed for MEDLINE]


205. Biosci Biotechnol Biochem. 2005 Jul;69(7):1262-9.

Gene cloning of an endoglucanase from the basidiomycete Irpex lacteus and its
cDNA expression in Saccharomyces cerevisiae.

Toda H, Takada S, Oda M, Amano Y, Kanda T, Okazaki M, Shimosaka M.

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu
University.

A gene (cen1) coding for an endoglucanase I (En-1) was isolated from white rot
fungus Irpex lacteus strain MC-2. The cen1 ORF was comprised of 399 amino acid
residues and interrupted by 14 introns. The deduced amino acid sequence of the
cen1 ORF revealed a multi-domain structure composed of a cellulose-binding
domain, a Ser-/Thr-rich linker, and a catalytic domain from the N-terminus. It
showed a significant similarity to those of other endoglucanases that belong to
family 5 of glycosyl hydrolases. cen1 cDNA was inserted into a yeast expression
vector, YEpFLAG-1, and introduced into Saccharomyces cerevesiae. The resulting S.
cerevisiae transformant secreted a recombinant En-1 that had enzymatic properties
similar to the original En-1. A strong synergistic effect for a degradation of
Avicel and phosphoric acid swollen cellulose was observed when recombinant En-1
was used together with a major exo-type cellobiohydrolase I of I. lacteus MC-2.

PMID: 16041128 [PubMed - indexed for MEDLINE]


206. Anal Biochem. 2005 Jul 15;342(2):246-53.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
protocol for monitoring the progress of enzymatic (13)C/(15)N-labeled DNA
syntheses.

Ambrus A, Chen D, Whatcott C, Somogyi A, Yang D.

Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ
85721, USA. ambrus@pharmacy.arizona.edu

We demonstrate that a simple matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry protocol provides a rapid and
accurate method for monitoring the stage and completeness of enzymatic DNA
syntheses. A crucial step of these syntheses is to quench the reaction at the
desired nucleotide length. This is especially important when expensive, e.g.,
(13)C/(15)N-labeled DNA segments, are synthesized for multinuclear magnetic
resonance purposes to reveal detailed structural information. The analyses of
three templates for a human telomeric 22-mer, a wild type, and a mutant human
c-MYC promoter (18- and 22-mer) DNA and their reactions with the 3'-5' exo(-)
Klenow fragment of DNA polymerase I demonstrate the usefulness of our protocol.
Small amounts of samples (ca. 1-2 microl each) were taken from the reaction
mixtures at different times and analyzed promptly by MALDI-TOF, applying our
successive on-plate desalting method that eliminates the insensitivity of the
MALDI technique at high salt content. The progress of the reaction was detected
by monitoring the relative intensity ratios of ions corresponding to the desired 
products and the primer-template complexes. The effectiveness of NH(3) cleavage
leading to final products was also followed by MALDI-TOF in successful enzymatic 
reactions.

PMID: 15989927 [PubMed - indexed for MEDLINE]


207. Org Lett. 2005 Jul 7;7(14):2985-8.

CO-trapping reaction under thermolysis of alkoxyamines: application to the
synthesis of 3,4-cyclopenta-1-tetralones.

Uenoyama Y, Tsukida M, Doi T, Ryu I, Studer A.

Department of Chemistry, Graduate School of Science, Osaka Prefecture University,
Sakai, Japan.

[reaction: see text] An efficient one-pot sequence comprising a PRE-mediated
radical 5-exo-cyclization, a radical carbonylation, a nitroxide trapping
reaction, and a subsequent acid-catalyzed Friedel-Craft-type acylation provides a
new entry into 3,4-cyclopenta-1-tetralones. Eight examples are presented.

PMID: 15987186 [PubMed]


208. Chemistry. 2005 Aug 19;11(17):4901-11.

2,5-Dioxido-1,4-benzoquinonediimine (H2L2-), a hydrogen-bonding noninnocent
bridging ligand related to aminated topaquinone: different oxidation state
distributions in complexes [{(bpy)2Ru}2(mu-H2L)]n (n=0,+,2+,3+,4+) and
[{(acac)2Ru}2(mu-H2L)]m (m=2-,-,0,+,2+).

Kar S, Sarkar B, Ghumaan S, Janardanan D, van Slageren J, Fiedler J, Puranik VG, 
Sunoj RB, Kaim W, Lahiri GK.

Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai
400076, India.

The symmetrically dinuclear title compounds were isolated as diamagnetic
[(bpy)2Ru(mu-H2L)Ru(bpy)2](ClO4)2 (1-(ClO4)2) and as paramagnetic
[(acac)2Ru(mu-H2L)Ru(acac)2] (2) complexes (bpy=2,2'-bipyridine; acac- =
acetylacetonate = 2,4-pentanedionato; H2L = 2,5-dioxido-1,4-benzoquinonediimine).
The crystal structure of 22 H2O reveals an intricate hydrogen-bonding network:
Two symmetry-related molecules 2 are closely connected through two
NH(H2L2-)O(acac-) interactions, while the oxygen atoms of H2L2- of two such pairs
are bridged by an (H2O)8 cluster at half-occupancy. The cluster consists of
cyclic (H2O)6 arrangements with the remaining two exo-H2O molecules connecting
two opposite sides of the cyclo-(H2O)6 cluster, and oxido oxygen atoms forming
hydrogen bonds with the molecules of 2. Weak antiferromagnetic coupling of the
two ruthenium(III) centers in 2 was established by using SQUID magnetometry and
EPR spectroscopy. Geometry optimization by means of DFT calculations was carried 
out for 1(2+) and 2 in their singlet and triplet ground states, respectively. The
nature of low-energy electronic transitions was explored by using time-dependent 
DFT methods. Five redox states were reversibly accessible for each of the
complexes; all odd-electron intermediates exhibit comproportionation constants
K(c)>10(8). UV-visible-NIR spectroelectrochemistry and EPR spectroscopy of the
electrogenerated paramagnetic intermediates were used to ascertain the
oxidation-state distribution. In general, the complexes 1n+ prefer the
ruthenium(II) configuration with electron transfer occurring largely at the
bridging ligand (mu-H2Ln-), as evident from radical-type EPR spectra for 13+ and 
(+. Higher metal oxidation states (iii, iv) appear to be favored by the complexes
2m; intense long-wavelength absorption bands and RuIII-type EPR signals suggest
mixed-valent dimetal configurations of the paramagnetic intermediates 2+ and 2-.

PMID: 15954152 [PubMed]


209. J Neurochem. 2005 Jul;94(1):276-87.

Proline mutations induce negative-dosage effects on uptake velocity of the
dopamine transporter.

Lin Z, Uhl GR.

Molecular Neurobiology Branch, NIDA-IRP, NIH, Baltimore, MD 21224, USA.
zlin@intra.nida.nih.gov

Ala and Gly substitutions for Pro 101 (P101) located in transmembrane domain 2 of
the dopamine transporter (DAT) abolished transport activity but did not disrupt
plasma membrane expression. Due to the high conservation of P101 in all
neurotransmitter transporters and the capability of Pro to add flexibility to
helices, we hypothesized that P101 contributes to the dynamic feature of
substrate translocation. To test this hypothesis, here we analysed transport
activity for DAT mutants where this Pro was mutated into different amino acids,
including Ser, Val, Leu and Phe. The transmembrane domain 2 helix of P101F,
unlike the other mutants, was computationally predicted to have a Van der Waals
energy threefold higher than the wild-type helix. P101F mutant expression was
consistently disrupted in COS cells. Among all the other mutants that express
normally, P101V, with a side-chain size close to that of Pro, restores the
transport activity of P101A by sevenfold. Most importantly, P101V, P101L and
P101S display negative-dosage effects on dopamine (DA) transport, i.e. the
velocity-concentration curve for DA uptake does not show a plateau with
increasing [DA] but rather peaks and then goes down. These data support the view 
that P101 of DAT plays an essential role in DA translocation.

PMID: 15953370 [PubMed - indexed for MEDLINE]


210. Appl Microbiol Biotechnol. 2005 Jun;67(5):577-91. Epub 2005 Jan 27.

Xylanases from fungi: properties and industrial applications.

Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS.

Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao
Preto-Universidade de Sao Paulo, Av. Bandeirantes, 3900, Bairro Monte Alegre ,
14040-901 Ribeirao Preto, Sao Paulo, Brazil. polizeli@ffclrp.usp.br

Xylan is the principal type of hemicellulose. It is a linear polymer of
beta-D-xylopyranosyl units linked by (1-4) glycosidic bonds. In nature, the
polysaccharide backbone may be added to 4-O-methyl-alpha-D-glucuronopyranosyl
units, acetyl groups, alpha-L-arabinofuranosyl, etc., in variable proportions. An
enzymatic complex is responsible for the hydrolysis of xylan, but the main
enzymes involved are endo-1,4-beta-xylanase and beta-xylosidase. These enzymes
are produced by fungi, bacteria, yeast, marine algae, protozoans, snails,
crustaceans, insect, seeds, etc., but the principal commercial source is
filamentous fungi. Recently, there has been much industrial interest in xylan and
its hydrolytic enzymatic complex, as a supplement in animal feed, for the
manufacture of bread, food and drinks, textiles, bleaching of cellulose pulp,
ethanol and xylitol production. This review describes some properties of xylan
and its metabolism, as well as the biochemical properties of xylanases and their 
commercial applications.

PMID: 15944805 [PubMed - indexed for MEDLINE]


211. Appl Microbiol Biotechnol. 2005 Oct;68(6):757-65. Epub 2005 Oct 13.

alpha-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for
the biosynthesis of complex carbohydrates.

Hung VS, Hatada Y, Goda S, Lu J, Hidaka Y, Li Z, Akita M, Ohta Y, Watanabe K,
Matsui H, Ito S, Horikoshi K.

Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, 
Kanagawa, 237-0061, Japan. vohungsi@jamstec.go.jp

An alpha-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea 
bacteria isolated from the sediment of the Mariana Trench, was purified to
homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion
strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo-
and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic
activity at 60 degrees C and pH 9.0 in 15 mM glycine-NaOH buffer. The enzyme
exclusively hydrolyzed alpha-1,4-glycosidic linkages of oligosaccharides in an
exo-type manner. The enzyme had an overwhelming transglycosylation activity and
glycosylated various non-sugar molecules when maltose was used as a sugar donor. 
It converted maltose to isomaltose. The gene encoding the enzyme was cloned and
sequenced. The recombinant enzyme could be extracellularly overproduced by
Bacillus subtilis harboring its gene and preserved the primary properties of the 
native enzyme. Site-directed mutagenesis experiments showed that Asp98 is
essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.

PMID: 15940457 [PubMed - indexed for MEDLINE]


212. J Pharmacol Exp Ther. 2005 Aug;314(2):575-83. Epub 2005 May 5.

Recognition of benztropine by the dopamine transporter (DAT) differs from that of
the classical dopamine uptake inhibitors cocaine, methylphenidate, and mazindol
as a function of a DAT transmembrane 1 aspartic acid residue.

Ukairo OT, Bondi CD, Newman AH, Kulkarni SS, Kozikowski AP, Pan S, Surratt CK.

Division of Pharmaceutical Sciences, Duquesne University, Mellon Hall, Room 453, 
600 Forbes Ave., Pittsburgh, PA 15282, USA.

Binding of cocaine to the dopamine transporter (DAT) protein blocks synaptic
dopamine clearance, triggering the psychoactive effects associated with the drug;
the discrete drug-protein interactions, however, remain poorly understood. A
longstanding postulate holds that cocaine inhibits DAT-mediated dopamine
transport via competition with dopamine for formation of an ionic bond with the
DAT transmembrane aspartic acid residue D79. In the present study, DAT mutations 
of this residue were generated and assayed for translocation of radiolabeled
dopamine and binding of radiolabeled DAT inhibitors under identical conditions.
When feasible, dopamine uptake inhibition potency and apparent binding affinity
K(i) values were determined for structurally diverse DAT inhibitors. The glutamic
acid substitution mutant (D79E) displayed values indistinguishable from wild-type
DAT in both assays for the charge-neutral cocaine analog 8-oxa-norcocaine, a
finding not supportive of the D79 "salt bridge" ligand-docking model. In
addressing whether the D79 side chain contributes to the DAT binding sites of
other portions of the cocaine pharmacophore, only inhibitors with modifications
of the tropane ring C-3 substituent, i.e., benztropine and its analogs, displayed
a substantially altered dopamine uptake inhibition potency as a function of the
D79E mutation. A single conservative amino acid substitution thus differentiated 
structural requirements for benztropine function relative to those for all other 
classical DAT inhibitors. Distinguishing the precise mechanism of action of this 
DAT inhibitor with relatively low abuse liability from that of cocaine may be
attainable using DAT mutagenesis and other structure-function studies, opening
the door to rational design of therapeutic agents for cocaine abuse.

PMID: 15879005 [PubMed - indexed for MEDLINE]


213. Inorg Chem. 2005 May 16;44(10):3746-54.

Structural increments for 11-vertex nido-phospha- and aza(carba)boranes and
-borates; dependence of energy penalties on the extent of Electron Localization.

Kiani FA, Hofmann M.

Anorganisch-Chemisches Institut, Ruprecht-Karls-Universitat Heidelberg, Germany.

Relevant structural features and corresponding energy penalties were determined
that allow to easily estimate the relative stabilities of 11-vertex nido-phospha-
and aza-substituted boranes, borates, carbaboranes, and carbaborates. For this
purpose, density functional theory computations at the
B3LYP/6-311+G(d,p)//B3LYP/6-31G(d)+ZPE level were carried out to determine the
relative energies of 95 phospha- and 46 aza(carba)boranes and -borates. Energy
penalties assigned to disfavoring structural features show additive behavior and 
excellent precision with respect to the computed results, as in the case of 6-
and 11-vertex nido-carboranes and -borates. An unsubstituted phosphorus atom was 
found to possess energy penalties quite similar to those of the
three-electron-donating H-C group. A bare nitrogen atom has energy penalties much
larger than those of a bare phosphorus atom. Four-electron-donating RP and RN
moieties, however, have even more adverse energy penalties. The disfavoring
effects of heteroatoms in a borane cluster are determined by the amount of
electron localization, that is, primarily by the number of skeletal electrons
that formally originate from the heterogroup and secondarily by the
electronegativity. Heteroatom energy penalties are independent of the type of the
other heteroatoms present in the same cluster. Some novel phospha(carba)borane
geometries with bare and exo-substituted phosphorus atoms in the same cluster
have favorable thermodynamic stabilities competitive with those of known isomers.

PMID: 15877459 [PubMed]


214. J Ind Microbiol Biotechnol. 2005 Apr;32(4):125-34. Epub 2005 Apr 22.

Comparison of wild-type and UV-mutant beta-glucanase-producing strains of
Talaromyces emersonii with potential in brewing applications.

McCarthy TC, Lalor E, Hanniffy O, Savage AV, Tuohy MG.

Department of Biochemistry, National University of Ireland, Galway, Ireland.

A screen of 46 UV-mutant strains of the moderately thermophilic fungus
Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross
morphological differences to the parent strain and enhanced activity against
mixed linkage cereal beta-glucans. Activity against beta-(1, 3)(1, 4)-D: -glucan 
from barley (BBGase) was measured during growth of the mutant and wild-type
strains on a variety of carbon sources, ranging from solka floc to crude cereal
fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase
than the parent strain and markedly less beta-glucosidase (exo-activity); enzyme 
levels were dependent on the carbon source. Cellulose induced high BBGase.
However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective
inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved 
similar end-points during the hydrolysis of commercial barley beta-glucan
(13.0-16.9%), but were more active against crude beta-glucan from barley
(16.0-24.2% hydrolysis). The products of hydrolysis were quantified by
high-performance anion-exchange chromatography. Mash trials indicated that enzyme
preparations from all three organisms effected a significant reduction in wort
viscosity and residual mash beta-glucan. Finally, TC2 and TC5 produce more
efficient beta-glucan-depolymerizing enzymes; and wheat bran and solka floc can
be used to provide inexpensive and potent enzyme cocktails with potential in
brewing applications.

PMID: 15856354 [PubMed - indexed for MEDLINE]


215. J Am Chem Soc. 2005 May 4;127(17):6178-9.

Intramolecular [4 + 2] cycloadditions of 1,3-enynes or arylalkynes with alkenes
with highly reactive cationic phosphine Au(I) complexes.

Nieto-Oberhuber C, Lopez S, Echavarren AM.

Institute of Chemical Research of Catalonia (ICIQ), 43007 Tarragona, Spain.

New Au(I) complexes with bulky, biphenyl phosphines are the most reactive
catalysts for the cyclizations of enynes. 1,6-Enynes with an aryl ring at the
alkyne give 2,3,9,9a-tetrahydro-1H-cyclopenta[b]naphthalenes by a 5-exo-dig
cyclization followed by a Nazarov-type ring expansion. 1,8-Dien-3-ynes also
cyclize by a 5-exo-dig pathway to form hydrindanes.

PMID: 15853316 [PubMed]


216. Archaea. 2004 Oct;1(4):285-92.

Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the
thermophile Pyrococcus furiosus.

van Lieshout J, Faijes M, Nieto J, van der Oost J, Planas A.

Laboratory for Microbiology, Department of Agrotechnology and Food Sciences,
Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT, Wageningen, The
Netherlands. johan.vanlieshout@wur.nl

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that
hydrolyzes beta-1,3-glucooligosaccharides, but not
beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small
saccharides by using 4-methylumbelliferyl beta-glucosides with different
linkages. Besides endo-activity, wild-type LamA has some exo-activity, and
catalyzes the hydrolysis of mixed-linked oligosaccharides
(Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with
both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely
reduced hydrolytic activity, which is consistent with Glu170 being the catalytic 
nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the
condensation of alpha-laminaribiosyl fluoride to different acceptors. The best
condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed
30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a
beta-1,4 linkage.

PMCID: PMC2685573
PMID: 15810439 [PubMed - indexed for MEDLINE]


217. Mol Microbiol. 2005 Mar;55(6):1867-82.

A new in vivo termination function for DNA polymerase I of Escherichia coli K12.

Markovitz A.

Department of Biochemistry and Molecular Biology, University of Chicago, Chicago,
IL 60637, USA. amarkovi@uchicago.edu

Escherichia coli deleted for the tus gene are viable. Thus there must be at least
one other mechanism for terminating chromosome synthesis. The tus deletion strain
yielded a small fraction of cells that overproduce DNA, as determined by flow
cytometry after run-out chromosome replication in the presence of rifampicin and 
cephalexin. A plasmid, paraBAD tus+, prevented the excess DNA replication only
when arabinose was added to the medium to induce the synthesis of the Tus
protein. Transduction studies were done to test whether or not additional
chromosomal deletions could enhance the excess chromosome replication in the tus 
deletion strain. A strain containing a second deletion in metE udp overproduced
DNA at a high level during run-out replication. Further studies demonstrated that
a spontaneous unknown mutation had occurred during the transduction. This
mutation was mapped and sequenced. It is polA(G544D). Transduction of polA(G544D)
alone into the tus deletion strain produced the high DNA overproduction
phenotype. The polA(G544D) and six other polA alleles were then tested in
wild-type and in tus deletion backgrounds. The two alleles with low levels of
5'-->3' exonuclease (exo) overproduced DNA while those with either high or normal
exo overproduce much less DNA in run-out assays in the wild-type background. In
contrast, all seven mutant polA alleles caused the high DNA overproduction
phenotype in a tus deletion background. To explain these results we propose a new
in vivo function for wild-type DNA polymerase I in chromosome termination at
site(s) not yet identified.

PMID: 15752206 [PubMed - indexed for MEDLINE]


218. Biochemistry. 2005 Mar 8;44(9):3338-46.

Base selectivity is impaired by mutants that perturb hydrogen bonding networks in
the RB69 DNA polymerase active site.

Yang G, Wang J, Konigsberg W.

Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar
Street, New Haven, Connecticut 06520, USA.

To investigate the molecular basis for the selective utilization of nucleoside
triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69
pol), we constructed a set of mutants that we predicted would perturb the "floor"
of the nascent base-pairing interface in the enzyme. We then determined the
pre-steady-state kinetic parameters for the incorporation of complementary and
noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found
that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C 
versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G
versus G incorporation with the Y567A mutant was 10 times higher than the
k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The
reduced level of discrimination by the Y567A mutant against incorporation of
mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence
was also employed to monitor rates of putative conformational changes with the
exo(-) RB69 pol and its mutants using a primer-template complex containing
2-aminopurine. The rates of fluorescence changes were equal to or greater than
the rates of the rapid chemical quench, indicating that we were monitoring a
process occurring before or during the phosphoryl transfer reaction. We have
interpreted our results within the context of the crystal structure of the RB69
pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].

PMID: 15736944 [PubMed - indexed for MEDLINE]


219. Diabetes. 2005 Mar;54(3):736-43.

Regulated exocytosis and kiss-and-run of synaptic-like microvesicles in INS-1 and
primary rat beta-cells.

MacDonald PE, Obermuller S, Vikman J, Galvanovskis J, Rorsman P, Eliasson L.

Section of Diabetes, Metabolism and Endocrinology, Lund University, Lund, Sweden.
patrick.macdonald@mphy.lu.se

We have applied cell-attached capacitance measurements to investigate whether
synaptic-like microvesicles (SLMVs) undergo regulated exocytosis in insulinoma
and primary pancreatic beta-cells. SLMV and large dense-core vesicle (LDCV)
exocytosis was increased 1.6- and 2.4-fold upon stimulation with 10 mmol/l
glucose in INS-1 cells. Exocytosis of both types of vesicles was coupled to
Ca(2+) entry through l-type channels. Thirty percent of SLMV exocytosis in INS-1 
and rat beta-cells was associated with transient capacitance increases consistent
with kiss-and-run. Elevation of intracellular cAMP (5 micromol/l forskolin)
increased SLMV exocytosis 1.6-fold and lengthened the duration of kiss-and-run
events in rat beta-cells. Experiments using isolated inside-out patches of INS-1 
cells revealed that the readily releasable pool (RRP) of SLMVs preferentially
undergoes kiss-and-run exocytosis (67%), is proportionally larger than the LDCV
RRP, and is depleted more quickly upon Ca(2+) stimulation. We conclude that SLMVs
undergo glucose-regulated exocytosis and are capable of high turnover. Following 
kiss-and-run exocytosis, the SLMV RRP may be reloaded with gamma-aminobutyric
acid and undergo several cycles of exo- and endocytosis. Our observations support
a role for beta-cell SLMVs in a synaptic-like function of rapid intra-islet
signaling.

PMID: 15734850 [PubMed - indexed for MEDLINE]


220. Carbohydr Res. 2005 Mar 21;340(4):637-44.

An original chemoenzymatic route for the synthesis of beta-D-galactofuranosides
using an alpha-L-arabinofuranosidase.

Remond C, Plantier-Royon R, Aubry N, O'Donohue MJ.

Laboratoire de Technologie Enzymatique et Physico-chimie des Agroressources, UMR 
URCA/INRA FARE, 8 rue Gabriel Voisin, BP 316, F-51688 Reims, France.

DGalactofuranose is a widespread component of cell wall polysaccharides in
bacteria, protozoa and fungi, but is totally absent in mammals. Importantly,
galactofuranose is a key constituent of major cell envelope polysaccharides in
pathogenic mycobacteria. In this respect, galactofuranose-based glycoconjugates
are interesting target molecules for drug design. O-Glycosidases and notably
beta-D-galactofuranosidases could be useful tools for the chemoenzymatic
synthesis of galactofuranosides, but to date no studies of this type have been
reported. Here we report the use of a GH 51 alpha-l-arabinofuranosidase for the
synthesis of beta-D-galactofuranosides. We have demonstrated that this enzyme can
catalyse both the autocondensation of p-nitrophenyl-beta-D-galactofuranoside and 
the transgalactofuranosylation of benzyl alpha-D-xylopyranoside, forming
p-nitrophenyl beta-D-galactofuranosyl-(1-->2)-beta-D-galactofuranoside and benzyl
beta-D-galactofuranosyl-(1-->2)-alpha-D-xylopyranoside, respectively. Both
reactions were very regiospecific and the reaction involving benzyl
alpha-D-xylopyranoside afforded very high yields (74.8%) of the major product. To
our knowledge, this demonstration of chemoenzymatic synthesis of
galactofuranosides constitutes the very first use of an O-glycosidase for the
synthesis of galactofuranosides.

PMID: 15721334 [PubMed - indexed for MEDLINE]


221. J Biol Chem. 2005 Apr 29;280(17):17180-6. Epub 2005 Feb 17.

Structural basis for the specificity of the reducing end xylose-releasing
exo-oligoxylanase from Bacillus halodurans C-125.

Fushinobu S, Hidaka M, Honda Y, Wakagi T, Shoun H, Kitaoka M.

Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan. asfushi@mail.ecc.u-tokyo.ac.jp

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125
(Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater
than or equal to 3, releasing the xylose unit at the reducing end. It is a unique
exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end 
in a very strict manner, even discriminating the beta-anomeric hydroxyl
configuration from the alpha-anomer or 1-deoxyxylose. We have determined the
crystal structures of Rex in unliganded and complex forms at 1.35-2.20-A
resolution and revealed the structural aspects of its three subsites ranging from
-2 to +1. The structure of Rex was compared with those of endo-type enzymes in
glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is
basically conserved with other GH-8a enzymes. However, subsite +2 is blocked by a
barrier formed by a kink in the loop before helix alpha10. His-319 in this loop
forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite +1,
contributing to the specific recognition of anomers at the reducing end.

PMID: 15718242 [PubMed - indexed for MEDLINE]


222. Langmuir. 2005 Feb 15;21(4):1412-5.

Diels-Alder chemistry on alkene functionalized films.

Roucoules V, Fail CA, Schofield WC, Teare DO, Badyal JP.

Department of Chemistry, Science Laboratories, Durham University, Durham DH1 3LE,
England.

A substrate-independent method has been devised for ring formation at solid
surfaces. This entails the aminolysis reaction of allylamine with maleic
anhydride pulsed plasma polymer films to yield terminal alkene groups at the
surface. Subsequent exposure to 1,3-cyclohexadiene leads to a Diels-Alder type (4
+ 2) cycloaddition reaction to give a mixture of endo- and
exo-bicyclo[2.2.2]oct-2-ene rings.

PMID: 15697288 [PubMed]


223. Biosci Biotechnol Biochem. 2005 Jan;69(1):45-50.

Mode of action of cellulases on dyed cotton with a reactive dye.

Yamada M, Amano Y, Horikawa E, Nozaki K, Kanda T.

Techno Center, Tokai Senko K.K., Aichi, Japan.

Cotton woven fabrics which were previously dyed with a reactive dye were treated 
with a commercial cellulase preparation. Dyeing with a reactive dye for cotton
apparently inhibited the weight loss activity and saccharification activity of
cellulase. In addition, dyed cotton was treated with highly purified cellulases
which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase
II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type
cellulases were inhibited more than endo-type cellulase by dyeing in the case of 
saccharification activity. CBH I was severely inhibited by dyeing as compared
with CBH II or EG II from the viewpoint of morphological changes in the fiber
surface. Dyes on the cellulose substrates severely influenced CBH I in spite of
the rare modification, because CBH I hydrolyzed cellulose with true-processive
action. The change in the activity of each cellulase component on dyed cotton can
affect the synergistic action of cellulases.

PMID: 15665466 [PubMed - indexed for MEDLINE]


224. Biochemistry. 2005 Jan 11;44(1):387-97.

Biochemical characterization and identification of the catalytic residues of a
family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6.

Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G,
Schomburg D, Baasov T, Shoham Y.

Department of Biotechnology and Food Engineering, Technion-Israel Institute of
Technology, Haifa 32000, Israel.

Beta-D-xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides 
into xylose units. Here, we describe the characterization and kinetic analysis of
a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3).
Enzymes in this family use an inverting single-displacement mechanism with two
conserved carboxylic acids, a general acid, and a general base. XynB3 was most
active at 65 degrees C and pH 6.5, with clear preference to xylose-based
substrates. Products analysis indicated that XynB3 is an exoglycosidase that
cleaves single xylose units from the nonreducing end of xylooligomers. On the
basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as
the general-base and general-acid catalytic residues, respectively. Kinetic
analysis with substrates bearing different leaving groups showed that, for the
wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected
by the leaving-group reactivity, whereas for the E187G mutant, both values
exhibited significantly greater dependency on the pK(a) of the leaving group. The
pH-dependence activity profile of the putative general-acid mutant (E187G)
revealed that the protonated catalytic residue was removed. Addition of the
exogenous nucleophile azide did not affect the activities of the wild type or the
E187G mutant but rescued the activity of the D15G mutant. On the basis of
thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was 
the only product of the accelerated reaction, suggesting that the azide ion does 
not attack the anomeric carbon directly but presumably activates a water
molecule. Together, these results confirm the suggested catalytic role of Glu187 
and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact
roles of the catalytic residues in an inverting GH43 glycosidase.

PMID: 15628881 [PubMed - indexed for MEDLINE]


225. BMC Genet. 2004 Dec 23;5(1):34.

The exceptionally high rate of spontaneous mutations in the polymerase delta
proofreading exonuclease-deficient Saccharomyces cerevisiae strain starved for
adenine.

Achilli A, Matmati N, Casalone E, Morpurgo G, Lucaccioni A, Pavlov YI, Babudri N.

Dipartimento di Genetica e Microbiologia, Universita di Pavia, Pavia, Italy.
achilli@ipvgen.unipv.it <achilli@ipvgen.unipv.it>

BACKGROUND: Mutagenesis induced in the yeast Saccharomyces cerevisiae by
starvation for nutrilites is a well-documented phenomenon of an unknown
mechanism. We have previously shown that the polymerase delta proofreading
activity controls spontaneous mutagenesis in cells starved for histidine. To
obtain further information, we compared the effect of adenine starvation on
mutagenesis in wild-type cells and, in cells lacking the proofreading activity of
polymerase delta (phenotype Exo-, mutation pol3-01). RESULTS: Ade+ revertants
accumulated at a very high rate on adenine-free plates so that their frequency on
day 16 after plating was 1.5 x 10(-4) for wild-type and 1.0 x 10(-2) for the Exo-
strain. In the Exo- strain, all revertants arising under adenine starvation are
suppressors of the original mutation, most possessed additional nutritional
requirements, and 50% of them were temperature sensitive. CONCLUSIONS: Adenine
starvation is highly mutagenic in yeast. The deficiency in the polymerase delta
proofreading activity in strains with the pol3-01 mutation leads to a further
66-fold increase of the rate of mutations. Our data suggest that adenine
starvation induces genome-wide hyper-mutagenesis in the Exo- strain.

PMCID: PMC544876
PMID: 15617571 [PubMed - indexed for MEDLINE]


226. J Biol Chem. 2005 Feb 25;280(8):6669-75. Epub 2004 Dec 21.

Molecular basis of exopeptidase activity in the C-terminal domain of human
angiotensin I-converting enzyme: insights into the origins of its exopeptidase
activity.

Naqvi N, Liu K, Graham RM, Husain A.

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales 2010,
Australia.

Proteolytic processing is a primary means of biological control. Exopeptidases
use terminal anchoring interactions to restrict cleavage at peptide substrate N
or C termini. In contrast, internal peptide bond targeting by endopeptidases is
through context-driven recognition. Angiotensin I-converting enzyme (ACE), a zinc
metalloproteinase, has tandem duplicate catalytic domains, N- and C-terminal,
each of which is a dual specificity enzyme with exo- and endocarboxypeptidase
activities. The mechanisms by which ACE evolved from its endopeptidase ancestors 
as a dual specificity enzyme have not been defined. Based on kinetic studies of
wild-type and mutant forms of the C-terminal catalytic domain of human ACE and of
the ACE substrates angiotensin I, substance P, and bradykinin, as well as
considerations of the ACE x-ray structure, we provide evidence that the
acquisition of its exopeptidase activity is due to novel evolutionary
specializations. These involve not only interactions between the S(2)' subsite
cognate for the C-terminal substrate P(2)' side chain, acting in concert with
carboxylate-docking interactions with Lys(1087) and Tyr(1096), but also
electrostatic selection against a cationic C-terminal substrate carboxylate. With
a blocked C terminus, substrate side chain interactions are dominant in cleavage 
site selection. In the evolution of obligate exopeptidases from endopeptidase
ancestors, mutations that destroy context-driven peptide bond targeting are
likely to have followed the acquisition of terminal docking interactions.
Evolutionary intermediates between endopeptidases and obligate exopeptidases
could therefore have been dual specificity proteinases like ACE.

PMID: 15615692 [PubMed - indexed for MEDLINE]


227. J Biochem Mol Biol. 2004 Nov 30;37(6):642-7.

Chemical modification of lysine residues in Bacillus licheniformis alpha-amylase:
conversion of an endo- to an exo-type enzyme.

Habibi AE, Khajeh K, Nemat-Gorgani M.

Institute of Biochemistry and Biophysics, University of Tehran. P. O. Box:
13145-1384, Tehran, Iran.

The lysine residues of Bacillus licheniformis alpha-amylase (BLA) were chemically
modified using citraconic anhydride or succinic anhydride. Modification caused
fundamental changes in the enzymes specificity, as indicated by a dramatic
increase in maltosidase and a reduction in amylase activity. These changes in
substrate specificity were found to coincide with a change in the cleavage
pattern of the substrates and with a conversion of the native endo- form of the
enzyme to a modified exo- form. Progressive increases in the productions of
rho-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble
starch, respectively, was used as substrate, were observed upon modification. The
described changes were affected by the size of incorporated modified reagent:
citraconic anhydride was more effective than succinic anhydride. Reasons for the 
observed changes are discussed and reasons for the effectivenesses of chemical
modifications for tailoring enzyme specificities are suggested.

PMID: 15607021 [PubMed - indexed for MEDLINE]


228. Microbiology. 2004 Dec;150(Pt 12):4115-23.

Characterization of Bacillus subtilis gamma-glutamyltransferase and its
involvement in the degradation of capsule poly-gamma-glutamate.

Kimura K, Tran LS, Uchida I, Itoh Y.

Division of Applied Microbiology, National Food Research Institute, Kannondai
2-1-12, Tsukuba, Ibaraki 305-8642, Japan.

During early stationary phase, Bacillus subtilis NAFM5 produces capsular
poly(gamma-glutamic acid) (gammaPGA, 2x10(6) Da), which contains D- and
L-glutamate, and then degrades it during late stationary phase. The
gamma-glutamyltransferase (EC 2.3.2.2; GGT) of this strain successively
hydrolysed gammaPGA from the amino-terminal end, to yield both D- and
L-glutamate. This enzyme was specifically synthesized during the stationary phase
through transcriptional activation of the corresponding ggt gene by the ComQXPA
quorum-sensing system. A ggt knockout mutant degraded gammaPGA into 1x10(5) Da
fragments, but not any further, indicating that the capsule gammaPGA is first
internally degraded by an endo-type of gammaPGA hydrolase into 1x10(5) Da
intermediates, then externally into glutamates via GGT. Due to its inability to
generate the glutamates from the capsule, the ggt mutant sporulated more
frequently than the wild-type strain. The results show that B. subtilis GGT has a
powerful exo-gamma-glutamyl hydrolase activity that participates in capsule
gammaPGA degradation to supply stationary-phase cells with constituent
glutamates.

PMID: 15583164 [PubMed - indexed for MEDLINE]


229. Brain Res Brain Res Rev. 2004 Dec;47(1-3):18-32.

Synaptic vesicle pools and plasticity of synaptic transmission at the Drosophila 
synapse.

Kidokoro Y, Kuromi H, Delgado R, Maureira C, Oliva C, Labarca P.

Institute for Behavioral Sciences, Gunma University of School of Medicine,
3-39-22 Showa-machi, Maebashi 371-8511, Japan. kidokoro@med.gunma-u.ac.jp

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus,
this synapse offers an excellent model for studies of the molecular mechanism of 
synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools
have been identified and characterized using a fluorescent styryl dye, FM1-43, to
stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to 
the readily releasable pool (RRP) defined electrophysiologically, and the reserve
pool (RP). These two pools were identified first in a temperature-sensitive
paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The
ECP participates in synaptic transmission during low frequency firing of
presynaptic nerves and locates in the periphery of presynaptic boutons in the
vicinity of release sites, while SVs in the RP spread toward the center of
boutons and are recruited only during tetanic stimulation. These two pools are
separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs 
from the RP to the ECP. Activation of presynaptic metabotropic glutamate
receptors recruits SVs from the RP and enhances SV release by elevation of the
cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and
rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to
the ECP and have limited short-term synaptic plasticity. SV mobilization between 
the two pools could be a key step for changes in synaptic efficacy. Since a
variety of mutants that have distinct defects in synaptic transmission are
available for detailed studies of synaptic function, this direction of approach
in Drosophila seems promising.

PMID: 15572160 [PubMed - indexed for MEDLINE]


230. Biochemistry. 2004 Dec 7;43(48):15217-22.

In vitro replication and repair of DNA containing a C2'-oxidized abasic site.

Greenberg MM, Weledji YN, Kroeger KM, Kim J.

Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street,
Baltimore, Maryland 21218, USA. mgreenberg@jhu.edu

Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides.
Nonetheless, polymerase and repair enzymes distinguish between various oxidized
abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion
is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases
and repair enzymes are unknown. A recently reported method for the chemical
synthesis of oligonucleotides containing C2-AP at a defined site was utilized for
studying the activity of Klenow exo(-) and repair enzymes on templates containing
the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and
C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but
extension of the primer past the lesion is strongly blocked. C2-AP is incised
less efficiently by exonuclease III and endonuclease IV than are other abasic
lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III
can be chemically trapped, the location of the 3'-phosphate alpha with respect to
the aldehyde prevents beta-elimination associated with the lyase activity of type
I base excision repair enzymes. The interactions of the C2'-oxidized abasic site 
with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic
and that it will be removed by strand displacement synthesis and flap
endonuclease processing via a long patch repair mechanism.

PMID: 15568814 [PubMed - indexed for MEDLINE]


231. Chem Commun (Camb). 2004 Dec 7;(23):2637-49. Epub 2004 Nov 4.

Dendrimers and gold nanoparticles as exo-receptors sensing biologically important
anions.

Astruc D, Daniel MC, Ruiz J.

Nanosciences and Catalysis Group, LCOO, UMR CNRS No. 5802, Universite Bordeaux I,
351 Cours de la Liberation, 33405 Talence Cedex, France.

Dendrimers, alkylthiol-gold nanoparticles and gold-nanoparticle-cored dendrimers 
containing tethers terminated by a redox group (typically an iron sandwich)
attached to a hydrogen-bonding group (amido, amino, silyl) are selective and
efficient exo-receptors for the recognition, sensing and titration of oxo-anions,
including ATP(2-), or halogens, mostly using cyclic voltammetry. Various positive
dendritic effects were disclosed (in contrast to catalysis), and large
gold-nanoparticle-cored redox dendrimers of this type that contain several
hundred equivalent ferrocenyl groups readily adsorb on Pt electrodes, providing
useful regenerable electrochemical sensors.

PMID: 15568050 [PubMed - indexed for MEDLINE]


232. Inorg Chem. 2004 Nov 29;43(24):7774-83.

Complexes having the fac-[M(CO)3]+ core (M=Tc, Re) useful in
radiopharmaceuticals: X-ray and NMR structural characterization and density
functional calculations of species containing two sp3 N donors and one sp3 O
donor.

Lipowska M, Cini R, Tamasi G, Xu X, Taylor AT, Marzilli LG.

Department of Radiology, Emory University, Atlanta, Georgia 30322, USA.

Radiopharmaceuticals containing the "fac-[M(CO)3]+ " core (M=99mTc, 186Re, or
188Re) have potential as diagnostic or therapeutic agents. Complexes with this
core with sp3 amine donors have received little attention. We have studied
adducts formed by ENDACH2 (HO2CCH2NHCH2CH2NHCH2CO2H) and ENACH
(NH2CH2CH2NHCH2CO2H). Re(CO)3(ENDACH)-A (1A) and Re(CO)3(ENDACH)-B (1B) isomers
were obtained by the reaction of ENDACH2 with Re(CO)5Cl. Re(CO)3(ENAC) (2) was
obtained by the reaction of ENACH with aqueous [Re(CO)3(H2O)3]+. From
single-crystal X-ray data, the three new neutral complexes, 1A, 1B, and 2, have a
six-coordinate, pseudo-octahedral Re center with facially coordinated carbonyl
ligands. ENDACH- and ENAC- bind facially to Re through both amine nitrogens and
one carboxyl oxygen, forming two five-membered chelate rings. The Re(CO)3(ENDACH)
isomers have an uncoordinated, dangling -CH2CO2H group, which is an ideal
coupling site for attachment to biomolecules. The isomers differ by the
configuration of the NH center bearing this dangling group. The H atom of the
amine (N2) is endo (near the carbonyl ligands in the basal plane) in 1A and exo
(away from carbonyl ligands) in isomer 1B. Isomers reach equilibrium (1A:1B,
70:30) after 3 days at high pH. Density functional structure optimizations were
performed for isolated molecules of the type Tc(I)/Re(I)(CO)3(N2O):
[Re(CO)3(NH3)2(H2O)]+, [Tc(CO)3(NH3)2(H2O)]+, [Re(CO)3(EN)(H2O)]+ (EN,
ethylenediamine), [Tc(CO)3(EN)(H2O)]+, and various models for 1A, 1B, and 2. The 
computed structures are in good agreement with the X-ray structures. The
theoretical and experimental Re-N bond distances usually agree within 0.045 A.
The total electronic energy values for the computed 1A and 1B models differ by
0.815 kcal mol(-1), giving an isomer ratio of 80:20, in good agreement with the
1A/1B ratio (70:30) found.

PMID: 15554642 [PubMed - indexed for MEDLINE]


233. Eur J Neurosci. 2004 Nov;20(10):2591-7.

Identified spinal motoneurons of young rats possess nicotinic acetylcholine
receptors of the heteromeric family.

Ogier R, Liu X, Tribollet E, Bertrand D, Raggenbass M.

Department of Physiology, University Medical Center, 1, rue Michel-Servet,
CH-1211 Geneva 4, Switzerland.

The aim of the present study was to determine whether, in young rats, spinal
motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons
were identified either by retrograde labelling or by choline acetyltransferase
immunohistochemistry. Whole-cell recordings were performed in spinal cord slices 
cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly
activating inward current. In current clamp, it depolarized the motoneuron
membrane and induced action potential firing. The acetylcholine-evoked current
was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad
spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine,
a nicotinic antagonist selective for receptors containing the alpha7 subunit.
Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific
agonist, was without effect. In young animals, light-microscopic autoradiography 
showed that in the central grey matter all laminae were intensely and equally
labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also
found in all laminae, with slightly lower levels in the superficial layers of the
dorsal horns and in the ventral part of the grey matter. In adults, the density
of [3H]epibatidine binding sites was much lower in the entire grey matter, except
in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were
present only in some selected areas. Our data indicate that spinal motoneurons
possess functional nicotinic receptors of the heteromeric type and suggest that
nicotinic cholinergic transmission may play a significant role in the developing 
spinal cord.

PMID: 15548202 [PubMed - indexed for MEDLINE]


234. Biochim Biophys Acta. 2004 Nov 1;1674(3):268-81.

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

Grishutin SG, Gusakov AV, Markov AV, Ustinov BB, Semenova MV, Sinitsyn AP.

Department of Chemistry, M.V. Lomonosov Moscow State University, Vorobyevy Gory, 
Moscow 119899, Russia.

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 
kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei
(75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their
high specific activity toward tamarind xyloglucan, whereas the activity against
carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low.
Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. 
reesei XG represents Cel74A, whose gene has been discovered recently (GenBank
accession no. AY281371 ), but the enzyme has not been characterized and described
elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases
did not show any identity to those from known glycoside hydrolases. All enzymes
produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of
xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the
polymeric substrate, while the mode of action of two other xyloglucanases was
similar to the exo-type, when oligosaccharides containing four glucose residues
in the main chain were split off the ends of xyloglucan molecules. These results 
together with growing literature data allow concluding that specific
xyloglucanases may represent a new class of glycoside hydrolases, which are
different from regular endo-1,4-beta-glucanases.

PMID: 15541296 [PubMed - indexed for MEDLINE]


235. Biomacromolecules. 2004 Nov-Dec;5(6):2258-68.

Novel biodegradable aliphatic poly(butylene succinate-co-cyclic carbonate)s
bearing functionalizable carbonate building blocks: II. Enzymatic biodegradation 
and in vitro biocompatibility assay.

Yang J, Tian W, Li Q, Li Y, Cao A.

Polymer Materials Laboratory, Modern Synthetic Chemistry Laboratory, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Road,
Shanghai 200032, PR China.

In a previous study, we have reported chemical synthesis of novel aliphatic
poly(butylene succinate-co-cyclic carbonate) P(BS-co-CC)s bearing various
functionalizable carbonate building blocks, and this work will continue to
present our new studies on their enzymatic degradation and in vitro cell
biocompatibility assay. First, enzymatic degradation of the novel P(BS-co-CC)
film samples was investigated with two enzymes of lipase B Candida Antartic
(Novozyme 435) and lipase Porcine Pancreas PPL, and it was revealed that
copolymerizing linear poly(butylene succinate) PBS with a functionalizable
carbonate building block could remarkably accelerate the enzymatic degradation of
a synthesized product P(BS-co-CC), and its biodegradation behavior was found to
strongly depend on the overall impacts of several important factors as the cyclic
carbonate (CC) comonomer structure and molar content, molar mass, thermal
characteristics, morphology, the enzyme-substrate specificity, and so forth.
Further, the biodegraded residual film samples and water-soluble enzymatic
degradation products were allowed to be analyzed by means of proton nuclear
magnetic resonance (1H NMR), gel permeation chromatograph (GPC), differential
scanning calorimeter (DSC), attenuated total reflection FTIR (ATR-FTIR), scanning
electron microscope (SEM), and liquid chromatograph-mass spectrometry (LC-MS). On
the experimental evidences, an exo-type mechanism of enzymatic chain hydrolysis
preferentially occurring in the noncrystalline domains was suggested for the
synthesized new P(BS-co-CC) film samples. With regard to their cell
biocompatibilities, an assay with NIH 3T3 mouse fibroblast cell was conducted
using the novel synthesized P(BS-co-CC) films as substrates with respect to the
cell adhesion and proliferation, and these new biodegradable P(BS-co-CC) samples 
were found to exhibit as low cell toxicity as the PLLA control, particularly the 
two samples of poly(butylene succinate-co-18.7 mol % dimethyl trimethylene
carbonate) P(BS-co-18.7 mol % DMTMC) and poly(butylene succinate-co-21.9 mol %
5-benzyloxy trimethylene carbonate) P(BS-co-21.9 mol % BTMC) were interestingly
found to show much better cell biocompatibilities than the PLLA reference.

PMID: 15530040 [PubMed - indexed for MEDLINE]


236. Biochem J. 2005 Mar 1;386(Pt 2):395-400.

Analysis of normal and mutant iduronate-2-sulphatase conformation.

Parkinson-Lawrence E, Turner C, Hopwood J, Brooks D.

Lysosomal Diseases Research Unit, Department of Genetic Medicine, Women's and
Children's Hospital, 72 King William Rd, North Adelaide, South Australia 5006,
Australia.

Mammalian sulphatases (EC 3.1.6) are a family of enzymes that have a high degree 
of similarity in amino acid sequence, structure and catalytic mechanism. IDS
(iduronate-2-sulphatase; EC 3.1.6.13) is a lysosomal exo-sulphatase that belongs 
to this protein family and is involved in the degradation of the
glycosaminoglycans heparan sulphate and dermatan sulphate. An IDS deficiency
causes the lysosomal storage disorder MPS II (mucopolysaccharidosis type II). To 
examine the structural alterations in heat-denatured and mutant IDS, a panel of
four monoclonal antibodies was raised to the denatured protein and used as probes
of protein conformation. The linear sequence epitope reactivity of a polyclonal
antibody raised against the native protein and the monoclonal antibodies were
defined and mapped to distinct regions on the IDS protein. The antigenicity of
native IDS was higher in regions without glycosylation, but reactivity was not
restricted to protein surface epitopes. One monoclonal epitope was relatively
surface accessible and in close proximity to an N-linked glycosylation site,
while three others required additional thermal energy to expose the epitopes. The
monoclonal antibodies demonstrated the capacity to differentiate progressive
structural changes in IDS and could be used to characterize the severity of MPS
type II in patients based on variable denatured microstates.

PMCID: PMC1134805
PMID: 15500445 [PubMed - indexed for MEDLINE]


237. J Biol Chem. 2004 Dec 24;279(52):54872-80. Epub 2004 Oct 18.

Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin 
is dependent on multimerization.

Grass I, Thiel S, Honing S, Haucke V.

Institut fur Chemie-Biochemie, Freie Universitat Berlin, Takustrasse 6, D-14195
Berlin, Germany.

Synaptotagmin is a multifunctional membrane protein that may regulate
exo-endocytic cycling of synaptic vesicles at the presynaptic plasmalemma. Its
C2B domain has been postulated to interact with a variety of effector molecules
including acidic phospholipids, phosphoinositides, SNAREs (soluble
N-ethylmaleimide-sensitive factor attachment protein receptors), calcium
channels, and the clathrin adaptor complex AP-2. Here we report that a basic
motif within the C2B domain is required and sufficient for binding to AP-2 via
its mu2 subunit and that this interaction is dependent on multimerization of the 
AP-2 binding site. Moreover, we show that upon fusion to a plasma membrane
reporter protein this sequence is sufficient to target the chimeric molecule for 
internalization. We hypothesize that basic motifs within multimeric membrane
proteins may represent a novel type of clathrin/AP-2-dependent endocytosis
signal.

PMID: 15491995 [PubMed - indexed for MEDLINE]


238. Biochemistry. 2004 Oct 26;43(42):13459-66.

Very efficient template/primer-independent DNA synthesis by thermophilic DNA
polymerase in the presence of a thermophilic restriction endonuclease.

Liang X, Jensen K, Frank-Kamenetskii MD.

Center for Advanced Biotechnology and Department of Biomedical Engineering,
Boston University, 36 Cummington Street, Boston, Massachusetts 02215, USA.
liangxg@bu.edu

We have found that, in the presence of a thermophilic restriction endonuclease,
thermophilic DNA polymerase efficiently synthesizes and amplifies DNA in the
absence of any added template and primer nucleic acid under isothermal
conditions. More than 10 microg of DNA can be synthesized by 1 unit of DNA
polymerase in 1 h, and the reaction proceeds until available dNTPs are consumed. 
We used mostly the Tsp509I restriction endonuclease (recognition sequence:
decreasing AATT), the TspRI restriction endonuclease (recognition sequence:
NNCA(G/C)TGNN decreasing), and Vent (exo(-)) and Vent DNA polymerase. The
synthesized double-stranded DNA has a highly repetitive palindromic sequence,
e.g. (AAAAATTTTT)(n) and (ATACACTGTATATACAGTGTAT)(n). In every repeating unit,
there are one or two recognition sites for the restriction enzyme. Our data show 
that the high efficiency of the restriction-endonuclease-DNA-polymerase (RE-pol) 
DNA synthesis results from an efficient exponential amplification involving
digestion-elongation cycles: a longer DNA with numerous recognition sites for the
restriction enzyme is digested to short fragments, and the short fragments are
used as seeds for elongation to synthesize longer DNA. A possible role of RE-pol 
DNA synthesis in the evolutionary development of genetic materials is briefly
discussed.

PMID: 15491153 [PubMed - indexed for MEDLINE]


239. Glycoconj J. 2004;21(5):243-50.

Chemoenzymatic synthesis of diverse asparagine-linked
alpha-(2,3)-sialyloligosaccharides.

Fukae K, Yamamoto N, Hatakeyama Y, Kajihara Y.

Otsuka Chemical Co., Ltd. 463, Kagasuno, Kawauchicho, Tokushima, 771-0193, Japan.

Partial sialyl transfer reaction by alpha-(2,3)-sialyltransferase toward
(Gal-beta-1,4-GlcNAc-beta-1,2-Man-alpha-1,6/1,3-)(2)Man-beta-1,4-GlcNAc-beta-1,4-
GlcNAc-beta-1-asparagine-Fmoc 1 was examined to obtain
mono-alpha-(2,3)-sialyloligosaccharides and then branch-specific exo-glycosidase 
digestion (beta-D-galactosidase, N -acetyl-beta-D-glucosaminidase and
alpha-D-mannosidase) toward the asialo-branch was performed to obtain diverse
asparagine-linked complex type alpha-(2,3)-sialyloligosaccharides. In addition,
two kinds of disialyloligosaccharides in which the sialyl linkage was a mixture
of alpha-(2,3)- and alpha-(2,6)-types were also specifically prepared by an
additional alpha-(2,6)-sialyltransferase reaction toward
mono-alpha-(2,3)-sialyloligosaccharides thus obtained.

PMID: 15486456 [PubMed - indexed for MEDLINE]


240. Inorg Chem. 2004 Oct 4;43(20):6228-37.

Synthesis and characterization of novel monocarbollide
exo-closo-(pi-arene)biruthenacarboranes
[(PPh3)mClRu(eta6-C6H5R)Ru'CB10H11-n(OMe)n] (where R = H, m = 2, n = 1; R =
mu-PPh2, m = 1, n = 0, 1).

Pisareva IV, Konoplev VE, Petrovskii PV, Vorontsov EV, Dolgushin FM, Yanovsky AI,
Chizhevsky IT.

A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of
Sciences, 28 Vavilov Street, 119991 Moscow, Russian Federation.

The monocarbon carborane [Cs][nido-7-CB(10)H(13)] reacts with the 16-electron
[RuCl(2)(PPh(3))(3)] in a solution of benzene/methanol in the presence of
N,N,N',N'-tetramethylnaphthalene-1,8-diamine as the base to give a series of
12-vertex monocarbon arene-biruthenacarborane complexes of two types:
[closo-2-[7,11-exo-RuClPPh(3)(mu,eta(6)-C(6)H(5)PPh(2))]-7,11-(mu-H)(2)-2,1-RuCB(
10)H(8)R] (5, R = H; 6, R = 6-MeO; 7, R = 3-MeO) and
[closo-2-(eta(6)-C(6)H(6))-10,11,12-[exo-RuCl(PPh(3))(2)]-10,11,12-(mu-H)(3)-2,1-
RuCB(10)H(7)R(1)] (8a, R(1) = 6-MeO; 8b, R(1) = 3-MeO, inseparable mixture of
isomers) along with trace amounts of 10-vertex mononuclear
hypercloso/isocloso-type complexes
[2,2-(PPh(3))(2)-2-H-3,9-(MeO)(2)-2,1-RuCB(8)H(7)] (9) and
[2,5-(Ph(3)P)-2-Cl-2-H-3,9-(MeO)(2)-2,1-RuCB(8)H(6)] (10). Binuclear
ruthenacarborane clusters of both series were characterized by a combination of
analytical and multinuclear NMR spectroscopic data and by single-crystal X-ray
diffraction studies of three selected complexes, 6-8. In solution, isomers 8a,b
have been shown to undergo the isomerization process through the scrambling of
the exo-[RuCl(PPh(3))(2)] fragment about two adjacent triangular cage boron faces
B(7)B(11)B(12) and B(8)B(9)B(12).

PMID: 15446868 [PubMed - indexed for MEDLINE]


241. J Am Chem Soc. 2004 Sep 29;126(38):11852-63.

The mechanism of nitrogenase. Computed details of the site and geometry of
binding of alkyne and alkene substrates and intermediates.

Dance I.

School of Chemical Sciences, University of New South Wales, Sydney 2052,
Australia. I.Dance@unsw.edu.au

The chemical mechanism by which the enzyme nitrogenase effects the remarkable
reduction of N(2) to NH(3) under ambient conditions continues to be enigmatic,
because no intermediate has been observed directly. Recent experimental
investigation of the enzymatic consequences of the valine --> alanine
modification of residue alpha-70 of the component MoFe protein on the reduction
of alkynes, together with EPR and ENDOR spectroscopic characterization of a
trappable intermediate in the reduction of propargyl alcohol or propargyl amine
(HCC[triple bond]C-CH(2)OH/NH(2)), has localized the site of binding and
reduction of these substrates on the FeMo-cofactor and led to proposed eta(2)-Fe 
coordination geometry. Here these experimental data are modeled using density
functional calculations of the allyl alcohol/amine intermediates and the
propargyl alcohol/amine reactants coordinated to the FeMo-cofactor, together with
force-field calculations of the interactions of these models with the surrounding
MoFe protein. The results support and elaborate the earlier proposals, with the
most probable binding site and geometry being eta(2)-coordination at Fe6 of the
FeMo-cofactor (crystal structure in the Protein Database), in a position that is 
intermediate between the exo and endo coordination extremes at Fe6. The models
described account for (1) the steric influence of the alpha-70 residue, (2) the
crucial hydrogen bonding with Nepsilon of alpha-195(His), (3) the spectroscopic
symmetry of the allyl-alcohol intermediate, and (4) the preferential
stabilization of the allyl alcohol/amine relative to propargyl alcohol/amine.
Alternative binding sites and geometries for ethyne and ethene, relevant to the
wild-type protein, are described. This model defines the location and scene for
detailed investigation of the mechanism of nitrogenase.

PMID: 15382920 [PubMed - indexed for MEDLINE]


242. Chemistry. 2004 Oct 4;10(19):4742-9.

Understanding the nature of the molecular mechanisms associated with the
competitive Lewis acid catalyzed [4+2] and [4+3] cycloadditions between
arylidenoxazolone systems and cyclopentadiene: a DFT analysis.

Arno M, Picher MT, Domingo LR, Andres J.

Instituto de Ciencia Molecular, Departamento de Quimica Organica, Universidad de 
Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain.

The molecular mechanisms of the reactions between aryliden-5(4H)-oxazolone 1, and
cyclopentadiene (Cp), in presence of Lewis acid (LA) catalyst to obtain the
corresponding [4+2] and [4+3] cycloadducts are examined through density
functional theory (DFT) calculations at the B3LYP/6-31G* level. The activation
effect of LA catalyst can be reached by two ways, that is, interaction of LA
either with carbonyl or carboxyl oxygen atoms of 1 to render [4+2] or [4+3]
cycloadducts. The endo and exo [4+2] cycloadducts are formed through a highly
asynchronous concerted mechanism associated to a Michael-type addition of Cp to
the beta-conjugated position of alpha,beta-unsaturated carbonyl framework of 1.
Coordination of LA catalyst to the carboxyl oxygen yields a highly functionalized
compound, 3, through a domino reaction. For this process, the first reaction is a
stepwise [4+3] cycloaddition which is initiated by a Friedel-Crafts-type addition
of the electrophilically activated carbonyl group of 1 to Cp and subsequent
cyclization of the corresponding zwitterionic intermediate to yield the
corresponding [4+3] cycloadduct. The next rearrangement is the nucleophilic
trapping of this cycloadduct by a second molecule of Cp to yield the final adduct
3. A new reaction pathway for the [4+3] cycloadditions emerges from the present
study.

PMID: 15372655 [PubMed]


243. Dalton Trans. 2004 Jan 21;(2):327-33. Epub 2003 Dec 11.

A paradigm shift in the construction of heterobimetallic complexes: synthesis of 
group 2 & 4 metal-calix[6]arene complexes.

Petrella AJ, Craig DC, Lamb RN, Raston CL, Roberts NK.

School of Chemical Sciences, University of New South Wales, Sydney 2052,
Australia.

Deprotonation of calix[6]arenes with barium in methanol followed by the addition 
of [Ti(OPr(i))(4)] or [Zr(OBu(n))(4)] is effective in the formation of novel
dimeric 2:1 barium-titanium(IV)/zirconium(IV) calix[6]arene complexes. In these
complexes a central Ti(IV)/Zr(IV) coordinated in the exo-position connects the
two calix[6]arenes in the 1,3-alternate conformation, each with an endo-barium
sharing common phenolate groups with the titanium/zirconium centre and
participating in cation-pi interactions. A homometallic barium calix[6]arene
dimer was also prepared wherein the calix[6]arenes are in the 1,3-alternate
conformation with each coordinating one endo- and one exo-barium centre. The
exo-barium cations connect the two calix[6]arenes through bridging methanol
ligands. In this and the heterometallic complexes, cation-pi complexation of the 
Ba(2+) ion within the 1,3 alternate conformation of calix[6]arene facilitates the
formation of the dimeric complexes in methanol. In contrast, the smaller Sr(2+)
ion did not form similar complexes in methanol, and the formation of an analogous
2:1 strontium-titanium calixarene complex required the use of the more sterically
demanding donor alcohol, isopropanol, the resulting complex being devoid of
cation-pi interaction. The results show (i) that a subtle interplay of solvation 
strength, coordination array type and cavity/cation size influences the
accessibility of heterobimetallic complexes based on calix[6]arenes, and (ii) a
synergistic endo-exo binding behaviour.

PMID: 15356731 [PubMed]


244. Genetics. 2004 Aug;167(4):1855-61.

In vivo interaction between mitochondria carrying mtDNAs from different mouse
species.

Sato A, Nakada K, Shitara H, Yonekawa H, Hayashi J.

Institute of Biological Sciences, University of Tsukuba, Ibaraki 305-8572, Japan.

Mitochondrial disease model mice, mitomice, were created using zygotes of B6mtspr
strain mice carrying mitochondrial DNA (mtDNA) from Mus spretus as recipients of 
exogenous mitochondria carrying wild-type and a deletion mutant mtDNA
(DeltamtDNA) of M. musculus domesticus. In these experiments, mtDNAs from
different mouse species were used for identification of exo- and endogenous
wild-type mtDNAs in the mitomice. Results showed transmission of exogenous
DeltamtDNA, but not exogenous wild-type mtDNA, of M. m. domesticus to following
generations through the female germ line. Complete elimination of exogenous
wild-type mtDNA would be due to stochastic segregation, whereas transmission of
exogenous DeltamtDNA would be due to its smaller size leading to a propagational 
advantage. Tissues in mitomice of the F3 generation carrying exogenous DeltamtDNA
showed protection from respiration defects until DeltamtDNA accumulated
predominantly. This protection from expression of mitochondrial dysfunction was
attained with the help of endogenous wild-type mtDNA of M. spretus, since
mitomice did not possess exogenous wild-type mtDNA of M. m. domesticus. These
observations provide unambiguous evidence for the presence of interaction between
exogenous mitochondria carrying DeltamtDNA and endogenous mitochondria carrying
M. spretus wild-type mtDNA. Copyright 2004 Genetics Society of America

PMCID: PMC1470990
PMID: 15342523 [PubMed - indexed for MEDLINE]


245. J Neurosci. 2004 Aug 11;24(32):7043-50.

Identification and characterization of metallothionein-1 and -2 gene expression
in the context of (+/-)3,4-methylenedioxymethamphetamine-induced toxicity to
brain dopaminergic neurons.

Xie T, Tong L, McCann UD, Yuan J, Becker KG, Mechan AO, Cheadle C, Donovan DM,
Ricaurte GA.

Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, 
Maryland 21224, USA.

In mice, the recreational drug (+/-)3,4-methylenedioxymethamphetamine [MDMA
("ecstasy")] produces a selective toxic effect on brain dopamine (DA) neurons.
Using cDNA microarray technology in combination with an approach designed to
facilitate recognition of relevant changes in gene expression, the present
studies sought to identify genes potentially involved in murine MDMA-induced
toxicity to DA neurons. Of 15,000 mouse cDNA fragments studied, metallothionein
(Mt)-1 and Mt2 emerged as candidate genes possibly involved in MDMA-induced
toxicity to DA neurons. Northern blot analysis confirmed the microarray findings 
and revealed a dynamic upregulation of Mt1 and Mt2 mRNA in the ventral midbrain
within 4-12 hr after MDMA treatment. Western blot analysis showed a similar
increase in MT protein levels, with peak times occurring subsequent to increases 
in mRNA levels. Mt1-2 double knock-out mice were more vulnerable to MDMA-induced 
toxicity to DA neurons than corresponding wild-type mice. Stimulation of
endogenous expression of MT protein with zinc acetate conferred complete
protection against MDMA-induced toxicity to DA neurons, and administration of
exogenous MT protein afforded partial protection. Collectively, these results
indicate that MDMA-induced toxicity to DA neurons is associated with increased
Mt1 and Mt2 gene transcription and translation, possibly as part of a
neuroprotective mechanism. The present findings may have therapeutic implications
for neuropathological conditions involving DA neurons.

PMID: 15306638 [PubMed - indexed for MEDLINE]


246. Chemistry. 2004 Aug 6;10(15):3615-21.

Synthesis, characterization, and electronic structure of Ba5In4Bi5: an acentric
and one-electron deficient phase.

Ponou S, Fassler TF, Tobias G, Canadell E, Cho A, Sevov SC.

Anorganisch-chemisches Institut der Technischen Universitat Munchen,
Lichtenbergstrasse 4, 85747 Garching, Germany.

The new ternary phase Ba(5)In(4)Bi(5) was synthesized by direct reaction of the
corresponding elements at high temperature. It crystallizes in a
noncentrosymmetric space group and represents a new structure type (tetragonal,
P4nc with a=10.620(2) and c=9.009(2) A, Z=2). The structure is built of
interconnected heteroatomic clusters of In(4)Bi(5), square pyramids with
In(4)-bases and four exo-bonded bismuth atoms (bond to the In atoms). According
to Wade's rule the compound is electron-deficient with one electron per cluster, 
that is, [In(4)Bi(5)](10-) instead of the expected [In(4)Bi(5)](11-) for a
closed-shell species. The clusters are discussed also in light of the known
heteroatomic deltahedral clusters with the same composition but different charge,
[In(4)Bi(5)](3-). Band structure calculations on the new compound suggest
substantial participation of barium in the overall bonding of the structure that 
"accounts" for the electron shortage

PMID: 15281144 [PubMed]


247. J Cell Biochem. 2004 Aug 1;92(5):869-81.

Metabolic pathways and physiological and pathological significances of lysolipid 
phosphate mediators.

Tokumura A.

Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima
770-8505, Japan. tokumura@ph.tokushima-u.ac.jp

Lysophosphatidic acid and sphingosine 1-phosphate are structurally simple and
physiologically very important lysophospholipids. Because they possess distinct
structural backbones (glycerol and sphingosine, respectively), there are
different metabolic pathways for their intracellular production. Recently,
several key enzymes that produce or degrade these lysolipid phosphate mediators
extracellularly have been characterized. This review focuses on the physiological
and pathophysiological significances of the extracellular metabolic pathways
involving recently characterized exo-type lysophospholipase D, ecto-type
phospholipase A, and ecto-type lipid phosphate phosphatase.

PMID: 15258912 [PubMed - indexed for MEDLINE]


248. J Biol Chem. 2004 Sep 10;279(37):38555-62. Epub 2004 Jul 7.

Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular
cloning, and characterization.

Ishimizu T, Sasaki A, Okutani S, Maeda M, Yamagishi M, Hase S.

Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, 
Osaka 560-0043, Japan.

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the
Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This
enzyme was partially purified and characterized in a previous report (Sasaki, A.,
Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J.
Biochem. 125, 363-367). Here we report the purification and molecular cloning of 
endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band
on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and
28 kDa. Amino acid sequence information from these three polypeptides allowed the
cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was
engineered for expression in Escherichia coli, and the recombinant protein
comprised a single polypeptide chain with a molecular mass of 112 kDa
corresponding to the sum of molecular masses of three polypeptides of the lily
enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of
Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and
GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM
hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not
PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the 
lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl
hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid
residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme
activity. The amino acid sequence of AtEBM has distinct differences from those of
the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like 
genes are only found in plants, indicating that endo-beta-mannosidase is a
plant-specific enzyme. The role of this enzyme in the processing and/or
degradation of N-glycan will be discussed.

PMID: 15247239 [PubMed - indexed for MEDLINE]


249. Acta Crystallogr C. 2004 Jul;60(Pt 7):o489-91. Epub 2004 Jun 22.

7-Deaza-2'-deoxy-7-propynylguanosine.

Seela F, Shaikh K, Eickmeier H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069, Germany.
frank.seela@uni-osnabrueck.de

The title compound, C14H16N4O4, adopts the anti conformation at the glycosylic
bond [chi -117.1 (5) degrees]. The sugar pucker of the 2'-deoxyribofuranosyl
moiety is C2'-endo-C3'-exo, 2T3 (S-type). The orientation of the exocyclic
C4'-C5' bond is +sc (gauche). The propynyl group is linear and coplanar with the 
nucleobase moiety. The structure of the compound is stabilized by several
hydrogen bonds (N-H...O and O-H...O), leading to the formation of a multi-layered
network. The nucleobases, as well as the propynyl groups, are stacked. This
stacking might cause the extraordinary stability of DNA duplexes containing this 
compound.

PMID: 15237172 [PubMed - indexed for MEDLINE]


250. J Steroid Biochem Mol Biol. 2004 May;89-90(1-5):31-4.

Synthesis and structure-activity relationships of TEI-9647 derivatives as Vitamin
D3 antagonists.

Takenouchi K, Sogawa R, Manabe K, Saitoh H, Gao Q, Miura D, Ishizuka S.

TEIJIN Institute for Bio-medical Research, 4-3-2 Asahigaoka, Hino, Tokyo
191-8512, Japan. k.takenouchi@teijin.co.jp

The Vitamin D(3) lactone analogues, (23S)- and
(23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647 and TEI-9648)
are antagonists of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3))
nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells. 
In order to clarify the structure-Vitamin D antagonistic activity relationship,
we paid attention to the unique lactone moiety of TEI-9647 and TEI-9648:
alpha-exo-methylene-gamma-lactone structure. We synthesized the
exo-methylene-modified analogues (methylene saturated, endo-methylene,
methylene-deleted, methyl-substituted, dimethyl-substituted, methylene-replaced
with dimethyl and cyclopropane) and oxygen-modified analogues (oxygen atom
replaced with nitrogen and carbon atom) by convergent method using
palladium-catalyzed coupling reaction or direct modification of VD(3) skeleton.
The antagonistic activity in HL-60 cell differentiation evaluating system of
these analogues revealed that any exo-methylene-modified analogues and nitrogen
analogue did not have the antagonistic activity, on the other hand carbon
analogue did show. The results suggest that "alpha-exo-methylene carbonyl"
structure of VD(3) side-chain is crucial for antagonistic activity. The structure
is integral building block of many natural products which have interesting
biological and it is thought that Michael-type addition of alpha-exo-methylene
carbonyl structure with protein nucleophiles such as cysteine would play an
important role for the activities. According to this theory, Michael-type
reaction of TEI-9647 and TEI-9648 with cysteine residue in protein related to
VDR/VDRE-mediated genomic actions such as VDR would be essential step of the
antagonistic action.

PMID: 15225742 [PubMed - indexed for MEDLINE]


251. J Biol Chem. 2004 Jul 30;279(31):32832-8. Epub 2004 May 25.

Biochemical and structural characterization of (South)-methanocarbathymidine that
specifically inhibits growth of herpes simplex virus type 1 thymidine
kinase-transduced osteosarcoma cells.

Schelling P, Claus MT, Johner R, Marquez VE, Schulz GE, Scapozza L.

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology
(ETH), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Two analogs of the natural nucleoside dT featuring a pseudosugar with fixed
conformation in place of the deoxyribosyl residue (carbathymidine analogs) were
biochemically and structurally characterized for their acceptance by both human
cytosolic thymidine kinase isoenzyme 1 (hTK1) and herpes simplex virus type 1
thymidine kinase (HSV1 TK) and subsequently tested in cell proliferation assays. 
3'-exo-Methanocarbathymidine ((South)-methanocarbathymidine (S)-MCT), which is a 
substrate for HSV1 TK, specifically inhibited growth of HSV1 TK-transduced human 
osteosarcoma cells with an IC(50) value in the range of 15 microM without
significant toxicity toward both hTK1-negative (TK(-)) and non-transduced cells. 
2'-exo-Methanocarbathymidine ((North)-methanocarbathymidine (N)-MCT), which is a 
weak substrate for hTK1 and a substantial one for HSV1 TK, induced a specific
growth inhibition in HSV1 TK-transfected cells comparable to that of (S)-MCT and 
ganciclovir. A growth inhibition activity was also observed with (N)-MCT and
ganciclovir in non-transduced cells in a cell line-dependent manner, whereas
TK(-) cells were not affected. The presented 1.95-A crystal structure of the
complex (S)-MCT.HSV1 TK explains both the more favorable binding affinity and
catalytic turnover of (S)-MCT for HSV1 TK over the North analog. Additionally the
plasticity of the active site of the enzyme is addressed by comparing the binding
of (North)- and (South)-carbathymidine analogs. The presented study of these two 
potent candidate prodrugs for HSV1 TK gene-directed enzyme prodrug therapy
suggests that (S)-MCT may be even safer to use than its North counterpart
(N)-MCT.

PMID: 15163659 [PubMed - indexed for MEDLINE]


252. J Am Chem Soc. 2004 May 26;126(20):6448-59.

Initial structure modification of tetrahedral to planar nickel(II) in a
nickel-iron-sulfur cluster related to the C-cluster of carbon monoxide
dehydrogenase.

Panda R, Zhang Y, McLauchlan CC, Venkateswara Rao P, Tiago de Oliveira FA, Munck 
E, Holm RH.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, USA.

A method has been devised that creates a planar Ni(II) site from a tetrahedral
site in a NiFe(3)S(4) cubane-type cluster. Reaction of
[(Ph(3)P)NiFe(3)S(4)(LS(3))](2)(-) (2) with 1,2-bis(dimethylphosphino)ethane
affords [(dmpe)NiFe(3)S(4)(LS(3))](2)(-) (3), isolated in ca. 45% yield as
(Et(4)N)(2)[3a].2.5MeCN and (Et(4)N)(2)[3b].0.25MeCN, both of which occur in
triclinic space group P. Each crystalline form contains two crystallographically 
inequivalent clusters with the same overall structure but slightly different
dimensions. The cluster is bound by three thiolate terminal ligands to semirigid 
cavitand ligand LS(3). The NiFe(3)S(4) core contains three tetrahedral sites, one
Fe(micro(3)-S)(3)(SR) and two Fe(micro(3)-S)(2)(micro(2)-S)(SR) with normal
metric features, and one distorted square planar Ni(micro(3)-S)(2)P(2) site in a 
Ni(micro(3)-S)(2)Fe face with mean bond lengths Ni-P = 2.147(9) A and Ni-S =
2.29(2) A. The opposite Fe(2)(micro(3)-S)(micro(2)-S) face places the micro(2)-S 
atom at nonbonding and variable distances (2.60-2.90 A) above the nickel atom.
Binding of the strong-field ligand dmpe results in a planar Ni(II) site and
deconstruction of the full cubane geometry. The structure approximates that
established crystallographically in the C-cluster of C. hydrogenoformans carbon
monoxide dehydrogenase whose NiFe(4)S(4) core contains a planar NiS(4) site and
three tetrahedral FeS(4) sites in a fragment that is bridged by sulfide atoms to 
an exo iron atom. Mossbauer studies of polycrystalline samples containing both
clusters 3a and 3b reveal the presence of at least two cluster types. The
spectroscopically best defined cluster accounts for ca. 54% of total iron and
exhibits hyperfine interactions quite similar to those reported for the S =
(5)/(2) state of the protein-bound cubane-type cluster [ZnFe(3)S(4)](1+), whose
Mossbauer spectrum revealed the presence of a high-spin Fe(2+) site and a
delocalized Fe(2.5+)Fe(2.5+) pair. Development of reactions leading to a planar
nickel and a sulfide-bridged iron atom is requisite to attainment of a synthetic 
analogue of this complex protein-bound cluster. This work demonstrates a
tetrahedral (2) --> planar (3) Ni(II) stereochemical conversion can be effected
by binding of ligands that generate a sufficiently strong in-plane ligand field
(dmpe = 1,2-bis(dimethylphosphino)ethane, LS(3) =
1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3-
)).

PMID: 15149242 [PubMed - indexed for MEDLINE]


253. J Neurochem. 2004 May;89(4):853-64.

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of 
binding interaction for GBR12909 and benztropine as opposed to cocaine.

Chen N, Zhen J, Reith ME.

Department of Psychiatry, New York University School of Medicine, New York 10016,
USA. nianhang.chen@med.nyu.edu

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine
may partially stem from their different molecular actions on the dopamine
transporter (DAT). To explore this possibility, we examined binding of these
inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and
with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3
beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact
cells, the mutation-induced change in the ability of Na(+) to enhance the
apparent affinity of CFT, cocaine, GBR12909, and benztropine was
inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus
apparent ligand affinity was steeper at W84L compared with wild type, shallower
at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and
cocaine was enhanced regardless of whether Na(+) was present. However, the
apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence
of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and 
W84LD313N was barely changed. At the single mutants, the alterations in Na(+)
dependence and apparent affinity of the four inhibitors were comparable between
[(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These
results demonstrate that DAT inhibitors producing different behavioral profiles
can respond in an opposite way when residues of the DAT protein are mutated. For 
GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest 
that they prefer a DAT state similar to that for cocaine. However, their
cocaine-unlike changes in apparent affinity argue that they, likely via their
diphenylmethoxy moiety, share DAT binding epitopes that are different from those 
for cocaine.

PMID: 15140185 [PubMed - indexed for MEDLINE]


254. J Bacteriol. 2004 May;186(9):2699-707.

Modulation of DNA repair and recombination by the bacteriophage lambda Orf
function in Escherichia coli K-12.

Poteete AR.

Department of Molecular Genetics and Microbiology, University of Massachusetts
Medical School, 55 Lake Avenue, Worcester, MA 01655, USA.
anthony.poteete@umassmed.edu

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK
locus of Escherichia coli K-12. Orf was found to suppress the recombination
deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB
recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, 
ruvAB, and ruvC functions. It also suppressed defects of these mutants in
establishing replication of a pSC101-related plasmid. Compared to orf, the
recA803 allele had only small effects on recF, recO, and recR mutant phenotypes
and no effect on a ruvAB mutant. In a fully wild-type background with respect to 
known recombination and repair functions, orf partially suppressed the UV
sensitivity of ruvAB and ruvC mutants.

PMCID: PMC387792
PMID: 15090511 [PubMed - indexed for MEDLINE]


255. J Biol Chem. 2004 Jun 18;279(25):26462-8. Epub 2004 Apr 6.

Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an
active site loop.

Geddie ML, Matsumura I.

Department of Biochemistry, Center for Fundamental and Molecular Evolution,
Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia
30322, USA.

Protein engineers have widely adopted directed evolution as a design algorithm,
but practitioners have not come to a consensus about the best method to evolve
protein molecular recognition. We previously used DNA shuffling to direct the
evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased
beta-galactosidase activity. Epistatic (synergistic) mutations in amino acids
557, 566, and 568, which are part of an active site loop, were identified in that
experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305,
331-339). Here we show that site saturation mutagenesis of these residues,
overexpression of the resulting library in E. coli, and high throughput screening
led to the rapid evolution of clones exhibiting increased activity in reactions
with p-nitrophenyl-beta-d-xylopyranoside (pNP-xyl). The xylosidase activities of 
the 14 fittest clones were 30-fold higher on average than that of the wild-type
GUS. The 14 corresponding plasmids were pooled, amplified by long PCR,
self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones
exhibiting an average of 80-fold improvement in xylosidase activity were isolated
in a second round of screening. One of the evolved proteins exhibited a
approximately 200-fold improvement over the wild type in reactivity (k(cat)/K(m))
with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of 
the 13 round 2 isolates suggested that all were products of intermolecular
recombination events that occurred during whole plasmid PCR. Further rounds of
evolution using DNA shuffling and staggered extension process (StEP) resulted in 
modest improvement. These results underscore the importance of epistatic
interactions and demonstrate that they can be optimized through variations of the
facile whole plasmid PCR technique.

PMID: 15069062 [PubMed - indexed for MEDLINE]


256. FEBS Lett. 2004 Apr 9;563(1-3):82-6.

EXORDIUM regulates brassinosteroid-responsive genes.

Coll-Garcia D, Mazuch J, Altmann T, Mussig C.

Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am Muhlenberg 1, 14476
Golm, Germany.

In a screen for potential mediators of brassinosteroid (BR) effects, the EXORDIUM
(EXO) protein was identified as a regulator of BR-responsive genes. The EXO gene 
was characterized as a BR-up-regulated gene. EXO overexpression under the control
of the 35SCaMV promoter resulted in increased transcript levels of the
BR-up-regulated KCS1, Exp5, delta-TIP, and AGP4 genes, which likely are involved 
in the mediation of BR-promoted growth. 35S::EXO lines grown in soil or in
synthetic medium showed increased vegetative growth in comparison to wild-type
plants, resembling the growth phenotype of BR-treated plants. Thus, the EXO
protein most likely promotes growth via the modulation of gene expression
patterns.

PMID: 15063727 [PubMed - indexed for MEDLINE]


257. Biosci Biotechnol Biochem. 2004 Mar;68(3):685-93.

Isolation and characterization of the K5-type yeast killer protein and its
homology with an exo-beta-1,3-glucanase.

Izgu F, Altinbay D.

Department of Biological Sciences, Middle East Technical University, Ankara,
Turkey. izgu@metu.edu.tr

K5-type yeast killer protein in the culture supernatant of Pichia anomala NCYC
434 cells was concentrated by ultrafiltration and purified to homogeneity by
ion-exchange chromatography with a POROS HQ/M column followed by gel filtration
with a TSK G2000SW column. The protein migrated as a single band on discontinuous
gradient SDS-PAGE and had a molecular mass of 49,000 Da. The pI value of the
K5-type killer protein was measured at pH 3.7 by high voltage vertical gel
electrofocusing. The result of an enzyme immuno assay revealed that it was a
glycosylated protein. Its internal amino acid sequencing yielded the sequences
LNDFWQQGYHNL, IPIGYWAFQLLDNDPY, and YGGSDYGDVVIGIELL, which are 100% identical to
exo-beta-1,3-glucanase (accession no. AJ222862) of Pichia anomala (strain K). The
purified protein was highly stable at pH values between 3 and 5.5 and
temperatures up to 37 degrees C.

PMID: 15056904 [PubMed - indexed for MEDLINE]


258. Biochemistry. 2004 Apr 6;43(13):3853-61.

Pre-steady-state kinetics of RB69 DNA polymerase and its exo domain mutants:
effect of pH and thiophosphoryl linkages on 3'-5' exonuclease activity.

Wang CX, Zakharova E, Li J, Joyce CM, Wang J, Konigsberg W.

Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar
Street, New Haven, Connecticut 06520, USA.

DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have
exonuclease domains with similar three-dimensional structures that require two
divalent metal ions for catalysis. B family DNA polymerases that are part of a
replicase generally have a more potent 3'-5' exonuclease (exo) activity than A
family DNA polymerases that mainly function in DNA repair. To investigate the
basis for these differences, we determined pH-activity profiles for the
exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives
of B family replicative DNA polymerases and the Klenow fragment (KF) as an
example of a repair DNA polymerase in the A family. We performed exo assays under
single-turnover conditions and found that excision rates exhibited by the B
family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5,
whereas the exo activity of KF increased 10-fold for each unit increase in pH.
Three exo domain mutants of RB69 polymerase had much lower exo activities than
the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On
the basis of pH versus activity data and elemental effects obtained using short
double-stranded DNA substrates terminating in phosphorothioate linkages, we
suggest that the rate of the chemical step is reduced to the point where it
becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to
the wild-type enzyme where chemistry is faster than the rate-determining step
that precedes it.

PMID: 15049692 [PubMed - indexed for MEDLINE]


259. J Org Chem. 2004 Apr 2;69(7):2469-77.

Synthesis of 4H-3,1-benzoxazines, quinazolin-2-ones, and quinoline-4-ones by
palladium-catalyzed oxidative carbonylation of 2-ethynylaniline derivatives.

Costa M, Ca ND, Gabriele B, Massera C, Salerno G, Soliani M.

Dipartimento di Chimica Organica e Industriale, Universita di Parma, Parco Area
delle Scienze 17/A, 43100 Parma, Italy. mirco.costa@unipr.it

An effective and straightforward approach to the synthesis of 4H-3,1-benzoxazines
3 and 4, quinazolin-2-ones 5, and quinoline-4-one derivatives 6 and 7 is provided
by palladium-catalyzed cyclization-alkoxycarbonylation of variously substituted
2-(trimethylsilanyl)ethynylaniline amide or urea derivatives 2. Reactions are
carried out in 7:1 MeCN/MeOH at 65 or 75 degrees C in the presence of catalytic
amounts of 10% Pd/C in conjunction with Bu(4)NI and KF and under 2.4 MPa of a 3:1
mixture of CO and air. Anti and syn 6-exo-dig cyclization modes account for the
formation of the two stereoisomers. Isomerization of the vinylpalladium
intermediate may occur as well. Formation of a double carbonylation product 7r
and of a gem-dimethoxycarbonylation product 6s, whose structures have been
determined by X-ray diffraction analysis, is justified through an unusual type of
rearrangement.

PMID: 15049647 [PubMed]


260. J Virol. 2004 Apr;78(8):4342-51.

Reptilian reovirus utilizes a small type III protein with an external
myristylated amino terminus to mediate cell-cell fusion.

Corcoran JA, Duncan R.

Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova
Scotia, Canada B3H 4H7.

Reptilian reovirus is one of a limited number of nonenveloped viruses that are
capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid
fusion protein encoded by the first open reading frame of a bicistronic viral
mRNA is responsible for this fusion activity. Sequence comparisons to previously 
characterized reovirus fusion proteins indicated that p14 represents a new member
of the fusion-associated small transmembrane (FAST) protein family. Topological
analysis revealed that p14 is a representative of a minor subset of integral
membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic)
(N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal
reverse signal-anchor sequence to direct membrane insertion and protein topology.
This topology results in the unexpected, cotranslational translocation of the
essential myristylated N-terminal domain of p14 across the cell membrane. The
topology and structural motifs present in this novel reovirus membrane fusion
protein further accentuate the diversity and unusual properties of the FAST
protein family and clearly indicate that the FAST proteins represent a third
distinct class of viral membrane fusion proteins.

PMCID: PMC374291
PMID: 15047847 [PubMed - indexed for MEDLINE]


261. Nucleosides Nucleotides Nucleic Acids. 2004;23(1-2):521-30.

Conformational flexibility in a triazole nucleoside derivative:
4-cyano-5-cyanomethyl-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-1,2,3-triazole.

Leban I, Jeselnik M, Sieler J, Kobe J.

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana,
Slovenia. ivan.leban@uni-lj.si

The crystal-structure determination of the molecular structure of the hydrophobic
compound,
4-cyano-5-cyanomethyl-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-1,2,3-triazole,
C16H17N5O7, provides us with two different conformations of ribofuranosyl
moieties [(C2'-exo, C3'-endo) and C2'-exo] with two markedly different
N-glycosidic angles. There are two molecules in the asymmetric unit of the
crystal and the overall stereochemistry of the molecules are influenced
predominantly by weak intramolecular bifurcated and trifurcated hydrogen bonds of
the type C-H...O and C-H...N, where endo-H atoms attached to C2' and C3' are
involved. The molecules in the crystal are interconnected with longer
intermolecular bonds of the same type. There are empty channels (occupying 14.0% 
of the whole volume of the unit cell), which are extended along b-axis of the
entire crystal.

PMID: 15043172 [PubMed - indexed for MEDLINE]


262. Physiol Plant. 2004 Apr;120(4):537-545.

Differences in structure of adventitious roots in Salix clones with contrasting
characteristics of cadmium accumulation and sensitivity.

Lux A, Sottnikova A, Opatrna J, Greger M.

Department of Plant Physiology, Faculty of Natural Sciences, Comenius University,
Mlynska dolina B2, 84215 Bratislava, Slovak Republic.

Various clones of Salix spp. have contrasting characteristics of accumulation,
translocation to shoots and of sensitivity to cadmium (Cd). The aim was to
investigate the structure of adventitious roots and find out if differences
between groups of clones in root anatomy accounted for differences in relation to
Cd. Stem cuttings of eight clones of Salix spp. with different combinations of
high or low Cd accumulation, translocation of Cd to shoots and sensitivity to Cd,
were cultivated for 3 weeks in hydroponics containing 100 micro M Ca(NO(3))(2).
No Cd was added in this experiment. Equal-sized roots were selected for
hand-sectioning and fluorescence staining to detect the beginning of Casparian
band formation and suberin lamellae deposition in endodermis. In addition, root
apices were fixed, embedded, sectioned longitudinally and transversally, and
stained. The image analysis system LUCIA was used for quantitative evaluation of 
tissue differences. The structure of adventitious root apices showed an
intermediate-open type of root apical meristem and the clones differed in
organization of root apices. Clones with low accumulation of Cd and high Cd
tolerance had smaller meristematic zones and more extensive vacuolation of cells 
in the root apices than clones characterized by high accumulation of, and high
sensitivity to, Cd. The apoplastic barriers, exo- and endodermis, were developed 
relatively close to the apex. In both layers the first ontogenetic stage, the
Casparian band development, was followed by the second stage, the suberin
lamellae deposition. This process started in the endodermis, preferentially
against phloem poles, which is a common phenomenon also in other plant species.
However, preferential development of exodermis in the sectors against phloem
poles was observed in this study for the first time in plants. Development of
endodermal Casparian bands in clones characterized by high accumulation of Cd
occurred more distant from the root tip than in clones with low accumulation.
Furthermore, the suberin lamellae were more distant from the root tip in clones
with high translocation of Cd compared with those with low translocation. This
indicates that apoplastic movement of Cd into the stele and the upward
translocation may vary due to the endodermal anatomy. The proportion of root
apoplastic barriers, exodermis and endodermis as well as epidermis to other root 
tissues was significantly increased in clones with higher tolerance to Cd ions,
indicating the importance of these tissues in protection of the root against
toxic effects of Cd.

PMID: 15032815 [PubMed - as supplied by publisher]


263. J Org Chem. 2004 Mar 5;69(5):1548-56.

Different reaction modes for the oxidative dimerization of epoxyquinols and
epoxyquinones. Importance of intermolecular hydrogen-bonding.

Shoji M, Imai H, Shiina I, Kakeya H, Osada H, Hayashi Y.

Department of Industrial Chemistry, Faculty of Engineering, Tokyo University of
Science, Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan.

An oxidative dimerization reaction, involving the three successive steps of
oxidation, 6 pi-electrocyclization, and Diels-Alder reaction, has been
experimentally and theoretically investigated for the three
2-alkenyl-3-hydroxymethyl-2-cyclohexen-1-one derivatives epoxyquinol 3,
epoxyquinone 6, and cyclohexenone 10. Of the sixteen possible modes of the
oxidation/6 pi-electrocylization/Diels-Alder reaction cascade for the
epoxyquinone 6, and eight for the cyclohexenone 10, only the
endo-anti(epoxide)-anti(Me)-hetero and endo-anti(Me)-hetero modes are,
respectively, observed, while both endo-anti(epoxide)-anti(Me)-hetero and
exo-anti(epoxide)-anti(Me)-homo reaction modes occur with the epoxyquinol 3.
Intermolecular hydrogen-bonding is found to be the key cause of formation of both
epoxyquinols A and B with 3, although epoxyquinone 6 and cyclohexenone 10 both
gave selectively only the epoxyquinol A-type product. In the dimerization of
epoxyquinol 3, two monomer 2H-pyrans 5 interact with each other to afford
intermediate complex 28 or 29 stabilized by hydrogen-bonding, from which
Diels-Alder reaction proceeds. Theoretical calculations have also revealed the
differences in the reaction profiles of epoxyquinone 6 and cyclohexenone 10.
Namely, the rate-determining step of the former is the Diels-Alder reaction,
while that of the latter is the 6 pi-electrocyclization.

PMID: 14987010 [PubMed]


264. J Biol Chem. 2004 Apr 30;279(18):18535-43. Epub 2004 Feb 23.

Contribution of the 3'- to 5'-exonuclease activity of herpes simplex virus type 1
DNA polymerase to the fidelity of DNA synthesis.

Song L, Chaudhuri M, Knopf CW, Parris DS.

Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State
University, Columbus, Ohio 43210, USA.

Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase
catalytic subunit (pol) is less faithful than for most replicative DNA
polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo)
activity. To determine the aspects of fidelity affected by the exo activity,
nucleotide incorporation and mismatch extension frequency for purified wild-type 
and an exo-deficient mutant (D368A) pol were compared using primer/templates that
varied at only a single position. For both enzymes, nucleotide discrimination
during incorporation occurred predominantly at the level of K(m) for nucleotide
and was the major contributor to fidelity. The contribution of the exo activity
to reducing the efficiency of formation of half of all possible mispairs was
6-fold or less, and 30-fold when averaged for the formation of all possible
mispairs. In steady-state reactions, mismatches imposed a significant kinetic
barrier to extension independent of exo activity. However, during processive DNA 
synthesis in the presence of only three nucleotides, misincorporation and
mismatch extension were efficient for both exo-deficient and wild-type pol
catalytic subunits, although slower kinetics of mismatch extension by the
exo-deficient pol were observed. The UL42 processivity factor decreased the
extent of misincorporation by both the wild-type and the exo-deficient pol to
similar levels, but mismatch extension by the wild-type pol.UL42 complex was much
less efficient than by the mutant pol.UL42. Thus, despite relatively frequent (1 
in 300) misincorporation events catalyzed by wild-type herpes simplex virus
pol.UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by 
the kinetic barrier to extending a mismatch. The kinetic barrier also increases
the probability that a mismatched primer terminus will be transferred to the exo 
site where it can be excised by the associated exo activity and subsequently
extended with correct nucleotide.

PMID: 14982924 [PubMed - indexed for MEDLINE]


265. Chemistry. 2004 Feb 20;10(4):971-85.

Prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its 
application to the solid-phase synthesis of a glycopeptide with Asn-linked
sialyl-undeca- and asialo-nonasaccharides.

Kajihara Y, Suzuki Y, Yamamoto N, Sasaki K, Sakakibara T, Juneja LR.

Graduate School of Integrated Science, Yokohama City University, 22-2, Seto,
Kanazawa-ku, Yokohama 236-0027, Japan. kajihara@yokohama-cu.ac.jp

We describe herein the preparation of 24 pure asparagine-linked oligosaccharides 
(Asn-oligosaccharides) from asparagine-linked biantennary complex-type
sialylundecasaccharide
[(NeuAc-alpha-2,6-Gal-beta-1,4-GlcNAc-beta-1,2-Man-alpha-1,6/1,3-)(2)-Man-beta-1,
4-GlcNAc-beta-1,4-GlcNAc-beta-1-asparagine, 2] obtained from egg yolk. Our
synthetic strategy aimed at adapting branch specific exo-glycosidases digestion
(beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and alpha-D-mannosidase)
of the individual asialo-branch after preparation of monosialyloligosaccharides
obtained from 2 by acid hydrolysis of NeuAc. In order to perform branch specific 
exo-glycosidase digestion, isolation of pure monosialyloligosaccharides obtained 
was essential. However, isolation of two kinds of monosialyloligosaccharides are 
difficult by HPLC due to their highly hydrophilic nature. Therefore, we examined 
chemical protection with hydrophobic protecting (Fmoc and benzyl) groups. These
chemical protection enabled us to separate the monosialyloligosaccharides by use 
of a HPLC column (ODS) on synthetic scales. Using these pure
monosialiloligosaccharides enable us to obtain 24 Asn-linked oligosaccharides
(100 mg scale) within a few weeks by branch specific exo-glycosidase digestions
(alpha-D-neuraminidase, beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and
alpha-D-mannosidase). In addition, solid-phase synthesis of glycopeptide having
Asn-linked sialyl-undeca- and asialo-nonasaccharides thus obtained, was also
performed on an acid labile HMPA-PEGA resin.

PMID: 14978824 [PubMed - indexed for MEDLINE]


266. Biochemistry. 2004 Feb 17;43(6):1715-23.

Folding of an abridged beta-lactamase.

Santos J, Gebhard LG, Risso VA, Ferreyra RG, Rossi JP, Ermacora MR.

Departamento de Ciencia y Tecnologia, Universidad Nacional de Quilmes, Roque
Saenz Pena 180, (1876) Bernal, Buenos Aires, Argentina.

The effects of C-terminal truncation on the equilibrium folding transitions and
folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have
been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no
enzymic activity. Deletion of the last 14 residues produces
ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues
(ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and
hydrodynamic properties, and is protease resistant, a distinguishing feature of
the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4
orders of magnitude slower than ES-betaL, making possible the isolation and
characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)).
Based on the analysis of folding rates and equilibrium constants, we propose that
equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow
folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are
helical and firmly integrated into the enzyme body through many van der Waals
interactions involving residues distant in sequence. The results suggest that the
deleted residues play a key role in the folding process and also the existence of
a modular organization of the protein matrix, at the subdomain level. The results
are compared with other examples of this kind in the folding literature.

PMID: 14769049 [PubMed - indexed for MEDLINE]


267. Biochemistry. 2004 Feb 10;43(5):1163-70.

Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from
Clostridium thermocellum.

Schubot FD, Kataeva IA, Chang J, Shah AK, Ljungdahl LG, Rose JP, Wang BC.

Department of Biochemistry & Molecular Biology, The University of Georgia,
Athens, Georgia 30602, USA.

Numerous bacterial and fungal organisms have evolved elaborate sets of modular
glycoside hydrolases and similar enzymes aimed at the degradation of polymeric
carbohydrates. Presently, on the basis of sequence similarity catalytic modules
of these enzymes have been classified into 90 families. Representatives of a
particular family display similar fold and catalytic mechanisms. However, within 
families distinctions occur with regard to enzymatic properties and type of
activity against carbohydrate chains. Cellobiohydrolase CbhA from Clostridium
thermocellum is a large seven-modular enzyme with a catalytic module belonging to
family 9. In contrast to other representatives of that family possessing only
endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an
exocellulase. The crystal structures of the combination of the
immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and
that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT)
are reported here. The detailed analysis of these structures reveals that, while 
key catalytic residues and overall fold are conserved in this enzyme and those of
other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off
after the -2 subsite. This feature which is created by an extension and altered
conformation of a single loop region explains the inability of the active site of
CbhA to accommodate a long cellulose chain and to cut it internally. This altered
loop region is responsible for the exocellulolytic activity of the enzyme.

PMID: 14756552 [PubMed - indexed for MEDLINE]


268. C R Biol. 2003 Dec;326(12):1175-84.

Replication of hexitol oligonucleotides as a prelude to the propagation of a
third type of nucleic acid in vivo.

Pochet S, Kaminski PA, Van Aerschot A, Herdewijn P, Marliere P.

Unite de chimie organique, URA CNRS 2128, Institut Pasteur, 28, rue du
Docteur-Roux, 75015 Paris, France.

No backbone motif other than phospho-ribose and phospho-deoxyribose has been
found in natural nucleic acids, currently restricting the molecular types of
replicable biopolymers to DNA and RNA. With the aim of propagating and expressing
a third type of nucleic acid in vivo, we assessed the replicability of
polynucleotides with a phospho-hexitol backbone (HNA) in vivo and in vitro.
Faithful polymerisation of up to four deoxynucleotides templated by hexitol
oligonucleotides was established in vitro using DNA polymerase from Escherichia
coli (PolA Klenow exo-fragment) and Thermus aquaticus (Taq polymerase).
Condensation of up to three successive hTTPs (hexitol thymidine triphosphate) in 
responses to a pentameric hexitol template (hA)5 could also be demonstrated in
vitro. Such a marginal HNA-dependent HNA polymerase activity of natural
polymerases may be evolved in the future to catalyse in vitro amplification of
HNA. The transmission of a two-codon-long genetic message carried on a hexameric 
hexitol template was also established using a selection screen for restoring
thymidylate synthase activity in E. coli. These results exemplify the potential
that can be explored by converting artificial substrates with natural enzymes in 
the field of informational polymer synthesis.

PMID: 14746272 [PubMed - indexed for MEDLINE]


269. J Biol Chem. 2004 Apr 16;279(16):16687-96. Epub 2004 Jan 23.

Aromatic residues located close to the active center are essential for the
catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon
Pyrococcus horikoshii.

Matsui E, Abe J, Yokoyama H, Matsui I.

Biological Information Research Center, National Institute of Advanced Industrial
Science and Technology, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan.

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3'
exonuclease activity plays important roles in DNA replication and repair. In this
study, the kinetic parameters of mutants at highly conserved aromatic residues,
Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers 
of FEN-1 were examined. The substitution of these aromatic residues with alanine 
led to a large reduction in kcat values, although these mutants retained Km
values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and
F79A decreased 333-fold and 71-fold, respectively, compared with that of the
wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster
Phe278-Phe279 mainly contributed to the recognition of the substrates without the
3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and
pseudo-Y substrates) for the both exo- and endo-activities, but played minor
roles in recognizing the substrates with the 3' projection (the double flap
substrate and the nick substrate with the 3' projection). The replacement of
Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with
aliphatic hydrophobic residues, recovered the kcat values almost fully for the
substrates without the 3' projection of the upstream strand, suggesting that the 
aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the
catalytic reaction, probably via multiple stacking interactions with nucleotide
bases. The stacking interactions of Tyr33 and Phe79 might play important roles in
fixing the template strand and the downstream strand, respectively, in close
proximity to the active center to achieve the productive transient state leading 
to the hydrolysis.

PMID: 14742430 [PubMed - indexed for MEDLINE]


270. Plant J. 2004 Feb;37(3):391-7.

Trafficking of the plant potassium inward rectifier KAT1 in guard cell
protoplasts of Vicia faba.

Hurst AC, Meckel T, Tayefeh S, Thiel G, Homann U.

Institute of Botany, Technical University of Darmstadt, 64287 Darmstadt, Germany.

Trafficking of K+ inward (Kin+) rectifying channels was analyzed in guard cells
of Vicia faba transfected with the Kin+ rectifier from Arabidopsis thaliana KAT1 
fused to the green fluorescent protein (GFP). Confocal images and whole-cell
patch-clamp measurements confirmed the incorporation of active KAT1 channels into
the plasma membrane of transfected guard cell protoplasts. The Kin+ rectifier
current density of the plasma membrane was much larger in transfected protoplasts
than in wild-type (wt) protoplasts. This shows a coupling between K+ channel
synthesis and incorporation of the channel into the plasma membrane.
Pressure-driven increase and decrease in surface area led to the incorporation
and removal of vesicular membrane carrying active Kin+ rectifier in wt and
transfected protoplasts. These vesicular membranes revealed a higher channel
density than the plasma membrane, suggesting that Kin+ rectifier remains in
clusters during trafficking to and from the plasma membrane. The observed results
can be explained by a model illustrating that vesicles of a pre-plasma membrane
pool carry K+ channels preferentially in clusters during constitutive and
pressure-driven exo- and endocytosis.

PMID: 14731259 [PubMed - indexed for MEDLINE]


271. Inorg Chem. 2004 Jan 26;43(2):584-92.

Platinum complexes with NH groups on the carrier ligand and with only one guanine
or hypoxanthine derivative. Informative models for assessing relative nucleobase 
and nucleotide hydrogen-bond interactions with amine ligands in solution.

Carlone M, Marzilli LG, Natile G.

Dipartimento Farmaco-Chimico, Universita degli Studi di Bari, Via E. Orabona 4,
70125 Bari, Italy.

Complexes of the type syn-(R,S)-Me(3)dienPtL (Me(3)dien = N,N',N'
'-trimethyldiethylenetriamine; L = guanine or hypoxanthine derivative) have two
rotamers, a feature useful for assessing hydrogen-bond interactions between a
Me(3)dien NH group and either the O6 or the phosphate group of the coordinated L.
The two rotamers are defined as endo and exo for the rotamer with the
six-membered ring of the purine on the same side and on the opposite side,
respectively, of the coordination plane as the N-Me's. For L = 5'-GMP and 5'-IMP 
the endo rotamer is the exclusive form (at neutral and basic pH) or is present at
90% and more (low pH where 5'-phosphate group is protonated). A 5'-phosphate
group can be positioned to form a direct H-bond with a Me(3)dien NH group only in
the endo form; such an H-bond explains this high endo preference. Such a direct
phosphate-NH H-bond is not possible for other complexes used in this study
because either L has no phosphate group (9-EtG, Guo) or the phosphate is at the
3'-position (3'-GMP and 3'-IMP), too far for H-bonding. Nevertheless, a
preference for the endo rotamer was observed for these L also. This result is
opposite to that expected both from potential steric repulsion of the L O6 with
the N-Me groups and also from the lack of a potential favorable H-bond
interaction between L O6 and a Me(3)dien NH. For the 9-EtG adduct, the
temperature dependence of the endo/exo equilibrium and the activation parameters 
for endo/exo interconversion suggest that the preference for the endo rotamer
arises from the hydration of the Me(3)dien NH groups; such hydration is favorable
in the endo rotamer. At basic pH, N1H deprotonation increases the H-bond capacity
of O6, and the exo rotamer increases in stability, becoming the dominant rotamer 
for the 9-EtG and Guo adducts. For L = 3'-GMP and 3'-IMP, stabilization of the
endo form upon phosphate deprotonation at neutral pH was observed. This result is
attributed to an H-bonding network involving water, the 3'-phosphate, and the
Me(3)dien NH groups.

PMID: 14731020 [PubMed - indexed for MEDLINE]


272. Org Lett. 2004 Jan 22;6(2):189-92.

Origin of stereochemical reversal in Meyers-type enolate alkylations. Importance 
of intramolecular Li coordination and solvent effects.

Ikuta Y, Tomoda S.

Department of Life Sciences, Graduate School of Arts and Sciences, The University
of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan.

[reaction: see text] The origin of exclusive exo-stereochemistry in the
alkylation of Meyers-type enolate 2 has been studied. It was found that the
intramolecular complex with a strong Li...O(ring) interaction (the O-complex) may
be responsible as the major enolate species in tetrahydrofuran (THF). The
transition state of the O-complex leading to exo-stereochemistry is found to be
the most favorable process in THF.

PMID: 14723525 [PubMed]


273. Curr Genet. 2004 Mar;45(3):140-8. Epub 2004 Jan 10.

Characterization of extracellular lytic enzymes produced by the yeast biocontrol 
agent Candida oleophila.

Bar-Shimon M, Yehuda H, Cohen L, Weiss B, Kobeshnikov A, Daus A, Goldway M,
Wisniewski M, Droby S.

Department of Postharvest Science, A.R.O., The Volcani Center, P.O. Box 6, 50250,
Bet Dagan, Israel.

The yeast Candida oleophila, the base of the commercial product Aspire, is
recommended for the control of postharvest decay of citrus and pome fruit.
Competition for nutrients and space is believed to be the major mode of action.
Involvement of fungal cell wall-degrading enzymes is also suggested to play a
role in the mechanism of action of yeast antagonists. The present study showed
that the yeast C. oleophila is capable of producing and secreting various cell
wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease.
Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early
stages of growth, whereas protease reached a maximum level only after 6-8 days.
Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by
the presence of cell wall fragments of Penicillium digitatum in the growth
medium, in addition to glucose. This study also provided evidence that C.
oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface
of grapefruit. The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol
activity of C. oleophila was tested using CoEXG1-knockouts and double- CoEXG1
over-producing transformants. In vitro bioassays showed that wild-type C.
oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar
inhibitory effects on spore germination and germ-tube elongation; and both were
more inhibitory to the fungus than the knockout transformant. In experiments
conducted on fruit to test the biocontrol activity against infection by P.
digitatum, no significant difference in inhibition was observed between
transformants and untransformed C. oleophila cells at the high concentrations of 
cells used, whereas at a lower concentration of yeast cells the knockout
transformants appeared to be less effective.

PMID: 14716497 [PubMed - indexed for MEDLINE]


274. Acta Crystallogr C. 2004 Jan;60(Pt 1):o94-7. Epub 2003 Dec 20.

Regioisomeric 4-nitroindazole N1- and N2-(beta-D-ribonucleosides).

Seela F, Peng X, Eickmeier H, Reuter H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.
frank.seela@uni-osnabrueck.de

The structures of the isomeric nucleosides
4-nitro-1-(beta-D-ribofuranosyl)-1H-indazole, C(12)H(13)N(3)O(6), (I), and
4-nitro-2-(beta-D-ribofuranosyl)-2H-indazole, C(12)H(13)N(3)O(6), (II), have been
determined. For compound (I), the conformation of the glycosylic bond is anti
[chi = -93.6 (6) degrees ] and the sugar puckering is C2'-exo-C3'-endo. Compound 
(II) shows two conformations in the crystalline state which differ mainly in the 
sugar pucker; type 1 adopts the C2'-endo-C3'-exo sugar puckering associated with 
a syn base orientation [chi = 43.7 (6) degrees ] and type 2 shows
C2'-exo-C3'-endo sugar puckering accompanied by a somewhat different syn base
orientation [chi = 13.8 (6) degrees ].

PMID: 14712059 [PubMed - indexed for MEDLINE]


275. Chem Commun (Camb). 2003 Dec 21;(24):2990-1.

Exo-metal coordination by a tricyclic [(P(mu-N-2-NC5H4))2(mu-O)]2 dimer in
[(P(mu-N-2-NC5H4))2(mu-O)]2(CuCl x (C5H5N)2)4 (2-NC5H4 = 2-pyridyl, C5H5N =
pyridine).

Bond AD, Doyle EL, Garcia F, Kowenicki RA, McPartlin M, Riera L, Wright DS.

Chemistry Department, Cambridge University, Lensfield Road, Cambridge, UK CB2
1EW.

The in situ reaction of the phosphazane dimer [CIP(mu-N-2-NC5H4)]2 (2) with CuCl 
in the presence of CsH5N/H2O gives the title complex
[(P(mu-N-2-NC5H4))2(mu-O)]2(CuCl x (C5H5N)2)4 (1), containing a tricyclic
[(P(mu-N-2-NC5H4))2(mu-O)]2 ligand which is isoelectronic with species of the
type [(P(mu-NR))2NR]2.

PMID: 14703822 [PubMed]


276. J Bacteriol. 2004 Jan;186(2):535-42.

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234.

Streit WR, Schmitz RA, Perret X, Staehelin C, Deakin WJ, Raasch C, Liesegang H,
Broughton WJ.

Institut fur Mikrobiologie und Genetik, Universitat Gottingen, Gottingen,
Germany. wstreit@gwdg.de

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to 
nodulate more than 112 genera of legumes. Since the overall organization of the
NGR234 genome is strikingly similar to that of the narrow-host-range symbiont
Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the
obvious question is why are the spectra of hosts so different? Study of the early
symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide
obvious answers. Yet, both rhizobia also possess second megaplasmids that bear,
among many other genes, those that are involved in the synthesis of extracellular
polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions
and thus may help answer the broad- versus narrow-host-range question.
Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open
reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high 
similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the
genetic information may have been acquired from a common ancestor. The largest
conserved cluster carried the exo and exs genes and contained 31 ORFs. In
addition, nine highly conserved regions with high similarity to Agrobacterium
tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain 
MAFF303099, as well as two conserved clusters that are highly homologous to
similar regions in the plant pathogen Erwinia carotovora, were identified.
Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b
genes are not strain specific and were probably acquired from a wide variety of
other microbes. The presence of 26 ORFs coding for transposases and site-specific
integrases supports this contention. Surprisingly, several genes involved in the 
degradation of aromatic carbon sources and genes coding for a type IV pilus were 
also found.

PMCID: PMC305759
PMID: 14702322 [PubMed - indexed for MEDLINE]


277. Biochem Pharmacol. 2004 Jan 15;67(2):293-302.

Structure-activity relationships for substrate recognition by the human dopamine 
transporter.

Appell M, Berfield JL, Wang LC, Dunn WJ 3rd, Chen N, Reith ME.

Department of Biomedical and Therapeutic Sciences, University of Illinois College
of Medicine, Box 1649, Peoria, IL 61656-1649, USA.

Information is available on the structure-activity relationships for dopamine as 
a substrate for uptake by the dopamine transporter. However, dopamine transport
is a complex process involving substrate binding, translocation, release as well 
as transporter reorientation. The present study examines only the substrate
recognition step by assessment of the potency of various dopamine-related
compounds in inhibiting the binding of the cocaine analog
[3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([3H]WIN 35,428) to human
dopamine transporters expressed in HEK-293 cells. alpha-Methylation of the side
chain, the presence of the amine, and the 2-carbon-length of the side chain were 
found to be important for binding affinity, whereas beta-hydroxylation of the
side chain and methoxylation at the phenyl ring generated weaker compounds. In
addition, the presence of both m- and p-OH at the phenyl ring bestowed an
increase in potency but the presence of p-OH alone a decrease. N-alkylation
(propylation or methylation) had little or an even slightly beneficial effect on 
affinity, whereas alpha-carbonylation and alpha-methanoylation reduced affinity. 
Amino naphthalene compounds with a fused benzenoid ring system retained some
potency consonant with the extended (i.e. beta-rotameric) trans (=anti) form of
the side chain in dopamine when interacting with the transporter. In a second
series of experiments, the interaction between dopamine and structural variants
was assessed by monitoring the capability of a compound to shift the dopamine
inhibition curve to the right as expected for a competitive inhibitor acting at
the same site. Appreciable deviation from competitive interaction was observed by
removal of the amine from the side chain, by alpha-carbonylation, and by
alpha-methanoylation. Two blocker-type compounds, semi-rigid variants of cocaine,
also displayed significant deviation. A substrate-based compound, inhibiting
cocaine analog binding without interfering with dopamine recognition, could be a 
cocaine antagonist allowing conformational changes to occur during dopamine
uptake.

PMID: 14698042 [PubMed - indexed for MEDLINE]


278. J Org Chem. 2003 Dec 26;68(26):10079-86.

Mechanistic studies of the biomimetic epoxy ester-orthoester and
orthoester-cyclic ether rearrangements.

Giner JL, Li X, Mullins JJ.

Department of Chemistry, State University of New York-ESF, Syracuse, New York
13210, USA. jlginer@syr.edu

The relative rates of acid-catalyzed rearrangements of epoxy esters to
[3.2.1]bicyclic orthoesters, the subsequent rearrangements of these ortho esters 
to substituted tetrahydrofurans, and the rates of orthoester hydrolysis at pH
4.75 were measured in NMR kinetics experiments. The ease of formation and
stabilities of these orthoesters compared favorably with the OBO-type
[2.2.2]bicyclic orthoesters typically used as protecting groups of carboxylic
acids. Studies with 13C NMR-detected 18O-labeling show that epoxy ester
rearrangement takes place preferentially via 6-exo cyclization, although the
7-endo process competes when the distal center of the epoxide is disubstituted.
The ortho ester-cyclic ether rearrangement was shown by 18O-labeling to occur
exclusively via intermediacy of a five-membered dioxonium ion. The structures of 
the hydrolysis products also indicate the intermediacy of a dioxolanium ion
during hydrolysis. The implications for a hypothetical biosynthesis of marine
polyether toxins are discussed.

PMID: 14682703 [PubMed - indexed for MEDLINE]


279. J Org Chem. 2003 Dec 26;68(26):9916-23.

Phosphonyl, phosphonothioyl, phosphonodithioyl, and phosphonotrithioyl radicals: 
generation and study of their addition onto alkenes.

Lopin C, Gouhier G, Gautier A, Piettre SR.

Laboratoire des Fonctions Azotees et Oxygenees Complexes, UMR CNRS 6014,
IRCOF-Universite de Rouen, Rue Tesnieres, F-76821 Mont Saint Aignan, France.

The treatment of benzyl dialkyl phosphites and dithiophosphites with
benzeneselanyl chloride generates an Arbuzov-type transformation leading to the
dialkyl selenophosphates 19a and 19b and to selenophosphorodithioates 21a and
21b. Interaction of these substrates with Lawesson's reagent yields the
corresponding selenophosphorothioates 20a and 20b and the
selenophosphorotrithioates 22a and 22b. When treated with a radical initiator in 
the presence of a hydrogen donor and an alkene, all eight phosphorus(V)
precursors undergo homolytic cleavage of the P-Se bond to generate the
phosphonyl, phosphonothioyl, phosphonodithioyl, or phosphonotrithioyl radicals.
Most of these are shown to add onto electron-rich and electron-poor alkenes to
deliver the expected adducts in fair to excellent yields. Cyclic precursor 19b
displays peculiar behavior and, under the reaction conditions, produces only the 
corresponding cyclic phosphite. Application of this radical chain process is
carried out on furanosyl 3-exo-methylene derivative 37 to diastereoselectively
furnish five new 3-phosphonomethyl-, 3-phosphonothiomethyl-, and
3-phosphonodithiomethyl-3-deoxofuranoses 38a-c and 38f,g. The possibility of
conducting tandem processes is also discussed through experiments involving
(1R)-(+)-alpha-pinene (39) and diallylamine 41.

PMID: 14682683 [PubMed - indexed for MEDLINE]


280. Anal Biochem. 2004 Jan 1;324(1):22-8.

Pyrophosphorolysis by Type II DNA polymerases: implications for
pyrophosphorolysis-activated polymerization.

Liu Q, Sommer SS.

Department of Molecular Genetics and Department of Molecular Diagnosis, City of
Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010-3000, USA.

We find that Type II DNA polymerases can catalyze pyrophosphorolysis, the reverse
reaction of DNA polymerization. This property is applied utilizing
pyrophosphorolysis-activated polymerization (PAP), a method of nucleic acid
amplification using serial coupling of pyrophosphorolysis and polymerization. PAP
can be used for ultrarare allele detection (detection of minimal residual disease
and cancer risk assessment through measurement of mutation load) and for
microarray-based scanning for unknown mutations. Herein, we show that Type II DNA
polymerases efficiently catalyze template-dependent pyrophosphorolysis to
activate oligonucleotides blocked at their 3' termini with acyclonucleotides in
which a 2-hydroxyethoxymethyl group substitutes for the 2'-deoxyribofuranosyl
sugar. Type II archeon DNA polymerases Vent (exo-) and Pfu (exo-) can be utilized
for PAP or a bidirectional form of PAP with acyclonucleotide-blocked
oligonucleotides, but not with dideoxynucleotide-blocked oligonucleotides. In
contrast, a Type I DNA polymerase, TaqFS, can utilize either
acyclonucleotide-blocked or dideoxynucleotide-blocked oligonucleotides. These
findings expand the potential of nascent PAP technology.

PMID: 14654041 [PubMed - indexed for MEDLINE]


281. J Am Chem Soc. 2003 Dec 10;125(49):15028-38.

What is so special about Arg 55 in the catalysis of cyclophilin A? insights from 
hybrid QM/MM simulations.

Li G, Cui Q.

Department of Chemistry and Theoretical Chemistry Institute, University of
Wisconsin, Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.

Potential of mean force (PMF) simulations with a hybrid QM/MM potential function 
were used to analyze the catalytic mechanism of human cyclophilin A (CypA). PMF
calculations were performed for proline isomerization of peptides in solution,
the wild-type CypA, and several CypA mutants. With an approximate density
functional theory, the self-consistent-charge density functional tight binding
(SCC-DFTB) as the QM level, and CHARMM 22 force field as MM, satisfactory
energetics compared to available experiments were obtained. Calculations for the 
Arg55Ala and zero-charge-Arg55 mutants clearly indicated that Arg 55
significantly stabilizes the isomerization transition state through electrostatic
interactions. However, the decrease in the average distance (thus the increase in
interaction) between Arg 55 and the substrate amide N in going from the stable
states to the transition state is mainly due to the pyramidalization of the amide
N rather than motions associated with Arg 55. Although the nanosecond simulations
cannot exclude the existence of sub-millisecond collective motions proposed on
the basis of recent elegant NMR relaxation and line-shape analyses, the
energetics obtained for the various enzyme systems here indicate that the
contribution from motions of active site residues to catalysis is expected to be 
small. Instead, the present simulations support that the structural stability
rather than mobility of the preorganized active site is more important. Through
hydrogen-bonding interactions among the substrate, Arg 55, Gln 63, and Asn 102,
the active site of the wild-type enzyme is structurally very stable and puts Arg 
55 in a favorable position to perform its catalytic role in the transition state.
This is further illustrated with the somewhat unexpected prediction that Arg55Lys
is largely catalytically inactive, because Lys does not have the unique
bifurcating construct of the guanidino group in Arg and thus the active site of
Arg55Lys cannot accommodate Lys in a position capable of providing electrostatic 
stabilization of the isomerization transition state. Among all the enzyme systems
studied, the wild-type CypA is the only one that selects the syn/exo transition
state, while the syn/endo conformation is also present in the mutants, which is
another reason for their higher barriers. Finally, the present analysis indicated
that the population of near-attack-conformations (NAC) is not relevant to
catalysis in CypA.

PMID: 14653737 [PubMed - indexed for MEDLINE]


282. J Am Chem Soc. 2003 Nov 19;125(46):14059-64.

Derivatization of deltahedral Zintl ions by nucleophilic addition:
[Ph-Ge9-SbPh2]2- and [Ph2Sb-Ge9-Ge9-SbPh2]4-.

Ugrinov A, Sevov SC.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame,
IN 46556, USA.

The type of the reactions of addition of exo-bonded groups to deltahedral Zintl
ions such as Ge9(n-) has been established as addition of anionic nucleophiles.
Various nucleophiles such as Ph2Bi-, Ph2Sb-, Ph- interact with the relatively
low-lying LUMO of Ge9(2-) and/or the half filled HOMO of Ge9(3-) and bond to the 
clusters. The title anions, characterized in their (K-crypt) salts where crypt = 
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo-[8.8.8]-hexacosane, and the previously 
characterized [Ph2Sb-Ge9-SbPh2](2-) are made by a reaction of K4Ge9 with SbPh3 in
ethylenediamine. [Ph-Ge9-SbPh2](2-) is the first ogranically functionalized
deltahedral Zintl ion, i.e., a deltahedral ion with a direct carbon-cluster
covalent bond, that can exists without the substituents as well. The Ge(9)
clusters resemble tricapped trigonal prisms with one elongated edge (one of the
three edges parallel to the pseudo 3-fold axis). The two substituents are always 
bonded to the vertexes of such an elongated edge. The same is true for the
intercluster bond in [Ph2Sb-Ge9-Ge9-SbPh2)](4-).

PMID: 14611242 [PubMed]


283. Can J Microbiol. 2003 Aug;49(8):514-24.

Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta,
Lepidoptera, Galleridae).

Dunphy GB, Oberholzer U, Whiteway M, Zakarian RJ, Boomer I.

Department of Natural Resource Sciences, Macdonald Campus of McGill University,
Sainte Anne de Bellevue, QC, Canada.

Culture medium affected the virulence of a strain of Candida albicans toward
Galleria mellonella larvae, but the yeast growth rates in yeast extract - peptone
- dextrose broth and synthetic Galleria serum were not correlated with yeast
virulence. Virulent C. albicans grew rapidly in larval serum, whereas, it limited
nodulation and continued development in vivo, producing toxins that damaged the
hemocytes and fat body. Nonpathogenic yeast-phase cells grew slowly in larval
serum but induced extensively melanized nodules in vivo and developed no further.
There was no discernible relationship in 14 exo-enzymes between the virulent and 
avirulent yeast strains and virulence. The avirulent myosin-I-defective yeast
cells were rapidly removed from the hemolymph in vivo because of
lysozyme-mediated yeast agglutination and the possible binding of the yeast cells
by lysozyme and apolipophorin-III. Both lysozyme and apolipophorin-III are
proteins that bind beta-1,3-glucan. Finally, insects with nonpathogenic C.
albicans exhibited induced immunity and were more resistant to candidiasis from
the wild-type yeast cells than were noninduced insects.

PMID: 14608387 [PubMed - indexed for MEDLINE]


284. J Org Chem. 2003 Nov 14;68(23):8798-807.

Photoactivated tungsten hexacarbonyl-catalyzed conversion of alkynols to glycals.

Wipf P, Graham TH.

Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
pwipf+@pitt.edu

The photoactivated W(CO)(6)/DABCO/THF system has been used for the formal
endo-cyclization of alkynes to pyran rings. We found that the regioselectivity of
ring closure depends on the relative configuration of the
3,5-dihydroxy-1-alkynes, as well as, more decisively, on the type of O-protective
group. Oxygen substitution at the propargylic carbon slows the rate of alkyne
insertion and allows for dihydrofuran formation through exo-cyclization. In
contrast, the use of bulky silyl ethers or carbon substituents leads to
dihydropyrans through endo-cyclization. Substrates bearing leaving groups such as
esters, phenols, or thiophenols at the propargylic site eliminate and thus
represent a limitation to the cycloisomerization methodology. Propargyl vinyl
ethers will rearrange to give dienals instead of glycals. 1,2-Wittig
rearrangement products of dihydropyrans are readily prepared and converted to
complex bicyclic building blocks for organic synthesis.

PMID: 14604347 [PubMed - indexed for MEDLINE]


285. Anal Sci. 2003 Oct;19(10):1461-2.

Crystal structure of 6-oxa-3,9-dithiabicyclo[9.2.2]pentadeca-1(15),11,13-triene.

Yoon I, Park KM, Kim J, Kim BG, Lee SS.

Department of Chemistry and Research Institute of Natural Sciences, Gyeongsang
National University, Chinju 66-701, S. Korea.

The structure of an S2O mixed-donor macrocycle incorporating para-substituted
xylyl subunit was characterized by a single crystal X-ray analysis. The crystal
structure shows an exo-dentate orientation of sulfur and oxygen donor atoms due
to the fully stretched aliphatic ring conformation. Thus all of torsional angles 
between donor atoms are arranged anti and S...S distance in a ring is 6.764(1)A, 
which is larger than those values of ortho- and meta-type analogs. The observed
high rigidity of the ring conformation may induce the extraordinary behaviors in 
chelation.

PMID: 14596418 [PubMed]


286. Prikl Biokhim Mikrobiol. 2003 Sep-Oct;39(5):542-8.

[O-glycosylhydrolases of marine filamentous fungi. beta-1,3-Glucanases of
Trichoderma aureviride]

[Article in Russian]

Burtseva IuV, Verigina NS, Sova VV, Pivkin MV, Zviagintseva TN.

Pacific Institute of Bioorganic Chemistry, Far-Eastern Division of the Russian
Academy of Sciences, Vladivostok, 690022 Russia.

The ability to produce extracellular O-glycosylhydrolases was studied in 14
strains of marine filamentous fungi sampled from bottom sediments of the South
China Sea. The following activities were detected in the culture liquids of the
fungi: N-acetyl-D-glucosaminidase, D-glucosidase, D-galactosidase,
beta-1,3-glucanase, amylase, and pustulanase. beta-1,3-Glucanases were isolated
by ultrafiltration, hydrophobic interaction chromatography, and ion-exchange
chromatography, and their properties were studied. Data on products of enzymatic 
digestion of laminaran, absence of transglycosylation activity, and the pattern
of action of natural inhibitors confirmed that beta-1,3-glucanase belonged to the
exo type. Inhibitor analysis demonstrated the role of a thiol group and
tryptophan and tyrosine residues in the catalytic activity.

PMID: 14593867 [PubMed - indexed for MEDLINE]


287. J Exp Bot. 2003 Dec;54(393):2615-22. Epub 2003 Oct 29.

Characterization of expression, and cloning, of beta-D-xylosidase and
alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon
esculentum Mill.) fruit.

Itai A, Ishihara K, Bewley JD.

Laboratory of Horticultural Science, Faculty of Agriculture, Tottori University, 
Tottori, 680-8553 Japan. itai@muses.tottori-u.ac.jp

Modifications to the cell wall of developing and ripening tomato fruit are
mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or
alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or 
arabinoxylans. The activity of both enzymes was highest during early fruit
growth, before decreasing during later development and ripening. Two
beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an
alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation 
of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined
during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively
highly expressed during fruit development and decreased after the onset of
ripening. By contrast, LeXYL1 was not expressed during fruit development, but was
expressed later, particularly during over-ripening. The expression of all three
genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of
cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the
mutants and its abundance was similar to that in mature green wild-type fruit. By
contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant,
suggesting that the two beta-d-xylosidase genes are subject to distinct
regulatory control during fruit development and ripening. LeARF1 mRNA was
detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr
mutant. The accumulation of LeARF1 in ripe fruit was restored by
1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no
effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1
expression is subject to negative regulation by ethylene and that the two
beta-d-xylosidase genes are independent of ethylene action.

PMID: 14585820 [PubMed - indexed for MEDLINE]


288. J Mol Biol. 2003 Oct 31;333(4):817-29.

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A
comparison with Phanerochaete chrysosporium Cel7D.

von Ossowski I, Stahlberg J, Koivula A, Piens K, Becker D, Boer H, Harle R,
Harris M, Divne C, Mahdi S, Zhao Y, Driguez H, Claeyssens M, Sinnott ML, Teeri
TT.

VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland.

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the 
active site tunnel at the catalytic centre. Mutants were designed to study the
role of this loop in crystalline cellulose degradation. A hydrogen bond to
substrate made by a tyrosine at the tip of the loop was removed by the Y247F
mutation. The mobility of the loop was reduced by introducing a new disulphide
bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant
Delta(G245-Y252). No major structural disturbances were observed in the mutant
enzymes, nor was the thermostability of the enzyme affected by the mutations. The
Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but
only a small effect on cellulose hydrolysis. Deletion of the tip of the loop
increased both k(cat) and K(M) and gave reduced product inhibition. Increased
activity was observed on amorphous cellulose, while only half the original
activity remained on crystalline cellulose. Stabilisation of the exo-loop by the 
disulphide bridge enhanced the activity on both amorphous and crystalline
cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is
indicative of processive action, was highest with Tr Cel7A wild-type enzyme and
smallest with the deletion mutant on both substrates. Based on these data it
seems that the exo-loop of Tr Cel7A has evolved to facilitate processive
crystalline cellulose degradation, which does not require significant
conformational changes of this loop.

PMID: 14568538 [PubMed - indexed for MEDLINE]


289. J Am Chem Soc. 2003 Oct 15;125(41):12584-605.

Organolathanide-catalyzed regioselective intermolecular hydroamination of
alkenes, alkynes, vinylarenes, di- and trivinylarenes, and
methylenecyclopropanes. Scope and mechanistic comparison to intramolecular
cyclohydroaminations.

Ryu JS, Li GY, Marks TJ.

Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, 
USA.

Organolanthanide complexes of the type Cp'(2)LnCH(SiMe(3))(2) (Cp' =
eta(5)-Me(5)C(5); Ln = La, Nd, Sm, Lu) and Me(2)SiCp' '(2)LnCH(SiMe(3))(2) (Cp' '
= eta(5)-Me(4)C(5); Ln = Nd, Sm, Lu) serve as efficient precatalysts for the
regioselective intermolecular hydroamination of alkynes R'Ctbd1;CMe (R' =
SiMe(3), C(6)H(5), Me), alkenes RCH=CH(2) (R = SiMe(3), CH(3)CH(2)CH(2)),
butadiene, vinylarenes ArCH=CH(2) (Ar = phenyl, 4-methylbenzene, naphthyl,
4-fluorobenzene, 4-(trifluoromethyl)benzene, 4-methoxybenzene,
4-(dimethylamino)benzene, 4-(methylthio)benzene), di- and trivinylarenes, and
methylenecyclopropanes with primary amines R' 'NH(2) (R' ' = n-propyl, n-butyl,
isobutyl, phenyl, 4-methylphenyl, 4-(dimethylamino)phenyl) to yield the
corresponding amines and imines. For R = SiMe(3), R = CH(2)=CH
lanthanide-mediated intermolecular hydroamination regioselectively generates the 
anti-Markovnikov addition products (Me(3)SiCH(2)CH(2)NHR' ',
(E)-CH(3)CH=CHCH(2)NHR' '). However, for R = CH(3)CH(2)CH(2), the Markovnikov
addition product is observed (CH(3)CH(2)CH(2)CHNHR' 'CH(3)). For internal
alkynes, it appears that these regioselective transformations occur under
significant stereoelectronic control, and for R' = SiMe(3), rearrangement of the 
product enamines occurs via tautomerization to imines, followed by a
1,3-trimethylsilyl group shift to stable N-SiMe(3)-bonded CH(2)=CMeN(SiMe(3))R' '
structures. For vinylarenes, intermolecular hydroamination with n-propylamine
affords the anti-Markovnikov addition product beta-phenylethylamine. In addition,
hydroamination of divinylarenes provides a concise synthesis of
tetrahydroisoquinoline structures via coupled intermolecular
hydroamination/subsequent intramolecular cyclohydroamination sequences.
Intermolecular hydroamination of methylenecyclopropane proceeds via highly
regioselective exo-methylene C=C insertion into Ln-N bonds, followed by
regioselective cyclopropane ring opening to afford the corresponding imine. For
the Me(2)SiCp' '(2)Nd-catalyzed reaction of Me(3)SiCtbd1;CMe and
H(2)NCH(2)CH(2)CH(2)CH(3), DeltaH() = 17.2 (1.1) kcal mol(-)(1) and DeltaS() =
-25.9 (9.7) eu, while the reaction kinetics are zero-order in [amine] and
first-order in both [catalyst] and [alkyne]. For the same substrate pair,
catalytic turnover frequencies under identical conditions decrease in the order
Me(2)SiCp' '(2)NdCH(SiMe(3))(2) > Me(2)SiCp' '(2)SmCH(SiMe(3))(2) > Me(2)SiCp'
'(2)LuCH(SiMe(3))(2) > Cp'(2)SmCH(SiMe(3))(2), in accord with documented steric
requirements for the insertion of olefinic functionalities into lanthanide-alkyl 
and -heteroatom sigma-bonds. Kinetic and mechanistic evidence argues that the
turnover-limiting step is intermolecular C=C/Ctbd1;C bond insertion into the Ln-N
bond followed by rapid protonolysis of the resulting Ln-C bond.

PMID: 14531704 [PubMed]


290. Chem Commun (Camb). 2003 Sep 21;(18):2346-7.

Synthesis of novel 2,6-disubstituted-3,4-dimethylidene tetrahydropyrans via
Prins-type cyclization.

Cho YS, Karupaiyan K, Kang HJ, Pae AN, Cha JH, Koh HY, Chang MH.

Biochemicals Research Center, Korea Institute of Science and Technology, P.O. Box
131, Cheongryang, Seoul 130-650, Korea.

Synthesis of novel substituted tetrahydropyrans with adjacent exo-methylene
groups at the C3 and C4 positions via Prins-type cyclization has been described.

PMID: 14518907 [PubMed]


291. Biosci Biotechnol Biochem. 2003 Aug;67(8):1852-6.

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of 
insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in
N-glycan processing of royal jelly glycoproteins.

Kimura Y, Tsumura K, Kimura M, Okihara K, Sugimoto H, Yamada H.

Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Tsushima-Naka 1-1-1, Okayama 700-8530, Japan. yosh8mar@cc.okayama-u.ac.jp

On a way of structural analysis of total N-glycans linked to glycoproteins in
royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120
(2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)),
we found that some complex type N-glycans containing a beta1-3galactose residue
occur on the insect glycoproteins. Up to date, it has been considered that
naturally occurring insect glycoproteins do not bear the galactose-containing
N-glycans, therefore, in this report we describe the structural analysis of the
complex type N-glycans of royal jelly glycoproteins. By a combination of endo-
and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the
structures of the beta1-3 galactose-containing N-glycan were identified as the
following;
GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbet
a1-4GlcNAcbeta1-4GlcNAc,
Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1
-4GlcNAcbeta1-4GlcNAc, and
Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalph
a1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report
showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect
glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc
transferase (GNT-IV) are expressed in the honeybee cells.

PMID: 12951530 [PubMed - indexed for MEDLINE]


292. J Virol. 2003 Sep;77(18):10147-53.

3' to 5' exonuclease activity of herpes simplex virus type 1 DNA polymerase
modulates its strand displacement activity.

Zhu Y, Trego KS, Song L, Parris DS.

Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State
University, 333 West Tenth Avenue, Columbus, OH 43210, USA.

Using a minicircle DNA primer-template, the wild-type catalytic subunit of herpes
simplex virus type 1 (HSV-1) DNA polymerase (pol) was shown to lack significant
strand displacement activity with or without its processivity factor, UL42.
However, an exonuclease-deficient (exo(-)) pol (D368A) was capable of slow strand
displacement. Although UL42 increased the rate (2/s) and processivity of strand
displacement by exo(-) pol, the rate was slower than that for gap-filling
synthesis. High inherent excision rates on matched primer-templates and rapid
idling-turnover (successive rounds of excision and polymerization) of
exo-proficient polymerases correlated with poor strand displacement activity. The
results suggest that the exo activity of HSV-1 pol modulates its ability to
engage in strand displacement, a function that may be important to the viability 
and genome stability of the virus.

PMCID: PMC224577
PMID: 12941927 [PubMed - indexed for MEDLINE]


293. Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3892-8.

Contribution of ExsA-regulated factors to corneal infection by cytotoxic and
invasive Pseudomonas aeruginosa in a murine scarification model.

Lee EJ, Cowell BA, Evans DJ, Fleiszig SM.

Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of
Optometry, University of California, Berkeley, California 94720, USA.

PURPOSE: The exoenzyme S regulatory protein ExsA regulates a type III secretion
system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to 
secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their
phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS,
but exsA mutation has little impact on their short-term interactions with
epithelia. In the present study, the contribution of these ExsA-regulated
proteins toward corneal infections in vivo was investigated. METHODS: After
anesthesia, the left cornea of C57BL/6 mice was scratch injured and then
inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with
isogenic mutants in exsA-related genes. Inocula of 10(3) to 10(6) bacteria/5
micro L were used, and at least five animals were assigned to each experimental
group. Corneal disease was quantified at regular intervals for 14 days in masked 
fashion with two different scoring systems. RESULTS: For the cytotoxic strain,
mutation of either exoU or exoT alone had little effect on virulence, whereas
simultaneous mutation of both exoT and exoU or of exsA resulted in a
significantly reduced capacity to cause corneal disease. Complementation of the
double exoUexoT mutant with exoU alone restored bacterial colonization levels
(>3-log increase) and disease severity to wild-type levels. Complementation with 
exoT alone increased colonization ( approximately 3-log increase) and increased
virulence to almost the same levels as wild-type or exoU-complemented infections.
Virulence of the invasive strain was not reduced by mutation of exsA or of genes 
encoding the ExsA-regulated secreted proteins. CONCLUSIONS: ExsA contributed to
corneal virulence of only cytotoxic P. aeruginosa, with contributions made by
both ExoU and ExoT to bacterial survival and disease severity. This differs from 
cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.

PMID: 12939306 [PubMed - indexed for MEDLINE]


294. J Biol Chem. 2003 Nov 7;278(45):44128-38. Epub 2003 Aug 22.

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals 
similarities to the DNA exonuclease of the human Werner syndrome protein.

Plchova H, Hartung F, Puchta H.

Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466
Gatersleben, Germany.

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and
exonuclease activities is essential for genome stability. Plants have no
homologue of this bifunctional protein, but surprisingly the Arabidopsis genome
contains a small open reading frame (ORF) (AtWRNexo) with homology to the
exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed
an exonuclease activity for AtWRN-exo-p with similarities but also some
significant differences to hWRN-p. The protein digests recessed strands of DNA
duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended
duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to
digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is
degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand
termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent
cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites,
cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and
8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including
uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of
a conserved residue within the exonuclease domain (E135A) completely abolished
the exonucleolytic activity. Our results indicate that a type of WRN-like
exonuclease activity seems to be a common feature of the DNA metabolism of
animals and plants.

PMID: 12937173 [PubMed - indexed for MEDLINE]


295. Org Biomol Chem. 2003 Jan 21;1(2):328-37.

The first synthetic studies on pestalotiopsin A. A stereocontrolled approach to
the functionalised bicyclic core.

Johnston D, Couche E, Edmonds DJ, Muir KW, Procter DJ.

Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow,
UK G12 8QQ.

Pestalotiopsin A is a structurally unique, caryophyllene-type sesquiterpene which
has shown immunosuppressive activity and cytotoxicity in preliminary assays. A
stereocontrolled approach to the functionalised 2-oxabicyclo[3.2.0]-heptane core 
of pestalotiopsin A is described. This constitutes the first synthetic studies on
pestalotiopsin A. Our approach includes a samarium(II)-mediated 4-exo-trig
cyclisation and a trans-lactonisation process triggered by the addition of
alkylytterbium reagents to a cyclobutanone intermediate. Further manipulation
provides access to advanced intermediates which are excellent precursors for the 
future construction of the final ring of the target.

PMID: 12929428 [PubMed - indexed for MEDLINE]


296. J Mol Biol. 2003 Aug 22;331(4):781-94.

A conserved insertion in protein-primed DNA polymerases is involved in primer
terminus stabilisation.

Dufour E, Rodriguez I, Lazaro JM, de Vega M, Salas M.

Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Instituto de Biologia
Molecular Eladio Vinuela (CSIC), Universidad Autonoma, Cantoblanco, 28049 Madrid,
Spain.

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA
polymerases family, also called family B or alpha-like. A multiple amino acid
sequence alignment of this subgroup of DNA polymerases led to the identification 
of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed
previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved
in the correct orientation of the terminal protein (TP) for the initiation of
replication. In this work, the functional role of two other conserved residues
from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives
constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5'
exonuclease activity on single-stranded DNA. However, when assayed on
double-stranded DNA such activity was higher than that of the wild-type enzyme.
This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising 
the primer terminus at the polymerase active site. On the other hand, although
mutant polymerases K305I and Y315A were able to couple processive DNA
polymerisation to strand displacement, they were severely impaired in phi29
TP-DNA replication. The possible role of the TPR-1 insertion in the set of
interactions with the nascent chain during the first steps of TP-DNA replication 
is discussed.

PMID: 12909010 [PubMed - indexed for MEDLINE]


297. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2000 Apr;22(2):111-4.

[Expression and deletion analysis of EcoR II endonuclease and methylase gene]

[Article in Chinese]

Liu J, Zhao X, Meng Y, Shen J, Xue Y, Shi S, Cai Y.

Department of Genetics, Institute of Laboratory Animal Science, PUMC, CAMS,
Beijing, 100021, China.

OBJECTIVE: To clone complete EcoR II restriction endonuclease gene (ecoR II R)
and methyl-transferase (ecoR II M) gene into one vector and to analyzing the
expression of the whole system. METHODS: Unidirective deletion subclones
constructed with Exo III, ecoR II R/M genes were preliminarily located in the
cloned fragments according to the enzyme activities of each subclone, exact
deletion sites were determined by sequencing, and transcriptional start sites
were mapped by S1 mapping. RESULTS: The DNA fragment which was cloned into
pBluescript SK+ contained the complete ecoR II R gene and ecoR II M gene, there
are two transcriptional start sites in ecoR II R gene, 132 bp to 458 bp from 3'
and of ecoR II R gene are indispensable to enzyme activities and deletion of 202 
bp from 3' end of ecoR II M gene made it lose the capability to resist specific
cut of EcoR II R enzyme, deletion of coding region and flanking sequence of one
gene did not affect the expression of the other gene, the recombinant only
containing ecoR II R gene appeared to be lethal to dcm + host. CONCLUSIONS: ecoR 
II M gene closely linking to ecoR II R gene was very important for the existence 
of the R-M system in process of evolution, but the key to control EcoR II R
enzyme acted later than EcoR II M enzyme did not exist in transcriptional level.

PMID: 12903509 [PubMed - indexed for MEDLINE]


298. Nucleic Acids Res Suppl. 2002;(2):191-2.

Theoretical study of substitution effect of the hydrogen bond stability of
9-methylguanine derivatives and 1-methylcytosine.

Kawahara S, Sekine M, Taira K, Kobayashi H, Uchimaru T.

National Institute of Advanced Industrial Science and Technology (AIST), National
Food Research Institute, Biological Function Division, Molecular Function
Laboratory, Tsukuba, Ibaraki, Japan.

The substitution effect on hydrogen bond stability of the Watson-Crick type base 
pair between 1-methylcytosine (C) and substitution-introduced 9-methylguanine
derivatives (Gx) was studied by an ab initio molecular orbital theory.
Introduction of an electron-withdrawing group on the 8-position or on the
exo-cyclic amino moiety enforced the base pair stability.

PMID: 12903170 [PubMed - indexed for MEDLINE]


299. J Infect Dis. 2003 Aug 15;188(4):512-8. Epub 2003 Jul 23.

Genotypic and phenotypic analysis of type III secretion system in a cohort of
Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association
between O serotypes and exo genes.

Berthelot P, Attree I, Plesiat P, Chabert J, de Bentzmann S, Pozzetto B, Grattard
F; Groupe d'Etudes des Septicemies a Pseudomonas aeruginosa.

Microbiology Laboratory, Groupe Immunite des Muqueuses et Agents Pathogenes,
University Hospital of Saint-Etienne, Saint-Etienne, France.

The type III secretion system (TTSS) of Pseudomonas aeruginosa was characterized 
genetically and phenotypically in 92 epidemiologically unrelated bacteremic
strains. Four groups of strains (TTSS types) were defined according to the level 
of type III protein secretion and kinetics of cytotoxicity. Type 1 strains (n=26)
were highly and rapidly cytotoxic and secreted ExoU, type 2 strains (n=48)
exhibited slower cytotoxic rates and expressed ExoS but not ExoU, type 3 strains 
(n=14) were poorly cytotoxic, and type 4 strains (n=4) were not cytotoxic. Type 3
and 4 strains did not have detectable secretion phenotype; however, some type 4
strains were able to reach a level of cytotoxicity similar to that of type 1 and 
type 2 strains when complemented in trans by a functional exsA gene. A
statistically significant association (P<.001) was found between TTSS types and
detection of the mutually exclusive exoU and exoS genes. In addition, 24 of 25
serotype O:1, O:10, and O:11 strains contained exoU, whereas 54 of 55 serotype
O:3, O:4, O:6, O:12, and O:16 strains contained exoS (P<.001). Our results
demonstrate correlations among exoU or exoS genotype, TTSS phenotype, and O
serotype in bacteremic P. aeruginosa isolates.

PMID: 12898437 [PubMed - indexed for MEDLINE]


300. J Exp Bot. 2003 Sep;54(390):2025-33. Epub 2003 Jul 16.

Antisense suppression of a beta-galactosidase gene (TB G6) in tomato increases
fruit cracking.

Moctezuma E, Smith DL, Gross KC.

Produce Quality and Safety Laboratory, USDA-ARS, Building 002, Henry A Wallace
Beltsville Area Research Center, 10300 Baltimore Avenue, Beltsville, MD
20705-2350, USA.

Antisense suppression of a tomato beta-galactosidase gene (TBG6) was used to
study its role in fruit development, cell wall-modification, and fruit firmness. 
TBG6 mRNA is highly abundant during the early stages of fruit development, but
the levels decline sharply after the breaker stage with the start of the
respiratory climacteric and a concomitant increase in ethylene production. Two
antisense lines were obtained with significantly reduced levels of TBG6 mRNA at
all stages of fruit development. At 30 d after pollination (dap), TBG6 mRNA
levels were reduced by up to 98% and 88% in lines 6-2 and 6-10, respectively.
Morphological phenotypes observed in the antisense lines included increased fruit
cracking, reduced locular space, and a doubling in the thickness of the fruit
cuticle. Two biochemical changes in antisense lines, compared with wild-type
lines, were a reduction of exo-galactanase activity at the breaker +3 d stage and
a reduction in the cell wall galactosyl content at the 20 dap stage. In addition,
transgenic lines exhibited a 35-39% reduction in fruit firmness at the 20 dap
stage, but their texture was equivalent to the wild type at 30 dap and beyond.
Although the exact function of the TBG6 product is still unknown, these results
implicate an important role for this enzyme in early fruit growth and development
in tomato.

PMID: 12867545 [PubMed - indexed for MEDLINE]


301. Acta Crystallogr C. 2003 Jul;59(Pt 7):o406-8. Epub 2003 Jun 30.

6-amino-3-bromo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1,5-dihydro-4H-pyraz
olo[3,4-d]pyrimidin-4-one-acetone-water (1/1/1): a fluorinated 2'-deoxyguanosine 
analogue with the sugar conformation of a ribonucleoside.

He J, Eickmeier H, Seela F.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.

In the title compound, C(10)H(11)BrFN(5)O(4).C(3)H(6)O.H(2)O, the N-glycosylic
bond torsion angle, chi, is anti [-108.0 (4) degrees ]. The sugar pucker is
N-type [C2'-exo, (2)E with P = 346.5 (4) degrees and tau(m) = 34.5 (2) degrees ],
and the conformation around the C-C bond linking the CH(2) group and the furan
ring is -sc [torsion angle gamma = -70.0 (4) degrees ].

PMID: 12855872 [PubMed - indexed for MEDLINE]


302. J Mol Model. 2003 Aug;9(4):230-4. Epub 2003 Jul 1.

A molecular mechanics and semiempirical molecular orbital study on the
conformation of polynorbornene chains.

Yilmaz SS, Abbasoglu R, Hazer B.

Department of Chemistry, Karadeniz Technical University, 61080 Trabzon, Turkey.
sevily@ktu.edu.tr

The conformational analysis of polynorbornene (PNB) chains was investigated with 
the AM1, MM2, AMBER and OPLS methods taking into consideration the possibility of
binding of norbornene monomers to each other at various positions, i.e. exo-exo, 
exo-endo, endo-endo. The chain that is formed by connecting exo-endo positions of
the monomers has lower torsional barrier energy than those formed with bonds at
other positions and has more flexibility. It is determined that the
thredisyndiotactic chain formed by exo-endo addition adopts a helix structure and
has a coil shape. The disyndiotactic chain formed by connecting norbornene
monomers in mixed type has a linear structure. It is found that the repeat unit
conformations of thredisyndiotactic and disyndiotactic chains of PNB are TGTG-
and (TGTG-)2, respectively.

PMID: 12838400 [PubMed - indexed for MEDLINE]


303. J Virol. 2003 Jul;77(14):7764-78.

Mutations within the ADP (E3-11.6K) protein alter processing and localization of 
ADP and the kinetics of cell lysis of adenovirus-infected cells.

Tollefson AE, Scaria A, Ying B, Wold WS.

Department of Molecular Microbiology and Immunology, St. Louis University Health 
Sciences Center, 1402 S. Grand Boulevard, St. Louis, MO 63104, USA.

ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus
infection and is required for efficient lysis of infected cells and release of
viral progeny at the end of the viral replication cycle. ADP is a type III
bitopic N(endo)C(exo) nuclear membrane and Golgi glycoprotein that is produced at
high levels in late adenovirus infection (>24 h postinfection). We show
pulse-chase and other studies indicating that ADP undergoes a complex process of 
N- and O-linked glycosylation and proteolytic cleavage. In order to further
characterize ADP, a series of 23 deletion and point mutations has been
constructed in the adenovirus serotype 2 adp gene and then built into a wild-type
adenovirus background. These mutants were analyzed for processing and
intracellular localization of ADP. Mutation of the single predicted N
glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich
in serine and threonine residues reduced O glycosylation. In general, mutations
within the lumenal domain of ADP resulted in lower protein stability;
immunofluorescence assays indicated that these ADPs were primarily present in the
Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic
domain of ADP showed normal glycosylation patterns and protein abundance for ADP,
but the protein was often found throughout cellular membranes rather than being
localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus
mutants were analyzed by cell viability assays to determine the kinetics of cell 
lysis following infection of human A549 cells. In general, viruses with mutations
within the lumenal domain of ADP display greatly reduced efficiencies of cell
lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of
ADP retain much of their ability to lyse infected cells.

PMCID: PMC161948
PMID: 12829816 [PubMed - indexed for MEDLINE]


304. Mol Genet Genomics. 2003 Aug;269(5):632-9. Epub 2003 Jun 25.

Enhanced expression of the DNA damage-inducible gene DIN7 results in increased
mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae.

Koprowski P, Fikus MU, Dzierzbicki P, Mieczkowski P, Lazowska J, Ciesla Z.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A
Pawinskiego St., 02-106 Warsaw, Poland.

We reported previously that the product of DIN7, a DNA damage-inducible gene of
Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are
involved in DNA repair and replication. This family includes the S. cerevisiae
protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1,
and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG
family which specifically functions in mitochondria. We reported previously that 
overexpression of DIN7 results in a mitochondrial mutator phenotype. In the
present study we wished to test the hypothesis that this phenotype is dependent
on the nuclease activity of Din7p. For this purpose, we constructed two alleles, 
din7-D78A and din7-D173A, which encode proteins in which highly conserved
aspartates important for the nuclease activity of the XPG proteins have been
replaced by alanines. Here, we report that overexpression of the mutant alleles, 
in contrast to DIN7, fails to increase the frequency of mitochondrial petite
mutants or erythromycin-resistant (Er) mutants. Also, overproduction of
din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial 
DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show
that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit
gross rearrangements of mtDNA, and that this correlates with enhanced
recombination within the mitochondrial cyt b gene. These results suggest that the
stability of the mitochondrial genome of S. cerevisiae is modulated by the level 
of the nuclease Din7p.

PMID: 12827502 [PubMed - indexed for MEDLINE]


305. Eur J Biochem. 2003 Jul;270(13):2913-9.

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A
unique type of cellulase among Bacillales.

Sanchez MM, Pastor FI, Diaz P.

Department of Microbiology, Faculty of Biology, University of Barcelona, Spain.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a
previously described endoglucanase revealed the presence of an additional
truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding
3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia
coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of
1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85,
displayed a multidomain organization bearing a canonical family 48 catalytic
domain, a bacterial type 3a cellulose-binding module, and a putative
fibronectin-III domain. The cloned cellulase, unique among Bacillales and
designated Cel48C, was purified through affinity chromatography using its ability
to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on
acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and
cellodextrins, whereas no activity was found on carboxy methyl cellulose,
cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside.
Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a
processive cellobiohydrolase. Although no chromogenic activity was detected from 
pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and
cellodextrins from these substrates, suggesting that Cel48C acts from the
reducing ends of the sugar chain. Presence of such a cellobiohydrolase in
Paenibacillus sp. BP-23 would contribute to widen up its range of action on
natural cellulosic substrates.

PMID: 12823562 [PubMed - indexed for MEDLINE]


306. Phytomedicine. 2003 May;10(4):318-24.

Immunologically active O6-branched (1-->3)-beta-glucan from the lichen Thamnolia 
vermicularis var. subuliformis.

Olafsdottir ES, Omarsdottir S, Paulsen BS, Wagner H.

Faculty of Pharmacy, University of Iceland, Hagi, Reykjavik, Iceland.
elinsol@hi.is

A lentinan-type gel-forming beta-glucan, Ths-2, has been isolated in about 1.5%
yield from the alkali extract of the lichen Thamnolia vermicularis var.
subuliformis, using ethanol fractionation, dialysis and gel filtration. The mean 
Mr of Ths-2 was determined by GP-HPLC to be 67 kD, and the optical rotation was
measured to be -14 degrees. The structure of Ths-2 was further elucidated by
methylation analysis by GC-MS, 1H- and 13C-NMR spectroscopy and selective
enzymatic hydrolysis with exo-(1 --> 3)-beta-D-glucanase followed by analysis of 
oligosaccharides by HPAEC-PAD. Ths-2 was found to be consisting of a (1 -->
3)-beta-D-glucopyranosyl main chain with branches of a (1 --> 6) linked
glucopyranosyl unit on every third unit of the main chain. Similar polysaccharide
structures have been described from fungi, but this is the first report of a
lentinan-type (1 --> 3)-beta-D-glucan from a lichen species. The immunomodulating
activity of Ths-2 was tested in an in vitro anti-complementary assay, and proved 
to be strongly active.

PMID: 12809362 [PubMed - indexed for MEDLINE]


307. Res Microbiol. 2003 May;154(4):259-67.

The diversity and evolution of the T4-type bacteriophages.

Desplats C, Krisch HM.

Laboratoire de Microbiologie et Genetique Moleculaire du CNRS, UMR 5100, 118
Route de Narbonne, 31062 Cedex, Toulouse, France.

Recent studies suggest that viruses are the most numerous entities in the
biosphere; bacteriophages, the viruses that infect Eubacteria and Archaea,
constitute a substantial fraction of this population. In spite of their ubiquity,
the vast majority of phages in the environment have never been studied and
nothing is known about them. For the last 10 years our research has focused on an
extremely widespread group of phages, the T4-type. It has now become evident that
phage T4 has a myriad of relatives in nature that differ significantly in their
host range. The genomes of all these phages have homology to the T4 genes that
determine virion morphology. Although phylogenetically related, these T4-type
phages can be subdivided into four groups that are increasingly distant from T4: 
the T-evens, the pseudo T-evens, the schizo T-evens and the exo T-evens. Genomic 
comparisons between the various T4-type phages and T4 indicate that these genomes
share homology not only for virion structural components but also for most of the
essential genes involved in the T4 life cycle. This suggests that horizontal
transmission of the genetic information may have played a less general role in
the evolution of these phages than has been supposed. Nevertheless, we have
identified several regions of the T4-type genome, such as the segment containing 
the tail fiber genes that exhibit evidence of extensive modular shuffling during 
evolution. The T4-type genomes appear to be a mosaic containing a large and fixed
group of essential genes as well as highly variable set of non-essential genes.
These non-essential genes are probably important for the adaptation of these
phages to their particular life-style. Furthermore, swapping autonomous domains
within the essential proteins may slightly modify their function(s) and
contribute to the adaptive ability of the T4-type phage family. Regulatory
sequences also display considerable evolutionary plasticity and this too may
facilitate the adaptation of phage gene expression to new environments and
stresses.

PMID: 12798230 [PubMed - indexed for MEDLINE]


308. Mol Plant Microbe Interact. 2003 Jun;16(6):536-44.

Characterization of a Ralstonia solanacearum operon required for
polygalacturonate degradation and uptake of galacturonic acid.

Gonzalez ET, Allen C.

Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden
Drive, Madison 53706, USA.

The bacterial wilt pathogen Ralstonia solanacearum produces three extracellular
polygalacturonases (PGs): PehA, PehB, and PehC. All three PGs hydrolyze pectin's 
polygalacturonic acid backbone, but each releases different reaction products.
PehA and PehB contribute significantly to pathogen virulence, probably by
facilitating root invasion and colonization. To determine the collective
contribution of PGs to virulence and saprophytic survival, we cloned,
characterized, and mutated the R. solanacearum pehC gene, which encodes a
distinctive monogalacturonate-releasing exo-PG. The virulence of a pehC mutant on
tomato was indistinguishable from that of its wild-type parent; thus, this exo-PG
alone does not contribute significantly to wilt pathogenesis. Unexpectedly, a
completely PG-deficient triple pehA/B/C mutant was slightly more virulent than a 
pehA/B mutant. PehC may degrade galacturonide elicitors of host defense, thereby 
protecting the pathogen from plant antimicrobial responses. A galacturonate
transporter gene, exuT, is immediately downstream of pehC and the two genes are
co-transcribed. It has been hypothesized that galacturonic acid released by PGs
from plant cell walls nourishes bacteria during pathogenesis. To separate the
pectolytic and nutrient-generating roles of the PGs, we made an exuT mutant,
which still produces all three isozymes of PG but cannot uptake PG degradation
products. This exuT mutant had wild-type virulence on tomato, demonstrating that 
metabolism of galacturonic acid does not contribute significantly to bacterial
success inside the plant.

PMID: 12795379 [PubMed - indexed for MEDLINE]


309. J Org Chem. 2003 Jun 13;68(12):4615-30.

Hydroxymethyl rotamer populations in disaccharides.

Roen A, Padron JI, Vazquez JT.

Instituto Universitario de Bio-Organica Antonio Gonzalez, Universidad de La
Laguna, Avda. Astrofisico Fco. Sanchez 2, 38206 La Laguna, Tenerife, Spain.

Sixteen methyl glucopyranosyl glucopyranoside disaccharides (methyl
beta-d-Glcp(p-Br-Bz)-(1-->x)-beta/alpha-d-Glcp) containing beta-glycosidic
linkages (1-->2, 1-->3, 1-->4, and 1-->6) were synthesized and analyzed by means 
of CD and NMR spectroscopy in three different solvents. For each of these four
types of disaccharides, a correlation was observed between the hydroxymethyl
rotational populations around the C5-C6 bond of the glucopyranosyl residue II
with the substituents and the anomeric configuration of the methoxyl group in
residue I, as well as with the solvent. Nonbonded interactions, the
stereoelectronic exo-anomeric effect, and hydrogen bonding were found to be
responsible for the observed rotameric differences. Whereas the rotational
populations of the (1-->6)-linked disaccharides are mainly dependent on the
exo-anomeric effect, the (1-->2)-bonded disaccharides are strongly dependent on
the anomeric configuration at C1, and the (1-->3)- and (1-->4)-linked
disaccharides are mainly dependent on the substituents and the solvent. The
population of the gt rotamer decreases as nonbonded interactions increase but
increases as the exo-anomeric effect becomes greater, as well as in the presence 
of intramolecular hydrogen bonding to the endocyclic oxygen O5'. Comparison of
the hydroxymethyl rotational preferences between our model disaccharides revealed
a dependence on the glycosidic linkage type. Thus the population of the gg and gt
rotamers decreases/increases from (1-->2)- (beta series), to (1-->6)-, to
(1-->2)- (alpha series), to (1-->4)-, and to (1-->3)-bonded disaccharides
respectively, while the tg rotamer population remains almost constant (around
20%), except for the (1-->3)- and (1-->4)-linked disaccharides with the
intramolecular hydrogen bonding to O5', where this population decreases to 10%.

PMID: 12790564 [PubMed - indexed for MEDLINE]


310. J Bacteriol. 2003 Jun;185(12):3485-90.

Purification and properties of an intracellular 3-hydroxybutyrate-oligomer
hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel
intracellular poly(3-hydroxybutyrate) depolymerase.

Kobayashi T, Shiraki M, Abe T, Sugiyama A, Saito T.

Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty 
of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293,
Japan.

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of
Ralstonia eutropha was purified from Escherichia coli harboring a plasmid
containing phaZ2(Reu). The purified enzyme hydrolyzed linear and cyclic
3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate)
(PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to
that of the previously identified intracellular PHB (iPHB) depolymerase
(PhaZ1(Reu)). The enzyme appeared to be an endo-type hydrolase, since it actively
hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear
3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase,
releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion
bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha;
in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha
H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was
observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14%
PHB of dry cell weight) and decreased to a very low level in the stationary phase
(ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the
PHB content in the cell increased to ca. 5% in the stationary phase. A double
mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase
(ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These 
results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in
the mobilization of PHB in R. eutropha along with PhaZ1.

PMCID: PMC156217
PMID: 12775684 [PubMed - indexed for MEDLINE]


311. J Am Chem Soc. 2003 Jun 4;125(22):6753-61.

Stereoselective association of binuclear metallacycles in coordination polymers.

Khlobystov AN, Brett MT, Blake AJ, Champness NR, Gill PM, O'Neill DP, Teat SJ,
Wilson C, Schroder M.

School of Chemistry, The University of Nottingham, University Park, Nottingham
NG7 2RD, U.K.

A series of structurally related binuclear metallacycles [Cd(NO(3))(2)L](2),
where L is an angular exo-bidentate ligand, have been synthesized. Each
metallacycle contains two coordinatively unsaturated, chiral metal centers within
a single molecule, and the assembly of these metallacycles into polymeric
framework structures has been studied systematically for the first time.
Stereoselective homochiral association of [Cd(NO(3))(2)L](2) leads to the
formation of helical coordination polymers, whereas meso type association results
in nonhelical chain structures. The type of stereoselective aggregation depends
on the conditions of self-assembly as well as on ligand functionality. Both
helical and nonhelical polymeric complexes have been isolated for the
metallacycle [Cd(NO(3))(2)(2,4'-pyacph)](2) (2,4'-pyacph =
2,4'-(4-ethynylphenyl)bipyridyl). Homochiral association results in the formation
of helical [Cd(NO(3))]( infinity ) chains which link the binuclear
[Cd(NO(3))(2)(2,4'-pyacph)](2) metallacycles into racemic two-dimensional sheets 
which contain both P and M [Cd(NO(3))]( infinity ) helices. In contrast,
meso-association leads to the formation of nonhelical one-dimensional chains. It 
is shown that the product of homochiral association is predominately formed at
room temperature and that of meso-association is generated at elevated
temperatures. Thus, it may be concluded that the homochiral association appears
to be energetically less favorable than the meso-association, a conclusion that
has been confirmed by theoretical calculations of the crystal lattice energy.
Several high-yield syntheses of bipyridyl-type ligands used for metallacyclic
assembly are also reported.

PMID: 12769586 [PubMed]


312. Biochimie. 2003 Jan-Feb;85(1-2):65-73.

The SPASIBA force field as an essential tool for studying the structure and
dynamics of saccharides.

Vergoten G, Mazur I, Lagant P, Michalski JC, Zanetta JP.

UMR CNRS 8576 Glycobiologie Structurale et Fonctionnelle, Universite des Sciences
et Technologies de Lille, 59655 Villeneuve d'Ascq, France.
gerard.vergoten@univ-lille1.fr

The SPASIBA force field has been applied to the determination of the structure
and dynamical properties of various disaccharides. It has been shown that the
experimental properties (structure, dipole moment, conformational relative
energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are
confidently reproduced without specific terms for the alpha and beta anomers and 
the type of glycosidic linkages.

PMID: 12765776 [PubMed - indexed for MEDLINE]


313. Mol Biotechnol. 2003 Jun;24(2):105-10.

Efficient mutagenesis method for producing the templates of single nucleotide
polymorphisms.

Zhang J, Li K, Deng Z, Liao D, Fang W, Zhang X.

Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua
University, Hengyang, China, 421001.

DNA templates harboring specific single nucleotide polymorphism (SNP) sites are
largely needed as positive controls in practical SNP analysis and in
determination of the reliability of newly developed methods in high-throughput
screening assays. Here we report a one-step method to produce SNP templates by
amplifying a wild-type sequence with primers having single nucleotide mismatches 
at or near their 3' ends. A short amplicon harboring an EcoRI site was used to
evaluate the feasibility of our strategy. Perfectly matched primers and primers
with a single base mismatch occurring from the first base to the sixth base of
the EcoRI site were used for primer extension. By using polymerase without a
proofreading function, we kept mismatched nucleotides from occurring in extended 
primer products, as confirmed by EcoRI digestion and sequencing analysis. The
strategy of using primers with a single mismatched base and exo- polymerase was
shown to be an efficient one-step method for preparing SNP templates, either for 
application in the development of SNP screening assays or as positive controls in
practical SNP assays.

PMID: 12746551 [PubMed - indexed for MEDLINE]


314. Neurochem Int. 2003 Sep-Oct;43(4-5):445-51.

Effects of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO) and
its N-substituted analogs on GABA transport in cultured neurons and astrocytes
and by the four cloned mouse GABA transporters.

Sarup A, Larsson OM, Bolvig T, Frolund B, Krogsgaard-Larsen P, Schousboe A.

Department of Pharmacology, Royal Danish School of Pharmacy, DK-2100 Copenhagen, 
Denmark. asa@dfh.dk

The system of GABA transporters in neural cells constitutes an efficient
mechanism for terminating inhibitory GABAergic neurotransmission. As such these
transporter are important therapeutical targets in epilepsy and potentially other
neurological diseases related to the GABA system. In this study a number of
analogs of 3-hydroxy-4-amino-4,5,6,7-tetrahydro-1,2-benzisoxazol (exo-THPO), a
promising lead structure for inhibitors of GABA uptake were investigated. It was 
found that the selectivity of N-acetyloxyethyl-exo-THPO for inhibition of the
astroglial GABA uptake system was 10-fold as compared to inhibition of the
neuronal GABA uptake system. Selectivity in this magnitude may provide potent
anti-convulsant activity as has recently been demonstrated with the likewise
glia-selective GABA uptake inhibitor, N-methyl-exo-THPO. In contrast to the
competitive inhibition of GABA uptake exhibited by N-substituted analogs of
4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO), nipecotic acid, and
guvacine, N-4,4-diphenyl-3-butenyl(DPB)-N-methyl-exo-THPO and
4-phenylbutyl-exo-THPO exhibited non-competitive type inhibition kinetics. The
lipophilic character of a number of GABA analogs was concluded by far to
constitute the determining factor for the potency of these compounds as
inhibitors of GAT1-mediated uptake of GABA. This finding underscores the
complexity of the pharmacology of the GABA transport system, since these
non-competitive inhibitors are structurally very similar to some competitive GABA
uptake inhibitors. Whether these structure-activity relationships for inhibition 
of GABA uptake may provide sufficient information for the development of new
structural leads and to what extent these compounds may be efficient as
therapeutical anti-convulsant agents remain to be elucidated.

PMID: 12742090 [PubMed - indexed for MEDLINE]


315. J Biol Chem. 2003 Jul 18;278(29):26742-9. Epub 2003 May 8.

Identification of the catalytic residues in family 52 glycoside hydrolase, a
beta-xylosidase from Geobacillus stearothermophilus T-6.

Bravman T, Belakhov V, Solomon D, Shoham G, Henrissat B, Baasov T, Shoham Y.

Department of Food Engineering and Biotechnology, Technion-Israel Institute of
Technology, Haifa 32000, Israel.

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze
short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the
glycosidic bond involves two carboxylic acid residues, and their identification, 
together with the stereochemistry of the reaction, provides crucial information
on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from
Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to
verify their role in catalysis. The activity of the E335G mutant decreased
approximately 106-fold, and this activity was enhanced 103-fold in the presence
of external nucleophiles such as formate and azide, resulting in a xylosyl-azide 
product with an opposite anomeric configuration. These results are consistent
with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was
subjected to detailed kinetic analysis using substrates bearing different leaving
groups (pKa). The mutant exhibited 103-fold reduction in activity, and the
Bronsted plot of log(kcat) versus pKa revealed that deglycosylation is the
rate-limiting step, indicating that this step was reduced by 103-fold. The rates 
of the glycosylation step, as reflected by the specificity constant (kcat/Km),
were similar to those of the wild type enzyme for hydrolysis of substrates
requiring little protonic assistance (low pKa) but decreased 102-fold for those
that require strong acid catalysis (high pKa). Furthermore, the pH dependence
profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the
presence of azide significantly enhanced the mutant activity accompanied with the
generation of a xylosyl-azide product with retained anomeric configuration. These
results are consistent with Asp495 acting as the acid-base in XynB2.

PMID: 12738774 [PubMed - indexed for MEDLINE]


316. Nucleic Acids Res. 2003 May 15;31(10):2636-46.

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II.
High-density labeling of natural DNA.

Tasara T, Angerer B, Damond M, Winter H, Dorhofer S, Hubscher U, Amacker M.

Gnothis SA, PSE-B, EPFL, CH-1015 Lausanne, Switzerland.

The modification of nucleic acids using nucleotides linked to detectable reporter
or functional groups is an important experimental tool in modern molecular
biology. This enhances DNA or RNA detection as well as expanding the catalytic
repertoire of nucleic acids. Here we present the evaluation of a broad range of
modified deoxyribonucleoside 5'-triphosphates (dNTPs) covering all four naturally
occurring nucleobases for potential use in DNA modification. A total of 30
modified dNTPs with either fluorescent or non-fluorescent reporter group
attachments were systematically evaluated individually and in combinations for
high-density incorporation using different model and natural DNA templates.
Furthermore, we show a side-by-side comparison of the incorporation efficiencies 
of a family A (Taq) and B (Vent(R) exo-) type DNA polymerase using the
differently modified dNTP substrates. Our results show superior performance by a 
family B-type DNA polymerase, Vent(R) exo-, which is able to fully synthesize a
300 bp DNA product when all natural dNTPs are completely replaced by their
biotin-labeled dNTP analogs. Moreover, we present systematic testing of various
combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling
of DNA with up to four differently modified dNTPs.

PMCID: PMC156052
PMID: 12736314 [PubMed - indexed for MEDLINE]


317. J Am Chem Soc. 2003 May 14;125(19):5632-3.

Broad-spectrum radical cyclizations boosted by polarity matching. Carbonylative
access to alpha-stannylmethylene lactams from azaenynes and CO.

Ryu I, Miyazato H, Kuriyama H, Matsu K, Tojino M, Fukuyama T, Minakata S, Komatsu
M.

Department of Chemistry, Faculty of Arts and Sciences, Osaka Prefecture
University, Sakai, Osaka 599-8531, Japan. ryu@ms.cias.osakafu-u.ac.jp

Free-radical mediated stannylcarbonylation of azaenynes provides a general [n +
1]-type annulation approach leading to alpha-stannylmethylene lactams. The
cyclization is unusual in its breadth, covering 4-exo, 5-exo, 6-exo, 7-exo, and
8-exo modes.

PMID: 12733892 [PubMed]


318. Curr Microbiol. 2003 Jun;46(6):391-7.

Identification of the gene for beta-fructofuranosidase of Bifidobacterium lactis 
DSM10140(T) and characterization of the enzyme expressed in Escherichia coli.

Ehrmann MA, Korakli M, Vogel RF.

Lehrstuhl fur Technische Mikrobiologie, Technische Universitat Munchen, 85350
Freising-Weihenstephan, Germany. M.ehrmann@lrz.tu-muenchen

Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium
often used in combination with fructooligosaccharides (FOS) as a probiotic
supplement in diverse dairy products. This is the first report describing the
gene structure and enzymatic properties of a beta-fructofuranosidase [EC
3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 
10140(T) and heterologously expressed in Escherichia coli. The G+C content was
identical with the G+C content as determined for the total genomic DNA (61.9 mol 
%). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly,
the deduced aa sequence revealed only minor similarity to other
fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity
after incorporation of a C-terminal 6 x HIS affinity tag. It hydrolased sucrose, 
1-kestose, Raftilose, Actilight, inulin, and raffinose (100%, 91%, 84%, 80%, 37%,
4%). Fructose moieties were released in an exo-type fashion. Substrates with
alpha-glycosidic linkages or residues other than fructose were not attacked. The 
kinetic parameters K(m) and V(max) for sucrose hydrolysis were 10.3 m M and 0.031
microM/min (pH 7.6; 37 degrees C). The activity was abolished by Zn(2+) (1 m M)
and significantly inhibited by Fe(2+) and Ni(2+) (10 m M). The enzyme showed its 
maximal activity at 40 degrees C.

PMID: 12732943 [PubMed - indexed for MEDLINE]


319. Mol Cell Neurosci. 2003 Apr;22(4):454-66.

Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving
botulinum toxins: the extent and duration are dictated by the sites of SNAP-25
truncation.

Meunier FA, Lisk G, Sesardic D, Dolly JO.

Centre for Neurobiochemistry, Department of Biological Sciences, Imperial College
of Science, Technology and Medicine, London SW7 2AY, UK.

Nerve sprouts emerge from motor nerve terminals following blockade of
exo-endocytosis for more than 3 days by botulinum neurotoxin (BoNT), and form
functional synapses, albeit temporary. Upon restoration of synaptic activity to
the parent terminal 7 and 90 days after exposure to BoNT/F or A respectively, a
concomitant retraction of the outgrowths was observed. BoNT/E caused short-term
neuroparalysis, and dramatically accelerated the recovery of BoNT/A-paralyzed
muscle by further truncation of SNAP-25 and its replenishment with functional
full-length SNARE. The removal of 9 C-terminal residues from SNAP-25 by BoNT/A
leads to persistence of the inhibitory product due to the formation of a
nonproductive SNARE complex(es) at release sites, whereas deletion of a further
17 amino acids permits replenishment and a speedy recovery.

PMID: 12727443 [PubMed - indexed for MEDLINE]


320. Biochim Biophys Acta. 2003 May 2;1621(2):204-10.

Purification and properties of two type-B alpha-L-arabinofuranosidases produced
by Penicillium chrysogenum.

Sakamoto T, Kawasaki H.

Division of Applied Biochemistry, Graduate School of Agriculture and Biological
Sciences, Osaka Prefecture University, Osaka 599-8531, Japan.
sakamoto@biochem.osakafu-u.ac.jp

Two distinct extracellular alpha-L-arabinofuranosidases (AFases; EC 3.2.1.55)
were purified from the culture filtrate of Penicillium chrysogenum 31B. The
molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa
(AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 degrees C and
were stable up to 50 degrees C. Enzyme activities of AFQ1 and AFS1 were highest
at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose 
to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1.
Both enzymes displayed broad substrate specificities; they released arabinose
from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and
arabino-oligosaccharides. AFS1 also showed low activity towards
p-nitrophenyl-beta-D-xylopyranoside. An exo-arabinanase, which catalyzes the
release of arabinobiose from linear arabinan at the nonreducing terminus, acted
synergistically with both enzymes to produce L-arabinose from branched arabinan.

PMID: 12726996 [PubMed - indexed for MEDLINE]


321. J Am Chem Soc. 2003 Apr 9;125(14):4271-8.

Theoretical elucidation of kinetic and thermodynamic control of radical addition 
regioselectivity.

Leach AG, Wang R, Wohlhieter GE, Khan SI, Jung ME, Houk KN.

Department of Chemistry and Biochemistry, University of California, Los Angeles, 
California 90095-1569, USA.

The cyclizations of two structurally similar 2-oxo-5-hexenyl-type radicals have
been investigated by ab initio and density functional
(UB3LYP/6-31+G**//UHF/6-31G* and UB3LYP/6-31G*//UB3LYP/6-31G*) calculations. The 
origin of apparently contradictory reports of 6-endo and 5-exo cyclizations is
determined. Kinetic control favors 6-endo cyclization, while thermodynamic
control gives 5-exo cyclization, and the observation of different products from
different research groups arises from the difference in experimental conditions
used by the two groups. The outcome of a new cyclization reaction was predicted
by using these theoretical techniques. Kinetic control is predicted to yield
exclusively the products of 6-endo cyclization, while thermodynamic control would
lead to an approximately equal mixture of one 6-endo and one 5-exo cyclized
product. Experimental studies revealed that the reaction yields only the products
of 6-endo cyclization through kinetic control.

PMID: 12670249 [PubMed - indexed for MEDLINE]


322. Carbohydr Res. 2003 Apr 4;338(8):697-703.

The presence of water improves reductive openings of benzylidene acetals with
trimethylaminoborane and aluminium chloride.

Sherman AA, Mironov YV, Yudina ON, Nifantiev NE.

N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Leninsky prospect 47, Moscow 119991, Russia.

The acidic reagent formed in situ from anhydrous AlCl(3) and H(2)O in 3:1 ratio
is much more efficient for the reductive openings of the cyclic benzylidene
acetals with Me(3)N x BH(3) in tetrahydrofurane than the AlCl(3) alone. Under
proposed conditions, the dioxane-type 4,6-O-bezylidene acetals of hexopyranosides
give regioselectively the corresponding 4-hydroxy,6-O-benzyl derivatives in
excellent yields. Reductive openings of the dioxolane-type 3,4-O-benzylidene
acetals of galactopyranoside are also very efficient and regioselective and give 
either 3-O-benzyl derivative (from 3,4-O-exo-benzylidene acetal) or 4-O-benzyl
derivative (from 3,4-O-endo-benzylidene acetal) depending on the configuration of
the acetal carbon atom.

PMID: 12668088 [PubMed - indexed for MEDLINE]


323. Org Lett. 2003 Apr 3;5(7):1087-9.

Stereoselective glycosylation of exo-glycals accelerated by Ferrier-type
rearrangement.

Lin HC, Yang WB, Gu YF, Chen CY, Wu CY, Lin CH.

Institute of Biological Chemistry, Academia Sinica, No. 128, Academia Road
Section 2, Nan-Kang, Taipei, 11529, Taiwan.

[reaction: see text] Owing to the driving force of the Ferrier-type
rearrangement, the exo-glycals are highly reactive with various alcohols to
afford glycosides and glycoconjugates with exclusive alpha-configuration. The
resulting vinyl group in these glycosylation products can be further elaborated
for general applications, including the synthesis of spiro derivatives and
glycosylation of 2-ketoaldonic acids.

PMID: 12659580 [PubMed - indexed for MEDLINE]


324. Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3995-4000. Epub 2003 Mar 19.

Phosphatidylinositol 4-kinase type IIalpha is responsible for the
phosphatidylinositol 4-kinase activity associated with synaptic vesicles.

Guo J, Wenk MR, Pellegrini L, Onofri F, Benfenati F, De Camilli P.

Howard Hughes Medical Institute and Department of Cell Biology, Yale University
School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

Phosphorylation of inositol phospholipids plays a key role in cellular regulation
via the generation of intracellular second messengers. In addition, it represents
a mechanism to regulate interactions of the lipid bilayer with proteins and
protein scaffolds involved in vesicle budding, cytoskeletal organization, and
signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from
phosphatidylinositol (PI) is an important step in this metabolic pathway because 
PI(4)P is a precursor of other important phosphoinositides and has protein
binding properties of its own. We report here that a PI 4-kinase (PI4K) activity 
previously reported on synaptic vesicles is accounted for by the alpha isoform of
the recently characterized type II PI4K (PI4KII) family. PI4KIIalpha, which also 
accounts for the bulk of PI4K activity in brain extracts, is concentrated at
synapses and in the region of the Golgi complex in neuronal perikarya. Our
results provide new evidence for the occurrence of a cycle of phosphoinositide
synthesis and hydrolysis nested within the exo-endocytic cycle of synaptic
vesicles and point to PI4KIIalpha as a critical player in this cycle.

PMCID: PMC153036
PMID: 12646710 [PubMed - indexed for MEDLINE]


325. Biosci Biotechnol Biochem. 2003 Jan;67(1):68-76.

beta-Galactosidase and its significance in ripening of "Saijyo" Japanese
Persimmon fruit.

Nakamura A, Maeda H, Mizuno M, Koshi Y, Nagamatsu Y.

Tsukuba R&D Center, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun,
Ibaraki 300-2497, Japan. 940080@so.fujioil.co.jp

The fruit extracts of ripening cv. Japanese Persimmon, "Saijyo", contained a
number of glycosidases and glycanases. Among them, beta-galactosidase appeared to
be the most significant, and the activity increased in parallel with tissue
ripening. Persimmon beta-galactosidase was presented in at least three isoforms, 
beta-galactosidase-I (pI = 4.88), beta-galactosidase-II (pI = 6.76), and
beta-galactosidase-III (pI = 7.05). beta-Galactosidase-III had exo-type
galactanase activity, while the others did not. The activity of endo-type
glycanases was a maximum in immature green or yellow fruits. The firmness of the 
pulp tissue decreased dramatically, and the amount of water-soluble
polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases,
especially beta-galactosidase, appeared maximal in mature red fruits. The amount 
of extractable pectin remained unchanged, although the galactose content of the
high-molecular-weight fraction in WSS decreased dramatically. These results
suggest that the ripening of persimmon was caused by the solubilization of pectic
polysaccharide by endo-type glycanases and digestion by exo-type glycosidases.
beta-Galactosidase, in particular, seemed to play a major role in ripening the
fruit.

PMID: 12619675 [PubMed - indexed for MEDLINE]


326. Chemistry. 2002 Nov 15;8(22):5228-40.

Experimental evidence for the existence of non-exo-anomeric conformations in
branched oligosaccharides: NMR analysis of the structure and dynamics of
aminoglycosides of the neomycin family.

Asensio JL, Hidalgo A, Cuesta I, Gonzalez C, Canada J, Vicent C, Chiara JL,
Cuevas G, Jimenez-Barbero J.

Instituto de Quimica Organica, CSIC Juan de la Cierva 3, 28006 Madrid, Spain.

It is commonly known that the exo-anomeric effect is a major factor governing the
conformational behavior of naturally occurring oligosaccharides. Conformational
flexibility in these molecules mainly concerns the aglycon psi angle since phi is
restricted by this stereo-electronic effect. In fact, to the best of our
knowledge no case of a natural glycoside adopting a non-exo-anomeric conformation
in solution has yet been reported. With respect to the flexibility among
naturally occurring carbohydrates, branched type oligosaccharides including sugar
residues glycosidated at contiguous positions (such as blood type carbohydrate
antigens Lewis X) have been considered as the paradigm of rigid saccharides--the 
rigidity being enhanced by van der Waals interactions. Herein, we demonstrate
unambiguously that both common beliefs are not to be generalized. For example in 
neomycin B, a branched oligosaccharide antibiotic, a large number of
non-exo-anomeric conformations was detected in solution for the first time in
naturally occurring sugars. This unusual behavior is attributed to branching.
Here, polar contacts between non-vicinal sugar units lead to an enhanced
flexibility of the ribose glycosidic torsion phi. The influence of sugar
flexibility on RNA recognition will also be discussed.

PMID: 12613042 [PubMed - indexed for MEDLINE]


327. J Biol Chem. 2003 May 9;278(19):16720-5. Epub 2003 Feb 27.

Mechanisms for the formation of isoprostane endoperoxides from arachidonic acid. 
"Dioxetane" intermediate versus beta-fragmentation of peroxyl radicals.

Yin H, Havrilla CM, Gao L, Morrow JD, Porter NA.

Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University,
Nashville, Tennessee 37235, USA.

The isoprostanes are a class of autoxidation products generated from arachidonic 
acid (or its esters) by a free radical initiated process. The potent biological
activity of these compounds has been attracting intense research interest since
they were detected in humans as well as animal models in the early 1990s. The
measurement of these compounds has been regarded as one of the most useful
non-invasive biomarkers for oxidative stress status. Two mechanisms for the
formation of these compounds have been proposed. In the first mechanism, a
peroxyl radical undergoes successive 5-exo cyclizations analogous to the
enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism
starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate
dioxetane, a mechanism that has also been proposed for prostaglandin biosynthesis
as well as for the formation of 4-hydroxy nonenal (HNE). Autoxidation of
cholesteryl-15-HpETE under free radical conditions provides Type IV isoprostanes.
The "dioxetane" mechanism for isoprostane generation from 15-HpETE requires that 
optically pure products are formed from an optically pure reactant, whereas an
alternate mechanism for the process involving beta-fragmentation of the
15-peroxyl would give racemic isoprostane products. We have carried out a test of
the mechanism based upon these stereochemical requirements. The results of
analysis of the product mixture derived from autoxidation of optically pure
Ch-15-HpETE by atmospheric pressure chemical ionization-mass spectrometry coupled
with chiral high performance liquid chromatography indicate that the major
isoprostane diastereomers are formed as a racemic mixture. These experimental
results are consistent with a mechanism for isoprostane formation involving
beta-fragmentation of the 15-peroxyl radical followed by re-addition of oxygen to
form the 11-HPETE peroxyl, and they exclude a mechanism proceeding through the
formation of a dioxetane intermediate.

PMID: 12609993 [PubMed - indexed for MEDLINE]


328. Plant J. 2003 Feb;33(4):677-90.

AtBXL1, a novel higher plant (Arabidopsis thaliana) putative beta-xylosidase
gene, is involved in secondary cell wall metabolism and plant development.

Goujon T, Minic Z, El Amrani A, Lerouxel O, Aletti E, Lapierre C, Joseleau JP,
Jouanin L.

Laboratoire de Biologie Cellulaire - Institut National de la Recherche
Agronomique, Route de St-Cyr 78026 Versailles Cedex, France.

Erratum in:
    Plant J. 2003 May;34(3):393.

To investigate mechanisms involved in cell wall development, an Arabidopsis T-DNA
insertion mutant collection was screened to identify mutants with
beta-glucuronidase fusion gene expression in tissues undergoing secondary cell
wall thickening. This promoter-trapping strategy allowed the isolation of a
transformant containing the GUS coding sequence inserted 700 bp upstream of the
ATG of a putative beta-xylosidase gene. The transformant has no phenotype as the 
expression of the gene was not disrupted by the insertion. The analysis of the
predicted protein, AtBXL1, suggests its targeting to the extracellular matrix and
its involvement in cell wall metabolism through a putative activity towards
xylans. The 2-kb promoter sequence of AtBXL1 was fused to the GUS coding sequence
and introduced into wild-type Arabidopsis thaliana. GUS expression was shown to
be restricted to tissues undergoing secondary cell wall formation.
Beta-xylosidase activity was associated with the cell wall-enriched fraction of
different organs of wild-type plants. The level of activity correlates with
transcript accumulation of AtBXL1 and other AtBXL1-related genes. Transgenic
plants expressing the AtBXL1 cDNA in antisense orientation were generated. Lines 
exhibiting the highest decrease in AtBXL1 transcript accumulation and
beta-xylosidase activity had phenotypic alterations. This newly identified gene
is proposed to be involved in secondary cell wall hemicellulose metabolism and
plant development.

PMID: 12609041 [PubMed - indexed for MEDLINE]


329. J Am Chem Soc. 2003 Feb 12;125(6):1492-3.

A new reaction manifold for the Barton radical intermediates: synthesis of
N-heterocyclic furanosides and pyranosides via the formation of the C(1)-C(2)
bond.

Rhee JU, Bliss BI, RajanBabu TV.

Department of Chemistry, 100 West 18th Avenue, The Ohio State University,
Columbus, Ohio 43210, USA.

The first radical intermediate in the thiourethane-mediated deoxygenation of an
alcohol (Barton-McCombie reaction) can participate in an exo-hex-5-enyl- or
exo-hept-6-enyl-type radical cyclization when a suitable radical acceptor (e.g., 
alpha,beta-unsaturated ester, oxime ether, or hydrazone) is appropriately placed.
Carbohydrate-derived imidazolyl and triazolyl thiourethanes with such acceptors, 
upon addition to excess of a good hydride donor (reverse addition), undergo
efficient cyclization reactions to give N-heterocyclic furanosides, and,
surprisingly even N-pyranosides. Depending on the acceptor, glycosides with
either a C(2)()-amino or a C(2)()-carbon substituent are formed.

PMID: 12568605 [PubMed]


330. Ophthalmic Physiol Opt. 2003 Jan;23(1):71-7.

The effect of changing from glasses to soft contact lenses on myopia progression 
in adolescents.

Fulk GW, Cyert LA, Parker DE, West RW.

College of Optometry, Northeastern State University, Tahlequah, OK, USA.
fulk@cherokee.nsuok.edu

At the end of a clinical trial of bifocals as myopia treatment, subjects were
allowed to select any type of optical correction they wished and were asked to
return in 1 year. This report gives results of that last examination with
emphasis on how progression rates differed between those remaining in their
original type of glasses compared with those who switched to soft contact lenses.
We found that myopia progressed at an age-adjusted average rate of 0.74 D in 19
children who switched to soft contact lens wear compared with 0.25 D for 24
children remaining in glasses (p < 0.0001). Increased growth of the vitreous
chamber appeared to account for much of this excess myopia progression, although 
the difference in that variable did not reach statistical significance (p =
0.101). We also noted a 0.203 D steepening in the corneal curvature in contact
lens wearers compared with spectacle wearers whose corneas steepened very little 
(0.014 D, p = 0.007). Soft contact lens wear was also accompanied by a greater
change in the near-point phoria which moved 4.5 prism dioptres in the exo
direction compared with spectacle wearers who experienced only a 1.4 prism
dioptre divergent shift (p = 0.048).

PMID: 12535059 [PubMed - indexed for MEDLINE]


331. J Am Chem Soc. 2003 Jan 22;125(3):705-14.

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam.

Bell SG, Chen X, Sowden RJ, Xu F, Williams JN, Wong LL, Rao Z.

Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford,
South Parks Road, UK.

Oxygenated derivatives of the monoterpene (+)-alpha-pinene are found in plant
essential oils and used as fragrances and flavorings. (+)-alpha-Pinene is
structurally related to (+)-camphor, the natural substrate of the heme
monooxygenase cytochrome P450(cam) from Pseudomonas putida. The aim of the
present work was to apply the current understanding of P450 substrate binding and
catalysis to engineer P450(cam) for the selective oxidation of (+)-alpha-pinene. 
Consideration of the structures of (+)-camphor and (+)-alpha-pinene lead to
active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and
V247L mutations. All mutants showed greatly enhanced binding and rate of
oxidation of (+)-alpha-pinene. Some mutants had tighter (+)-alpha-pinene binding 
than camphor binding by the wild-type. The most active was the Y96F/V247L mutant,
with a (+)-alpha-pinene oxidation rate of 270 nmol (nmol of P450(cam))(-)(1)
min(-)(1), which was 70% of the rate of camphor oxidation by wild-type P450(cam).
Camphor is oxidized by wild-type P450(cam) exclusively to 5-exo-hydroxycamphor.
If the gem dimethyl groups of (+)-alpha-pinene occupied similar positions to
those found for camphor in the wild-type structure, (+)-cis-verbenol would be the
dominant product. All P450(cam) enzymes studied gave (+)-cis-verbenol as the
major product but with much reduced selectivity compared to camphor oxidation by 
the wild-type. (+)-Verbenone, (+)-myrtenol, and the (+)-alpha-pinene epoxides
were among the minor products. The crystal structure of the Y96F/F87W/V247L
mutant, the most selective of the P450(cam) mutants initially examined, was
determined to provide further insight into P450(cam) substrate binding and
catalysis. (+)-alpha-Pinene was bound in two orientations which were related by
rotation of the molecule. One orientation was similar to that of camphor in the
wild-type enzyme while the other was significantly different. Analysis of the
enzyme/substrate contacts suggested rationalizations of the product distribution.
In particular competition rather than cooperativity between the F87W and V247L
mutations and substrate movement during catalysis were proposed to be major
factors. The crystal structure lead to the introduction of the L244A mutation to 
increase the selectivity of pinene oxidation by further biasing the binding
orientation toward that of camphor in the wild-type structure. The
F87W/Y96F/L244A mutant gave 86% (+)-cis-verbenol and 5% (+)-verbenone. The
Y96F/L244A/V247L mutant gave 55% (+)-cis-verbenol but interestingly also 32%
(+)-verbenone, suggesting that it may be possible to engineer a P450(cam) mutant 
that could oxidize (+)-alpha-pinene directly to (+)-verbenone. Verbenol,
verbenone, and myrtenol are naturally occurring plant fragrance and flavorings.
The preparation of these compounds by selective enzymatic oxidation of
(+)-alpha-pinene, which is readily available in large quantities, could have
applications in synthesis. The results also show that the protein engineering of 
P450(cam) for high selectivity of substrate oxidation is more difficult than
achieving high substrate turnover rates because of the subtle and dynamic nature 
of enzyme-substrate interactions.

PMID: 12526670 [PubMed - indexed for MEDLINE]


332. Inorg Chem. 2003 Jan 13;42(1):60-9.

"True" inorganic heterocycles: structures and stability of group 13-15 analogues 
of benzene and their dimers.

Timoshkin AY, Frenking G.

Inorganic Chemistry Group, Department of Chemistry, St. Petersburg State
University, University Pr. 26, Old Peterhof 198504, Russia.
alextim@chemie.uni-marburg.de

Group 13-15 inorganic analogues of benzene, [HMYH](3) (M = B, Al, Ga; Y = N, P,
As), mixed heterocycles of the type [BAlGaNPAs]H(6) and their dimers have been
theoretically examined at the B3LYP/TZVP level of theory. Six different isomers
have been structurally characterized for the mixed compounds [BAlGaNPAs]H(6). B-N
bonding strongly (about approximately 90-100 kJ mol(-)(1)) stabilizes the mixed
heterocycles, followed by the preference of the Al-N bonded structures over Ga-N 
bonded ( approximately 30-40 kJ mol(-1)), while B-P bonding is slightly (5-10 kJ 
mol(-1)) more favorable compared to B-As. Thus, the bonding pattern is predicted 
to be the most stable, followed by the core. Processes of [HMYH](3) formation
from donor-acceptor complexes H(3)MYH(3) are predicted to be thermodynamically
favorable for all MY combinations. Dimerization reactions of the coordinationally
unsaturated [HMYH](3) heterocycles yielding hexamer clusters [HMYH](6) are found 
to be exothermic, with the exception of borazine, for which, as for benzene,
dimerization is strongly endothermic due to the aromaticity of C(6)H(6) and
[HBNH](3). Despite the high endothermicity of [HBNH](3) dimerization, the B-N
bond formation is the driving force of the dimerization of mixed species
[BAlGaNPAs]H(6). The dimerization enthalpies of [BAlGaNPAs]H(6) may be both exo- 
and endothermic, depending on the bonding pattern of the isomers. A complete set 
of mean MY bond energies in four- and six-membered cycles of [HMYH](6) was
derived. The MY energies were found to be transferable quantities and may serve
for a qualitative prediction of the relative stability of different isomers of
mixed cluster compounds. [BAlGaNPAs](2)H(12) clusters are promising synthetic
targets, they are expected to serve as single-source precursors for the
stoichiometry-controlled CVD processes of the group 13-15 composites. A strategy 
of their synthesis and the most suitable starting systems have been also
predicted.

PMID: 12513078 [PubMed]


333. Bioorg Med Chem Lett. 2003 Jan 20;13(2):223-8.

Anti-hyperlipidemic sesquiterpenes and new sesquiterpene glycosides from the
leaves of artichoke (Cynara scolymus L.): structure requirement and mode of
action.

Shimoda H, Ninomiya K, Nishida N, Yoshino T, Morikawa T, Matsuda H, Yoshikawa M.

Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan.

The methanolic extract from the leaves of artichoke (Cynara scolymus L.) was
found to suppress serum triglyceride elevation in olive oil-loaded mice. Through 
bioassay-guided separation, sesquiterpenes (cynaropicrin, aguerin B, and
grosheimin) were isolated as the active components together with new
sesquiterpene glycosides (cynarascolosides A, B, and C). The oxygen functional
groups at the 3- and 8-positions and exo-methylene moiety in
alpha-methylene-gamma-butyrolactone ring were found to be essential for the
anti-hyperlipidemic activity of guaiane-type sesquiterpene. In addition,
inhibition of gastric emptying was shown to be partly involved in
anti-hyperlipidemic activity.

PMID: 12482428 [PubMed - indexed for MEDLINE]


334. J Mol Biol. 2003 Jan 3;325(1):85-97.

phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is 
required for a stable and functional interaction with the terminal protein.

Rodriguez I, Lazaro JM, Salas M, de Vega M.

Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma,
Cantoblanco, E-28049 Madrid, Spain.

Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the
eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as
primer for initiation of DNA replication. By multiple sequence alignments of DNA 
polymerases from such a family, we have been able to identify two amino acid
residues specifically conserved in the protein-priming subgroup of DNA
polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate 
belonging to the Exo III motif. Here, we have studied the functional role of
these residues in reactions that are specific for DNA polymerases that use a
protein-primed DNA replication mechanism, by site-directed mutagenesis in the
corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase.
Mutations introduced at residue Phe128 severely impaired the protein-primed
replication capacity of the polymerase, being the interaction with the terminal
protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As 
a consequence, very few initiation products were obtained, and essentially no
transition products were detected. Interestingly, phi29 DNA polymerase mutant
F128Y showed a decreased binding affinity for short template DNA molecules. These
results, together with the high degree of conservation of Phe128 residue among
protein-primed DNA polymerases, suggest a functional role for this amino acid
residue in making contacts with the TP during the first steps of genome
replication and with DNA in the further replication steps.

PMID: 12473453 [PubMed - indexed for MEDLINE]


335. Genetics. 2002 Nov;162(3):1003-18.

Dissecting the fidelity of bacteriophage RB69 DNA polymerase: site-specific
modulation of fidelity by polymerase accessory proteins.

Bebenek A, Carver GT, Dressman HK, Kadyrov FA, Haseman JK, Petrov V, Konigsberg
WH, Karam JD, Drake JW.

Laboratory of Molecular Genetics, National Institute of Environmental Health
Sciences, National Institutes of Health, Research Triangle Park, North Carolina
27709-2233, USA.

Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with
an associated proofreading 3' exonuclease. Crystal structures have been
determined for this enzyme with and without DNA substrates. We previously
described the mutation rates and kinds of mutations produced in vivo by the
wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant
(Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the
polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-).
Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes
revealed the patterns and efficiencies of proofreading, while Tyr(567) was
identified as an important determinant of base-selection fidelity. Here, we
sought to determine how well the fidelities of the same enzymes are reflected in 
vitro. Compared to their behavior in vivo, the three mutator polymerases
exhibited modestly higher mutation rates in vitro and their mutational
predilections were also somewhat different. Although the RB69 gp43 accessory
proteins exerted little or no effect on total mutation rates in vitro, they
strongly affected mutation rates at many specific sites, increasing some rates
and decreasing others.

PMCID: PMC1462346
PMID: 12454051 [PubMed - indexed for MEDLINE]


336. Carbohydr Res. 2002 Nov 19;337(21-23):1941-51.

Stereoselective (2-naphthyl)methylation of sugar hydroxyls by the hydrogenolysis 
of diastereoisomeric dioxolane-type (2-naphthyl)methylene acetals.

Borbas A, Szabo ZB, Szilagyi L, Benyei A, Liptak A.

Research Group for Carbohydrates of the Hungarian Academy of Sciences, Debrecen, 
PO Box 55, H-4010, Hungary.

The cis axial/equatorial OH groups of methyl alpha-L- and ethyl
1-thio-alpha-L-rhamnopyranoside, 1,6-anhydro-beta-D-mannopyranose, and
1,6-anhydro-beta-D-galactopyranose were reacted with 2-naphthaldehyde dimethyl
acetal to diastereomeric dioxolane-type 2,3-O-(2-naphthyl)methylene or
3,4-O-(2-naphthyl)methylene acetals. The glycosides yielded the exo- and
endo-isomers in nearly 1:1 ratio, 1,6-anhydro-beta-D-mannopyranose gave
predominantly the endo-, and 1,6-anhydro-beta-D-galactopyranose exclusively
endo-isomer. The acetals and some of their fully protected derivatives bearing
benzyl or tert-butyldimethylsilyl groups were hydrogenolised with AlH(3)
(3LiAlH(4)-AlCl(3)) or with Me(3)N.BH(3)-AlCl(3) reagents. The endo-isomers were 
cleaved by both reagents to give axial NAP ethers, the exo-isomers of pyranosides
furnished equatorial NAP ethers. However, the exo-isomers of pyranoses gave
irregular axial ethers with a > 30-fold enhancement of the reaction rates with
respect to the endo-isomer. Copyright 2002 Elsevier Science Ltd.

PMID: 12433460 [PubMed - indexed for MEDLINE]


337. J Biol Chem. 2003 Jan 17;278(3):1626-33. Epub 2002 Nov 6.

Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 
3'-5'-exonuclease activities of Pol delta in the creation of a ligatable nick.

Jin YH, Ayyagari R, Resnick MA, Gordenin DA, Burgers PM.

Laboratory of Molecular Genetics, National Institute for Environmental and Health
Sciences, National Institutes of Health, North Carolina 27709, USA.

To address the different functions of Pol delta and FEN1 (Rad27) in Okazaki
fragment maturation, exonuclease-deficient polymerase Pol delta-01 and Pol
delta-5DV (corresponding to alleles pol3-01-(D321A, E323A) and pol3-5DV-(D520V), 
respectively) were purified and characterized in this process. In the presence of
the replication clamp PCNA, both wild-type and exo(-) Pol delta carried out
strand displacement synthesis with similar rates; however, initiation of strand
displacement synthesis was much more efficient with Pol delta-exo(-). When Pol
delta-exo(-) encountered a downstream primer, it paused with 3-5 nucleotides of
the primer displaced, whereas the wild type carried out precise gap filling.
Consequently, in the absence of FEN1, Pol delta exonuclease activity was
essential for closure of simple gaps by DNA ligase. Compared with wild type,
Okazaki fragment maturation with Pol delta-exo(-) proceeded with an increased
duration of nick translation prior to ligation. Maturation was efficient in the
absence of Dna2 and required Dna2 only when FEN1 activity was compromised. In
agreement with these results, the proposed generation of double strand breaks in 
pol3-exo(-) rad27 mutants was suppressed by the overexpression of DNA2. Further
genetic studies showed that pol3-exo(-) rad27 double mutants were sensitive to
alkylation damage consistent with an in vivo defect in gap filling by
exonuclease-deficient Pol delta.

PMID: 12424237 [PubMed - indexed for MEDLINE]


338. Biochim Biophys Acta. 2002 Dec 19;1573(3):225-35.

Golgi alpha-mannosidase II deficiency in vertebrate systems: implications for
asparagine-linked oligosaccharide processing in mammals.

Moremen KW.

Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602,
USA. moremen@arches.uga.edu

The maturation of N-glycans to complex type structures on cellular and secreted
proteins is essential for the roles that these structures play in cell adhesion
and recognition events in metazoan organisms. Critical steps in the biosynthetic 
pathway leading from high mannose to complex structures include the trimming of
mannose residues by processing mannosidases in the endoplasmic reticulum (ER) and
Golgi complex. These exo-mannosidases comprise two separate families of enzymes
that are distinguished by enzymatic characteristics and sequence similarity.
Members of the Class 2 mannosidase family (glycosylhydrolase family 38) include
enzymes involved in trimming reactions in N-glycan maturation in the Golgi
complex (Golgi mannosidase II) as well as catabolic enzymes in lysosomes and
cytosol. Studies on the biological roles of complex type N-glycans have employed 
a variety of strategies including the treatment of cells with glycosidase
inhibitors, characterization of human patients with enzymatic defects in
processing enzymes, and generation of mouse models for the enzyme deficiency by
selective gene disruption approaches. Corresponding studies on Golgi mannosidase 
II have employed swainsonine, an alkaloid natural plant product that causes
"locoism", a phenocopy of the lysosomal storage disease, alpha-mannosidosis, as a
result of the additional targeting of the broad-specificity lysosomal mannosidase
by this compound. The human deficiency in Golgi mannosidase II is characterized
by congenital dyserythropoietic anemia with splenomegaly and various additional
abnormalities and complications. Mouse models for Golgi mannosidase II deficiency
recapitulate many of the pathological features of the human disease and confirm
that the unexpectedly mild effects of the enzyme deficiency result from a
tissue-specific and glycoprotein substrate-specific alternate pathway for
synthesis of complex N-glycans. In addition, the mutant mice develop symptoms of 
a systemic autoimmune disorder as a consequence of the altered glycosylation.
This review will discuss the biochemical features of Golgi mannosidase II and the
consequences of its deficiency in mammalian systems as a model for the effects of
alterations in vertebrate N-glycan maturation during development.

PMID: 12417404 [PubMed - indexed for MEDLINE]


339. Bioorg Med Chem. 2002 Dec;10(12):4083-90.

Folded conformation of an immunostimulating tetrapeptide rigin: high temperature 
molecular dynamics simulation study.

Ashish A, Kishore R.

Institute of Microbial Technology, Chandigarh, India.

Employing high temperature quenched molecular dynamics (QMD) stimulations the
conformational energy space of an immunostimulating tetrapeptide rigin:
H-Gly341-Gln-Pro-Arg344-OH, is explored. Using distance dependent dielectric
(epsilon =r(ij)) 31 different low energy starting structures with identical
sequence were computed for their conformational preferences. According to the
hypothesis of O'Connors et al. [J. Med. Chem. 35 (1992), 2870], 83 low-energy
conformers resulted from unrestrained molecular dynamics (MD) simulations, could 
be classified into two energy minimized families: A and B, comprised of 64 (Pro
C(gamma)-endo orientation) and 19 (Pro C(gamma)-exo orientation) structures,
respectively. An examination of these families revealed the existence of a
remarkably similar folded backbone conformation: torsion angles being phi(i+1)
approximately -65 degrees, psi(i+1) approximately -65 degrees, phi(i+2)
approximately -65 degrees, psi(i+2) approximately -60 degrees, characterizing a
distorted type III beta-turn structure across the central Gln-Pro segment. The
folded conformation of rigin is devoid of a classical 1 <-- 4 intra-molecular
hydrogen bond nevertheless, the conformation is stabilized by an effective
'salt-bridge', i.e., Gly H(3)N(+)...C(alpha)OO(-) Arg interaction. Surprisingly, 
in both the families the unusual folded side-chain dispositions of the Gln
residue favor the formation of a unique intra-residue 'main-chain to side-chain' 
H-bond, i.e., N(alpha)-H...N(epsilon) interaction, encompassing a seven-membered 
ring motif. The conformational attributes may be valuable in de novo construction
of structure-based drug candidates having sufficient stimulating activity.

PMID: 12413862 [PubMed - indexed for MEDLINE]


340. Arch Microbiol. 2002 Nov;178(5):325-30. Epub 2002 Aug 6.

Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme
from an epsilon-poly-L-lysine-producing strain of Streptomyces albulus.

Kito M, Takimoto R, Yoshida T, Nagasawa T.

Department of Biomolecular Science, Faculty of Engineering, Gifu University,
Yanagido, Gifu 501-1193, Japan.

An epsilon-poly-L-lysine-degrading enzyme of an epsilon-poly-L-lysine-producing
strain of Streptomyces albulus was purified and characterized. The enzyme was
tightly bound to the cell membrane. After solubilization with NaSCN, the enzyme
was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography. The 
subunit molecular mass of the purified enzyme was 54 kDa. Enzyme activity was
inhibited by o-phenanthroline, and could be restored in the presence of 1 mM
Mg(2+), Ca(2+), Fe(3+) or Zn(2+). The mode of epsilon-poly-L-lysine degradation
was of the exo-type, and the enzyme released N-terminal L-lysines one by one. The
enzyme acted on various peptides possessing L-lysine residues at the N-terminus
and was classified as an aminopeptidase. Epsilon-Poly-L-lysine-degrading activity
was found in the membrane fraction of some other Streptomyces strains as well as 
that of Streptomyces albulus. Streptomyces virginiae IFO 12827 and Streptomyces
norsei IFO 15452 exhibited high epsilon-poly-L-lysine-degrading activity, and
both strains could produce epsilon-poly-L-lysine, indicating a correlation
between the distribution of membrane-bound epsilon-poly-L-lysine-degrading enzyme
and epsilon-poly-L-lysine-producing activity.

PMID: 12375099 [PubMed - indexed for MEDLINE]


341. J Am Chem Soc. 2002 Oct 16;124(41):12192-9.

Stereochemical memory in the temperature-dependent photodenitrogenation of
bridgehead-substituted DBH-type azoalkanes: inhibition of inverted-housane
formation in the diazenyl diradical through the mass effect (inertia) and steric 
hindrance.

Adam W, Garcia H, Diedering M, Marti V, Olivucci M, Palomares E.

Institut fur Organische Chemie, Universitat Wurzburg, Am Hubland, D-97074
Wurzburg, Germany. adam@chemie.uni-wuerzburg.de

The photochemical denitrogenation of the cyclopentene-annelated DBH-type
azoalkanes 1 has been examined in solution as a function of bridgehead
substitution and temperature. For all derivatives, namely, the unsubstituted
1a(H/H), monomethyl 1b(Me/H) dimethyl 1c(Me/Me), monophenyl 1d(Ph/H), and
diphenyl 1e(Ph/Ph), the temperature-dependent ratio of syn and anti housanes 2
provides experimental support for a competition between the singlet (high
temperature) and triplet (low temperature) reaction channels in the direct
photolysis. The syn/anti ratio of the housanes 2 depends on the extent and type
of bridgehead substitution; the amount of the anti diastereomer (retention)
follows the order Ph > Me > H, and double substitution is more effective than
single. This stereochemical memory is interpreted in terms of the mass effect
(inertia) of the substituents and steric interaction (size) between the
substituents at the bridgehead and the methylene bridge during the deazetation
step of the transient diazenyl diradical conformations (1)DZ (exo-ax) and (1)DZ
(exo-eq). These conformers are impulsively generated upon decay of the
(1)(n,pi)-excited azoalkane, a trajectory assessed through computational work.
The new mechanistic feature disclosed by the unprecedented anti stereoselectivity
(retention) is the intervention of a puckered 1,3-cyclopentanediyl singlet
diradical (1)DR as product bifurcation step, whose conformational relaxation to
the planar species (loss of stereochemical memory) is encumbered by bridgehead
substitution.

PMID: 12371859 [PubMed]


342. Acta Crystallogr C. 2002 Oct;58(Pt 10):o593-5. Epub 2002 Sep 21.

The regioisomeric 1H(2H)-pyrazolo[3,4-d]pyrimidine N1- and
N2-(2'-deoxy-beta-D-ribofuranosides).

He J, Seela F, Eickmeier H, Reuter H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.

In the title regioisomeric nucleosides, alternatively called
1-(2-deoxy-beta-D-erythro-furanosyl)-1H-pyrazolo[3,4-d]pyrimidine,
C(10)H(12)N(4)O(3), (II), and
2-(2-deoxy-beta-D-erythro-furanosyl)-2H-pyrazolo[3,4-d]pyrimidine,
C(10)H(12)N(4)O(3), (III), the conformations of the glycosylic bonds are anti
[-100.4 (2) degrees for (II) and 15.0 (2) degrees for (III)]. Both nucleosides
adopt an S-type sugar pucker, which is C2'-endo-C3'-exo ((2)T(3)) for (II) and
3'-exo (between (3)E and (4)T(3)) for (III).

PMID: 12359936 [PubMed]


343. J Pharmacol Exp Ther. 2002 Oct;303(1):99-103.

Muscarinic agonist-mediated increases in serum corticosterone levels are
abolished in m(2) muscarinic acetylcholine receptor knockout mice.

Hemrick-Luecke SK, Bymaster FP, Evans DC, Wess J, Felder CC.

Neuroscience Division, Lilly Research Laboratories, Eli Lilly and Co., Lilly
Corporate Center, Indianapolis, Indiana 46285, USA. luecke_susan_h@lilly.com

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the
central and peripheral nervous system. Due to the lack of receptor
subtype-selective ligands, however, the physiological roles of individual
muscarinic receptor subtypes remain to be determined. In this study, we examined 
the effects of the muscarinic M(2)/M(4) receptor-preferring agonist
[5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-octane
(BuTAC) on serum corticosterone levels in M(2) and M(4) receptor single knockout 
(KO) and M(2,4) receptor double KO mice. Responses were compared with those
obtained with the corresponding wild-type (WT) mice. BuTAC (0.03-0.3 mg/kg s.c.) 
dose dependently and significantly increased serum corticosterone concentrations 
in WT mice to 5-fold or greater levels compared with vehicle controls. In
muscarinic M(2) and M(2,4) KO mice, however, BuTAC had no significant effect on
corticosterone concentrations at doses of 0.1, 0.3, and 1 mg/kg s.c. In both WT
and muscarinic M(4) KO mice increases in serum corticosterone concentrations
induced by BuTAC (0.1 and 0.3 mg/kg) were not significantly different and were
blocked by scopolamine. In summary, the muscarinic M(2,4)-preferring agonist
BuTAC had no effect on corticosterone levels in mice lacking functional
muscarinic M(2) receptors. These data suggest that the muscarinic M(2) receptor
subtype mediates muscarinic agonist-induced activation of the
hypothalamic-pituitary-adrenocortical axis in mice.

PMID: 12235238 [PubMed - indexed for MEDLINE]


344. J Org Chem. 2002 Sep 20;67(19):6690-8.

Asymmetric total syntheses of (+)-cheimonophyllon e and (+)-cheimonophyllal.

Takao K, Tsujita T, Hara M, Tadano K.

Department of Applied Chemistry, Keio University, Hiyoshi, Kohoku-ku, Yokohama
223-8522, Japan.

The highly enantiocontrolled total syntheses of natural (+)-cheimonophyllon E (5)
and (+)-cheimonophyllal (6), biologically intriguing oxygenated bisabolane-type
sesquiterpenoids, have been completed. The present synthetic strategy featured
the use of an asymmetric aldol-type reaction for preparing in the first synthetic
step an optically active 6-C-substituted 3-methyl-2-cyclohexenone derivative.
Thus, a Mukaiyama aldol reaction of 1-methyl-3-silyloxy-1,3-cyclohexadiene 31
with alpha,beta-unsaturated aldehyde 11 in the presence of a chiral
(acyloxy)borane (CAB)-type Yamamoto catalyst 33 proceeded with high levels of
both diastereo- and enantioselectivities. The predominant aldol adduct, syn-9,
was transformed into gamma,delta-epoxy allylic alcohol 8 by a nine-step sequence,
including the substrate-controlled 1,2-reduction of enone, syn-12, also the
epoxidation of allylic alcohol 15. Epoxy-alcohol 8 underwent 5-exo-cyclization in
a high regioselective manner under acidic conditions to produce a bicyclic key
intermediate (+)-7, which was eventually efficiently converted to
(+)-cheimonophyllon E (5) or (+)-cheimonophyllal (6).

PMID: 12227798 [PubMed]


345. J Mol Biol. 2002 Sep 6;322(1):41-52.

Acetylation and accessibility of rDNA chromatin in Saccharomyces cerevisiae in
(Delta)top1 and (Delta)sir2 mutants.

Cioci F, Vogelauer M, Camilloni G.

Dipartimento di Genetica e Biologia Molecolare, Universita di Roma La Sapienza,
P. le A. Moro 5, 00185 Rome, Italy.

The insertion of reporter genes in the ribosomal DNA (rDNA) locus of
Saccharomyces cerevisiae causes their transcriptional repression. This kind of
transcriptional silencing depends on proteins such as Sir2p and Top1p, and has
been shown to be mediated by chromatin. While Sir2p modifies nucleosomes directly
through its histone deacetylase activity, little is known about changes in the
chromatin structure that occur at the rDNA locus when TOP1 is deleted. Here, we
show that the absence of Top1p causes increased histone acetylation at the rDNA
locus. Moreover, rDNA chromatin becomes more accessible in a similar manner in
both top1 and sir2 mutant strains.

PMID: 12215413 [PubMed - indexed for MEDLINE]


346. Appl Environ Microbiol. 2002 Sep;68(9):4233-9.

Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25/EC
3.2.1.33) and its potential for cycloamylose production.

Yanase M, Takata H, Takaha T, Kuriki T, Smith SM, Okada S.

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Nishiyodogawa-ku, Osaka
555-8502, Japan. yanase-michiyo@glico.co.jp

Glycogen debranching enzyme (GDE) has 4-alpha-glucanotransferase and
amylo-1,6-glucosidase activities in the single polypeptide chain. We analyzed the
detailed action profile of GDE from Saccharomyces cerevisiae on amylose and
tested whether GDE catalyzes cyclization of amylose. GDE treatment resulted in a 
rapid reduction of absorbance of iodine-amylose complex and the accumulation of a
product that was resistant to an exo-amylase (glucoamylase [GA]) but was degraded
by an endo-type alpha-amylase to glucose and maltose. These results indicated
that GDE catalyzed cyclization of amylose to produce cyclic alpha-1,4 glucan
(cycloamylose). The formation of cycloamylose was confirmed by high-performance
anion-exchange chromatography, and the size was shown to range from a degree of
polymerization of 11 to a degree of polymerization around 50. The minimum size
and the size distribution of cycloamylose were different from those of
cycloamylose produced by other 4-alpha-glucanotransferases. GDE also efficiently 
produced cycloamylose even from the branched glucan substrate, starch,
demonstrating its potential for industrial production of cycloamylose.

PMCID: PMC124075
PMID: 12200270 [PubMed - indexed for MEDLINE]


347. Plant Cell Physiol. 2002 Aug;43(8):923-31.

AtMRD1 and AtMRU1, two novel genes with altered mRNA levels in the methionine
over-accumulating mto1-1 mutant of Arabidopsis thaliana.

Goto DB, Naito S.

Laboratory of Molecular Biology, Division of Applied Bioscience, Graduate School 
of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan.

The mto1-1 mutant of Arabidopsis thaliana over-accumulates soluble methionine
(Met) up to 40-fold higher than that in its Col-0 wild type. In order to identify
genes regulated by altered Met concentrations, microarray analysis of gene
expression in young rosettes and developing siliques of the mto1-1 mutant were
performed. Expression of selected genes was then examined in detail in three
developmental stages of the mto1-1 mutant using a combination of Northern
hybridisation analysis and real-time PCR. Eight genes were identified that had
altered mRNA accumulation levels in the mto1-1 mutant compared to that in
wild-type plants. Three of the genes have known roles in plant development
unrelated to amino acid biosynthesis. One other gene up-regulated specifically in
mto1-1 rosettes shared similarity with the embryo-specific protein 3 (ATS3). Two 
novel genes, referred to as AtMRD1 and AtMRU1, were also identified that were
expressed in a developmental manner in wild-type Col-0 and do not share sequence 
similarity with genes of known function. AtMRD1 was strongly down-regulated in
both rosette and young silique tissues of the mto1-1 mutant. AtMRU1 was
up-regulated approximately 3-fold in young mto1-1 rosettes and exhibited a
developmental response to the mto1-1 mutation.

PMID: 12198195 [PubMed - indexed for MEDLINE]


348. J Biol Chem. 2002 Nov 1;277(44):41379-89. Epub 2002 Aug 23.

Weak strand displacement activity enables human DNA polymerase beta to expand
CAG/CTG triplet repeats at strand breaks.

Hartenstine MJ, Goodman MF, Petruska J.

Department of Biological Sciences, Hedco Molecular Biology Laboratories,
University of Southern California, Los Angeles 90089-1340, USA.

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta
effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at 
nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes
under physiological conditions. In the same constructs, Escherichia coli DNA
polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, 
because its much stronger strand displacement activity inhibits slippage by
enabling rapid extension through two downstream repeats into flanking non-repeat 
sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min
period at rates of approximately 1 triplet added per min, reveal significant
effects of break type (nick versus gap), strand composition (CTG versus CAG), and
dNTP substrate concentration, on repeat expansions at strand breaks. At
physiological substrate concentrations (1-10 microm of each dNTP), polymerase
beta expands triplet repeats with the help of weak strand displacement limited to
the two downstream triplet repeats in our constructs. Such weak strand
displacement activity in DNA repair at strand breaks may enable short tracts of
repeats to be converted into longer, increasingly mutable ones associated with
neurological diseases.

PMID: 12196536 [PubMed - indexed for MEDLINE]


349. Chemistry. 2002 Jun 3;8(11):2464-75.

Enantioselective Norrish-Yang cyclization reactions of
N-(omega-oxo-omega-phenylalkyl)-substituted imidazolidinones in solution and in
the solid state.

Bach T, Aechtner T, Neumuller B.

Lehrstuhl fur Organische Chemie I Technische Universitat Munchen
Lichtenbergstrasse 4, 85747 Garching, Germany. thorsten.bach@ch.tum.de

The four N-(omega-oxo-omega-phenyl-alkyl)-substituted imidazolidinones 5-8 were
prepared from N-acetylimidazolidinone (4). Upon irradiation, these substrates
underwent Norrish-Yang cyclization to the racemic products rac-9-rac-12 (51-75%).
The reactions of the N-2-oxoethylimidazolidinones 5 and 6 were conducted in
tBuOH, and yielded 1:1 mixtures of exo/endo diastereoisomers rac-9a/rac-9b and
rac-10a/rac-10b, accompanied by Norrish type II cleavage products. The reactions 
of the N-3-oxopropylimidazolidinones 7 and 8 were performed in toluene. The exo
diastereoisomers rac-11a and rac-12a were the major diastereoisomers (d.r.
approximately equal to 4:1). In the presence of the chiral compounds 1-3, the
photocyclization of substrate 8 proceeded with significant enantiomeric excess
(5-60% ee). The more sophisticated complexing agents 3 and ent-3 provided better 
enantiofacial differentiation (up to 60% ee) than the lactams 1 and 2 (up to 26% 
ee). Low temperatures and an excess of the complexing agent helped to increase
the enantioselectivity. The absolute configuration of the major exo product 12a
obtained from compound 8 in the presence of complexing agent 3 was unambiguously 
established by single-crystal X-ray crystallography of its chiral
N-methoxyphenylacetyl derivative 15a. In a similar fashion, the absolute
configurations of the endo products 12b and ent-12b were established. The
N-2-oxoethylimidazolidinone 5, which crystallized in a chiral space group, was
irradiated in the solid state. At low levels of conversion, the product 9a/ent-9a
was formed with high enantiomeric excess (78% ee). The enantioselectivity
deteriorated at higher levels of conversion.

PMID: 12180325 [PubMed]


350. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1998;30(1):47-52.

Analysis of the Exo(-) Mutant of Rhizobium huakuii and Their Exopolysaccharide
Component.

Su WY, Song HY, Wang KY.

Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences, Shanghai
200032, China.

We have subcloned two plasmids, pWS-6BP1 and pWS-6BP2, from the 8.5 kb DNA
fragment of plasmid pJB22-B6. The plasmid of pWS-6BP1, with a 5.8 kb DNA
fragment, can complement the exopolysaccharide-deficient mutant NA06 and NA12 of 
Rhizobium huakuii strain 107, but the plasmid pWS-6BP2, with a 2.6 kb DNA
fragment can only complement NA12. The effective nodules formed by the wild type 
strain 107 (Rh107) contain lots of bacteriods; the ineffective nodules formed by 
the mutant NA06 and NA12 contain few bacteroids. Although the nodule formed by
Exo(+) transconjugants are ineffective, the bacteria can be successfully released
into the cells and form lots of bacteroids. The Rh107 produces acidic
exopolysaccharides (EPS) of high molecular weight (HMW) in large amount with only
small amount of low molecular weight (LMW) forms. EPS secreted by mutants NA06
and NA12 are just the other way round with LMW in higher amount than HMW ones.
They also produce an EPS with molecular weight in between. The EPS secreted by
the Exo(+) transconjugants is more like the EPS of Rh107 than that of mutants,
although their component and structure are still different from that of Rh107.

PMID: 12174297 [PubMed - as supplied by publisher]


351. J Biol Chem. 2002 Oct 4;277(40):37840-7. Epub 2002 Jul 29.

Molecular structure and novel DNA binding sites located in loops of flap
endonuclease-1 from Pyrococcus horikoshii.

Matsui E, Musti KV, Abe J, Yamasaki K, Matsui I, Harata K.

Biological Information Research Center and the Gene Discovery Research Center,
National Institute of Advanced Industrial Science and Technology, Higashi 1-1-1, 
Tsukuba, Ibaraki 305-566, Japan.

The crystal structure of flap endonuclease-1 from Pyrococcus horikoshii (phFEN-1)
was determined to a resolution of 3.1 A. The active cleft of the phFEN-1 molecule
is formed with one large loop and four small loops. We examined the function of
the conserved residues and positively charged clusters on these loops by kinetic 
analysis with 45 different mutants. Arg(40) and Arg(42) on small loop 1, a
cluster Lys(193)-Lys(195) on small loop 2, and two sites, Arg(94) and
Arg(118)-Lys(119), on the large loop were identified as binding sites. Lys(87) on
the large loop may play significant roles in catalytic reaction. Furthermore, we 
successfully elucidated the function of the four DNA binding sites that form
productive ES complexes specific for each endo- or exo-type hydrolysis, probably 
by bending the substrates. For the endo-activity, Arg(94) and Lys(193)-Lys(195)
located at the top and bottom of the molecule were key determinants. For the
exo-activity, all four sites were needed, but Arg(118)-Lys(119) was dominant. The
major binding sites for both the nick substrate and double-stranded DNA might be 
the same.

PMID: 12147694 [PubMed - indexed for MEDLINE]


352. Biochemistry. 2002 Aug 6;41(31):9736-46.

A case for reverse protonation: identification of Glu160 as an acid/base catalyst
in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic
analysis of a site-directed mutant.

Vocadlo DJ, Wicki J, Rupitz K, Withers SG.

Protein Engineering Network of Centres of Excellence of Canada, Vancouver,
British Columbia, Canada V6T 1Z1.

The catalytic mechanism of the family 39 Thermoanaerobacterium saccharolyticum
beta-xylosidase (XynB) involves a two-step double-displacement mechanism in which
a covalent alpha-xylosyl-enzyme intermediate is formed with assistance from a
general acid and then hydrolyzed with assistance from a general base. Incubation 
of recombinant XynB with the newly synthesized active site-directed inhibitor,
N-bromoacetyl-beta-D-xylopyranosylamine, resulted in rapid, time-dependent
inactivation of the enzyme (k(i)/K(i) = 4.3 x 10(-4) s(-1)mM(- 1)). Protection
from inactivation using xylose or benzyl 1-thio-beta-xyloside suggested that the 
inactivation was active site-directed. Mass spectrometric analysis indicated that
incubation of the enzyme with the inactivator resulted in the stoichiometric
formation of a new enzyme species bearing the label. Comparative mapping of
peptic digests of both the labeled and unlabeled enzyme by HPLC coupled to an
electrospray ionization mass spectrometer permitted the identification of a
labeled peptide. Sequencing of this peptide by tandem mass spectrometry
identified Glu160 within the sequence (157)IWNEPNL(164) as the site of attachment
of the N-acetyl-beta-D-xylopyranosylamine moiety. Kinetic analysis of the
Glu160Ala mutant strongly suggests that this residue is involved in acid/base
catalysis as follows. First, a significant difference in the dependence of
k(cat)/K(m) on pH as compared to that seen for the wild-type enzyme was found, as
expected for a residue that is involved in acid/base catalysis. The changes,
however, were not as simple as those seen in other cases. Second, a dramatic
decrease (up to 10(5)-fold) in the catalytic efficiency (k(cat)/K(m)) of the
enzyme with a substrate requiring protonic assistance is observed upon such
mutation. In contrast, the catalytic efficiency of the enzyme with substrates
bearing a good leaving group, not requiring acid catalysis, is only moderately
impaired relative to that of the wild-type enzyme (8-fold). Surprisingly,
however, the glycosylation step was rate-limiting for all but the most reactive
substrates. Last, the addition of azide as a competitive nucleophile resulted in 
the formation of a beta-xylosyl azide product and increased the k(cat) and K(m)
values up to 8-fold while k(cat)/K(m) remained relatively unchanged. Such kinetic
behavior is consistent with azide acting competitively with water as a
nucleophile in the second step of the enzyme catalyzed reaction involving
breakdown of the xylosyl-enzyme intermediate. Together, these results provide
strong evidence for a role of Glu160 in acid/base catalysis but suggest that it
may be partnered by a second carboxylic acid residue and that the enzyme may
function through using acid catalysis involving reverse protonation of active
site residues.

PMID: 12146939 [PubMed - indexed for MEDLINE]


353. J Chromatogr A. 2002 Jun 14;959(1-2):121-9.

Micropreparative fractionation of DNA fragments on metathesis-based monoliths:
influence of stoichiometry on separation.

Lubbad S, Mayr B, Huber CG, Buchmeiser MR.

Institute of Analytical Chemistry and Radiochemistry, University of Innsbruck,
Austria.

Applying Grubbs' first generation benzylidene-type catalyst Cl2Ru(PCy3)2(CHPh) in
ring opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and
1,4,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonapthalene (DMN-H6), various
monoliths were prepared within the confines of silanized borosilicate columns
(100x3 mm I.D.) and investigated for the micropreparative separation of pBR322
DNA-Hae III restriction fragments ranging in size from 51 to 587 base pairs (bp),
as a sample of double-stranded (ds) DNA. The approach to good resolution of dsDNA
on monolithic columns entailed the modulation of the polymer morphology in terms 
of structure and porosity to suit such an analysis. Structural variations were
achieved by changing the relative ratios of comonomers (NBE+DMN-H6) at the
expense of porogens, and by increasing the DMN-H6 to NBE mass ratio. For dsDNA
separations, eluents comprised 0.1 M aqueous triethylammonium acetate, pH 7.0,
and acetonitrile. Alternatively, methanol was introduced in this study as a less 
polar gradient former. In terms of column evaluation, each column prepared was
first tested in the separation of 5'-phosphorylated oligodeoxythymidylic acids
[p(dT)(12-18)], since good separation of oligodeoxynucleotides indicates the
potential liability of the column tested for dsDNA analysis, and vice versa. It
was noted that monoliths with combinations of 25:25:40:10, 28:28:35:9, and
30:30:32:8 (as weight% of NBE/DMN-H6/2-propanol/toluene) showed good resolution
of p(dT)(12-18). Moreover, they demonstrated good separation of the first 12
fragments (51-267 bp) of the pBR322 DNA-Hae III digest; however, reduced
resolution in the separation of the last five highest molecular mass fragments
(434-587 bp) was experienced. The best separation of these fragments was
accomplished on a 25:25:40:10 NBE/DMN-H6/2-propanol/toluene combination at a
flow-rate of 2 ml/min, a temperature of 50 degrees C, and a gradient of 4-10%
acetonitrile in 1 min, then 10-16% in 14 min. The total amount of pBR322 HaeIII
digest that may be fractionated on these systems is 0.5-2.5 microg.

PMID: 12141537 [PubMed - indexed for MEDLINE]


354. Biochem Biophys Res Commun. 2002 Jul 26;295(4):818-25.

A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase
and alpha-glucosidase, that liberates glucose from the reducing end of the
substrates.

Lee MH, Kim YW, Kim TJ, Park CS, Kim JW, Moon TW, Park KH.

Research Center for New Bio-Materials in Agriculture and Department of Food
Science and Technology, School of Agricultural Biotechnology, Seoul National
University, Suwon, Republic of Korea.

The gene previously designated as putative cyclodextrinase from Thermotoga
maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant 
TMG was partially purified and its enzymatic characteristics on various
substrates were examined. The enzyme hydrolyzes various maltodextrins including
maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and
maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose,
a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG
initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and
p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from 
the reducing end of maltooligosaccharides unlike to other glucosidases. Since its
enzymatic activity is negligible if alpha-methylglucoside is present in the
reducing end, the type of the residue at the reducing end of the substrate is
important for the TMG activity. These results support the fact that TMG is a
novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan 
hydrolyzing enzyme and alpha-glucosidase.

PMID: 12127967 [PubMed - indexed for MEDLINE]


355. J Org Chem. 2002 Jul 26;67(15):5197-201.

Reaction of allylsilanes and allylstannanes with alkynes catalyzed by
electrophilic late transition metal chlorides.

Fernandez-Rivas C, Mendez M, Nieto-Oberhuber C, Echavarren AM.

Departamento de Quimica Organica, Universidad Autonoma de Madrid, Cantoblanco,
28049 Madrid, Spain.

The intramolecular reaction of allylsilanes and allylstannanes with alkynes
proceeds catalytically in the presence of Pt(II), Pd(II), Ru(II), and Au(III)
chlorides. Although more limited, AgOTf also catalyzes the cyclization. Usually, 
PtCl2 as the catalyst in methanol or acetone gives the best results. The reaction
proceeds by exo attack of the allyl nucleophile on the alkyne to form five- or
six-membered ring carbocycles. The reaction generally proceeds with anti
stereoselectivity. However, a terminally substituted trimethylsilyl derivative
reacts by a syn-type addition. The intermediate alkenylpalladium complex has been
trapped with allyl chloride to form an allylated derivative with an additional
carbon-carbon bond.

PMID: 12126406 [PubMed]


356. J Neurosci. 2002 Jul 1;22(13):5516-24.

The Role of gp130 in cerebral cortical development: in vivo functional analysis
in a mouse exo utero system.

Hatta T, Moriyama K, Nakashima K, Taga T, Otani H.

Department of Anatomy, Shimane Medical University, Izumo 693-8501, Japan.
thatta@shimane-med.ac.jp

The role of gp130 in cerebral cortical histogenesis remains unknown. Mice lacking
gp130 showed a hypoplastic cortical plate and decreased incorporation of
5-bromo-2'-deoxyuridine (BrdU) in progenitor cells of the developing cerebrum. In
contrast, injection of leukemia inhibitory factor (LIF), a gp130 ligand, into the
lateral cerebral ventricle of wild-type embryos exo utero induced hyperplasia of 
the cerebral cortex and increased the incorporation of BrdU in progenitor cells. 
Furthermore, chronologically controlled injection of LIF followed or preceded by 
BrdU revealed that gp130-mediated signals promote the progenitor cells to reenter
the stem cell cycle without affecting the duration of cell cycle and enhance the 
migration of postmitotic neurons in the developing cerebrum.

PMID: 12097503 [PubMed - indexed for MEDLINE]


357. Comp Biochem Physiol A Mol Integr Physiol. 2002 May;132(1):1-8.

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in
GcMAF-related macrophage activation.

Mohamad SB, Nagasawa H, Uto Y, Hori H.

Department of Biological Science and Technology, Faculty of Engineering, The
University of Tokushima, Tokushima 770-8506, Japan.

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate
in serum of cancer patients and be responsible for deglycosylation of Gc protein,
which is a precursor of GcMAF-mediated macrophage activation cascade, finally
leading to immunosuppression in advanced cancer patients. We studied the
biochemical characterization of alpha-NaGalase from several human tumor cell
lines. We also examined its effect on the potency of GcMAF to activate mouse
peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage
activation cascade. The specific activity of alpha-NaGalases from human colon
tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver
cells (Chang liver cell line) were evaluated using two types of substrates;
GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type
substrate). Tumor-derived alpha-NaGalase having higher activity than normal
alpha-NaGalase, had higher substrate specificity to the exo-type substrate than
to the endo-type substrate, and still maintained its activity at pH 7. GcMAF
enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF
with tumor cell lysate reduce the activity. We conclude that tumor-derived
alpha-NaGalase is different in biochemical characterization compared to normal
alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived
alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

PMID: 12062184 [PubMed - indexed for MEDLINE]


358. J Org Chem. 2002 May 31;67(11):3773-82.

Stereochemistry in the synthesis and reaction of exo-glycals.

Yang WB, Yang YY, Gu YF, Wang SH, Chang CC, Lin CH.

Institute of Biological Chemistry, Academia Sinica, No. 128, Academia Road
Section 2, Nan-Kang, Taipei 11529, Taiwan.

Two general methods are explored for the stereoselective synthesis of
exo-glycals. One method utilizes a nucleophilic addition of fully protected sugar
lactones of gluco-, galacto-, and manno-types, followed by the subsequent
dehydration, to give the desired exo-glycals with (Z)-configuration. The other
method proceeds with selenylation of C-glycosides in a stereoselective manner.
The subsequent selenoxide elimination also provides (Z)-exo-glycals. The prepared
exo-glycal conjugated esters of either gluco- or manno-type react with allyl
alcohol to give exclusively alpha-anomers.

PMID: 12027693 [PubMed - indexed for MEDLINE]


359. J Am Chem Soc. 2002 May 15;124(19):5514-7.

Directing power of cyclobutenoid annelations on the double bonds of planar
cyclooctatetraenes.

Baldridge KK, Siegel JS.

San Diego Supercomputer Center, La Jolla, California 92093-0505, USA.

Ab initio and hybrid density functional quantum mechanical computations are
applied to the structure and energetics of a series of two-atom-bridge annelated 
cyclooctatetraenes. The contribution of each annelation to the exo/endo relative 
energy is estimated. Key directing factors for a given type of annelation, such
as strain, electronegativity, or cyclic electron count, can be sorted out by
comparison of various bridge compositions. Overall, electron count and the
essential components of the Clar/Robinson rule work well to predict the exo/endo 
preferences. Specifically, three 4-e(-) Huckel systems (CH-CH, NH-BH and NH-C(O))
display dominant exo forms whereas the three 4n + 2 Huckel counterparts
(C(O)-C(O), BH-BH, and planar NH-NH) display a common preference for endo. These 
endo systems act like four independent four-membered "aromatic" rings linked by
"single" bonds. An analysis based on the effective hybridization of carbon atoms 
in the annulene (Bent's rule) provides a rationale for subtle trends in their
specific annulene geometry.

PMID: 11996594 [PubMed - indexed for MEDLINE]


360. J Biol Chem. 2002 Jul 12;277(28):25096-105. Epub 2002 May 1.

Isolation of cyanophycin-degrading bacteria, cloning and characterization of an
extracellular cyanophycinase gene (cphE) from Pseudomonas anguilliseptica strain 
BI. The cphE gene from P. anguilliseptica BI encodes a cyanophycinhydrolyzing
enzyme.

Obst M, Oppermann-Sanio FB, Luftmann H, Steinbuchel A.

Institut fur Mikrobiologie, Westfalische Wilhelms-Universitat Munster,
Corrensstrasse 3, Germany.

Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide
(CGP)) as a carbon source for growth were isolated. One isolate was taxonomically
affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular
cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon 
source and extracellular cyanophycinases were hitherto not described. CphE was
detected in supernatants of CGP cultures and purified from a corresponding
culture of strain BI employing chromatography on the anion exchange matrix
Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the
inducible enzyme consisted of one type of subunit with M(r) = 43,000 and
exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic
acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products
of the enzyme reaction were identified as aspartic acid-arginine dipeptides
(beta-Asp-Arg) by high performance liquid chromatography and electrospray
ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was 
identified in subclones of a cosmid gene library of strain BI by heterologous
active expression in Escherichia coli, and its nucleotide sequence was
determined. The enzyme exhibited only 27-28% amino acid sequence identity to
intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino
acid sequence of cphE revealed a putative catalytic triad consisting of the motif
GXSXG plus a histidine and most probably a glutamate residue. In addition, the
strong inhibition of the enzyme by Pefabloc((R)) and phenylmethylsulfonyl
fluoride indicated that the catalytic mechanism of CphE is related to that of
serine type proteases. Quantitative analysis on the release of beta-Asp-Arg
dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation
mechanism.

PMID: 11986309 [PubMed - indexed for MEDLINE]


361. Biochemistry. 2002 Apr 30;41(17):5462-72.

Mispairing of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct with
deoxyadenosine results in extrusion of the mismatched dA toward the major groove.

Giri I, Johnston DS, Stone MP.

Department of Chemistry and Center in Molecular Toxicology, Vanderbilt
University, Nashville, Tennessee 37235, USA.

The G --> T transversion is the dominant mutation induced by the cationic
trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct. The structure of
d(ACATC(AFB)GATCT).d(AGATAGATGT), in which the cationic adduct was mismatched
with deoxyadenosine, was refined using molecular dynamics calculations restrained
by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained
molecular dynamics calculations refined structures with pairwise rmsd <1 A and a 
sixth root R1x factor between the refined structure and NOE data of 10.5 x 10-2. 
The mismatched duplex existed in a single conformation at neutral pH. The
aflatoxin moiety intercalated above the 5' face of the modified (AFB)G. The
mismatched dA was in the anti conformation about the glycosyl bond. It extruded
toward the major groove and did not participate in hydrogen bonding with (AFB)G. 
The structure was compared with that of d(ACATCGATCT).d(AGATAGATGT) containing
the corresponding unmodified G.A mismatch and with
d(ACATC(AFB)GATCT).d(AGATCGATGT) containing the aflatoxin lesion in the correctly
paired (AFB)G.C context. The correctly paired oligodeoxynucleotide exhibited
Watson-Crick-type geometry at the (AFB)G.C pair. It melted at higher temperature 
than the mismatched (AFB)G.A duplex. The unmodified mismatched G.A duplex
exhibited spectral line broadening at neutral pH, suggesting a mixture of
conformations. It exhibited a lower melting temperature than did the mismatched
(AFB)G.A duplex. These differences correlated with replication bypass experiments
performed in vitro utilizing DNA polymerase I exo- [Johnston, D. S., and Stone,
M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164]. Those experiments showed that
correct insertion of dC opposite (AFB)G blocked replication by the enzyme,
whereas incorrect insertion of dA opposite (AFB)G allowed full-length replication
of the adducted template strand.

PMID: 11969407 [PubMed - indexed for MEDLINE]


362. FEMS Microbiol Lett. 2002 Feb 5;207(2):147-51.

Occurrence of epsilon-poly-L-lysine-degrading enzyme in
epsilon-poly-L-lysine-tolerant Sphingobacterium multivorum OJ10: purification and
characterization.

Kito M, Onji Y, Yoshida T, Nagasawa T.

Department of Biomolecular Science, Faculty of Engineering, Gifu University,
Yanagido, 501-1193, Gifu, Japan.

Epsilon-poly-L-lysine (epsilon-PL)-degrading enzyme was found in the
epsilon-PL-tolerant strain Sphingobacterium multivorum OJ10 and purified to
homogeneity. The purified enzyme has a molecular mass of approximately 80 kDa.
The enzyme catalyzed exo-type degradation of epsilon-PL and released L-lysine.
The enzyme was a Co2+ or Ca2+ ion-activated aminopeptidase.

PMID: 11958932 [PubMed - indexed for MEDLINE]


363. Chemistry. 2002 Apr 2;8(7):1649-62.

Fluxional processes in diamagnetic and paramagnetic allyl dicarbonyl and
2-methylallyl dicarbonyl molybdenum histidinato complexes as revealed by
spectroscopic data and density functional calculations.

van Staveren DR, Bill E, Bothe E, Buhl M, Weyhermuller T, Metzler-Nolte N.

Max-Planck-Institut fur Strahlenchemie Stiftstrasse 34-36, 45 470 Mulheim/Ruhr,
Germany.

This work describes a detailed study on the structure and dynamics of
pseudooctahedral low-valent complexes of the type
[Mo(His-N(epsilon)-R)(eta-2-R'-allyl)(CO)(2)] (His=N(delta),N,O-L-histidinate;
R=H, R'=H (1); R=C(2)H(4)CO(2)Me, R'=H (2); R=H, R'=Me (3); R=C(2)H(4)CO(2)Me,
R'=Me (4)). These diamagnetic 18-electron complexes were comprehensively
characterized spectroscopically and by X-ray crystallography. In the solid state,
the (substituted) allyl ligand is in an endo position in all compounds, but it is
trans to the His-N(delta) atom in 1 and 2, whereas it is trans to the carboxylate
O atom for the 2-Me-allyl compounds 3 and 4. In solution, both isomers are
present in a solvent-dependent equilibrium. The third isomer (allyl trans to
His-NH(2)) is not spectroscopically observed in solution. This is in agreement
with the results from density functional (DFT) computations (BPW 91 functional)
for 1 and 3, which predict a considerably higher energy (+6.3 and +5.9 kJ
mol(-1), respectively) for this isomer. A likely path for isomerization is
calculated, which is consistent with the activation energy determined by variable
temperature NMR measurements. At least for 3, the preferred path involves several
intermediates and a rotation of the 2-Me-allyl ligand. For the paramagnetic
17-electron congeners, DFT predicts the exo isomer of 3(+) with the 2-Me-allyl
ligand trans to the carboxylate O atom to be by far the most stable isomer. For
1(+), an endo-exo equilibrium between the isomers with the allyl ligand trans to 
the carboxylate O atom is suggested. These suggestions are confirmed by EPR
spectroscopy on the electrochemically generated species, which show signals for
one- (4) and two- (2) metal-containing compounds. The appearance of the EPR
spectra may be rationalized by inspection of the SOMOs from DFT calculations of
the species in question. The notion of a metal-centered oxidation is also
substantiated by IR spectroelectrochemistry and by UV/Vis spectra of the
17-electron complexes. Upon depleting the metal of electron density, the
stretching vibrations of the carbonyl ligands shift more than 100 cm(-1) to
higher wavenumbers, and the carbonyl vibration of the metal-coordinated
carboxylate shifts by about 50 cm(-1). A color change from yellow to green upon
oxidation is observed visually and quantified by the appearance of a new band at 
622 nm (2(+)) and 546 nm (4(+)), respectively.

PMID: 11933093 [PubMed - indexed for MEDLINE]


364. Naunyn Schmiedebergs Arch Pharmacol. 2002 Apr;365(4):303-11. Epub 2002 Feb 20.

Is Na(+) required for the binding of dopamine, amphetamine, tyramine, and
octopamine to the human dopamine transporter?

Li LB, Cui XN, Reith MA.

Department of Biology, Illinois State University, Normal, IL 61790, USA.

The role of Na(+) in the recognition of blockers by the dopamine transporter is
accomodated by a model with a cation site that overlaps with the blocker binding 
domain, and a distal Na(+) site that interacts with this cation site and perhaps 
with the blocker binding domain itself. The present study addresses the
application of this model to the recognition of substrates by the dopamine
transporter, focusing on conditions that should reveal a stimulatory effect, if
present, of Na(+) on substrate binding. Recognition was studied via the
inhibition of binding of [(3)H]WIN 35,428
(2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to
the human dopamine transporter in human embryonic kidney 293 cells. Little or no 
changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or
dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-),
both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and
d-amphetamine showed binding increases upon raising Na(+), leveling off with
NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+),
consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM
Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and,
with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed.
This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by
Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of
Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50)
values in Na(+) free buffer were not lower for dopamine and d-amphetamine than
p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory
effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+)
disinhibition. All results together support the existence of a substrate domain
overlapping with a cation site that also binds Tris(+); a distal Na(+) site
interacts with this cation site and with the substrate domain by negative
allosterism and is additionally impacted by Cl(-). Importantly, interactions
between sites vary with the type of substrate, and, in membrane preparations,
Na(+) is not required for, or stimulatory to, the binding of any of the four
substrates studied unlike the binding of the cocaine analog WIN 35,428.

PMID: 11919655 [PubMed - indexed for MEDLINE]


365. Infect Immun. 2002 Apr;70(4):1807-15.

Purification and characterization of arginine carboxypeptidase produced by
Porphyromonas gingivalis.

Masuda K, Yoshioka M, Hinode D, Nakamura R.

Department of Preventive Dentistry, School of Dentistry, The University of
Tokushima, Tokushima, Japan.

Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas
gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by
high-performance liquid chromatography on DEAE-5PW and TSK G2000SW(XL). Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme
revealed the presence of three major bands at 42, 33, and 32 kDa with identical
N-terminal sequences. By Western blotting analysis and immunoelectron microscopy,
the arginine carboxypeptidase was found to be widely distributed in the cytoplasm
and on the surface of the outer membrane. The open reading frame corresponding to
the N-terminal amino acids of the arginine carboxypeptidase was detected by a
search of the sequence of the P. gingivalis W83 genome. This sequence showed
homology with mammalian carboxypeptidases (M, N, and E/H) and included a
zinc-binding region signature, suggesting that the enzyme is a member of the zinc
carboxypeptidase family. The purified enzyme was inhibited by EGTA,
o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and
some metal ions, such as Cu(2+), Zn(2+), and Cd(2+). On the other hand, Co(2+)
activated the enzyme. The enzyme released arginine and/or lysine from
biologically active peptides containing these amino acids at the C terminus but
did not cleave substrates when proline was present at the penultimate position.
These results indicate that the arginine carboxypeptidase produced by P.
gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to
release arginine in collaboration with an arginine aminopeptidase, e.g.,
Arg-gingipain, to obtain specific amino acids from host tissues during the growth
of P. gingivalis.

PMCID: PMC127852
PMID: 11895942 [PubMed - indexed for MEDLINE]


366. J Am Chem Soc. 2002 Mar 20;124(11):2497-505.

Collagen stability: insights from NMR spectroscopic and hybrid density functional
computational investigations of the effect of electronegative substituents on
prolyl ring conformations.

DeRider ML, Wilkens SJ, Waddell MJ, Bretscher LE, Weinhold F, Raines RT, Markley 
JL.

Department of Chemistry, Graduate Program in Biophysics, University of
Wisconsin-Madison, Madison, WI 53706, USA.

Collagen-like peptides of the type (Pro-Pro-Gly)(10) fold into stable triple
helices. An electron-withdrawing substituent at the H(gamma)(3) ring position of 
the second proline residue stabilizes these triple helices. The aim of this study
was to reveal the structural and energetic origins of this effect. The approach
was to obtain experimental NMR data on model systems and to use these results to 
validate computational chemical analyses of these systems. The most striking
effects of an electron-withdrawing substituent are on the ring pucker of the
substituted proline (Pro(i)) and on the trans/cis ratio of the Xaa(i-1)-Pro(i)
peptide bond. NMR experiments demonstrated that N-acetylproline methyl ester
(AcProOMe) exists in both the C(gamma)-endo and C(gamma)-exo conformations (with 
the endo conformation slightly preferred), N-acetyl-4(R)-fluoroproline methyl
ester (Ac-4R-FlpOMe) exists almost exclusively in the C(gamma)-exo conformation, 
and N-acetyl-4(S)-fluoroproline methyl ester (Ac-4S-FlpOMe) exists almost
exclusively in the C(gamma)-endo conformation. In dioxane, the K(trans/cis)
values for AcProOMe, Ac-4R-FlpOMe, and Ac-4S-FlpOMe are 3.0, 4.0, and 1.2,
respectively. Density functional theory (DFT) calculations with the (hybrid)
B3LYP method were in good agreement with the experimental data. Computational
analysis with the natural bond orbital (NBO) paradigm shows that the pucker
preference of the substituted prolyl ring is due to the gauche effect. The
backbone torsional angles, phi and psi, were shown to correlate with ring pucker,
which in turn correlates with the known phi and psi angles in collagen-like
peptides. The difference in K(trans/cis) between AcProOMe and Ac-4R-FlpOMe is due
to an n-->pi interaction associated with the Burg-Dunitz trajectory. The decrease
in K(trans/cis) for Ac-4S-FlpOMe can be explained by destabilization of the trans
isomer because of unfavorable electronic and steric interactions. Analysis of the
results herein along with the structures of collagen-like peptides has led to a
theory that links collagen stability to the interplay between the pyrrolidine
ring pucker, phi and psi torsional angles, and peptide bond trans/cis ratio of
substituted proline residues.

PMID: 11890798 [PubMed - indexed for MEDLINE]


367. Biophys Chem. 2002 Jan 23;95(1):69-77.

X-Ray analysis of d(CGCGAATTXGCG)(2) containing a 2' or
minute-deoxy-N(4)-methoxycytosine residue at X: a characteristic pattern of sugar
puckers in the crystalline state of the Dickerson-Drew type DNA dodecamers.

Hossain MT, Kondo J, Ueno Y, Matsuda A, Takenaka A.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, 226-8501, Yokohama, Japan.

In a series of structural studies on damaged DNA, a modified Dickerson-Drew
dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is
2'-deoxy-N(4)-methoxycytidine, was synthesized and its structure in a new crystal
form has been determined by the X-ray diffraction method. The two dodecamers form
a B-form duplex, in which the two mo(4)C residues, respectively, form a wobble
pair and a Watson-Crick type pair with the guanine residues of the opposite
strand. A comparison of the sugar conformations with those of the other related
Dickerson-Drew dodecamers indicates a common feature of their puckering patterns.
The sugar pucker of the third residue always adopts an intermediate state
(C4'-exo-O4'-endo) between the A-form and B-form. This deviation is ascribed to
the stacking interaction of the ribose ring at the third residue with the guanine
base at the 12th residue, which is brought about by an extra G12:G2 interaction
between two duplexes related by a crystallographic 2(1) symmetry.

PMID: 11880174 [PubMed - indexed for MEDLINE]


368. Acta Crystallogr C. 2002 Mar;58(Pt 3):o142-4. Epub 2002 Feb 13.

The alpha-D anomer of 5-aza-7-deaza-2'-deoxyguanosine.

Seela F, Rosemeyer H, Melenewski A, Heithoff EM, Eickmeier H, Reuter H.

Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie,
Universitat Osnabruck, Barbarastrasse 7, 49069 Osnabruck, Germany.
frank.seela@uni-osnabrueck.de

In the monohydrate of
2-amino-8-(2-deoxy-alpha-D-erythro-pentofuranosyl)-8H-imidazo[1,2-a][1,3,5]triazi
n-4-one, C(10)H(13)N(5)O(4) x H(2)O, denoted (I) or alpha Z(d), the conformation 
of the N-glycosylic bond is in the high-anti range [chi = 87.5 (3)]. The
2'-deoxyribofuranose moiety adopts a C2'-endo,C3'-exo((2')T(3')) sugar puckering 
(S-type sugar) and the conformation at the C4'-C5' bond is -sc (trans).

PMID: 11870307 [PubMed - indexed for MEDLINE]


369. Z Naturforsch C. 2001 Nov-Dec;56(11-12):1022-8.

Production and properties of a bacterial thermostable exo-inulinase.

Uzunova K, Vassileva A, Kambourova M, Ivanova V, Spasova D, Mandeva R, Derekova
A, Tonkova A.

Department of Extremophilic Bacteria, Institute of Microbiology, Bulgarian
Academy of Sciences, Sofia.

Inulinase and Invertase Activities, Thermophilic Bacilli, Enzyme Thermostability 
Enzyme production of newly isolated thermophilic inulin-degrading Bacillus sp. 11
strain was studied by batch cultivation in a fermentor. The achieved inulinase
and invertase activities after a short growth time (4.25 h) were similar or
higher compared to those reported for other mesophilic aerobic or anaerobic
thermophilic bacterial producers and yeasts. The investigated enzyme belonged to 
the exo-type inulinases and splitted-off inulin, sucrose and raffinose. It could 
be used at temperatures above 65 degrees C and pH range 5.5-7.5. The obtained
crude enzyme preparation possessed high thermostability. The residual inulinase
and invertase activities were 92-98% after pretreatment at 65 degrees C for 60
min in the presence of substrate inulin.

PMID: 11837654 [PubMed - indexed for MEDLINE]


370. Biosci Biotechnol Biochem. 2001 Dec;65(12):2613-21.

Stable isotope-labeling studies on the oxidative coupling of caffeic acid via
o-quinone.

Tazaki H, Taguchi D, Hayashida T, Nabeta K.

Department of Bioresource Science, Obihiro University of Agriculture and
Veterinary Medicine, Japan. tazaki@obihiro.ac.jp

The formation of ortho-quinone from ortho-diphenol is a key step in its
dimerization. An NMR analysis of the oxidation of 3,4-dihydroxycinnamic acid
(caffeic acid) by NaIO4 revealed the formation of
3-(3',4'-dioxo-1',5'-cyclohexadienyl) propenoic acid (o-quinone) prior to the
formation of furofuran-type lignan 4,8-exo-bis
(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione. Both electrolytic 
and enzymatic oxidation of caffeic acid also generated o-quinone. The yields of
o-quinone from caffeic acid were quantified by NMR and HPLC analyses. A stable
isotope-labeling study of the formation of lignans directly proved the random
radical coupling of semiquinone radicals formed from a set of caffeic acid and
o-quinone.

PMID: 11826955 [PubMed - indexed for MEDLINE]


371. Clin Chim Acta. 2002 Mar;317(1-2):77-84.

Residual galactosylsphingosine (psychosine) beta-galactosidase activities and
associated GALC mutations in late and very late onset Krabbe disease.

Harzer K, Knoblich R, Rolfs A, Bauer P, Eggers J.

Institut fur Hirnforschung, Universitat Tubingen, Calwer Strasse 3, D-72076,
Tubingen, Germany. Harzer-Rottenburg@t-online.de

BACKGROUND: Krabbe disease (globoid-cell leukodystrophy; GLD) is caused by
mutations in the GALC gene. Beta-galactocerebrosidase (GALC) is a specific
beta-galactosidase which is defective in GLD. About 90% of GLD patients have an
infantile course by fatal cerebral demyelination, but 10% have a later onset
(LOGLD) of symptoms and survive for one or several decades. METHODS: Activities
of GALC towards galactosylceramide (GC) and galactosylsphingosine (psychosine;
PS) were determined in white blood cells and cultured fibroblasts derived from
GLD patients and controls using tritium-labelled natural substrates. In the
galactosylsphingosine (psychosine) beta-galactosidase (GALC-PS) assay, a thin
layer chromatographic technique was used to separate enzymatically released
radioactive galactose. RESULTS: Both galactosylceramide beta-galactosidase
(GALC-GC) and GALC-PS activities were reduced by at least 85% of the normal in
all but 2 of the 10 GLD patients studied. In particular, one 23-year-old severely
demyelinated LOGLD patient was strongly deficient (11% of the normal) in GALC-GC 
but apparently normal for GALC-PS activity. This patient's GALC genotype was the 
30-kb-deleted/502T allele combined with a wild-type allele in the 1637C
background known to slightly reduce GALC-GC activity. Further, of six LOGLD
patients, both of 62- and 63-year-old brothers had the deleted allele combined
with an 809G>A mutated 1637C allele. The sibs had strongly reduced GALC-GC and
GALC-PS activities but became clinically remarkable only in their 50s with a
severe mental downhill course in one of them. CONCLUSIONS: A GALC genotype with
one deleted and one polymorphic GALC activity-reducing allele can lead to
enzymatic and clinical signs of LOGLD in the absence of marked GALC-PS
deficiency. If an active PS hydrolysis in the fibroblasts of a LOGLD patient also
reflected such hydrolysis in the brain, the psychosine hypothesis for GLD may
need to be revised.

PMID: 11814461 [PubMed - indexed for MEDLINE]


372. Nucleic Acids Res. 2002 Feb 1;30(3):719-25.

Crystal structure of 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide
bound to d(CGTACG)2: implications for structure-activity relationships of
acridinecarboxamide topoisomerase poisons.

Adams A, Guss JM, Denny WA, Wakelin LP.

Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia.
a.adams@biochem.usyd.edu.au

The structure of the complex formed between d(CGTACG)2 and
9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide, an inactive derivative 
of the antitumour agents N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) 
and 9-amino-DACA, has been solved to a resolution of 1.8 A using X-ray
crystallography. The complex crystallises in the space group P6(4 )and the final 
structure has an overall R factor of 21.9%. A drug molecule intercalates between 
each of the CpG dinucleotide steps with its side chain lying in the major groove,
and its protonated morpholino nitrogen partially occupying positions close to the
N7 and O6 atoms of guanine G2. The morpholino group is disordered, the major
conformer adopting a twisted boat conformation that makes van der Waals contact
with the O4 oxygen of thymine T3. A water molecule forms bridging hydrogen bonds 
between the 4-carboxamide NH and the phosphate group of guanine G2. Sugar rings
are found in alternating C3'-exo/C2'-endo conformations except for cytosine C1
which is C3'-endo. Intercalation perturbs helix winding throughout the
hexanucleotide compared with B-DNA, steps 1 and 2 being unwound by 10 and 8
degrees, respectively, while the central TpA step is overwound by 11 degrees. An 
additional drug molecule lies at the end of each DNA helix linking it to the next
duplex to form a continuously stacked structure. The protonated morpholino
nitrogen of this 'end-stacked' drug hydrogen bonds to the N7 atom of guanine G6, 
and its conformationally disordered morpholino ring forms a C-H...O hydrogen bond
with the guanine O6 oxygen. In both drug molecules the 4-carboxamide group is
internally hydrogen bonded to the protonated N10 atom of the acridine ring. We
discuss our findings with respect to the potential role played by the interaction
of the drug side chain and the topoisomerase II protein in the poisoning of
topoisomerase activity by the acridinecarboxamides.

PMCID: PMC100304
PMID: 11809884 [PubMed - indexed for MEDLINE]


373. Mol Pharmacol. 2002 Feb;61(2):436-45.

Cocaine induction of dopamine transporter trafficking to the plasma membrane.

Little KY, Elmer LW, Zhong H, Scheys JO, Zhang L.

Department of Psychiatry, University of Michigan, Ann Arbor, Michigan, USA.
kylittle@umich.edu

Several previous human postmortem experiments have detected an increase in
striatal [(3)H]WIN 35428 binding to the dopamine transporter (DAT) in chronic
cocaine users. However, animal experiments have found considerable variability in
DAT radioligand binding levels in brain after cocaine administration, perhaps
caused by length and dose of treatment and type of radioligand used. The present 
experiments tested the hypothesis that [(3)H]WIN 35428 binding and [(3)H]dopamine
uptake would be increased by exposure to cocaine through alterations in DAT
cellular trafficking, rather than increased protein synthesis. Experiments were
conducted in stably hDAT-transfected N2A cells and assessed the dose response and
time course of cocaine effects on [(3)H]WIN 35428 binding to the DAT,
[(3)H]dopamine uptake, measures of DAT protein and mRNA, as well as DAT
subcellular location. Cocaine doses of 10(-6) M caused statistically significant 
increases in [(3)H]WIN 35428 binding and [(3)H]dopamine uptake after 12 and 3 h, 
respectively. Despite these increases in DAT function, there was no change in DAT
total protein or mRNA. Immunofluorescence and biotinylation experiments indicated
that cocaine treatment induced increases in plasma membrane DAT immunoreactivity 
and intracellular decreases. The present model system may further our
understanding of regulatory alterations in DAT radioligand binding and function
caused by cocaine exposure.

PMID: 11809869 [PubMed - indexed for MEDLINE]


374. J Pediatr Ophthalmol Strabismus. 2001 Nov-Dec;38(6):327-30; quiz 354-5.

Sensory strabismus--eso or exo?

Havertape SA, Cruz OA, Chu FC.

Saint Louis University Health Sciences Center, Missouri, USA.

PURPOSE: The type of horizontal strabismus from loss or impairment of vision is
thought to depend on patient age at the time of vision loss. Association between 
the age at onset of vision loss and development of esotropia vs exotropia will be
determined. METHODS: Patients with a diagnosis of sensory strabismus and visual
acuity of 20/40 or poorer were reviewed as well as patients with diagnoses
consistent with the development of sensory strabismus. Parameters considered were
age at onset of vision loss and type of strabismus. Patients were excluded if the
age at onset was not clear. RESULTS: Of 123 patients with sensory strabismus
reviewed: 82 (67%) had unilateral vision loss; 41 (33%) had bilateral vision
loss; 75 (61%) had congenital vision loss; 50 (67%) developed esotropia; 25 (33%)
developed exotropia; 48 (39%) had acquired vision loss; 5 (10%) developed
esotropia; and 43 (90%) developed exotropia. A significant difference was noted
between age at onset and type of horizontal strabismus (X2= 37.44; P <.0001).
CONCLUSION: Of patients with congenital vision loss, 67% developed sensory
esotropia and 33% developed sensory exotropia. Of those with acquired vision
loss, 10% developed sensory esotropia and 90% developed sensory exotropia.
Patients with congenital vision loss are significantly more likely to develop
esotropia, P <.005, and those with acquired vision loss are significantly more
likely to develop exotropia, P <.001.

PMID: 11759769 [PubMed - indexed for MEDLINE]


375. Proc Natl Acad Sci U S A. 2002 Jan 8;99(1):60-5. Epub 2001 Dec 26.

Integration of Shaker-type K+ channel, KAT1, into the endoplasmic reticulum
membrane: synergistic insertion of voltage-sensing segments, S3-S4, and
independent insertion of pore-forming segments, S5-P-S6.

Sato Y, Sakaguchi M, Goshima S, Nakamura T, Uozumi N.

Graduate School of Bioagricultural Sciences, and Bioscience Center, Nagoya
University, Nagoya 464-8601, Japan.

KAT1 is a member of the Shaker family of voltage-dependent K(+) channels, which
has six transmembrane segments (called S1-S6), including an amphipathic S4 with
several positively charged residues and a hydrophobic pore-forming region (called
P) between S5 and S6. In this study, we systematically evaluated the function of 
individual and combined transmembrane segments of KAT1 to direct the final
topology in the endoplasmic reticulum membrane by in vitro translation and
translocation experiments. The assay with single-transmembrane constructs showed 
that S1 possesses the type II signal-anchor function, whereas S2 has the
stop-transfer function. The properties fit well with the results derived from
combined insertion of S1 and S2. S3 and S4 failed to integrate into the membrane 
by themselves. The inserted glycosylation sequence at the S3-S4 loop neither
prevented the translocation of S3 and S4 nor impaired the function of
voltage-dependent K(+) transport regardless of the changed length of the S3-S4
loop. S3 and S4 are likely to be posttranslationally integrated into the membrane
only when somewhat specific interaction occurs between them. S5 had the ability
of translocation reinitiation, and S6 had a strong preference for N(exo)/C(cyt)
orientation. The pore region resided outside because of its lack of its
transmembrane-spanning property. According to their own topogenic function,
combined constructs of S5-P-S6 conferred the membrane-pore-membrane topology.
This finding supports the notion that a set of S5-P-S6 can be independently
integrated into the membrane. The results in this study provide the fundamental
topogenesis mechanism of transmembrane segments involving voltage sensor and pore
region in KAT1.

PMCID: PMC117514
PMID: 11756658 [PubMed - indexed for MEDLINE]


376. Protein Eng. 2001 Oct;14(10):797-802.

Engineering the CYP101 system for in vivo oxidation of unnatural substrates.

Bell SG, Harford-Cross CF, Wong LL.

Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford,
South Parks Road, Oxford OX1 3QR, UK.

The protein engineering of CYP enzymes for structure-activity studies and the
oxidation of unnatural substrates for biotechnological applications will be
greatly facilitated by the availability of functional, whole-cell systems for
substrate oxidation. We report the construction of a tricistronic plasmid that
expresses the CYP101 monooxygenase from Pseudomonas putida, and its physiological
electron transfer co-factor proteins putidaredoxin reductase and putidaredoxin in
Escherichia coli, giving a functional in vivo catalytic system. Wild-type CYP101 
expressed in this system efficiently transforms camphor to 5-exo-hydroxycamphor
without further oxidation to 5-oxo-camphor until >95% of camphor has been
consumed. CYP101 mutants with increased activity for the oxidation of
diphenylmethane (the Y96F-I395G mutant), styrene and ethylbenzene (the Y96F-V247L
mutant) have been engineered. In particular, the Y96F-V247L mutant shows coupling
efficiency of approximately 60% for styrene and ethylbenzene oxidation, with
substrate oxidation rates of approximately 100/min. Escherichia coli cells
transformed with tricistronic plasmids expressing these mutants readily gave
100-mg quantities of 4-hydroxydiphenylmethane and 1-phenylethanol in 24-72 h.
This new in vivo system can be used for preparative scale reactions for product
characterization, and will greatly facilitate directed evolution of the CYP101
enzyme for enhanced activity and selectivity of substrate oxidation.

PMID: 11739899 [PubMed - indexed for MEDLINE]


377. Biochimie. 2001 Oct;83(10):961-7.

Chemical mechanism of beta-xylosidase from Trichoderma reesei QM 9414:
pH-dependence of kinetic parameters.

Gomez M, Isorna P, Rojo M, Estrada P.

Departamento de Bioquimica y Biologia Molecular I, Facultad de Biologia,
Universidad Complutense, 28040 Madrid, Spain.

The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM
9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl
beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a
group on the enzyme with a pK value of 3.20 must be unprotonated and a group with
a pK value of 5.20 must be protonated for activity and both are involved in
catalysis. Solvent-perturbation studies indicated that these groups are neutral
acid type. Temperature dependence of kinetic parameters suggested the stickiness 
of the substrate at lower temperatures than the optimum and the calculated
ionization enthalpies pointed to carboxyl groups as responsible for both pKs.
Chemical modification with triethyloxonium tetrafluoroborate and protection with 
the substrate studies demonstrated essential carboxyl groups on the enzyme.
Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK
value of 3.45 must be protonated for binding and it has been assigned to the
carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.

PMID: 11728634 [PubMed - indexed for MEDLINE]


378. Appl Environ Microbiol. 2001 Dec;67(12):5833-9.

An antifungal exo-alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus
Trichoderma harzianum.

Ait-Lahsen H, Soler A, Rey M, de La Cruz J, Monte E, Llobell A.

Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla-CSIC,
Seville, Spain.

Trichoderma harzianum secretes alpha-1,3-glucanases when it is grown on
polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source
(simulated antagonistic conditions). We have purified and characterized one of
these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic.
AGN13.1 was an exo-type alpha-1,3-glucanase and showed lytic and antifungal
activity against fungal plant pathogens. Northern and Western analyses indicated 
that AGN13.1 is induced by conditions that simulated antagonism. We propose that 
AGN13.1 contributes to the antagonistic response of T. harzianum.

PMCID: PMC93379
PMID: 11722942 [PubMed - indexed for MEDLINE]


379. J Biol Chem. 2002 Jan 4;277(1):746-54. Epub 2001 Oct 30.

Defective flap endonuclease 1 activity in mammalian cells is associated with
impaired DNA repair and prolonged S phase delay.

Shibata Y, Nakamura T.

Department of Radiology and Cancer Biology, Nagasaki University School of
Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.

Flap endonuclease 1 (FEN-1) is a 5'-3' flap exo-/endonuclease that plays an
important role in Okazaki fragment maturation, nonhomologous end joining of
double-stranded DNA breaks, and long patch base excision repair. Here, we
demonstrate that the wild type FEN-1 binds tightly to chromatin in conjunction
with proliferating cell nuclear antigen (PCNA) recruitment after MMS treatment,
and the nuclease-defective FEN-1 increased the sensitivity of the cells to
methylmethane sulfonate (MMS) and to UV light but not to ionizing radiation. In
contrast, the cells expressing the nuclease-defective and PCNA binding-defective 
double mutant FEN-1 exhibited sensitivities similar to those in the cells
expressing the wild type FEN-1. MMS treatment caused a prolonged delay of S phase
progression and impairment in colony-forming activity of cells expressing
nuclease-defective FEN-1. A comet assay demonstrated that DNA repair after MMS or
UV treatment was impaired in the cells expressing nuclease-deficient FEN-1 but
not in the cells with double-mutated FEN-1. Taken together, these findings
suggest that FEN-1 plays an essential role in the DNA repair processes in
mammalian cells and that this activity of FEN-1 is PCNA-dependent.

PMID: 11687589 [PubMed - indexed for MEDLINE]


380. J Cell Sci. 2001 Oct;114(Pt 19):3455-62.

UV-induced binding of ING1 to PCNA regulates the induction of apoptosis.

Scott M, Bonnefin P, Vieyra D, Boisvert FM, Young D, Bazett-Jones DP, Riabowol K.

Department of Biochemistry, Faculty of Medicine, The University of Calgary, 3330 
Hospital Drive, NW, Calgary, Alberta T2N 4N1, Canada.

Previous studies have shown that UV-induced binding of p21(WAF1) to PCNA through 
the PCNA-interacting protein (PIP) domain in p21(WAF1) promotes a switch from DNA
replication to DNA repair by altering the PCNA protein complex. Here we show that
the p33(ING1b) isoform of the ING1 candidate tumour suppressor contains a PIP
domain. UV rapidly induces p33(ING1b) to bind PCNA competitively through this
domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG 
exo/endonucleases, and DNA methyltransferase. Interaction of p33(ING1b) with PCNA
occurs between a significant proportion of ING1 and PCNA, increases more than
tenfold in response to UV and is specifically inhibited by overexpression of
p21(WAF1), but not by p16(MTS1), which has no PIP sequence. In contrast to
wild-type p33(ING1b), ING1 PIP mutants that do not bind PCNA do not induce
apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for
this PCNA-p33(ING1b) interaction in eliminating UV-damaged cells through
programmed cell death. These data indicate that ING1 competitively binds PCNA
through a site used by growth regulatory and DNA damage proteins, and may
contribute to regulating the switch from DNA replication to DNA repair by
altering the composition of the PCNA protein complex.

PMID: 11682605 [PubMed - indexed for MEDLINE]


381. J Org Chem. 1999 Mar 5;64(5):1626-1629.

Synthesis of Bridged Bicyclic Ethers and Fused Oxetanes from Pyran-4-ones via
Tandem Solvent Trapping and Norrish Type II Cyclization(1).

Fleming M, Basta R, Fisher PV, Mitchell S, West FG.

Department of Chemistry, University of Utah, 315 S. 1400 East, Rm. Dock, Salt
Lake City, Utah 84112-0850.

Polyalkyl pyran-4-ones 1a-c were irradiated in methanol or ethanol. Although the 
expected solvent trapping products 3 could be observed, extended irradiation
times allowed exclusive formation of secondary photoproducts 4 and 5 in combined 
yields of 37-64%. These bicyclic compounds are believed to arise from
gamma-hydrogen abstraction by the excited enone chromophore of 3, followed by
closure of the resulting biradical through one of two possible pathways. Moderate
stereoselectivity was observed in the radical coupling to produce 4, whereas the 
analogous closure to 5 was completely diastereoselective. Tautomerization of the 
enol precursors to 5 also occurred with complete selectivity for protonation from
the exo face. Overall, this process converts simple, planar heterocycles and
alkanols into complex products in a single transformation.

PMID: 11674228 [PubMed - as supplied by publisher]


382. Inorg Chem. 1998 Dec 28;37(26):6587-6596.

Coordination Chemistry of N-Alkylbenzamide-2,3-dithiolates as an Approach to
Poly(dithiolate) Ligands: 1,4-Bis[(2,3-dimercaptobenzamido)methyl]benzene and Its
Chelate Complex with the (C(5)H(5))Ti Fragment.

Seidel WW, Hahn FE, Lugger T.

Institut fur Anorganische und Analytische Chemie der Freien Universitat Berlin,
Fabeckstrasse 34-36, D-14195 Berlin, Germany.

The bidentate dithiolate ligands N,N-diethyl-2,3-dimercaptobenzamide (H(2)-1),
bis(N,N-diethyl-2,3-dimercaptoterephthalamide (H(2)-2), and
1,4-bis(hydroxymethyl)-2,3-dimercaptobenzene (H(4)-3) were synthesized from
2,3-dimercaptobenzoic acid or 2,3-dimercaptoterephthalic acid. The air-sensitive 
ligands form metallocene complexes of the type [(eta(5)-C(5)H(5))(2)Ti(1)] (13), 
[(eta(5)-C(5)H(5))(2)Mo(1)] (15), [(eta(5)-C(5)H(5))(2)Ti(3)] (16), and
[(eta(5)-C(5)H(5))(2)Ti(2)] (17). Complexes 15 and 16 were characterized by X-ray
diffraction. Selected crystallographic details for 15 are as follows: formula
C(21)H(23)MoNOS(2); M = 465.49; Pbca; a = 12.536(3), b = 14.313(3), c = 22.463(3)
A; V = 4031(2) A(3); Z = 8; R = 3.56 and R(w) = 4.49 for 2111 structure factors
(F(o)(2) >/= 3sigma(F(o)(2))) and 254 refined parameters. The molybdenum complex 
15 shows an almost planar Mo(&mgr;-S)(2)C(2) chelate ring. Selected
crystallographic details for 16 are as follows: formula C(18)H(18)O(2)S(2)Ti; M =
378.35; P&onemacr;; a = 10.1778(11), b = 11.5806(14), c = 22.967(3) A; alpha =
96.42(1), beta = 101.74(1), gamma = 108.82(1) degrees; V = 2462.3(5) A(3); Z = 6;
R = 4.79 and R(w) = 13.27 for 5426 structure factors (I >/= 2sigma(I)) and 766
refined parameters. All titanocene derivatives assume the envelope conformation. 
The free activation energy for the flip around the S-S axis was determined for 13
to be 69 kJ/mol. The bis(dithiolate) ligand
1,4-bis[(2,3-dimercaptobenzamido)methyl]benzene (H(4)-4) was prepared from
2,3-dimercaptobenzoic acid and converted into the dinuclear air-stable titanocene
complex [{(eta(5)-C(5)H(5))(2)Ti}(2)(4)] (19). Complex 19 reacts with HCl/CHCl(3)
with liberation of free H(4)-4 while reaction with NMe(4)Cl results in an
intramolecular dithiolate shift under formal liberation of [(C(5)H(5))(3)TiCl]
and formation of the square-pyramidal chelate complex
(NMe(4))[(eta(5)-C(5)H(5))Ti(4)], (NMe(4))[20]. Crystallographic details for
(NMe(4))[20].CH(2)Cl(2) are as follows: formula C(32)H(35)Cl(2)N(3)O(2)S(4)Ti; M 
= 740.67; P&onemacr;; a = 11.579(4), b = 12.210(4), c = 14.016(4) A; alpha =
112.28(2), beta = 94.46(3), gamma = 104.22(3) degrees; V = 1744.9(10) A(3); Z =
2; R = 5.35 and R(w) = 12.84 for 2870 structure factors (I >/= 2sigma(I)) and 397
refined parameters. The chelate rings in [20](-) assume an endo/exo conformation.

PMID: 11670790 [PubMed - as supplied by publisher]


383. AIDS Res Hum Retroviruses. 2001 Sep 20;17(14):1345-56.

Stepwise deletion of the HIV type 1 glycoprotein 41 N terminus leads to an
increasing export of microvesicles containing uncleaved Env glycoprotein.

Adams O, Scheid A.

Institut fur Medizinische Mikrobiologie und Virologie,
Heinrich-Heine-Universitat, D-40225 Dusseldorf, Germany.

Deletion of two or more amino acid residues from the N terminus of HIV-1 gp41
leads to an increasing loss of cleavability of the envelope (Env) precursor on
introduction of an env-expressing vector into HeLa-T4+ cells. In protein
analysis, this is paralleled by the appearance of a second form of uncleaved Env 
precursor that is terminally sialylated. Cell-derived microvesicles that
preferentially incorporate this form of Env precursor were found in the culture
medium. The same applies to a mutant with a nonfunctional cleavage site,
indicating that a pathway by which uncleaved Env glycoprotein leaves the cell
exists. The amount of exported glycoprotein is augmented as compared with
wild-type Env. Transfection with a wild-type Env-expressing vector leads to the
presence of extracellular microvesicles that contain only the transmembrane
domain of HIV-1 Env. Microvesicles derived from wild-type Env and mutant Env
contain sialylated glycoproteins that are resistant to exo- and endoglycosidase
treatment unless the particles have been previously lysed by detergent. This
raises the possibility that the C-terminal domains of the glycoproteins are
exposed on the surface of the exported microvesicles.

PMID: 11602045 [PubMed - indexed for MEDLINE]


384. Tanpakushitsu Kakusan Koso. 2001 Aug;46(11 Suppl):1717-25.

[Structural motifs in DNA cleaving enzymes]

[Article in Japanese]

Nishino T, Ishino Y.

nishino@beri.co.jp

PMID: 11579571 [PubMed - indexed for MEDLINE]


385. Biosci Biotechnol Biochem. 2001 Aug;65(8):1824-31.

Cloning, sequencing, and expression of the gene encoding an intracellular
beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

Tsujibo H, Takada C, Tsuji A, Kosaka M, Miyamoto K, Inamori Y.

Osaka University of Pharmaceutical Sciences, Takatsuki, Japan.
tsujibo@oysun01.oups.ac.jp

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus
OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or
xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was
partially purified and its N-terminal amino acid sequence was analyzed. The gene 
encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The
bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The
deduced amino acid sequence of the bxlA gene product had significant similarity
with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA
gene was expressed in E. coli, and the recombinant protein was purified to
homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The
purified enzyme showed hydrolytic activity towards only
p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested.
Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme
that hydrolyze xylooligosaccharides, but had no activity toward xylan. High
activity against pNPX occurred in the pH range 6.0-7.0 and temperature range
40-50 degrees C.

PMID: 11577723 [PubMed - indexed for MEDLINE]


386. Klin Med (Mosk). 2001;79(3):26-30.

[Inheritance of Wolff-Parkinson-White syndrome and evolution of its clinical
symptoms in families of patients in a prospective trial]

[Article in Russian]

Fomina IG, Logunova LV, Kuleshov NP, Markova ZS.

Medicogenetic examination was conducted in families of 46 patients (21 women and 
25 men aged 16-74 years) with Wolff-Parkinson-White (WPW) syndrome. A total of
256 relatives were investigated (136 women and 120 men aged 2 to 85 years). As a 
result, the diagnosis of preexcitation syndrome and phenomenon was made initially
in 75(29.3%) of the relatives: WPW syndrome, Clerc-Levy-Cristesco (CLC) syndrome,
CLC phenomenon was made in 6(2.3%), 27(10.6%) and 42(16.4%) relatives,
respectively. Additional conduction pathways in the families with WPW syndrome
are inherited by the autosome-dominant type with penetrability 0.94(94%) and
clinical polymorphism. Prospective observation of the families revealed evolution
of the clinical symptoms (development of arrhythmia) in the relative with CLC or 
WPW phenomenon in unfavorable exo- and endogenic factors. WPW syndrome evolution 
in the patients ran with aggravation of arrhythmia though 12 patients showed
improvement.

PMID: 11490411 [PubMed - indexed for MEDLINE]


387. Insect Biochem Mol Biol. 2001 Sep;31(10):937-48.

Partial characterization of oligosaccharides expressed on midgut microvillar
glycoproteins of the mosquito, Anopheles stephensi Liston.

Wilkins S, Billingsley PF.

Department of Biology, Imperial College of Science Technology and Medicine, SW7
2BB, London, UK.

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were
homogenized and microvillar membranes prepared by calcium precipitation and
differential centrifugation. Oligosaccharides present on the microvillar
glycoproteins were identified by lectin blotting before and after in vitro and in
situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins
expressed a structurally restricted range of terminal sugars and oligosaccharide 
linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid
N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue.
Complex-type N-linked oligosaccharides on eight glycoproteins all possessed
terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine.
Eight glycoproteins expressed O-linked oligosaccharides all containing
N-acetylgalactosamine with or without further substitutions of fucose and/or
galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic
acidalpha2-3/6galactose-, fucosealpha1-2galactose- and
galactosealpha1-3galactose- were not detected. Terminal alpha-linked
N-acetylgalactosamine residues on N-linked oligosaccharides are described for the
first time in insects. The nature and function of these midgut glycoproteins have
yet to be identified, but the oligosaccharide side chains are candidate receptors
for ookinete binding and candidate targets for transmission blocking strategies.

PMID: 11483430 [PubMed - indexed for MEDLINE]


388. Physiol Plant. 2001 Jul;112(3):308-314.

Small complex-type N-linked glycans are attached to cell-wall bound
exo-beta-glucanases of both mung bean and barley seedlings.

Kotake T, Tonari A, Ohta M, Matsuura F, Sakurai N.

Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi Hiroshima,
739-8521, Japan; Department of Biotechnology, Fukuyama University, Fukuyama,
Hiroshima 729-0292, Japan.

N-linked glycans of wall-bound exo-beta-glucanases from mung bean and barley
seedlings, namely Mung-ExoI and Barley-ExoII, were characterized. The N-linked
glycans of Mung-ExoI and Barley-ExoII were liberated by gas-phase hydrazinolysis 
followed by re-N-acetylation. Their structures were determined by two-dimensional
sugar-mapping analysis and MALDI-TOF mass spectrometry. N-glycans from both
glucanases were of paucimannosidic-type (small complex-type) structures,
Manalpha1-6(+/-Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(+/-Fucalpha1-3)
GlcNAc, which are known as typical vacuole-type N-glycans. The results suggest
that N-glycans of cell-wall glucanase were produced by partial trimming of
complex-type N-glycans by exoglycosidases during its transport from Golgi
apparatus to cell walls or in the cell walls.

PMID: 11473686 [PubMed - as supplied by publisher]


389. J Anim Sci. 2001 Jul;79(7):1905-16.

Influence of supplementary fibrolytic enzymes on the fermentation of corn and
grass silages by mixed ruminal microorganisms in vitro.

Wallace RJ, Wallace SJ, McKain N, Nsereko VL, Hartnell GF.

Rowett Research Institute, Bucksburn, Aberdeen, UK. rjw@rri.sari.ac.uk

This study was done to determine the effectiveness of supplementary enzymes at
increasing the fiber digestion by ruminal microorganisms and to assess whether
enzyme activity limits the rate of fiber digestion in ruminal digesta. In vitro
comparisons of enzyme activities in two feed enzyme preparations (A and B) with
enzyme activities extracted from ruminal fluid indicated that the addition of
fibrolytic enzymes at the application rates recommended by the manufacturers
would not be expected to increase significantly glycanase and polysaccharidase
activities in ruminal fluid. Preparations A and B both increased (P < 0.001) the 
rate of gas production from freeze-dried corn and grass silages in in vitro
incubations with ruminal fluid, but only at concentrations much higher than
recommended application rates. Autoclaved controls had little or no effect.
Ultrafiltration of enzyme B indicated that most stimulation was due to components
>100 kDa, which is consistent with the cause of the stimulation being enzyme
activity. Fibrolytic enzymes from other sources were also able to stimulate gas
production: increased rates of gas production were observed in seven out of eight
combinations of "cellulase" and corn or grass silage (P < 0.05). The comparison
of glycanase and polysaccharidase activities with gas-stimulatory activity in the
different enzyme preparations indicated that the highest correlation was between 
increased gas production and enzyme activity against microgranular cellulose (P <
0.05). In a wider range of fibrolytic enzyme preparations, those with
endo-(beta-1,4)- or exo-(beta-1,4)-xylanase activity equal to that of preparation
A did not produce similar increased rates of fermentation of corn silage when
glucanase activity was low (P > 0.05). In contrast, preparations with glucanase
activity similar to enzyme A gave at least as great (P < 0.05) an improvement in 
gas production than enzyme A, irrespective of xylanase activity. It was concluded
that enzyme activity, probably a type of endo-(beta-1,4)-glucanase activity,
limits the rate of fermentation of corn and grass silage in the rumen. Enzyme
supplements of the type used in these experiments are unlikely to possess
sufficient activity to overcome this limitation by direct application to ruminal 
digesta, implying that treatment of the ration prefeeding will be key to
harnessing the potential of exogenous fibrolytic enzymes in ruminant nutrition.

PMID: 11465379 [PubMed - indexed for MEDLINE]


390. Curr Opin Cell Biol. 2001 Aug;13(4):461-9.

Endocytotic mechanisms in synapses.

Jarousse N, Kelly RB.

Department of Biochemistry and Biophysics, University of California, San
Francisco, 94143-0448, USA.

Nerve terminals are highly enriched in proteins needed for endocytosis. Although 
constitutive and ligand-stimulated endocytosis take place in nerve terminals, the
primary type is compensatory endocytosis--the process by which a cell retrieves
the additional membrane added to cell surface by a regulated secretory event.
This process has been extensively characterized using electrophysiological
techniques. Except for an unusual form of coupled exo- and endocytosis called
kiss-and-run release, compensatory endocytosis appears to use basically the same 
clathrin-mediated mechanisms as the constitutive and ligand stimulated type. The 
remarkable speed and selectivity of compensatory endocytosis may be achieved by
concentrating the machinery at specialized sites in the nerve terminal adjacent
to exocytosis sites and by the use of neuronal isoforms of the proteins that
mediate endocytosis.

PMID: 11454453 [PubMed - indexed for MEDLINE]


391. Carbohydr Res. 2001 Jul 3;333(2):123-8.

Synthesis of beta-D-galactofuranosyl nucleoside analogues. A new type of
beta-D-galactofuranosidase inhibitor.

Marino C, Herczegh P, de Lederkremer RM.

CIHIDECAR, Departamento de Quimica Organica, Facultad de Ciencias Exactas y
Naturales, Universidad de Buenos Aires, Pabellon II, Ciudad Universitaria, 1428
Buenos Aires, Argentina. cmarino@qo.fcen.uba.ar

The development of beta-D-galactofuranosidase inhibitors provides a good
chemotherapeutic target for treatment of major human diseases, because
beta-D-galactofuranose is a constituent of important pathogen microorganisms but 
is absent in mammals. With this purpose we have prepared beta-D-galactofuranosyl 
nucleoside analogues, derived by the addition of nucleophiles to perbenzoylated
beta-D-galactofuranosyl isothiocyanate, a compound previously prepared in this
laboratory. N-beta-D-Galactofuranosyl-O-ethylthiourethane,
N-beta-D-galactofuranosyl-4-oxoimidazolidine-2-thione,
N-beta-D-galactofuranosyl-4-imidazoline-2-thione, and
N-beta-D-galactofuranosyl-4-methoxyimidazolidine-2-thione, were prepared. The
biological assays showed that imidazoline and imidazolidine-2-thione derivatives 
act as a new type of exo beta-D-galactofuranosidase inhibitor.

PMID: 11448672 [PubMed - indexed for MEDLINE]


392. Chemistry. 2001 Jun 1;7(11):2332-40.

An efficient method for the preparation of 1'alpha-branched-chain sugar
pyrimidine ribonucleosides from uridine: the first conversion of a natural
nucleoside into 1'-substituted ribonucleosides.

Kodama T, Shuto S, Nomura M, Matsuda A.

Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

The 1'alpha-phenylselenouridine derivative 13 was successfully synthesized by
enolization of the 3',5'-O-TIPDS-2'-ketouridine 8, and was subjected to a radical
reaction with a vinylsilyl tether--an efficient procedure for preparing
1'alpha-branched-chain sugar pyrimidine nucleosides. Successive treatment of 8
with LiHMDS and PhSeCl in THF at < -70 degrees C gave the desired 1'-phenylseleno
products in 85% yield as an anomeric mixture of the 1'alpha-product 11 and the
1'beta-product 12 (11/12= 2.5:1). Highly stereoselective reduction at the
2'-carbonyl of the 1'alpha-product 11 occurred from the beta-face by using
NaBH4/CeCl3 in MeOH, and subsequent introduction of a dimethylvinylsilyl tether
at the 2'-hydroxyl gave the radical reaction substrate 14. The photochemical
radical atom-transfer reaction of 14 by using a high-pressure mercury lamp
proceeded effectively in benzene to give the exo-cyclized PhSe-transferred
product 18, in which (PhSe)2 proved to be essential as an additive for radical
atom-transfer cyclization reactions. Subsequent phenylseleno-group elimination of
18 gave the sugar-protected 1'alpha-vinyluridine. With this procedure,
1'alpha-vinyluridine (22) and -cytidine (25), designed to be potential antitumor 
agents, were successfully synthesized. This study is the first example of
functionalization at the anomeric 1'-position of a nucleoside by starting from a 
natural nucleoside to produce a ribo-type 1'-modified nucleoside.

PMID: 11446636 [PubMed - indexed for MEDLINE]


393. J Mol Biol. 2001 Jul 13;310(3):549-62.

Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted
linear molecules, head-to-tail concatemers, and a 5' protein.

Oldenburg DJ, Bendich AJ.

Department of Botany, University of Washington, Seattle, WA 98195-5325, USA.

Mapping predicts that the mitochondrial genome of the liverwort Marchantia
polymorpha exists as a circular molecule, although nearly all the mitochondrial
DNA (mtDNA) is found as genome-sized and multigenomic molecules in linear and
branched form. We used restriction enzymes with one recognition site per genome, 
end-specific exonucleases and pulsed-field gel electrophoresis (PFGE) to analyze 
the arrangement of genomic units and the terminal structure of the molecules. We 
find a head-to-tail arrangement in the concatemers and circular permutation in
both the monomeric and multigenomic molecules. The termini contain covalently
bound protein at the 5' end and an open (unblocked) 3' end. We find that the
standard in-gel procedure used to prepare large DNA molecules for PFGE may
introduce extraction artifacts leading to erroneous conclusions about the
termini. These artifacts can be reduced by omitting high salt (high EDTA) and
protease during mitochondrial lysis. Our results suggest that the mtDNA may use a
T4 phage-like mechanism of replication and that the linear molecules may be due
to strand breaks mediated by type II topoisomerase. Copyright 2001 Academic
Press.

PMID: 11439023 [PubMed - indexed for MEDLINE]


394. Org Lett. 2001 Jun 28;3(13):2001-4.

Samarium(II)-mediated 4-exo-trig cyclization. A stereocontrolled approach to the 
core of pestalotiopsin A.

Johnston D, Francon N, Edmonds DJ, Procter DJ.

Department of Chemistry, The Joseph Black Building, University of Glasgow,
Glasgow, G12 8QQ Scotland.

[reaction: see text] Pestalotiopsin A is a structurally unique caryophyllene-type
sesquiterpene which has shown immunosuppressive activity and cytotoxicity in
preliminary assays. A stereocontrolled approach to the functionalized
2-oxabicyclo[3.2.0]heptane core of pestalotiopsin A is described. The approach
includes a samarium(II)-mediated 4-exo-trig cyclization and a trans-lactonization
process triggered by the addition of alkylytterbium reagents to a cyclobutanone
intermediate.

PMID: 11418034 [PubMed - indexed for MEDLINE]


395. Mutat Res. 2001 Jul 1;478(1-2):141-51.

Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension
using Vent(R)(exo-) DNA polymerase and RFLP analysis.

Jacobi FK, Meyer J, Pusch CM, Wissinger B.

Molekulargenetisches Labor, Universitats-Augenklinik, Auf der Morgenstelle 15,
72076 Tubingen, Germany. felix.k.jacobi@augen.med.uni-giessen.de

In this report we describe a simple and rapid protocol for reliable quantitation 
of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the 
traditional polymerase chain reaction (PCR)/restriction fragment length
polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been
of limited use for the accurate determination of ratios of mutant and wild type
molecules, largely owing to the formation of heteroduplex molecules by PCR and
incompleteness of restriction digestion. In order to overcome this problem, we
have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA
polymerase and a fluorescence-labeled primer to the standard assay. The labeled
homoduplex molecules are then digested with a restriction endonuclease, and the
nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN
analysis software. The amount of mutant mtDNA is readily estimated from
fluorescence intensities of the wild-type and mutant mtDNA fragments corrected
for incomplete digestion as monitored by a homologous control fragment. The
accuracy of the improved protocol was determined by constructing standard curves 
obtained from defined mixtures of genomic DNA containing homoplasmic wild-type
and mutant mtDNA. The expected values were obtained, with an observed correlation
coefficient of 0.997 and a typical variability of +/-5% between repeated
measurements. Further validation of the protocol is provided by the screening of 
five patients and unaffected subjects carrying the guanine to adenine transition 
at the nucleotide 3460 of the mitochondrial genome responsible for the
mitochondrial disorder of Leber's hereditary optic neuropathy.

PMID: 11406178 [PubMed - indexed for MEDLINE]


396. J Biotechnol. 2001 Jun 15;88(2):141-9.

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease
deficient mutant enzyme.

Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H,
Takagi M, Imanaka T.

Department of Biotechnology, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, 565-0871, Osaka, Japan.

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp.
KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher
accuracy and elongation velocity than any other commercially available DNA
polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510].
However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase,
amplification efficiency (product yield) becomes lower because of its strong
3'-5' exonuclease activity for proof-reading. In order to improve a target length
limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease
activity were designed by substituting amino acid residues in conserved
exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), 
and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification
fidelity (error rate) of the DNA polymerases were altered by mutagenesis.
However, long and accurate PCR by a single-type of mutant DNA polymerase was very
difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant
(N210D) were mixed in different ratio and their characteristics in PCR were
examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40,
long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.

PMID: 11403848 [PubMed - indexed for MEDLINE]


397. J Biol Chem. 2001 Aug 3;276(31):29012-8. Epub 2001 Jun 6.

The uptake inhibitors cocaine and benztropine differentially alter the
conformation of the human dopamine transporter.

Reith ME, Berfield JL, Wang LC, Ferrer JV, Javitch JA.

Department of Biomedical and Therapeutic Sciences, University of Illinois College
of Medicine, Peoria, Illinois 61656, USA. MaartenR@uic.edu

The binding affinity of the cocaine analog [(3)H]2
beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine
transporter (DAT) is increased by the reaction of Cys-90, at the extracellular
end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents.
Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby
augmenting the increase in binding. In contrast, cocaine decreases the reaction
of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS
reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects
against the loss of binding caused by reaction of these cysteines. In the present
work, we compare the abilities of DAT inhibitors and substrates to affect the
reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET).
The results indicate that the different abilities of compounds to protect against
the MTSET-induced inhibition of binding are attributable to differences in their 
abilities to attenuate the inhibitory effects of modification of Cys-135 and
Cys-342 as well as to enhance the reaction with Cys-90 and the resulting
potentiation of binding. The inhibitor benztropine was unique in its inability to
protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced 
the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction.
These two features combine to give benztropine its weak potency in protecting
ligand binding to wild-type DAT from MTSET. These results indicate that different
inhibitors of DAT, such as cocaine and benztropine, produce different
conformational changes in the transporter. There are differences in the
psychomotor stimulant-like effects of these compounds, and it is possible that
the different behavioral effects of these DAT inhibitors stem from their
different molecular actions on DAT.

PMID: 11395483 [PubMed - indexed for MEDLINE]


398. Hua Xi Yi Ke Da Xue Xue Bao. 1999 Dec;30(4):375-8.

[Construction of nested set of unidirectional deletion of recombinant plasmid DNA
for sequencing]

[Article in Chinese]

Qin Y, Burtonboy G.

Department of Biochemistry, School of Basic Medical Sciences, WCUMS, Chengdu,
610041.

In order to rapidly sequence a batch of large DNA fragments, we developed a
method for the construction of nested set of unidirectional deletions. A nested
set of unidirectional deletions of the large DNA fragment of a c-type retrovirus 
gene was successfully constructed by the adoption of this method. The recombinant
plasmid DNA was excised by BamHI and SphI at on end of the target DNA to create
5' and 3' overhanging ends. The DNA was digested with Exo III from 5' overhanging
end to generate a set of unidirectional deletion. With the use of this method a
set of deleted plasmid DNA and the deleted target DNA excised from the deleted
constructs were observed on agrose gel electrophoresis. We sequenced all of 24
deleted DNA fragments (forward and reverse orientation). All of them contained
overlapping sequences at their ends. These sequences were readily aligned. The
results demonstrate this is an efficient method for sequencing large DNA
fragment. The influential factors in this method were discussed in this report.

PMID: 11387944 [PubMed - indexed for MEDLINE]


399. J Neurochem. 2001 May;77(4):1116-27.

The role of conserved tryptophan and acidic residues in the human dopamine
transporter as characterized by site-directed mutagenesis.

Chen N, Vaughan RA, Reith ME.

Department of Biomedical and Therapeutic Sciences, University of Illinois College
of Medicine, Peoria, Illinois, USA nhc@uic.edu

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and
acidic residues that are completely or highly conserved among
Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues 
using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and 
W184L) lacked plasma membrane immunostaining and were not functional. Both DA
uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane,
CFT) binding were abolished in W63L and severely damaged in W311L. Four of five
aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative
selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, 
mutation of D345 in the third intracellular loop still allowed considerable
[(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon
anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, 
but this band could not be photoaffinity labeled with cocaine analog
[(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82).
Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of
[(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT,
but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms
of inhibition. The data taken together indicate that mutation of conserved
tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and
substrate transport. Additionally, binding of cocaine to the transporter may not 
be the only way by which cocaine analogs inhibit DA uptake.

PMID: 11359877 [PubMed - indexed for MEDLINE]


400. Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):5122-7. Epub 2001 Apr 17.

The 3'-->5' exonuclease of DNA polymerase delta can substitute for the 5' flap
endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome
instability.

Jin YH, Obert R, Burgers PM, Kunkel TA, Resnick MA, Gordenin DA.

Laboratory of Molecular Genetics, National Institute of Environmental Health
Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA.

Many DNA polymerases (Pol) have an intrinsic 3'-->5' exonuclease (Exo) activity
which corrects polymerase errors and prevents mutations. We describe a role of
the 3'-->5' Exo of Pol delta as a supplement or backup for the Rad27/Fen1 5' flap
endonuclease. A yeast rad27 null allele was lethal in combination with Pol delta 
mutations in Exo I, Exo II, and Exo III motifs that inactivate its exonuclease,
but it was viable with mutations in other parts of Pol delta. The rad27-p allele,
which has little phenotypic effect by itself, was also lethal in combination with
mutations in the Pol delta Exo I and Exo II motifs. However, rad27-p Pol delta
Exo III double mutants were viable. They exhibited strong synergistic increases
in CAN1 duplication mutations, intrachromosomal and interchromosomal
recombination, and required the wild-type double-strand break repair genes RAD50,
RAD51, and RAD52 for viability. Observed effects were similar to those of the
rad27-null mutant deficient in the removal of 5' flaps in the lagging strand.
These results suggest that the 3'-->5' Exo activity of Pol delta is redundant
with Rad27/Fen1 for creating ligatable nicks between adjacent Okazaki fragments, 
possibly by reducing the amount of strand-displacement in the lagging strand.

PMCID: PMC33174
PMID: 11309502 [PubMed - indexed for MEDLINE]


401. J Org Chem. 2001 Mar 23;66(6):1966-83.

Preparation of polycyclic systems by sequential 5-exo-digonal radical
cyclization, 1,5-hydrogen transfer from silicon, and 5-endo-trigonal cyclization.

Clive DL, Yang W, MacDonald AC, Wang Z, Cantin M.

Chemistry Department, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
derrick.clive@ualberta.ca

Radicals of type 1 undergo 5-exo diagonal cyclization, and the resulting vinyl
radical abstracts hydrogen from silicon to afford a silicon-centered radical.
This radical closes in a 5-endo trigonal manner to generate radicals of type 4,
which are reduced (4 --> 5) by stannane, except when the starting acetylene
carries a terminal trimethylstannyl group. In this case, radicals 4 expel
trimethylstannyl radical to afford vinyl silanes 6. The stereochemical outcome of
the radical cascade 1 --> 5 is controlled by the stereochemistry of the
oxygen-bearing carbon in 1 (see starred atom). The sequence can be initiated by
carbon-, alpha-substituted carbon-, oxyacyl-, and carbamoyl radicals and
generates a silicon-containing ring fused onto a carbocycle or heterocycle.
Numerous examples are described, as well as a number of transformations of the
final cyclization products, especially their response to n-Bu(4)NF and to
BF(3).OEt(2), reagents that cleave the newly formed carbon-silicon bond.

PMID: 11300889 [PubMed]


402. J Mol Biol. 2001 Apr 6;307(4):987-99.

Sequence dependence of translational positioning of core nucleosomes.

Negri R, Buttinelli M, Panetta G, De Arcangelis V, Di Mauro E, Travers A.

Centro di studio per gli Acidi Nucleici, CNR and Fondazione Istituto
Pasteur-Fondazione Cenci-Bolognetti, c/o Dipartimento di Genetica e Biologia
Molecolare, Universita di Roma "La Sapienza", Piazzale le Aldo Moro 5, Roma,
00185, Italy.

The basis for the choice of translational position of a histone octamer on DNA is
poorly understood. To gain further insights into this question we have studied
the translational and rotational settings of core particles assembled on a simple
repeating 20 bp positioning sequence. We show that the translational positions of
the core particles assembled on this sequence are invariant with respect to the
DNA sequence and occur at 20 bp intervals. Certain modifications of the original 
sequence reduce the spacing of possible dyads to 10 bp. At least one of these
alters both the translational and rotational settings. We conclude that the
translational position of a core particle is specified by sequence determinants
additional to those specifying rotational positioning. The rotational settings on
either side of the dyads of core particles assembled on the wild-type and a
mutant sequence differ by +2 bp, corresponding to an overall helical periodicity 
of approximately 10.15 bp. The average helical periodicity of the central two to 
four turns is 10.5-11 bp whilst that of the flanking DNA is closer to 10 bp. The 
DNA immediately flanking the dyad is also characterised by a more extensive
susceptibility to cleavage by hydroxyl radical. Copyright 2001 Academic Press.

PMID: 11286550 [PubMed - indexed for MEDLINE]


403. Front Biosci. 2001 Apr 1;6:D630-59.

Regulation of glucose uptake in differentiated cells.

Lange K.

Kladower Damm 25b, D-14089 Berlin, Germany. piotr222@zedat.fu-berlin.de

Glucose uptake into the cell is mediated by a family of glycosylated membrane
proteins, called glucose transporters (GTs) that are able to facilitate passive
hexose transfer across the lipid plasma membrane. The tissue-specific transporter
isoforms generally differ in their affinity to the natural substrate D-glucose
according to the specific functions of the respective organ. The mechanisms by
which external and internal signals regulate glucose uptake into the cells belong
to one of the most extensively studied fields of cell physiology. However, in
spite of significant progress in identifying the involved molecular components
and signaling pathways, the final cellular mechanism responsible for the
short-term regulation of glucose uptake is still a matter of intense debate. The 
widely accepted translocation hypothesis, which explains transport regulation by 
exo- and endocytic modulation of the number of GTs in the plasma membrane,
insufficiently accounts for the whole insulin-induced transport stimulation and
is insufficient to integrate the wide variety of different transport-modulating
signals in differentiated tissue cells into a common mechanistic concept. Some
time ago, a novel type of glucose transport regulation has been proposed
prevailing in differentiated tissue cells. This mechanism depends on the presence
of glucose transporters on microvilli of differentiated cells. The basic
framework for this theory was provided by a recently presented novel concept of
ion channel regulation via microvillar structures [Lange, K. (1999): Microvillar 
Ca++ signaling: A new view on an old problem. J. Cell. Physiol. 180, 19-35;
Lange, K. (2000): Regulation of cell volume via microvillar ion channels. J.
Cell. Physiol. 185, 21-35; Lange, K. (2000): Microvillar ion channels -
Cytoskeletal regulation of ion fluxes. J. Theor. Biol. 206, 561-584], earlier
studies on glucose transport regulation and a number actual biochemical findings.
Here, a survey on both concepts is given and the ability of the novel mechanism
of microvillar transport regulation to integrate a large body of experimental
data into the common concept of cellular regulation via microvillar pathways is
discussed.

PMID: 11282567 [PubMed - indexed for MEDLINE]


404. Chemistry. 2001 Feb 2;7(3):700-10.

"Dimers" and "trimers" of tetrahydroindenes and hexahydroazulenes, respectively
generated from [2-(1-cycloalkenyl)ethynyl]carbene complexes (M = W, Cr) by
cascade cyclization/cycloaddition reactions.

Wu HP, Aumann R, Frohlich R, Saarenketo P.

Organisch-Chemisches Institut der Universitat Munster, Germany.

A cascade of cyclization/cycloaddition reactions was triggered by addition of
protic oxygen nucleophiles ROH 2 (RO = CH3CO2, PhCO2, PhO) to
[2-(1-cyclohexenyl)ethynyl]carbene complexes 1b and 1c (M=W, Cr, respectively),
affording highly strained "dimers" 11/11' and "trimers" 12 of the carbene ligand.
The first reaction step involved the formation of 1-metalla1,3,5-hexatrienes 7,
which readily gave tetrahydroindenes 8 by pi cyclization and extrusion of the
metal unit. "Dimers" 11/11' were generated from tetrahydroindenes 8 by a highly
exo selective [4+2] cycloaddition of compounds 1b and 1c to afford
1-metalla-1,3,5-hexatriene intermediates 9, and a spontaneous pi cyclization of
the latter compounds involving the disengagement of the metal unit. Propenylidene
cyclohexenes 13/13' were formed in "ene"-type side reactions to the pi
cyclization of 1-metalla-1,3,5-hexatrienes 7, by loss of the metal unit. "Dimers"
11 were transformed into "trimers" 12 by a [4+2] cycloaddition and subsequent
pi-cyclization of the resulting 1-metalla-1,3,5-hexatriene system. The course of 
the reaction was elucidated by means of model reactions with
(2-phenylethynyl)carbene complex 14, in which 1-metalla-1,3,5-hexatriene
intermediates 16 and 17 were isolated and characterized. Alkynyl benzene
derivatives 19 were obtained by an unprecedented ring-expansion of a
cyclopentadiene unit of "dimers" 11a and 11c, involving the insertion of a
carbene carbon atom of compound 14 into a C=C bond. A reaction cascade leading to
"dimers" 24/24' could also be triggered by treatment of compounds 2 with
[2-(1-cycloheptenyl)ethynyl]carbene tungsten complex 1d.

PMID: 11261668 [PubMed]


405. J Biomol NMR. 2001 Jan;19(1):19-31.

Sugar conformation of a stereospecific 2'-R or 2'-S deuterium-labeled DNA decamer
studied with proton-proton J coupling constants.

Kojima C, Kawashima E, Sekine T, Ishido Y, Ono A, Kainosho M, Kyogoku Y.

Institute for Protein Research, Osaka University, Suita, Japan.

The sugar conformation of a DNA decamer was studied with proton-proton 3J
coupling constants. Two samples, one comprising stereospecifically labeled
2'-R-2H for all residues and the other 2'-S-2H, were prepared by the method of
Kawashima et al. [J. Org. Chem. (1995) 60, 6980-6986; Nucleosides Nucleotides
(1995) 14, 333-336], the deuterium labeling being highly stereospecific (> or =
99% for all 2''-2H, > or = 98% for 2'-2H of A, C, and T, and > or = 93% for 2'-2H
of G). The 3J values of all H1'-H2' and H1'-H2'' pairs, and several H2'-H3' and
H2''-H3' pairs were determined by line fitting of 1D spectra with 0.1-0.2 Hz
precision. The observed J coupling constants were explained by the rigid sugar
conformation model, and the sugar conformations were found to be between C3'-exo 
and C2'-endo with phi(m) values of 26 degrees to 44 degrees, except for the
second and 3' terminal residues C2 and C10. For the C2 and C10 residues, the
lower fraction of S-type conformation was estimated from JH1'H2' and JH1'H2''
values. For C10, the N-S two-site jump model or Gaussian distribution of the
torsion angle model could explain the observed J values, and 68% S-type
conformation or C1'-exo conformation with 27 degrees distribution was obtained,
respectively. The differences between these two motional models are discussed
based on a simple simulation of J-coupling constants.

PMID: 11246851 [PubMed - indexed for MEDLINE]


406. J Neurochem. 2001 Feb;76(4):1242-51.

Reaction of oxidized dopamine with endogenous cysteine residues in the human
dopamine transporter.

Whitehead RE, Ferrer JV, Javitch JA, Justice JB.

Department of Chemistry, Emory University, Atlanta, Georgia 30322, USA.

There is evidence to suggest that dopamine (DA) oxidizes to form dopamine
ortho-quinone (DAQ), which binds covalently to nucleophilic sulfhydryl groups on 
protein cysteinyl residues. This reaction has been shown to inhibit dopamine
uptake, as well as other biological processes. We have identified specific
cysteine residues in the human dopamine transporter (hDAT) that are modified by
this electron-deficient substrate analog. DAQ reactivity was inferred from its
effects on the binding of
[(3)H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (beta-CFT) to hDAT
cysteine mutant constructs. One construct, X5C, had four cysteines mutated to
alanine and one to phenylalanine (Cys(90)A, Cys(135)A, C306A, C319F and
Cys(342)A). In membrane preparations 1 mM DAQ did not affect [(3)H]beta-CFT
binding to X5C hDAT, in contrast to its effect in wild-type hDAT in which it
reduced the B:(max) value by more than half. Wild-type cysteines were substituted
back into X5C, one at a time, and the ability of DAQ to inhibit [(3)H]beta-CFT
binding was assessed. Reactivity of DAQ with Cys(90) increased the affinity of
[(3)H]beta-CFT for the transporter, whereas reactivity with Cys(135) decreased
the affinity of [(3)H]beta-CFT. DAQ did not change the K:(D) for [(3)H]beta-CFT
binding to wild-type. The reactivity of DAQ at Cys(342) decreased B:(max) to the 
same degree as wild-type. The latter result suggests that Cys(342) is the
wild-type residue most responsible for DAQ-induced inhibition of [(3)H]beta-CFT
binding.

PMID: 11181843 [PubMed - indexed for MEDLINE]


407. Eur J Biochem. 2001 Feb;268(4):1020-9.

Structural study of N-linked oligosaccharides of human intercellular adhesion
molecule-3 (CD50).

Funatsu O, Sato T, Kotovuori P, Gahmberg CG, Ikekita M, Furukawa K.

Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology,
Itabashi-ku, Japan.

The N-linked oligosaccharides were released from purified human intercellular
adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of
oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced
with NaB[3H]4 were separated into neutral and acidic fractions by paper
electrophoresis. Most of the acidic oligosaccharides were converted to neutral
ones by digestion with sialidase, indicating that they are sialyl derivatives.
The neutral and sialidase-treated acidic oligosaccharides were fractionated by
serial lectin column chromatography followed by Bio-Gel P-4 column
chromatography. Structural studies of each oligosaccharide by sequential exo- and
endo-glycosidase digestion and by methylation analysis revealed that N-linked
oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type,
about 60% of which contain two to three poly N-acetyllactosamine chains
terminated with the type 1 structure and those without the type 1 structure per
oligosaccharide. In addition, a small amount of the high mannose-type
oligosaccharide with six alpha-mannose residues, which could act as a ligand for 
the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.

PMID: 11179968 [PubMed - indexed for MEDLINE]


408. Mol Cell Neurosci. 2001 Jan;17(1):208-25.

An alternative amino-terminus expressed in the central nervous system converts
agrin to a type II transmembrane protein.

Neumann FR, Bittcher G, Annies M, Schumacher B, Kroger S, Ruegg MA.

Department of Pharmacology/Neurobiology, Biozentrum, University of Basel,
Klingelbergstrasse 70, Basel, CH-4056, Switzerland.

Agrin is a basal lamina-associated heparansulfate proteoglycan that is a key
molecule in the formation of the vertebrate neuromuscular junction. The
carboxy-terminal part of agrin is involved in its synaptogenic activity. The
amino-terminal end of chick agrin consists of a signal sequence, required for the
targeting of the protein to the secretory pathway, and the amino-terminal agrin
(NtA) domain that binds to basal lamina-associated laminins. The cDNA encoding
rat agrin lacks this NtA domain and instead codes for a shorter amino-terminal
end. While the NtA domain is conserved in several species, including human,
sequences homologous to the amino-terminus of rat agrin have not been described. 
In this work, we have characterized these amino-terminal sequences in mouse and
chick. We show that they all serve as a noncleaved signal anchor that immobilizes
the protein in a N(cyto)/C(exo) orientation in the plasma membrane. Like the
secreted form, this transmembrane form of agrin is highly glycosylated indicative
of a heparansulfate proteoglycan. The structure of the 5' end of the mouse agrin 
gene suggests that a distinct promoter drives expression of the transmembrane
form. Agrin transcripts encoding this form are enriched in the embryonic brain,
particularly in neurons. To our knowledge, this is the first example of a
molecule that is synthesized both as a basal lamina and a plasma membrane
protein.

PMID: 11161480 [PubMed - indexed for MEDLINE]


409. J Neurosci. 2001 Feb 15;21(4):1218-27.

Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis.

Fergestad T, Broadie K.

Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840, USA.

The Drosophila dicistronic stoned locus encodes two distinctive presynaptic
proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein without
homology to known synaptic proteins, and STNB contains a domain with homology to 
the endocytotic protein AP50. Both Stoned proteins colocalize precisely with
endocytotic proteins including the AP2 complex and Dynamin in the "lattice
network" characteristic of endocytotic domains in Drosophila presynaptic
terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking
decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of
spatial regulation of the vesicular recycling intermediates. Mutant synapses
display a significant delay in vesicular membrane retrieval after depolarization 
and neurotransmitter release. These studies suggest that the Stoned proteins play
a role in mediating synaptic vesicle endocytosis. We have documented previously a
highly specific synaptic mislocalization and degradation of Synaptotagmin I in
stoned mutants. Here we show that transgenic overexpression of Synaptotagmin I
rescues stoned embryonic lethality and restores endocytotic recycling to normal
levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type
animals results in increased synaptic dye uptake, indicating that Synaptotagmin I
directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel
with recent biochemical studies, this genetic analysis strongly suggests that
Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic
vesicle endocytosis. We conclude that Stoned proteins control synaptic
transmission strength by mediating the retrieval of Synaptotagmin I from the
plasma membrane.

PMID: 11160392 [PubMed - indexed for MEDLINE]


410. J Med Chem. 2001 Jan 4;44(1):47-57.

exo-2-(Pyridazin-4-yl)-7-azabicyclo[2.2.1]heptanes: syntheses and nicotinic
acetylcholine receptor agonist activity of potent pyridazine analogues of
(+/-)-epibatidine.

Che D, Wegge T, Stubbs MT, Seitz G, Meier H, Methfessel C.

Institut fur Pharmazeutische Chemie, Universitat Marburg, Marbacher Weg 6,
D-35032 Marburg/Lahn, Germany.

A new strategy for the straightforward synthesis of novel racemic epibatidine
analogues is presented, in which the 2-chloropyridinyl moiety of epibatidine is
bioisosterically replaced by differently substituted pyridazine rings. A key step
of the new syntheses is the inverse type Diels-Alder reaction of the
electron-rich enol ether 13 with the electron-deficient diazadiene systems of the
1,2,4, 5-tetrazines 14a-d to yield the novel pyridazine analogues of
(+/-)-epibatidine 18, 19, 22, and 24. In addition preparation of the
N-substituted derivatives, such as 26 and 28, is described. The structures of the
novel epibatidine analogues were assigned on the basis of spectral data, that of 
compound 24 being additionally verified by X-ray crystallography exhibiting two
racemic solid-state conformations in the crystal lattice and representing the
first X-ray structure of an unprotected 7-azabicyclo[2.2.1]heptane moiety. The
nAChR agonist activity of the racemic compounds 18, 19, 22, 24, and 28 was
assayed in vitro by whole-cell current recordings from Xenopus oocytes expressing
different recombinant nicotinic receptors from the rat. Among the compounds
synthesized and tested, the pyridazine analogue 24 of (+/-)-epibatidine and its
N-methyl derivative 28 were found to be the most active ones retaining much of
the potency of natural epibatidine but with a substantially improved selectivity 
ratio between the alpha4beta2 and alpha3beta4 subtypes.

PMID: 11141087 [PubMed - indexed for MEDLINE]


411. Chemistry. 2000 Dec 1;6(23):4343-7.

Modular chemistry with aluminum phosphanides: cluster formation of (AlP)n
(n=3,6,7), Al4P3, and Al4Li4P6 frameworks.

Driess M, Kuntz S, Monse C, Merz K.

Lehrstuhl fur Anorganische Chemie I, Fakultat fur Chemie, Ruhr-Universitat
Bochum, Germany. mathias.driess@ac1.ruhr-uni-bochum.de

The Al3P3 heterocycle 1 is formed in 94% yield by the reaction of the primary
silylphosphane 6a with Me3Al in toluene at 70 degrees C. While 1 crystallizes in 
an isomerically pure form, in which the six-membered Al3P3 ring prefers the chair
conformation and the P-H hydrogen atoms adopt exo positions, it isomerizes in
solution to give different diastereomers as shown by 1H and 31P NMR spectroscopy.
Intermolecular cyclocondensation of 1 at 110 degrees C in toluene leads, under
liberation of methane, to the distorted hexameric-prismatic (AlP)6 cluster 2 in
98% yield. The capability of 1 to function as a building block was further used
for the synthesis of the solvent-separated ion pair [Li(thf)2]+ [(Me2Al)4(PR)3]- 
(3) which was prepared by a one-pot reaction of 1 with nBuLi and Me2AlCl in 15%
yield. The structure of 3 was established by an X-ray diffraction analysis.
Double deprotonation at phosphorus in 1 with RPLi2 (R = iPr3Si) (molar ratio
1:2), and subsequent transformation of the reaction mixture with Me3Al afforded
the novel donor-solvent-free cluster 4 in 62% yield. The latter consists of a
rhombododecahedral Al4Li4P6 framework, in which the Li centers are
three-coordinate. The reaction of the silylphosphane 6b with the trimethylamine
adduct of alane furnishes not only the hexamer (RPAIH)6 (R = (iPrMe2C)-Me2Si) but
also the corresponding heptamer 5, which has a nonregular polyhedral (AIP)7
framework and represents the first cluster of this type.

PMID: 11140963 [PubMed]


412. Praxis (Bern 1994). 2000 Nov 16;89(46):1918-23.

[Chronic abdominal pain in a young diabetic patient]

[Article in German]

Weber C, Czerwenka W, Schulthess G.

Medizinische Poliklinik, Departement fur Innere Medizin, Universitatsspital
Zurich.

We report on a 33-year-old patient from Sri Lanka who had been suffering from
recurrent episodes of abdominal cramps since he was ten years old. He
additionally suffered from postprandial flatulence and an increased frequency of 
bowel movements. By the age of 24, his condition had worsened with polyuria and
polydipsia and he was diagnosed with type II diabetes mellitus. Recently, the
patient's compliance deteriorated steadily and his diabetes mellitus was
uncontrolled. His flatulence continued and he had six to seven bowel movements
daily. He presented to us with renewed bouts of severe stomach cramps, similar to
the painful episodes that the patient experienced in his youth. After exclusion
of other etiologies and judging by the clinical picture, the patient's origin and
the sonographically and radiologically verified pancreatic calcification, we
rendered the diagnosis of a tropic calcifying pancreatitis with secondary
diabetes mellitus. According to the literature, malignant neoplasia may develop
on the basis of this disease. However, we were able to rule out a carcinoma as
the cause of the current pain episodes in this patient based on clinical findings
and course. We attributed the stomach cramps to compression of the common bile
duct by the fibrotic head of pancreas. Pain and cholestasis regressed, thus
obviating the need for surgical intervention (pancreaticojejunostomy). On therapy
with enzyme substitution and insulin, the patient's exo- and endocrine pancreatic
insufficiency was asymptomatic.

PMID: 11111410 [PubMed - indexed for MEDLINE]


413. Arch Biochem Biophys. 2000 Nov 1;383(1):28-37.

Chemoenzymatic synthesis of glycopolypeptides carrying
alpha-Neu5Ac-(2-->3)-beta-D-Gal-(1-->3)-alpha-D-GalNAc,
beta-D-Gal-(1-->3)-alpha-D-GalNAc, and related compounds and analysis of their
specific interactions with lectins.

Zeng X, Nakaaki Y, Murata T, Usui T.

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
University, Japan.

Glycopolypeptide (1) carrying the beta-D-Gal-(1-->3)-alpha-D-GalNAc unit as a
kind model of asialo-type mucin was synthesized through three steps: enzymatic
synthesis of p-nitrophenyl disaccharide glycoside, reduction of the p-nitrophenyl
group, and coupling of the amino group with the carboxyl group of poly(L-glutamic
acid)s (PGA). In a similar manner, glycopolypeptides (2-7) carrying
beta-D-Gal-(1-->3)-beta-D-GalNAc, beta-D-Gal-(1-->3)-beta-D-GlcNAc,
beta-D-Gal-(1-->6)-alpha-D-GalNAc, beta-D-Gal-(1-->6)-beta-D-GalNAc,
alpha-D-GalNAc, and beta-D-GalNAc, respectively, were synthesized as analogous
polymers of polymer 1. Glycopolypeptides 8 and 9 as a mimic of sialo-type mucin
were further prepared from polymers 1 and 2 as the acceptor of CMP-Neu5Ac by
alpha2,3-(O)-sialyltransferase, respectively. Interactions of these
glycopolypeptides with lectins were investigated with the double-diffusion test
and the hemagglutination-inhibition assay and in terms of an optical biosensor
based on surface plasmon resonance. Polymers 1 and 2 reacted strongly with peanut
(Arachis hypogaea) agglutinin (PNA) and Agaricus bisporus agglutinin (ABA). On
the other hand, polymers 8 and 9 through sialylation from polymers 1 and 2
reacted with ABA, but did not with PNA. Other polymers 3-7 did not show any
reactivity for both the lectins. These results show that PNA acts precisely in an
exo manner on the beta-D-Gal-(1-->3)-D-GalNAc sequence, while ABA acts in an endo
manner. Polymers 6 and 7 substituted with GalNAc reacted strongly with soybean
(Glycine max) agglutinin and Vicia villosa agglutinin B4, regardless of the
configuration of the glycosidic linkage. The interaction of all polymers with
Bauhinia purpurea agglutinin was much stronger than that of the corresponding
sugars. Polymers 8 and 9 reacted with wheat germ (Triticum vulgaris) agglutinin
(WGA), to which Neu5Ac residues are needed for binding, but polymers 1 and 2 did 
not. These sugar-substituted glycopolypeptides interacted specifically with the
corresponding lectins. Furthermore, polymers 4-7 reacted with WGA, but the
corresponding sugars did not. It suggests that the N-acetyl group along the PGA
backbone has a cluster effect for WGA. The artificial glycopolypeptides were
shown to be useful as tools and probes of carbohydrate recognition and modeling
in the analysis of glycoprotein-lectin interactions.

PMID: 11097173 [PubMed - indexed for MEDLINE]


414. Microb Pathog. 2000 Dec;29(6):329-43.

Triggering the ExoS regulon of Pseudomonas aeruginosa: A GFP-reporter analysis of
exoenzyme (Exo) S, ExoT and ExoU synthesis.

Hornef MW, Roggenkamp A, Geiger AM, Hogardt M, Jacobi CA, Heesemann J.

Max von Pettenkofer Institut, Ludwig Maximilian-Universitat, Munich, Germany.

The ExoS regulon of Pseudomonas aeruginosa encodes diverse type III secreted
effector proteins which have been shown to exert cytotoxic effects in cell
culture experiments. However, little information exists about the environmental
conditions and stimuli for upregulation of the ExoS regulon. Translational
reporter fusion proteins of exoenzyme (Exo) S, ExoT and ExoU, as well as the type
II secreted exotoxin A (ETA) to the green fluorescent protein (GFP), were
constructed in order to compare exoprotein production under diverse growth
conditions. Reporter protein activity was recorded by FACS-analysis and by
conventional and confocal laser scanning microscopy. Low ion concentration
induced co-ordinated upregulation of ExoS, ExoT and ExoU with a maximum effect at
37 degrees C. A dose-dependent upregulation was seen with human serum or
increasing NaCl concentrations. A type III secretion-negative pcrD mutant of P.
aeruginosa showed a weak ExoS response to environmental stimuli, compared with
the parental strain, suggesting a negative regulatory mechanism. Co-culture with 
the mammalian cell lines J774A.1 or HeLa led to rapid upregulation of ExoS, ExoT 
and ExoU synthesis. These data suggest that the ExoS regulon of P. aeruginosa can
be triggered by a variety of environmental signals as well as by cell contact
with eukaryotic cells. Copyright 2000 Academic Press.

PMID: 11095918 [PubMed - indexed for MEDLINE]


415. Carbohydr Res. 2000 Oct 20;329(1):97-107.

Purification and characterisation of a malto-oligosaccharide-forming amylase
active at high pH from Bacillus clausii BT-21.

Duedahl-Olesen L, Kragh KM, Zimmermann W.

Department of Civil Engineering, Aalborg University, Denmark.

Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming
amylase active at high pH when grown on starch substrates. The enzyme was
purified to homogeneity by affinity and anion-exchange chromatography. The
molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide
electrophoresis was 101 kDa. The enzyme showed an optimum of activity at pH 9.5
and 55 degrees C. Maltohexaose was detected as the main initially formed starch
hydrolysis product. Maltotetraose and maltose were the main products obtained
after hydrolysis of starch by the enzyme for an extended period of time and were 
not further degraded. The enzyme readily hydrolysed soluble starch, amylopectin
and amylose, while cyclodextrins, pullulan or dextran were not degraded. The mode
of action during hydrolysis of starch indicated an exo-acting type of amylolytic 
enzyme mainly producing maltohexaose and maltotetraose. Amino acid sequencing of 
the enzyme revealed high homology with the maltohexaose-forming amylase from
Bacillus sp. H-167.

PMID: 11086690 [PubMed - indexed for MEDLINE]


416. Arch Biochem Biophys. 2000 Oct 15;382(2):275-83.

Structural analysis of the asparagine-linked glycan units of the ZP2 and ZP3
glycoproteins from mouse zona pellucida.

Tulsiani DR.

Department of Obstetrics & Gynecology, Vanderbilt University School of Medicine, 
Nashville, Tennessee 37232-2633, USA. daulat.tulsiani@mcmail.vanderbilt.edu

Zona pellucida (ZP), the extracellular glycocalyx that surrounds the mammalian
egg plasma membrane, is a relatively simple structure consisting of three to four
glycoproteins. In the mouse, the ZP is composed of three glycoproteins, namely
ZP1 (200 kDa), ZP2 (120 kDa), and ZP3 (83 kDa). Extensive studies in this species
have resulted in the identification of primary (mZP3) and secondary (mZP2)
binding sites for spermatozoa. The two zona components are highly glycosylated
containing N-linked and O-linked glycan units. In an attempt to characterize
N-linked glycan units, mZP2 and mZP3 were purified and the N-linked carbohydrate 
chains were released by exhaustive digestion with N-glycanase. The released
oligosaccharides (OSs) were radiolabeled by reduction with NaB3H4 and resolved by
gel filtration on a column of Bio-Gel P-4. The OSs separated into several peaks
indicating the presence of a variety of N-linked glycans. Interestingly, the
radioactive peaks resolved from mZP2 and mZP3 were quite different, a result
suggesting qualitative and quantitative differences in the glycans. The
[SH]-labeled glycans present in mZP2 and mZP3 were pooled separately and
fractionated by serial lectin chromatography. Experimental evidence included in
this report strongly suggests that mZP3 (but not mZP2) contains polylactosaminyl 
glycan with terminal, nonreducing alpha-galactosyl residues. The mZP3 glycans
eluted from the immobilized lectin columns were further characterized by lectin
and sizing column chromatography before or after digestion with endo-/
exo-glycohydrolases. Data revealed the presence of a variety of OSs, including
poly-N-acetyllactosaminyl, bi-, tri-, and tetraantennary complex-type, and
high-mannose-type glycans. Taken together, these results provide additional
evidence on the complex nature of the glycan chains present on mZP
glycoconjugates.

PMID: 11068879 [PubMed - indexed for MEDLINE]


417. Biosci Biotechnol Biochem. 2000 Sep;64(9):1896-902.

Purification and characterization of chitosanase and Exo-beta-D-glucosaminidase
from a Koji mold, Aspergillus oryzae IAM2660.

Zhang XY, Dai AL, Zhang XK, Kuroiwa K, Kodaira R, Shimosaka M, Okazaki M.

Department of Applied Biology, Faculty of Textile Science and Technology, and
Shinshu University, Ueda, Nagano, Japan.

Chitosan-degrading activity was detected in the culture fluid of Aspergillus
oryzae, A. sojae, and A. flavus among various fungal strains belonging to the
genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher
level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a 
carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular
masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric 
assay and an analysis of reaction products by thin-layer chromatography clearly
indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa
enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a
hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan,
and chitosan with a low degree of acetylation (0-30%). The 135-kDa
exo-beta-D-glucosaminidase,enzyme,named released a single GlcN residue from the
GlcN oligomers and chitosan, but did not release GlcNAc residues from either
GlcNAc oligomer or colloidal chitin.

PMID: 11055393 [PubMed - indexed for MEDLINE]


418. Exp Parasitol. 2000 Sep;96(1):23-31.

Trypanosoma cruzi: cloning and characterization of a RAB7 gene.

Leal ST, Araripe JR, Urmenyi TP, Cross GA, Rondinelli E.

Instituto de Biofisica Carlos Chagas Filho, CCS, UFRJ, Rio de Janeiro, RJ,
21949-900, Brazil.

The small monomeric GTP-binding proteins of the RAB subfamily are key regulatory 
elements of the machinery that controls membrane traffic in eukaryotic cells.
These proteins have been localized to many different intracellular organelles, on
both endocytic and exocytic compartments, suggesting that each step of vesicular 
traffic can involve a specific RAB protein. The presence of conserved amino acid 
domains in these proteins has allowed the cloning of their genes from several
organisms, including yeast, plants, humans, and parasites. In this work we
describe the identification, cloning, and characterization of a RAB7 gene
homologue in Trypanosoma cruzi (TcRAB7). Our data indicate that this gene is
present as a single copy in the T. cruzi genome, located on a 2.25-Mb chromosomal
DNA. TcRAB7 is expressed in T. cruzi epimastigotes, metacyclic trypomastigotes,
and spheromastigotes. We established transformed cell lines that express two
versions of an epitope-tagged TcRAB7 protein: one wild type (pTAG) and one
deleted at the C-terminal cysteines (pDeltaCXC). Wild-type TcRAB7 protein (pTAG) 
appears to be localized exclusively in the membrane fraction, while the mutated
TcRAB7 protein (pDeltaCXC) loses the ability to associate with the membrane,
showing only cytosolic localization. Also, we produced the recombinant TcRAB7
protein and demonstrated that it binds GTP. The identification of exo- and
endocytic machinery components in T. cruzi and their function would provide
specific markers of these subcellular compartments, thereby unveiling important
aspects of vesicular traffic in this parasite. Copyright 2000 Academic Press.

PMID: 11038317 [PubMed - indexed for MEDLINE]


419. J Org Chem. 2000 Oct 20;65(21):7119-23.

Radical and anionic response of N-(Bromomethanesulfonyl)-substituted
alpha,alpha'-bridged piperidine substrates

Schloss JD, Leit SM, Paquette LA.

Evans Chemical Laboratories, The Ohio State University, Columbus, Ohio 43210,
USA.

The N-(bromomethanesulfonyl) azabicyclic ketones 2a-c and their exo-methylene
analogues 1a-c were prepared. Our examination of the radical-induced behavior of 
the latter triad provided experimental evidence for the propensity of the b and c
systems to engage in 7-endo cyclization. For 1a, only reductive debromination was
observed. In no instance was ring closure by the 6-exo radical mode seen. As
concerns ketones 2a-c, all three showed a remarkable facility for engaging in
intramolecular S(N)2 displacement in the presence of KHMDS. Yields at the mid-80%
level were realized irrespective of the value of n. Molecular mechanics
calculations of the Monte Carlo type were performed on several conformational
isomers and product classes in an effort to provide support for the explanatory
conclusions offered as rationale for the collective experimental observations.

PMID: 11031038 [PubMed - as supplied by publisher]


420. J Bacteriol. 2000 Sep;182(18):5059-69.

The redox-sensitive transcriptional activator OxyR regulates the peroxide
response regulon in the obligate anaerobe Bacteroides fragilis.

Rocha ER, Owens G Jr, Smith CJ.

Department of Microbiology and Immunology, East Carolina University School of
Medicine, Greenville, North Carolina 27858-4354, USA.

The peroxide response-inducible genes ahpCF, dps, and katB in the obligate
anaerobe Bacteroides fragilis are controlled by the redox-sensitive
transcriptional activator OxyR. This is the first functional oxidative stress
regulator identified and characterized in anaerobic bacteria. oxyR and dps were
found to be divergently transcribed, with an overlap in their respective promoter
regulatory regions. B. fragilis OxyR and Dps proteins showed high identity to
homologues from a closely related anaerobe, Porphyromonas gingivalis. Northern
blot analysis revealed that oxyR was expressed as a monocistronic 1-kb mRNA and
that dps mRNA was approximately 500 bases in length. dps mRNA was induced over
500-fold by oxidative stress in the parent strain and was constitutively induced 
in the peroxide-resistant mutant IB263. The constitutive peroxide response in
strain IB263 was shown to have resulted from a missense mutation at codon 202
(GAT to GGT) of the oxyR gene [oxyR(Con)] with a predicted D202G substitution in 
the OxyR protein. Transcriptional fusion analysis revealed that deletion of oxyR 
abolished the induction of ahpC and katB following treatment with hydrogen
peroxide or oxygen exposure. However, dps expression was induced approximately
fourfold by oxygen exposure in DeltaoxyR strains but not by hydrogen peroxide.
This indicates that dps expression is also under the control of an
oxygen-dependent OxyR-independent mechanism. Complementation of DeltaoxyR mutant 
strains with wild-type oxyR and oxyR(Con) restored the inducible peroxide
response and the constitutive response of the ahpCF, katB, and dps genes,
respectively. However, overexpression of OxyR abolished the catalase activity but
not katB expression, suggesting that higher levels of intracellular OxyR may be
involved in other physiological processes. Analysis of oxyR expression in the
parents and in DeltaoxyR and overexpressing oxyR strains by Northern blotting and
oxyR'::xylB fusions revealed that B. fragilis OxyR does not control its own
expression.

PMCID: PMC94652
PMID: 10960088 [PubMed - indexed for MEDLINE]


421. J Org Chem. 2000 Jul 28;65(15):4523-8.

Stereoselective synthesis of cyclic ethers using vinylogous sulfonates as radical
acceptors: effect of E/Z geometry and temperature on diastereoselectivity.

Evans PA, Manangan T.

Department of Chemistry and Biochemistry, University of Delaware, Newark, 19716, 
USA. paevans@udel.edu

Erratum in:
    J Org Chem 2000 Sep 22;65(19):6291.

Treatment of the E-vinylogous sulfonates 1a-g with tris(trimethylsilyl)silane and
triethylborane, in the presence of air, furnished the cyclic ethers 2/3a-g with
good to excellent diastereoselectivity favoring the cis-isomer 2. This study
demonstrated the level of stereocontrol in a 6-exo radical cyclization and may be
attributed to the type of radical intermediate. Hence, the modest selectivity
obtained for the cyclization of 1e may be a function of the acyl radical geometry
(sp2) and high inversion barrier (29 kcal/mol) as compared to the alkyl (1
kcal/mol) and vinyl (2.9 kcal/mol) radicals. This is consistent with the acyl
radical cyclization having an earlier transition state than the corresponding
alkyl and vinyl radicals. The modest diastereoselectivity can be improved
dramatically using the Z-vinylogous sulfonate (> or =34:1; R = Ph) to promote
kinetic trapping of the s-trans rotamer I and III, respectively (Figure 1). The
5-exo alkyl radical cyclization reaction under nonreductive Keck-allylation
conditions was also examined, in which 8 was formed in 91% overall yield. This
transformation provides a convenient method for in situ homologation and should
be applicable to target directed synthesis.

PMID: 10959853 [PubMed - indexed for MEDLINE]


422. Chemistry. 2000 Jul 3;6(13):2435-48.

Catalytic enantioselective aza-Diels-alder reactions of imines--an approach to
optically active nonproteinogenic alpha-amino acids

Yao S, Saaby S, Hazell RG, Jorgensen KA.

Center for Metal Catalyzed Reactions, Department of Chemistry, Aarhus University,
Denmark.

A catalytic enantioselective aza-Diels-Alder reaction of imines has been
developed. The reaction of N-tosyl alpha-imino ester with different dienes
including activated, non-activated, cyclic, and acyclic dienes has been
investigated in the presence of various chiral Lewis acids. A series of
phosphino-oxazoline ligands have been synthesized and evaluated for the reaction.
It was found that the combination of phosphino-oxazoline ligands with copper(I)
salts gives the best results for the activated dienes, while BINAP-copper(I)
complexes are good catalysts for all the dienes studied. In the case of activated
acyclic dienes the aza-Diels-Alder products can be obtained in higher than 80%
isolated yield and 96% ee, while for the unactivated cyclic dienes the exo
diastereomer is formed as the major product in up to 95 % ee. For an activated
cyclic conjugated diene, 2-trimethylsilyloxy-1,3-cyclohexadiene, the reaction
proceeds as a Mannich-type addition reaction giving optically active gamma-oxo
alpha-amino acid derivatives in good yields and up to 96% ee. The reaction of an 
unactivated acyclic diene, 2,3-dimethyl-1,3-butadiene, with the N-tosyl
alpha-imino ester gives both the aza-Diels-Alder and aza-ene products, in a ratio
of 9:1 favoring the aza-Diels-Alder product. Furthermore, a series of different
imines have been synthesized and investigated as possible substrates for the
present catalytic enantioselective aza-Diels-Alder reaction in order to obtain
mechanistic insight. All imines studied gave moderate to high ee. Particularly,
the reaction of the N-phenyl and N-p-methoxyphenyl substituted glyoxylate imines 
with Danishefsky's diene proceeded well affording the corresponding
aza-Diels-Alder product in high yield with up to 91% ee at room temperature. The 
present catalytic enantioselective reaction of imines provided an effective route
to optically active nonproteinogenic alpha-amino acids. The products of the
catalytic enantioselective aza-Diels-Alder reaction of the cyclic dienes can be
used for the preparation of key compounds such as natural products and compounds 
of pharmaceutical interest. The absolute configurations of five products have
been solved by X-ray structural analysis, and it is found that the absolute
configuration of the aza-Diels-Alder adduct is dependent on the substituent on
the imine nitrogen atom. It turned out that the N-tosyl glyoxylate imine and
N-p-methoxyphenyl glyoxylate imine give the aza-Diels-Alder adduct with opposite 
absolute configuration using the same enantiomer of the catalyst. On the basis of
the results the mechanistic aspects for the reactions are discussed.

PMID: 10939745 [PubMed - as supplied by publisher]


423. Biochemistry. 2000 Aug 8;39(31):9597-603.

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant
of Herpes simplex virus type 1 thymidine kinase interacting with
(North)-methanocarba-thymidine.

Prota A, Vogt J, Pilger B, Perozzo R, Wurth C, Marquez VE, Russ P, Schulz GE,
Folkers G, Scapozza L.

Department of Applied BioSciences, Swiss Federal Institute of Technology (ETH),
Zurich, Switzerland.

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1
thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the 
potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed.
The kinetic study reveals a 12-fold K(M) increase for thymidine processed with
Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for 
both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and
TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively,
whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM,
rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover,
TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency
(k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT
when compared to the wild-type enzyme. Crystal structures of wild-type and mutant
TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4
A, respectively. The thymine moiety of MCT binds like the base of dT while the
conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety,
assumes a 2'-exo envelope conformation that is flatter than the one observed for 
the free compound. The hydrogen bond pattern around the sugar-like moiety differs
from that of thymidine, revealing the importance of the rigid conformation of MCT
with respect to hydrogen bonds. These findings make MCT a lead compound in the
design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in 
combination with MCT, opens new possibilities for gene therapy.

PMID: 10924157 [PubMed - indexed for MEDLINE]


424. J Nutr Sci Vitaminol (Tokyo). 2000 Feb;46(1):23-9.

Preventive effect of soybean resistant proteins against experimental
tumorigenesis in rat colon.

Azuma N, Machida K, Saeki T, Kanamoto R, Iwami K.

Department of Biological Resource Chemistry, Kyoto Prefectural University, Japan.

The insoluble 'high-molecular-weight' fraction (HMF) centrifugally separable
after digestion of soy protein isolate with a microbial protease of the exo-type,
of which about a quarter is regarded as an indigestible 'resistant protein,' was 
examined for its preventive effect against colonic tumorigenesis in a model
system with male F-344 rats. The rats were intraperitoneally injected with
azoxymethane (15 mg/kg BW) once a week for 3 wk and were fed a 20.6% HMF diet
(+0.4% DL-Met) or 14.7% casein diet (+0.3% DL-Met) supplemented with 0.2% sodium 
deoxycholate (DCA) or without supplementation. Twelve wk later, 5 rats of each
group were inspected for formation of tumors but no tumors were visible to the
naked eye. The DCA-fed casein group was conspicuous for a low count of aberrant
crypt foci. At 39 wk, 6 rats of the DCA-fed casein group (n = 10) and 3 rats of
the DCA-fed HMF group (n = 9) had a total of 18 tumors with a major axis of 4.0
+/- 0.4 mm and 3 tumors with an axis of 2.0 +/- 0.1 mm, respectively, in contrast
to only a single tumor for the DCA-unfed casein group (nil for the DCA-unfed HMF 
group). The difference in tumor number and size was considered significant
between these DCA-fed casein and HMF groups; that is to say, HMF feeding retarded
tumor development despite the frequent occurrence of pre-neoplastic lesions. In
addition, fecal bile acid excretion was much more elevated by HMF feeding than by
casein feeding. It can be assumed from these observations that the
antitumorigenicity of HMF is due to the inhibitory effect of soybean resistant
proteins on reabsorption as well as the mucosal contact of bile acids in the
intestine.

PMID: 10868349 [PubMed - indexed for MEDLINE]


425. J Org Chem. 2000 Feb 11;65(3):847-60.

Enzyme-assisted asymmetric total synthesis of (-)-podophyllotoxin and
(-)-picropodophyllin.

Berkowitz DB, Choi S, Maeng JH.

Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska
68588-0304, USA.

Described is the first catalytic, asymmetric synthesis of (-)-podophyllotoxin and
its C(2)-epimer, (-)-picropodophyllin. Asymmetry is achieved via the enzymatic
desymmetrization of advanced meso diacetate 20, through PPL-mediated ester
hydrolysis. A second key feature of the synthesis is the strategically late
introduction of the highly oxygenated natural ring E through an arylcopper
species. The successful implementation of this approach augers well for the
introduction of other functionalized rings E for future SAR work. The synthesis
begins from piperonal, which is fashioned into isobenzofuran (IBF) precursor 14
in three steps (bromination, acetalization, and halogen-metal
exchange/hydroxymethylation). Interestingly, treatment of 14 with HOAc in
commerical dimethyl maleate (contains 5% dimethyl fumarate) leads to a nearly
equimolar mixture of fumarate- (15) and maleate-IBF Diels-Alder adducts (16 and
17), indicating that IBF 11 reacts about 15 times faster with dimethyl fumarate
than with dimethyl maleate. With scrupulously pure dimethyl maleate a 2.8:1
endo:exo mixture of maleate DA adducts is still obtained. On the other hand, the 
desired meso diester 16 is obtained pure and in nearly quantitative yield by
employing neat dimethyl acetylene dicarboxylate as the dienophile, followed by
catalytic hydrogenation. Reduction (LiAlH(4)) of 16 provides meso diol 19, which 
is then treated with Ac(2)O, BzCl, and PhCH(2)COCl to provide the corresponding
meso diesters, 20-22. Screening of these meso benzoxabicyclo[2.2.1]heptyl
substrate candidates across a battery of acyl transfer enzymes leads to an
optimized match of diacetate 20 with PPL. Even on 10-20 g scales, asymmetry is
efficiently introduced here, yielding the key chiral intermediate, monoacetate 25
(66% isolated yield, 83% corrected yield, 95% ee). Protecting group manipulation 
and oxidation (Swern) provide aldehyde 27b, which undergoes efficient
retro-Michael ring opening to produce dihydronaphthalene 30, in which the C(3)
and C(4) stereocenters are properly set. Following several unsuccessful
approaches to the intramolecular delivery of ring E (via Claisen rearrangement,
Heck-type cyclization, or radical cyclization), a highly diastereoselective,
intermolecular conjugate addition of the arylcopper reagent derived from
(3,4,5-trimethoxy)phenylmagnesium bromide and CuCN to acyl oxazolidinone 50 was
developed (85% yield, only the required alpha-stereochemistry at C(1) is
observed). The conjugate addition product is converted to (-)-picropodophyllin in
two steps (lactonization, SEM deprotection) or to (-)-podophyllotoxin, in three
steps, through the introduction of a C(2)-epimerization step, under Kende
conditions, prior to the final conjugate addition.

PMID: 10814019 [PubMed - indexed for MEDLINE]


426. Exp Nephrol. 2000 May-Jun;8(3):135-43.

Inhibiting albumin glycation ameliorates diabetic nephropathy in the db/db mouse.

Cohen MP, Masson N, Hud E, Ziyadeh F, Han DC, Clements RS.

University City Science Center, Philadelphia, PA, USA. drmpcohen@AOL.com

Albumin modified by Amadori glucose adducts stimulates the expression of
extracellular matrix proteins by glomerular mesangial and endothelial cells, and 
has been mechanistically linked to the pathogenesis of diabetic nephropathy. To
test the hypothesis that inhibiting the formation of glycated albumin might
beneficially influence the development of kidney disease in diabetes, we treated 
diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226)
that impedes the condensation of free glucose with lysine epsilon-amino groups in
albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the
plasma concentration of glycated albumin within days after initiation of
treatment and maintained glycated albumin within the normal range throughout the 
study, despite persistent and severe hyperglycemia. Urine albumin excretion,
which was markedly increased at the start of the study (age 12 weeks), was
significantly reduced in treated diabetic animals compared with their untreated
diabetic littermates. The fall in creatinine clearance that was observed in
untreated diabetic animals was prevented in diabetic littermates that received
treatment. Compared with the nondiabetic controls, the amount of glomerular
mesangial matrix was threefold greater in untreated diabetic mice; in contrast,
the mesangial matrix fraction was only 1. 5 times that of nondiabetic controls in
the treated diabetic animals, representing a reduction in mesangial matrix
accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA
encoding for alpha1 (IV) collagen in renal cortex of db/db mice. We conclude that
normalization of plasma glycated albumin concentrations with the glycation
inhibitor EXO-226 ameliorates the glomerular structural and functional
abnormalities associated with diabetic nephropathy in the db/db mouse. Copyright 
2000 S. Karger AG, Basel.

PMID: 10810230 [PubMed - indexed for MEDLINE]


427. Nucleosides Nucleotides Nucleic Acids. 2000 Jan-Feb;19(1-2):13-29.

Conformation and local environment of nucleotides bound to HIV type 1 reverse
transcriptase (HIV-1 RT) in the ground state.

Painter GR, Andrews CW, Furman PA.

Triangle Pharmaceuticals, Inc. Durham, NC, USA.

Nuclear Overhauser effect experiments were used to characterize the protein
environment and conformations of dTTP, dATP and AZTTP bound to HIV-RT in the
ground state. The results show the initial binding sites for the nucleotides
overlap but are not completely coincident. All of the bound nucleotides assume
the same anti C4'-exo conformation.

PMID: 10772700 [PubMed - indexed for MEDLINE]


428. Mol Plant Microbe Interact. 2000 Apr;13(4):359-65.

Cloning and disruption of pgx4 encoding an in planta expressed
exopolygalacturonase from Fusarium oxysporum.

Garcia-Maceira FI, Di Pietro A, Roncero MI.

Departamento de Genetica, Facultad de Ciencias, Universidad de Cordoba, Spain.

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, 
produces an array of pectinolytic enzymes, including at least two
exo-alpha1,4-polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was
isolated with degenerate polymerase chain reaction primers derived from amino
acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid
polypeptide with nine potential N-glycosylation sites and a putative 21 amino
acid N-terminal signal peptide. The deduced mature protein has a calculated
molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs 
of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is
present in a single copy in different formae speciales of F. oxysporum.
Expression of pgx4 was detected during in vitro growth on pectin,
polygalacturonic acid, and tomato vascular tissue and in roots and stems of
tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F.
oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement 
were as virulent on tomato plants as the wild-type strain.

PMID: 10755298 [PubMed - indexed for MEDLINE]


429. J Appl Physiol. 2000 Apr;88(4):1239-46.

Glucose ingestion and substrate utilization during exercise in boys with IDDM.

Riddell MC, Bar-Or O, Hollidge-Horvat M, Schwarcz HP, Heigenhauser GJ.

Children's Exercise and Nutrition Centre, McMaster University, Hamilton, Ontario,
Canada L8N 3Z5.

This study was intended to compare exogenous [(13)C]glucose (Glu(exo)) oxidation 
in boys with insulin-dependent diabetes mellitus (IDDM) and healthy boys of
similar age, weight, and maximal O(2) uptake. In a control trial with water
intake (CT) and in a (13)C-enriched glucose trial (GT), subjects cycled for 60
min (58.8 +/- 0.9% maximal O(2) uptake) while the utilization of total glucose,
total fat, and Glu(exo) was assessed. In CT, total glucose was 84.7 +/- 9.2 vs.
91.3 +/- 6.6 g/60 min (not significantly different) and total fat was 13.3 +/-
2.2 vs. 11.1 +/- 1.7 g/60 min (not significantly different) in IDDM vs. healthy
boys, respectively. In GT, Glu(exo) was 10.4 +/- 1.7 vs. 14.8 +/- 1.1 g/60 min,
corresponding to 9.0 +/- 1.0 vs. 12.4 +/- 0.5% of the total energy supply in IDDM
and healthy boys, respectively (P < 0.05). Endogenous glucose was spared in both 
groups by 12.6 +/- 3.5% (P < 0.05). Blood glucose and plasma insulin
concentrations were two- to threefold higher in IDDM vs. healthy boys in both
trials. In conclusion, Glu(exo) is impaired in exercising boys with IDDM, even
when plasma insulin levels are elevated.

PMID: 10749813 [PubMed - indexed for MEDLINE]


430. FASEB J. 2000 Apr;14(5):715-28.

Dopamine transporter proline mutations influence dopamine uptake, cocaine analog 
recognition, and expression.

Lin Z, Itokawa M, Uhl GR.

Molecular Neurobiology Branch, NIDA-IRP, National Institutes of Health, Johns
Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.

Analyses of mutation effects can aid in understanding how large proteins act. The
dopamine transporter (DAT) mediates complex actions in recognizing cocaine and in
recognizing and translocating dopamine, sodium, and chloride. DAT proline
residues, especially those in transmembrane (TM) domains, are good candidates for
involvement in these DAT actions. We now report production of mutants
substituting alanine and/or glycine residues for 16 prolines located in or near
putative DAT TM domains. We examine effects of these modifications on DAT
expression, dopamine uptake, and cocaine analog binding. Mutants in prolines
located in five DAT TM domains and four connecting loops alter apparent DAT
membrane targeting. Five mutations decrease dopamine affinities more than
threefold without significantly decreasing cocaine analog affinities. One
decreases cocaine analog affinity without decreasing dopamine affinity. Two
mutations decrease affinities for both dopamine and cocaine analog. P101 is
especially implicated in dopamine uptake. Alanine substitution for this proline
yields dopamine V(max) values of less than 3% of wild-type values despite
dopamine affinities more than fourfold higher than wild-type and normal Na(+) and
Cl(-) dependence. These DAT proline mutants identify DAT regions likely for
dopamine translocation and for recognition of dopamine and cocaine.

PMID: 10744628 [PubMed - indexed for MEDLINE]


431. Bioconjug Chem. 2000 Mar-Apr;11(2):202-11.

Facile synthesis of stable and lectin-recognizable DNA-carbohydrate conjugates
via diazo coupling.

Matsuura K, Akasaka T, Hibino M, Kobayashi K.

Department of Molecular Design and Department of Biotechnology, Graduate School
of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan.

Stable and lectin-recognizable DNA-carbohydrate conjugates were prepared by diazo
coupling of lactose and cellobiose derivatives to fragmented salmon testes DNA.
The diazo coupling is suggested to take place selectively to guanine bases since 
the amount of lactose moiety introduced was directly proportional to the G
content of various DNAs with different G contents. According to the CD spectra,
the conjugates bearing carbohydrate less than 25% content kept a typical B-type
conformation similar to native DNA. The conjugates possessed higher melting
temperature and stronger nuclease resistance both to exo- and endonucleases than 
native DNA. Gel shift assay and fluorescence binding assay showed that the
DNA-lactose conjugates were specifically bound to galactose-specific lectin
RCA(120) with strong binding affinity (Ka = 10(4)-10(5) M(-1)) due to glycoside
cluster effect. This facile method will be a useful protocol of molecular design 
for cell-targeted gene therapy.

PMID: 10725097 [PubMed - indexed for MEDLINE]


432. J Biomol NMR. 2000 Feb;16(2):147-64.

Determination of the populations and structures of multiple conformers in an
ensemble from NMR data: multiple-copy refinement of nucleic acid structures using
floating weights.

Gorler A, Ulyanov NB, James TL.

Department of Pharmaceutical Chemistry, University of California, San Francisco
94143-0466, USA.

A new algorithm is presented for determination of structural conformers and their
populations based on NMR data. Restrained Metropolis Monte Carlo simulations or
restrained energy minimizations are performed for several copies of a molecule
simultaneously. The calculations are restrained with dipolar relaxation rates
derived from measured NOE intensities via complete relaxation matrix analysis.
The novel feature of the algorithm is that the weights of individual conformers
are determined in every refinement step, by the quadratic programming algorithm, 
in such a way that the restraint energy is minimized. Its design ensures that the
calculated populations of the individual conformers are based only on
experimental restraints. Presence of internally inconsistent restraints is the
driving force for determination of distinct multiple conformers. The method is
applied to various simulated test systems. Conformational calculations on nucleic
acids are carried out using generalized helical parameters with the program
DNAminiCarlo. From different mixtures of A- and B-DNA, minor fractions as low as 
10% could be determined with restrained energy minimization. For B-DNA with three
local conformers (C2'-endo, O4'-exo, C3'-endo), the minor O4'-exo conformer could
not be reliably determined using NOE data typically measured for DNA. The other
two conformers, C2'-endo and C3'-endo, could be reproduced by Metropolis Monte
Carlo simulated annealing. The behavior of the algorithm in various situations is
analyzed, and a number of refinement protocols are discussed. Prior to
application of this algorithm to each experimental system, it is suggested that
the presence of internal inconsistencies in experimental data be ascertained. In 
addition, because the performance of the algorithm depends on the type of
conformers involved and experimental data available, it is advisable to carry out
test calculations with simulated data modeling each experimental system studied.

PMID: 10723994 [PubMed - indexed for MEDLINE]


433. J Mol Biol. 2000 Feb 18;296(2):385-401.

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking
activity of RecBCD and together with lambda bet, promotes Chi-independent
recombination.

Murphy KC.

Department of Molecular Genetics, University of Massachusetts Medical School,
Worcester, MA, 01655, USA.

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli
to promote phage growth and recombination. Overproduction of the RecC subunit in 
vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization,
revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking
experiments revealed that Abc2 protein does not interfere with the binding of ATP
to either the RecB or RecD subunits in the absence of DNA, though it partially
inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type
Salmonella typhimurium correlates with the presence of Abc2, but is independent
of the absolute level of ATP-dependent nuclease activity, suggesting a
qualitative change in the nature of Abc2-modified RecBCD nuclease activity
relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD
could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam
could not. In exonuclease assays designed to examine the polarity of digestion,
Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA
exonuclease by increasing the rate of digestion of the 5' strand. In this
respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered
the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified
RecBCD still possesses 3' exonuclease activity. These results are discussed in
terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22
phage recombination pathway. This mechanism of P22-mediated recombination
distinguishes it from phage lambda recombination, in which the phage
recombination system (Red) and its anti-RecBCD function (Gam) work independently.
Copyright 2000 Academic Press.

PMID: 10669596 [PubMed - indexed for MEDLINE]


434. Biol Chem. 1999 Dec;380(12):1421-30.

Dipeptidyl peptidase III from rat liver cytosol: purification, molecular cloning 
and immunohistochemical localization.

Ohkubo I, Li YH, Maeda T, Yamamoto Y, Yamane T, Du PG, Nishi K.

Department of Medical Biochemistry, Shiga University of Medical Science, Seta,
Otsu, Japan.

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver
cytosol. The calculated molecular weight of the purified enzyme was 82845.6
according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in
the absence or presence of beta-mercaptoethanol. These findings suggest that the 
enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly
hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Gly-Arg-MCA in the
pH range of 7.5 to 9.5. The Km, k(cat) and k(cat)/Km values of DPP III at optimal
pH (pH 8.5) were 290 microM, 18.0 s(-1) and 62.1 s(-1) x nM(-1) for Arg-Arg-MCA
and 125 microM, 4.53 s(-1) and 36.2 s(-1) x nM(-1) for Ala-Arg-MCA, respectively.
DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. 
These findings suggest that DPP III is an exo-type peptidase with characteristics
of a metallo- and serine peptidase. For further information on the molecular
structure, we screened a rat liver cDNA library using affinity-purified anti-rat 
DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the
amino acid sequence. The cDNA, designated as lambdaRDIII-11, is composed of 2640 
bp and encodes 738 amino acids in the coding region. Although the enzyme has a
novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in
clan MA in the metalloprotease kingdom. The DPP III antigen was detected in
significant amounts in the cytosol of various rat tissues by immunohistochemical 
examination.

PMID: 10661869 [PubMed - indexed for MEDLINE]


435. FASEB J. 2000 Feb;14(2):407-17.

In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini 
in ischemic mouse brain.

Huang D, Shenoy A, Cui J, Huang W, Liu PK.

Department of Neurosurgery, Baylor College of Medicine, Houston, Texas 77030,
USA.

Our aims were to examine whether oxidative DNA damage was elevated in brain cells
of male C57BL/6 mice after oxidative stress, and to determine whether neuronal
nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was
induced by occluding both common carotid arteries for 90 min, followed by
reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or
apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and
converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can 
then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and
detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with 
the non-ischemia controls, the density of EXOSS-positive cells was elevated at
least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for
another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and
neurons. Signs of cell death were detected at 24 h of reperfusion and occurred
mostly in the neurons. Both DNA damage and cell death in the cerebral cortical
neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg,
intraperitoneal), which specifically inhibited nNOS. Our results suggest that
nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage 
after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ 
detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in
ischemic mouse brain.

PMCID: PMC2746459
PMID: 10657997 [PubMed - indexed for MEDLINE]


436. Biochem J. 2000 Feb 15;346 Pt 1:9-15.

Purification, characterization and gene cloning of two
alpha-L-arabinofuranosidases from streptomyces chartreusis GS901.

Matsuo N, Kaneko S, Kuno A, Kobayashi H, Kusakabe I.

Central Research Laboratory, Godo Shusei Co. Ltd., Kamihongo, Matsudo, Chiba
271-0064, Japan. 23209331@people.or.jp

alpha-L-Arabinofuranosidases I and II were purified from the culture filtrate of 
Streptomyces chartreusis GS901 and were found to have molecular masses of 80 and 
37 kDa and pI values of 6.6 and 7.5 respectively. Both enzymes demonstrated
slight reactivity towards arabinoxylan and arabinogalactan as substrates but did 
not hydrolyse gum arabic or arabinoxylo-oligosaccharides.
alpha-L-Arabinofuranosidase I hydrolysed all of the alpha-linkage types that
normally occur between two alpha-L-arabinofuranosyl residues, with the following 
decreasing order of reactivity being observed for the respective disaccharide
linkages: alpha-(1-->2) alpha-(1-->3) alpha-(1-->5). This enzyme cleaved the
(1-->3) linkages of the arabinosyl side-chains of methyl 3,
5-di-O-alpha-L-arabinofuranosyl-alpha-L-arabinofuranoside in preference to the
(1-->5) linkages. alpha-L-Arabinofuranosidase I hydrolysed approx. 30% of the
arabinan but hydrolysed hardly any linear arabinan. In contrast,
alpha-L-Arabinofuranosidase II hydrolysed only (1-->5)-arabinofuranobioside among
the regioisomeric methyl arabinobiosides and did not hydrolyse the
arabinotrioside. Linear 1-->5-linked arabinan was a good substrate for this
enzyme, but it hydrolysed hardly any of the arabinan. Synergism between the two
enzymes was observed in the conversion of arabinan and debranched arabinan into
arabinose. Complete amino acid sequencing of alpha-L-arabinofuranosidase I
indicated that the enzyme consists of a central catalytic domain that belongs to 
family 51 of the glycoside hydrolases and additionally that unknown functional
domains exist in the N-terminal and C-terminal regions. The amino acid sequence
of alpha-L-arabinofuranosidase II indicated that this enzyme belongs to family 43
of the glycoside hydrolase family and, as this is the first report of an exo-1,
5-alpha-L-arabinofuranosidase, it represents a novel type of enzyme.

PMCID: PMC1220816
PMID: 10657233 [PubMed - indexed for MEDLINE]


437. Hum Mutat. 1998;12(3):218.

The minisatellite expansion mutation in EPM1: resolution of an initial
discrepancy. Mutatations in brief no. 186. Online.

Virtaneva K, Paulin L, Krahe R, de la Chapelle A, Lehesjoki AE.

Department of Medical Genetics, University of Helsinki and the Folkhalsan
Institute of Genetics, Helsinki, Finland.

Mutations in the cystatin B (CSTB) gene underlie progressive myoclonus epilepsy
of Unverricht-Lundborg type (EPM1) (Pennacchio et al., 1996). We previously
described an unstable minisatellite expansion mutation in the putative promoter
region of CSTB that accounts for the majority of EPM1 patients. Sequencing of a
genomic lambda clone, generated from a Finnish EPM1 patient homozygous for an
enlarged restriction fragment, revealed a 15- to 18-mer minisatellite repeat
expansion (Virtaneva et al., 1997). Later, sequencing of plasmid clones generated
from Swiss and French patients revealed a dodecamer repeat expansion (Lalioti et 
al., 1997a). By restriction enzyme analysis of our original patient clone and a
clone generated from an Italian patient, we now show that the expansion is
neither a 15-mer nor an 18-mer contrary to our initial results. Moreover, direct 
sequencing of the Finnish patient clone with Pfu exo polymerase confirmed that
the expanded repeat is a dodecamer. Based on this finding and additional
experiments, we suggest that the discrepancy between the two studies was due to
errors caused by the combination of native Pfu polymerase and modified guanosine 
deaza-7-dGTP used in the PCR reaction.

PMID: 10660338 [PubMed - indexed for MEDLINE]


438. Pancreas. 2000 Jan;20(1):47-54.

Rel B is an early marker of autoimmune islet inflammation in the biobreeding (BB)
rat.

Bieg S, Simonson W, Ellefsen K, Lernmark A.

Robert H. Williams Laboratory, Department of Medicine, University of Washington, 
Seattle, USA. bieg@mail.uni-mainz.de

Because the development of insulitis and diabetes is predictable in Lyp/Lyp
congenic BB rats, we have characterized early islet inflammation in these rats to
determine the cell subsets involved in the onset of autoimmune insulitis.
Pancreas sections from prediabetic Lyp/Lyp, Lyp/+ and +/+ rats were analyzed by
immunohistochemistry. We found W3/25+ cells in the exo- and endocrine tissue from
all three genotypes, but intraislet insulitis was never found in Lyp/+ or +/+
rats. The onset of massive, intraislet B- and T-cell infiltration in Lyp/Lyp rats
was preceded by Rel B+ cells in and around the islets, followed by ED1+
monocytes/macrophages. Rel B+ cells were more frequent in the parafollicular
cortex of pancreatic lymph nodes from Lyp/Lyp than from Lyp/+ and +/+ rats. In
the Lyp/Lyp thymus, we found significantly increased expression of IL-12p40
messenger RNA (mRNA; p<0.001), located in the Rel B-protein-rich corticomedullary
junction. The NF-KB/Rel B complex specifically transactivates genes involved in
antigen presentation in dendritic cells. Rel B+ cells in the islets may therefore
mark the onset of autoimmune insulitis and antigen-specific activation of
autoreactive T cells in the lymph nodes of diabetes prone Lyp/Lyp BB rats. In the
thymus, Rel B+ cells may support the Lyp-dependent development of self-reactive
thymocytes by activation of cytokine expression.

PMID: 10630383 [PubMed - indexed for MEDLINE]


439. J Cell Biol. 1999 Dec 13;147(6):1249-60.

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at 
the nerve terminal.

Neale EA, Bowers LM, Jia M, Bateman KE, Williamson LC.

Laboratory of Developmental Neurobiology, National Institute of Child Health and 
Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. 
eneale@codon.nih.gov

The supply of synaptic vesicles in the nerve terminal is maintained by a
temporally linked balance of exo- and endocytosis. Tetanus and botulinum
neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins 
identified as critical for synaptic vesicle exocytosis. We show here that
botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis
does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell
cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter 
release nor synaptic currents can be detected, twice the ordinary number of
synaptic vesicles are docked at the synaptic active zone, and its protein
substrate is cleaved, which is similar to observations with tetanus and other
botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence
of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum
neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic
terminals and uptake of HRP into synaptic vesicles. These experiments are the
first demonstration that botulinum neurotoxin A uncouples vesicle exo- from
endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle
membrane retrieval.

PMCID: PMC2168097
PMID: 10601338 [PubMed - indexed for MEDLINE]


440. Biosci Biotechnol Biochem. 1999 Sep;63(9):1582-8.

Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var.
dextranlyticum strain 0407 and its expression in Escherichia coli.

Mizuno T, Mori H, Ito H, Matsui H, Kimura A, Chiba S.

Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido
University, Sapporo, Japan.

The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT,
was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum 
strain 0407, and expressed in Escherichia coli. A single open reading frame
consisting of 1923 base pairs that encoded a polypeptide composed of a signal
peptide of 37 amino acids and a mature protein of 604 amino acids (M(r), 68,300) 
was found. The primary structure had no significant similarity with the structure
of two other reported exo-type dextranases (glucodextranase and
isomalto-dextranase), but had high similarity with that of an endo-dextranase
isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene
encoding mature protein of IMTD overproduced IMTD under the control of the T7
phage promoter induced by IPTG. The purified recombinant enzyme showed the same
optimum pH, lower specific activity, and similar hydrolytic pattern, as to those 
of native IMTD.

PMID: 10540747 [PubMed - indexed for MEDLINE]


441. J Agric Food Chem. 1999 Apr;47(4):1530-2.

Autolysis of lentinan, an antitumor polysaccharide, during storage of Lentinus
edodes, shiitake mushroom.

Minato K, Mizuno M, Terai H, Tsuchida H.

Division of Science of Biological Resources, Graduate School of Science and
Technology, Kobe University, Japan.

The lentinan contents in the Lentinus edodes fruit body during storage were
examined by ELISA method using anti-lentinan antibodies. The lentinan content
(12.8 mg.g(-)(1) dw) before storage decreased to 3.7 mg.g(-)(1) dw over 7 days at
20 degrees C. However, it only slightly decreased at 1 degrees C and only
decreased to 9.3 mg. g(-)(1) dw at 5 degrees C. Glucanase activity, which seems
to be associated with lentinan degradation, increased more during storage of L.
edodes at 20 degrees C than it did at lower temperatures. In addition, only
glucose was detected as a degraded product from lentinan by the glucanase. This
suggested that this enzyme would fit the profile of an exo-type glucanase. Also, 
polyphenol oxidase activity, known as an index of freshness reduction in the
mushroom, increased approximately 2.7-fold (to 61.5 units.mg(-)(1)) over 7 days
during storage at 20 degrees C. However, its activity changed little during
storage at lower temperatures. These results indicate that the reduction during
storage of the quality of L. edodes as a functional food is accompanied by the
decrease of lentinan, and by browning, and that exo-glucanase plays an important 
role in the decrease of lentinan content.

PMID: 10564011 [PubMed - indexed for MEDLINE]


442. Biochim Biophys Acta. 1999 Nov 16;1472(3):447-54.

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda.
Activation, inactivation and the kinetic properties of the enzyme.

Yoshinaga K, Fujisue M, Abe J, Hanashiro I, Takeda Y, Muroya K, Hizukuri S.

The United Graduate School of Agricultural Sciences, Kagoshima University,
Korimoto 1-21-4, Kagoshima, Japan.

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria
chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-)
ions. The ions also stabilized the enzyme increasing the temperature of its
maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by
chemical modification with carbodiimide and a carboxyl group of the enzyme was
shown essential to the lyase activity. A tryptophanyl residue(s) was also shown
to be important for the activity and was probably involved in substrate binding. 
K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1
mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0)
values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these
kinetic parameters showed that the enzyme has four subsites to accommodate
oligosaccharides. The subsite map of GLase was unique, since subsite 1 and
subsite 2 have large positive and small negative affinities, respectively. The
subsite map of this type has not been found in other enzymes with exo-action on
alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower
(0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of
another affinity site specific to the polysaccharides.

PMID: 10564758 [PubMed - indexed for MEDLINE]


443. Glycobiology. 1999 Dec;9(12):1295-305.

Sialoforms of dipeptidylpeptidase IV from rat kidney and liver.

Schmauser B, Kilian C, Reutter W, Tauber R.

Institut fur Molekularbiologie und Biochemie der Freien Universitat Berlin,
Arnimallee 22, D-14195 Berlin-Dahlem, Germany.

Dipeptidylpeptidase IV (DPP IV, CD26), a serine-type exo- and endopeptidase found
in the cell surface membrane of many tissues, was employed as a model membrane
glycoprotein to study the expression of sialoforms on cell surface glycoproteins.
Native, enzymatically active DPP IV was purified from plasma membranes of kidney 
and liver by lectin affinity chromatography in conjunction with crown ether anion
exchange chromatography. The enzyme was gradient-eluted in continuous fractions, 
all showing a single polypeptide band of about 100 kDa when separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing,
denaturing conditions. Analysis of the purified DPP IV by isoelectric focusing
(IEF) showed that it consists of several polypeptides of different isoelectric
points (IP) ranging from 5.5 to 7.0. In vitro- desialylation of the enzyme and
subsequent isoelectric focusing revealed that the differences in isoelectric
points were due to differences in the degree of sialylation. Differences in the
degree of sialylation between the fractions were also demonstrated by SDS-PAGE
under nonreducing and nondenaturing conditions. Increased sialylation of the
enzyme as demonstrated by isoelectric focusing resulted in increased migration
velocity in nonreducing and nondenaturing SDS-polyacrylamide gels. In vitro
-desialylation of the enzyme and its resialylation confirmed that sialylation was
responsible for this extraordinary migration behavior. The native enzyme was
predominantly sialylated via alpha 2, 6-linkage, as shown by lectin affinity
blotting employing Sambucus nigra agglutinin (SNA) and Maackia amurensis
agglutinin (MAA). These findings demonstrate that a distinct membrane
glycoprotein may exist in various sialoforms, distinguished from each other by a 
different number of sialic acid residues. Moreover, these sialoforms can be
individually purified by crown ether anion exchange chromatography.

PMID: 10561454 [PubMed - indexed for MEDLINE]


444. Biochemistry. 1999 Oct 12;38(41):13717-24.

Preparation and characterization of DNA containing a site-specific nonadjacent
cyclobutane thymine dimer of the type implicated in UV-induced -1 frameshift
mutagenesis.

Lingbeck JM, Taylor JS.

Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.

One mechanism for the origin of UV-induced -1 deletion mutations involves the
bypass of a nonadjacent cis-syn cyclobutane pyrimidine dimer containing a single 
intervening nucleotide. To begin to investigate this mechanism, we required a
method for obtaining a single, site-specific, nonadjacent dimer. One approach to 
the preparation of a nonadjacent dimer is to irradiate a DNA duplex containing a 
centrally located TNT sequence in which the two T's are paired to an AA sequence 
in an otherwise fully complementary strand. Triplet-sensitized irradiation of the
duplex formed between the 13-mer d(GAGTATCTATGAG) and the 12-mer d(CTCATAATACTC) 
on ice gave a major product that could be reverted to the parent 13-mer by 254 nm
irradiation. Proton NMR experiments established the major product to be the
nonadjacent cis-syn cyclobutane dimer formed between the two T's of the TCT
sequence. Melting temperature studies show that the nonadjacent dimer is more
destabilizing to DNA duplex structure than a normal cis-syn dimer and is as
stable as the parental bulged DNA duplex. The nonadjacent dimer-containing 13-mer
was ligated into a 51-mer and used as a template for primer-extension studies by 
DNA polymerases. The nonadjacent dimer could not be bypassed by Sequenase Version
2.0 and terminated synthesis primarily prior to and opposite the 3'-T of the
dimer. In contrast, approximately 30% of the dimer was bypassed by an
exonuclease-deficient (exo-) Klenow fragment, and termination occurred primarily 
opposite the 3'- and 5'-T's of the dimer. Bypass of the nonadjacent dimer by
exo(-) Klenow fragment led primarily to a single-nucleotide deletion mutation as 
well as small amounts of a full-length product and a four-nucleotide deletion
that could be explained by a primer misalignment mechanism.

PMID: 10521279 [PubMed - indexed for MEDLINE]


445. Eur J Biochem. 1999 Nov;265(3):929-35.

Mutations of glutamate-84 at the putative potassium-binding site affect camphor
binding and oxidation by cytochrome p450cam.

Westlake AC, Harford-Cross CF, Donovan J, Wong LL.

Department of Chemistry, Inorganic Chemistry Laboratory, Oxford, UK.

Cytochrome P450cam (CYP101) from Pseudomonas putida is unusual among P450 enzymes
in that it exhibits co-operative binding between the substrate camphor and a
potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a
surface residue which forms part of the cation-binding site. Substitutions that
neutralize or reverse the charge of this side chain are shown to disrupt the
co-operativity of potassium and camphor binding by P450cam, and also to influence
the catalytic activity. In particular, replacement of Glu84 by positively charged
residues such as lysine results in increased high-spin haem fractions and camphor
turnover activities in the absence of potassium, along with decreased camphor
dissociation constants. However, in the presence of potassium the camphor
dissociation constants of these mutants are significantly increased compared with
the wild-type, although the camphor turnover activities remain marginally higher.
In contrast, substitution by aspartate results in tighter binding of both
potassium and camphor, but has little effect on the enzymatic activity. In all
cases the reaction remains essentially 100% coupled and gives
5-exo-hydroxycamphor as the only product. These results suggest that an anionic
side chain at the 84 position is crucial for the co-operativity of camphor and
cation binding, and that the physiological role for potassium binding by
cytochrome P450cam is to promote camphor binding even at the expense of turnover 
rate, thus allowing the organism to utilize low environmental concentrations of
this substrate for growth.

PMID: 10518786 [PubMed - indexed for MEDLINE]


446. Farmaco. 1999 Aug 30;54(8):497-516.

Bioconjugation in pharmaceutical chemistry.

Veronese FM, Morpurgo M.

Department of Pharmaceutical Sciences, University of Padua, Italy.
veronese@pdfar3.dsfarm.unipd.it

Polymer conjugation is of increasing interest in pharmaceutical chemistry for
delivering drugs of simple structure or complex compounds such peptides, enzymes 
and oligonucleotides. For long time drugs, mainly with antitumoral activity, have
been coupled to natural or synthetic polymers with the purpose of increasing
their blood permanence time, taking advantage of the increased mass that reduces 
kidney ultrafiltration. However only recently complex constructs were devised
that exploit the 'enhanced permeability and retention' (EPR) effect for an
efficient tumor targeting, the high molecular weight for adsorption or receptor
mediated endocytosis and finally a lysosomotropic targeting, taking advantage of 
acid labile bonds or cathepsin susceptible polypeptide spacers between polymer
and drug. New original, very active conjugates of this type, as those based on
poly(hydroxyacrylate) polymers, are already in advanced state of development.
Labile oligonucleotides, including antisense drugs, were also successfully
coupled to polymers in view of an increased cell penetration and stabilization
towards nucleases. However, the most active research activity resides in the
field of polypeptides and proteins delivery, mainly for the two following
reasons: first of all because a great number of therapeutically interesting
compounds are now being produced by genetic engineering in large quantity and,
secondly, because these products are difficult to administer to patients for
several inherent drawbacks. Proteins are in fact easily digested by many endo-
and exo-peptidases present in blood or in other body districts; most of them are 
immunogenic to some extent and, finally, they are rapidly excreted by kidney
ultrafiltration. Covalent polymer conjugation at protein surface was demonstrated
to reduce or eliminate these problems, since the bound polymer behaves like a
shield hindering the approach of proteolytic enzymes, antibodies, or antigen
processing cell. Furthermore, the increase of the molecular weight of the
conjugate allows to overcome the kidney elimination threshold. Many successful
results were already obtained in peptides and proteins, conjugated mainly to
water soluble or amphiphilic polymers like poly(ethylene glycol) (PEG), dextrans,
or styrenemaleic acid anhydride. Among the most successful are the conjugates of 
asparaginase, interleukin-2 or -6 and neocarcinostatin, to remind some antitumor 
agents, adenosine deaminase employed in a genetic desease treatment, superoxide
dismutase as scavenger of toxic radicals, hemoglobin as oxygen carrier and
urokinase and streptokinase as proteins with antithrombotic activity. In
pharmaceutical chemistry the conjugation with polymers is also of great
importance for synthetic applications since many enzymes without loss of
catalytic activity become soluble in organic solvents where many drug precursors 
are. The various and often difficult chemical problems encountered in conjugation
of so many different products prompted the development of many synthetic
procedures, all characterized by high specificity and mild condition of reaction,
now known as 'bioconjugation chemistry'. Bioconjugation developed also the design
of new tailor-made polymers with the wanted molecular weight, shape, structure
and with the functional groups needed for coupling at the wanted positions in the
chain.

PMID: 10510847 [PubMed - indexed for MEDLINE]


447. Carcinogenesis. 1999 Oct;20(10):1971-7.

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B(1) in
human lung: differential cellular distribution and lack of significance of the
GSTM1 genetic polymorphism.

Stewart RK, Smith GB, Donnelly PJ, Reid KR, Petsikas D, Conlan AA, Massey TE.

Department of Pharmacology, Queen's University, Kingston, Ontario K7L 3N6,
Canada.

Epidemiological studies suggest that aflatoxin B(1) (AFB(1)), a mycotoxin
produced by certain Aspergillus species, may play a role in human respiratory
cancers in occupationally-exposed individuals. AFB(1) requires bioactivation to
the corresponding exo-8,9-epoxide for carcinogenicity, and glutathione
S-transferase (GST)-catalyzed conjugation of the epoxide with glutathione (GSH)
is a critical determinant of susceptibility to AFB(1). Of the purified human GST 
enzymes studied, the polymorphic hGSTM1-1 has the highest activity towards AFB(1)
exo-epoxide. The influence of the GSTM1 polymorphism on AFB(1)-GSH formation, as 
well as the abilities of cytosols from preparations enriched in different
isolated lung cell types to conjugate AFB(1)-epoxides, were examined. In
whole-lung cytosols from patients undergoing clinically indicated lobectomy,
GSTM1 genotype correlated with GSTM1 phenotype as determined by
[(3)H]trans-stilbene oxide conjugation: GSTM1-positive = 295 +/- 31 pmol/mg/h (n 
= 6); GSTM1-negative = 92.8 +/- 23.3 pmol/mg/h (n = 4) (P < 0.05). In contrast,
conjugation of microsome-generated [(3)H]AFB(1)-epoxides with GSH was low and
variable between patients, and did not correlate with GSTM1 genotype:
GSTM1-positive = 11.9 +/- 8.1, 111 +/- 66 and 510 +/- 248 fmol/mg/h (n = 6);
GSTM1-negative = 15.3 +/- 16.7, 167 +/- 225 and 540 +/- 618 fmol/mg/h (n = 4)
(for 1, 10 and 100 microM [(3)H]AFB(1), respectively). GSH conjugates of AFB(1)
exo-epoxide and the much less mutagenic stereoisomer AFB(1) endo-epoxide were
produced in a ratio of approximately 1:1 in cytosols from both whole lung and
isolated cells. Total cytosolic AFB(1)-epoxide conjugation was significantly
higher in fractions enriched in alveolar type II cells (3.07 +/- 1.61 pmol/mg/h) 
than in unseparated lung cells (0.143 +/- 0.055 pmol/mg/h) or fractions enriched 
in alveolar macrophages (0. 904 +/- 0.319 pmol/mg/h; n = 4) (P < 0.05).
Furthermore, AFB(1)-GSH formation and percentage of alveolar type II cells in
different cell fractions were correlated (r = 0.78, P < 0.05). These results
demonstrate that human lung GSTs exhibit very low conjugation activity for both
AFB(1)-8,9-epoxide stereoisomers, and that this activity is heterogeneously
distributed among cell types, with alveolar type II cells exhibiting relatively
high activity. Of the GSTs present in human peripheral lung which contribute to
AFB(1) exo- and endo-epoxide detoxification, hGSTM1-1 appears to play at most
only a minor role.

PMID: 10506113 [PubMed - indexed for MEDLINE]


448. J Biol Chem. 1999 Sep 24;274(39):27481-90.

Replication slippage of different DNA polymerases is inversely related to their
strand displacement efficiency.

Canceill D, Viguera E, Ehrlich SD.

Laboratoire de Genetique Microbienne, Institut National de la Recherche
Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.
canceill@biotec.jouy.inra.fr

Replication slippage is a particular type of error caused by DNA polymerases
believed to occur both in bacterial and eukaryotic cells. Previous studies have
shown that deletion events can occur in Escherichia coli by replication slippage 
between short duplications and that the main E. coli polymerase, DNA polymerase
III holoenzyme is prone to such slippage. In this work, we present evidence that 
the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II,
as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo
slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not.
Furthermore, we have measured the strand displacement activity of the different
polymerases tested for slippage in the absence and in the presence of the E. coli
single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases
having a strong strand displacement activity cannot slip (DNA polymerase from
Phi29); (ii) polymerases devoid of any strand displacement activity slip very
efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand
displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB
(T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA
polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of
slippage. We propose that these observations can be interpreted in terms of a
model, for which we have shown that high strand displacement activity of a
polymerase diminishes its propensity to slip.

PMID: 10488082 [PubMed - indexed for MEDLINE]


449. Mol Gen Genet. 1999 Jul;261(6):1032-44.

The eff-482 locus of Sinorhizobium meliloti CXM1-105 that influences symbiotic
effectiveness consists of three genes encoding an endoglycanase, a
transcriptional regulator and an adenylate cyclase.

Sharypova LA, Yurgel SN, Keller M, Simarov BV, Puhler A, Becker A.

All-Russia Research Institute for Agricultural Microbiology, St.
Petersburg-Pushkin.

The mutant T482 of Sinorhizobium meliloti CXM1-105, which carries a Tn5 insertion
on megaplasmid 1, exhibits an enhanced symbiotic efficiency phenotype. Three
genes, eglC, cya3 and syrB2, were identified in the eff-482 region tagged by the 
Tn5 insertion in T482. The eglC gene encodes an endoglycanase which contributes
to the depolymerization of the exo-polysaccharide succinoglycan. The N-terminal
region of the predicted cya3 gene product was similar to eukaryotic-type
adenylate cyclases from Brevibacterium liquefaciens and Streptomyces coelicolor. 
Four contiguous tetratricopeptide repeats which are known to mediate
protein-protein interactions were identified in the C-terminal portion of Cya3.
Complementation analysis demonstrated that cya3 indeed encodes a functional
adenylate cyclase. A central helix-turn-helix DNA-binding motif and a putative
C-terminal coiled-coil structure implicated in protein oligomerization were found
in SyrB2. Extra copies of the syrB2 gene negatively affect transcription of both 
syrB2 itself and cya3. The Tn5 insertion in T482 was localized between the
divergently transcribed genes eglC and syrB2. It eliminated eglC function and
slightly stimulated transcription of both syrB2 and cya3, which lies downstream
of syrB2. Mutants carrying insertions of the lacZ-Gm interposon in the genes
eglC, syrB2 and cya3 exhibit the same phenotype as mutant T482, indicating that
these three genes influence symbiotic efficiency.

PMID: 10485295 [PubMed - indexed for MEDLINE]


450. J Biochem. 1999 Sep;126(3):559-65.

Crystal structure of prolyl aminopeptidase from Serratia marcescens.

Yoshimoto T, Kabashima T, Uchikawa K, Inoue T, Tanaka N, Nakamura KT, Tsuru M,
Ito K.

School of Pharmaceutical Sciences, Nagasaki University, Nagasaki, 852-8521,
Japan. t-yoshimoto@cc.nagasaki-u.ac.jp

Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal
of N-terminal proline residues from peptides. We have solved its
three-dimensional structure at 2.3 A resolution by the multiple isomorphous
replacement method. The enzyme consists of two contiguous domains. The larger
domain shows the general topology of the alpha/beta hydrolase fold, with a
central eight-stranded beta-sheet and six helices. The smaller domain consists of
six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the
large cavity at the interface between the two domains. Cys271, which is sensitive
to SH reagents, is located near the catalytic residues, in spite of the fact that
the enzyme is a serine peptidase. The specific residues which make up the
hydrophobic pocket line the smaller domain, and the specificity of the exo-type
enzyme originates from this smaller domain, which blocks the N-terminal of P1
proline.

PMID: 10467172 [PubMed - indexed for MEDLINE]


451. J Mol Biol. 1999 Aug 27;291(4):877-98.

High resolution crystal structures of the Escherichia coli lytic transglycosylase
Slt70 and its complex with a peptidoglycan fragment.

van Asselt EJ, Thunnissen AM, Dijkstra BW.

University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands.

The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an
exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N
-acetylmuramic acid and N -acetylglucosamine residues in peptidoglycan, the main 
structural component of the bacterial cell wall. This cleavage is accompanied by 
the formation of a 1,6-anhydro bond between the C1 and O6 atoms in the N
-acetylmuramic acid residue (anhMurNAc). Crystallographic studies at medium
resolution revealed that Slt70 is a multi-domain protein consisting of a large
ring-shaped alpha-superhelix with on top a catalytic domain, which resembles the 
fold of goose-type lysozyme. Here we report the crystal structures of native
Slt70 and of its complex with a 1,6-anhydromuropeptide solved at nominal
resolutions of 1.65 A and 1.90 A, respectively. The high resolution native
structure reveals the details on the hydrogen bonds, electrostatic and
hydrophobic interactions that stabilise the catalytic domain and the
alpha-superhelix. The building-block of the alpha-superhelix is an
"up-down-up-down" four-alpha-helix bundle involving both parallel and
antiparallel helix pairs. Stabilisation of the fold is provided through an
extensive packing of apolar atoms, mostly from leucine and alanine residues. It
lacks, however, an internal consensus sequence that characterises other
super-secondary helical folds like the beta-helix in pectate lyase or the
(beta-alpha)-helix in the ribonuclease inhibitor. The 1, 6-anhydromuropeptide
product binds in a shallow groove adjacent to the peptidoglycan-binding groove of
the catalytic domain. The groove is formed by conserved residues at the interface
of the catalytic domain and the alpha-superhelix. The structure of the Slt70-1,
6-anhydromuropeptide complex confirms the presence of a specific binding-site for
the peptide moieties of the peptidoglycan and it substantiates the notion that
Slt70 starts the cleavage reaction at the anhMurNAc end of the peptidoglycan.
Copyright 1999 Academic Press.

PMID: 10452894 [PubMed - indexed for MEDLINE]


452. Microbiology. 1999 Jul;145 ( Pt 7):1649-59.

The Shigella flexneri bacteriophage Sf6 tailspike protein (TSP)/endorhamnosidase 
is related to the bacteriophage P22 TSP and has a motif common to exo- and
endoglycanases, and C-5 epimerases.

Chua JE, Manning PA, Morona R.

Department of Microbiology and Immunology, University of Adelaide, South
Australia.

The temperate bacteriophage Sf6 infects Shigella flexneri strains of serotype X
or Y, converting them into serotypes 3a or 3b, respectively. The tailspike
protein (TSP) of Sf6 possesses endo-1,3-alpha-L-rhamnosidase (endorhamnosidase)
activity which results in cleavage of the lipopolysaccharide O-antigen receptor
during the adsorption of the phage to the cell surface. When used in Southern
hybridization, a P22 gene 9 (encoding P22 TSP) DNA probe hybridized with
restriction fragment Pstl-7 of Sf6. DNA sequencing and analysis of Pstl-7 and the
adjacent Pstl-8 fragment revealed an open reading frame (ORF1) of 1872 bp (624
amino acids) bearing amino acid sequence homology to the bacteriophage P22 TSP
N-terminal head-binding domain. High conservation of key residues was suggestive 
of similar secondary and tertiary N-terminal protein structure and a similar
function of the Sf6 TSP in this region. In addition, an amino acid sequence motif
(DFGX3DGX6AX3A) was identified between residues 164 and 184 which was also found 
to exist in various prokaryotic and eukaryotic exo-/endoglycanases, C-5
epimerases and bacteriophage proteins. Expression of ORF1 from a T7 promoter
produced a 67 kDa protein (detected by L-[35S]methionine labelling and SDS-PAGE).
Assay of heat-treated cytoplasmic extracts containing the ORF1-encoded protein by
incubation with whole Sh. flexneri Y cells demonstrated that O-antigen hydrolysis
activity was present; ORF1 therefore encodes Sf6 TSP. Sf6 TSP exhibited specific 
and preferential activity for long-chain Sh. flexneri serotype X or Y O-antigen, 
cleavage of which resulted in the release of oligosaccharide fragments,
consistent with octasaccharides in size, as detected by fluorophore-assisted
carbohydrate electrophoresis (FACE).

PMID: 10439404 [PubMed - indexed for MEDLINE]


453. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8455-60.

The kinetoplast structure-specific endonuclease I is related to the 5'
exo/endonuclease domain of bacterial DNA polymerase I and colocalizes with the
kinetoplast topoisomerase II and DNA polymerase beta during replication.

Engel ML, Ray DS.

Molecular Biology Institute, Cell and Developmental Biology, University of
California, Los Angeles, Los Angeles, CA 90095-1570, USA.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia
fasciculata has an unusual structure composed of minicircles and maxicircles
topologically interlocked into a single network and organized in a disc-shaped
structure at the base of the flagellum. We previously purified a
structure-specific endonuclease (SSE1), based on its RNase H activity, that is
enriched in isolated kinetoplasts. The endonuclease gene has now been cloned,
sequenced, and found to be closely related to the 5' exonuclease domain of
bacterial DNA polymerase I proteins. Although the protein does not contain a
typical mitochondrial leader sequence, the enzyme is shown to colocalize with a
type II DNA topoisomerase and a DNA polymerase beta at antipodal sites flanking
the kinetoplast disc. Cell synchronization studies with an epitope-tagged
construct show that the localization of the endonuclease to the antipodal sites
varies in a cell cycle-dependent manner similar to that of the DNA polymerase
beta [Johnson, C. E. & Englund, P. T. (1998) J. Cell Biol. 143, 911-919].
Immunofluorescent localization of SSE1 to the antipodal sites is only observed
during kinetoplast replication. Together, these results suggest a point of
control for kinetoplast DNA replication through the regulation of the
availability of DNA replication proteins and a possible role for the antipodal
sites in removal of RNA primers and the repair of gaps in newly replicated
minicircles.

PMCID: PMC17537
PMID: 10411896 [PubMed - indexed for MEDLINE]


454. Microbiology. 1999 May;145 ( Pt 5):1089-95.

A unique eukaryotic beta-xylosidase gene from the phytopathogenic fungus
Cochliobolus carbonum.

Wegener S, Ransom RF, Walton JD.

Department of Energy, Michigan State University, East Lansing 48824, USA.

The plant-pathogenic fungus Cochliobolus carbonum secretes one major
beta-xylosidase (Xyp1) when grown on xylan or maize cell walls. cDNA and genomic 
DNA encoding Xyp1 were isolated using PCR primers based on peptide sequences from
the purified protein. XYP1 contains three introns, has 5' and 3' untranslated
regions of 74 and 145 bp, respectively, and is predicted to encode a protein of
328 amino acids (Mr 36700) with four N-glycosylation sites. Although it is
secreted, Xyp1 has no predicted signal peptide. Furthermore, Xyp1 appears not to 
be processed at the N-terminus because one of the peptides isolated from the
mature protein is located only six amino acids downstream of the translational
start methionine. The primary sequence of Xyp1 is unrelated to any known
eukaryotic beta-xylosidase but has 35% overall identity to two bacterial
bifunctional beta-xylosidase/alpha-arabinosidases. Mutation of XYP1 by targeted
gene replacement resulted in the loss of the major beta-xylosidase activity
corresponding to the product of XYP1, but a significant amount of secreted
beta-xylosidase activity (25% of wild-type) remained in the culture filtrates.
The xyp1 mutant was still fully pathogenic on maize.

PMID: 10376824 [PubMed - indexed for MEDLINE]


455. J Am Soc Mass Spectrom. 1999 Jun;10(6):521-8.

Identification of single stranded regions of DNA by enzymatic digestion with
matrix-assisted laser desorption/ionization analysis.

Bartolini WP, Bentzley CM, Johnston MV, Larsen BS.

Department of Chemistry and Biochemistry, University of Delaware, Newark 19716,
USA.

Elucidating structure function relationships of DNA in cellular processes
requires fast, reliable methods that can be applied to picomole amounts of
sample. Higher order structure can be inferred by distinguishing paired and
unpaired regions. It is shown here that enzymatic digestion coupled with product 
analysis by matrix-assisted laser desorption ionization (MALDI) is able to
identify unpaired bases within structured DNA regions. The method is demonstrated
with DNA duplexes having a five nucleotide mismatch as a 5' overhang, a 3'
overhang, and an internal loop. Exo- and endonuclease digestions are performed
under solution conditions (temperature, annealing, and enzyme buffers) which
promote base pairing and specific enzyme activity. For each type of mismatch, the
length and sequence of the single stranded region can be inferred from MALDI
spectra taken as a function of digestion time.

PMID: 10368947 [PubMed - indexed for MEDLINE]


456. Med Mycol. 1999 Apr;37(2):123-9.

Humoral and cellular immune response to a crude exo-antigen and purified
keratinase of Microsporum canis in experimentally infected guinea pigs.

Mignon BR, Leclipteux T, Focant C, Nikkels AJ, Pierard GE, Losson BJ.

Faculty of Veterinary Medicine, Department of Parasitology and Parasitic
Diseases, University of Liege, Liege, Belgium. bmignon@ulg.ac.be

In order to understand better the host-parasite relationship and to compare with 
previous observations in Microsporum canis naturally infected cats, the humoral
and cellular immune responses to both a crude exo-antigen and a 31.5 kDa purified
keratinase were evaluated in 12 M. canis experimentally infected guinea pigs.
Humoral and cellular responses were assessed by ELISA from days 0 to 56
postinfection (PI) and by measurement of delayed-type hypersensitivity (DTH)
responses on days 14 and 57 PI, respectively. Additionally, immunohistochemical
staining was performed and demonstrated that the keratinase was produced in
infected guinea pig skin, as previously reported in cats. Despite a marked
interindividual variation, all the guinea pigs produced specific IgG to the crude
exo-antigen from day 21 PI onwards, but no anti-keratinase IgG was detected.
Strongly positive DTH responses to the exo-antigen were observed on both dates,
whereas the keratinase elicited no and weak DTH on days 14 and 57 PI,
respectively. These results are in agreement with those previously described for 
naturally infected cats, and indicate that the 31.5 kDa keratinase is not a major
antigen in M. canis infection.

PMID: 10361268 [PubMed - indexed for MEDLINE]


457. J Biomol Struct Dyn. 1999 Apr;16(5):1075-85.

NMR structures of a nonapeptide from DNA binding domain of human polymerase-alpha
determined by iterative complete-relaxation-matrix approach.

Bose RN, Li D, Yang WW, Basu S.

Department of Chemistry, Kent State University, OH 44242, USA.
rbose@platinum.kent.edu

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from 
the DNA binding domain of human polymerase-alpha, were determined by complete
relaxation matrix analysis of transverse NOE data. The structures exhibit a type 
III turn with residues KCPC, and the remaining residues exhibit non-ordered
structures. The turn was confirmed by alpha, N (i,i+3) connectivity, a low
temperature coefficient of NH chemical shift (-3.1 x 10(-3)) of the fourth
residue, 3J(NHalpha) coupling constants, and characteristic CD peaks at 228 and
200 nm. Furthermore, phi and psi dihedral angles for the i + 1, and i + 2
residues of the turn are found to be -80 and -41 and -60 and -40 degrees. The
first proline residue is trans- while the second exists in both cis- and trans-
configurations, with trans- being more than 80% populated. The
trans-configuration was established from C5alpha-P6alpha correlation and phi and 
psi angles of the proline. The five-membered proline ring is in DOWN puckered
(C-beta-exo/C-gamma-endo) conformation. The structure of the peptide reveals that
the two cysteine thiols are approximately 5 A(o) apart and appropriately
positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used
anti-cancer drug.

PMID: 10333177 [PubMed - indexed for MEDLINE]


458. Bioorg Med Chem. 1999 Mar;7(3):467-79.

Synthesis of a fluorine-18 labeled derivative of epibatidine for in vivo
nicotinic acetylcholine receptor PET imaging.

Dolci L, Dolle F, Valette H, Vaufrey F, Fuseau C, Bottlaender M, Crouzel C.

Service Hospitalier Frederic Joliot, Departement de Recherche Medicale, CEA,
Orsay, France.

Epibatidine (exo-2-(2'-chloro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural 
compound isolated from the skin of the Ecuadorian poison frog Epipedobates
tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist
reported to date. In order to visualize and quantify in vivo these receptors in
human brain using Positron Emission Tomography (PET),
[18F]norchlorofluoroepibatidine
(exo-2-(2'-[18F]fluoro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a fluorine-18
(t(1/2): 110 min) radiolabeled derivative of epibatidine has been designed. The
corresponding 2'-bromo-, 2'-iodo- and 2'-nitro
exo-2-(5'-pyridyl)-7-azabicyclo[2.2.1]heptane analogues as labeling precursors,
as well as norchlorofluoroepibatidine as a reference compound have been
synthesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling
between an N-Boc protected azanorbornene and the corresponding halopyridine.
[18F]Norchlorofluoroepibatidine has been radiolabeled with fluorine-18 by
nucleophilic aromatic substitution from the corresponding Boc-protected halo- and
nitro precursors using [18F]FK-K222 complex in DMSO by conventional heating (at
150-180 degrees C for 10 min) or microwave activations (at 100 Watt, for 1 to 2.5
min), followed by TFA-removal of the protective group. Typically, using the
microwave activation procedure, 60-80 mCi (2.22-2.96 GBq) of pure
[18F]norchlorofluoroepibatidine could be obtained in less than 2 h (110-115 min) 
from the bromo labeling precursor, with specific radioactivities of 1.5-2.5
Ci/micromol (55.5-92.5 GBq/micromol) calculated for End of Bombardment. The
preliminary PET experiments in baboon (Papio papio) with
[18F]norchlorofluoroepibatidine show a high uptake and a rapid accumulation of
the radiotracer into the brain within 30 min. In the thalamus, a nAChR rich area,
uptake of radioactivity reached a maximum at 40 min (10% I.D./100 mL tissue). The
ratio of radioactivity thalamus/cerebellum (the latter being a nAChR poor area)
was 2 at 40 min and increased with time, up to 4.3 at 160 min. Its specific
regiodistribution and its high ratio of specific-to-nonspecific binding confirm
the ideal profile of [18F]norchlorofluoroepibatidine as a suitable radioligand
for PET imaging of nAChRs in the brain.

PMID: 10220033 [PubMed - indexed for MEDLINE]


459. Glycobiology. 1999 May;9(5):469-79.

Sequential deglycosylation and utilization of the N-linked, complex-type glycans 
of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis.

Byers HL, Tarelli E, Homer KA, Beighton D.

Joint Microbiology Research Unit, Faculty of Clinical Dentistry, King's College
School of Medicine and Dentistry, Caldecot Road, London SE5 9RW, UK.

Streptococcus oralis is the agent of a large number of infections in
immunocompromised patients, but little is known regarding the mechanisms by which
this fermentative organism proliferates in vivo. Glycoproteins are widespread
within the circulation and host tissues, and could provide a source of
fermentable carbohydrate for the growth of those pathogenic organisms with the
capacity to release monosaccharides from glycans via the production of specific
glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to
support growth of S.oralis in vitro has been examined as a model for growth of
this organism on N-linked glycoproteins. Growth was accompanied by the production
of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and
beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked
substrates. The residual glycoprotein glycans remaining during growth of this
organism were released by treatment with hydrazine and their analysis by
HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with 
only terminal N-acetylglucosamine residues attached to asparagines of the protein
backbone remaining when growth was complete. Monosaccharides were released
sequentially from the glycans by S.oralis glycosidases in the order sialic acid, 
galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the
actions of exo-glycosidic activities, including mannosidases which have not
previously been reported for S.oralis. All released monosaccharides were
metabolized during growth with the exception of fucose which remained free in
culture supernatants. Direct release of oligosaccharides was not observed,
indicating the absence of endo-glycosidases in S.oralis. We propose that this
mechanism of deglycosylation of host glycoproteins and the subsequent utilization
of released monosaccharides is important in the survival and persistence of this 
and other pathogenic bacteria in vivo.

PMID: 10207179 [PubMed - indexed for MEDLINE]


460. Science. 1999 Mar 12;283(5408):1668-9.

Watching DNA at work.

Service RF.

PMID: 10189320 [PubMed - indexed for MEDLINE]


461. FEMS Microbiol Lett. 1999 Mar 15;172(2):231-7.

Cloning and characterization of a new exo-cellulase gene, cel3, in Irpex lacteus.

Hamada N, Fuse N, Shimosaka M, Kodaira R, Amano Y, Kanda T, Okazaki M.

Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu
University, Ueda, Japan.

A new cellulose-inducible gene (named cel3) was isolated from a strain of the
white rot basidiomycete, Irpex lacteus MC-2. The cel3 open reading frame,
containing two introns, encodes a polypeptide of 526 amino acids residues with a 
molecular mass of 55794 Da. Expression of the cel3 gene was induced by various
insoluble celluloses and CM-cellulose. Transcription of cel3 was abolished when
cells were cultivated in media containing the above cellulosic substrates, but
added with glucose, fructose or lactose, while addition of glycerol or mannitol
did not affect the cel3 mRNA level. The amino acid sequence of the catalytic
domain of the Cel3 protein was homologous to that of fungal exo-type cellulases
belonging to family 7 of the glycosyl hydrolases. A phylogenetic study showed
that these exo-type cellulases can be clearly separated from family 7 endo-type
cellulases.

PMID: 10188251 [PubMed - indexed for MEDLINE]


462. Biotechnol Appl Biochem. 1999 Apr;29 ( Pt 2):133-40.

Comparative study of intracellular and extracellular pectinases produced by
Penicillium frequentans.

Kawano CY, Chellegatti MA, Said S, Fonseca MJ.

Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo,
Ribeirao Preto, S.P., Avenida do Cafe s/n, CEP 14049-903, Ribeirao Preto, S.P.,
Brazil.

The filamentous fungus Penicillium frequentans synthesized eleven
polygalacturonases (PGs) and two pectinesterases (PEs) when grown in liquid
culture supplemented with pectin. Seven PGs and the two PEs were secreted in the 
medium, whereas four PGs were not secreted. Among the secreted PGs, the endo-PG
(band 10) and exo-PGs (band 5) were the enzymes secreted at the highest levels.
All secreted PGs bound to lectin and their secretion and/or enzymic activities
were inhibited by tunicamycin (TM), except for the constitutive and inducible
endo-PG (band 10). Studies on the affinity for concanavalin A (ConA) and the
effect of TM suggested that the secreted endo-PG and exo-PG differed in level and
process of glycosylation. The exo-PG was characterized as a N-glycoprotein,
whereas the endo-PG is probably an O-glycoprotein. The PGs (bands 3 and 4) were
neither bound to ConA nor secreted and their enzymic activities were inhibited by
TM, suggesting that they are probably N-glycoproteins with complex
oligosaccharides of type three and tetra-antennary structure. The other PGs
(bands 6 and 8) that were not secreted and did not bind to ConA were not
inhibited by TM. These enzymes presented chromatographic characteristics and
effects with TM that were similar to endo-PG (band 10), because these PGs might
be unglycosylated or/and aggregate forms of the endo-PG (band 10).

PMID: 10075909 [PubMed - indexed for MEDLINE]


463. J Biomol Struct Dyn. 1998 Dec;16(3):547-68.

The solution conformation of a carbocyclic analog of the Dickerson-Drew
dodecamer: comparison with its own X-ray structure and that of the NMR structure 
of the native counterpart.

Denisov AY, Zamaratski EV, Maltseva TV, Sandstrom A, Bekiroglu S, Altmann KH,
Egli M, Chattopadhyaya J.

Department of Bioorganic Chemistry, Biomedical Center, University of Uppsala,
Sweden.

The NMR conformation of a carbocyclic analog of the Dickerson-Drew dodecamer
[d(CGC-GAAT*T*CGCG)]2 containing 6'-alpha-Me carbocyclic thymidines (T*) has been
determined and compared with that of its X-ray structure. The solution structure 
of the 6'-alpha-Me carbocyclic thymidine modified duplex has also been compared
with the solution structure of the corresponding unmodified Dickerson-Drew duplex
solved by us under the same experimental conditions. The NMR structures have been
based on 24 experimental distance and torsion constraints per residue for
[d(CGCGAAT*T*CGCG)]2 (1) and on 21 constraints per residue for the natural
counterpart. In general, both final NMR structures are more close to the B-type
DNA. The cyclopentane moieties of the carbocyclic thymidine residues adopt
C1'-exo B-DNA type puckers (the phase angles P = 136-139 degrees and the
puckering amplitudes psi = 36-37 degrees) that are close to their previously
published crystal C1'-exo or C2'-endo puckers. The main differences between the
two NMR structures are for beta(T*8) and epsilon, xi(T*7) backbone torsions
(27-50 degrees ), for basepair twist for the 7-8 and 8-9 basepair steps (5-6
degrees), tilt for the 8-9 step (7 degrees), roll for the 7-8 step (7 degrees),
shift for the 7-8 step (0.9A) and slide for the 9-10 step (0.6A). The relatively 
small deviations of helical structure parameters lead to structural isomorphism
of these duplexes in aqueous solutions (atomic RMSD = 1.0A). The difference of
the minor groove widths (less than 0.7A) in the core part of the modified duplex 
in comparison with the native one is much smaller than the difference between the
X-ray structures of these duplexes. A detailed comparison of NMR and X-ray
structure parameters showed significant monotonic differences (0.9-2.5A) for all 
basepair slides in both duplexes. Deviations between NMR and X-ray structure
parameters for the modified duplex were also found for basepair tilt of the 4-5
step (13 degrees), rolls for the 8-9 and 10-11 steps (16 and 19 degrees), twist
of the 3-4 step (8 degrees) and shift of the 9-10 step (0.9A).

PMID: 10052613 [PubMed - indexed for MEDLINE]


464. Yeast. 1999 Jan 30;15(2):91-109.

Cloning and characterization of 1,3-beta-glucanase-encoding genes from
non-conventional yeasts.

Esteban PF, Vazquez de Aldana CR, del Rey F.

Departamento de Microbiologia, Universidad de Salamancal/CSIC, Spain.

The molecular cloning of 1,3-beta-glucanase-encoding genes from different yeast
species was achieved by screening genomic libraries with DNA probes obtained by
PCR-amplification using oligonucleotides designed according to conserved regions 
in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. The nucleotide
sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 (Hansenula polymorpha) and 
SoEXG1 (Schwanniomyces occidentalis) genes was determined. K1EXG1 consists of a
1287 bp open reading frame encoding a protein of 429 amino acids (49,815 Da).
HpEXG1 specifies a 435-amino acid polypeptide (49,268 Da) which contains two
potential N-glycosylation sites. SoEXG1 encodes a protein of 425 residues (49,132
Da) which contains one potential site for N-linked glycosylation. Expression in
S. cerevisiae of KlEXG1, SoEXG1 or HpEXG1 under control of their native promoters
resulted in the secretion of active 1,3-beta-glucanases. Disruption of KlEXG1 did
not result in a phenotype under laboratory conditions. Comparison of the primary 
translation products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously
characterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveals 
that enzymes with this type of specificity constitute a family of highly
conserved proteins in yeasts. KlExg1p, HpExg1p and SoExg1p contain the invariant 
amino acid positions which have been shown to be important in the catalytic
function of family 5 glycosyl hydrolases.

PMID: 10029988 [PubMed - indexed for MEDLINE]


465. J Mol Biol. 1999 Feb 12;286(1):57-69.

Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication.

Truniger V, Blanco L, Salas M.

Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autonoma,
Canto Blanco, Madrid, 28049, Spain.

We have analyzed the functional significance of the phi29 DNA polymerase "YxGG/A"
motif in initiation and replication reactions involving the terminal protein (TP)
as a primer. This motif, located between the proposed limits of the polymerase
and exonuclease domains, has been shown to be very important for the coordination
between synthesis and degradation in phi29 DNA polymerase. Mutations in this
region affected the polymerization/exonucleolysis (pol/exo) balance, due to its
importance for DNA template binding stability at both active sites. Here, we show
that the YxGG/A motif of phi29 DNA polymerase is necessary for the formation of a
stable complex between TP and phi29 DNA polymerase, affecting initiation and
transition during replication of phi29 TP-DNA. The phenotypes in TP-primed
reactions in nine of 11 mutant polymerases, showed reduced initiation and/or
replication activities using TP-DNA as template. High dATP concentrations allowed
the reduced initiation activities of some of these mutant polymerases to reach
the wild-type level. The reduction in their affinity for the initiating
nucleotide is likely due to their reduced interaction with the TP. Besides, the
YxGG/A motif of phi29 DNA polymerase controls the pol/exo balance in the
transition step immediately after TP-primed initiation, before DNA polymerase and
TP dissociate. Thus, from the first elongation step, the phenotypes of the mutant
polymerases parallel those obtained in DNA-primed replication: wild-type, high
and low pol/exo balance. A detailed analysis of different transition
intermediates suggests that mutants at the YxGG/A motif switch from interaction
with TP to DNA once the TP has been extended with six nucleotides. Copyright 1999
Academic Press.

PMID: 9931249 [PubMed - indexed for MEDLINE]


466. J Nat Prod. 1999 Jan;62(1):5-21.

Occurrence and significance of decahydroquinolines from dendrobatid poison frogs 
and a myrmicine ant: use of 1H and 13C NMR in their conformational analysis

Spande TF, Jain P, Garraffo HM, Pannell LK, Yeh HJC, Daly JW, Fukumoto S, Imamura
K, Tokuyama T, Torres JA, Snelling RR, Jones TH.

Department of Chemistry, Virginia Military Institute, Lexington, Virginia
24450-0304, USA.

Structures for 2,5-disubstituted decahydroquinolines (DHQs) are reported for the 
two diastereomeric pairs cis-275B (14) and cis-275B' (15) and 5-epi-trans-269AB
(18) and trans-269AB (19), all isolated from skin extracts of dendrobatid frogs, 
and for 5-epi-cis-275B' (16) and 5-epi-trans-275B (17) found in the extracts of
virgin queens of a myrmicine ant [Solenopsis (Diplorhoptrum) azteca]. Detection
of such DHQs in an ant, their first reported occurrence, strengthens a dietary
hypothesis for the origin of the approximately 30 DHQs that have been detected in
extracts of frog skin. NMR data on the two conformers of cis-decahydroquinoline
permit assignment of ring conformations and stereochemistry to cis-DHQs of the
"N-endo" type or the "N-exo" type. These conformations are also assigned on
whether H-8a is equatorial or axial as determined with E-COSY or 1D-HOHAHA
spectra.

PMID: 9917275 [PubMed - as supplied by publisher]


467. Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43.

Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease
suggests separate mechanisms of endo-and exonucleolytic cleavage.

Garforth SJ, Ceska TA, Suck D, Sayers JR.

Division of Molecular and Genetic Medicine, University of Sheffield, Sheffield
S10 2JF, United Kingdom.

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. 
These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates
exonucleolytically, and they are structure-specific endonucleases. The 5'-3'
exonucleases are conserved in organisms as diverse as bacteriophage and mammals. 
Crystal structures of three representative enzymes identify two
divalent-metal-binding sites typically separated by 8-10 A. Site-directed
mutagenesis was used to investigate the roles of three lysine residues (K83,
K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3'
exonuclease. Neither K196 nor K215 was essential for either the exo- or the
endonuclease activity, but mutation of these residues increased the dissociation 
constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A).
Biochemical analysis demonstrated that K83 is absolutely required for
exonucleolytic activity on single-stranded DNA but is not required for
endonucleolytic cleavage of flap structures. Structural analysis of this mutant
by x-ray crystallography showed no significant perturbations around the
metal-binding sites in the active site. The wild-type protein has different pH
optima for endonuclease and exonuclease activities. Taken together, these results
suggest that different mechanisms for endo- and exonucleolytic hydrolysis are
used by this multifunctional enzyme.

PMCID: PMC15089
PMID: 9874768 [PubMed - indexed for MEDLINE]


468. J Biol Chem. 1998 Oct 30;273(44):28966-77.

phi29 DNA polymerase residue Ser122, a single-stranded DNA ligand for 3'-5'
exonucleolysis, is required to interact with the terminal protein.

de Vega M, Blanco L, Salas M.

Centro de Biologia Molecular "Severo Ochoa", Universidad Autonoma, Canto Blanco, 
28049 Madrid, Spain.

Three amino acid residues highly conserved in most proofreading DNA polymerases, 
a phenylalanine contained in the Exo II motif and a serine and a leucine
belonging to the S/TLx2h motif, were recently shown to be critical for 3'-5'
exonucleolysis by acting as single-stranded DNA ligands (de Vega, M., Lazaro,
J.M., Salas, M. and Blanco, L. (1998) J. Mol. Biol. 279, 807-822). In this paper,
site-directed mutants at these three residues were used to analyze their
functional importance for the synthetic activities of phi29 DNA polymerase, an
enzyme able to start linear phi29 DNA replication using a terminal protein (TP)
as primer. Mutations introduced at Phe65, Ser122, and Leu123 residues of phi29
DNA polymerase severely affected the replication capacity of the enzyme. Three
mutants, F65S, S122T, and S122N, were strongly affected in their capacity to
interact with a DNA primer/template structure, suggesting a dual role during both
polymerization and proofreading. Interestingly, mutant S122N was not able to
maintain a stable interaction with the TP primer, thus impeding the firsts steps 
(initiation and transition) of phi29 DNA replication. The involvement of Ser122
in the consecutive binding of TP and DNA is compatible with the finding that the 
TP/DNA polymerase heterodimer was not able to use a DNA primer/template
structure. Assuming a structural conservation among the eukaryotic-type DNA
polymerases, a model for the interactions of phi29 DNA polymerase with both TP
and DNA primers is presented.

PMID: 9786901 [PubMed - indexed for MEDLINE]


469. J Med Chem. 1998 Oct 22;41(22):4378-84.

Muscarinic agonists with antipsychotic-like activity: structure-activity
relationships of 1,2,5-thiadiazole analogues with functional dopamine antagonist 
activity.

Sauerberg P, Jeppesen L, Olesen PH, Rasmussen T, Swedberg MD, Sheardown MJ,
Fink-Jensen A, Thomsen C, Thogersen H, Rimvall K, Ward JS, Calligaro DO, DeLapp
NW, Bymaster FP, Shannon HE.

Health Care Discovery, Novo Nordisk A/S, Novo Nordisk Park, 2760 Malov, Denmark, 
and Neuroscience Research, Lilly Research Laboratories, Indianapolis, Indiana
46285, USA. psa@novo.dk

Muscarinic agonists were tested in two models indicative of clinical
antipsychotic activity: conditioned avoidance responding (CAR) in rats and
inhibition of apomorphine-induced climbing in mice. The standard muscarinic
agonists oxotremorine and pilocarpine were both active in these tests but showed 
little separation between efficacy and cholinergic side effects.
Structure-activity relationships of the alkylthio-1,2,5-thiadiazole azacyclic
type muscarinic partial agonists are shown, revealing the
exo-6-(3-propyl/butylthio-1,2, 5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane
analogues (4a,b and 9a, b) to be the most potent antipsychotic agents with large 
separation between efficacy and cholinergic side effects. The lack of
enantiomeric selectivity suggests the pharmacophoric elements are in the mirror
plane of the compounds. A model explaining the potency differences of closely
related compounds is offered. The data suggest that muscarinic agonists act as
functional dopamine antagonists and that they could become a novel treatment of
psychotic patients.

PMID: 9784113 [PubMed - indexed for MEDLINE]


470. J Biol Chem. 1998 Oct 16;273(42):27250-8.

Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor,
(R)-9-(2-Phosphonylmethoxypropyl)adenine.

Suo Z, Johnson KA.

Department of Biochemistry and Molecular Biology, the Pennsylvania State
University, University Park, Pennsylvania 16802, USA.

(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside
phosphonate that has been shown to be effective in the treatment of AIDS although
it has a shorter separation between the adenine and phosphorus than dideoxy-AMP
and dAMP. By using pre-steady state kinetic methods, we examined the
incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate 
(ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1
(HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7
exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity
of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex,
the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed 
by HIV-1 reverse transcriptase in that both analogs have similar substrate
specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast,
PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the 
DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than
ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower
incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, 
PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA
22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA
(22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel
studies will need to be performed using appropriate human polymerases, these
results begin to define the mechanistic basis for the reported lower toxicity of 
PMPA in the treatment of AIDS.

PMID: 9765248 [PubMed - indexed for MEDLINE]


471. Appl Microbiol Biotechnol. 1998 Jul;50(1):55-64.

Analysis of the gene for beta-fructosidase (invertase, inulinase) of the
hyperthermophilic bacterium Thermotoga maritima, and characterisation of the
enzyme expressed in Escherichia coli.

Liebl W, Brem D, Gotschlich A.

Institut fur Mikrobiologie und Genetik, Georg-August-Universitat, Gottingen,
Germany.

This is the first report describing the gene structure and the enzymatic
properties of a beta-fructosidase of a hyperthermophilic organism. The bfrA gene 
of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue,
polypeptide of about 50 kDa, with significant sequence similarity to other
beta-fructosidases. On the basis of its primary structure, BfrA can be assigned
to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli 
and the recombinant enzyme was purified and characterised. BfrA was specific for 
the fructose moiety and the beta-anomeric configuration of the glycosidic
linkages of its substrates. The enzyme released fructose from sucrose and
raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an
exo-type fashion. BfrA displayed similar catalytic efficiencies for the
hydrolysis of sucrose and inulin with Kcat/K(m) values (at 75 degrees C, pH 5.5) 
of about 4.1 x 10(4) M-1S-1 and 3.1 x 10(4) M-1S-1 respectively. BfrA had an
optimum temperature of 90-95 degrees C (10-min assay) and was extremely
insensitive to thermo-inactivation. During 5 h at temperatures up to 80 degrees C
at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is 
the most thermostable beta-fructosidase and also the most thermostable inulinase 
described to date. In conclusion, the T. maritima enzyme can be classified as an 
exo-beta-D-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase
activity. Its catalytic properties along with the extreme thermostability
recommend it for use in biotechnology.

PMID: 9720201 [PubMed - indexed for MEDLINE]


472. FEMS Microbiol Lett. 1998 Aug 1;165(1):215-20.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with 
enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Krishnan HB, Pueppke SG.

Department of Plant Pathology, University of Missouri, Columbia 65211, USA.
krishnan@psu.missouri.edu

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms
nodules on roots of soybean and other legume plants. We previously found that the
Tn5-containing mutant 208T3, which was derived from strain USDA208, is both
deficient in production of exopolysaccharides and more competitive than the
wild-type strain in competing against other rhizobia for nodulation of soybean.
We now demonstrate that the transposon insertion of the mutant lies in a locus
that is highly homologous to a portion of the exo region, which functions in
exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp
surrounding the insertion site and identified three genes: exoA, exoM, and exoO. 
The transposon lies within exoM, a glucosyl transferase. A cosmid containing
exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 
to wild-type levels. Although exo mutants of S. meliloti are defective in their
abilities to form indeterminate nodules, the capacities of mutant 208T3 and its
wild-type parent to form such nodules on five legume species are
indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii 
appears to differ fundamentally from that in S. meliloti.

PMID: 9711859 [PubMed - indexed for MEDLINE]


473. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9238-43.

Cocaine alters the accessibility of endogenous cysteines in putative
extracellular and intracellular loops of the human dopamine transporter.

Ferrer JV, Javitch JA.

Center for Molecular Recognition and Departments of Pharmacology, College of
Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, NY
10032, USA.

Cocaine and other psychostimulants act by blocking the dopamine transporter.
Binding of the cocaine analog, [3H]2-beta-carbomethoxy-3-beta-(4-fluorophenyl)
tropane (CFT) to the dopamine transporter is sensitive to polar
sulfhydryl-specific derivatives of methanethiosulfonate (MTS). These reagents
preferentially react with water-accessible, reduced cysteines. The human dopamine
transporter has 13 cysteines. Their topology is not completely determined. We
sought to identify those cysteine residues the modification of which affects CFT 
binding and to determine the topology of these reactive cysteines. We mutated
each of the cysteines, one at a time and in various combinations, to residues
that preserved binding and transport, and we tested the sensitivity of each of
the mutant transporters to the reagents. One construct, X5C, had five mutated
cysteines (C90A, C135A, C306A, C319F, and C342A). Using a membrane preparation in
which both extracellular and intracellular cysteines could be accessible, we
found that CFT binding in X5C, as compared with wild-type transporter, was two
orders of magnitude less sensitive to MTS ethylammonium (MTSEA). The wild-type
cysteines were substituted back into X5C, one at a time, and these constructs
were tested in cells and in membranes. Cys-90 and Cys-306 appear to be
extracellular, and Cys-135 and Cys-342 appear to be intracellular. Each of these 
residues is predicted to be in extramembranous loops. The binding of cocaine
increases the rate of reaction of MTSEA and MTS ethyltrimethylammonium with the
extracellular Cys-90 and therefore acts by inducing a conformational change.
Cocaine decreases the rate of reaction of MTSEA with Cys-135 and Cys-342, acting 
either directly or indirectly on these intracellular residues.

PMCID: PMC21322
PMID: 9689064 [PubMed - indexed for MEDLINE]


474. Virology. 1998 Jul 5;246(2):298-305.

Mutations in the Exo III motif of the herpes simplex virus DNA polymerase gene
can confer altered drug sensitivities.

Hwang YT, Smith JF, Gao L, Hwang CB.

Department of Microbiology, and Immunology, College of Medicine, State University
of New York, Syracuse 13210, USA. hwangc@vax.cs.hscsyr.edu

Two herpes simplex virus mutants containing mutated residues within the conserved
Exo III motif of the polymerase gene were previously shown to be defective in
3'-5' exonuclease activity and exhibited extremely high mutation frequencies. In 
this study, we have shown that these mutants also exhibited higher resistance to 
phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs
tested, including acyclovir and gangciclovir, compared to wild-type virus. Marker
transfer experiments and sequencing analyses demonstrated that these altered
phenotypes were the result of mutations within the Exo III motif. The data
indicate that, aside from leading to exonuclease deficiency, mutations in the Exo
III motif may also affect interaction of nucleoside triphosphates with the
catalytic sites of polymerase activity.

PMID: 9657948 [PubMed - indexed for MEDLINE]


475. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7893-7.

Structural, functional, and evolutionary relationships between lambda-exonuclease
and the type II restriction endonucleases.

Kovall RA, Matthews BW.

Institute of Molecular Biology, Howard Hughes Medical Institute and Department of
Physics, University of Oregon, Eugene, OR 97403, USA.

lambda-exonuclease participates in DNA recombination and repair. It binds a free 
end of double-stranded DNA and degrades one strand in the 5' to 3' direction. The
primary sequence does not appear to be related to any other protein, but the
crystal structure shows part of lambda-exonuclease to be similar to the type II
restriction endonucleases PvuII and EcoRV. There is also a weaker correspondence 
with EcoRI, BamHI, and Cfr10I. The structure comparisons not only suggest that
these enzymes all share a similar catalytic mechanism and a common structural
ancestor but also provide strong evidence that the toroidal structure of
lambda-exonuclease encircles its DNA substrate during hydrolysis.

PMCID: PMC20900
PMID: 9653111 [PubMed - indexed for MEDLINE]


476. J Lipid Res. 1998 May;39(5):1039-45.

Stereochemical structures of synthesized and natural plasmalogalactosylceramides 
from equine brain.

Yachida Y, Kashiwagi M, Mikami T, Tsuchihashi K, Daino T, Akino T, Gasa S.

Department of Chemistry, Sapporo Medical University School of Medicine, Japan.

Modified galactosylceramide with a long-chain cyclic acetal at the sugar moiety, 
plasmalogalactosylceramide, was isolated from equine brain. To identify the
isomeric stereostructure of the natural product, the plasmalo derivative was
chemically synthesized from galactosylceramide through acetalization. The
presence of cyclic acetal linkage, the linked position and length of the acetal
chain of the synthesized and natural products were determined by proton nuclear
magnetic resonance spectroscopy and fast-atom bombardment-mass spectrometry, as
well as gas chromatography-mass spectrometry and gas-liquid chromatography. The
orientation of the acetal chain linked to galactoside was characterized by
connectivity between the cyclic acetal proton and ring proton(s) on the sugar
moiety using the homonuclear Overhauser effect. This revealed that, of the two
positional isomers of the acetal linkage with 4,6-O-acetal and 3,4-O-acetal
derivatives obtained from the acetalization reaction, the former positional
isomer, separated into two spots, was identified to 'endo'- and 'exo'-type acetal
chains. In comparison to the NMR data of the synthesized derivative, equine brain
acetalized lipid was found to be an 'endo'-type 4,6-O-acetal derivative.

PMID: 9610771 [PubMed - indexed for MEDLINE]


477. Biochemistry. 1998 May 19;37(20):7608-16.

The conformation of NADH bound to inosine 5'-monophosphate dehydrogenase
determined by transferred nuclear Overhauser effect spectroscopy.

Schalk-Hihi C, Zhang YZ, Markham GD.

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia,
Pennsylvania 19111, USA.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine
5-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The reaction proceeds
with concomitant conversion of NAD+ to NADH and is the rate-limiting step in the 
de novo biosynthesis of guanosine nucleotides. IMPDH is a target for numerous
chemotherapeutic agents. The conformations of enzyme-bound substrates,
enzyme-bound products and enzyme-bound ligands in general, are of interest for
the understanding of the catalytic mechanism of the enzyme and the design of new 
inhibitors. Although several of the chemotherapeutic inhibitors of IMPDH are NAD+
or NADH analogues, no structural data for IMPDH-bound NAD+ (or NADH) are
available. In the present work, we have used transferred nuclear Overhauser
effect spectroscopy (TRNOESY) to determine the conformation of NADH bound to the 
active site of human type II IMPDH (IMPDH-h2). The inter-proton distances
determined from TRNOESY data indicate that NADH binds to the enzyme active site
in an overall extended conformation. The adenosine moiety and the nicotinamide
riboside moiety are both in the anti conformation about the glycosidic bond, and 
both ribose rings are in approximately C4'-exo conformations. The nicotinamide
amide group was found to be in a cis conformation. The anti conformation of the
nicotinamide riboside moiety is in accord with the preferred conformations of
several potent and selective dinucleotide inhibitors and is consistent with that 
implied by the stereospecificity of hydride transfer in the enzymatic reaction.
The implications of this conformation for the catalytic mechanism of IMPDH-h2 are
discussed.

PMID: 9585576 [PubMed - indexed for MEDLINE]


478. Bratisl Lek Listy. 1998 Feb;99(2):99-103.

The effect of ambroxol on the vascular reactivity in the rabbit.

Holecyova A, Kriska M.

Institute of Normal and Pathological Physiology, Slovak Academy of Sciences,
Bratislava, Slovakia.

Experiments were designed to determine whether ambroxol, a drug used for the
treatment of respiratory disorders, affects the basal tension and/or contractions
due to adrenergic stimuli in the isolated rabbit portal vein and pulmonary
artery. Ambroxol in concentrations of 10(-6)-10(-4) mol/l produced a
concentration-dependent increase in basal tension, but a decrease in spontaneous 
mechanical activity of portal vein. The same concentrations of ambroxol failed to
influence basal tension of pulmonary artery. However, when the vessel tone was
increased by exogenous noradrenaline, ambroxol elicited concentration-dependent
contractions also in this vessel. Moreover, ambroxol in the concentration of
10(-5) mol/l significantly enhanced vascular contractions due to both exo- and
endogenous noradrenaline. The results suggest that ambroxol, in biologically
relevant concentrations, may influence or even induce vascular contractions to
adrenergic stimuli. Their expression, however, depend on the type of vascular
smooth muscle. (Fig. 7, Ref. 25.)

PMID: 9588086 [PubMed - indexed for MEDLINE]


479. Appl Environ Microbiol. 1998 Apr;64(4):1497-503.

Targeted mutants of Cochliobolus carbonum lacking the two major extracellular
polygalacturonases.

Scott-Craig JS, Cheng YQ, Cervone F, De Lorenzo G, Pitkin JW, Walton JD.

Department of Energy Plant Research Laboratory, Michigan State University, East
Lansing 48824, USA.

The filamentous fungus Cochliobolus carbonum produces endo-alpha
1,4-polygalacturonase (endoPG), exo-alpha 1,4-polygalacturonase (exoPG), and
pectin methylesterase when grown in culture on pectin. Residual activity in a
pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible
protein has now been purified. After chemical deglycosylation, the molecular mass
of the purified protein decreased from greater than 60 to 45 kDa. The gene that
encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences
from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of
48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an
exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum
mutated in PGX1 were constructed by targeted gene disruption and by gene
replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and 
they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was
constructed by crossing. The double mutant grew as well as the pgx1 single mutant
on pectin and was still pathogenic despite having less than 1% of total wild-type
PG activity. Double mutants retained a small amount of PG activity with the same 
cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG
activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant
on pectin could be due to one or more of these residual activities.

PMCID: PMC106176
PMID: 9546185 [PubMed - indexed for MEDLINE]


480. Biosci Biotechnol Biochem. 1998 Feb;62(2):393-5.

Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme 
of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme.

Mizuno K, Tachiki T.

Department of Bioscience and Biotechnology, Faculty of Science and Engineering,
Ritsumeikan University, Kusatsu, Japan.

p-NP-alpha-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus
circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme,
increased remarkably when the bacterium was grown on dextran as a carbon source. 
It was suggested that the increase of the activity was caused by increases of two
major species, alpha-D-glucosidase I and alpha-D-glucosidase II.
alpha-D-Glucosidase I, which showed a certain reactivity toward dextran, was
isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery). The enzyme was
stable around pH 6.5-7.5 and showed its highest activity at pH 6.5. The enzyme
preparation inactivated with p-chloromerucuribenzoic acid recovered its activity 
by incubating with ditiothereitol. Its substrate specificity suggested that the
enzyme was an exo-type enzyme with certain affinity toward alpha-1,6-glucosidic
linkage.

PMID: 9532804 [PubMed - indexed for MEDLINE]


481. Mol Gen Genet. 1998 Feb;257(4):433-41.

The Sinorhizobium meliloti MucR protein, which is essential for the production of
high-molecular-weight succinoglycan exopolysaccharide, binds to short DNA regions
upstream of exoH and exoY.

Bertram-Drogatz PA, Quester I, Becker A, Puhler A.

Universitat Bielefeld, Biologie VI (Genetik), Germany.

Sinorhizobium meliloti (Rhizobium meliloti) is able to produce two different
exopolysaccharides, succinoglycan and galactoglucan. Mutations in the mucR gene
of S. meliloti result in the stimulation of galactoglucan synthesis, while the
type of succinoglycan produced is modified. In culture supernatants of a mucR
mutant, low-molecular-weight succinoglycan is present, whereas no
high-molecular-weight succinoglycan could be detected. The biosynthesis of
succinoglycan is directed by the products of the exo gene cluster. Two DNA
fragments from this cluster, one located in front of the exoH gene and one in the
intergenic region between the divergently transcribed genes exoX and exoY, were
shown to represent effective binding sites for MucR. Whereas the latter binding
site contains an inverted repeat motif, the former does not. However, the binding
of MucR did not strongly modify the transcription of the exo genes involved. In
the mucR mutant the expression levels of exoH-lacZ and exoX-lacZ transcriptional 
fusions were found to be increased 1.5- and 1.7-fold, respectively. On the other 
hand, the expression level of an exoY-lacZ transcriptional fusion was found to be
1.5-fold lower in the mucR mutant than in the wild-type background. Comparison of
the DNA sequences of MucR-binding sites provides insight into the structural
requirements for binding of MucR.

PMID: 9529524 [PubMed - indexed for MEDLINE]


482. Biotechniques. 1998 Mar;24(3):392-6.

Use of the restriction enzyme AvaI and exo- Bst polymerase in strand displacement
amplification.

Milla MA, Spears PA, Pearson RE, Walker GT.

Becton Dickinson Research Center, Research Triangle Park, NC, USA.

PMID: 9526646 [PubMed - indexed for MEDLINE]


483. Eur J Cell Biol. 1998 Jan;75(1):46-53.

Parafusin is a membrane and vesicle associated protein that cycles at exocytosis.

Zhao H, Satir BH.

Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, New York, NY 10461, USA.

In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis
leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that
dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process
fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation
suggesting that PFUS dephosphoglucosylation may be causally related to
exocytosis. To further corroborate the hypothesis that PFUS is involved in the
molecular events in exocytosis, we used laser confocal scanning microscopy and a 
PFUS specific peptide antibody to perform localization studies of PFUS in wild
type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated
both with the exocytic site of the cell membrane and with the membrane of the
dense core secretory vesicles. Localization at these two sites was shown to be
independent of each other since the exocytosis mutant (exo-) tam8, in which
docking of its vesicles is blocked, still showed cell membrane staining.
Immunofluorescence and immunoblotting of isolated intact secretory vesicles also 
revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from 
both the dense core secretory vesicles and the cell membrane in a
Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated 
with the newly formed secretory vesicles in the cytoplasm prior to their docking 
at the exocytic sites. Immunoblot analysis of PFUS during this time showed no
changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer 
or in the exo- temperature sensitive mutant (nd9) at the non-permissive
temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a 
novel critical component whose cycling probably participates in the molecular
exocytic fusion machinery in these cells.

PMID: 9523154 [PubMed - indexed for MEDLINE]


484. Toxicology. 1997 Dec 26;124(2):153-62.

Toxaphene congeners differ from toxaphene mixtures in their dysmorphogenic
effects on cultured rat embryos.

Calciu C, Chan HM, Kubow S.

School of Dietetics and Human Nutrition, Macdonald Campus of McGill University,
Ste-Anne-de-Bellevue, Quebec, Canada.

The presence of persistent organic pollutants, including the pesticide toxaphene 
has been reported even in remote regions such as the Arctic and is becoming a
health concern. The technical mixture of toxaphene contains over 800 different
congeners. The numbers of prevalent congeners, however, decrease along the food
chain. About 20 major congeners are found in fish, eight in marine mammals and
only two major ones in human,
2-exo,3-endo,5-exo,6-endo,8,8,10,10-octachlorobornane (T2) and
2-exo,3-endo,5-exo,6-endo,8,8,9,10,10-nonachlorobornane (T12). Embryotoxicity of 
these individual congeners is not known, as previous studies focused on the
toxaphene technical mixture. We studied the relative dysmorphogenic activity of
toxaphene technical mixture and individual congeners (T2 and T12) using rat
embryo culture. Explanted embryos (0-2 somites) were treated for 48 h with
concentrations of 0 (DMSO 0.01%), 100, 1000 and 5000 ng/ml of either (a)
toxaphene technical mixture; (b) T2; (c) T12; or (d) a 50:50 mixture of T2 and
T12. The treatment period corresponds to gestational days (GD) 10-12, a period
within the critical time of morphogenesis and organogenesis. Both the technical
mixture and the two individual congeners had a significant adverse effects on the
total morphological score, somite number, head and crown rump length and the
central nervous system scores of embryos. All treatments caused a high incidence 
of central nervous system defects. The T2 and T12 congeners differed in their
spectrum of abnormalities as exposure to T2 caused limb and flexion defects which
were not observed with the T12 congener. Differences were also observed in the
type of toxicity and the target sites between the technical mixture and the
congeners. T2 showed a more potent adverse effect on the morphological score as
compared to the technical mixture. Both T2 and T12 were less inhibitory on growth
than the technical mixture as indicated by crown-rump length but they showed a
stronger inhibitory effect on otic system development. The mixture of T2 + T12
showed a synergistic effect on decreasing crown-rump and head length. Conversely,
the combination of T2 and T12 inhibited the strong adverse effect of the
individual congeners on otic development. The results suggest environmentally
predominant toxaphene congeners can have organ specific embryotoxic effects not
predicted by the toxaphene technical mixture.

PMID: 9458005 [PubMed - indexed for MEDLINE]


485. J Physiol. 1997 Nov 1;504 ( Pt 3):501-15.

Kinetic analysis of the phosphorylation-dependent interactions of synapsin I with
rat brain synaptic vesicles.

Stefani G, Onofri F, Valtorta F, Vaccaro P, Greengard P, Benfenati F.

Department of Experimental Medicine, University of Roma Tor Vergata, Italy.

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein, is
involved in the regulation of neurotransmitter release and synapse formation. By 
binding to both phospholipid and protein components of SV with high affinity and 
in a phosphorylation-dependent fashion, synapsin I is believed to cluster SV and 
to attach them to the actin-based cytoskeleton of the nerve terminal. 2. In the
present study we have investigated the kinetic aspects of synapsin I-SV
interactions and the mechanisms of their modulation by ionic strength and
site-specific phosphorylation, using fluorescence resonance energy transfer
between suitable fluorophores linked to synapsin I and to the membrane bilayer.
3. The binding of synapsin I to the phospholipid and protein components of SV has
fast kinetics: mean time constants ranged between 1 and 4 s for association and 9
and 11's for ionic strength-induced dissociation at 20 degrees C. The interaction
with the phospholipid component consists predominantly of a hydrophobic binding
with the core of the membrane which may account for the membrane stabilizing
effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or
purified exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII)
inhibited the association rate and the binding to SV at steady state by acting on
the ionic strength-sensitive component of the binding. When dephosphorylated
synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the
presence of ATP triggered a prompt dissociation of synapsin I with a time
constant similar to that of ionic strength-induced dissociation. 5. In
conclusion, the reversible interactions between synapsin I and SV are highly
regulated by site-specific phosphorylation and have kinetics of the same order of
magnitude as the kinetics of SV recycling determined in mammalian neurons under
comparable temperature conditions. These findings are consistent with the
hypothesis that synapsin I associates with, and dissociates from, SV during the
exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the
SV-associated CaMPKII, and the subsequent dissociation of the protein from the
vesicle membrane, though not involved in mediating exocytosis of primed vesicles 
evoked by a single stimulus, may represent a prompt and efficient mechanism for
the modulation of neurotransmitter release and presynaptic plasticity.

PMCID: PMC1159955
PMID: 9401959 [PubMed - indexed for MEDLINE]


486. J Bacteriol. 1997 Dec;179(23):7369-78.

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence 
of Ralstonia solanacearum.

Huang Q, Allen C.

Department of Plant Pathology, University of Wisconsin-Madison, 53706, USA.

Ralstonia solanacearum, which causes bacterial wilt disease of many plant
species, produces several extracellular plant cell wall-degrading enzymes that
are suspected virulence factors. These include a previously described
endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the
exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying
PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa
with a typical N-terminal signal sequence. The cloned pehB gene product cleaves
polygalacturonic acid into digalacturonic acid units. The amino acid sequence of 
pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2%
identity at the amino acid level. PehB also has limited similarity to plant
exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. 
solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced
with an insertionally inactivated copy of pehB. The resulting mutants were
deficient in the production of PehB and of both PehA and PehB, respectively. The 
pehB mutant was significantly less virulent than the wild-type strain in eggplant
virulence assays using a soil inoculation method. However, the pehA mutant was
even less virulent, and the pehA pehB double mutant was the least virulent of
all. These results suggest that PehB is required for a wild-type level of
virulence in R. solanacearum although its individual role in wilt disease
development may be minor. Together with endo-PG PehA, however, PehB contributes
substantially to the virulence of R. solanacearum.

PMCID: PMC179687
PMID: 9393701 [PubMed - indexed for MEDLINE]


487. Mol Plant Microbe Interact. 1997 Dec;10(9):1054-64.

A regulatory locus, pehSR, controls polygalacturonase production and other
virulence functions in Ralstonia solanacearum.

Allen C, Gay J, Simon-Buela L.

Department of Plant Pathology, University of Wisconsin-Madison 53706, USA.
cza@plantpath.wisc.edu

We previously identified a locus that regulates production of polygalacturonase
(PG), an extracellular plant cell wall-degrading enzyme important in bacterial
wilt of plants caused by Ralstonia (Pseudomonas) solanacearum. The DNA sequence
of this locus, called pehSR, was determined and two consecutive open reading
frames (ORFs) of 1,905 and 1,680 bp were identified. The amino acid sequences
predicted to be encoded by these ORFs are similar to those of regulators of pilin
synthesis in Pseudomonas aeruginosa and Myxococcus xanthus and to a regulator of 
flagellin synthesis and adhesion in P. aeruginosa, as well as to other
two-component regulators of the NtrB/C subfamily. pehSR mutants produced
negligible levels of endo-PG activity, while exo-PG activity was reduced by 50%. 
Northern (RNA) blot analysis showed that PehSR regulates endo-PG expression at
the transcriptional level. pehSR mutants grew normally in culture and in planta
but were dramatically reduced in virulence; this loss of virulence was
substantially greater than that observed for endo-PG structural gene mutants,
suggesting that pehSR regulates additional factors important in virulence.
Although pehSR mutants were essentially nonmotile, like the wild-type strain,
multiple copies of pehSR conferred motility on the bacterium. Reporter gene
studies indicated that pehSR expression increased when bacteria grew in plant
tissue, and that the pehSR locus was itself negatively regulated by the global
virulence gene regulator PhcA.

PMID: 9390420 [PubMed - indexed for MEDLINE]


488. J Nucl Med. 1997 Nov;38(11):1737-41.

Imaging nicotinic acetylcholine receptors with fluorine-18-FPH, an epibatidine
analog.

Villemagne VL, Horti A, Scheffel U, Ravert HT, Finley P, Clough DJ, London ED,
Wagner HN Jr, Dannals RF.

Division of Nuclear Medicine, The Johns Hopkins Medical Institutions, Baltimore, 
Maryland 21205-2179, USA.

Nicotinic acetylcholine receptors (nAChRs) have been implicated in a variety of
central processes, such as learning and memory and analgesia. These receptors
also mediate the reinforcing properties of nicotine in tobacco products and are
increased in postmortem samples of brains of smokers. On the other hand, brains
of individuals who have died from dementia of the Alzheimer type show abnormally 
low densities of nAChRs. In this study, the distribution and kinetics of
[(+/-)-exo-2-(2-[18F] fluoro-5-pyridyl)-7-azabicyclo[2.2.1]heptane (18F-FPH), a
high-affinity nAChR agonist, was evaluated in a baboon using PET. METHODS: After 
intravenous injection of 5 mCi [185 MBq] 18F-FPH into a 25-kg anesthetized
baboon, sequential quantitative tomographic data were acquired over a period of
150 min. Regions of interest were placed and time-activity curves were generated.
Brain kinetics of the radiotracer were calculated, and the in vivo regional
binding in the baboon brain was compared with the known in vitro regional
distribution of nAChRs in the rat and human brain. RESULTS: Brain activity
reached a plateau within 60 min after injection of the tracer, and the binding
was reversible. Elimination of 18F-FPH was relatively rapid from the cerebellum
(clearance t[1/2] = 3 hr), intermediate from the hypothalamus/midbrain (t[1/2] = 
7 hr) and slow from the thalamus (t[1/2] = 16 hr). Radioactivity due to 18F-FPH
at 130 min postinjection was highest in the thalamus and hypothalamus/midbrain,
intermediate in the neocortex and hippocampus and lowest in the cerebellum.
Subcutaneous injection of 1 mg/kg cytisine 45 min after injection of the
radiotracer reduced brain activity at 130 min by 67%, 64%, 56% and 52% of control
values in the thalamus, hypothalamus/midbrain, hippocampus and cerebellum,
respectively. The regional binding of 18F-FPH at 130 min was highly correlated
with the known densities of nAChR measured in vitro in human (r = 0.81) and rat
brain (r = 0.90). CONCLUSION: These results demonstrate the feasibility of
imaging nAChRs in vivo. Fluorine-18-FPH appears to be a suitable tracer to study 
nAChRs in the human brain.

PMID: 9374343 [PubMed - indexed for MEDLINE]


489. J Mol Biol. 1997 Aug 29;271(4):619-28.

Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized
with maltopentaose.

Yoshioka Y, Hasegawa K, Matsuura Y, Katsube Y, Kubota M.

Institute for Protein Research, Osaka University, Osaka, Suita, 565, Japan.

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of 
Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex
with carbohydrate obtained by cocrystallization with maltopentaose were
determined. Two crystal forms were obtained for the complexed enzyme, and a bound
maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A 
resolution, respectively for the uncomplexed and complexed mutant. These
structures were compared with the wild-type enzyme structure. In the complexed
crystals, the maltotetraose was firmly bound, extensively interacting with the
amino acid environments in the active cleft. The non-reducing end glucose unit
was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of
Gly158, which seem to be predominantly required for the recognition of the
non-reducing end of the substrate that determines the exo-wise degradation of
this enzyme. The reducing end glucose unit of bound maltotetraose showed clear
deformation, adopting a half-chair conformation with extensive hydrogen bonds to 
surrounding polypeptides. The C1-atom of this deformed glucose unit lies very
close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is
firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing 
end glucose unit. Upon binding of the carbohydrate, small but significant induced
fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible
roles of the three catalytic residues are also discussed.

PMID: 9281429 [PubMed - indexed for MEDLINE]


490. Biochemistry. 1997 Aug 26;36(34):10474-81.

Residues at the carboxy terminus of T4 DNA polymerase are important determinants 
for interaction with the polymerase accessory proteins.

Goodrich LD, Lin TC, Spicer EK, Jones C, Konigsberg WH.

Protein Science Corporation, Meriden, Connecticut 06450, USA.

Three T4 DNA polymerase accessory proteins (44P/62P and 45P) stimulate the
polymerase (pol) activity and the 3'-5' exonuclease (exo) activity of T4 DNA
polymerase (43P) on long, double-stranded DNA substrates. The 44P/62P "clamp
loader" facilitates the binding of 45P, the "sliding clamp", to DNA that is
primed for replication. Using a series of truncated 43P mutants, we identified a 
region at the extreme carboxy terminus of the DNA polymerase that is required for
its interaction with accessory proteins. Truncation mutants of 43P lacking the
carboxy-terminal 3, 6, or 11 residues retained full pol and exo activity on short
synthetic primer-templates. However, the ability of the accessory proteins to
enhance these activities on long double-stranded DNA templates was drastically
reduced, and the extent of the reduction in activity was greater as more residues
were deleted. One of the truncation mutants (N881), which had 17 residues removed
from the carboxy terminus, showed reduced binding affinity and diminished pol
activity but enhanced exo activity upon incubation with a small primer-template. 
The exo activity of the N881 mutant, on short, single-stranded DNA was unchanged,
however, compared to the wild-type enzyme. These results are consistent with
inferences drawn from the crystal structure of a DNA polymerase from a related
T-even phage, RB69, where the carboxy-terminal 12 residues (equivalent to the 11 
residues of 43P from phage T4) protrude from the thumb domain and are free to
interact with complementary surfaces of the accessory proteins. The structural
integrity of the thumb region in the N881 mutant is probably perturbed and could 
account for its reduced binding affinity and pol activity when incubated with
short, double-stranded DNA substrates.

PMID: 9265627 [PubMed - indexed for MEDLINE]


491. J Neurocytol. 1997 Jul;26(7):455-65.

Large dense-core vesicles at the frog neuromuscular junction: characteristics and
exocytosis.

Pecot-Dechavassine M, Brouard MO.

Departmet de Neurobiologie des Signaux Intercellulaires (URA CNRS 1488),
Universite Pierre et Marie Curie, Paris, France.

The population of large dense-core vesicles (LDCVs) in motor nerve terminals of
the frog cutaneous pectoris muscle was analysed after various experimental
protocols leading to large acetylcholine release. Three types of LDCVs classified
according to their size and the core density were detected. Vesicles, 100-150 nm 
in diameter, with a large and very dense core (type 1) or with an irregular and
diffuse dense core (type 2) were present in similar proportions (45 and 50%
respectively) in controls. Smaller vesicles, 50-80 nm in diameter, with a very
dense core (type 3) were rare, representing around 5% of the cored vesicles. The 
relative proportion of type 1 and type 2 LDCVs was not modified after prolonged
treatment with 25 mM K+. In contrast, the proportion of type 2 LDCVs
significantly increased whereas that of type 1 LDCVs decreased after two or three
series of 20 Hz electrical stimuli applied to the nerve at 5 s intervals. These
changes suggest that type 2 LDCVs are newly recycled LDCVs in the process of
reloading. Images of fusion of LDCVs with the axolemma in regions facing Schwann 
cell digitations were observed both in K(+)- and in electrically stimulated
preparations. They indicate that exocytosis of LDCVs at the frog neuromuscular
junction takes place preferentially away from the active zones. The presence of a
clathrin-like coat on large pockets still containing a core and of both type 1
and type 2 LDCVs in the vicinity of coated pockets strongly suggests that LDCVs
might undergo a combined process of exo-endocytosis at the same site.

PMID: 9306244 [PubMed - indexed for MEDLINE]


492. J Virol. 1997 Jul;71(7):5355-60.

Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia
virus: identification of differences in processing in vitro and in vivo.

Fujisawa R, McAtee FJ, Zirbel JH, Portis JL.

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National
Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840, USA.

The neuroinvasiveness of a chimeric murine retrovirus, CasFrKP (KP), is dependent
on the expression of glycosylated Gag (gp85gag). This viral protein is the
product of alternate translation initiation 88 codons upstream of and in frame
with the initiation codon of pr65gag, the precursor of the viral core proteins.
Although expression of glycosylated Gag affects virus spread in the spleen, it
appears not to affect virus spread in vitro in fibroblast cell lines (J. L.
Portis et al., J. Virol. 68:3879-3887, 1994). The differential effects of this
protein in vitro and in vivo have not been explained, and its function is
unknown. We have here compared the in vitro processing of this molecule with that
expressed in spleens of infected mice. In vitro, gp85gag was cleaved near the
middle of the molecule, releasing the C-terminal half (containing capsid and
nucleocapsid domains of pr65gag) as a secreted glycoprotein. The N-terminal half 
of the protein was associated with the plasma membrane as a approximately 55-kDa 
glycoprotein bearing the matrix domain of pr65gag as well as the N-terminal 88
residue L domain. This processing scheme was also observed in vivo, although two 
differences were seen. There were differences in N-linked glycosylation of the
secreted form of the protein expressed in the spleen. In addition, whereas the
membrane-associated species assumed the orientation of a type II integral
membrane protein (N(cyto) C(exo)) in fibroblasts in vitro, a subpopulation of
spleen cells was detected in which the N terminus of the protein was exposed at
the cell surface. These results suggest that the differential effects of
glycosylated Gag expression in vivo and in vitro may be related to differences in
posttranslational processing of the protein.

PMCID: PMC191773
PMID: 9188605 [PubMed - indexed for MEDLINE]


493. J Org Chem. 1997 Jun 27;62(13):4418-4427.

An Expedient Route for the Stereoselective Construction of Bridged
Polyheterocyclic Ring Systems Using the Tandem "Pincer" Diels-Alder Reaction.

Lautens M, Fillion E.

Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.

The tandem "pincer" Diels-Alder reaction, consisting of two consecutive [4 + 2]
cycloadditions between two dienes and an acetylenic bis-dienophile, has been
applied toward the rapid construction of bridged polyoxacyclic ring systems when 
furan derivatives are used as the diene components. The study has demonstrated
the stereoselectivity (exo-exo adduct), the chemoselectivity ("pincer" vs
"domino"), as well as the regioselectivity of the reaction. The reaction has been
successfully applied to a variety of 2-substituted furans and tethered bis-furans
in combination with monoactivated and diactivated dienophiles. The synthesis of
unsymmetrical cycloadducts starting from the aza- and oxanorbornadiene-type
intermediate has also been realized.

PMID: 11671769 [PubMed - as supplied by publisher]


494. Eur J Biochem. 1997 Jun 15;246(3):681-9.

Man9-mannosidase from pig liver is a type-II membrane protein that resides in the
endoplasmic reticulum. cDNA cloning and expression of the enzyme in COS 1 cells.

Bieberich E, Treml K, Volker C, Rolfs A, Kalz-Fuller B, Bause E.

Institut fur Physiologische Chemie, Bonn, Germany.

Man9-mannosidase, one of three different alpha 1,2-exo-mannosidases known to be
involved in N-linked oligosaccharide processing, has been cloned in lambda gt10, 
using a mixed-primed pig liver cDNA library. Three clones were isolated which
allowed the reconstruction of a 2731-bp full-length cDNA. The cDNA construct
contained a single open reading frame of 1977 bp, encoding a 659-residue
polypeptide with a molecular mass of approximately 73 kDa. The Man9-mannosidase
specificity of the cDNA construct was verified by the observation that all
peptide sequences derived from a previously purified, catalytically active 49-kDa
fragment were found within the coding region. The N-terminus of the 49-kDa
fragment aligns with amino acid 175 of the translated cDNA, indicating that the
catalytic activity is associated with the C-terminus. Transfection of COS 1 cells
with the Man9-mannosidase cDNA gave rise to a > 30-fold over-expression of a
73-kDa protein whose catalytic properties, including substrate specificity,
susceptibility towards alpha-mannosidase inhibitors and metal ion requirements,
were similar to those of the 49-kDa enzyme fragment. Thus deletion of 174
N-terminal amino acids in the 73-kDa protein appears to have only marginal
influence on the catalytic properties. Structural and hydrophobicity analysis of 
the coding region, as well as the results from tryptic degradation studies, point
to pig liver Man9-mannosidase being a non-glycosylated type-II transmembrane
protein. This protein contains a 48-residue cytosolic tail followed by a
22-residue membrane anchor (which probably functions as internal and
non-cleavable signal sequence), a lumenal approximately 100-residue-stem region
and a large 49-kDa C-terminal catalytic domain. As shown by immuno-fluorescence
microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the
endoplasmic reticulum, in contrast to COS 1 Man9-mannosidase from human kidney
which is Golgi-located [Bieberich, E. & Bause, E. (1995) Eur. J. Biochem. 233,
644-649]. Localization of the porcine enzyme in the endoplasmic reticulum is
consistent with immuno-electron-microscopic studies using pig hepatocytes. The
different intracellular distribution of pig liver and human kidney
Man9-mannosidase is, therefore, enzyme-specific rather than a COS-1-cell-typical 
phenomenon. Since we observe approximately 81% sequence similarity between the
two alpha-mannosidases, we deduce that the localization in either endoplasmic
reticulum or Golgi is likely to be sequence-dependent.

PMID: 9219526 [PubMed - indexed for MEDLINE]


495. Biochemistry. 1997 May 20;36(20):6059-68.

Exploiting nucleotide thiophosphates to probe mechanistic aspects of Escherichia 
coli DNA gyrase.

Cullis PM, Maxwell A, Weiner DP.

Department of Chemistry, Leicester University, U.K.

The interaction of DNA gyrase with ATP has been probed using a range of
thiophosphate ATP analogs. ATP gammaS is not detectably hydrolyzed by gyrase but 
can support limited, probably catalytic, DNA supercoiling. ATP gammaS is a good
inhibitor of both ATP hydrolysis and ATP-supported supercoiling. In contrast,
both ATP alphaS(Rp) and ATP betaS(Rp) have been shown to be good substrates for
the ATPase reaction of gyrase and to support catalytic DNA supercoiling. The
corresponding Sp diastereoisomers do not support significant levels of
supercoiling and are not readily hydrolyzed, but are shown to be reasonable
inhibitors of gyrase. For ATP alphaS(Rp), the supercoiling and ATPase activities 
appear to be tightly coupled with the thionucleotide being apparently a better
substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis.
In the case of ATP betaS(Rp), DNA supercoiling and nucleotide hydrolysis appear
to be uncoupled in that ATP betaS(Rp) is almost as good a substrate as ATP for
the ATPase reaction of both intact gyrase and the 43 kDa GyrB fragment, whereas
it only supports slow DNA supercoiling; the mechanistic consequences of these
observations are discussed in terms of a new model for energy coupling in gyrase.
DNA gyrase has been shown to be capable of catalyzing DNA supercoiling in the
presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2+, or Cd2+. The
pronounced diastereoselectivity seen in both the DNA supercoiling and ATPase
activity with ATP alphaS and ATP betaS together with evidence from the X-ray
structure of the 43 kDa GyrB-ADPNP-Mg complex is consistent with metal ion
coordination at both of these sites, and probably to the gamma-phosphoryl center 
during turnover. Thus, the absolute configuration of the catalytically active
Mg2+-ATP complex is likely to involve coordination to the pro-S oxygens at both P
alpha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP complex with 
the lambda-exo configuration.

PMID: 9166776 [PubMed - indexed for MEDLINE]


496. Gene. 1997 May 6;190(2):257-61.

Cloning and characterization of a dextranase gene (dexS) from Streptococcus suis.

Serhir B, Dugourd D, Jacques M, Higgins R, Harel J.

Departement de pathologie et microbiologie, Faculte de medecine veterinaire,
Universite de Montreal, Quebec, Canada.

A gene (dexS) coding for a Streptococcus suis capsular type 2 dextranase (DexS)
was detected in a recombinant gene library constructed in phage lambda ZapII, and
its nucleotide sequence was determined. Sequence comparison showed that the dexS 
gene product had significant similarities with enzymes which hydrolyze glucose
polymers. Moreover, conserved amino acids that are suggested to be part of the
active site of the glucosidases are also found in DexS. The dexS gene, adjacent
to the gene encoding a S. suis IgG-binding protein, encoded a protein of
approximately 62 kDa which exhibited DexS activity.

PMID: 9197542 [PubMed - indexed for MEDLINE]


497. J Org Chem. 1997 Apr 4;62(7):2113-2122.

Palladium(II)-Catalyzed Intramolecular Aminocarbonylation of endo-Carbamates
under Wacker-Type Conditions.

Harayama H, Abe A, Sakado T, Kimura M, Fugami K, Tanaka S, Tamaru Y.

Department of Applied Chemistry, Faculty of Engineering, Nagasaki University,
1-14 Bunnkyo, Nagasaki 852, Japan.

Pd(II)-catalyzed intramolecular aminocarbonylation of olefins bearing many types 
of nitrogen nucleophiles has been examined under two typical conditions: acidic
conditions [conditions A, typically PdCl(2) (0.1 equiv) and CuCl(2) (3.0 equiv)
under 1 atm of CO at room temperature in methanol] and buffered conditions
[conditions B, typically PdCl(2) (0.1 equiv) and CuCl(2) (2.3 equiv) under 1 atm 
of CO at 30 degrees C in trimethyl orthoacetate]. Among nitrogen nucleophiles,
endo-carbamates 7 display distinctive reactivity: endo-carbamates 7a-k smoothly
undergo intramolecular aminocarbonylation under conditions B to furnish
4-[(methoxycarbonyl)methyl]-2-oxazolidinones 8a-k in good yields, while they
would not undergo the expected reaction under conditions A. Other nitrogen
nucleophiles (exo-ureas 1, endo-ureas 3, exo-carbamates 5, and exo-tosylamides
9), on the other hand, satisfactorily undergo aminocarbonylation only under
conditions A to give rise to 2, 4, 6, and 10, respectively, in good yields. Under
conditions B, they are unreactive and provide either the expected products in
poor yields or intractable mixtures of products. On the basis of this contrasting
reactivity between endo-carbamates and other nitrogen nucleophiles, the
chemoselective aminocarbonylation of 7l-o has been achieved; aminocarbonylation
takes place at the endo-carbamate moieties to furnish 8l-o exclusively under
conditions B, and aminocarbonylation occurs at the exo-carbamate, exo-urea, and
exo-tosylamide moieties to yield 13a-d exclusively under conditions A.

PMID: 11671516 [PubMed - as supplied by publisher]


498. J Mol Biol. 1997 Apr 4;267(3):661-72.

Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas
stutzeri.

Morishita Y, Hasegawa K, Matsuura Y, Katsube Y, Kubota M, Sakai S.

Institute for Protein Research, Osaka University, Suita, Japan.

The three-dimensional structure of an exo-type alpha-amylase from Pseudomonas
stutzeri which degrades starch from its non-reducing end to produce maltotetraose
has been determined by X-ray structure analysis. The catalytic domain of this
enzyme (G4-2), whose structure was determined, is a product of spontaneous
limited proteolysis in culture broth. It has 429 amino acid residues and a
molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH
7.5. The structure was elucidated by the multiple isomorphous replacement method 
and refined at 2.0 A resolution, resulting in a final R-factor of 0.178 for
significant reflections with a root-mean-square deviation from ideality in bond
distances of 0.013 A. The polypeptide chain of this molecule folds into three
domains; the first with a (beta/alpha)8 barrel structure, the second with an
excursed part from the first one, and the third with five-stranded antiparallel
beta-sheets. The active cleft is formed on the C-terminal side of the beta-sheets
in the (beta/alpha)8 barrel as in the known endo-type alpha-amylases. A histidine
side-chain nitrogen ND1 is coordinated to one of the bound calcium ion. The
recognition site of the non-reducing end of the amylose that determines exo-wise 
degradation is presumed to be at one end of this cleft where there is a
disordered loop consisting of the 66th to 72nd residues, and a loop carrying an
aspartic acid (Asp160). These structural features may be responsible for the
binding of the non-reducing end of the substrate amylose.

PMID: 9126844 [PubMed - indexed for MEDLINE]


499. Semin Cell Dev Biol. 1997 Apr;8(2):121-31.

Membrane and soluble proteins of adrenal chromaffin granules.

Apps DK.

Department of Biochemistry, University of Edinburgh, Hugh Robson Building, George
Square, Edinburgh, EH8 9XD, Scotland, UK.

Bovine adrenal chromaffin granules are useful 'model' neurosecretory vesicles,
particularly for biochemical studies. The granule matrix contains three major
secretory proteins (chromogranin A and secretogranins I and II) together with
peptides derived from them, and smaller amounts of neuropeptides (enkephalins and
neuropeptide Y). Several different endo- and exo-proteinases are also present in 
both soluble and membrane-bound forms. The major membrane proteins are those
involved in catecholamine biosynthesis (dopamine beta-monooxygenase and
cytochrome b(561)), active transport of granule components (vacuolar-type
proton-translocating ATPase, and carriers for monoamines, nucleotides and small
ions) and exocytosis (synaptotagmin, synaptobrevin and other proteins). In
addition, the functions of a number of major granule membrane proteins remain
unknown.

PMID: 15001087 [PubMed]


500. Neurology. 1997 Apr;48(4):1109-11.

Striatal dopamine nerve terminal markers but not nigral cellularity are reduced
in spinocerebellar ataxia type 1.

Kish SJ, Guttman M, Robitaille Y, el-Awar M, Chang LJ, Levey AI.

Human Neurochemical Pathology Laboratory, Clarke Institute of Psychiatry,
Toronto, ON, Canada.

We previously reported markedly reduced (-76%) dopamine (DA) levels in the
putamen of seven patients with spinocerebellar ataxia type 1 (SCA1) who had no
evidence of nigral cell loss or parkinsonism. To determine whether the DA
reduction was accompanied by loss of DA nerve terminals, we measured levels of
the DA transporter ([3H]WIN, 35,428 binding; DA transporter protein) and the
vesicular monoamine transporter ([3H]DTBZ binding) in the putamen of these
patients. As compared with the controls (n = 14), mean putamen concentrations of 
[3H]WIN 35,428 binding (-45%), dopamine transporter protein (-61%), and [3H]DTBZ 
binding (-48%) were significantly reduced in this SCA1 subgroup. We conclude that
the degeneration in nigrostriatal DA neurons begins at the nerve ending in SCA1.

PMID: 9109912 [PubMed - indexed for MEDLINE]


501. Microbiology. 1997 Mar;143 ( Pt 3):891-8.

Structure of the Clostridium stercorarium gene celY encoding the
exo-1,4-beta-glucanase Avicelase II.

Bronnenmeier K, Kundt K, Riedel K, Schwarz WH, Staudenbauer WL.

Institute for Microbiology, Technical University Munich, Eederal Republic of
Germany.

The nucleotide sequence of the celY gene coding for the thermostable
exo-1,4-beta-glucanase Avicelase II of Clostridium stercorarium was determined.
The gene consists of an ORF of 274Z bp which encodes a preprotein of 914 amino
acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was
identified by comparison with the N-terminal amino acid sequence of Avicelase II 
purified from C stercorarium. The celY gene is located in close vicinity to the
celZ gene coding for the endo-1,4-beta-glucanase Avicelase I. The CelY-encoding
sequence was isolated from genomic DNA of C. stercorarium with the PCR technique.
The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion
protein could be purified using a simple two-step procedure. The properties of
CelY proved to be consistent with those of Avicelase II purified from C.
stercorarium. Sequence comparison revealed that CelY consists of an N-terminal
catalytic domain flanked by a domain of 95 amino acids with unknown function
joined to a type III cellulose-binding domain. The catalytic domain belongs to
the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).

PMID: 9084173 [PubMed - indexed for MEDLINE]


502. Mol Plant Microbe Interact. 1997 Mar;10(2):290-301.

Cloning and characterization of four genes of Rhizobium leguminosarum bv.
trifolii involved in exopolysaccharide production and nodulation.

van Workum WA, Canter Cremers HC, Wijfjes AH, van der Kolk C, Wijffelman CA,
Kijne JW.

Institute of Molecular Plant Sciences, Leiden University, The Netherlands.
workum@rulsfb.LeidenUniv.nl

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599
involved in exopolysaccharide (EPS) production were identified by complementation
of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one
cosmid pssA was located, which was found to be almost identical to the pss4 gene 
from R. leguminosarum bv. viciae VF39 and highly homologous to a family of
glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and
found to produce 19 and 1% of the wild-type amount of EPS, respectively. The
three other genes were found to be closely linked on a different complementing
cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding
glucosyl transferases involved in the synthesis of succinoglycan. A mutation in
this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount.
We found an operon closely linked to pssC, consisting of two overlapping genes,
pssD and pssE, that is essential for EPS production. Homology of pssD and pssE
was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two
genes responsible for the second step in capsule polysaccharide synthesis.
Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, 
of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase.
Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed
it to be identical to that of the parental strain RBL5599, with the exception of 
acetyl groups esterified to one of the glucose residues being absent.

PMID: 9057334 [PubMed - indexed for MEDLINE]


503. J Med Chem. 1997 Feb 14;40(4):486-94.

Novel 3'-C/N-substituted 2',3'-beta-D-dideoxynucleosides as potential
chemotherapeutic agents. 1. Thymidine derivatives: synthesis, structure, and
broad spectrum antiviral properties.

Fedorov II, Kazmina EM, Gurskaya GV, Jasko MV, Zavodnic VE, Balzarini J, De
Clercq E, Faraj A, Sommadossi JP, Imbach JL, Gosselin G.

Moscow Medical Sechenov Academy, Russia.

A synthetic scheme for the 3'-oxime derivatives 3E, 5E, 5Z, 7E and 7Z of
1-(2,3-dideoxy-beta-D-glycero-pentofuranosyl)thymine and for
1-(2,3-dideoxy-3-nitro-beta-D-erythro-pentofuranosyl)-thymine (10) has been
developed starting from appropriately 5'-protected 3'-ketothymidine. X-ray
analysis showed that 3'-N-hydroxyimino 3E and 3'-N-methoxyimino 5Z derivatives
have close molecular conformations: anti about the N1-C1' bond, and gauche+ about
the C4'-C5' exocyclic bond. Their sugar conformations are C1'-exo-O4'-endo and
C1'-exo-C2'-endo, respectively. The antiviral assays in cell cultures
demonstrated that 3'-N-hydroxyimino 3E and 3'-N-acetoxyimino 7E + 7Z derivatives 
are endowed with significant activity against human immunodeficiency virus (HIV) 
with EC50 values ranging between 0.02 and 0.40 microgram/mL for both HIV-1 and
HIV-2. The other compounds 5E + 5Z and 10 were at least 2 orders of magnitude
less active. The 3'-N-hydroxyimino derivative 3E also shows promising activity
against hepatitis B virus (HBV) (EC50 = 0.25 microgram/mL) and against herpes
simplex virus type 1 (HSV-1) and HSV-2.

PMID: 9046339 [PubMed - indexed for MEDLINE]


504. J Biol Chem. 1997 Feb 7;272(6):3161-7.

Identification of Glu-330 as the catalytic nucleophile of Candida albicans
exo-beta-(1,3)-glucanase.

Mackenzie LF, Brooke GS, Cutfield JF, Sullivan PA, Withers SG.

Department of Chemistry, University of British Columbia, Vancouver, British
Columbia V6T 1Z1, Canada.

The exo-beta-(1,3)-glucanase from Candida albicans hydrolyzes cell wall
beta-glucans via a double-displacement mechanism involving a glycosyl enzyme
intermediate. Reaction of the enzyme with
2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside resulted in the
time-dependent inactivation of this enzyme via the accumulation of a
2-deoxy-2-fluoro-glycosyl-enzyme intermediate as monitored also by electrospray
mass spectrometry. The catalytic competence of this intermediate is demonstrated 
by its reactivation through hydrolysis (kreact = 0.0019 min-1) and by
transglycosylation to benzyl thio-beta-D-glucopyranoside (kreact = 0.024 min-1;
Kreact = 56 mM). Peptic digestion of the labeled enzyme followed by tandem mass
spectrometric analysis in the neutral loss mode allowed detection of two
glycosylated active site peptides, the sequences of which were identified as
NVAGEW and NVAGEWSAA. A crucial role for Glu-330 is confirmed by site-directed
mutagenesis at this site and kinetic analysis of the resultant mutant. The
activity of the Glu-330 --> Gln mutant is reduced over 50,000-fold compared to
the wild type enzyme. The glutamic acid, identified in the exoglucanase as
Glu-330, is completely conserved in this family of enzymes and is hereby
identified as the catalytic nucleophile.

PMID: 9013549 [PubMed - indexed for MEDLINE]


505. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):946-51.

Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA
synthesis in vivo.

Rangarajan S, Gudmundsson G, Qiu Z, Foster PL, Goodman MF.

Department of Biological Sciences, University of Southern California, Los Angeles
90089-1340, USA.

We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in
copying chromosomal and episomal DNA in dividing cells in vivo. Forward mutation 
frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and
base substitution and frameshift mutation frequencies were measured on an
F'(lacZ) episome. To amplify any differences in polymerase error rates,
methyl-directed mismatch repair was inactivated. When wild-type Pol II (polB+)
was replaced on the chromosome by a proofreading-defective Pol II exo- (polBex1),
there was a significant increase in mutation frequencies to rifampicin resistance
(RifR) (rpoB) and nalidixic acid resistance (NalR) (gyrA). This increased
mutagenesis occurred in the presence of an antimutator allele of E. coli DNA
polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III 
(dnaE+), suggesting that Pol II can compete effectively with DnaE915 but not with
DnaE+. Sequencing the RifR mutants revealed a G --> A hot spot highly specific to
Pol II exo-. Pol II exo- caused a significant increase in the frequency of base
substitution and frameshift mutations on F' episomes, even in dnaE+ cells,
suggesting that Pol II is able to compete with Pol III for DNA synthesis on F
episomes.

PMCID: PMC19619
PMID: 9023362 [PubMed - indexed for MEDLINE]


506. J Med Chem. 1997 Jan 3;40(1):35-43.

Highly selective, novel analogs of 4-[2-(diphenylmethoxy)ethyl]-
1-benzylpiperidine for the dopamine transporter: effect of different aromatic
substitutions on their affinity and selectivity.

Dutta AK, Coffey LL, Reith ME.

Organix Inc., Woburn, Massachusetts 01801, USA.

Several analogs of the potent and selective dopamine transporter (DAT) ligand
4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine, 1a, were prepared and
biologically evaluated at the dopamine and serotonin transporter (SERT) sites.
Several substituents were introduced in the aromatic rings to evaluate the
influences of electronic and steric interactions in their binding to the DAT. All
the novel analogs showed preferential interaction at the DAT compared with the
SERT. Different aromatic substitutions in the phenyl ring of the N-benzyl part of
the molecule played a key role in the selectivity. In general, compounds with
strong electron-withdrawing substituents were most active and selective at the
DAT. Thus, compounds 5a (R = F) and 11b (R = NO2) were among the most potent
(IC50 = 17.2 and 16.4 nM, respectively) and most selective (SERT/DAT = 112 and
108, respectively) and were far more selective than GBR 12909 (SERT/DAT = 6).
Bioisosteric replacement of one of the phenyl rings of the diphenylmethoxy moiety
by a thiophene ring was tolerated well and produced the most potent compound 13b 
(IC50 = 13.8 nM) in the series. Our current structure-activity studies of these
piperidine analogs resulted in the generation of second generation of GBR-type
compounds, and all of these new compounds reported here were more selective than 
GBR 12909 in interacting with the DAT over the SERT.

PMID: 9016326 [PubMed - indexed for MEDLINE]


507. Morfologiia. 1997;111(1):85-90.

[The endocrine apparatus of the epithelium of the gastric mucosa in the steppe
turtle (Testudo horsfieldi)]

[Article in Russian]

Ivanova VF, Rossol'ko GN, Puzyrev AA.

Endocrinocytes of stomach mucosal epithelium were studied using light and
electron microscopy in steppe turtle (Testudo horsfieldi). Endocrine apparatus of
stomach in turtle was shown to retain the pattern characteristic for other
representatives of vertebrates. Difference is observed in endocrinocytes
localization. The latter are accumulated predominantly in upper and middle
regions of glandulae, sometimes being encountered also in surface epithelium. 8
cell types were identified according to size and structure: EC, G, D, D1, A-like,
X, ECL and P. Relating to glandular lumen, endocrinocytes are divided into
elements of closed (D, D1, A-like, ECL, X, and P) and open types (EC, G). In the 
latter apico-basal differentiation is expressed in cytoplasmic structure.
Endocrinocytes are unevenly distributed throughout the stomach regions. Greatest 
number is concentrated in pyloric part, EC-cells being the predominant type. EC, 
ECL-cells and less A-like and D-cells are mostly encountered in fundal part, EC, 
G and single D and P cells--in pyloric part and, agranular and exo-endocrine
cells--in both stomach parts.

PMID: 9156762 [PubMed - indexed for MEDLINE]


508. EMBO J. 1996 Dec 2;15(23):6460-75.

Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in
yeast.

Benli M, Doring F, Robinson DG, Yang X, Gallwitz D.

Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry,
Gottingen, Germany.

In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as
regulators at defined steps of vesicular transport in exo- and endocytosis. Here 
we report on the isolation and characterization of two genes (YPT31 and YPT32) of
the yeast Saccharomyces cerevisiae which encode members of the Ypt family
exhibiting >80% sequence identity. Whereas the disruption of one of the two genes
was phenotypically neutral, the disruption of both YPT31 and YPT32 led to
lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in 
a ypt32 null background led to a massive accumulation of Golgi-like membranes, an
inhibition of invertase secretion and defects in vacuolar protein maturation.
Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30
min after shift to the non-permissive temperature. According to subcellular
fractionation, a significant part of Ypt31p appeared to be located in
Golgi-enriched membrane fractions. In accordance with this, indirect
immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate
staining similar to that observed with Golgi-located proteins. From the
phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that 
the two GTPases are involved in intra-Golgi transport or in the formation of
transport vesicles at the most distal Golgi compartment.

PMCID: PMC452471
PMID: 8978673 [PubMed - indexed for MEDLINE]


509. J Biochem. 1996 Dec;120(6):1123-9.

Fine substrate specificities of four exo-type cellulases produced by Aspergillus 
niger, Trichoderma reesei, and Irpex lacteus on (1-->3), (1-->4)-beta-D-glucans
and xyloglucan.

Amano Y, Shiroishi M, Nisizawa K, Hoshino E, Kanda T.

Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu
University, Nagano. yoamano@gipwc.shinshu-u.ac.jp

To investigate the fine substrate specificities of four highly purified exo-type 
cellulases (Exo-A from Aspergillus niger, CBHI and CBHII from Trichoderma reesei,
and Ex-1 from Irpex lacteus), water-soluble substrates such as barley glucan,
xyloglucan from tamarind (Tamarindus indica L.), and their oligosaccharides were 
employed. Four exo-type cellulases immediately hydrolyzed
3-O-beta-D-cellotriosylglucose to produce cellobiose and laminaribiose. In
contrast, CBHII showed no hydrolytic activity towards
3(2)-O-beta-D-cello-biosylcellobiose, which was hydrolyzed to cellobiose by the
other exo-type cellulases. These cellulases hydrolyzed the internal linkages of
barley glucan and lichenan in an endo-type fashion to produce cellobiose and
mix-linked oligosaccharides as main products. The DP-lowering activities of the
four exo-type cellulases on barley glucan were in the order of Ex-1, CBHII,
Exo-A, and CBHI. Based on gel permeation chromatography analysis of the
hydrolysates, Ex-1 seemed to attack the internal cellobiosyl unit adjacent to
beta-1,3-glucosidic linkages in barley glucan molecule more frequently than did
the other cellulases. Xyloglucan was hydrolyzed only by CBHI and CBHII, and
produced hepta-, octa-, and nona-saccharides. In addition, a xyloglucan
tetradecasaccharide (XG14) was split only to heptasaccharide (XG7) by CBHI and
CBHII.

PMID: 9010760 [PubMed - indexed for MEDLINE]


510. Biosci Biotechnol Biochem. 1996 Dec;60(12):2099-102.

N-linked sugar chain of 55-kDa royal jelly glycoprotein.

Kimura Y, Kajiyama S, Kanaeda J, Izukawa T, Yonekura M.

Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Japan.

An N-linked sugar chain from 55-kDa royal jelly glycoprotein (RJGP), which
maintains the high viability of rat liver primary cultured cell and is a
different molecular species from 350-kDa RJGP [Kimura et al., Biosci. Biotech.
Biochem., 59, 507-509 (1995)], has been identified. The sugar chains were
released by hydrazinolysis followed by N-acetylation and pyridylamination. The
structural analysis of the pyridylaminated sugar chain was done by a combination 
of sequential exo-mannosidase digestions, MALDI-TOF MS, and 500 MHz 1H-NMR. For
the carbohydrate moiety of 55-kDa RJGP, only one N-linked sugar chain has been
detected. The structure has been found to be Man alpha 1-->2Man alpha 1-->6(Man
alpha 1--> 2Man alpha 1-->3)Man alpha 1-->6(Man alpha 1-->2Man alpha 1-->2Man
alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc, which is a non-processed high 
mannose type structure.

PMID: 8988648 [PubMed - indexed for MEDLINE]


511. J Biol Chem. 1996 Nov 15;271(46):29245-54.

Herpes simplex virus type 1 DNA polymerase. Mutational analysis of the
3'-5'-exonuclease domain.

Kuhn FJ, Knopf CW.

Department of Genomforschung und Bioinformatik, Deutsches Krebsforschungszentrum,
Im Neuenheimer Feld 506, D-69120 Heidelberg, Federal Republic of Germany.
c.knopf@dkfz-heidelberg.de

Like true DNA replicases, herpes simplex virus type 1 DNA polymerase is equipped 
with a proofreading 3'-5'-exonuclease. In order to assess the functional
significance of conserved residues in the putative exonuclease domain, we
introduced point mutations as well as deletions within and near the conserved
motifs' exonuclease (Exo) I, II, and III of the DNA polymerase gene from a
phosphonoacetic acid-resistant derivative of herpes simplex virus-1 strain ANG.
We examined the catalytic activities of the partially purified enzymes after
overexpression by recombinant baculovirus. Mutations of the motifs' Exo I (D368A,
E370A) and Exo III (Y577F, D581A) yielded enzymes without detectable and severely
impaired 3'-5'-exonuclease activities, respectively. Except for the Exo I
mutations, all other Exo mutations examined affected both exonuclease and
polymerization activities. Mutant enzymes D368A, E370A, Y557S, and D581A showed a
significant ability to extend mispaired primer termini. Mutation Y557S resulted
in a strong reduction of the 3'-5'-exonuclease activity and in a polymerase
activity that was hyperresistant to phosphonoacetic acid. The results of the
mutational analysis provide evidence for a tight linkage of polymerase and
3'-5'-exonuclease activity in the herpesviral enzyme.

PMID: 8910584 [PubMed - indexed for MEDLINE]


512. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12822-7.

The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for
holoenzyme complex formation.

Berdis AJ, Soumillion P, Benkovic SJ.

Pennsylvania State University, Department of Chemistry, University Park
16802-6300, USA.

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme
formation, a mutant polymerase in which the last six carboxyl-terminal amino
acids are deleted, was constructed, overexpressed, and purified to homogeneity.
The mutant polymerase, designated delta C6 exo-, is identical to wild-type exo-
polymerase with respect to kcat, kpol, and dissociation constants for nucleotide 
and DNA substrate. However, unlike wild-type exo- polymerase, the delta C6 exo-
polymerase is unable to interact with the 45 protein to form the stable
holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the
wild-type exo- polymerase was tested as an in vitro inhibitor of bacteriophage T4
DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme
complex formation by disrupting the interaction of the polymerase with the 45
protein. On the contrary, the peptide appears to disrupt the interaction of the
44/62 protein with the 45 protein, suggesting that the 44/62 protein and the
polymerase use the same site on the 45 protein for functional interactions. Data 
presented are discussed in terms of a model correlating the functionality of the 
carboxyl terminus of the polymerase for productive interactions with the 45
protein as well as in terms of the 45 protein concomitantly interacting with the 
44/62 protein and polymerase.

PMCID: PMC24004
PMID: 8917503 [PubMed - indexed for MEDLINE]


513. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12229-34.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas
fimi to insoluble microcrystalline cellulose is entropically driven.

Creagh AL, Ong E, Jervis E, Kilburn DG, Haynes CA.

Department of Chemical Engineering, University of British Columbia, Vancouver,
Canada.

Isothermal titration microcalorimetry is combined with solution-depletion
isotherm data to analyze the thermodynamics of binding of the cellulose-binding
domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to
insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration 
microcalorimetry data against two putative binding models indicates that the
bacterial microcrystalline cellulose surface presents two independent classes of 
binding sites, with the predominant high-affinity site being characterized by a
Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of
1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is
mainly driven by a large positive change in entropy. This differs from protein
binding to soluble carbohydrates, which is usually driven by a relatively large
exothermic standard enthalpy change for binding. Differential heat capacity
changes are large and negative, indicating that sorbent and protein dehydration
effects make a dominant contribution to the driving force for binding.

PMCID: PMC37972
PMID: 8901562 [PubMed - indexed for MEDLINE]


514. J Biol Chem. 1996 Oct 4;271(40):24408-12.

Stimulation of tumor cell motility linked to phosphodiesterase catalytic site of 
autotaxin.

Lee HY, Clair T, Mulvaney PT, Woodhouse EC, Aznavoorian S, Liotta LA, Stracke ML.

Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland
20892, USA.

A family of extracellular type I phosphodiesterases has recently been isolated by
cDNA cloning, but a physiological function linked to the phosphodiesterase active
site has remained unknown. We now present evidence that the phosphodiesterase
catalytic site, 201YMRPVYPTKTFPN213, is essential for the motility stimulating
activity of autotaxin (ATX), one member of the exophosphodiesterase family.
Native ATX possesses phosphodiesterase activity at neutral and alkaline pH, binds
ATP noncovalently, and undergoes threonine phosphorylation. Homogeneously
purified recombinant ATX, based on the teratocarcinoma sequence, retains these
same activities. A single amino acid in the phosphodiesterase catalytic site,
Thr210, is found to be necessary for motility stimulation, phosphodiesterase
activity, and phosphorylation. Two mutant recombinant proteins, Ala210- and
Asp210-ATX, lack motility stimulation and lack both enzymatic activities;
Ser210-ATX possesses intermediate activities. Another mutation, with the adjacent
lysine (Lys209) changed to Leu209-ATX, possesses normal motility stimulation with
sustained phosphodiesterase activity but exhibits no detectable phosphorylation. 
This mutation eliminates the phosphorylation reaction and indicates that the
dephosphorylated state is an active motility-stimulating form of the ATX
molecule. By demonstrating that the phosphodiesterase enzymatic site is linked to
motility stimulation, these data reveal a novel role for this family of
exo/ecto-enzymes and open up the possibility of extracellular enzymatic cascades 
as a regulatory mechanism for cellular motility.

PMID: 8798697 [PubMed - indexed for MEDLINE]


515. Mol Gen Genet. 1996 Sep 25;252(4):493-6.

Characterization of genes from Thermoanaerobacterium thermosulfurigenes EM1 that 
encode two glycosyl hydrolases with conserved S-layer-like domains.

Matuschek M, Sahm K, Zibat A, Bahl H.

Institut fur Mikrobiologie, Georg-August-Universitat Gottingen, Germany.

Two genes from Thermoanaerobacterium thermosulfurigenes EM1 were identified which
are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase
(Pg1A). The xynA gene has the potential to encode a 1234-amino acid product
consisting of a signal peptide followed by a repeated domain, a xylanase family F
domain, two cellulose-binding domains and a triplicated sequence at its
C-terminus. The gene pglA is predicted to encode a product of 1148 amino acids
consisting of a signal sequence followed by a fibronectin type III-like domain
(Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a
triplicated domain. The triplicated segments at the C-termini of deduced XynA and
Pg1A are about 95% identical to each other and to the S-layer-like domains of the
previously characterized pullulanase (AmyB) from the same organism. In contrast, 
sequence comparisons revealed only distant amino acid sequence similarities
between the fibronectin type III-like domains of Pg1A and AmyB from T.
thermosulfurigenes EM1.

PMID: 8879252 [PubMed - indexed for MEDLINE]


516. J Biol Chem. 1996 Sep 13;271(37):22462-9.

Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein.
Construction of a self-sufficient Escherichia coli catalytic system.

Sibbesen O, De Voss JJ, Montellano PR.

Department of Pharmaceutical Chemistry, School of Pharmacy, University of
California, San Francisco, 94143-0446, USA.

Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin
reductase (PdR), the two proteins required to transfer electrons from NADH to
P450cam, were constructed by fusing cDNAs encoding the three proteins in the
expression vector pCWori+. Several fusion proteins, in which the order of the
three protein domains and the linkers between them were varied, were expressed in
Escherichia coli, purified, and characterized. The highest activity (kcat = 30
min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS
and PLEL were used, respectively, to link the PdR to the Pd and the Pd to the
P450cam domains. Oxygen and NADH consumption is tightly coupled to substrate
oxidation in the fusion proteins. The rate-limiting step in the catalytic
turnover of these fusion proteins is electron transfer from Pd to P450cam. This
is indicated by high rates of electron transfer from the PdR and Pd domains to
exogenous electron acceptors, by an increase in the activity of the P450cam
domain upon addition of exogenous Pd, and by the high activity of wild-type
P450cam when incubated with a PdR-Pd fusion protein. E. coli cells expressing the
PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor
and 5-oxocamphor. E. coli cells expressing the triple fusion protein thus
constitute the first heterologous self-sufficient catalytic system for the
oxidation of camphor and other substrates by P450cam.

PMID: 8798411 [PubMed - indexed for MEDLINE]


517. J Biol Chem. 1996 Sep 6;271(36):21891-7.

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as 
revealed by affinity labeling with a photoreactive dNTP analog.

Lavrik OI, Prasad R, Beard WA, Safronov IV, Dobrikov MI, Srivastava DK, Shishkin 
GV, Wood TG, Wilson SH.

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian
Academy of Sciences, 630090 Novosobirsk, Russia.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse
transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a
photoreactive analog of dCTP,
exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate
(FABdCTP). Two approaches of photolabeling were utilized. In one approach,
photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the 
presence of each enzyme and resulted in polymerase radiolabeling. In an alternate
approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled
primer-template was added, and the enzyme-linked dCTP analog was incorporated
onto the 3'-end of the radiolabeled primer. The results showed strong labeling of
the p66 subunit of RT, with only minor labeling of p51. No difference in the
intensity of cross-linking was observed with either approach. FABdCTP
cross-linking was increased in the presence of a dideoxyterminated
primer-template with RT, but not with beta-pol, suggesting a significant
influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of
beta-pol residues observed to interact with the incoming dNTP in the crystal
structure of the ternary complex resulted in labeling consistent with kinetic
characterization of these mutants and indicated specific labeling of the dNTP
binding pocket.

PMID: 8702991 [PubMed - indexed for MEDLINE]


518. J Biol Chem. 1996 Aug 23;271(34):20885-94.

The dopamine transporter carboxyl-terminal tail. Truncation/substitution mutants 
selectively confer high affinity dopamine uptake while attenuating recognition of
the ligand binding domain.

Lee FJ, Pristupa ZB, Ciliax BJ, Levey AI, Niznik HB.

Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8,
Canada.

In order to delineate structural motifs regulating substrate affinity and
recognition for the human dopamine transporter (DAT), we assessed [3H]dopamine
uptake kinetics and [3H]CFT binding characteristics of COS-7 cells transiently
expressing mutant DATs in which the COOH terminus was truncated or substituted.
Complete truncation of the carboxyl tail from Ser582 allowed for the expression
of biphasic [3H]dopamine uptake kinetics displaying both a low capacity (Vmax
approximately 0.4 pmol/10(5) cells/min) high affinity (Km approximately 300 nM)
component and one exhibiting low affinity (Km approximately 15 microM] and high
capacity (Vmax approximately 5 pmol/10(5)cells/min) with a concomitant 40%
decrease in overall apparent Vmax relative to wild type (WT) DAT. Truncation of
the last 22 amino acids or substitution of the DAT-COOH tail with sequences
encoding the intracellular carboxyl-terminal of either dopamine D1 or D5
receptors produced results that were identical to those with the fully truncated 
DAT, suggesting that the induction of biphasic dopamine uptake kinetics is likely
conferred by removal of DAT-specific sequence motifs distal to Pro597. The
attenuation of WT transport activity, either by lowering levels of DAT expression
or by pretreatment of cells with phorbol 12-myristate 13-acetate (1 microM), did 
not affect the kinetics of [3H]dopamine transport. The estimated affinity of
dopamine (Ki approximately 180 nM) for all truncated/substituted DAT mutants was 
10-fold lower than that of WT DAT (approximately 2000 nM) and appears selective
for the endogenous substrate, since the estimated inhibitory constants for
numerous putative substrates or uptake inhibitors were virtually identical to
those obtained for WT DATs. In marked contrast, DAT truncation/substitution
mutants displayed significantly reduced high affinity [3H]CFT binding
interactions with estimated Ki values for dopamine and numerous other substrates 
and inhibitors tested from 10-100-fold lower than that observed for WT DAT.
Moreover, co-expression of truncated and/or substituted DATs with WT transporter 
failed to reconstitute functional or pharmacological activities associated with
both transporters. Instead, complete restoration of uniphasic low affinity
[3H]dopamine uptake kinetics (Km approximately 2000 nM) and high affinity
substrate and inhibitor [3H]CFT binding interactions attributable to WT DATs were
evident. These data clearly suggest the functional independence and differential 
regulation of the dopamine translocation process from the characteristics
exhibited by its ligand binding domain. The lack of functional phenotypic
expression of mutant DAT activities in cells co-expressing WT transporter is
consistent with the contention that native DATs may exist as multisubunit
complexes, the formation and maintenance of which is dependent upon sequences
encoded within the carboxyl tail.

PMID: 8702845 [PubMed - indexed for MEDLINE]


519. Mol Cell Probes. 1996 Aug;10(4):247-56.

Detection of M. tuberculosis DNA using thermophilic strand displacement
amplification.

Spargo CA, Fraiser MS, Van Cleve M, Wright DJ, Nycz CM, Spears PA, Walker GT.

Department of Molecular Biology, Becton Dickinson Research Center, Research
Triangle Park, NC 27709, USA.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of
amplifying DNA that is based upon the combined action of a DNA polymerase and
restriction enzyme. Previously, a form of SDA was developed which utilizes the
exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and
the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37
degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new
thermophilic form of SDA is reported here which uses a restriction endonuclease
from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient
polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from
Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved
after 15 min of SDA at 60 degrees C. The new thermophilic system is much more
specific than the previous mesophilic system as evidenced by a dramatic decrease 
in background amplification products. Thermophilic SDA was also optimized with
dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA
glycosylase.

PMID: 8865173 [PubMed - indexed for MEDLINE]


520. DNA Cell Biol. 1996 Jul;15(7):589-94.

Fidelity and predominant mutations produced by deep vent wild-type and
exonuclease-deficient DNA polymerases during in vitro DNA amplification.

Huang H, Keohavong P.

Department of Environmental and Occupational Health, University of Pittsburgh, PA
15238, USA.

Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates
and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent
DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in
the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was
found to decrease the error rate of the wt, but not that of the exo-, Deep Vent. 
The average errors per base duplication for the native form were 8.0 x 10(-5) and
6.0 x 10(-5) in the absence and presence of gp32, respectively. For the exo-
form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base
duplication in the absence and presence of gp32, respectively. Examination of
mutations produced by native Deep Vent showed that A/T to G/C transition
predominated, consistent with the results of our earlier studies with DNA
polymerases derived from other thermophilic bacteria. These results indicate that
PCR with high fidelity can be achieved by using wt Deep Vent in combination with 
gp32.

PMID: 8756340 [PubMed - indexed for MEDLINE]


521. Am J Respir Cell Mol Biol. 1996 Jul;15(1):132-40.

Pseudomonas aeruginosa and epithelial permeability: role of virulence factors
elastase and exotoxin A.

Azghani AO.

Department of Biochemistry, University of Texas Health Science Center at Tyler
75710, USA.

Lung injury in bacterial infection is a multifactorial phenomenon that involves
bacterial metabolites and host factors. Primary isolates of type II pneumocytes
and established cultures of Madin-Darby canine kidney (MDCK) cells were used to
study effects of Pseudomonas aeruginosa exoproducts on epithelial paracellular
permeability. The results indicate that elastase (PE) and exotoxin A (Exo A) have
different, but complementary, actions that diminish epithelial barrier function. 
We measured transepithelial electrical resistance (TER) and permeability
coefficient for mannitol (Pm) across cell monolayers plated on tissue culture
membranes. Application of 100 ng/ml of Exo A to the basal side decreased TER from
1,405 +/- 106 to 462 +/- 50 ohm (omega) and increased Pm for mannitol 6-fold in
16 h (P < 0.05). Application of Exo A to the apical side did not affect either
TER or Pm. In contrast, PE (6.5 U/ml) applied either apically or basolaterally
reduced TER to 353 +/- 66 omega and increased Pm by 10-fold within 90 min (P <
0.05). The increase in permeability correlated with the number of bacteria that
traversed the epithelial monolayers. Fluorescent staining and western immunoblot 
analysis of toxin-treated cells showed that two tight junctional proteins, ZO-1
and ZO-2, were depleted in monolayers treated with enzymatically active PE. The
junctional proteins decreased in cells treated overnight with Exo A but were not 
depleted. Neither agent diminished cell viability as measured by trypan blue
staining or release of radioactivity from 51 Cr-labeled cells. Elastase from P.
aeruginosa thus seems to increase alveolar epithelial permeability by damaging
tight junction-associated proteins. Exo A, through its effect on protein
synthesis, may render the cells unable to restore the junctional proteins and
thus the functional junctions.

PMID: 8679217 [PubMed - indexed for MEDLINE]


522. EMBO J. 1996 Jul 1;15(13):3430-41.

A DNA binding motif coordinating synthesis and degradation in proofreading DNA
polymerases.

Truniger V, Lazaro JM, Salas M, Blanco L.

Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma,
Madrid, Spain.

The functional significance of the conserved motif 'YxGG/A', located between the 
3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, 
has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single
substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant
derivatives were overproduced in Escherichia coli and purified to homogeneity.
Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a
template/primer DNA structure, giving rise to three different mutant phenotypes: 
(i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low
pol/exo ratio); and (iii) favored exonucleolysis and null polymerization.
Interestingly, these three different phenotypes could be obtained by mutating a
single amino acid at the 'YxGG/A' motif. All different phenotypes could be
directly related to defects in DNA binding at a particular active site. Thus, a
high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease
active site. On the contrary, a low pol/exo ratio or null polymerization capacity
was related to a poor stability at the polymerization active site and either a
normal or an increased accessibility to the exonuclease active site. These
results allow us to propose that this motif, located in the connecting region
between the N-terminal and C-terminal domains, has a primary role in DNA binding,
playing a critical role in the coordination or cross-talk between synthesis and
degradation.

PMCID: PMC451907
PMID: 8670845 [PubMed - indexed for MEDLINE]


523. J Org Chem. 1996 Jun 26;61(13):4361-4368.

Stereochemical Aspects in the Asymmetric Michael Addition of Chiral Imines to
Substituted Electrophilic Alkenes.

Cave C, Desmaele D, d'Angelo J, Riche C, Chiaroni A.

Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse, 91198 
Gif-sur-Yvette, France.

The Michael-type addition of chiral imines, derived from racemic
alpha-substituted cyclanones and optically active 1-phenylethylamine, to
electrophilic alkenes, in neutral conditions, constitutes one of the most
efficient methods for the stereocontrolled construction of quaternary carbon
centers. In order to create an additional stereogenic center at the alpha- or
beta-position to the quaternary one, the behavior of a variety of alpha- and
beta-substituted alkenyl acceptors was examined. In general, these additions are 
highly regioselective, the alkylation taking place predominantly, if not
exclusively, at the more substituted alpha-side of the imine function; however,
in some cases (electrophilic alkenes 28 and 49), significant amounts (10-15%) of 
regioisomeric adducts were obtained. With the exception of methyl propiolate 52, 
a remarkable control of the absolute configuration of the adducts were always
observed with these Michael acceptors. According to the general rule we have
previously proposed, the alkylation process takes place preferentially on the
less hindered pi-face of the more substituted secondary enamine, in tautomeric
equilibrium with the starting imine. An excellent diastereocontrol was always
obtained by using the present alpha- and beta-substituted alkenes. These
stereochemical outcomes can be interpreted by invoking that the reaction proceeds
through a compact approach of the reactants, the hydrogen atom at the nitrogen
center of the enamine being transferred to the alpha-vinylic carbon atom of the
acceptor, concertedly with the creation of the C-C bond. In this respect the
"endo-approach" 58, in which the electron-withdrawing group of the acceptor faced
to the nitrogen atom of the enamine (case of acceptors 10, methyl methacrylate,
24, 28, 43, 47, and 49) largely prevails over the "exo-approach" 59 (case of
acceptor 38). This predominant "endo-preference" can be reasonably interpreted in
terms of a cooperative effect between steric and stereoelectronic factors.

PMID: 11667338 [PubMed - as supplied by publisher]


524. J Org Chem. 1996 May 31;61(11):3806-3814.

Synthesis of Tricyclic beta-Methylene Spiro Lactones Related to Bakkenolides by
Successive Radical Cyclization-High Pressure Diels-Alder Reactions.

Back TG, Gladstone PL, Parvez M.

Department of Chemistry, University of Calgary, Calgary, Alberta, Canada T2N 1N4.

The synthesis of some novel bakkenolides and their epi-spiro analogues was
achieved by a new approach. Photolysis of allyl
1-(phenylseleno)-2-oxocyclopentanecarboxylates 7-9 afforded the corresponding
spiro lactones 10-12 by radical cyclization via group transfer of the
phenylseleno group. Selenoxide elimination of 11 and 12 produced the
corresponding beta-methylene lactones 14 and 15. Diels-Alder cycloaddition of
lactone11 with piperylene failed at ambient pressure, but proceeded in generally 
good yield in the presence of various Lewis acids at pressures of ca. 16 kbar, to
give mixtures of beta-exo, alpha-endo, and beta-endo cycloadducts 19, 21, and 23,
respectively. The preponderance of endo products 21 and 23, formed via highly
hindered, but more compact, transition states was attributed to the high pressure
and resulted in trans-dimethyl configurations of the products. The facial
selectivity was dependent upon the Lewis acids, and the greatest alpha:beta ratio
was observed with catalysts of the type TiCl(2)(OR)(2). Epimerization of the C-4 
methyl group in 21 and 23 to furnish the corresponding cis-dimethyl analogues was
achieved via exo-epoxidation, regioselective reduction, oxidation to the
corresponding 3-keto derivatives, and base-catalyzed equilibration, thereby
affording (+/-)-3,6-dioxobakkenolide-A (39) and its epi-spiro derivative 28,
respectively. When the radical cyclization step was performed subsequent to the
Diels-Alder cycloaddition by photolysis of perhydrindane 43, only the epi-spiro
product 44 was obtained.

PMID: 11667233 [PubMed - as supplied by publisher]


525. FEBS Lett. 1996 May 6;385(3):244.

Commentary on: 'Thermostability of beta-xylosidase from Aspergillus sydowii MG49'
by M. Ghosh and G. Nanda, FEBS Letters 330, 275-278 (1993)

Ananthapadmanaban D.

Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial
Technology, Chandigarh, India.

Comment on:
    FEBS Lett. 1993 Sep 20;330(3):275-8.

PMID: 8647261 [PubMed - indexed for MEDLINE]


526. Appl Environ Microbiol. 1996 May;62(5):1774-80.

Production and processing of a 59-kilodalton exochitinase during growth of
Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or
fungal chitin.

Vionis AP, Niemeyer F, Karagouni AD, Schrempf H.

Universitat Osnabruck, Germany.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces
olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa
exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin
type III-like module, and a chitin-binding domain (12 kDa). The propagation rate 
of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal
mycelia than in those containing crab chitin. Comparative biochemical and
immunological studies allowed the following conclusions to be drawn. Within soil 
microcosm systems amended with crab shell chitin or chitin-containing Aspergillus
proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced 
high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached 
via its binding domain to the pellet from soil or liquid cultures. In contrast,
truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The
relative proportions of the 59-kDa enzyme and its truncated forms varied
depending on the source of chitin and differed in soil and in liquid cultures.

PMCID: PMC167953
PMID: 8633877 [PubMed - indexed for MEDLINE]


527. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2856-61.

Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and
inviability due to error catastrophe.

Fijalkowska IJ, Schaaper RM.

Laboratory of Molecualr Genetics, NationalInstitute of Enviromental Health
Sciences, Research Triangle Park, NC 27709, USA.

The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon
subunit) of DNA polymerase III holoenzyme and is a critical determinant of
chromosomal replication fidelity. We constructed by site-specific mutagenesis a
mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and 
Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading
exonucleases, is essential for the catalytic activity. When residing on a
plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that
the protein, although deficient in exonuclease activity, still binds to the
polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was
transferred to the chromosome, replacing the wild-type gene, the cells became
inviable. However, viable dnaQ926 strains could be obtained if they contained one
of the dnaE alleles previously characterized in our laboratory as antimutator
alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene.
These results suggest that loss of proofreading exonuclease activity in dnaQ926
is lethal due to excessive error rates (error catastrophe). Error catastrophe
results from both the loss of proofreading and the subsequent saturation of DNA
mismatch repair. The probability of lethality by excessive mutation is supported 
by calculations estimating the number of inactivating mutations in essential
genes per chromosome replication.

PMCID: PMC39723
PMID: 8610131 [PubMed - indexed for MEDLINE]


528. FEMS Microbiol Lett. 1996 Mar 15;137(1):75-8.

Purification and characteristics of cytosolic chitinase from Piromyces communis
OTS1.

Sakurada M, Morgavi DP, Komatani K, Tomita Y, Onodera R.

Faculty of Agriculture, Miyazaki University, Japan.

A chitinase was purified from the cytosolic fraction of the anaerobic rumen
fungus Piromyces communis OTS1 by affinity chromatography using regenerated
chitin, gel filtration and chromatofocusing. The chitinase was most active at pH 
6.2 and at 60 degrees C in a 20-min assay. The molecular mass of the purified
protein was estimated by SDS-PAGE to be 42 kDa and its pI was 4.9. The enzyme
activity, which was of the 'endo' type, was inhibited by Ag+, Hg2+ and
allosamidin. N-Acetyl-beta-glucosaminidase and 'exo' type chitinase activity were
absent from the purified preparation.

PMID: 8935660 [PubMed - indexed for MEDLINE]


529. J Chem Inf Comput Sci. 1996 Mar-Apr;36(2):286-93.

Development of neural network simulator for structure--activity correlation of
molecules (NECO). Prediction of endo/exo substitution of norbornane derivatives
and of carcinogenic activity of PAHs from 13C-NMR shifts.

Isu Y, Nagashima U, Aoyama T, Hosoya H.

Department of Information Sciences, Ochanomizu University, Tokyo, Japan.

A perceptron type neural network simulator for structure--activity correlation of
molecules has been developed with two different learning methods, i.e.,
back-propagation and reconstruction methods. First by use of the back-propagation
method the exo/endo branching of norbornane and norbornene derivatives was
correctly predicted from the set of 13C NMR chemical shifts for various ring
carbon atoms. Then the obtained correlation was analyzed by the reconstruction
learning method. It was shown in this case that the NMR shifts for two carbon
atoms out of seven have strong correlation with the exo/endo branching. Further, 
structure--activity correlation between the 13C NMR chemical shifts and
carcinogenicity of 11 polycyclic aromatic hydrocarbons was also analyzed using
the reconstruction method. It was demonstrated that neural network analysis is
suitable for the elucidation of complicated structure--activity problems where
many factors are nonlinearly entangled.

PMID: 8882811 [PubMed - indexed for MEDLINE]


530. J Bacteriol. 1996 Mar;178(6):1600-6.

Cyclization reaction catalyzed by branching enzyme.

Takata H, Takaha T, Okada S, Takagi M, Imanaka T.

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Nishiyodogawa-ku, Osaka, 
Japan.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on 
amylose was analyzed. The enzyme reduced the molecular size of amylose without
increasing the reducing power. This result could not be explained by the normal
branching reaction model. When the product was treated with glucoamylase (an
exo++-type amylase), a resistant component remained. The glucoamylase-resistant
component was easily digested by an endo-type alpha-amylase or by isoamylase plus
glucoamylase. These results suggested that the glucoamylase-resistant component
was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In 
other words, it was suggested that branching enzyme catalyzed cyclization of the 
alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic
linkage, thereby forming two smaller molecules. Mass spectrometry also supported 
the cyclic nature of the product.

PMCID: PMC177844
PMID: 8626287 [PubMed - indexed for MEDLINE]


531. J Med Chem. 1996 Feb 16;39(4):834-41.

Tetrahydronaphthalenes: influence of heterocyclic substituents on inhibition of
steroid enzymes P450 arom and P450 17.

Wachter GA, Hartmann RW, Sergejew T, Grun GL, Ledergerber D.

Universitat de Saarlandes, Saarbrucken, Germany.

In search of new leads for selective inhibition of estrogen and androgen
biosynthesis, respectively, heterocyclic substituted
2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones
(5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and
3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized
and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, 
P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) 
(TXA(2)) synthase. The test compounds inhibited human placental P450 arom,
showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was
the most potent inhibitor, with a relative potency (rp) of 110 [rp of
aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of
inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other
hand some of these compounds inhibited rat testicular P450 17. Maximum activity
was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was 
the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) 
= 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward
the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom,
whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m)
testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17:
K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were
not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM,
K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM).

PMID: 8632407 [PubMed - indexed for MEDLINE]


532. Biochemistry. 1996 Feb 6;35(5):1485-99.

Improved binding of cytochrome P450cam substrate analogues designed to fill extra
space in the substrate binding pocket.

Helms V, Deprez E, Gill E, Barret C, Hui Bon Hoa G, Wade RC.

European Molecular Biology Laboratory, Heidelberg, Germany.

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor
analogues were designed to fill an empty region of the substrate binding pocket
with the expectation that they would bind more tightly than camphor itself due to
increased van der Waals interactions with the protein and the displacement of any
solvent occupying this site. A series of compounds (endo-borneol methyl ether,
endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl
allyl ether) were synthesized with substituents at the camphor carbonyl oxygen.
The spin conversion and thermodynamic properties of this series of compounds were
measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in
the context of molecular dynamics simulations of the camphor analogues in the
P450 binding site and in solution. Compounds with a 3-carbon chain substituent
were predicted to match the size of the unoccupied region most optimally and thus
bind best. Consistent with this prediction, the borneol allyl ether binds to
cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared
to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions).
Binding of the camphor analogues to the Y96F mutant is much enhanced over the
binding of camphor, indicating that hydrogen bonding plays a less important role 
in binding of these analogues. Binding enthalpies calculated from the
simulations, taking all solvent contributions into account, agree very well with 
experimental binding enthalpies. Binding affinity is not however correlated with 
the calculated binding enthalpy because the binding of the substrate analogues is
characterized by enthalpy-entropy compensation. The new compounds are useful
probes for further studies of the mechanism of cytochrome P450cam due to their
high binding affinities and high spin properties.

PMID: 8634279 [PubMed - indexed for MEDLINE]


533. Biosci Biotechnol Biochem. 1996 Feb;60(2):194-9.

Purification and characterization of beta-N-acetylhexosaminidase from the liver
of a prawn, Penaeus japonicus.

Koga D, Hoshika H, Matsushita M, Tanaka A, Ide A, Kono M.

Laboratory of Biochemistry, Yamaguchi University, Yoshida, Japan.

Beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn,
Penaeus japonicus, by ammonium sulfate fractionation and chromatography with
Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m.
The purified enzyme showed a single band keeping the potential activity on both
native PAGE and SDS-PAGE. The apparent molecular weight was 64,000 and 110,000 by
SDS-PAGE and gel filtration, respectively. The pI was less than 3.2 by
chromatofocusing. The amino-terminal amino acid sequence was
NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly-
Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50 degrees C,
respectively. The enzyme was stable from pH 4 to 11, and below 55 degrees C. It
was 39% inhibited by 10 mM HgCl2. Steady-state kinetic analysis was done with the
purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and
p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAcn, n = 1 to 3) as the
substrates. The enzyme hydrolyzed all of these substrates to release monomeric
GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat 
at 25 degrees C and pH 5.5 were 0.137 mM and 598 s-1 for pNp-beta-GlcNAc, 0.117
mM and 298 s-1 for GlcNAc2, 0.055 mM and 96.4 s-1 for GlcNAc3, 0.044 mM and 30.1 
s-1 for GlcNAc4, 0.045 mM and 14.7 s-1 for GlcNAc5, and 0.047 mM and 8.3 s-1 for 
GlcNAc6, respectively. These results suggest that this
beta-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin
degradation, and prefers the shorter substrates.

PMID: 9063965 [PubMed - indexed for MEDLINE]


534. Chem Pharm Bull (Tokyo). 1996 Feb;44(2):307-13.

Synthesis and pharmacological evaluation of
N-(6-functionalized-amino-3-pyridyl)-N'-bicycloalkyl-N''-cyanoguanidine s as
antihypertensive agents.

Eda M, Takemoto T, Okada T, Sakashita H, Matzno S, Gohda M, Hayashi K, Nakamura
N, Fukaya C.

Research Division, Green Cross Corporation, Osaka, Japan.

A series of amino acid conjugates of
N-(6-amino-3-pyridyl)-N'-[exo-bicyclo[2.2.1]hept-2-yl]-N''- cyanoguanidine (4)
were prepared and evaluated as antihypertensive agents. The parent compound 4
showed potent potassium channel-opening and antihypertensive activities, but with
undesirable changes of the urinary balance of electrolytes. However, alanine and 
histidine congeners (9,19) reduced this undesirable side effect of 4 through
improved pharmacokinetics without loss of antihypertensive activity. They also
provided additional information on the structural requirements for pinacidil-type
potassium channel openers.

PMID: 8998837 [PubMed - indexed for MEDLINE]


535. Electrophoresis. 1996 Feb;17(2):372-8.

Detection and differentiation of pectic enzyme activity in vitro and in vivo by
capillary electrophoresis of products from fluorescent-labeled substrate.

Zhang Z, Pierce ML, Mort AJ.

Department of Biochemistry and Molecular Biology, Oklahoma State University,
Stillwater 74078-3035, USA.

A sensitive assay is described for the detection of pectate-depolymerizing
enzymes using capillary electrophoresis of a fluorescent end-labeled pectate
oligomer. The labeled oligomer is allowed to react with the enzyme either in
vitro or in vivo, such as inside the intercellular spaces of a cotton cotyledon, 
and after an appropriate incubation time the products are analyzed by capillary
electrophoresis. The site and mode of action of the pectate-depolymerizing
activity can be inferred from the products. Both endo- and exopolygalacturonase
activity, and lyase activity, were distinguished. Since only the fluorescent
oligomer and products from its labeled reducing end are detected, there is no
interference from other compounds; only pectic enzyme activity is detected. By
this type of analysis we can show that there is considerable endo- and
exo-polygalacturonase activity in the intercellular spaces of cotton cotyledons.

PMID: 8900945 [PubMed - indexed for MEDLINE]


536. Analyst. 1996 Feb;121(2):105-9.

Raman spectroscopic determination of thymidine nucleoside structures in
nucleotides.

Escobar R, Carmona P, Molina M.

Departamento de Quimica Analitica, Universidad de Sevilla, Spain.

Several spectral differences in Raman spectra were observed for thymidine (TM),
3'-O-acetylthymidine (3'OATM), 3',5'-di-O-acetylthymidine (3'5'DOATM), thymidine 
5'-monophosphoric acid (H2TMP) and its Ca2+, Sr2+ and Ba2+ salts owing to
nucleoside conformational differences in the thymidine moiety. The spectroscopic 
characterization of the nucleoside structures was made on the basis of previous
crystallographic works relative to TM, 3'OATM, 3'5'DOATM and Ba(TMP).6H2O. The
crystal structures of the other compounds reported here are, as yet, unknown.
However, their nucleoside structures were inferred from the position of the
phosphoester stretching band. The results show that C(2')-endo-anti,
C(3')-endo-anti and C(2')-endo/C(3')-exo-anti puckers generate marker bands
useful to characterize and titrate these thymidine nucleoside conformations. The 
furanose sugar puckering of the C(2')-endo-anti geometry has been suggested to
account for the enhanced activity of thymidine derivatives against human
immunodeficiency virus-type 1. A Raman spectroscopic method is described for the 
determination of this structure in thymidine nucleosides and nucleotides.

PMID: 8849034 [PubMed - indexed for MEDLINE]


537. Int Urol Nephrol. 1996;28(4):485-94.

Stress urinary incontinence in women. II. Abnormalities of glycogenolysis in
tissues related to the lower urinary tract.

Jozwik M Jr, Lotocki W, Jozwik M.

Department of Gynaecology and Septic Obstetrics, School of Medicine, Bialystok,
Poland.

The aim of the study was the investigation of the biochemical condition of
elements likely to directly participate in active closing of the urethral lumen. 
We estimated glycogenolysis in urinary bladder, perivesical connective tissue and
levator ani muscle (LAM) samples obtained intraoperatively from 80 stress
incontinent women. Glycogen content as well as activities of active and total
glycogen phosphorylase and acid exo-1,4-alpha-glucosidase were measured. Material
from the urinary bladder and perivesical connective tissue was insignificantly
altered, and glycogen contents in the bladder (2.03 +/- 1.38 g/100 g wet tissue) 
were considered to be normal. In the LAM glycogenolysis was much more activated
than in other tissues (p < 0.001 by Fischer's exact test). Of LAM specimens 78%
(22/28) revealed imbalanced biochemistry of glycogen with activation of
hydrolytic decomposition. We conclude that stress urinary incontinence in women
is frequently associated with metabolic alterations in the periurethral striated 
fibres. This study indirectly supports our recent hypothesis on the pathogenesis 
of the disease in terms of muscle fibre type transitions.

PMID: 9119633 [PubMed - indexed for MEDLINE]


538. Biochimie. 1996;78(8-9):763-70.

NMR studies of recombinant cytochrome P450cam mutants.

Wakasugi K, Ishimori K, Morishima I.

Division of Molecular Engineering, Graduate School of Engineering, Kyoto
University, Japan.

In the active center of cytochrome P450cam, Thr-252 is one of the conserved amino
acid residues in the cytochrome P450 superfamily and plays a key role in the
hydroxylation of camphor. T252A mutant, in which Thr-252 is replaced by alanine, 
consumed O2 at a rate comparable to that of the wild-type enzyme, whereas the
amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the
wild-typed enzyme and H2O2 is the main product in the hydroxylation reaction.
H2O2 was also yielded by the valine mutant and the consumption rate of O2 was
much lower than that for the wild-type enzyme (Imai et al (1989) Proc Natl Acad
Sci USA 86, 7823-7827). On the basis of the 1H- and 15N-NMR spectra, it was
revealed that the anionic nature of the axial thiolate and the heme-environmental
structures were substantially affected in the absence of d-camphor by the amino
acid substitution at 252 Thr. In T252A mutant, however, the binding of camphor
reduced these conformational alterations in the heme vicinity, probably due to
the formation of interactions between camphor and enzyme. On the other hand,
T252V mutant still exhibited large reduction of the anionic nature of the axial
ligand in the presence of d-camphor and structural changes around heme were also 
enhanced, since the affinity of the valine mutant to d-camphor was low. These
results imply that the hydrophobic and/or steric effects of the valine residue at
252 interfere with interactions around heme and camphor binding sites, which
corresponds to the larger functional defects for T252V mutant.

PMID: 9010605 [PubMed - indexed for MEDLINE]


539. Prostate Suppl. 1996;7:25-9.

Analysis of glycosylation of prostate-specific membrane antigen derived from
LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.

Holmes EH, Greene TG, Tino WT, Boynton AL, Aldape HC, Misrock SL, Murphy GP.

Pacific Northwest Cancer Foundation, Department of Cell Surface Biochemistry,
Seattle, Washington, USA.

BACKGROUND: Prostate-specific membrane antigen (PSMA) has been detected in human 
prostatic cancer tissues, serum, and seminal fluid based on Western blot data
with the monoclonal antibody 7E11.C5. The reactive protein is very similar in
size to that from human prostatic carcinoma LNCaP cells and corresponds to a
protein with a molecular size of about 110,000 daltons. Given that PSMA is known 
to be a 750 amino acid protein of about 84,000 daltons, a substantial portion,
perhaps 20-25% of the native molecular weight, is composed of carbohydrates.
METHODS: In this study, we have begun initial analyses of the glycosylation of
the PSMA protein from multiple sources using a variety of exo- and
endoglycosidase treatments. RESULTS: The results indicate that the carbohydrate
is primarily N-linked and in each case the deglycosylated protein has an apparent
molecular weight of about 86,000 daltons. The glycan present on in vivo-derived
PSMA from tumor tissue or serum was found to be primarily N-linked complex type. 
A small amount of O-linked glycan also appears to be present. In contrast, only
high mannose-type N-linked glycans are present on the PSMA from LNCaP cells.
CONCLUSIONS: Oligosaccharides present on PSMA derived from both tissue culture
LNCaP cells and in vivo specimens are primarily N-linked and comprise about
20-25% of the native molecular weight. N-linked glycans of PSMA derived from in
vivo sources were found to be complex type, lacking polylactosamine structures.
In contrast, LNCaP cells express only high mannose-type structures. These results
will be useful in our ongoing efforts to develop monoclonal antibodies which are 
specific for protein epitopes present in the extracellular domain of the protein.

PMID: 8950359 [PubMed - indexed for MEDLINE]


540. Chem Res Toxicol. 1996 Jan-Feb;9(1):241-6.

Retro-Diels-Alder reaction: possible involvement in the metabolic activation of
7-oxabicyclo[2.2.1]hepta-2(3),5(6)-diene-2,3-dicarboxylates and a phosphonate
analog.

Mahajna M, Quistad GB, Casida JE.

Department of Environmental Science, Policy, and Management, University of
California, Berkeley 94720-3112, USA.

A discontinuous structure-activity relationship signaled a change in mode of
action and led to the discovery of a possible novel metabolic activation
mechanism. The toxicity of the herbicide endothal
(exo,exo-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid) to mice (ip LD50 = 14 
mg/kg) is attributed to the inhibition of protein phosphatase 2A (PP2A) at the
cantharidin binding site. The potency is reduced by the introduction of a 2,3- or
5,6-double bond. Surprisingly, high toxicity (ip LD50's = 15-50 mg/kg) is
restored in oxabicyclohepta-2(3),5(6)-dienes substituted in the 2- and
3-positions with bis(methyl carboxylate), bis(ethyl carboxylate), and diethyl
phosphonate/ethyl carboxylate, whereas the dicarboxylic acid, bis(tert-butyl
carboxylate), and bis(dimethyl phosphonate) are inactive. The diene adducts do
not inhibit the cantharidin binding site of PP2A. Two observations provided an
alternative working hypothesis that the active but not the inactive diene adducts
are protoxicants: GC analyses revealed that selected bicyclic dienes readily
undergo thermal dissociation by retro-Diels-Alder reactions to liberate
disubstituted acetylenes; the liberated acetylenes have mouse ip LD50's of 8-25
mg/kg. Apparent exceptions to this hypothesis are that bicyclic dienes with
bis(tert-butyl carboxylate) and bis(dimethyl phosphonate) substituents are not
toxic, yet their corresponding acetylenes are quite toxic. These apparent
anomalies are resolved by finding that only the toxic bicyclic dienes readily
react with albumin and 4-nitrobenzenethiol and that their low-toxicity analogs
are much less reactive. Albumin can be replaced by hemoglobin but not by
myoglobin or chymotrypsin in reaction with a bicyclic diene indicating the
importance of the free thiol group. Diethyl oxabicycloheptadienedicarboxylate
readily reacts with GSH to give two products, which are also formed from the
corresponding acetylene, identified as the cis and trans isomers of the
GSH-acetylene conjugate. This is the first proposal, to our knowledge, that a
retro-Diels-Alder-type reaction is involved in the metabolic activation of a
toxicant.

PMID: 8924598 [PubMed - indexed for MEDLINE]


541. Tsitologiia. 1996;38(2):115-8.

[The ultrastructure of the epithelial endocrinocytes of the appendiceal mucosa in
appendicitis patients]

[Article in Russian]

Kostiukevich SV.

The electron microscopic study of the vermiform process epithelium under
inflammation showed five types of endocrinocytes, based on the ultrastructure of 
secretory granules: EC, DI, L, I, P. The overwhelming majority of endocrinocytes 
form EC-cells. Besides, cells of a "mixed" type, involving exo- and
endo-endocrinic cells, were revealed. Morphologic and functional alteration of
the human vermiform process epithelium endocrinocytes under inflammation have
been noticed.

PMID: 8754127 [PubMed - indexed for MEDLINE]


542. Biochemistry. 1995 Dec 19;34(50):16306-12.

Structure of binary and ternary complexes of zinc and cobalt carboxypeptidase A
as determined by X-ray absorption fine structure.

Zhang K, Auld DS.

Biostructure Institute, University City Science Center, University of
Pennsylvania, Philadelphia 19104, USA.

Carboxypeptidase A, ZnCPD, is typical of a wide range of exo- and
endo-metalloproteases that have three protein ligands and a water molecule bound 
to a catalytic zinc atom and a glutamate residue in the active site that likely
acts in conjunction with the Zn-bound water to bring about catalysis. Such
enzymes generally have bell-shaped pH-activity profiles (EH2 in equilibrium with 
EH in equilibrium with E) where the concentration of the catalytic species EH is 
regulated by pKEH2 and pKEH. The present X-ray absorption fine structure, XAFS,
study has determined the structure and pH behavior of the binary and ternary
product complexes in order to examine the role of the Zn-bound water in
catalysis. Increasing the pH from 7 to 10 of the ZnCPD.L-Phe complex results in
the same type of progressive spectral changes in the near-edge XAFS spectrum as
is seen for ZnCPD, but the changes are complete by more than 1 pH value below
that observed for ZnCPD. The results are in agreement with kinetic studies that
show E binds the protonated form of L-Phe more tightly than EH, thus in effect
decreasing the value of pKEH. The XAFS results show the average interatomic
distance. R. for the zinc ligands of the EH.L-Phe complex decreases by 0.02 A
upon formation of the E.L-Phe complex, essentially identical to that obtained for
the EH and E forms of the native enzyme. Addition of azide to ZnCPD.L-Phe at pH 7
markedly changes the zinc coordination sphere from 4 N/O atoms at 2.021 +/- O.06 
A and 1.4 +/- 0.5 N/O atoms at 2.54 +/- 0.5 A to 3.9 N/O atoms at 1.995 +/- 0.006
A. The decrease in R of about 0.03 A in both the Zn- and CoCPD.L-Phe.N3-complexes
is likely due to the ligand exchange from a neutral water to an anion. The XAFS
spectra of the ternary complex is pH independent from 7 to 9, in agreement with
the ionization of the water being the source of the spectral changes in the free 
enzyme and its binary L-Phe complex. The enzyme.azide.L-Phe complex is likely
bound in a manner analogous to that expected for a post-transition state in a
biproduct complex for peptide hydrolysis--that is, the carboxylate anion of the
peptide bound to the Zn and the protonated form of L-Phe H-bonded to the
catalytic Glu-270 carboxylate. The XAFS results on the "spectroscopically silent"
ZnCPD are compared to nuclear magnetic resonance and electron absorption studies 
on CoCPD.

PMID: 8845355 [PubMed - indexed for MEDLINE]


543. Mol Gen Genet. 1995 Dec 15;249(5):487-97.

Extension of the Rhizobium meliloti succinoglycan biosynthesis gene cluster:
identification of the exsA gene encoding an ABC transporter protein, and the exsB
gene which probably codes for a regulator of succinoglycan biosynthesis.

Becker A, Kuster H, Niehaus K, Puhler A.

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Federal
Republic of Germany.

Two new genes, designated exsA and exsB, were identified adjacent to the 24 kb
exo gene cluster of Rhizobium meliloti, which is involved in succinoglycan (EPS
I) biosynthesis. The derived amino acid sequence of ExsA displayed significant
homologies to ATP binding cassette (ABC) transporter proteins. R. meliloti
strains mutated in exsA were characterized by a decreased ratio of HMW to LMW EPS
I, indicating a function for ExsA in EPS I biosynthesis. The R. meliloti NdvA
protein, which is involved in the transport of cyclic beta-(1,2)-glucans, was
identified as the closest homologue of ExsA. R. meliloti exsB mutants produced a 
three-fold increased amount of EPS I in comparison to the wild-type strain. In
contrast, high copy number of exsB resulted in a decrease in the EPS I level to
20% of wild type, indicating that the exsB gene product can negatively influence 
EPS I biosynthesis. It was demonstrated that this influence is not due to
transcriptional regulation of the exo genes by the exsB gene product. By plasmid 
integration it was shown that exsA and exsB represent monocistronic transcription
units.

PMID: 8544814 [PubMed - indexed for MEDLINE]


544. J Mol Biol. 1995 Dec 15;254(5):856-68.

Modelling viral evolution in vitro using exo- Klenow polymerase: continuous
selection of strand displacement amplified DNA that binds an oligodeoxynucleotide
to form a triple-helix.

Walter NG.

Max-Planck-Institute for Biophysical Chemistry, Department of Biochemical
Kinetics, Gottingen, Germany.

Evolution comprises cycles of amplification, mutagenesis and selection. To study 
evolutionary phenomena, isothermal strand displacement amplification (SDA) of
double-stranded DNA as an in vitro model for rolling-circle replication of
viruses has been coupled to a positive selection procedure. First, two subsequent
amplification reactions utilizing exo- Klenow polymerase were performed under
direct observation using the fluorescent dye thiazole orange. Under the chosen
conditions, the mutation rate was 1.5 x 10(-3) and 0.4 x 10(-3) for base
substitutions and deletions, respectively. Then, a 16mer oligodeoxynucleotide
with an acridine moiety coupled to its 5' end was used to select for double
strands that retained their ability to form a triple-helix with the
oligodeoxynucleotide. Conditions for triple-helix formation were chosen such that
only 10 to 40% of the SDA products were allowed to bind the third strand.
Non-denaturing polyacrylamide gel electrophoresis was used to separate
triple-helices from unmodified double strands, and only triplex strands were used
to initiate a new round of error-prone amplification and selection. Nine such
rounds with about 270 molecular generations were performed. The final mutant
spectrum was characterized and compared with those of amplification reactions
without additional selection pressure. While without selection pressure base
substitutions and deletions throughout the initial wild-type rapidly produce a
diverse mutant distribution, the consensus after nine selection rounds clearly
shows two mutational hotspot positions. Using gel shift assays and a newly
developed non-radioactive DNase I footprinting technique, it could be shown that 
both the initial wild-type and the final consensus do not differ significantly in
their triplex formation ability. As opposed to this, they do show different
amplification efficiencies. The final consensus sequence is amplified with the
highest rate in the exponential reaction phase, while the most abundant clone,
which is characterized by two additional point deletions, is the sequence with
the highest amplification rate in the linear growth phase.

PMID: 7500356 [PubMed - indexed for MEDLINE]


545. Gene. 1995 Oct 16;164(1):41-4.

Detection of exonuclease activities in restriction endonuclease preparations
using an enforcement plasmid for kanamycin-resistance selection.

Hashimoto-Gotoh T, Tsujimura A, Ogasahara Y.

Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University
of Medicine, Japan.

A new enforcement (kyosei-) cloning plasmid vector, designated pKF4, was
constructed which confers kanamycin resistance (KmR) and enforces streptomycin
sensitivity (SmS). Since it is important to employ restriction endonuclease
(ENase) preparations free of exonuclease (Exo) activities for effective use of
the kyosei-cloning procedure [Hashimoto-Gotoh et al., Gene 137 (1993) 211-216],
ENases such as HpaI and SmaI purchased from four different suppliers were
examined for possible contamination by exonucleases using pKF4. The plasmid DNA
was digested with either ENase, ligated and transformed into Escherichia coli
mutants, rpsL, supE, trpR. With pKF4 intact DNA (approx. 8 ng), 2.3 x 10(5) KmR
transformant and four KmRSmR transformant colonies were obtained; the efficiency 
of transformation plating (ETP) of the intact DNA was approx. 2 x 10(-5). On the 
other hand, the ETP values were significantly higher by one to three orders of
magnitude when cut and re-joined DNAs were used under the same conditions in six 
out of eight ENase samples examined. The results indicate that even commercially 
supplied ENases, that should have passed their quality control test, could have
been contaminated with Exo sufficient to interfere with effective use of the
kyosei-cloning method. Therefore, it is advisable to examine ENase samples for
possible contamination with Exo activities, in order to choose the right
preparations for this method at the beginning of the experiments.

PMID: 7590318 [PubMed - indexed for MEDLINE]


546. Eur J Biochem. 1995 Oct 15;233(2):644-9.

Man9-mannosidase from human kidney is expressed in COS cells as a Golgi-resident 
type II transmembrane N-glycoprotein.

Bieberich E, Bause E.

Institut fur Physiologische Chemie, Bonn, Germany.

Man9-mannosidase, an alpha 1,2-specific exo-enzyme involved in N-linked
oligosaccharide processing, has been cloned recently from a human kidney cDNA
library [Bause, E., Bieberich, E., Rolfs, A., Volker, C. & Schmidt, B. (1993)
Eur. J. Biochem. 217, 533-540]. Transient expression in COS 1 cells of the enzyme
resulted in a more than 20-fold increase of a catalytic activity cleaving
specifically alpha 1,2-mannosidic linkages in [14C]Man9-GlcNAc2 or
[14C]Man5-GlcNAc2. Man9-mannosidase is expressed as a N-glycoprotein with a
molecular mass of 73 kDa. Its enzymic activity is metal ion dependent and
inhibited strongly by 1-deoxymannojirimycin (50% at 100 microM). Proteolytic
studies with the membrane-associated form of Man9-mannosidase support the view
that the enzyme is a type II transmembrane protein as predicted from its cDNA
sequence. Several lines of evidence suggest that Man9-mannosidase, as expressed, 
is N-glycosylated at one of three potential Asn-Xaa-Thr/Ser/Cys acceptor sites.
Approximately 50% of the N-linked oligosaccharide chains are removed by
endoglycosidase H treatment, whereas complete deglycosylation of the enzyme is
observed, when transfected cells were cultured in the presence of the Golgi
mannosidase II inhibitor swainsonine, indicating that the sugar moiety of
Man9-mannosidase is processed partially by Golgi-resident enzymes. This
observation is consistent with the results of indirect immunofluorescence
studies, pointing to a localization of the Man9-mannosidase predominantly in the 
juxtanuclear Golgi region. This localization clearly differs from that of pig
liver Man9-mannosidase which appears to be located in the endoplasmic reticulum
and transient vesicles.

PMID: 7588811 [PubMed - indexed for MEDLINE]


547. Genomics. 1995 Oct 10;29(3):616-22.

Isolation of murine telomere-proximal sequences by affinity capture and PCR.

Rounds D, Brueckner M, Ward DC.

Yale University School of Medicine, Department of Molecular Biophysics and
Biochemistry, New Haven, Connecticut 06510, USA.

We describe a method of selectively enriching for murine telomere-proximal
sequences using affinity capture followed by PCR amplification. The telomeric
fragments were selected from NotI-digested and lambda exonuclease-resected mouse 
genomic DNA by annealing to a biotinylated riboprobe containing multiple copies
of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively 
retained on a matrix with covalently bound avidin. The captured DNA was then
specifically released by ribonuclease action, and PCR amplification was performed
using mouse repeat primers. The PCR products were cloned and used to screen a
mouse genomic cosmid library, and the resultant cosmid clones were analyzed by
fluorescence in situ hybridization. Ten of 70 clones analyzed gave
telomere-proximal hybridization signals, indicating an at least 500-fold
enrichment for telomere-proximal sequences.

PMID: 8575753 [PubMed - indexed for MEDLINE]


548. Appl Microbiol Biotechnol. 1995 Oct;43(5):808-14.

Production and properties of three pectinolytic activities produced by
Aspergillus niger in submerged and solid-state fermentation.

Acuna-Arguelles ME, Gutierrez-Rojas M, Viniegra-Gonzalez G, Favela-Torres E.

Departamento de Biotecnologia, Universidad Autonoma, D.F., Mexico.

Three extracellular pectinases were produced by Aspergillus niger CH4 by
submerged and solid-state fermentation, and their physicochemical and kinetic
properties were studied. The highest productivities of endo- and exo-pectinase
and pectin lyase were obtained with solid-state fermentation. The kinetic and
physicochemical properties of these enzymes were influenced by the type of
culture method used. All activities were very different in terms of pH and
temperature optima, stability at different pH and temperature values and affinity
for the substrate (Km values). In solid-state fermentation, all pectinase
activities were more stable at extreme pH and temperature values but the Km
values of endo-pectinase and pectin lyase were higher with respect to those
activities obtained by the submerged-culture technique. The pectin lyase activity
obtained by the submerged-culture technique showed substrate inhibition but the
enzyme obtained by solid-state fermentation did not. Electrophoresis, using
sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for
both culture methods, showed the same number of protein bands but some
differences were found in their electrophoretic position. The results obtained in
this work suggest that the culture method (submerged or solid-state) may be
responsible for inducing changes in some of the pectinolytic enzymes produced by 
A. niger.

PMID: 7576547 [PubMed - indexed for MEDLINE]


549. Biochem J. 1995 Oct 1;311 ( Pt 1):67-74.

Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic
bacterium Cellulomonas fimi.

Shen H, Gilkes NR, Kilburn DG, Miller RC Jr, Warren RA.

Department of Microbiology and Immunology, University of British Columbia,
Vancouver, Canada.

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a
polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids
long, with a calculated molecular mass of 109765 Da. The enzyme comprises five
domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin
type III repeats of 97 amino acids each, and a C-terminal cellulose-binding
domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl 
hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of
CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose 
is the major product of hydrolysis of cellulose. In common with two other
exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity.
CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the
cellulase system of C. fimi resembles those of fungi in comprising multiple
endoglucanases and cellobiohydrolases.

PMCID: PMC1136120
PMID: 7575482 [PubMed - indexed for MEDLINE]


550. Mol Pharmacol. 1995 Oct;48(4):774-82.

Comparative pharmacology of epibatidine: a potent agonist for neuronal nicotinic 
acetylcholine receptors.

Gerzanich V, Peng X, Wang F, Wells G, Anand R, Fletcher S, Lindstrom J.

Department of Neuroscience, University of Pennsylvania Medical School,
Philadelphia 19104-6074, USA.

Pharmacological properties of the (+)- and (-)-isomers of synthetic epibatidine, 
exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1]heptane, were compared with
nicotine and acetylcholine on several subtypes of chicken and human nicotinic
acetylcholine receptors (AChRs). Both isomers of epibatidine behaved as extremely
potent full agonists on chicken (alpha 3 beta 2, alpha 3 beta 4, alpha 4 beta 2, 
alpha 7, and alpha 8) and human (alpha 3 beta 2, alpha 3 beta 4, and alpha 7)
neuronal AChRs expressed in Xenopus oocytes. Currents induced by epibatidine were
effectively blocked by the nicotinic antagonists hexamethonium and mecamylamine. 
Apparent affinity was 100 to 1000-fold higher for epibatidine than for nicotine
or acetylcholine. EC50 values ranged from 1 nM (for homomeric chicken alpha 8) to
2 microM (for homomeric chicken alpha 7). Epibatidine showed comparatively lower 
affinity for muscle-type AChRs from Torpedo and humans (EC50 values, 1.6 and 16
microM respectively). In binding assays, epibatidine was used on AChR subtypes
immunoisolated from chicken brain and retina (alpha 4 beta 2, alpha 7, and alpha 
8), the human neuronal cell line SH-SY5Y (alpha 3 and alpha 7), Torpedo electric 
organ (alpha 1 beta 1 gamma delta), or the human rhabdomyosarcoma cell line TE671
(alpha 1 beta 1 gamma delta). Both isomers of epibatidine exhibited extremely
high affinity for all neuronal AChRs tested, with KI values ranging from 0.6 pM
(human alpha 3 AChRs) to 0.6 microM (chicken alpha 7 AChRs). In contrast,
epibatidine had lower affinity for Torpedo muscle-type AChRs (KI approximately 5 
microM). Racemic [3H]epibatidine was an effective labeling reagent for human
alpha 3 beta 2 AChRs, exhibiting a KD (0.14 nM) similar to the KI values observed
for unlabeled (+)-epibatidine (0.23 nM) or (-)-epibatidine (0.16 nM).

PMID: 7476906 [PubMed - indexed for MEDLINE]


551. Mol Plant Microbe Interact. 1995 Sep-Oct;8(5):747-54.

Pleiotropic effects of regulatory ros mutants of Agrobacterium radiobacter and
their interaction with Fe and glucose.

Brightwell G, Hussain H, Tiburtius A, Yeoman KH, Johnston AW.

School of Biological Sciences, University of East Anglia, Norwich, U.K.

Four exo mutants of Agrobacterium radiobacter, defective in the synthesis of
acidic exopolysaccharide were complemented by a gene from that species, which is 
similar to the transcriptional regulator, ros, of A. tumefaciens. It was
confirmed that this A. radiobacter gene, which we term rosAR, like ros, repressed
its own transcription as well as that of virC and virD, two loci involved in
tumorigenesis. The sequence of RosAR suggested that it might bind to a transition
metal and its repressor abilities were shown to require Fe in the medium;
repression was also enhanced with increasing levels of glucose. Certain rosAR
mutants, in which its 3' end was removed were dominant; i.e., when plasmids
containing such mutant forms of the gene were introduced into wild-type A.
radiobacter, the transconjugants were nonmucoid. Such effects were also seen in a
wide range of bacteria, including Escherichia coli and Xanthomonas. Several
mutants that were complementd by rosAR also accumulated protoporphyrin,
suggesting a defect in haem synthesis.

PMID: 7579618 [PubMed - indexed for MEDLINE]


552. Mol Gen Genet. 1995 Aug 21;248(3):260-9.

Regulation of cell wall beta-glucan assembly: PTC1 negatively affects PBS2 action
in a pathway that includes modulation of EXG1 transcription.

Jiang B, Ram AF, Sheraton J, Klis FM, Bussey H.

Department of Biology, McGill University, Montreal, Quebec, Canada.

Analysis of genes involved in yeast cell wall beta-glucan assembly has led to the
isolation of EXG1, PBS2 and PTC1. EXG1 and PBS2 were isolated as genes that, when
expressed from multicopy plasmids, led to a dominant killer toxin-resistant
phenotype. The PTC1 gene was cloned by functional complementation of the
calcofluor white-hypersensitive mutant cwh47-1. PTC1/CWH47 is the structural gene
for a type 2C serine/threonine phosphatase, EXG1 codes for an exo-beta-glucanase,
and PBS2 encodes a MAP kinase kinase in the Pbs2p-Hog1p signal transduction
pathway. Overexpression of EXG1 on a 2 mu plasmid led to reduction in a cell wall
beta 1,6-glucan and caused killer resistance in wild type cells; while the exg1
delta mutant displayed modest increases in killer sensitivity and beta 1,6-glucan
levels. Disruption of PTC1/CWH47 and overexpression of PBS2 gave rise to similar 
beta-glucan related phenotypes, with higher levels of EXG1 transcription,
increased exo-beta-glucanase activity, reduced beta 1,6-glucan levels, and
resistance to killer toxin. Genetic analysis revealed that loss of function of
the PBS2 gene was epistatic to PTC1/CWH47 disruption, indicating a functional
role for the Ptc1p/Cwh47p phosphatase in the Pbs2p-Hog1p signal transduction
pathway. These results suggest that Ptc1p/Cwh47p and Pbs2p play opposing
regulatory roles in cell wall glucan assembly, and that this is effected in part 
by modulating Exg1p activity.

PMID: 7565587 [PubMed - indexed for MEDLINE]


553. Optom Vis Sci. 1995 Aug;72(8):580-8.

Comparison of fixation disparity curve variables measured with the Sheedy
Disparometer and the Wesson Fixation Disparity Card.

Goss DA, Patel J.

School of Optometry, Indiana University, Bloomington, USA.

BACKGROUND. Previous studies suggest differences in the fixation disparity curves
obtained with the Sheedy Disparometer and the Wesson Fixation Disparity Card, the
two most commonly used methods for measuring fixation disparity. In one study the
investigators proposed that the differences do not exist for subgroups divided by
phoria. The purpose of this paper is to try to clarify this issue by use of two
large sets of data. METHODS. Dissociated phorias were measured by the von Graefe 
method. Fixation disparity curves were plotted using the Disparometer and the
Wesson card. RESULTS. Type I fixation disparity curves were most common with the 
Wesson card. Type II curves were found more often with the Disparometer than with
the Wesson card. The x-intercepts were shifted in the base-in (BI) direction with
the Wesson card compared to the Disparometer. The y-intercepts were shifted in
the exo direction with the Wesson card compared to the Disparometer. The
differences were statistically significant regardless of whether the dissociated 
phoria was exo or eso. The slope of the fixation disparity curve was steeper with
the Wesson card than with the Disparometer. The difference was statistically
significant for exophores but not for esophores. The differences between results 
obtained with the two instruments are not consistent from one subject to another 
as shown by high standard deviations of the differences. CONCLUSIONS. The
fixation disparity curves measured with these two instruments are different.
Fixation disparity parameters obtained from one of these instruments cannot be
used with normative findings from the other.

PMID: 8539027 [PubMed - indexed for MEDLINE]


554. Nucleic Acids Res. 1995 Jul 11;23(13):2427-33.

X-ray crystal structure of a dimethylene sulfone-bridged ribonucleotide dimer in 
a single-stranded state.

Hyrup B, Richert C, Schulte-Herbruggen T, Benner SA, Egli M.

Department of Chemistry, Swiss Federal Institute of Technology, Zurich.

A crystal structure has been solved for an analog of the r(ApU) ribodinucleotide,
r(Aso2U), where a bridging non-ionic dimethylene sulfone linker replaces the
phosphodiester linking group found in natural RNA. Crystals of the
single-stranded state of r(Aso2U) were obtained from water at 50 degrees C. In
these crystals, one hydrogen bond is formed between bases from different strands 
and base stacking occurs in intermolecular 'homo-A' and 'homo-U' stacks. Similar 
to typical oligoribonucleotides, the ribose rings adopt N-type conformations and 
dihedral angles chi are in the anti range. The all-trans rotamer of the
CH2-SO2-CH2-CH2 bridge was found, which leads to a large adenine-uracil distance.
Qualitative analysis of a NOESY spectrum of the Aso2U part in r(Uso2Cso2Aso2U)
dissolved in a dimethylsulfoxide-D2O mixture indicates that the conformation
observed in the crystal is also populated in solution. Comparison with the
structure of r(Gso2C), which has been crystallized in the Watson-Crick paired
state, shows that a rotation around zeta by +112 degrees leads from the observed,
single-stranded state to a conformation that is compatible with formation of a
duplex. A concerted trans/gauche flip of alpha and gamma then yields the standard
conformer of A-type RNA helices. From the observed structure of r(Gso2C) and
other oligonucleotides it is anticipated that this flip will also revert the
ribose pucker from C2'-exo to C3'-endo.

PMCID: PMC307047
PMID: 7543197 [PubMed - indexed for MEDLINE]


555. Chem Res Toxicol. 1995 Jul-Aug;8(5):792-9.

Type I benzophenone-mediated nucleophilic reaction of
5'-amino-2',5'-dideoxyguanosine. A model system for the investigation of
photosensitized formation of DNA-protein cross-links.

Morin B, Cadet J.

CEA/Departement de Recherche Fondamentale sur la Matiere Condensee, SESAM/LAN,
Grenoble, France.

5'-Amino-2',5'-dideoxyguanosine has been synthesized in order to investigate the 
intramolecular reactivity of an amino group toward the guanine radical produced
by type I photosensitization mechanism. Benzophenone-mediated photosensitization 
of 5'-amino-2',5'-dideoxyguanosine in aerated aqueous solution results in the
formation of a predominant cyclic nucleoside together with an unstable nucleoside
precursor. The two modified nucleosides have been isolated by reverse phase high 
performance liquid chromatography and characterized by spectroscopic measurements
including 13C and 1H NMR, fast atom bombardment mass spectroscopy, and UV
absorption. The stable photoproduct has been identified as
9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-7-ol, 3-amino- (1R-exo), whereas its
precursor has been assigned as acetic acid,
[(7-hydroxy-9-oxa-2,4-diazabicyclo[4.2.1]non-2-en-3-yl)amino]oxo- (1R-exo). A
reaction mechanism, involving nucleophilic addition of the sugar amino group to
guanine radical intermediates, is proposed to explain the formation of the two
photoproducts.

PMID: 7548763 [PubMed - indexed for MEDLINE]


556. Proteins. 1995 Jul;22(3):245-58.

The catalytic domain of a bacterial lytic transglycosylase defines a novel class 
of lysozymes.

Thunnissen AM, Isaacs NW, Dijkstra BW.

Laboratory of Biophysical Chemistry, University of Groningen, The Netherlands.

The 70-kDa soluble lytic transglycosylase (SLT70) from Escherichia coli is a
bacterial exo-muramidase that cleaves the cell wall peptidoglycan, producing
1,6-anhydro-muropeptides. The X-ray structure of SLT70 showed that one of its
domains is structurally related to lysozyme, although there is no obvious
similarity in amino acid sequence. To relate discrete structural features to
differences in reaction mechanism and substrate/product specificity, we compared 
the three-dimensional structure of the catalytic domain of SLT70 with the
structures of three typical representatives of the lysozyme superfamily:
chicken-type hen egg-white lysozyme, goose-type swan egg-white lysozyme, and
phage-type lysozyme from bacteriophage T4. We find a particularly close
relationship between the catalytic domain of SLT70 and goose-type lysozyme, with 
not only a significant similarity in overall structure, but even a weak homology 
in amino acid sequence. This finding supports the notion that the goose-type
lysozyme takes up a central position in the lysozyme superfamily and that it is
structurally closest to the lysozyme ancestors. The saccharide-binding groove is 
the most conserved part in the four structures, but only two residues are
absolutely preserved: the "catalytic" glutamic acid and a structurally required
glycine. The "catalytic" aspartate is absent in SLT70, a difference that can be
related to a different mechanism of cleavage of the beta-1,4-glycosidic bond. The
unique composition of amino acids at the catalytic site, and the observation of a
number of differences in the arrangements of secondary structure elements, define
the catalytic domain of SLT70 as a novel class of lysozymes. Its fold is expected
to be exemplary for other bacterial and bacteriophage muramidases with lytic
transglycosylase activity.

PMID: 7479697 [PubMed - indexed for MEDLINE]


557. J Med Chem. 1995 Jun 9;38(12):2103-11.

Pyridyl-substituted tetrahydrocyclopropa[a]naphthalenes: highly active and
selective inhibitors of P450 arom.

Hartmann RW, Bayer H, Grun G, Sergejew T, Bartz U, Mitrenga M.

Fachrichtung 12.1 Pharmazeutische Chemie, Universitat des Saarlandes,
Saarbrucken, Germany.

The synthesis and biological evaluation of substituted
exo-1-(4-pyridyl)-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalene s as
inhibitors of estrogen biosynthesis is described [H (1); 4-OCH3 (2); 5-OCH3 (3); 
6-OCH3 (4); 1-CH3, 6-OCH3 (5); 4-OCH3, 7-Br (6); 6-OCH3, 5-Br (7); 4-OH (8); 5-OH
(9); 6-OH (10)]. The synthetic key step--the formation of the cyclopropyl
ring--was accomplished using the conditions of a modified Wolff-Kishner reduction
(N2H5OH/KOH; delta T) and yielded exclusively the exo-configurated diastereomers.
The racemic compounds 1-10 showed an inhibition of human placental aromatase
(P450 arom) exhibiting relative potencies (rp) from 3.7 to 303 (compounds 8 and
4, respectively; rp of aminoglutethimide (AG) identical to 1, fadrozole = 359).
The enantiomers of 4 and 7 were separated by LPLC on tribenzoyl cellulose and by 
crystallization of the diastereomeric tartrates (4). (1aS,2S,7bS)-(+)-4 (absolute
configuration determined by X-ray crystallographic analysis) is the active P450
arom inhibiting enantiomer of 4 and shows a rp value of 617. Compound 4 is a
reversible inhibitor showing a competitive type of inhibition and a type II
difference spectrum. In vitro 4 influenced other steroidogenic P450 enzymes
either not at all (bovine adrenal P450 scc) or only marginally (rat testicular
P450 17, bovine adrenal P450 18). In ACTH-stimulated rat adrenal tissue, 4 was
less active, inhibiting corticosterone and aldosterone formation compared to AG
and fadrozole, respectively. In vivo 4 was not superior to AG as far as the
inhibition of the uterotrophic activity of androstenedione (juvenile SD rats) and
the reduction of the plasma estradiol concentration (pregnant mares' serum
gonadotropin-primed SD rats) are concerned. Compound 4 shows marked antitumor
activity in the dimethylbenzanthracene-induced mammary carcinoma of the SD rat:
in the postmenopausal model it is at least as active as AG; in the premenopausal 
experiment it is clearly superior to AG. No induction of hepatic P450 enzymes was
observed in the latter experiment. The rp value of 4 toward rat ovarian P450
arom, i.e., 23 (rp of AG identical to 1), is markedly decreased compared to the
human enzyme (rp value of 303). From this fact it must be concluded that 4 should
be more active in the human than in the rat.

PMID: 7783141 [PubMed - indexed for MEDLINE]


558. J Neurochem. 1995 Mar;64(3):1416-9.

Dopamine transporter cysteine mutants: second extracellular loop cysteines are
required for transporter expression.

Wang JB, Moriwaki A, Uhl GR.

Division of Intramural Research, National Institute on Drug Abuse, Baltimore, MD 
21224.

Studies with thiol-modifying reagents have suggested that cysteines might play
important roles in the function of the dopamine transporter (DAT). To identify
DAT cysteines with important thiol groups, we have studied six mutant dopamine
transporters in which cysteines were replaced by alanines. Substitutions of
cysteines assigned to the DAT's second putative extracellular loop--positions 180
and 189--dramatically decreased the expression of the mutant transporters.
Substitutions at positions 90, 242, 305, and 345 had no significant effect in
decreasing dopamine uptake, MPP+ uptake, or cocaine analogue binding.
Immunostaining COS cells transfected with Cys180 and Cys189 to Ala mutants
revealed reduced membrane staining and prominent staining in perinuclear regions 
consistent with Golgi apparatus. These results suggest that cysteines i the DAT
second extracellular loop may provide sulfide residues crucial to full
transporter expression, at least in part, through interference with membrane
insertion. Conceivably, they might also provide the targets for the influences of
thiol-modifying reagents in modifying the function of the wild-type DAT expressed
in striatal membranes.

PMID: 7861176 [PubMed - indexed for MEDLINE]


559. Eur J Cell Biol. 1995 Mar;66(3):246-56.

A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro.

Mundigl O, Verderio C, Krazewski K, De Camilli P, Matteoli M.

Department of Cell Biology, Yale University School of Medicine, Boyer Center for 
Molecular Medicine, New Haven, CT 06510, USA.

Exocytosis of synaptic vesicles (SV) results in the surface exposure of lumenal
epitopes of SV proteins. We have recently described the use of antibodies
directed against the lumenal N-terminus of synaptotagmin I (Sytlum-Abs) to
morphologically monitor exo-endocytic recycling of SVs. We report here that a
radioimmunoassay based on these antibodies can be used to quantify levels of
synaptic activity in primary neuronal cultures. High density cultures of
hippocampal neurons grown in the absence of glia were used for these studies. A
significant cell surface pool of synaptotagmin I immunoreactivity was detectable 
by Sytlum-Abs at steady state. The increase in the amount of Sytlum-Abs which
became cell bound during a 3 min incubation at 37 degrees C over the Ab bound to 
this cell surface pool, was substantially higher in depolarizing media containing
extracellular Ca2+ than in Ca(2+)-free media. Incubation of the cultures with
Sytlum-Abs for longer time periods indicated a sustained increase in the rate of 
SV exocytosis in depolarizing media which lasted for at least 1 h. This increase 
was completely abolished by pretreating the neurons with tetanus toxin and this
block correlated with a disappearance of synaptobrevin immunoreactivity. This
radioimmunoassay therefore offers a new way to monitor SV exocytosis of neuronal 
populations in vitro irrespective of the type of neurotransmitter secreted and of
postsynaptic effects.

PMID: 7774610 [PubMed - indexed for MEDLINE]


560. Mol Plant Microbe Interact. 1995 Mar-Apr;8(2):267-77.

Molecular analysis of the Rhizobium meliloti mucR gene regulating the
biosynthesis of the exopolysaccharides succinoglycan and galactoglucan.

Keller M, Roxlau A, Weng WM, Schmidt M, Quandt J, Niehaus K, Jording D, Arnold W,
Puhler A.

Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Federal
Republic of Germany.

The Rhizobium meliloti Tn5 mutant Rm3131, producing galactoglucan (EPS II)
instead of succinoglycan (EPS I), was complemented by a 3.6-kb EcoRI-fragment of 
the Rhizobium meliloti genome. Sequencing of this fragment revealed six open
reading frames (ORFs). The ORF found to be affected in the mutant Rm3131 codes
for a putative protein of 15.7 kDa and forms a monocistronic transcriptional
unit. Further genetic analysis revealed that the gene mutated in Rm3131 is
identical to the previously described R. meliloti mucR gene (H. Zhan, S.B.
Levery, C. C. Lee, and J.A. Leigh, 1989, Proc. Natl. Acad. Sci. USA
86:3055-3059). By hybridization it was shown that a mucR homologous gene is
present in several rhizobacteria. The deduced amino acid sequence of MucR showed 
nearly 80% identity to the Agrobacterium tumefaciens Ros protein, a negative
regulator of vir genes and necessary for succinoglycan production. MucR contains 
like Ros a putative zinc finger sequence of the C2H2 type. Transcriptional
fusions of genes for EPS I and EPS II synthesis, the so-called exo and exp genes,
with the marker gene lacZ were used to delineate the role of mucR for exo and exp
gene expression. It was found that exp genes are negatively regulated by MucR on 
the transcriptional level, whereas a posttranscriptional regulation by MucR is
assumed for exo genes. Furthermore, mucR is negatively regulating its own
transcription.

PMID: 7756693 [PubMed - indexed for MEDLINE]


561. Glycobiology. 1995 Feb;5(1):105-15.

Carbohydrate structures of a normal counterpart of the carcinoembryonic antigen
produced by colon epithelial cells of normal adults.

Fukushima K, Ohkura T, Kanai M, Kuroki M, Matsuoka Y, Kobata A, Yamashita K.

Department of Biochemistry, Sasaki Institute, Tokyo, Japan.

Normal faecal antigen-2 (NFA-2) and non-specific cross-reacting antigen-2
(NCA-2), cross-reacting with anticarcinoembryonic antigen (CEA) antibodies, were 
found in normal human faeces and meconium, respectively. Because NFA-2, NCA-2 and
CEA are considered as the same gene products, NFA-2 and NCA-2 should be normal
counterparts of CEA produced by colon epithelial cells of normal adults and
fetuses, respectively. Comparison of sugar chain structures of these three
antigens is indispensable in order to unravel the structural alteration induced
by malignant transformation and development of colon epithelial cells. The sugar 
chain structures of CEA (Yamashita, K. et al., Cancer Res., 47, 3451-3459, 1987) 
and NCA-2 (Yamashita, K. et al., J. Biol. Chem., 264, 17873-17881, 1989) were
previously reported. In this paper, the structures of the oligosaccharides
released from four NFA-2 samples by hydrazinolysis were studied by means of
lectin-affinity column chromatography, endo- and exo-glycosidase digestion,
methylation analysis, hydrazinolysis-nitrous acid deamination and electrospray
ionization mass spectrometry. NFA-2 contains 24-27 mol of N-linked sugar
chains/molecule, which is similar to NCA-2 (27 mol) and CEA (24-27 mol). In
contrast to CEA, which contains approximately 8% high-mannose-type sugar chains, 
all sugar chains of NFA-2 are mono- to tetra-antennary complex-type chains having
four types of tri-mannosyl cores, with or without bisecting N-acetylglucosamine
and fucose residues. The structures of their outer chain moieties comprise Gal
beta 1-->3(HSO(3-)-->6)GlcNAc, Neu5Ac alpha 2-->3Gal beta 1-->3GlcNAc, Type 1,
repeating chain, Type 2, Type 2H, Type 1H, Lex, Lea and Leb antigenic
determinants. Approximately 50% of the outer chain moieties of the
oligosaccharides of NFA-2 contain Type 1 chain, in contrast to those of CEAs
produced by the liver metastases of colon tumours in which only a trace amount of
Type 1 chain was detected.

PMID: 7772858 [PubMed - indexed for MEDLINE]


562. Tsitologiia. 1995;37(3):187-92.

[The ultrastructure of the epithelial endocrinocytes of the appendiceal mucosa in
the human fetus]

[Article in Russian]

Kostiukevich SV.

An electron microscope study of the epithelium of vermiform processes of 11-26
week old human fetuses showed seven types of endocrinocytes differing in
ultrastructure and dimensions of secretory granules. In addition to the six known
types of endocrinocytes (EC, D, D1, I, L and P), endocrinocytes of a 7th type
were discovered which are beyond the International classification of
endocrinocytes of the gastroenteropancreatic system. The overwhelming majority of
endocrinocytes of the vermiform process epithelium form EC-cells. Besides, cells 
of a "mixed" type, both exo-endocrine and endo-endocrine cells, were revealed.

PMID: 8553456 [PubMed - indexed for MEDLINE]


563. Life Sci. 1995;56(23-24):1983-9.

Rat brain cannabinoid receptors are N-linked glycosylated proteins.

Song C, Howlett AC.

Department of Pharmacological and Physiological Science, Saint Louis University
School of Medicine, MO 63104, USA.

To study the N-linked glycosylation properties of the CB1 receptor, rat brain
membranes were treated with exo- and endoglycosidases. For visualizing CB1
receptors, an antipeptide antibody was raised against the N-terminal 14 amino
acids and used to specifically detect the protein by Western blotting. We found
that the apparent molecular weight of mature CB1 receptors was 64 kDa. Treatment 
of membranes with endoglycosidase F shifted the 64 kDa band to the 59 kDa and 53 
kDa bands. The latter is consistent with the calculated molecular weight of
deglycosylated CB1 receptors. Treatment of membranes with endoglycosidase H and
alpha-mannosidase partially shifted the 64 kDa band to 53 kDa band, indicating a 
portion of the oligosaccharides was of the high mannose type. These data
confirmed that the CB1 receptors in brain are N-linked glycoproteins with
heterogeneous carbohydrate composition. Among three potential N-linked
glycosylation sites on the N-terminus of the CB1 receptor, only two sites are
actually glycosylated.

PMID: 7776822 [PubMed - indexed for MEDLINE]


564. Biochemistry. 1994 Dec 13;33(49):14908-17.

Mutants affecting nucleotide recognition by T7 DNA polymerase.

Donlin MJ, Johnson KA.

Department of Biochemistry and Molecular Biology, Pennsylvania State University, 
University Park 16802.

Analysis of two mutations affecting nucleotide selection by the DNA polymerase
from bacteriophage T7 is reported here. Two conserved residues (Glu480 and
Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA
polymerase were mutated using site-directed mutagenesis (Glu480-Asp and
Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding,
nucleotide incorporation, and pyrophosphorolysis were measured with the mutants
E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical
quench-flow methods. Both mutants have slightly lower Kd values for DNA binding
compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide
binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower
for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding
constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP
binding compared to those constants for wild-type(exo-). The significance of a
specific destabilization of dTTP binding by these amino acids was examined using 
a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number
of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was
unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454
microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with
wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly
higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480
selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding 
to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs 
were consistent with the observation of a selective stabilization of dTTP by both
residues. Pyrophosphorolysis experiments revealed that neither mutation had a
significant effect on the chemistry of polymerization. The fidelity of the
mutants were examined in misincorporation assays. Both E480D(exo-) and
Y530F(exo-) showed saturation kinetics with the wrong nucleotide, with binding
constants of 1-3 mM compared to the estimated binding affinity of 6-8 mM with
wild-type(exo-). Accordingly, both mutants showed slightly lower selectivity
against misincorporation. Taken together, these results indicate that E480 and
Y530 each contribute to ground state nucleotide binding and suggest that the E480
may serve to specifically stabilize the incoming dTTP of A:T base pairs to
compensate for the fewer hydrogen bonds compared to G:C base pairs.

PMID: 7993917 [PubMed - indexed for MEDLINE]


565. Biochemistry. 1994 Dec 6;33(48):14443-51.

Design of potent bivalent thrombin inhibitors based on hirudin sequence:
incorporation of nonsubstrate-type active site inhibitors.

Tsuda Y, Cygler M, Gibbs BF, Pedyczak A, Fethiere J, Yue SY, Konishi Y.

Biotechnology Research Institute, National Research Council of Canada, Montreal, 
Quebec.

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor
from medicinal leech with a K(i) value of 2.2 x 10(-14) M. It consists of an
active site blocking moiety, hirudin1-48, a fibrinogen-recognition exo-site
binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these
inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal
portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was
replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g.,
dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by omega-amino
acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin55-65 was
used as a fibrinogen-recognition exo-site binding moiety in most of the
inhibitors. The crystal structure of the inhibitor in complex with human
alpha-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site
blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and
were therefore designated as P3, P1, and P2 residues. The use of
dansyl-Arg-(D-pipecolic acid) improved the affinity (K(i)) of the inhibitor
10-100-fold (down to 1.70 x 10(-11) M) compared to that of the similar compounds 
having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K(i) =
10(-9)-10(-10) M). The linker connected to P2 residue eliminated the scissile
peptide bond. The inhibitor was also stable against human plasma proteases.
Further inhibitor design revealed that the toxic dansyl group could be replaced
by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in
vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7981204 [PubMed - indexed for MEDLINE]


566. J Biol Chem. 1994 Dec 2;269(48):30479-84.

cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals 
a homology with phosphodiesterases.

Murata J, Lee HY, Clair T, Krutzsch HC, Arestad AA, Sobel ME, Liotta LA, Stracke 
ML.

Laboratory of Pathology, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892.

A human cDNA clone encoding autotaxin, a tumor cell motility-stimulating protein,
reveals that this protein is an ecto/exo-enzyme with significant homology to the 
plasma cell membrane differentiation antigen PC-1. ATX is a 125-kDa glycoprotein,
previously isolated from a human melanoma cell line (A2058), which elicits
chemotactic and chemokinetic responses at picomolar to nanomolar concentrations. 
Affinity-purified antipeptide antibodies to the ATX peptide, ATX-102, were
employed to screen an A2058 cDNA expression library made in lambda gt11. The
partial cDNA sequence which was obtained was then extended by utilizing reverse
transcriptase on total cellular RNA followed by polymerase chain reaction
amplification. The isolated cDNA clone contained 3251 base pairs, and the mRNA
message size was approximately 3.3 kilobases. The deduced amino acid sequence of 
autotaxin matched 30 previously sequenced peptides and comprised a protein of 915
amino acids. Data base analysis of the ATX sequence revealed a 45% amino acid
identity (including 30 out of 33 cysteines) with PC-1, a pyrophosphatase/type I
phosphodiesterase expressed on the surface of activated B cells and plasma cells.
ATX, like PC-1, was found to hydrolyze the type I phosphodiesterase substrate
p-nitrophenyl thymidine-5'-monophosphate. Autotaxin now defines a novel
motility-regulating function for this class of ecto/exo-enzymes.

PMID: 7982964 [PubMed - indexed for MEDLINE]


567. J Mol Evol. 1994 Dec;39(6):631-43.

Tracing the spread of fibronectin type III domains in bacterial glycohydrolases.

Little E, Bork P, Doolittle RF.

Center for Molecular Genetics, University of California, San Diego, La Jolla
92093-0634.

The evolutionary spread of 22 fibronectin type III (Fn3) sequences among a dozen 
bacterial enzymes has been traced by searching databases with the non-Fn3 parts
of the enzyme sequences. Numerous homologues were found that lacked the Fn3
domains. In each case the related sequences were aligned, phylogenetic trees were
constructed, and the occurrences of Fn3 units on the trees were noted. Comparison
with phylogenetic trees prepared from the Fn3 segments themselves allowed
inferences to be made about when the Fn3 units were shuffled into their present
positions.

PMID: 7528812 [PubMed - indexed for MEDLINE]


568. Carbohydr Res. 1994 Nov 15;264(2):281-91.

Purification and properties of an exo-(1-->3)-beta-D-galactanase from Aspergillus
niger.

Pellerin P, Brillouet JM.

Institut National de la Recherche Agronomique, Laboratoire des Polymeres et des
Techniques Physico-Chimiques, Montpellier, France.

An exo-(1-->3)-beta-D-galactanase was purified by six chromatographic steps from 
a culture supernatant of Aspergillus niger. Its apparent molecular mass was 66
kDa, as estimated by SDS-PAGE analysis. The purified enzyme had no detectable
activity on various p-nitrophenyl glycosides and on native plant polysaccharides 
but exhibited a high activity on a (1-->3)-beta-D-linked galactan backbone
obtained after partial acid hydrolysis and two Smith degradations of gum arabic. 
The optimum conditions were pH 4.5 and 40-50 degrees C. The enzyme had a
Michaelis constant (Km) of 1.9 mg/mL for the beta-(1-->3)-D-galactan with a
maximum reaction velocity (Vmax) of 1380 nkat/mg. The study of the reaction
products obtained after enzyme treatment of two galactans derived from gum arabic
through one or two Smith degradations showed that it was an
exo-(1-->3)-beta-D-galactanase able to by-pass the branching points of galactan
backbones and thus to release the side-chains of type II arabinogalactans in an
undegraded form.

PMID: 7805066 [PubMed - indexed for MEDLINE]


569. Microbiology. 1994 Nov;140 ( Pt 11):3007-13.

Gellan lyases--novel polysaccharide lyases.

Kennedy L, Sutherland IW.

Institute of Cell and Molecular Biology, Edinburgh University, UK.

A number of bacterial strains capable of degrading the bacterial
exopolysaccharide gellan have been isolated by standard enrichment procedures.
They include several pink-pigmented Gram-negative rod-shaped bacteria. A
red-pigmented Gram-positive bacillus earlier found to degrade the
exopolysaccharide xanthan from Xanthomonas campestris also showed slight
gellanase activity. All the Gram-negative bacteria are non-fermentative, motile
and amylase-producing. The gellan degradation in each case is due to
eliminase-type enzymes (lyases) which appear to be extracellular enzymes cleaving
the sequence... beta-D-glucosyl-(1-->4)-beta-D-glucuronosyl... in the
tetrasaccharide repeat unit of the substrate polysaccharides. Although in some
isolates these enzymes appear to be exo-acting, it appears from the loss in
viscosity of the alternative substrate deacetylated rhamsan that they are
predominantly endoenzymes. The enzyme activity is inducible: it is almost absent 
from glucose-grown cells. Associated with the 'gellanase' activity, all the
Gram-negative bacterial isolates possess intracellular alpha-L-rhamnosidase and
beta-D-glucosidase activities apparently located in the periplasm. The enzymes
are highly specific and fail to cause significant degradation of most of the
other bacterial exopolysaccharides which have been shown to be structurally
related to gellan. As well as acting on gellan, they exert similar degradative
activity against the chemically deacylated form of polysaccharide S194 (rhamsan
gum), which is effectively a gentiobiosylated form of gellan. The enzymes only
have relatively slight activity against the natural, acylated gellan-like
polysaccharides from the bacteria now designated as strains of Sphingomonas
paucimobilis.

PMID: 7812440 [PubMed - indexed for MEDLINE]


570. Appl Environ Microbiol. 1994 Oct;60(10):3592-3596.

A Sensitive Method Using 4-Methylumbelliferyl-beta-Cellobiose as a Substrate To
Measure (1,4)-beta-Glucanase Activity in Sediments.

Boschker HT, Cappenberg TE.

Centre for Limnology, Netherlands Institute of Ecology, 3631 AC Nieuwersluis, The
Netherlands.

A sensitive method to measure (1,4)-beta-glucanase activity in organic
matter-rich sediments, using 4-methyl-umbelliferyl-beta-cellobiose as a
substrate, is described. beta-Glucosidases, which were also able to hydrolyze
this substrate, were inhibited with d-glucono-delta-lactone. The produced
4-methylumbelliferone was recovered quantitatively out of the sediment by an
extraction with 80% ethanol. An inhibition experiment with known substrates or
inhibitors suggested that at least 59% of the measured activity could be
explained by enzymes of the exo-(1,4)-beta-glucanase type and that the
contribution of endo-(1,4)-beta-glucanases was minor. Results of the inhibition
experiment also suggested that the measured activity was of bacterial origin in
the sediment used. First results of field measurements are given for the sediment
from the reed bed of Lake Gooimeer.

PMCID: PMC201860
PMID: 16349405 [PubMed - as supplied by publisher]


571. EMBO J. 1994 Sep 15;13(18):4412-20.

Active site of the replication protein of the rolling circle plasmid pC194.

Noirot-Gros MF, Bidnenko V, Ehrlich SD.

Laboratoire de Genetique Microbienne, Institut National de la Recherche
Agronomique, Jouy en Josas, France.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA
of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and
aspartate) which are well conserved among numerous related plasmids. The effect
of mutations was examined by an in vivo activity test. Mutations of one tyrosine 
and two glutamate residues were found to greatly impair or abolish activity,
without affecting affinity for the origin, as deduced from in vitro gel mobility 
assays. We conclude that all three amino acids have a catalytic role. Tyrosine
residues were found previously in active sites of different RC plasmid Rep
proteins and topoisomerases, but not in association with acidic residues, which
are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo-
and endonucleases. We propose that the active site of RepA contains two different
catalytic centers, corresponding to a tyrosine and a glutamate. The former may be
involved in the formation of the covalent DNA-protein intermediate at the
initiation step of RC replication, and the latter may catalyze the release of the
protein from the intermediate at the termination step.

PMCID: PMC395368
PMID: 7925284 [PubMed - indexed for MEDLINE]


572. J Biol Chem. 1994 Sep 9;269(36):22712-8.

Identification of polysialic acid-containing glycoprotein in the jelly coat of
sea urchin eggs. Occurrence of a novel type of polysialic acid structure.

Kitazume S, Kitajima K, Inoue S, Troy FA 2nd, Cho JW, Lennarz WJ, Inoue Y.

Department of Biophysics and Biochemistry, Graduate School of Science, University
of Tokyo, Japan.

Sea urchin eggs are surrounded by a gelatinous layer (called the jelly coat) that
consists of mixture of fucoserich polysaccharides and sialic acid-rich
glycoproteins. Chemical and 500 MHz 1H NMR spectroscopic studies revealed for the
first time the presence of a novel polysialic acid (polySia) structure in the
jelly coat glycoproteins isolated from Hemicentrotus pulcherrimus (designated
polySia-gp(H)). The structure of the polySia chains was thoroughly characterized 
as (-->5-Oglycolyl-Neu5Gc alpha 2-->)n, where n ranges from 4 to more than 40
Neu5Gc residues. The polyNeu5Gc chains were attached to core oligosaccharides
that were O-glycosidically linked to threonine residues on a core polypeptide.
Each polypeptide contained about 17 O-linked polysialylglycan chains. The
apparent molecular weight of polySia-gp(H) was 180,000. The expression of this
new polySia structure in place of alpha 2-->8-linked polySia is the main
structural feature that distinguishes polySia-gp from other known polysialylated 
glycoproteins. The (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains were resistant to 
exo- and endosialidases from Arthrobacter ureafaciens and bacteriophage K1F,
respectively. Discovery of these (-->5-Ogly-colyl-Neu5Gc alpha 2-->)n chains adds
a new class of naturally occurring polySia to the structurally diverse family of 
polysialylated glycoproteins. The structure of a poly-Sia-gp from a different sea
urchin species, Stronglyocentrotus purpuratus (designated polySia-gp(S)), was
also determined to ascertain if there were any species-specific differences. The 
500 MHz 1H NMR spectra of the two polySia-gps were identical, indicating that at 
this level of molecular detail the structures were the same. The molecular weight
of polySiagp(S) was larger, however (250,000), and it contained about 25 polySia 
chains O-glycosidically linked to both threonine (two-thirds) and serine
(one-third) residues.

PMID: 8077223 [PubMed - indexed for MEDLINE]


573. Mol Microbiol. 1994 Sep;13(5):843-54.

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the 
enzyme and its structural gene, polC.

Barnes MH, Tarantino PM Jr, Spacciapoli P, Brown NC, Yu H, Dybvig K.

Department of Pharmacology, University of Massachusetts Medical School, Worcester
01655.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative
evolution. The results of previous investigations of mycoplasmal DNA polymerases 
suggest that the process of evolution has wrought a major simplification of the
typical Gram-positive bacterial DNA polymerase profile, reducing it from three
exonuclease (exo)-positive enzymes to a single exo-negative species. The
objective of this work was to rigorously investigate this suggestion, focusing on
the evolutionary fate of DNA polymerase III (Pol III), the enzyme which
Gram-positive bacteria specifically require for replicative DNA synthesis. The
approach used Mycoplasma pulmonis as the model organism and exploited structural 
gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as
investigative tools. Our results indicate that M. pulmonis has strongly conserved
a single copy of a structural gene homologous to polC, the Gram-positive
bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA
polymerase that displays the size, primary structure, exonuclease activity, and
level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The 
high level of sensitivity of M. pulmonis growth to Gram-positive Pol
III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has 
conserved not only the basic structure of Pol III, but also its essential
replicative function. Evidence for a second, HPUra-resistant polymerase activity 
in M. pulmonis is also described, indicating that the DNA polymerase composition 
of Mycoplasma is complex and closer to that of Gram-positive bacteria than
previously thought.

PMID: 7815943 [PubMed - indexed for MEDLINE]


574. Anal Biochem. 1994 Sep;221(2):340-7.

Homogeneous fluorescence detection method for human leukocyte antigen-DR typing
following polymerase chain reaction amplification with sequence-specific primer.

Okamoto N, Lee A, Kano T, Lee TD.

Biomedical Research Center, Olympus America Inc., East Setauket, New York 11733.

A fluorescent homogeneous method for the detection of sequence-specific
amplification of human leukocyte antigen (HLA) alleles has been developed. In
this approach, polymerase chain reaction sequence-specific primers (PCR-SSP) are 
used to amplify DRB1, DRB3, and DRB4 alleles. Lambda exonuclease and Exonuclease 
I are added to reduce background by digesting template DNA, partial primer dimer,
and primer. PCR amplicons are then detected following the addition of a
fluorescent dye (thiazole yellow dimer) which binds to double-stranded DNA. No
transfer or wash steps are required. Thus, the risk of sample contamination,
which is a major source of inaccuracy for DNA amplification methods, is greatly
reduced. This approach is also faster and more easily automated than the standard
approach using gel electrophoresis and ethidium bromide staining. Speed and
automation are important considerations for HLA typing since the number of
possible alleles for each HLA type is substantial. A homogeneous HLA-SSP typing
method may be especially useful for clinical labs doing large numbers of samples 
and for the eventual automation of HLA DNA typing.

PMID: 7810876 [PubMed - indexed for MEDLINE]


575. Diabetes. 1994 Aug;43(8):1020-6.

Insulin sensitivity in cystic fibrosis.

Moran A, Pyzdrowski KL, Weinreb J, Kahn BB, Smith SA, Adams KS, Seaquist ER.

Diabetes Center, University of Minnesota, Minneapolis.

Cystic fibrosis (CF) patients demonstrate a spectrum of pancreatic beta-cell
abnormalities. Those with no exocrine insufficiency (NEXO) have normal insulin
secretion. Exocrine-insufficient CF patients with overt diabetes (EXO-IT) have
impaired insulin secretion and fasting hyperglycemia. Exocrine-insufficient
patients without diabetes (EXO) have impaired insulin secretion but maintain
normoglycemia. We postulated that EXO individuals compensate for insulin
deficiency by increasing insulin sensitivity and investigated glucose utilization
in CF. To examine hepatic and peripheral insulin sensitivity,
euglycemic-hyperinsulinemic clamp studies were performed by using the hot GINF
isotope dilution technique. Insulin was sequentially infused at 0.25, 1.0, and
10.0 mU.kg-1.min-1. Glucose-mediated glucose uptake (GMGU) was assessed on
another day with hyperglycemic clamp studies, during which insulin and
somatostatin were infused to hold insulin-mediated glucose uptake constant
between the two clamp studies. Skeletal muscle GLUT4 levels were assessed in EXO 
and control patients with Western blotting. Three patterns of peripheral and
hepatic insulin sensitivity were seen that were related to the degree of
pancreatic beta-cell dysfunction. NEXO individuals had normal peripheral and
hepatic insulin sensitivity. EXO individuals had enhanced peripheral insulin
sensitivity that was not associated with a change in skeletal muscle glucose
transporter abundance compared with control patients; paradoxically, EXO subjects
demonstrated hepatic insulin resistance. EXO-IT had peripheral and hepatic
insulin resistance. GMGU was diminished in both EXO and EXO-IT subjects. The
unique combination of increased hepatic glucose production and increased
peripheral glucose utilization seen in EXO may be a metabolic adaptation to
increased peripheral energy needs.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8039595 [PubMed - indexed for MEDLINE]


576. J Mol Biol. 1994 Jul 15;240(3):226-42.

Production of UV-induced frameshift mutations in vitro by DNA polymerases
deficient in 3'-->5' exonuclease activity.

Sagher D, Turkington E, Acharya S, Strauss B.

Department of Molecular Genetics and Cell Biology, University of Chicago, IL
60637.

In order to study the conversion of UV lesions into frameshift and base
substitution mutations, M13mp2 phage DNA was altered by the addition of extra
pyrimidines, or by construction of a nonsense codon preceded by a run of
pyrimidines within the beta-galactosidase complementing region. The normal
sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT
C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study
reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA
polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced
UV-induced revertants at higher frequency than did their exonuclease proficient
counterparts. Removal of cyclobutane dimers with photolyase before in vitro
synthesis did not greatly affect mutant frequency although such treatment led to 
significantly increased DNA synthesis by the wild-type T7 DNA polymerase on
UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA
produced by the delta 28 T7 DNA polymerase were mainly by base substitution in
the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a
TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity
gave revertant frequencies that correlated better with published values of enzyme
velocity than with exonuclease activity or with measured bypass synthesis. Our
data indicate that loss of proofreading activity increases the frequency of
UV-induced frameshifts, but lack of such activity is not sufficient for their
production. We suggest that frameshifts occur more frequently when nucleotide
addition opposite the lesion is slow. The same lesion can give rise to a
different spectrum of mutations depending on the polymerase.

PMID: 8028006 [PubMed - indexed for MEDLINE]


577. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6574-8.

Disruption of hydrogen bonding between plant cell wall polymers by proteins that 
induce wall extension.

McQueen-Mason S, Cosgrove DJ.

Department of Biology, Pennsylvania State University, University Park, PA 16802, 
USA.

Plant cell enlargement is controlled by the ability of the constraining cell wall
to expand. This ability has been postulated to be under the control of
polysaccharide hydrolases or transferases that weaken or rearrange the
loadbearing polymeric networks in the wall. We recently identified a family of
wall proteins, called expansins, that catalyze the extension of isolated plant
cell walls. Here we report that these proteins mechanically weaken pure cellulose
paper in extension assays and stress relaxation assays, without detectable
cellulase activity (exo- or endo- type). Because paper derives its mechanical
strength from hydrogen bonding between cellulose microfibrils, we conclude that
expansins can disrupt hydrogen bonding between cellulose fibers. This conclusion 
is further supported by experiments in which expansin-mediated wall extension (i)
was increased by 2 M urea (which should weaken hydrogen bonding between wall
polymers) and (ii) was decreased by replacement of water with deuterated water,
which has a stronger hydrogen bond. The temperature sensitivity of
expansin-mediated wall extension suggests that units of 3 or 4 hydrogen bonds are
broken by the action of expansins. In the growing cell wall, expansin action is
likely to catalyze slippage between cellulose microfibrils and the polysaccharide
matrix, and thereby catalyze wall stress relaxation, followed by wall surface
expansion and plant cell enlargement.

PMCID: PMC44245
PMID: 11607483 [PubMed]


578. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6486-90.

Interaction of Grb2 via its Src homology 3 domains with synaptic proteins
including synapsin I.

McPherson PS, Czernik AJ, Chilcote TJ, Onofri F, Benfenati F, Greengard P,
Schlessinger J, De Camilli P.

Department of Cell Biology, Howard Hughes Medical Institute, Yale University
School of Medicine, New Haven, CT 06536.

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and
two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple
tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors
(through its SH3 domains). Using an overlay assay, we have identified four major 
Grb2-binding proteins in synaptic fractions. These proteins interact with
wild-type Grb2 but not with Grb2 containing point mutations in each of its two
SH3 domains corresponding to the loss of function mutants in the Caenorhabditis
elegans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were
previously shown to bind Grb2. The third protein of 145 kDa is brain specific and
to our knowledge has not been previously described. The fourth protein is
synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I
is thought to mediate the interaction of synaptic vesicles with the presynaptic
cytomatrix. These data suggest that Grb2, or other proteins containing SH3
domains, may play a role in the regulation of the exo/endocytotic cycle of
synaptic vesicles and therefore of neurotransmitter release.

PMCID: PMC44227
PMID: 8022809 [PubMed - indexed for MEDLINE]


579. J Bacteriol. 1994 Jul;176(14):4437-43.

Isolation, characterization, and nucleotide sequence of IS1202, an insertion
sequence of Streptococcus pneumoniae.

Morona JK, Guidolin A, Morona R, Hansman D, Paton JC.

Department of Microbiology, Women's and Children's Hospital, North Adelaide,
Australia.

A comparative hybridization protocol was used to isolate a small segment of DNA
present in the Streptococcus pneumoniae type 19F strain SSZ but absent from
strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment
of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp
imperfect inverted repeats and containing a single open reading frame sufficient 
to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either
end of the element. IS1202 is not related to any of the currently known insertion
elements and is the first reported for S. pneumoniae. Although found
predominantly in type 19F strains in up to five copies, it has also been shown to
be present in the chromosomes of pneumococci belonging to other serotypes. One of
the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp
downstream of the dexB gene, and transformation studies reveal that it is also
closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.

PMCID: PMC205658
PMID: 8021229 [PubMed - indexed for MEDLINE]


580. Biochim Biophys Acta. 1994 Jun 22;1192(2):263-71.

Structural characteristics of thylakoid membranes of Arabidopsis mutants
deficient in lipid fatty acid desaturation.

Tsvetkova NM, Brain AP, Quinn PJ.

Department of Biochemistry, King's College London, UK.

The ultrastructure of thylakoid membranes from Arabidopsis thaliana wild-type,
JB67 and LK3 fatty acid desaturation deficient mutants was studied by
thin-section and freeze-fracture electron microscopy. There was a decrease in the
amount of the appressed and non-appressed membranes in JB67 and LK3 Arbidopsis
mutants when compared to the wild type, resulting in a reduction in the length of
photosynthetic membrane per plastid. The results from freeze-fracture showed a
decrease in size and a marked increase in packing density of membrane-associated 
particles on the exo- and endoplasmic fracture faces of the mutants. In addition,
areas of the appressed membranes of the mutants contained particles in regular
arrays under conditions where no such arrays were observed in wild-type thylakoid
membranes. These observations suggest, that the decreased level of lipid fatty
acid unsaturation affects the ability of the lipid matrix to mediate the assembly
of chloroplast membrane components. The role of polyunsaturated membrane lipids
is considered in terms of their ability to promote functional oligomeric
assemblies of components of the photosynthetic apparatus.

PMID: 8018707 [PubMed - indexed for MEDLINE]


581. J Biomol Struct Dyn. 1994 Jun;11(6):1161-74.

Molecular dynamics simulations of a r(GA12G).d(CT12C) hybrid duplex.

Fritsch V, Wolf RM.

Central Research Laboratories Ciba-Geigy Ltd, Basel, Switzerland.

RNA.DNA hybrid duplexes are relevant in various biological mechanisms like
transcription and replication. Enzymes like RNase H cleave specifically the RNA
strand in RNA.DNA duplexes. In antisense technology the complexation of mRNA with
"modified" oligo(deoxy)-nucleotides leads to new hybrid duplexes. The knowledge
about structure and dynamical behavior on an atomic level is fundamental for the 
understanding of any process involving hybrid duplexes. Therefore, molecular
dynamics studies (200 picoseconds of trajectory) on a hybrid duplex structure
r(GA12G).d(CT12C) were performed. During the stimulations, the deoxyribose
residues assumed a puckering state between C2'-endo and C3'-endo, with an average
mode around O4'-endo-C1'-exo, whereas the riboses of the RNA strand remained in
the C3'-endo puckering domain. The results are compared to those obtained for the
DNA.DNA duplex d(GA12G).d(CT12C) under identical simulation conditions. The DNA
strand in the hybrid duplex behaves similar to that in a standard B-type DNA
duplex. The helical parameters of the hybrid duplex however are closer to A- than
to B-type. These observations suggest that RNA.DNA hybrid double helices are
neither clearly A-form nor B-form. The furanoses in both strands can assume
different puckering modes without the appearance of major geometrical
constraints. The simulation results are in excellent agreement with recent
experimental data.

PMID: 7524540 [PubMed - indexed for MEDLINE]


582. Biol Mass Spectrom. 1994 May;23(5):262-6.

Analysis of a bioactive synthetic analogue of tuftsin by tandem mass
spectrometry: anomalous fast atom bombardment activated processes.

De Angelis F, Nicoletti R, Kuster T, Heizmann CW, Pinori M, Verdini AS.

Dip. di Chimica, Univ. de l'Aquila, Italy.

Fast atom bombardment (FAB) tandem mass spectrometry has been used to analyse the
biologically potent, partially modified retro-inverso (PMRI) synthetic isomer of 
tuftsin: this compound represents the active peptide of the fraction of
gamma-globulin (leukokinin) which binds specifically to blood neutrophilic
leukocytes and monocytes. Protonated molecules and fragment ions were
collisionally dissociated at low energies in a triple-quadrupole mass
spectrometer to yield a complete picture of the reactions that occur in the
condensed and in the gas phase. The study shows that, when retro-inversion is
within the N-terminal amino acid, charge localization at the basic sites
(possibly at the N-terminus) induces a marked decomposition of the molecule, the 
loss of ammonia being the most favourable fragmentation process. Also, artifacts 
are formed in the liquid phase via bimolecular reactions promoted by the
high-energy beam. The findings indicate that despite the fact that PMRI isomers
of this type are stable against exo-peptidases and also stable under acidic
conditions, they appear to be labile under conditions where the energy
deposition, due to FAB is necessarily high.

PMID: 8204682 [PubMed - indexed for MEDLINE]


583. J Biochem. 1994 May;115(5):837-42.

Hydrolysis of cotton cellulose by Exo- and endo-type cellulases from Irpex
lacteus: differential scanning calorimetric study.

Hoshino E, Kubota Y, Okazaki M, Nisizawa K, Kanda T.

Household Products Research Laboratories, Kao Corporation, Wakayama.

The mode of hydrolysis of cotton cellulose by two highly purified exo- and
endo-type cellulases from Irpex lacteus was investigated by differential scanning
calorimetry, to measure changes in the size of the amorphous region in cotton
fibers with the enzymatic reaction. The cellulases induced entirely different
changes in the size of the amorphous region, particularly at earlier stages of
reaction. Exo-type cellulase gradually reduced the amorphous region with release 
of cellobiose from the initial stage of hydrolysis, but began to increase the
amorphous region at more advanced stages of hydrolysis. By contrast, endo-type
cellulase caused no liberation of reducing sugar at the initial stage of
hydrolysis but caused a sharp increase in the amorphous region, and it thereafter
caused a rapid decrease of the amorphous region, accompanied with the production 
of various kinds of cellooligosaccharides. The rate of size reduction of the
amorphous region caused by endo-type cellulase was much higher than that by
exo-type cellulase. Convergence of the decrease in the size of amorphous region
during hydrolysis by endo-type cellulase is followed by the increase in this
region being influenced by further hydrolysis of remained crystalline region.
Substantial changes in the morphology of cotton occurred with the two cellulases 
after the hydrolysis stages at which the size of the amorphous region was
minimum.

PMID: 7961594 [PubMed - indexed for MEDLINE]


584. Mol Pharmacol. 1994 Mar;45(3):555-60.

Role of tyrosine 337 in the binding of huperzine A to the active site of human
acetylcholinesterase.

Ashani Y, Grunwald J, Kronman C, Velan B, Shafferman A.

Israel Institute for Biological Research, Ness-Ziona.

Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of
antiacetylcholinesterase (AChE), has been suggested to be superior to
antiacetylcholinesterase drugs now being used for management of Alzheimer's
disease. To delineate the binding site of human AChE (HuAChE) for HUP, the
biochemical constants kon, koff, and Ki were determined for complexes formed
between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or
double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic
and dissociation constants were compared with those obtained for wild-type
rHuAChE and AChE from Torpedo californica. Results demonstrate that the
inhibition of AChE by HUP occurs through association with residues located inside
the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at
position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM).
An aromatic array constituted from residues Y337, F295, and probably W86 is
likely to offer a multicontact subsite that interacts with the ammonium group and
with both the exo-and endocyclic double bond moieties of HUP. Lack of the
aromatic side chain in the position homologous to Y337 explains the poor
inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM).
Replacement of the carboxylate-containing E202 by glutamine had only marginal
effect on the stability of the complex formed between HUP and rHuAChE. The
pH-rate profiles suggest that destabilization of the complex after proton gain
cannot be attributed solely to protonation of E202. These findings are expected
to establish HUP as a lead compound for the design of new anti-AChE drugs.

PMID: 8145739 [PubMed - indexed for MEDLINE]


585. Microbiology. 1994 Mar;140 ( Pt 3):631-6.

Presence in rumen bacterial and protozoal populations of enzymes capable of
degrading fungal cell walls.

Morgavi DP, Sakurada M, Tomita Y, Onodera R.

Laboratory of Animal Nutrition and Biochemistry, Faculty of Agriculture, Miyazaki
University Japan.

Ruminal bacteria and protozoa, and cell-free rumen fluid, were tested for the
presence of enzymes involved in the degradation of the fungal cell wall.
Protozoal homogenate obtained by ultrasonication showed chitinase (EC 3.2.1.14)
and N-acetyl-beta-glucosaminidase (EC 3.2.1.52) activities when assayed with
fluorogenic 4-methylumbelliferyl substrates. The chitinase activity was
predominantly of the 'exo'-type. Lysozyme (EC 3.2.1.17) and 1,3-beta-glucanase
(EC 3.2.1.39) activities were also present in this fraction. All these
activities, except lysozyme activity, were recovered mainly in the supernatant
fraction of the homogenate (approximately 85% of the total activity). Lysozyme
showed the same amount of activity in the precipitate and supernatant fractions. 
Bacterial homogenates had N-acetyl-beta-glucosaminidase activity in both
supernatant and precipitate fractions. The specific activity was one-third that
of the protozoa. Bacteria able to grow in a medium with chitin as the sole carbon
source were recognized and counted. Cell-free rumen fluid was unable to degrade
any of the substrates tested.

PMID: 8012585 [PubMed - indexed for MEDLINE]


586. J Biol Chem. 1994 Feb 11;269(6):4641-7.

A beta turn in the cytoplasmic tail of the integrin alpha v subunit influences
conformation and ligand binding of alpha v beta 3.

Filardo EJ, Cheresh DA.

Scripps Research Institute, Department of Immunology, La Jolla, California 92037.

Integrins undergo conformational alterations in response to extracellular or
intracellular stimuli, suggesting that structural elements within their exo- and 
cytoplasmic domains cooperate during transmembrane signaling. In this report, we 
identify a beta turn in the cytoplasmic tail of the alpha v subunit that impacts 
the ligand binding and conformation of the alpha v beta 3 heterodimer. Cells
expressing a mutant alpha v beta 3 heterodimer composed of a truncated alpha v
subunit, alpha v1000, lacking 18 carboxyl-terminal amino acids exhibits wild-type
receptor structure and function. However, a truncation mutant, alpha v995,
lacking five additional residues (PPQEE), which define a beta turn, is deficient 
in vitronectin and fibrinogen adhesion. This alteration in adhesive function is
associated with two detectable structural changes in the alpha v beta 3
heterodimer. First, the alpha v995 membrane-spanning light chain exhibits
retarded electrophoretic mobility on SDS-polyacrylamide gels. Second, the alpha
v995 beta 3 receptor shows an altered chymotryptic profile as measured by the
loss of a 39-kDa proteolytic fragment from its ectodomain. These findings
demonstrate that the ligand binding and structural properties of the intact alpha
v beta 3 heterodimer can be influenced by a beta turn within the cytoplasmic tail
of its alpha v subunit. The presence of homologous beta turns within other alpha 
subunit cytoplasmic tails suggests that this structural motif may play a role in 
regulating integrin-mediated bidirectional transmembrane signals.

PMID: 7508446 [PubMed - indexed for MEDLINE]


587. Appl Environ Microbiol. 1994 Feb;60(2):594-8.

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase,
in the phytopathogenic fungus Cochliobolus carbonum.

Schaeffer HJ, Leykam J, Walton JD.

Department of Energy Plant Research Laboratory, Michigan State University, East
Lansing 48824.

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme
capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of
partial amino acid sequences of the purified enzyme, two degenerate
oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb
fragment of corresponding genomic DNA. The PCR product was used to isolate the
genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA
confirmed that the PCR product corresponded to EXG1. A strain of the fungus
specifically mutated in the EXG1 gene was constructed by homologous integration
of an internal fragment of EXG1. In the mutant, enzymatic activity and the
corresponding peak of UV absorption during high-pressure liquid chromatography
purification were reduced by at least 98%. However, crude culture filtrates of
the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This
residual activity was due to two additional activities which were
chromatographically separable from the product of EXG1 and which were coeluted
with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal
on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 
mutant was still pathogenic to maize.

PMCID: PMC201354
PMID: 8135518 [PubMed - indexed for MEDLINE]


588. J Neurochem. 1994 Feb;62(2):645-55.

Influence of region-specific alterations of neuropeptidase content on the
catabolic fates of neuropeptides in Alzheimer's disease.

Ichai C, Chevallier N, Delaere P, Dournaud P, Epelbaum J, Hauw JJ, Vincent JP,
Checler F.

Institut de Pharmacologie Moleculaire et Cellulaire, UPR 411 CNRS, Valbonne,
France.

We established the cartography of 11 exo- and endopeptidases in the frontal and
parietal cortices and in the cerebellum of brains of patients diagnosed with a
senile dementia of the Alzheimer's type (SDAT). Comparison with those of four
subjects who had died without known neurologic or psychiatric illness indicated
that there existed a region-specific alteration of the peptidase contents in the 
disease. In the frontal area of SDAT brains, postproline dipeptidyl
aminopeptidase and aminopeptidase M activities were significantly reduced. In the
parietal cortex of SDAT brain, activities of three additional
endopeptidases--angiotensin-converting enzyme, proline endopeptidase, and
endopeptidase 24.15--were also drastically reduced. In contrast, the cerebellum
displayed a set of proteolytic activities that remained unaffected in SDAT brain.
The putative influence of the disease on the catabolic fates of neurotensin,
neuropeptide Y, and somatostatin(1-14) was investigated. Neurotensin was
catabolized at identical rates in the frontal and parietal cortices in
nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed
down in SDAT brains in the frontal but not in the parietal cortex. Finally, the
degradation velocities of somatostatin(1-14) were lowered in both cortical areas 
of SDAT brains. It is interesting that, by means of specific peptidase
inhibitors, we demonstrated that endopeptidase 24.15 participated in
somatostatin(1-14) inactivation in the parietal but not in the frontal cortex. It
is suggested that the lowering of the rate of somatostatin(1-14) inactivation in 
the parietal cortex of SDAT brains likely results from the depletion of
endopeptidase 24.15 in this brain region.

PMID: 7905027 [PubMed - indexed for MEDLINE]


589. Carbohydr Res. 1994 Jan 3;251:89-98.

A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization,
and mode of action.

Maruyama Y, Nakajima T, Ichishima E.

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku
University, Sendai-shi, Japan.

A 1,2-alpha-D-mannosidase was purified to homogeneity from the culture
supernatant of Bacillus sp. M-90, which was isolated from soil by enrichment
culture on baker's yeast mannan. The purified enzyme had M(r) 380,000 Da, and was
comprised of two apparently identical 190,000 Da subunits. It had a neutral
optimum pH (7.0) and an isoelectric point of 3.6. The enzyme was highly specific 
for alpha 1,2-linked D-mannose oligosaccharides. An N-linked high-mannose type
oligosaccharide, Man9GlcNAc2, was a good substrate, yielding Man5GlcNAc2, and the
alpha 1,2-linked side chains of Saccharomyces cerevisiae mannan were also
specifically hydrolyzed by the enzyme. p-Nitrophenyl alpha-D-mannopyranoside and 
1,2-alpha-D-mannobiitol were not hydrolyzed at all. Calcium ion,
1-deoxyman-nojirimycin, and swainsonine had no effect on the enzyme, but the
activity was completely inhibited by EDTA. The mode of action on alpha 1,2-linked
mannotetraose indicated that the enzyme is an exo-1,2-alpha-D-mannanase.

PMID: 8149382 [PubMed - indexed for MEDLINE]


590. Biochemistry. 1993 Nov 30;32(47):12694-704.

Structural study of the sugar chains of human leukocyte common antigen CD45.

Sato T, Furukawa K, Autero M, Gahmberg CG, Kobata A.

Department of Biochemistry, University of Tokyo, Japan.

The leukocyte cell surface glycoprotein CD45 is a protein tyrosine phosphatase
and is involved in signal transduction mediated by the T cell antigen receptor.
The asparagine-linked sugar chains were released as oligosaccharides from
purified CD45 by hydrazinolysis. Approximately 6 mol of sugar chains was released
from 1 mol of CD45. These sugar chains were converted to radioactive
oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic
fractions by paper electrophoresis. All of the acidic oligosaccharides were
converted to neutral ones by digestion with sialidase, indicating that they are
sialyl derivatives. Binding of the sialylated oligosaccharides to an SNA-agarose 
column as well as methylation analysis revealed that the oligosaccharides have
only alpha-2,6-linked sialic acid residues. The neutral and sialidase-treated
acidic oligosaccharides were fractionated by serial lectin column chromatography 
followed by Bio-Gel P-4 column chromatography. Structural studies of each
oligosaccharide by sequential exo- and endoglycosidase digestion and by
methylation analysis revealed that CD45 contains mainly bi-, tri-, and
tetraantennary complex-type sugar chains. About 46% of the tetraantennary
complex-type sugar chains had the poly(N-acetyllactosamine) groups and 18% of the
2,4-branched triantennary complex-type sugar chains had the fucosyl
N-acetyllactosamine group. A portion of the bi- and 2,4-branched triantennary
complex-type sugar chains were bisected. In addition to these sugar chains, a
small amount of high mannose-type and hybrid-type sugar chains were detected.

PMID: 8251489 [PubMed - indexed for MEDLINE]


591. Morfologiia. 1993 Nov-Dec;105(11-12):96-105.

[The structure and cytophysiology of the endocrinocytes of the gastric epithelium
in a disordered food regimen]

[Article in Russian]

Rossol'ko GN, Ivanova VF.

By means of the light and electron microscopic methods the differentiation and
the genesis of the epithelium endocrinocytes of the stomach mucous membrane were 
studied in the albino rats, fasted for 72-144 hours. The deprivation of food is
followed by the increase of the number of endocrinocytes and by the change of
their ultrastructure. The latter demonstrates the delay of the elimination of the
secretory material in some cells (ECL, G, D, D1 cells) while the active
functional condition was found in the others (ECL, A-like cells). Exo-endocrine
cells with the EC or A-type granules are found in the epithelium, which is
regarded as a mechanism aimed at the increase of the number of the appropriate
(ECL or A-type of endocrinocytes, participating in the general metabolism of the 
organism.

PMID: 7874299 [PubMed - indexed for MEDLINE]


592. J Biol Chem. 1993 Sep 25;268(27):20037-45.

Enhanced oxidation of aniline derivatives by two mutants of cytochrome c
peroxidase at tryptophan 51.

Roe JA, Goodin DB.

Department of Molecular Biology, Scripps Research Institute, La Jolla, California
92037.

Two hyperactive mutants of cytochrome c peroxidase (CCP), W51F and W51A, catalyze
the enhanced oxidation of a number of substituted anilines. The reaction of CCP
compound ES with mesidine is biphasic, while similar reactions using compound II 
give monophasic kinetics. These data, in addition to the ratio of the Fe4+ = O
and free-radical species observed during steady-state turnover, indicate that
reduction of the Trp-191 free radical of compound ES is more rapid than the
reduction of the Fe4+ = O species. Transient kinetics were examined for the
oxidation of eight mono-substituted anilines by CCP, W51F, and W51A. Each of the 
aniline derivatives were oxidized by the mutants at rates that exceeded that of
the wild-type enzyme, and the rate constant for m-chloroaniline was 400-fold
faster for W51F than for wild-type CCP. Variations in the rate constants for the 
different substrates follow a linear free-energy relationship using the Hammet
substituent effect parameter sigma +, implicating electron transfer from the
aniline ring in the transition state. For aniline oxidation, the free energy of
activation is 3 kcal/mol lower for the mutants than for wild-type CCP, and this
is due primarily to an increase in the activation entropy. These results indicate
that the enhanced kinetics of W51F and W51A result from a generalized increase in
enzyme reactivity characterized by an exo-entropic transition state such as
dissociation of bound H2O from the Fe4+ = O center.

PMID: 8397197 [PubMed - indexed for MEDLINE]


593. J Cell Sci. 1993 Sep;106 ( Pt 1):31-43.

Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent
urothelial cell surface glycoprotein.

Wu XR, Sun TT.

Ronald O. Perelman Department of Dermatology, New York University School of
Medicine, NY 10016.

Despite the fact that bladder epithelium has many interesting biological features
and is a frequent site of carcinoma formation, relatively little is known about
its biochemical differentiation. We have shown recently that a 47 kDa
glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa)
and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized
biomembrane characteristic of the apical surface of bladder epithelium.
Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of 
N-linked sugars attached to a core protein of 28.9 kDa. The presence of an
N-terminal signal peptide sequence and a single transmembrane domain located near
the C terminus, plus the N-terminal location of all the potential N-glycosylation
sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the
extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds
that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution,
a feature shared by the other two major uroplakins, provides a molecular
explanation as to why the luminal leaflet of AUM is almost twice as thick as the 
cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has 
a significant cytoplasmic domain suggests that this molecule may play an
important role in AUM-cytoskeleton interaction in terminally differentiated
urothelial cells.

PMID: 8270634 [PubMed - indexed for MEDLINE]


594. Izv Akad Nauk Ser Biol. 1993 Sep-Oct;(5):670-83.

[The concept of receptor sorption as a basis for the development of
nontraditional methods of therapy, prevention and diagnosis in pathological
states]

[Article in Russian]

Piruzian LA, Mikhailovskii EM.

A potential use of receptor sorption for medical purposes is discussed. This type
of sorption is based on selective fixation of exo and endogenous chemical
substances and viruses by their cellular receptors, which serve as sorbents,
"landing grounds" ("chemical traps"). We propose to elaborate non-traditional
methods of therapy, prophylaxis and diagnosis of various pathologies. These
methods include: "dialysis by receptors", peroral and local use of isolated
receptors, blocking of receptors by specific antireceptor antibodies, and
analysis of binding properties of the receptors in various pathologies. We
propose to carry out "receptor mapping" ("receptor classification") of the human 
population. This mapping consists in drawing up a "personal receptor map"
carrying information about biochemical properties and binding parameters of the
person's cellular receptors. The necessity of careful laboratory and clinical
approbation of the proposed methods is emphasized.

PMID: 8220078 [PubMed - indexed for MEDLINE]


595. J Gen Microbiol. 1993 Sep;139(9):2019-26.

Metabolism of polysaccharides by the Streptococcus mutants dexB gene product.

Whiting GC, Sutcliffe IC, Russell RR.

Department of Oral Biology, Dental School, University of Newcastle upon Tyne, UK.

The Streptococcus mutans dexB gene, a member of the multiple sugar metabolism
(msm) operon, encodes an intracellular glucan 1,6-alpha-glucosidase which
releases glucose from the non-reducing terminus of alpha-1,6-linked
isomaltosaccharides and dextran. Comparison of primary amino acid sequences
showed strong homology to Bacillus oligo-1,6-glucosidases and, like these
enzymes, DexB was able to release free glucose from the alpha-1,4,6-branch point 
in panose. This suggested a role for DexB in the metabolism of either starch or
intracellular polysaccharide, which contain such branch points. However, purified
intracellular polysaccharide from the wild-type S. mutans strain LT11 and a
mutant deficient in dexB revealed no substantial differences in the extent of
branching as demonstrated by iodine staining spectra and the degree of
polymerization. Furthermore, thin layer chromatography of radiolabelled
intracellular polysaccharide digested with S. mutans wild-type and mutant cell
extracts showed no differences in the products obtained. The involvement of DexB 
in dietary starch metabolism was investigated using alpha-limit dextrins produced
from the action of alpha-amylase on starch. These induced the msm operon,
including dexB, and the DexB enzyme was able to act on the alpha-limit dextrins
to give further fermentable substrates. The transport system encoded by the msm
operon can also transport alpha-limit dextrin. DexB may therefore be important in
the metabolism of extracellular starch.

PMID: 7504068 [PubMed - indexed for MEDLINE]


596. J Med Microbiol. 1993 Aug;39(2):141-6.

Mortality rates amongst mice with endogenous septicaemia caused by Pseudomonas
aeruginosa isolates from various clinical sources.

Furuya N, Hirakata Y, Tomono K, Matsumoto T, Tateda K, Kaku M, Yamaguchi K.

Department of Laboratory Medicine, Nagasaki University School of Medicine, Japan.

Mice that had been treated with cyclophosphamide and ampicillin were fed with
Pseudomonas aeruginosa. These procedures induced an endogenous septicaemia under 
conditions mimicking the pathophysiology of the disease in man. This model was
used to compare the mortality rates in mice infected with P. aeruginosa isolates 
from various clinical sources. Mortality rates in mice given isolates from blood 
cultures had a broad range (0-100%), but the mean rate was significantly higher
than with isolates from other infection sites. Moreover, blood isolates persisted
in the intestines of mice after oral inoculation, whereas most isolates from
other sources were gradually eliminated. Most P. aeruginosa isolates from blood
culture produced significantly higher levels of exotoxin A and total proteases
than isolates from other infection sites. Amongst the blood isolates, all but one
of the lethal strains produced large quantities of exotoxin A or total proteases 
or both. Taken together, the results suggest that the ability of P. aeruginosa to
adhere to the intestinal tract and to produce high levels of exo-enzymes may
contribute to the development of fatal septicaemia.

PMID: 8345509 [PubMed - indexed for MEDLINE]


597. J Biochem. 1993 Aug;114(2):236-45.

Electron microscopic observation of cotton cellulose degradation by exo- and
endo-type cellulases from Irpex lacteus.

Hoshino E, Sasaki Y, Mori K, Okazaki M, Nisizawa K, Kanda T.

Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu
University, Nagano.

The interaction of two highly purified cellulases, exo- and endo-type cellulases 
from Irpex lacteus, with pure cotton and amorphous cellulose was investigated by 
electron microscopy. The morphological observations indicated that exo- and
endo-type cellulases are both strongly adsorbed on the internal microfibril of
cotton fiber before enzymatic hydrolysis, and then initiate their action toward
the internal cellulose microfibrils with retention of the original shape. The two
cellulases, however, caused considerably different morphological changes in
cotton cellulose, and each cellulase seems to degrade native cellulose with a
distinct mode of action. In the hydrolysis of cotton with exo-type cellulase,
deep transverse cracks were produced and they extended from the fiber surface to 
the lumen structure located inside the fibers. In contrast, it was found that
there were no deep cracks on fibers treated with endo-type cellulase, but severe 
internal erosion and cavitation occurred along fibril or microfibril layers
inside the fibers. Thus, the degradation of cotton by exo- and endo-type
cellulases yielded quite different morphological patterns, while little
difference was found for regenerated celluloses. The mode of enzymatic hydrolysis
of cellulose shown by cellulases with different degrees of randomness (exo and
endo types) appears to be markedly affected by the fine structure of cellulose
fibers.

PMID: 8262905 [PubMed - indexed for MEDLINE]


598. J Biochem. 1993 Aug;114(2):230-5.

Mode of action of exo- and endo-type cellulases from Irpex lacteus in the
hydrolysis of cellulose with different crystallinities.

Hoshino E, Sasaki Y, Okazaki M, Nisizawa K, Kanda T.

Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu
University, Nagano.

The mode of action of two highly purified cellulases of exo- and endo-types from 
Irpex lacteus was investigated by using pure cellulosic substrates with different
crystallinities derived from cellulose I. Exo-type cellulase saccharified all
celluloses more effectively than endo-type enzyme, and the saccharification
activities of both cellulases similarly increased with decreasing crystallinity
of cellulose. The DP-lowering activity of exo-type cellulase remained similar for
celluloses with higher crystallinity, while this cellulase showed a degradation
mode resembling that of the endo-type enzyme for the substrates with lower
crystallinity. Compared with exo-type cellulase, endo-type cellulase remarkably
decreased the DP of cellulose with higher crystallinity, while this activity was 
abated for cellulose with lower crystallinity. Thus, the effects of both
cellulases became similar in the degradation of amorphous substrates such as
H3PO4-treated cellulose. Endo-type cellulase produced several kinds of
cellooligosaccharide from all kinds of cellulose used, while the product of the
exo-type enzyme was only cellobiose from crystalline cellulose such as cotton and
cotton linter even after a 12-h incubation period. The results indicate that each
cellulase shows a typical mode of action (exo or endo) for crystalline cellulose,
but that their characteristic modes of attack may change with decreasing
crystallinity of cellulose.

PMID: 8262904 [PubMed - indexed for MEDLINE]


599. Biochemistry. 1993 Jul 13;32(27):6836-45.

The 2.0-A resolution structure of soybean beta-amylase complexed with
alpha-cyclodextrin.

Mikami B, Hehre EJ, Sato M, Katsube Y, Hirose M, Morita Y, Sacchettini JC.

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 
10461.

New crystallographic findings are presented which offer a deeper understanding of
the structure and functioning of beta-amylase, the first known exo-type
starch-hydrolyzing enzyme. A refined three-dimensional structure of soybean
beta-amylase, complexed with the inhibitor alpha-cyclodextrin, has been
determined at 2.0-A resolution with a conventional R-value of 17.5%. The model
contains 491 amino acid residues, 319 water molecules, 1 sulfate ion, and 1
alpha-cyclodextrin molecule. The protein consists of a core with an (alpha/beta)8
supersecondary structure, plus a smaller globular region formed by long loops
(L3, L4, and L5) extending from beta-strands beta 3, beta 4, and beta 5. Between 
the two regions is a cleft that opens into a pocket whose floor contains the
postulated catalytic center near the carboxyl group of Glu 186. The annular
alpha-cyclodextrin binds in (and partly projects from) the cleft with its
glucosyl O-2/O-3 face abutting the (alpha/beta)8 side and with its alpha-D(1 --> 
4) glucosidic linkage progression running clockwise as viewed from that side. The
ligand does not bind deeply enough to interact with the carboxyl group of Glu
186. Rather, it occupies most of the cleft entrance, strongly suggesting that
alpha-cyclodextrin inhibits catalysis by blocking substrate access to the more
deeply located reaction center. Of the various alpha-cyclodextrin interactions
with protein residues in loops L4, L5, L6, and L7, most notable is the shallow
inclusion complex formed with Leu 383 (in L7, on the core side of the cleft)
through contacts of its methyl groups with the C-3 atoms of four of the ligand's 
D-glucopyranosyl residues. All six residues of the bound alpha-cyclodextrin are
of 4C1 conformation and are joined by alpha-1,4 linkages with similar torsional
angles to form a nearly symmetrical torus as reported for crystalline inclusion
complexes with alpha-cyclodextrin. We envision a significant role for the methyl 
groups of Leu 383 at the cleft entrance with respect to the productive binding of
the outer chains of starch.

PMID: 8334116 [PubMed - indexed for MEDLINE]


600. J Mol Biol. 1993 Jun 5;231(3):605-20.

Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme,
which lacks chi-specific, but not non-specific, nuclease activity.

Eggleston AK, Kowalczykowski SC.

Department of Cell, Molecular & Structural Biology, Northwestern University
Medical School, Chicago, IL 60611.

RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses
ATP-dependent nuclease activities. We have purified a mutant recBCD enzyme,
designated recB2109CD enzyme, and have examined the nuclease activities of this
protein in vitro to determine whether any alteration in these activities is
responsible for the recombination-deficient phenotype of the recB2109 strain. The
recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA
exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD
enzyme although they are reduced approximately 2 to 3-fold relative to the
wild-type enzyme. The ATP-dependent dsDNA exonuclease activity of recB2109CD
enzyme requires significantly higher ATP concentrations for optimal activity when
compared to the wild-type enzyme. The ATP-independent ssDNA endonuclease activity
of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and
ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced
relative to those of wild-type recBCD enzyme. Despite its ability to degrade
linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking 
activity at chi sites, which are hotspots for genetic recombination. Since this
interaction with chi significantly attenuates the non-specific dsDNA exonuclease 
activity of wild-type recBCD enzyme, these results suggest that the non-specific 
dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the
consequence that a DNA substrate which is suitable for recombination is not
produced.

PMID: 8390577 [PubMed - indexed for MEDLINE]


601. Mol Microbiol. 1993 Jun;8(6):1083-94.

The presence of a novel type of surface polysaccharide in Rhizobium meliloti
requires a new fatty acid synthase-like gene cluster involved in symbiotic nodule
development.

Petrovics G, Putnoky P, Reuhs B, Kim J, Thorp TA, Noel KD, Carlson RW, Kondorosi 
A.

Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences,
Szeged.

Bacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have
been shown to play important roles in plant-bacterium interactions. Here we have 
demonstrated that the fix-23 loci, which compensate for exo mutations during
symbiotic nodule development, are involved in the production of a novel
polysaccharide that is rich in 3-deoxy-D-manno-2-octulosonic acid (Kdo) but is
not the classical LPS. This molecule is likely to be a surface antigen since
antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since
mutations in fix-23 result in an inability to produce this polysaccharide and to 
bind bacteriophage 16-3. It is likely that this Kdo-rich polysaccharide is
analogous to certain Escherichia coli K-antigens which are anchored to the
membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of 
this region revealed that the predicted protein products of six genes exhibit a
high degree of homology and similar organization to those of the rat fatty acid
synthase multifunctional enzyme domains.

PMID: 8361353 [PubMed - indexed for MEDLINE]


602. Biosci Biotechnol Biochem. 1993 Jun;57(6):965-8.

Purification and characterization of beta-N-acetyl-D-hexosaminidase from the
mid-gut gland of scallop (Patinopecten yessoensis).

Sakai T, Nakanishi Y, Kato I.

Research Institute for Glycotechnology Co., Ltd., Aomori, Japan.

beta-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of
Patinopecten yessoensis. The enzyme was purified by making an acetone-dried
preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer 
(pH 4.0) (about 13% of the extracted proteins was
beta-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column
chromatographies on CM-Sepharose and DEAE-Sepharose. The purified
beta-N-acetyl-D-hexosaminidase was homogeneous on SDS-PAGE, and sufficiently free
from other exo-type glycosidases. The molecular weight was 56,000 by SDS-PAGE.
The enzyme hydrolyzed both p-nitrophenyl beta-N-acetyl-D-glucosaminide and
p-nitrophenyl beta-N-acetyl-D-galactosaminide. For p-nitrophenyl
beta-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature
was 45 degrees C, and the Km was 0.24 mM. For p-nitrophenyl
beta-N-acetyl-D-galactosaminide, these were pH 3.4, 45 degrees C, and 0.15 mM,
respectively. The enzyme liberated non-reducing terminal beta-linked
N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl
derivatives of oligosaccharides of N-glycan or glycolipid type except of
GM2-tetrasaccharide. As the enzyme was stable around pH 3.5-5.5, it may be useful
for long time reactions around the optimum pH.

PMID: 7763885 [PubMed - indexed for MEDLINE]


603. Nucleic Acids Res. 1993 May 11;21(9):2201-8.

Crystal structure of a Z-DNA hexamer d(CGCICG) at 1.7 A resolution:
inosine.cytidine base-pairing, and comparison with other Z-DNA structures.

Kumar VD, Weber IT.

Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson
University, Philadelphia, PA 19107.

The crystal structure of the deoxyhexamer, d(CGCICG), has been determined and
refined to a resolution of 1.7A. The DNA hexamer crystallises in space group
P2(1)2(1)2(1) with unit cell dimensions of a = 18.412 +/- .017 A, b = 30.485 +/- 
.036A, and c = 43.318 +/- .024 A. The structure has been solved by rotation and
translation searches and refined to an R-factor of 0.148 using 2678 unique
reflections greater than 1.0 sigma (F) between 10.0-1.7 A resolution. Although
the crystal parameters are similar to several previously reported Z-DNA hexamers,
this inosine containing Z-DNA differs in the relative orientation, position, and 
crystal packing interactions compared to d(CGCGCG) DNA. Many of these differences
in the inosine form of Z-DNA can be explained by crystal packing interactions,
which are responsible for distortions of the duplex at different locations. The
most noteworthy features of the inosine form of Z-DNA as a result of such
distortions are: (1) sugar puckers for the inosines are of C4'-exo type, (2) all 
phosphates have the Zl conformation, and (3) narrower minor grove and compression
along the helical axis compared to d(CGCGCG) DNA. In addition, the substitution
of guanosine by inosine appears to have resulted in Watson-Crick type
base-pairing between inosine and cytidine with a potential bifurcated hydrogen
bond between inosine N1 and cytidine N3 (2.9 A) and O2 (3.3-3.A).

PMCID: PMC309485
PMID: 8502562 [PubMed - indexed for MEDLINE]


604. J Bacteriol. 1993 May;175(10):2826-32.

Characterization and symbiotic importance of acidic extracellular polysaccharides
of Rhizobium sp. strain GRH2 isolated from acacia nodules.

Lopez-Lara IM, Orgambide G, Dazzo FB, Olivares J, Toro N.

Department of Microbiology, Estacion Experimental del Zaidin, Consejo Superior de
Investigaciones Cientificas, Granada, Spain.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of 
the leguminous tree Acacia cyanophylla and has a broad host range which includes 
herbaceous legumes, e.g., Trifolium spp. We examined the extracellular
exopolysaccharides (EPSs) produced by strain GRH2 and found three independent
glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which 
is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar
trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a
dimer of the repeat unit of the high-molecular-weight EPS, and
low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant
derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was
obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but
infected a smaller proportion of cells in the central zone of the nodules than
did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several
herbaceous legume hosts that are nodulated by wild-type strain GRH2.

PMCID: PMC204597
PMID: 8491702 [PubMed - indexed for MEDLINE]


605. Biotechnol Appl Biochem. 1993 Apr;17 ( Pt 2):239-50.

Exo-glucosidase activity and substrate specificity of the beta-glycosidase
isolated from the extreme thermophile Sulfolobus solfataricus.

Nucci R, Moracci M, Vaccaro C, Vespa N, Rossi M.

Institute of Protein Biochemistry and Enzymology, Naples, Italy.

The enzyme with beta-galactosidase activity from Sulfolobus solfataricus strain
MT-4, like other enzymes of this type isolated from thermophilic sources, has
broad specificity for beta-D-gluco-, fuco- and galacto-sides. The
beta-galactosidase activity was purified by a new procedure that improved yields 
(44%) and final specific activity (182 units mg-1 at 75 degrees C using
chromogenic beta-D-galactoside as substrate). The enzyme hydrolysed a large
number of beta-linked glycoside dimers and oligomers; chromogenic beta-glucosides
and beta-fucosides are the preferred substrates, and kinetic analysis indicated
that they bind to a common catalytic site. The order of catalytic efficiency was 
beta 1-3 > beta 1-4 > beta 1-6 and cellotetraose > cellotriose > cellobiose for
glucose dimers and oliogomers respectively. The cleavage occurred at the
non-reducing end of the oligosaccharide, and the enzyme showed noticeable
specificity also for the aglycone part of substrates. From these results the
enzyme from S. solfataricus strain MT-4 is defined as a true glycosyl hydrolase
with remarkable exo-glucosidase activity and it is designated S beta-gly.

PMID: 8484908 [PubMed - indexed for MEDLINE]


606. Biol Chem Hoppe Seyler. 1993 Apr;374(4):255-63.

Oligosaccharide reprocessing and recycling of a cell surface glycoprotein in
cultured rat hepatocytes.

Kreisel W, Hildebrandt H, Mossner W, Tauber R, Reutter W.

Medizinische Klinik, Klinikum der Albert-Ludwigs-Universitat, Freiburg, Germany.

The metabolism of the cell surface glycoprotein dipeptidyl peptidase IV(DPPIV)
was studied in cultured rat hepatocytes. In pulse-chase labelling experiments
using L-[35S]methionine a 100-kDa high-mannose precursor polypeptide is converted
into the mature complex-type 110-kDa glycoprotein. Digestion with exo- and
endoglycosidases and metabolic labelling with radioactive sugars demonstrate that
the 110-kDa form contains about 6 complex-type oligosaccharides which are
fucosylated and sialylated. About 25 min after the beginning of the
pulse-labelled glycoprotein appears in the sinusoidal membrane. Physiologically
only the 110-kDa form is found in the cell surface. If cell surface DPP IV was
desialylated by sialidase at 4 degrees C, it is resialylated during incubation at
37 degrees C. This oligosaccharide reprocessing indicates that the surface
glycoprotein has been recycled to the cell compartment containing terminal
glycosyltransferases (presumably the trans Golgi system). Two different methods
demonstrate internalization of cell surface DPP IV: 1) The complex cell surface
DPPIV -anti-DPP IV-antibody -L-[35S]methionine-labelled secondary
goat-anti-mouse-antibody formed at 4 degrees C becomes less accessible to trypsin
during incubation at 37 degrees C. 2) Part of the complex plasma membrane DPP
IV-anti-DPP IV-antibody formed in the cold cannot be recognized by the
radioactive secondary antibody after rewarming. Internalization is not blocked by
inhibition of protein synthesis with cycloheximide. During internalization of
plasma membrane DPP IV its concentration in the membrane remains
constant.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8101088 [PubMed - indexed for MEDLINE]


607. J Pharm Sci. 1993 Mar;82(3):276-81.

Solution- and solid-state structures of N-desmethylnefopam hydrochloride, a
metabolite of the analgesic drug.

Glaser R, Geresh S, Blumenfeld J, Donnell D, Sugisaka N, Drouin M, Michel A.

Department of Chemistry, Ben-Gurion University of the Negev, Beersheva, Israel.

The solid-state structure of (+/-)-N-desmethylnefopam hydrochloride (1), a
metabolite of the analgesic drug, was determined by single-crystal X-ray
diffraction analysis. Compound 1 gave crystalline prisms belonging to the
orthorhombic Pcab space group, and at ambient temperature (293 K), a = 9.939(2), 
b = 14.479(1), c = 20.148(3) A, V = 2899.5(8) A3, Z = 8, R(F) = 0.045, and Rw(F) 
= 0.025. The benzoxazocine ring of crystalline 1 is twisted into the
boat-flattened (chair) [BfC] conformation, the phenyl ring resides in a
relatively sterically unhindered exo-type ring position, whereas the O atom and
NCH2Ar occupy sterically hindered positions between "boat" and "chair" regions.
Dissolution of BfC crystalline 1 in CD2Cl2 solvent affords a dynamic
conformational equilibrium (involving the putative twist-chair-flattened (chair) 
conformer) as shown by line broadening and weighted time-averaged vicinal
coupling constants [-OCH2CH2N- segment] in the 1H NMR spectrum. The
solution-state weighted time-averaged 50(1) degrees O-CH2-CH2-N dihedral angle,
calculated by the R-ratio method, shows that the BfC conformation is the major
contributor to time-averaged structure.

PMID: 8450422 [PubMed - indexed for MEDLINE]


608. FEMS Microbiol Lett. 1993 Mar 1;107(2-3):307-12.

The spoOA and degU genes of Bacillus subtilis show genetic homology.

Cucchi A, Sanchez-Rivas C.

Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales,
Ciudad Universitaria, Buenos Aires, Argentina.

The transformation efficiency of competent Bacillus subtilis degU32(Hy) strains
was found to depend on the marker that was selected. Prototrophic transformants
were obtained at frequencies similar to those in the wild type control, but Spo- 
transformants were rare also when a spoOA::erm insertion that produces a
selectable marker (ErmR) was used. The ErmR transformants obtained within the
degU32(Hy) background were Spo+ and had lost the characteristics of the DegU(Hy) 
parental recipient strain i.e., secretion of exo-enzymes and sporulation
resistance to catabolites. The spoOA::erm insertion was mapped to a location near
degU. The similarities between the spoOA and degU sequences and the metabolic
interferences between the mutated products which result in this unexpected
recombination, are discussed.

PMID: 8386124 [PubMed - indexed for MEDLINE]


609. Neuropharmacology. 1993 Mar;32(3):229-34.

Ion channel inhibitors may function as potential modulators of cocaine binding.

Boja JW, Kopajtic TA.

Neuroscience Branch, National Institute on Drug Abuse, Addiction Research Center,
Baltimore, MD 21224.

The effects of NaCl, KCl and Ca2Cl were determined in a binding assay, using the 
analog of cocaine [3H]WIN 35,428. The addition of NaCl had no effect upon
specific binding; however, Ca2Cl and to a lesser extent, KCl decreased binding.
Various Na+, K+, Cl- and Ca2+ channel antagonists were tested for their ability
to inhibit specific binding of [3H]WIN 35,428. Most of the Na+ channel blockers
tested were of moderate affinity, the exceptions being benzamil and flunarizine. 
Both of these agents also show activity at the Ca2+ channel. The K+ channel
blockers were of low and moderate affinity, while the Cl- channel blockers had no
effect. Of the Ca2+ channel blockers tested, only pimozide demonstrated a high
affinity. This was postulated to be due to its ability to act upon both L and
T-type channels. These results suggest that the Ca2+ channel blockers deserve
further study as potential useful therapeutic agents in the treatment of cocaine 
addiction.

PMID: 7682676 [PubMed - indexed for MEDLINE]


610. Biochemistry. 1993 Jan 26;32(3):739-44.

Sugar conformations at hybrid duplex junctions in HIV-1 and Okazaki fragments.

Salazar M, Champoux JJ, Reid BR.

Department of Chemistry, University of Washington, Seattle 98195.

We have carried out a solution study of the local conformation in a
hybrid-chimeric duplex of the [sequence: see text] type (where r and D represent 
RNA and DNA). The object of this study was to investigate the sugar conformations
at the internal junction in the hybrid-DNA octamer duplex (gccaCTGC).
(GCAGTGGC)--where the lower-case letters represent RNA residues. Such duplexes
represent good models for Okazaki fragments in which RNA primers are covalently
extended into DNA strands during DNA replication of the lagging strand.
Furthermore, this particular sequence occurs during HIV-1 retrovirus reverse
transcription. The chimeric RNA-DNA strand and the complementary pure DNA strand 
chosen for this study result from the priming of (-)-strand DNA synthesis by
tRNA(Lys) and subsequent (+)-strand DNA synthesis by reverse transcriptase prior 
to HIV-1 retrovirus integration. Despite the unusual specificity of the RNase H
activity of reverse transcriptase, which cleaves the RNA c-a phosphodiester
rather than the junction a-C linkage, we found no major structural differences
among the RNA c-a phosphodiester rather than the junction a-C linkage, we found
no major structural differences among the RNA sugar conformations--all RNA sugars
were found in the normal C3'-endo A-form conformation. Instead, we find that the 
first DNA residue of the chimeric strand (5C) assumes a sugar conformation in the
C4'-exo to O4'-endo range (P = 54-90 degrees). Furthermore, the hybrid segment of
this duplex is more heteronomous than previously assumed for duplexes of the
[sequence: see text] type.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 8380708 [PubMed - indexed for MEDLINE]


611. J Bacteriol. 1993 Jan;175(2):386-94.

The Saccharomyces cerevisiae SPR1 gene encodes a sporulation-specific
exo-1,3-beta-glucanase which contributes to ascospore thermoresistance.

Muthukumar G, Suhng SH, Magee PT, Jewell RD, Primerano DA.

Department of Methods Development and Scale-Up, Enzon, Inc., Piscataway, New
Jersey 08854-3998.

A number of genes have been shown to be transcribed specifically during
sporulation in Saccharomyces cerevisiae, yet their developmental function is
unknown. The SPR1 gene is transcribed during only the late stages of sporulation.
We have sequenced the SPR1 gene and found that it has extensive DNA and protein
sequence homology to the S. cerevisiae EXG1 gene which encodes an
exo-1,3-beta-glucanase expressed during vegetative growth (C. R. Vasquez de
Aldana, J. Correa, P. San Segundo, A. Bueno, A. R. Nebrada, E. Mendez, and F. del
Ray, Gene 97:173-182, 1991). We show that spr1 mutant cells do not hydrolyze
p-nitrophenyl-beta-D-glucoside or laminarin in a whole-cell assay for
exo-1,3-beta-glucanases. In addition to the absence of this enzymatic activity,
spr1 mutant spores exhibit reduced thermoresistance relative to isogenic
wild-type spores. These observations are consistent with the notion that SPR1
encodes a sporulation-specific exo-1,3-beta-glucanase.

PMCID: PMC196152
PMID: 8419289 [PubMed - indexed for MEDLINE]


612. Appl Biochem Biotechnol. 1993 Jan-Feb;38(1-2):69-81.

Immobilization of Aspergillus niger NRC 107 Xylanase and beta-Xylosidase, and
Properties of the Immobilzed Enzymes.

Abdel-Naby MA.

Department of Chemistry of Natural and Microbial Products, National Research
Center, Dokki, Cairo, Egypt.

Aspergillus niger NRC 107 xylanase and beta-xylosidase were immobilized on
various carriers by different methods of immobilization, including physical
absorption, covalent binding, ionic binding, and entrapment. The immobilized
enzymes were prepared by physical adsorption on tannin-chitosan, ionic binding
onto Dowex-50W, covalent binding on chitosan beads through glutaraldehyde, and
entrapment in polyacrylamide had the highest activities. In most cases, the
optimum pH of the immobilized enzymes were shifted to lower than those of free
enzymes. The optimum reaction temperature of immobilized xylanase was shifted
from 50C to 52.5-65C, whereas that of immobilized beta-xylosidase was shifted
from 45C to 50-60C. The Km values of immobilized enzymes were higher than those
of native enzymes. The operational stability of the immobilized enzymes was
evaluated in continuous operation in packed-bead column-type reactors. The
enzymes covalently bounded to chitosan showed the highest operational stability. 
However, the enzymes immobilized by physical absorption or by ionic binding
showed a low operational stability. The enzymes entrapped in polyacrylamide
exhibited lower activity, but better operational stability.

PMID: 8346906 [PubMed - indexed for MEDLINE]


613. Acta Biochim Pol. 1993;40(4):477-82.

Homology of genes for exopolysaccharide synthesis in Rhizobium leguminosarum and 
effect of cloned exo genes on nodule formation.

Skorupska A, Derylo M.

Department of General Microbiology, M. Curie-Sklodowska University, Lublin,
Poland.

A 5.4 kb BamHI fragment of R. leguminosarum bv. trifolii TA1 was found to carry
genes involved in exopolysaccharide synthesis (exo genes). This fragment was
strongly hybridized to the total DNA from R. l. bv. viciae and bv. phaseoli
digested with EcoRI. No homology was found with total DNA of R. meliloti and
Rhizobium sp. NGR 234. The exo genes from R. l. bv. trifolii TA1 conjugally
introduced into R. l. bv. viciae 1302 considerably affected the symbiosis: the
nodules induced on vetch were abortive and did not fix nitrogen. On the other
hand, Phaseolus beans infected with R. l. bv. phaseoli harbouring R. l. bv.
trifolii exo genes formed the nitrogen-fixing nodules. It can be concluded that
additional copies of exo genes introduced into wild type Rhizobium leguminosarum 
strains can disturb the synthesis of acidic exopolysaccharides and affect
symbiosis of the plants forming indeterminate nodules, but do not affect
symbiosis of the plants forming the determinate nodules.

PMID: 8140821 [PubMed - indexed for MEDLINE]


614. Biochem J. 1992 Dec 1;288 ( Pt 2):539-44.

A cDNA clone for human glucosamine-6-sulphatase reveals differences between
arylsulphatases and non-arylsulphatases.

Robertson DA, Freeman C, Morris CP, Hopwood JJ.

Department of Chemical Pathology, Adelaide Children's Hospital, South Australia.

Glucosamine-6-sulphatase is an exo-hydrolase required for the lysosomal
degradation of heparan sulphate and keratan sulphate. Deficiency of
glucosamine-6-sulphatase activity leads to the lysosomal storage of the
glycosaminoglycan, heparan sulphate and the monosaccharide sulphate
N-acetylglucosamine 6-sulphate and the autosomal recessive genetic disorder
mucopolysaccharidosis type IIID. Glucosamine-6-sulphatase can be classified as a 
non-arylsulphatase since, relative to arylsulphatase B, it shows negligible
activity toward 4-methylumbelliferyl sulphate. We have isolated human cDNA clones
and derived amino acid sequence coding for the entire glucosamine-6-sulphatase
protein. The predicted sequence has 552 amino acids with a leader peptide of 36
amino acids and contains 13 potential N-glycosylation sites, of which it is
likely that 10 are used. Glucosamine-6-sulphatase shows strong sequence
similarity to other sulphatases such as the family of arylsulphatases, although
the degree of similarity is not as high as that between members of the
arylsulphatase family. This pattern of inter- and intra-family similarity
delineates regions and amino acid residues that may be critical for sulphatase
function and substrate specificity.

PMCID: PMC1132044
PMID: 1463457 [PubMed - indexed for MEDLINE]


615. Mol Cell Biol. 1992 Dec;12(12):5499-507.

Unstable amplification of two extrachromosomal elements in
alpha-difluoromethylornithine-resistant Leishmania donovani.

Hanson S, Beverley SM, Wagner W, Ullman B.

Department of Biochemistry and Molecular Biology, Oregon Health Sciences
University, Portland 97201-3098.

We describe the first example of unstable gene amplification consisting of linear
extrachromosomal DNAs in drug-resistant eukaryotic cells.
alpha-Difluoromethylornithine (DFMO)-resistant Leishmania donovani with an
amplified ornithine decarboxylase (ODC) gene copy number contained two new
extrachromosomal DNAs, both present in 10 to 20 copies. One of these was a 140-kb
linear DNA (ODC140-L) on which all of the amplified copies of the odc gene were
located. The second was a 70-kb circular DNA (ODC70-C) containing an inverted
repeat but lacking the odc gene. Both ODC140-L and ODC70-C were derived from a
preexisting wild-type chromosome, probably by a conservative amplification
mechanism. Both elements were unstable in the absence of DFMO, and their
disappearance coincided with a decrease in ODC activity and an increase in DFMO
growth sensitivity. These results suggest the possibility that ODC70-C may play a
role in DFMO resistance. These data expand the diversity of known amplification
mechanisms in eukaryotes to include the simultaneous unstable amplification of
both linear and circular DNAs. Further characterization of these molecules will
provide insights into the molecular mechanisms underlying gene amplification,
including the ability of linear amplified DNAs to acquire telomeres and the
determinants of chromosomal stability.

PMCID: PMC360487
PMID: 1448081 [PubMed - indexed for MEDLINE]


616. J Biochem Biophys Methods. 1992 Dec;25(4):253-72.

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine,
2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy
and ab-initio molecular orbital calculations.

Plavec J, Koole LH, Chattopadhyaya J.

Department of Bioorganic Chemistry, University of Uppasala, Sweden.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been
assessed by NMR spectroscopy and pseudorotational analysis in conjunction with
its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and
2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds
has prompted us to establish the relationship between proton-proton and
corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety
on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A 
subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4)
shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred 
(75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and
South conformers with the corresponding puckering amplitude (psi m) for ddI (1), 
ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m 
= 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2
degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6
degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate
between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a
psi m of 34.7 degrees. Note that these preponderant North-sugar structures
(approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and
ddC (4) are not reflected in the X-ray crystal structures of
2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues 
in both of these crystal structures denosine and 2',3'-dideoxycytidine. The
constituent sugar residues in both of these crystal structures are found to be in
the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC
adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo
twist form). This means that X-ray structures of ddA (2) and ddC (4) only
represent the minor conformer of the overall pseudorotamer population in
solution. An assumption that the structure of the pentofuranose sugar (i.e. P and
psi m) participating in conformational equilibrium described by the two-state
model remains unchanged at different temperatures has been experimentally
validated by assessing five unknown pseudorotational parameters with eight unique
observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4')
for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 1337354 [PubMed - indexed for MEDLINE]


617. Biochem J. 1992 Dec 1;288 ( Pt 2):475-82.

Partial purification and characterization of an
endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. 
OH-11242.

Ishii-Karakasa I, Iwase H, Hotta K, Tanaka Y, Omura S.

Department of Biochemistry, School of Medicine, Kitasato University, Kanagawa,
Japan.

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from
the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase,
Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151,
422-428], a method for assaying enzyme activity was established. Using purified
pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated
from PGM were pyridylaminated, and the reducing terminal sugars of
oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The
crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate 
precipitate from the concentrated culture medium. The enzyme was partially
purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography.
Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and
eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction
obtained, no exo-glycosidases or proteases could be detected. The molecular mass 
of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 
5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser
(Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the
disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta
1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from
Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled
asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

PMCID: PMC1132035
PMID: 1281406 [PubMed - indexed for MEDLINE]


618. J Med Chem. 1992 Nov 27;35(24):4567-75.

3'-C-branched 2'-deoxy-5-methyluridines: synthesis, enzyme inhibition, and
antiviral properties.

Fedorov II, Kazmina EM, Novicov NA, Gurskaya GV, Bochkarev AV, Jasko MV,
Victorova LS, Kukhanova MK, Balzarini J, De Clercq E, et al.

Moscow Medical Sechenov Academy, Russia.

A synthesis scheme for 3'-C-methyl-2'-deoxynucleosides and
3'-C-methylidene-2',3'-dideoxy-5-methyluridine has been proposed with
2-deoxyribose as the starting material. Methyl 5-O-benzoyl-2-deoxyribofuranose
was oxidized and the mixture of the 3'-keto derivatives was separated into the
alpha- and beta-anomers. The beta-keto derivative was converted by reaction with 
MeMgBr, and after reaction with thymine and subsequent deprotection
1-(3'-C-methyl-2'-alpha-deoxy-alpha-D-threo-pentofuranosyl)thymine and its
beta-anomer were obtained. The same reactions with the alpha-keto sugar gave
1-(3'-C-methyl-2'-deoxy-alpha-D-erythro-pentofuranosyl)thymine and its
beta-anomer.
1-(5-O-Benzoyl-3'-C-methyl-2'-deoxy-alpha-D-threo-pentofuranosyl)thymine was
converted to a mixture of 3'-C-methylidene-2',3'-dideoxy-5-methyluridine and
3'-C-methyl-2',3'-dideoxy-2',3'-didehydro-5-methyluridine, which were separated. 
The stereoselectivity of the Grignard reagent's attachment to 2-deoxyfuranose
3-ulosides has been ruled by the substitute configuration at Cl. Also, the effect
of the hydroxyl or OBz group configuration at C3 on the condensation
stereoselectivity of 3-C-methyl-2-deoxyfuranosides with silylated thymine has
been studied. The structure of the obtained compounds was proved by 1H NMR UV,
13C NMR, and CD spectroscopy, as well as elemental (C, H, N) analysis. The
C2'-endo-C1'-exo conformation, the anti conformation of thymine in relation to
the glycosidic bond, and the gauche+conformation in relation to the C4'-C5' bond 
are characteristic for the 3'-C-methyl-2'-deoxythymidine structure in the
crystals. 3'-C-Methyl-2'-deoxythymidine 5'-triphosphate was synthesized and
proved to be a competitive inhibitor, with respect to dTTP, of a number of DNA
polymerases, including the reverse transcriptases of human immunodeficiency virus
type 1 (HIV-1) and avian myeloblastosis virus (AMV). None of the DNA polymerases 
examined were able to incorporate this compound into the growing DNA chain. In
contrast, 3'-C-methylidene-2',3'-dideoxy-5-methyluridine 5'-triphosphate was
found to be incorporated at the 3'-end of the DNA chain by HIV-1 reverse
transcriptase, albeit with very low efficiency.
3'-C-Methyl-2'-deoxy-5-methyluridine did not suppress HIV-1 replication in MT-4
cells at 500 microM while its 5'-phosphite derivative exhibited modest anti-HIV-1
activity.

PMID: 1281882 [PubMed - indexed for MEDLINE]


619. Int J Pept Protein Res. 1992 Nov;40(5):383-94.

Conformational studies on beta-bend containing a cis peptide unit.

Nagarajaram HA, Paul PK, Ramanarayanan K, Soman KV, Ramakrishnan C.

Molecular Biophysics Unit, Indian Institute of Science, Bangalore.

Conformational studies have been carried out on the X-cis-Pro tripeptide system
(a system of three linked peptide units, in the trans-cis-trans configuration)
using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro
have been used. The energy minima have been classified into different groups
based upon the conformational similarity. There are 15, 20, 18 and 6 minima that 
are possible for the four cases respectively and these fall into 11 different
groups. A study of these minima shows that, (i) some minima contain hydrogen
bonds--either 4-->1 or 1-->2 type, (ii) the low energy minima qualify themselves 
as bend conformations, (iii) cis' and trans' conformations are possible for the
prolyl residue as also the C gamma-endo and C gamma-exo puckerings, and (iv) for 
Pro-cis-Pro, cis' at the first prolyl residue is ruled out, due to the high
energy. The available crystal structure data on proteins and peptides, containing
cis-Pro segment have been examined with a view to find the minima that occur in
solid state. The data from protein show that they fall under two groups. The
conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. 
In both peptides and proteins there exists a preference for trans' conformation
at prolyl residue over cis' when X is a non-glycyl residue. The minima obtained
can be useful in modelling studies.

PMID: 1483833 [PubMed - indexed for MEDLINE]


620. EMBO J. 1992 Nov;11(11):4227-37.

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase;
overlapping structural domains for the 3'-5' exonuclease and strand-displacement 
activities.

Soengas MS, Esteban JA, Lazaro JM, Bernad A, Blasco MA, Salas M, Blanco L.

Centro de Biologia Molecular (CSIC-UAM), Universidad Autonoma, Madrid, Spain.

In this report we present the alignment of one of the most conserved segments
(Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences,
including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the 
two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 
DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization,
processivity or protein-primed initiation. In contrast, substitution of the
highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease
activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the 
highly conserved Asp residue into Ala resulted in almost complete inactivation
(0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5'
exonuclease to the fidelity of DNA replication, the three mutations in the Exo
III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency
of misinsertion and extension of DNA polymerization errors. Surprisingly, the
three mutations in the Exo III motif strongly decreased (80- to 220-fold) the
ability to replicate phi 29 DNA, this behaviour being due to a defect in the
strand displacement activity, an intrinsic property of phi 29 DNA polymerase
required for this process. Taking these results into account, we propose that the
strand displacement activity of phi 29 DNA polymerase resides in the N-terminal
domain, probably overlapping with the 3'-5' exonuclease active site.

PMCID: PMC556934
PMID: 1396603 [PubMed - indexed for MEDLINE]


621. Biosci Biotechnol Biochem. 1992 Oct;56(10):1523-8.

An Avicel-affinity site in an Avicel-digesting exocellulase from a Trichoderma
viride mutant.

Goto M, Furukawa K, Hayashida S.

Department of Agricultural Chemistry, Kyushu University, Fukuoka, Japan.

A single form of exo-type cellulase (Exo I; MW, 65,000), purified from a
Trichoderma viride protease-depressed mutant, HK-75, digested Avicel to
cellobiose exowise, and hydrolyzed cellotriose, cellotetraose, and cellopentaose 
in the strict manner of splitting off by cellobiose units. Exo I, however,
hydrolyzed cellohexaose by both cellobiose and cellotriose units. Exo I was
proteolyzed by papain into two fragments; GPExo (MW, 9,000) and Exo I' (MW,
56,000). The GPExo intensively adsorbed onto Avicel but did not hydrolyze it. Exo
I' had nearly identical activity to that of intact Exo I toward
cellooligosaccharides but was almost inert to Avicel in digestion and adsorption.
Sequence analysis of N-terminal and C-terminal amino acids showed that GPExo was 
between Gly435 and Leu496 and Exo I' between Glu1 and Gly434 in Exo I. Exo I
therefore consists of two domains, one for adsorption to Avicel, as demonstrated 
by the Avicel-affinity site, GPExo and the other for the cleavage of glycosidic
linkages as demonstrated in Exo I'.

PMID: 1369052 [PubMed - indexed for MEDLINE]


622. Biochemistry. 1992 Sep 29;31(38):9132-40.

Conformationally restricted thrombin inhibitors resistant to proteolytic
digestion.

Szewczuk Z, Gibbs BF, Yue SY, Purisima EO, Konishi Y.

Biotechnology Research Institute, National Research Council of Canada, Montreal, 
Quebec.

A new type of thrombin exo-site inhibitor has been designed with enhanced
inhibitory potency and increased metabolic stability. With the aid of the model
of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio,
J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5,
77-85], cyclic analogs of the hirudin fragment (hirudin55-65) were designed and
synthesized. In these analogs, the side chains of appropriately substituted
residues, 58 and 61, were joined in order to restrict the conformation of the
inhibitor. An analog with an 18-membered lactam ring showed higher antithrombin
activity (IC50 = 0.57 microM) than the corresponding analogs with 17- or
16-membered rings and was 2-fold more potent than its linear counterpart. Even
4-fold greater enhancement was obtained when a shorter fragment, hirudin 55-62,
was cyclized. This cyclization not only improved the potency but, more
importantly, dramatically increased the resistance to proteolytic digestion.
Remarkable enhancement of stability to proteolysis was observed for peptide bonds
located in the exocyclic linear peptide segments. These results are discussed
using molecular modeling.

PMID: 1390700 [PubMed - indexed for MEDLINE]


623. Carbohydr Res. 1992 Jul 2;231:105-16.

Structure of the D-mannan of the pathogenic yeast, Candida stellatoidea ATCC
20408 (type II) strain, in comparison with that of C. stellatoidea ATCC 36232
(type I) strain.

Kobayashi H, Takaku M, Nishidate Y, Takahashi S, Takikawa M, Shibata N, Suzuki S.

Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, Japan.

Acid treatment of the cell-wall D-mannas of Candida stellatoidea strains ATCC
36232 (Type I, A3 strain) and ATCC 20408 (Type II, A2 strain) gave
(1----2)-linked beta-D-manno-oligosaccharides (dp 2-5), whereas treatment with
alkali gave the (1----2)-linked alpha-D-mannobiose. Conventional acetolysis of
the acid- and alkali-treated D-mannan of the A3 strain gave oligosaccharides
consisting of (1----2)- and (1----3)-linked alpha-D-mannopyranose residues,
similar to those of Candida albicans serotype B strain. Mild acetolysis of the
acid- and alkali-treated D-mannan of the A2 strain gave higher oligosaccharides
that were digested by the Arthrobacter GJM-1 strain exo-alpha-D-mannosidase. The 
results of 1H- and 13C-NMR analyses indicated this D-mannan to contain branches
with the following structures:
beta-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp++
+-(1----2)-alpha-D-Manp- (1----2)-D-Man,
beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-alpha-D-Manp -(1----2)-
alpha-D-Manp-(1----2)-D-Man, and beta-D-Manp-(1----2)-beta-D-Manp-(1----2)-beta- 
D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp-(1- ---2)-alpha-D-Manp-
(1----2)-D-Man, in common with the D-mannans of C. albicans serotype A strains.

PMID: 1394307 [PubMed - indexed for MEDLINE]


624. Carbohydr Res. 1992 Jun 16;230(2):223-44.

Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose
using MM3.

Dowd MK, Zeng J, French AD, Reilly PJ.

Department of Chemical Engineering, Iowa State University, Ames 50011.

Energy surfaces were computed for relative orientations of the relaxed pyranosyl 
rings of the two anomeric forms of kojibiose, nigerose, and maltose, the
(1----2)-alpha, (1----3)-alpha, and (1----4)-alpha-linked D-glucosyl
disaccharides, respectively. Twenty-four combinations of starting conformations
of the rotatable side-groups were considered for each disaccharide. Optimized
structures were calculated using MM3 on a 20 degree grid spacing of the torsional
angles about the glycosidic bonds. The energy surfaces of the six disaccharides
were similar in many respects but differed in detail within the low-energy
regions. The maps also illustrate the importance of the exo-anomeric effect and
linkage type in determining the conformational flexibility of disaccharides.
Torsional conformations of known crystal structures of maltosyl-containing
molecules lie in a lower MM3 energy range than previously reported.

PMID: 1394298 [PubMed - indexed for MEDLINE]


625. Can J Microbiol. 1992 Jun;38(6):555-62.

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 
dicarboxylates and symbiotic nitrogen fixation.

Rastogi V, Labes M, Finan T, Watson R.

Plant Research Centre, Agriculture Canada, Ottawa, Ont.

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates 
into bacteroids in the nodule for use as a carbon and energy source. In an
attempt to increase dicarboxylate transport, a plasmid was constructed in which
the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan
operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional
dctA gene that was no longer under the control of the dctBD regulatory genes, but
the recombinant plasmid was found to be unstable in R. meliloti. To stably
integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into 
the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed
constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that
found in fully induced wild-type cells. Uptake assays showed that [14C]succinate 
transport by the trpPO-dctA fusion strain was constitutive, and the transport
rate was the same as that of induced control cells. Acetylene reduction assays
indicated a significantly higher rate of nitrogen fixation in plants inoculated
with the trpPO-dctA fusion strain compared with the control. Despite this
apparent increase, the plants had the same top dry weights as those inoculated
with control cells.

PMID: 1504920 [PubMed - indexed for MEDLINE]


626. J Biochem. 1992 May;111(5):600-5.

Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus
tulipiferae) on cellulose with different crystallinities.

Hoshino E, Kanda T, Sasaki Y, Nisizawa K.

Household Products Research Laboratories, Kao Corporation, Wakayama.

The adsorption mode of two highly purified cellulases, exo- and endo-type
cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using 
pure cellulosic materials with different crystallinity as substrates. Adsorption 
of the two enzymes on the substrates was found to fit the Langmuir-type
adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir
plots showed an inverse correlation to the crystallinity of the substrate with
both enzymes, and this value of endo-type cellulase was less dependent on the
degree of crystallinity of substrates than that of exo-type cellulase, whose
isotherms reached saturation in the range of low enzyme concentrations. The two
enzymes showed relatively high affinities for all the substrates and their
affinities increased with increasing crystallinity, but this tendency was less
marked with endo-type cellulase than with exo-type one. In addition, large
negative values of free energy change were observed on the adsorption of both
enzymes, and the values became more negative with increasing crystallinity.
Consequently, both cellulases showed high adsorption on crystalline cellulose and
the adsorption process became smoother with increasing crystallinity. The
adsorption of the two types of cellulases was endothermic with an increase in
entropy, especially for amorphous cellulose, suggesting the occurrence of water
release from the substrates during enzyme adsorption. In addition, the changes in
thermodynamic parameters (delta H, delta S, and delta G) in adsorption of
exo-type cellulase were larger than in that of endo-type enzyme.

PMID: 1639755 [PubMed - indexed for MEDLINE]


627. J Bacteriol. 1992 May;174(10):3403-6.

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo
mutants.

Urzainqui A, Walker GC.

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during
symbiosis with Medicago sativa has been shown to be required for the proper
development of nitrogen-fixing nodules. Cloned DNA from the exo region of R.
meliloti is shown to stimulate production of the low-molecular-weight form of
this exopolysaccharide, and in this report we show that the symbiotic
deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be
rescued by the addition of this low-molecular-weight material at the time of
inoculation. For exoA and exoH mutants, rescue with a preparation containing
low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing
nodules which appear somewhat later and at a reduced efficiency compared with
wild-type-induced nodules; however, microscopic analysis of these nodules reveals
similar nodule morphology and the presence of large numbers of bacteroids in
each.

PMCID: PMC206013
PMID: 1577707 [PubMed - indexed for MEDLINE]


628. J Biol Chem. 1992 Apr 25;267(12):8012-20.

Comparative study of the sugar chains of factor VIII purified from human plasma
and from the culture media of recombinant baby hamster kidney cells.

Hironaka T, Furukawa K, Esmon PC, Fournel MA, Sawada S, Kato M, Minaga T, Kobata 
A.

Department of Biochemistry, University of Tokyo, Japan.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations 
purified from human plasma of blood group A donors and from the culture media of 
recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These 
sugar chains were converted to radioactive oligosaccharides by reduction with
sodium borotritide and separated into neutral and acidic fractions by paper
electrophoresis. Most of the acidic oligosaccharides were converted to neutral
ones by sialidase digestion, indicating that they are sialyl derivatives. The
neutral and sialidase-treated acidic oligosaccharides were fractionated by serial
chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural
study of each oligosaccharide by sequential exo- and endoglycosidase digestion
and by methylation analysis revealed that both factor VIII preparations contain
mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar
chains. Some of the biantennary complex-type sugar chains from human plasma
factor VIII contain blood group A and/or H determinant, while those from
recombinant product do not. Some of the bi-, tri- and tetra-antennary
complex-type sugar chains of the recombinant factor VIII contain the Gal alpha
1----3Gal group. A small number of the triantennary complex-type sugar chains
from both preparations was found to contain the Gal beta 1----4(Fuc alpha
1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies
of pharmacokinetic parameters of the recombinant factor VIII infused into baboons
revealed that its half-life in blood circulation is similar to that of plasma
derived factor VIII, suggesting that the oligosaccharide structural differences
between them do not affect the fate of factor VIII in vivo.

PMID: 1569060 [PubMed - indexed for MEDLINE]


629. J Biol Chem. 1992 Apr 5;267(10):6855-8.

Substrate specificity and reaction mechanism of human glycoasparaginase. The
N-glycosidic linkage of various glycoasparagines is cleaved through a reaction
mechanism similar to L-asparaginase.

Kaartinen V, Mononen T, Laatikainen R, Mononen I.

Department of Clinical Chemistry, Kuopio University Central Hospital, Finland.

Human glycoasparaginase (N4-(beta-N-acetyl-D-glucosaminyl)-L-asparaginase, EC
3.5.1.26) hydrolyzes a series of compounds that contain L-asparagine residue with
free alpha-amino and alpha-carboxyl groups. Substrates include high mannose and
complex type glycoasparagines as well as those that lack the
di-N-acetylchitobiose moiety, L-aspartic acid beta-methyl ester and L-aspartic
acid beta-hydroxamate. The enzyme is inactive toward L-asparagine and L-glutamine
and glycoasparagines containing substituted alpha-amino and/or alpha-carboxyl
groups. In the presence of the acyl acceptor hydroxylamine, glycoasparaginase
catalyzes the synthesis of L-aspartic acid beta-hydroxamate from
aspartyl-glucosamine, L-aspartic acid beta-methyl ester, and L-aspartic acid. 13C
NMR studies using 18O-labeled L-aspartic acid demonstrate that glycoasparaginase 
catalyzes an oxygen exchange between water and the carboxyl group at C-4 of
L-aspartic acid. These results indicate that glycoasparaginase reacts as an
exo-hydrolase toward the L-asparagine moiety of the substrates and the free
alpha-amino and alpha-carboxyl groups are required for the enzyme reaction. The
results are consistent with an L-asparaginase-like reaction pathway which
involves a beta-aspartyl enzyme intermediate. Since glycoasparaginase is active
toward a series of structurally different glycoasparagines, we suggest the
revised systematic name of N4-(beta-glycosyl)-L-asparaginase for the enzyme.

PMID: 1551892 [PubMed - indexed for MEDLINE]


630. Appl Environ Microbiol. 1992 Apr;58(4):1095-1101.

Extracellular Polysaccharide Is Not Responsible for Aluminum Tolerance of
Rhizobium leguminosarum bv. Phaseoli CIAT899.

Kingsley MT, Bohlool BB.

Department of Microbiology, University of Hawaii, Honolulu, Hawaii 96822.

Strain UHM-5, a pSym Exo derivative of the aluminum-tolerant Rhizobium
leguminosarum bv. phaseoli strain CIAT899, was equally tolerant of aluminum (Al) 
as the parental culture. Dialyzed culture supernatants of the wild-type cells
grown in YEM broth (10 cells ml) contained 185 mug of glucose equivalents ml
whereas UHM-5 culture supernatants yielded 2 mug of glucose ml. The Exo
derivative and the parental strain gave essentially similar growth in medium
containing from 0 to 300 muM Al, indicating that the pSym of CIAT899, and
extracellular polysaccharide, were not involved in the aluminum tolerance of this
strain. However, increasing the level of Al from 80 to 150 muM increased the lag 
phase, induced a slight killing of the inoculum, and depressed the final
populations by about fivefold. Doubling the aluminum concentration from 150 to
300 muM presented a severe aluminum stress to CIAT899 and UHM-5: the inoculum
level dropped 10-fold, indicating killing of the inoculum, and remained depressed
for ca. 4 days before continuing to grow slowly; the final population was
decreased 15-fold relative to that of cultures grown in medium containing 80 muM 
Al. The production by CIAT899 of other extracellular or intracellular aluminum
tolerance factors was investigated in culture by using aluminum-sensitive
rhizobia as stress indicators. These experiments, conducted at 80 muM Al,
demonstrated that CIAT899 produced neither extracellular nor intracellular
products that could alleviate toxicity for the Al-sensitive indicator rhizobia.

PMCID: PMC195560
PMID: 16348680 [PubMed - as supplied by publisher]


631. Biull Eksp Biol Med. 1992 Mar;113(3):263-8.

[Changes in the organization of the intermediate filament system of human
fibroblasts in lysosomal storage diseases and their modeling]

[Article in Russian]

Ivleva TS, Tint IS, Bershadskii AD, Vidershain GIa.

The organization of the system of the vimentin intermediate filaments (IFs) in
human fibroblasts in lysosomal storage diseases (Fabry's disease, mannosidosis)
and their modelling has been studied in vitro. It was shown that during
accumulation of nonhydrolyzable compounds, hypertrophy of the lysosomal
compartment is accompanied by formation of ring-shaped bundles IFs, surrounding
apparently these increased organelles. The changed organization of IFs is
characteristic of polarised pathological cells in monolayer, and after repassage 
it is retained only at the spreading state; on transition from the discoid to
extended cellular form there occurred the centrifugal shift of ring-shaped
structures of IFs to active cell border and gradual restoration of radial
fibrillar state of IFs. It is suggested that on intralysosomal storage of unsplit
compounds reorganization of the vimentin-type IFs in ring-shaped structure is
necessary for optimal distribution and stabilization into the cytoplasm of large 
amounts of increased lysosomes with exo- and endogenous contents. In condition of
free spreading (i. e. with diminished cell density) the restoration of normal
fibrillar IF organization may be due to the loss of considerable number of
hypertrophied lysosomes; the involvement of lysosomal membrane in formation of
active cellular border is not to be ruled out.

PMID: 1421222 [PubMed - indexed for MEDLINE]


632. J Mol Biol. 1992 Feb 20;223(4):1013-28.

Three-dimensional structure of a mutant ribonuclease T1 (Y45W) complexed with
non-cognizable ribonucleotide, 2'AMP, and its comparison with a specific complex 
with 2'GMP.

Hakoshima T, Itoh T, Tomita K, Goda K, Nishikawa S, Morioka H, Uesugi S, Ohtsuka 
E, Ikehara M.

Faculty of Pharmaceutical Sciences, Osaka University, Japan.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a
non-cognizable ribonucleotide, 2'AMP, has been determined and refined to an
R-factor of 0.159 using X-ray diffraction data at 1.7 A resolution. A specific
complex of the enzyme with 2'GMP was also determined and refined to an R-factor
of 0.173 at 1.9 A resolution. The adenine base of 2'AMP was found at a
base-binding site that is far apart from the guanine recognition site, where the 
guanine base of 2'GMP binds. The binding of the adenine base is mediated by a
single hydrogen bond and stacking interaction of the base with the imidazole ring
of His92. The mode of stacking of the adenine base with His92 is similar to the
stacking of the guanine base observed in complexes of ribonuclease T1 with
guanylyl-2',5'-guanosine, reported by Koepke et al., and two guanosine bases,
reported by Lenz et al., and in the complex of barnase with d(GpC), reported by
Baudet & Janin. These observations suggest that the site is non-specific for base
binding. The phosphate group of 2'AMP is tightly locked at the catalytic site
with seven hydrogen bonds to the enzyme in a similar manner to that of 2'GMP. In 
addition, two hydrogen bonds are formed between the sugar moiety of 2'AMP and the
enzyme. The 2'AMP molecule adopts the anti conformation of the glycosidic bond
and C-3'-exo sugar pucker, whereas 2'GMP is in the syn conformation with
C-3'-endo-C'-2'-exo pucker. The mutation enhances the binding of 2'GMP with
conformational changes of the sugar ring and displacement of the phosphate group 
towards the interior of the catalytic site from the corresponding position in the
wild-type enzyme complex. Comparison of two crystal structures obtained provides 
a solution to the problem that non-cognizable nucleotides exhibit unexpectedly
strong binding to the enzyme, compared with high specificity in nucleolytic
activity. The results indicate that the discrimination of the guanine base from
the other nucleotide bases at the guanine recognition site is more effective than
that estimated from nucleotide-binding experiments so far.

PMID: 1311385 [PubMed - indexed for MEDLINE]


633. Mol Microbiol. 1992 Feb;6(4):479-88.

Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo
genes.

Nagpal P, Khanuja SP, Stanfield SW.

Center for Molecular Genetics, University of California, San Diego, La Jolla
92093-0634.

The ndvA and ndvB genes of Rhizobium meliloti are involved in the export and
synthesis, respectively, of the small cyclic polysaccharide beta(1,2)glucan. We
have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants 
do not produce periplasmic beta(1,2)glucan. Here we show that the
pseudorevertants also do not produce extracellular beta(1,2)glucan, but do show
alterations in the amount of the major acidic exopolysaccharide produced. This
exopolysaccharide is not detectably different from that produced by the wild type
or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both
ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA 
pseudorevertant. This cosmid contains a number of exo genes, including exoH and
exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated 
exoH and exoF genes are both involved in suppression of the ndv mutant phenotype 
and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the 
mutation responsible for second site suppression.

PMID: 1560776 [PubMed - indexed for MEDLINE]


634. Plant Physiol. 1992 Jan;98(1):143-151.

Nodules Initiated by Rhizobium meliloti Exopolysaccharide Mutants Lack a
Discrete, Persistent Nodule Meristem.

Yang C, Signer ER, Hirsch AM.

Department of Biology, Peking University, Beijing, China.

Infection of alfalfa with Rhizobium meliloti exo mutants deficient in
exopolysaccharide results in abnormal root nodules that are devoid of bacteria
and fail to fix nitrogen. Here we report further characterization of these
abnormal nodules. Tightly curled root hairs or shepherd's crooks were found after
inoculation with Rm 1021-derived exo mutants, but curling was delayed compared
with wild-type Rm 1021. Infection threads were initiated in curled root hairs by 
mutants as well as by wild-type R. meliloti, but the exo mutant-induced threads
aborted within the peripheral cells of the developing nodule. Also, nodules
elicited by Rm 1021-derived exo mutants were more likely to develop on secondary 
roots than on the primary root. In contrast with wild-type R. meliloti-induced
nodules, the exo mutant-induced nodules lacked a well defined apical meristem,
presumably due to the abortion of the infection threads. The relationship of
these findings to the physiology of nodule development is discussed.

PMCID: PMC1080161
PMID: 16668605 [PubMed - as supplied by publisher]


635. Microbiol Immunol. 1992;36(6):643-7.

Characterization of an exo-beta-D-fructosidase from Streptococcus mutans
Ingbritt.

Igarashi T, Yamamoto A, Goto N.

Department of Oral Microbiology, Showa University School of Dentistry, Tokyo,
Japan.

An extracellular enzyme beta-D-fructosidase was purified from the culture
supernatant of Streptococcus mutans Ingbritt and characterized. The molecular
weight of the enzyme was 127,000 as determined by SDS-polyacrylamide gel
electrophoresis. The enzyme was specific for levan which mainly consists of
beta-(2,6)-linked D-fructose and was also able to hydrolyze inulin, sucrose and
raffinose at the activities of 13, 9 and 5% of that hydrolyzing levan,
respectively. The pH optima for levan, inulin and sucrose were approximately 5.5,
6.0 and 5.0, respectively. The enzyme was optimally reactive at 55 C for levan.
The enzyme was inhibited by Fe3+, Hg2+ and Zn2+ and not by either anionic or
non-ionic detergents. Paper chromatographic analysis revealed that the enzyme
attacked levan by an exo-type mechanism.

PMID: 1522814 [PubMed - indexed for MEDLINE]


636. Acta Biochim Pol. 1992;39(2):177-91.

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned
exo region.

Urbanik-Sypniewska T, Skorupska A, Derylo M.

Department of General Microbiology, University M. Curie-Sklodowska, Lublin,
Poland.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS)
which plays an important role in the development of nitrogen-fixing nodules. Tn5 
mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective
(Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 
and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the
complementation is not correlated with restoration of Fix+ phenotype.
Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its 
ability to form nitrogen-fixing nodules. These results might suggest that
bacteria with cosmids carrying the exo region form EPS of altered structure. It
has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii
93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl 
and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the
biological activity of the exopolysaccharide of R. trifolii depends on the
contents of the non-carbohydrate substitutions.

PMID: 1441845 [PubMed - indexed for MEDLINE]


637. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):392-6.

Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase
system.

Walker GT, Little MC, Nadeau JG, Shank DD.

Department of Molecular Biology, Becton Dickinson Research Center, Research
Triangle Park, NC 27709.

An isothermal in vitro DNA amplification method was developed based upon the
following sequence of reaction events. Restriction enzyme cleavage and subsequent
heat denaturation of a DNA sample generates two single-stranded target DNA
fragments (T1 and T2). Present in excess are two DNA amplification primers (P1
and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5'
overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a
recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An
exonuclease-deficient form of the large fragment of Escherichia coli DNA
polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S.,
Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988)
Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, 
and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces
hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the
unprotected primer strands of the hemiphosphorothioate recognition sites, leaving
intact the modified complementary strands. The exo- Klenow polymerase extends the
3' end at the nick on P1.T1 and displaces the downstream strand that is
functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results
in displacement of a downstream strand functionally equivalent to T1. Nicking and
polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because
extension at a nick regenerates a nickable HincII recognition site. Target
amplification is exponential because strands displaced from P1.T1 serve as
targets for P2 and strands displaced from P2.T2 serve as targets for P1. A
10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or
Mycobacterium bovis was achieved in 4 h at 37 degrees C.

PMCID: PMC48243
PMID: 1309614 [PubMed - indexed for MEDLINE]


638. Biochemistry. 1991 Dec 3;30(48):11420-9.

Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala.

Raag R, Martinis SA, Sligar SG, Poulos TL.

Maryland Biotechnology Institute, University of Maryland, Shady Grove, Rockville,
Maryland 20850.

The crystal structure of a cytochrome P-450CAM site-directed mutant in which the 
active site Thr252 has been replaced with an Ala (Thr252Ala) has been refined to 
an R factor of 0.18 at 2.2 A. According to sequence alignments (Nelson & Strobel,
1989), Thr252 is highly conserved among P-450 enzymes. The crystallographic
structure of ferrous camphor- and carbon monoxide-bound P-450CAM (Raag & Poulos, 
1989b) suggests that Thr252 is a key active site residue, forming part of the
dioxygen-binding site. Mutation of the active site threonine to alanine produces 
an enzyme in which substrate hydroxylation is uncoupled from electron transfer.
Specifically, hydrogen peroxide and "excess" water are produced instead of the
product, 5-exo-hydroxycamphor. The X-ray structure has revealed that a local
distortion in the distal helix between Gly248 and Thr252 becomes even more severe
in the Thr252Ala mutant. Furthermore, a solvent molecule not present in the
native enzyme is positioned in the dioxygen-binding region of the mutant enzyme
active site. In this location, the solvent molecule could sterically interfere
with and destabilize dioxygen binding. In addition, the active site solvent
molecule is connected, via a network of hydrogen bonds, with an internal solvent 
channel which links distal helix residues to a buried Glu side chain. Thus,
solvent protons appear to be much more accessible to dioxygen in the mutant than 
in the wild-type enzyme, a factor which may promote hydrogen peroxide and/or
water production instead of substrate hydroxylation. On the basis of
crystallographic and mutagenesis data, a proton delivery pathway involving
residues Lys178/Arg186, Asp251, and Thr252 is proposed for wild-type P-450CAM.
Coordinates of structures discussed in this paper have been submitted to the
Brookhaven Protein Data Bank (Bernstein et al., 1977).

PMID: 1742281 [PubMed - indexed for MEDLINE]


639. Glycoconj J. 1991 Dec;8(6):456-83.

Data bank of three-dimensional structures of disaccharides: Part II,
N-acetyllactosaminic type N-glycans. Comparison with the crystal structure of a
biantennary octasaccharide.

Imberty A, Delage MM, Bourne Y, Cambillau C, Perez S.

Laboratoire de Synthese Organique, Faculte des Sciences, Nantes, France.

Conformational energy maps and descriptions of structures at the local minima are
presented for the following fragments found in N-acetyllactosaminic type glycans 
of N-glycoproteins: GlcNAc beta(1-2)Man, GlcNAc beta(1-4)Man, GlcNAc
beta(1-6)Man, Gal beta(1-4)GlcNAc, GlcNAc beta(1-3)Gal, Fuc alpha(1-6)GlcNAc, Fuc
alpha(1-3)GlcNAc, Xyl beta(1-2)Man, Gal beta(1-3)GlcNAc and GlcNAc beta(1-6)Gal. 
These results are the second part of a data bank on glycoprotein moieties; five
disaccharides found in oligomannose type N-glycans were analysed earlier (Imberty
et al., 1990, Glycoconjugate J 7:27-54). In the present study, three to seven
minima are found for each dimer. Conformations of disaccharide fragments found in
the crystal structure of the complex of a biantennary octasaccharide with
Lathyrus ochrus lectin are plotted on these energy maps. While the observed
conformations are at predicted minima, they are not always at the minimum
predicted to have the lowest energy. Further, not all observed conformations are 
stabilized by the exo-anomeric effect. We conclude that these oligosaccharides
are highly flexible.

PMID: 1823622 [PubMed - indexed for MEDLINE]


640. Biochemistry. 1991 Nov 5;30(44):10601-6.

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme
MboI.

Winkle SA, Aloyo MC, Morales N, Zambrano TY, Sheardy RD.

Department of Chemistry, Florida International University, Miami 33199.

We have been investigating the structure, dynamics, and ligand-binding properties
of the interface that exists between a right-handed conformation and a
left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since
exo- and endonuclease activity is known to be sensitive to the conformation of
the template DNA, we have designed and synthesized a DNA oligonucleotide of 20
base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the
location of a potential B-Z junction. The activity of the MboI enzyme toward this
molecule and DNA oligomers that contain multiple MboI sites located at B-Z
junctions was monitored in the absence and presence of the
Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of 
the enzyme was enhanced in the presence of cobalt hexaammine. The activity of
MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also
examined when the DNA oligomer is also in the presence of the DNA binding drugs
actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of
the enzyme was inhibited in the presence of drug. The results suggest that B-Z
junctions are structurally unique and that this uniqueness may alter nuclease
activity at sites in or near the junction.

PMID: 1931982 [PubMed - indexed for MEDLINE]


641. J Biol Chem. 1991 Oct 25;266(30):20244-61.

Structural characterization of the asparagine-linked oligosaccharides from
Trypanosoma brucei type II and type III variant surface glycoproteins.

Zamze SE, Ashford DA, Wooten EW, Rademacher TW, Dwek RA.

Department of Biochemistry, University of Oxford, United Kingdom.

The complete primary structures of the major Asn-linked oligosaccharides from the
type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the
type III VSG, MITat 1.5, were determined using a combination of exo- and
endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR 
spectroscopy. Each variant contained classical branched oligomannose-type and
biantennary complex oligosaccharides, a proportion of the latter substituted with
terminal alpha(1-3)-linked galactose residues, the first report of the presence
of this epitope in Trypanosoma brucei. In addition both the type II variants
contained relatively large amounts of the unusual small oligomannose-type
oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel
branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to 
those from mammalian glycoproteins. These latter structures were also partially
substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in 
the type II variants showed site specificity in that the poly-N-acetyllactosamine
and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at
Asn-glycosylation site 1 very close to the C terminus, whereas the
Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively
at site 2. This is the first report of the presence of poly-N-acetyllactosamine
oligosaccharides in protozoa.

PMID: 1939085 [PubMed - indexed for MEDLINE]


642. J Biol Chem. 1991 Oct 5;266(28):18684-90.

Oxidation of deuterated compounds by high specific activity methane monooxygenase
from Methylosinus trichosporium. Mechanistic implications.

Rataj MJ, Kauth JE, Donnelly MI.

Biotechnology Division, Amoco Technology Company, Naperville, Illinois 60566.

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high
specific activity from the type II methanotroph Methylosinus trichosporium OB3b
were compared to the same reactions catalyzed by methane monooxygenase from the
type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome
P-450. The two methane monooxygenases produced nearly identical product
distributions, in accord with physical studies of the enzymes which have shown
them to be very similar. The products obtained from the oxidation of a series of 
deuterated substrates by the M. trichosporium methane monooxygenase were very
similar to those reported for the same reaction catalyzed by liver microsomal
cytochrome P-450, suggesting that the enzymes use similar mechanisms. However,
differences in the product distributions and other aspects of the reactions
indicated the mechanisms are not identical. Methane monooxygenase epoxidized
propene in D2O and d6-propene in H2O without exchange of substrate protons or
deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T.,
Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J.
Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of
olefins by methane monooxygenase differs at least in part from that of cytochrome
P-450. Hydroxylation of alkanes by methane monooxygenase revealed close
similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of
3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in
the case of methane monooxygenase, whereas 33% was reported for this reaction
catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J.
Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of
exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with
epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves,
J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys.
Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo
greater than or equal to 5.5, was calculated for this reaction. However, the
intermolecular kinetic isotope effect on Vm for methane oxidation was small,
suggesting that steps other than C-H bond breakage were rate limiting in the
overall enzymatic reaction. Similar isotope effects have been observed for
cytochrome P-450. These observations indicate a stepwise mechanism of
hydroxylation for methane monooxygenase analogous to that proposed for cytochrome
P-450.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 1917992 [PubMed - indexed for MEDLINE]


643. Eur J Biochem. 1991 Sep 1;200(2):379-85.

Purification and properties of a novel type of exo-1,4-beta-glucanase (avicelase 
II) from the cellulolytic thermophile Clostridium stercorarium.

Bronnenmeier K, Rucknagel KP, Staudenbauer WL.

Institute for Microbiology, Technical University Munich, Federal Republic of
Germany.

Avicelase II was purified to homogeneity from culture supernatants of Clostridium
stercorarium. A complete separation from the major cellulolytic enzyme activity
(avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective
retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa 
and a pI of 3.9. Determination of the N-terminal amino acid indicates that
avicelase II is not a proteolytically processed product of avicelase I. Maximal
activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+,
the enzyme is highly thermostable, exhibiting a temperature optimum around 75
degrees C. Hydrolysis of avicel occurs at a linear rate for three days at 70
degrees C. Avicelase II is active towards unsubstituted celluloses, cellotetraose
and larger cellodextrins. It lacks activity towards carboxymethylcellulose and
barley beta-glucan. Unlike other bacterial exoglucanases, avicelase II does not
hydrolyze aryl-beta-D-cellobiosides. Avicel is degraded to cellobiose and
cellotriose at a molar ratio of approximately 4:1. With acid-swollen avicel as
substrate, cellotetraose is also formed as an intermediary product, which is
further cleaved to cellobiose. The degradation patterns of reduced cellodextrins 
differ from that expected for a cellobiohydrolase attacking the non-reducing ends
of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while
cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These
findings, taken together, indicate that avicelase II represents a novel type of
exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of
the substrate, releases cellotetraose, cellotriose, or cellobiose from the
non-reducing end of the cellulose chains.

PMID: 1909625 [PubMed - indexed for MEDLINE]


644. Biochem Biophys Res Commun. 1991 Aug 30;179(1):386-91.

1H NMR study of the sugar pucker of 2',3'-dideoxynucleosides with anti-human
immunodeficiency virus (HIV) activity.

Jagannadh B, Reddy DV, Kunwar AC.

Indian Institute of Chemical Technology, Hyderabad, India.

The sugar ring conformations of 2',3'-dideoxyribosyladenine (ddA),
2',3'-dideoxyribosylcytosine (ddC), 2',3'-dideoxyribosylguanine (ddG),
2',3'-dideoxyribosylhypoxanthine (ddI), 3'-azido-2',3'-dideoxyribosylthymine
(AZT), 3'-azido-2',3'-dideoxyribosyluracil (AZU) and
3'-fluoro-2',3'-dideoxyribosylthymine (FddT) have been investigated by 1H NMR
spectroscopy. While the sugar ring in FddT exists almost totally in C2'-endo
geometry, other nucleosides show equilibrium between sugar puckers of C3'-endo
family (N-type) and C2'-endo family (S-type). For unsubstituted
dideoxynucleosides C3'-endo conformer is favoured (congruent to 75%), whereas for
AZT and AZU both the conformers have almost equal populations. Unlike X-ray
diffraction studies, the NMR results do not support the suggestion that C3'-exo
sugar puckers are desirable for the anti-HIV activity of these nucleosides.

PMID: 1883367 [PubMed - indexed for MEDLINE]


645. J Mol Biol. 1991 Aug 5;220(3):801-18.

Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and 
endonuclease activities.

Wohrl BM, Volkmann S, Moelling K.

Max Planck Institut fur Molekulare Genetik, Berlin, Germany.

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a
protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA
polymerase and an RNase H at the carboxy terminus of the molecule. In order to
characterize the mode of action of the RNase H, two previously described mutant
enzymes were used, with substitutions in the highly conserved histidine 539,
which was mutated to the neutral amino acid asparagine and to the negatively
charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities 
are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA
that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides
complementary to the PPT or to other regions of the RNA. Analysis of the
radioactively labeled RNA of these model hybrids after RNase H treatment
indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an
endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease
activity. They are more sensitive towards salt and competition with excess of
unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation
by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase
H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive
3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic
endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities
of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence
of DNA synthesis different from those of the wt enzyme. They cannot recognize
specific sequences required to generate a PPT-primer and therefore cannot
initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is
essential for viral replication.

PMID: 1714505 [PubMed - indexed for MEDLINE]


646. J Gen Microbiol. 1991 Aug;137(8):1815-23.

Pectinase production by Neurospora crassa: purification and biochemical
characterization of extracellular polygalacturonase activity.

Polizeli Mde L, Jorge JA, Terenzi HF.

Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao
Preto, Universidade de Sao Paulo, Brazil.

The production of pectinase was studied in Neurospora crassa, using the
hyperproducer mutant exo-1, which synthesized and secreted five to six times more
enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were
induced by pectin, and this induction was glucose-repressible. Polygalacturonase 
was induced by galactose four times more efficiently than by pectin; in contrast 
the activity of lyases was not affected by galactose. The inducing effect of
galactose on polygalacturonase was not glucose-repressible. Extracellular
pectinases were separated by ion exchange chromatography. Pectate and pectin
lyases eluted into three main fractions containing both activities;
polygalacturonase eluted as a single, symmetrical peak, apparently free of other 
protein contaminants, and was purified 56-fold. The purified polygalacturonase
was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular
mass 36.6-37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme
hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of
the rate for polypectate. Km and Vmax for polypectate hydrolysis were 5.0 mg ml-1
and 357 mumol min-1 (mg protein)-1, respectively. Temperature and pH optima were 
45 degrees C and 6.0, respectively. The purified polygalacturonase reduced the
viscosity of a sodium polypectate solution by 50% with an increase of 7% in
reducing sugar groups. The products of hydrolysis at initial reaction times
consisted of oligogalacturonates without detectable monomer. Thus, the purified
Neurospora crassa enzyme was classified as an endopolygalacturonase
[poly(1,4-alpha-D-galacturonide) glycanohydrolase; EC 3.2.1.15].

PMID: 1835496 [PubMed - indexed for MEDLINE]


647. Eur J Biochem. 1991 Aug 1;199(3):637-41.

Comparative study of the structure/function relationship of wild-type and
structurally modified maltopentaose-producing amylase.

Candussio A, Schmid G, Bock A.

Lehrstuhl fur Mikrobiologie der Universitat Munchen, Federal Republic of Germany.

Amylase A-180, which is secreted by a new alkaliphilic organism, isolate 163-26, 
consists of a single type of polypeptide chain of 186.5 kDa and hydrolyses starch
by exo-attack releasing malto-pentaose as preferential product. The
structure/function relationship of this unusual starch-degrading enzyme was
analysed by introducing 3' deletions into the structural gene. It was found that 
removal of up to a 110-kDa portion from the C-terminus leaving 563 N-terminal
amino acids still led to the formation of a fully active enzyme. The part of the 
structural gene coding for these 563 N-terminal amino acids was fused with the
signal peptide-encoding segment of the cyclodextrin glucanotransferase gene from 
Klebsiella oxytoca and was cloned into an expression vector. The resulting
truncated A-180 derivative, A-180/21, was efficiently transported through the
cytoplasmic membrane and released into the medium by an Escherichia coli strain
which 'leaks' periplasmatic components. A-180/21 was purified and its catalytic
properties, i.e. specific activity and product specificity, proved to be
identical to those of the wild-type enzyme; however, in contrast to the wild-type
enzyme, it was unable to bind to raw starch and it displayed an altered
temperature and pH dependence of activity.

PMID: 1714389 [PubMed - indexed for MEDLINE]


648. Carbohydr Res. 1991 Jul 18;214(1):131-45.

Structural determination of D-mannans of pathogenic yeasts Candida stellatoidea
type I strains: TIMM 0310 and ATCC 11006 compared to IFO 1397.

Kobayashi H, Kojimahara T, Takahashi K, Takikawa M, Takahashi S, Shibata N, Okawa
Y, Suzuki S.

Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, Miyagi,
Japan.

The structures of the cell-wall D-mannans of pathogenic yeasts of Candida
stellatoidea Type I strains, IFO 1397, TIMM 0310, and ATCC 11006, were
investigated by mild acid and, alkaline hydrolysis, by digestion with the
Arthrobacter GJM-1 strain exo-alpha-D-mannosidase, and by acetolysis. The
modified D-mannans and their degradation products were studied by 1H- and
13C-n.m.r. analyses. D-Manno-oligosaccharides released by acid treatment from the
parent D-mannans were identified as the homologous beta-(1----2)-linked
D-manno-oligosaccharides from biose to hexaose, whereas those obtained by
alkaline degradation were the homologous alpha-(1----2)-linked D-mannobiose and
D-mannotriose. The acid- and alkali-modified D-mannans lacking 1H-n.m.r. signals 
above 4.900 p.p.m. [corresponding to beta-(1----2)-linked D-mannopyranose units] 
were acetolyzed with 10:10:1 (v/v) Ac2O-AcOH-H2SO4, and the resultant
D-manno-oligosaccharides were also analyzed. It was found that the longest
branches of these D-mannans, corresponding to hexaosyl residues, had the
following structures: alpha-D-Manp-(1----3)-alpha-D-Manp-(1----2)-alpha-D-Manp+
++-(1----2)-alpha-D-Manp- (1----2)-alpha-D-Manp-(1----2)-D-Man and
alpha-D-Manp-(1----2)-alpha-D-Manp-(1----3)-alpha-D-Manp+
++-(1----2)-alpha-D-Manp- (1----2)-alpha-D-Manp-(1----2)-D-Man. These results
indicate that the D-mannans of C. stellatoidea Type I strains possess structures 
in common with the D-mannans of Candida albicans serotype B strain (see ref. 4)
containing phosphate-bound beta-(1----2)-linked oligo-D-mannosyl residues.

PMID: 1954627 [PubMed - indexed for MEDLINE]


649. J Bacteriol. 1991 Jul;173(13):3981-92.

ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium
meliloti, is required for normal nodule development.

Charles TC, Newcomb W, Finan TM.

Department of Biology, McMaster University, Hamilton, Ontario, Canada.

Rhizobium meliloti strains carrying either of two overlapping deletions (delta
5408 and delta F114) of the megaplasmid pRmeSU47b form nodules on alfalfa which
fail to fix N2 (Fix-). Strains carrying these deletions also fail to fluoresce on
media containing calcofluor, indicating a defect in synthesis of the acidic
exopolysaccharide (Exo-) of R. meliloti. We have isolated cosmid clones (pTH21
and pTH22) which complement the Fix- but not the Exo- phenotype of the strains
carrying the delta 5408 and delta F114 deletions. In addition, cosmid clones
which complement the Exo- phenotype fail to complement the Fix- phenotype of
these deletions; thus, the Exo- phenotype is not related to the Fix- phenotype. A
5-kb region within a 7.3-kb BamHI restriction fragment was found to be required
for complementation of the Fix- phenotype of the delta 5408 and delta F114
deletion strains. Tn5 insertions in the 5-kb region generated a Fix- phenotype
when recombined into the wild-type genome. We have designated this locus ndvF,
for nodule development. TnphoA mutagenesis of this region generated active
alkaline-phosphatase gene fusions, indicating that ndvF encodes extracytoplasmic 
protein(s). Induction of nodules by the ndvF mutants was delayed by 2 to 3 days
compared with induction by the wild-type strain. Light microscopy of nodules
elicited by strains carrying the large 150-kb delta F114 deletion, a 12-kb
deletion removing ndvF, or an individual ndvF::Tn5 insertion mutation
demonstrated that many nodules contained few infected cortical cells, indicating 
that nodule development was blocked early in the infection process, before the
release of bacteria from the infection threads.

PMCID: PMC208044
PMID: 1648074 [PubMed - indexed for MEDLINE]


650. J Neurocytol. 1991 Jun;20(6):504-17.

Periaxonal ensheathment of lobster giant nerve fibres as revealed by
freeze-fracture and lanthanum penetration.

Villegas GM, Sanchez F.

Centro de Biociencias, Instituto Internacional de Estudios Avanzados (IDEA),
Caracas, Venezuela.

Sheath structure and permeability have been studied in the nerve fibres of
lobster (Panulirus argus) walking limbs, in particular the individually
ensheathed larger giant fibres, 100-150 microns in diameter, of which there are
five or six in a peripheral bundle. They are easily distinguished and can be
separated from neighbouring fibre bundles in which smaller giant axons (65-80
microns diameter) and many axons of much smaller diameter (5-15 microns) are
ensheathed together. Each of the larger giant axons is enveloped by a Schwann
cell layer outside of which is a multilayered sheath consisting of one-cell thick
belts of flattened cells and interleaved zones of collagen fibrils and
extracellular matrix. The cells in each belt lack basal lamina and, after
freeze-fracture, as well as in thin sections, exhibit intercellular gap junctions
and incomplete, fascia type, tight junctions; their most striking aspect is an
exceedingly large number of exo-endocytic profiles. Permeability to lanthanum
chloride in the bathing medium studied before or during fixation both in intact
nerves and in nerves with surgically breached (slit) epineurium showed
penetration of lanthanum tracer between the cells around the giant fibres, but
the electron-dense tracer was excluded from the Schwann cell layer and the
periaxonal space unless the epineurium had been slit. The extent of lanthanum
diffusion was evaluated by transmission electron microscopy of thin sections and 
confirmed by X-ray microanalysis (EDAX) of comparable selected areas in such
sections. The results indicate structural similarities but distinct permeability 
differences between the multilayered sheath surrounding the lobster giant axons
and the vertebrate nerve perineurium. Other ultrastructural details provided by
the freeze-fracture replicas concern the distribution of intramembrane particles 
in the axolemma and the Schwann and sheath cell membranes.

PMID: 1869886 [PubMed - indexed for MEDLINE]


651. J Bacteriol. 1991 Jun;173(12):3789-94.

The exoR gene of Rhizobium meliloti affects RNA levels of other exo genes but
lacks homology to known transcriptional regulators.

Reed JW, Glazebrook J, Walker GC.

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

Rhizobium meliloti strains mutant in the exoR gene overproduce an
exopolysaccharide called succinoglycan or EPS I. Protein fusions to several
different exo genes required for EPS I biosynthesis are expressed at a higher
level in an exoR strain than in a wild-type strain, showing that the
overproduction of EPS I in exoR strains results at least in part from increased
gene expression. This regulation is important to nodulation, since exoR mutants
fail to invade alfalfa nodules unless secondary suppressor mutations that cause a
decrease in EPS I production occur. Here, we show that an exoR strain contains
higher levels of mRNA for other exo genes than does the wild-type parental
strain. ExoR therefore most probably exerts its regulatory effect at the level of
transcription. In addition, we have localized, subcloned, and sequenced the exoR 
gene. A newly constructed insertion allele of exoR has the same phenotype as the 
original mutant. The deduced sequence of ExoR is 268 amino acids long but does
not show homology to other sequenced genes.

PMCID: PMC208009
PMID: 1711027 [PubMed - indexed for MEDLINE]


652. J Bacteriol. 1991 May;173(10):3066-77.

Heterologous exopolysaccharide production in Rhizobium sp. strain NGR234 and
consequences for nodule development.

Gray JX, Zhan HJ, Levery SB, Battisti L, Rolfe BG, Leigh JA.

Plant Microbe Interactions Group, Research School of Biological Sciences,
Australian National University, Canberra, A.C.T.

Rhizobium sp. strain NGR234 produces large amounts of acidic exopolysaccharide.
Mutants that fail to synthesize this exopolysaccharide are also unable to
nodulate the host plant Leucaena leucocephala. A hybrid strain of Rhizobium sp.
strain NGR234 containing exo genes from Rhizobium meliloti was constructed. The
background genetics and nod genes of Rhizobium sp. strain NGR234 are retained,
but the cluster of genes involved in exopolysaccharide biosynthesis was deleted. 
These exo genes were replaced with genes required for the synthesis of
succinoglycan exopolysaccharide from R. meliloti. As a result of the genetic
manipulation, the ability of these hybrids to synthesize exopolysaccharide was
restored, but the structure was that of succinoglycan and not that of Rhizobium
sp. strain NGR234. The replacement genes were contained on a cosmid which encoded
the entire known R. meliloti exo gene cluster, with the exception of exoB.
Cosmids containing smaller portions of this exo gene cluster did not restore
exopolysaccharide production. The presence of succinoglycan was indicated by
staining with the fluorescent dye Calcofluor, proton nuclear magnetic resonance
spectroscopy, and monosaccharide analysis. Although an NGR234 exoY mutant
containing the R. meliloti exo genes produced multimers of the succinoglycan
repeat unit, as does the wild-type R. meliloti, the deletion mutant of Rhizobium 
sp. strain NGR234 containing the R. meliloti exo genes produced only the monomer.
The deletion mutant therefore appeared to lack a function that affects the
multiplicity of succinoglycan produced in the Rhizobium sp. strain NGR234
background. Although these hybrid strains produced succinoglycan, they were still
able to induce the development of an organized nodule structure on L.
leucocephala. The resulting nodules did not fix nitrogen, but they did contain
infection threads and bacteroids within plant cells. This clearly demonstrated
that a heterologous acidic exopolysaccharide structure was sufficient to enable
nodule development to proceed beyond the developmental barrier imposed on mutants
of Rhizobium sp. strain NGR234 that are unable to synthesize any acidic
exopolysaccharide.

PMCID: PMC207899
PMID: 2022612 [PubMed - indexed for MEDLINE]


653. Cell. 1991 Feb 22;64(4):777-87.

Topology of eukaryotic type II membrane proteins: importance of N-terminal
positively charged residues flanking the hydrophobic domain.

Parks GD, Lamb RA.

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern
University, Evanston, Illinois 60208-3500.

We have tested the role of different charged residues flanking the sides of the
signal/anchor (S/A) domain of a eukaryotic type II (N(cyt)C(exo)) integral
membrane protein in determining its topology. The removal of positively charged
residues on the N-terminal side of the S/A yields proteins with an inverted
topology, while the addition of positively charged residues to only the
C-terminal side has very little effect on orientation. Expression of chimeric
proteins composed of domains from a type II protein (HN) and the oppositely
oriented membrane protein M2 indicates that the HN N-terminal domain is
sufficient to confer a type II topology and that the M2 N-terminal ectodomain can
direct a type II topology when modified by adding positively charged residues.
These data suggest that eukaryotic membrane protein topology is governed by the
presence or absence of an N-terminal signal for retention in the cytoplasm that
is composed in part of positive charges.

PMID: 1997206 [PubMed - indexed for MEDLINE]


654. Biochemistry. 1991 Feb 12;30(6):1561-71.

Structural study of the sugar chains of human leukocyte cell adhesion molecules
CD11/CD18.

Asada M, Furukawa K, Kantor C, Gahmberg CG, Kobata A.

Department of Biochemistry, University of Tokyo, Japan.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell
surface glycoproteins that are involved in various leukocyte functions. The
asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by
hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs.
These sugar chains were converted to radioactive oligosaccharides by reduction
with sodium borotritide and separated into neutral and acidic fractions by paper 
electrophoresis. All of the acidic oligosaccharides were converted to neutral
ones by digestion with sialidase, indicating that they are sialyl derivatives.
The neutral and sialdase-treated acidic oligosaccharides were fractionated by
chromatography on lectin columns followed by Bio-Gel P-4 column chromatography.
Structural studies of each oligosaccharide by sequential exo- and endoglycosidase
digestion and by methylation analysis revealed that Leu-CAMs contain mainly high 
mannose type and high molecular weight complex type sugar chains. The latter
sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal
beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc
beta 1----groups together with the Gal beta 1----4GlcNAc group in their
outer-chain moieties. In addition to these sugar chains, a small amount of
monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs.
Furthermore, analysis of the asparagine-linked sugar chains released from the
beta-subunit of Leu-CAMs by a series of lectin chromatography showed that
subunit-specific glycosylation is not observed between the alpha- and
beta-subunits of Leu-CAMs.

PMID: 1671554 [PubMed - indexed for MEDLINE]


655. Res Microbiol. 1991 Feb-Apr;142(2-3):325-32.

Murein chemistry of cell division in Escherichia coli.

Romeis T, Kohlrausch U, Burgdorf K, Holtje JV.

Max-Planck-Institut fur Entwicklungsbiologie, Abteilung Biochemie, Tubingen,
Germany.

The length distribution of the glycan strands of murein has been analysed with a 
novel method in filamentous and spherical cells of Escherichia coli, as well as
during septum formation and cell separation. A shift to the longer glycan strands
was observed in the murein of furazlocillin-induced filaments. In contrast,
shorter glycan strands were increased in the murein of mecillinam-induced
spherical cells. During septum formation in a chain-forming envA mutant that is
defective in the splitting process of the septum, a shift to the shorter glycan
strands was detected that was not seen in wild type E. coli cells. It is
concluded that septum-specific murein structures of rather short glycan strands
are released during splitting of the septum. This intermediate material remains
present in the septum of the envA mutant. The splitting process of the septum was
investigated by analysing the murein during penicillin-induced bacteriolysis,
which is known to take place by strictly localized murein degradation in the
equatorial zone of the cell. No changes in the length distribution of the glycan 
strands could be detected during penicillin-induced lysis, with the exception of 
an increase in disaccharides, the shortest glycan strands possible. This is
explained by the action of exo-muramidases progressively digesting glycan
strands, leaving disaccharide units covalently linked to the remaining murein at 
the sites of murein cross-linkage. It is proposed that this "zipper-like"
mechanism represents the normal cutting process of the septum during cell
separation.

PMID: 1925031 [PubMed - indexed for MEDLINE]


656. Chem Biol Interact. 1991;79(3):335-47.

Amphiphile-induced antihaemolysis is not causally related to shape changes and
vesiculation.

Hagerstrand H, Isomaa B.

Department of Biology, Abo Akademi University, Turku, Finland.

A wide variety of structurally different antihaemolytic amphiphiles were tested
for their ability to induce exovesiculation (acetylcholinesterase (AChE) release,
transmission electron microscopic (TEM) studies), endovesiculation (fluorescein
isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies)
and shape changes in human erythrocytes at concentrations where they exert
maximum protection against hypotonic haemolysis. The results show that
vesiculation is a common phenomenon induced by amphiphiles in erythrocytes.
Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas
stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency
of the amphiphiles was not related to their ability to induce exo- or
endovesiculation, or to the type or extent of shape changes induced, and it could
not be ascribed to any molecular feature of the amphiphiles or to their charge.
It is proposed that amphiphiles, when intercalated into the lipid bilayer of the 
membrane, rapidly induce rearrangements within the bilayer and that these
rearrangements are associated with an increase in the permeability of the
membrane; it is suggested that a rapid efflux of ions decreases the difference in
osmotic pressure between cell interior and hypotonic buffer, thereby protecting
cells from being lysed.

PMID: 1717169 [PubMed - indexed for MEDLINE]


657. Plant Physiol. 1990 Nov;94(3):1033-1039.

Purification of a beta-Amylase that Accumulates in Arabidopsis thaliana Mutants
Defective in Starch Metabolism.

Monroe JD, Preiss J.

Department of Biochemistry, Michigan State University, East Lansing, Michigan
48824.

Amylase activity is elevated 5- to 10-fold in leaves of several different
Arabidopsis thaliana mutants defective in starch metabolism when they are grown
under a 12-hour photoperiod. Activity is also increased when plants are grown
under higher light intensity. It was previously determined that the elevated
activity was an extrachloroplastic beta-(exo)amylase. Due to the location of this
enzyme outside the chloroplast, its function is not known. The enzyme was
purified to homogeneity from leaves of both a starchless mutant deficient in
plastid phosphoglucomutase and from the wild type using polyethylene glycol
fractionation and cyclohexaamylose affinity chromatography. The molecular mass of
the beta-amylase from both sources was 55,000 daltons as determined by denaturing
gel electrophoresis. Gel filtration studies indicated that the enzyme was a
monomer. The specific activities of the purified protein from mutant and
wild-type sources, their substrate specificities, and K(m) for amylopectin were
identical. Based on these results it was concluded that the mutant contained an
increased level of beta-amylase protein. Enzyme neutralization studies using a
polyclonal antiserum raised to purified beta-amylase showed that in each of two
starchless mutants, one starch deficient mutant and one starch overproducing
mutant, the elevated amylase activity was due to elevated beta-amylase protein.

PMCID: PMC1077338
PMID: 16667793 [PubMed - as supplied by publisher]


658. Mutat Res. 1990 Nov-Dec;233(1-2):45-51.

Site-directed mutagenesis for quantitation of base-base interactions at defined
sites.

Singer B, Dosanjh MK.

Lawrence Berkeley Laboratory, University of California, Berkeley 94720.

Two alkylation products implicated in initiation of carcinogenesis are
O6-alkylguanine (m6G) and O4-alkylthymine (m4T). We have used site-specific
insertion of these derivatives into oligonucleotides and measured the kinetic
constants of various pairings, using both prokaryotic and eukaryotic polymerases 
for replication. Preliminary data are also reported for another carcinogen
product, N2,3-ethenodeoxyguanosine ( epsilon G). The immediate neighbor bases
play an important role in determining the frequency of specific changed
basepairing and subsequent elongation of the annealed primer. However, both m4T
and m6G prefer to form a type of G.T pairing which would lead to the transitions:
G.C----A.T or T.A----C.G. The enzymes were the Klenow fragment of E. coli DNA
polymerase I (Kf), engineered 3'----5' exonuclease-free Kf (exo-free Kf),
polymerase alpha-primase complex from Drosophila melanogaster or calf thymus, and
human immunodeficient virus-I reverse transcriptase (HIV-I RT). All enzymes led
to approximately the same frequency of transitions. It is postulated that the
mutation frequency at a given site is primarily a function of the structure of
the sequence around the target site.

PMID: 2233812 [PubMed - indexed for MEDLINE]


659. Biochemistry. 1990 Oct 2;29(39):9126-34.

Structures of the asparagine-linked sugar chain of glucose transporter from human
erythrocytes.

Endo T, Kasahara M, Kobata A.

Department of Biochemistry, University of Tokyo, Japan.

The asparagine-linked sugar chain of glucose transporter from human erythrocytes 
was quantitatively released as oligosaccharides from the polypeptide backbone by 
hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4
reduction after N-acetylation and fractionated by anion-exchange column
chromatography and Bio-Gel P-4 column chromatography after sialidase treatment.
Structural study of each oligosaccharide by exo- and endoglycosidase digestion
and methylation analysis indicated that the glycoprotein contains a
high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary
complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man
alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc 
as their cores and the poly-N-acetyllactosamine composed of about 16
N-acetyllactosaminyl units as their outer chains. These structural features of
the sugar moiety of glucose transporter are quite different from those of two
major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.

PMID: 2271582 [PubMed - indexed for MEDLINE]


660. Aust Dent J. 1990 Oct;35(5):468-71.

Utilization of nitrogenous compounds by oral bacteria.

Rogers AH.

Department of Dentistry, The University of Adelaide.

In terms of the crucial acid-base balance in dental plaque, the bacterial
catabolism of nitrogenous compounds, such as peptides and amino acids, is of
importance because the end-products can raise plaque pH. Of particular
significance is the fermentation of arginine by bacteria such as Streptococcus
sanguis, a numerically important plaque organism. Aspects of the uptake of this
amino acid were studied and it was also shown the organism can obtain arginine
from small peptides, since it possesses cell-associated exo-peptidases.
Furthermore, it could grow in media containing whole protein (casein), or one of 
its fractions, as the sole source of organic nitrogen. The studies thus showed
that S. sanguis is well equipped, in terms of endo- and exo-peptidase activities,
to obtain the metabolically important arginine from whole protein. It is
suggested that knowledge of this type should lead to a better understanding of
overall plaque metabolism--of relevance to both cariogenic and periodontopathic
plaques.

PMID: 2073196 [PubMed - indexed for MEDLINE]


661. J Bacteriol. 1990 Sep;172(9):5254-9.

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

Zhan HJ, Leigh JA.

Department of Microbiology SC-42, University of Washington, Seattle 98195.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of 
the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R.
leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in
its ability to inhibit exopolysaccharide synthesis when present in multiple
copies, exoX did not appear to regulate the expression of exoP. The effect of
exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to
Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2
in its mutant phenotype and in portions of its deduced amino acid sequence. The
effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the
relative copy numbers of the two genes. exoX-lacZ fusions manifested
threefold-higher beta-galactosidase activities in exoF backgrounds than in the
wild-type background. exoX mutants produced increased levels of succinoglycan.
However, the exoF gene was required for succinoglycan synthesis even in an exoX
mutant background. exoF did not affect the expression of exoP. Strains containing
multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled
nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants 
formed nitrogen-fixing nodules, indicating that, if the inhibition of
succinoglycan synthesis within the nodule is necessary for nitrogen fixation,
then exoX is not required for this inhibition. We present indirect evidence that 
succinoglycan synthesis within the nodule is not necessary for bacteroid
function.

PMCID: PMC213187
PMID: 2118508 [PubMed - indexed for MEDLINE]


662. Mol Pharmacol. 1990 Aug;38(2):177-83.

Glycoprotein nature of the A2-adenosine receptor binding subunit.

Barrington WW, Jacobson KA, Stiles GL.

Department of Medicine, Cardiology, Duke University Medical Center, Durham, North
Carolina 27710.

Mammalian A2-adenosine receptor binding subunits (A2AR) can be visualized by
covalent labeling with the photoaffinity crosslinking ligand
125I-2-[4-[2-[2-[(4-aminophenyl)methylcarbonylamino]
ethylaminocarbonyl]ethyl]phenyl]ethylamino-5'-N-ethylcarboxamidoad enosine or
directly with the azide derivative described in this paper. The protein
comprising the A2-adenosine receptor binding subunit migrates with a Mr of 45,000
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the 
glycoproteins representing the radiolabeled A1- and A2-adenosine receptor binding
subunit from bovine brain were compared by partial peptide maps and following
treatment with exo- and endoglycosidases. Peptide maps using two separate
proteases reveal that the A1- and A2-adenosine receptor binding subunits share no
common peptide fragments by two-dimensional gel electrophoresis. Endoglycosidase 
F treatment of labeled A2AR results in a single labeled peptide of Mr 38,000
without intermediate peptides, suggesting a single N-linked carbohydrate chain.
The labeled A2AR demonstrates a sensitivity to neuraminidase, as evidenced by an 
increased mobility on gel electrophoresis, suggesting the receptors contain a
glycan component containing terminal sialic acid. Treatment of the labeled A2AR
with alpha-mannosidase reveals two distinct populations of A2ARs, one of which is
sensitive and the other resistant to the enzyme. The nonadditivity of sequential 
treatments with the two exoglycosidases suggests, a heterogeneous population of
A2AR containing either complex- or high mannose-type carbohydrate chains. These
data suggest the A2AR is a Mr 45,000 glycoprotein with a single carbohydrate
chain of either the complex or high mannose type. In addition, the A1- and A2ARs 
are distinct glycoproteins, as evidenced by their differing molecular weights
(before and after deglycosylation) and distinct peptide maps.

PMID: 2385230 [PubMed - indexed for MEDLINE]


663. J Biol Chem. 1990 Jun 15;265(17):10088-94.

Purification and characterization of an exo-beta-D-glucosaminidase, a novel type 
of enzyme, from Nocardia orientalis.

Nanjo F, Katsumi R, Sakai K.

NFI Laboratories, Yaizu Suisan Kagaku Industry Co., Ltd., Shizuoka, Japan.

A new enzyme capable of hydrolyzing chitobiose, which is an induced enzyme, was
purified to apparent homogeneity from the culture filtrate of Nocardia orientalis
IFO 12806. Biospecific affinity chromatography on chitotriitol-Sepharose CL-4B
was effective for purification of this enzyme. It is clearly demonstrated that
the enzyme is an exo-hydrolase, removing single glucosamine residues from the
nonreducing terminal of a sequence of beta-(1----4)-linked glucosamine chain,
such as chitosan and chitooligosaccharides, and therefore characterized as an
exo-beta-D-glucosaminidase. The enzyme was found to show maximum activity on
chitotetraose, chitopentaose, and their corresponding alcohols and a slight
decrease in rate on longer chain lengths of substrates. A significant decrease in
rate was observed using p-nitrophenyl beta-D-glucosaminide and chitobiitol as
substrates. In the hydrolysis of partially acetylated chitosans, the enzyme
appeared to be effective in cleaving glucosamine from the GlcN beta 1----4GlcNAc 
beta 1----sequence as well as the GlcN beta 1----4GlcN beta 1----sequence. These 
observations suggest that the second residue from the terminal plays an important
role in enzyme activity, but the enzyme permits the replacement of glucosamine at
the second residue by N-acetylglucosamine.

PMID: 2351651 [PubMed - indexed for MEDLINE]


664. Neuron. 1990 Jun;4(6):833-45.

Lineage-independent determination of cell type in the embryonic mouse retina.

Turner DL, Snyder EY, Cepko CL.

Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.

We previously used a retroviral vector to mark clones in the postnatal rodent
retina and showed that at least two types of neurons and Muller glia can arise
from a common progenitor. Here we describe the use of exo utero surgery to
introduce a marker retrovirus into the proliferative zone of the retinas of
embryonic day 13 and 14 mice. Analysis of marked clones in the resulting adult
retinas shows that almost all progenitor cells that continued mitosis were
multipotential and that a single progenitor can generate most retinal cell types.
The size of marked clones indicates that retinal cells do not employ a stem cell 
mode of division, but instead, both daughter cells of a progenitor can continue
to divide. These results suggest that cell type determination in the rodent
retina is independent of lineage. We propose a model for the generation of
retinal cell types in which the cessation of mitosis and cell type determination 
are independent events, controlled by environmental interactions.

PMID: 2163263 [PubMed - indexed for MEDLINE]


665. Biochim Biophys Acta. 1990 May 24;1049(1):69-77.

Sequence specific conformation of a DNA decamer containing an adenine tract
studied in solution by H-NMR spectroscopy.

Searle MS, Wakelin LP.

Molecular Pharmacology Group, Peter MacCallum Cancer Institute, Melbourne,
Australia.

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive
two-dimensional (2D) NMR experiments have been used to investigate the solution
conformation of an adenine-tract and its junction with another DNA sequence. 2D
nuclear Overhauser effect data confirm that the oligonucleotide has a general
B-type DNA morphology but an array of unusual correlations implies that the
adenine tract and the 5'-ApC junction have conformations more compatible with the
modified X-ray structures recently reported for DNAs of similar sequence (Nelson,
H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The
pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's 
of the complementary base to the 5'-neighbouring residue indicate that the A-T
basepairs are highly propeller twisted and that the minor groove is narrowed,
showing its greatest compression at the 3'-end of the tract at the 5'-ApC step.
Quantifying spin-coupling interactions within the deoxyribose rings by analysing 
both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the
purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in
the range 140-180 degrees. For the pyrimidines, however, there are distortions
away from this standard B-type geometry with the data being best described by P
values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar
puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of 
their positions at, or close to, the ends of the duplex. Thus the conformation of
the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are
different with an abrupt change in sugar puckering occurring at the 5'-ApC
(5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', 
H2' and H2", together with a number of interresidue NOEs with unusual
intensities, imply that there are also substantial modifications to basepair
stacking interactions at this step. Taken as a whole, our data are consistent
with the view that the conformational dislocation at the 5'-ApC dinucleotide
results from a combination of slide and roll manoeuvres and that the junction
between the AAAA and CG sequences is a potential nucleation site for DNA bending.

PMID: 2357466 [PubMed - indexed for MEDLINE]


666. J Biol Chem. 1990 May 5;265(13):7207-15.

Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus
tulipiferae) and its action on arabinogalactan-proteins.

Tsumuraya Y, Mochizuki N, Hashimoto Y, Kovac P.

Department of Biochemistry, Faculty of Science, Saitama University, Urawa, Japan.

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme
preparation from Irpex lacteus (Polyporus tulipiferae) has been purified
166-fold. Apparent molecular weights of the purified enzyme, estimated by
denaturing gel electrophoresis and gel filtration, were found to be 51,000 and
42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of
(1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal
activity toward these substrates at pH 4.6. Based on the mode of the liberation
of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of
beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase
capable of catalyzing the sequential hydrolytic release of single D-galactosyl
residues from the nonreducing termini. The extent of the hydrolysis of the
carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased 
with their decreasing branching. Isolation and characterization of the major
products formed from the proteoglycans indicated the action pattern of the enzyme
to include the capability of bypassing the branching points. Consequently, the
side chains carrying an additional D-galactosyl group at the reducing termini are
released as neutral (1----6)-linked beta-D-galactooligosaccharides and their
acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the
nonreducing end-group. The specificity and the mode of action showed the enzyme
to be a useful tool for analyzing the fine structure of type II arabinogalactans 
and arabinogalactan-protein conjugates.

PMID: 2158993 [PubMed - indexed for MEDLINE]


667. J Biol Chem. 1990 Apr 5;265(10):5390-7.

A new type of exo-beta-glucuronidase acting only on non-sulfated
glycosaminoglycans.

Nakamura T, Takagaki K, Majima M, Kimura S, Kubo K, Endoss M.

Department of Biochemistry, Hirosaki University School of Medicine, Japan.

Using chondroitin as a substrate, a new type of exo-beta-glucuronidase (EC
3.2.1.31) from rabbit liver was purified using a combination of ammonium sulfate 
fractionation, DEAE-cellulose chromatography, gel filtration on Sephracryl S-300,
affinity chromatography through heparin-Sepharose CL-6B, and preparative
polyacrylamide gel electrophoresis. This enzyme acts only on non-sulfated
glycosaminoglycans and their oligosaccharides and was shown to be quite different
from exo-beta-glucuronidase, which does act on p-nitro-phenyl-beta-D-glucuronide 
with regard to the following properties. 1) Neither sulfated
glycosaminoglycanoligosaccharides nor p-nitrophenyl-beta-D-glucuronide were
substrates for the enzyme. 2) The molecular weight was found to be about 130,000 
by gel filtration, compared with a molecular weight of 280,000-300,000 for
beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 3) The
enzyme showed maximal activity at pH 5.0, compared with an optimum pH of 4.5 for 
beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 4) The
enzyme showed maximal activity in 0.075 M NaCl but no activity above 0.25 M NaCl.
5) The enzyme was inhibited strongly by compounds bearing a sulfate group. 6) The
enzyme did not react with an antibody against beta-glucuronidase acting on
p-nitrophenyl-D-glucuronide. It is suggested that the enzyme may be involved in
the catabolism of glycosaminoglycans, acting especially on chondroitin after the 
desulfation reaction and/or hyaluronic acid, but showing little involvement with 
the detoxification system.

PMID: 2108135 [PubMed - indexed for MEDLINE]


668. J Antibiot (Tokyo). 1990 Apr;43(4):372-82.

Synthesis and cytostatic activity of the antitumor antibiotic chartreusin
derivatives.

Kon K, Sugi H, Tamai K, Ueda Y, Yamada N.

Biochemistry Research Laboratory, Ishihara Sangyo Kaisha, Ltd., Shiga-ken, Japan.

In order to overcome the rapid biliary excretion of chartreusin, which diminished
its activity when administered iv, a series of 3',4'-O-substituted derivatives of
chartreusin were synthesized. Exo-type of 3',4'-O-benzylidene-chartreusin was
found active both by ip and iv administration. Therefore, this compound was
selected for further modification on its 6-phenol to obtain broader spectra and
better pharmacokinetic parameters than the original compound. Several
6-O-acyl-3',4'-O-exo-benzylidene-chartreusins had high antitumor activity against
some murine tumors both by iv and po administration.

PMID: 2351612 [PubMed - indexed for MEDLINE]


669. Biotechnol Appl Biochem. 1990 Apr;12(2):150-60.

Purification and characterization of polygalacturonases produced by the hyphal
fungus Aspergillus niger.

Kester HC, Visser J.

Department of Genetics, Agricultural University, Wageningen, The Netherlands.

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase,
EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide)
galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase
preparation derived from Aspergillus niger. All five endo-enzymes could be
purified to homogeneity by affinity chromatography on cross-linked alginate,
ion-exchange chromatography, chromatofocusing, and gel permeation chromatography.
The exo-polygalacturonase was only partially purified but free from
endo-polygalacturonase activity. The two most abundant endo-polygalacturonases
(endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are
quite different with respect to their isoelectric point, specific activity, mode 
of action on oligomeric substrates, and amino acid composition. The
physicochemical properties of the other three endo-polygalacturonases (endo-IIIA,
endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of
the endo-I type. The pH optima of all these endo-polygalacturonases are in the
range of 4.3-4.9.

PMID: 2331322 [PubMed - indexed for MEDLINE]


670. J Cell Sci. 1990 Apr;95 ( Pt 4):667-74.

DNA-RNA complexes that might represent transient attachment sites of nuclear DNA 
to the matrix.

Patriotis C, Andreeva M, Pascaleva M, Ivanov V, Djondjurov L.

Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.

In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The
presence of such unusual structures is confirmed by the following evidence. When 
a preparation of matrix DNA is electrophoresed in agarose an RNA component always
migrates together with DNA. There should be a close interaction between DNA and
RNA in such a preparation because the presence of the RNA component causes
resistance of DNA to DNase I and Exo III. An intimate, hybrid-type association of
part of the RNA component with DNA is indicated also by the fact that about 20%
of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis 
with alpha-amanitin and actinomycin D it was shown that the bulk of associated
RNA is transcribed by RNA polymerase III. Hybridization experiments showed
similarity between the DNA sequences isolated from the complexes and those from
the base of dehistonized DNA loops obtained by high-salt extraction of nuclei.
This observation suggests that the complexes might represent attachment sites of 
nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA
component. Experiments with induction of erythroid differentiation indicated that
a profound reorganization of the nucleus, accompanying terminal differentiation, 
leads to a striking reduction in the number of complexes and thus in the number
of attachment sites. This suggests that the complexes should function as
transient attachment sites.

PMID: 1696580 [PubMed - indexed for MEDLINE]


671. Biopolymers. 1990 Mar-Apr;29(4-5):823-36.

Theoretical studies of cis-Pt(II)-diammine binding to duplex DNA.

McCarthy SL, Hinde RJ, Miller KJ, Anderson JS, Basch H, Krauss M.

Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180.

The binding of cis-Pt(II) diammine (cis-DP) to double-stranded DNA was studied
with several kinked conformations that can accommodate the formation of a square 
planar complex. Molecular mechanics (MM) calculations were performed to optimize 
the molecular fit. These results were combined with quantum mechanical (QM)
calculations to ascertain the relative energetics of ligand binding through water
vs direct binding of the phosphate to the ammine and platinum, and to guide the
selection of DNA conformations to model complex formation. Based on QM and MM
calculations, models are proposed that may be characterized by several general
features. A structure involving hydrogen bonding between each ammine and distinct
adjacent phosphate groups, referred to as closed conformation (CC), has already
been reported. This is also found in the crystal structure of small dimers. We
report alternative conformations that may be important in platination of duplex
DNA. They are characterized by an intermediate conformation (IC), involving
hydrogen bonding between one ammine and phosphate group, and an open conformation
(OC), without ammine phosphate hydrogen bonding. The IC and OC can be stabilized 
by water bridges in the space between the ammine and the phosphate groups. Sugar 
puckers alternate from the type C(2')-endo or C(1')-exo (S), to the type
C(3')-endo or C(2')-exo (N), with intermediate types near O(1')-endo (O). In
general, the sugar puckers alternate from S to N to S through the platinated
region (3'-TpG*pG*p-5'), with the complexed strand exhibiting, (3')-S*-N*-S-(5') 
alternation, while the complementary strand shows either (3')-S*-N*-S-(5') or
(3')-S*-N*-O-(5') alternation. In both the OC and IC, a hydrogen bond is found
between the ammine and O4(T) on thymine (T) at the (3') end, adjacent to the
complex site. There is a continuous range of backbone conformations through the
platinated region which relate the OC to the IC. The models presented suggest
that the dynamics of the binding of the cis-Pt(II)-diammines to adjacent N7(G) in
double-stranded DNA may encompass several conformational possibilities, and that 
water bridges may play a roll in supporting open and intermediate conformations. 
Proton-proton distances are reported to assist in the experimental determination 
of conformations.

PMID: 2383646 [PubMed - indexed for MEDLINE]


672. Biochemistry. 1990 Feb 6;29(5):1271-5.

Tyrosine-96 as a natural spectroscopic probe of the cytochrome P-450cam active
site.

Atkins WM, Sligar SG.

Department of Biochemistry, University of Illinois, Urbana 61801.

The previously described correlation between the ferric spin equilibrium of
cytochrome P-450cam and the environmental polarity of tyrosine residues (Fisher
et al., 1986) has been further examined with the use of site-directed mutagenesis
and active-site affinity reagents. Whereas the wild-type demonstrates an increase
in environmental polarity of approximately one tyrosine residue, the mutant
protein Y96F, in which Tyr-96 has been changed to Phe-96, demonstrates a lack of 
spin-state-dependent change in the second-derivative ultraviolet absorption
spectrum. This suggests that the active-site Tyr-96 serves as a ultraviolet
spectroscopic probe which can be utilized to determine the relative degree of
water access to the active site for various substrate/protein complexes. The
affinity reagent isobornyl mercaptan has been used to demonstrate the utility of 
this probe in determining the active-site polarity when substrate analogues are
bound at the active site. In addition, the sensitivity of Tyr-96 to environmental
polarity has been used to demonstrate that the product/enzyme complex, formed
with 5-exo-hydroxycamphor, may be associated with increased water access to the
heme iron. This may provide a means for turning off electron transfer when the
product, instead of the substrate, is bound at the active site.

PMID: 2182119 [PubMed - indexed for MEDLINE]


673. Plant Physiol. 1990 Feb;92(2):368-374.

Rhizobium meliloti exopolysaccharide Mutants Elicit Feedback Regulation of Nodule
Formation in Alfalfa.

Caetano-Anolles G, Lagares A, Bauer WD.

Department of Agronomy, Ohio State University, Columbus, Ohio 43210-1086.

Nodule formation by wild-type Rhizobium meliloti is strongly suppressed in
younger parts of alfalfa (Medicago sativum L.) root systems as a feedback
response to development of the first nodules (G Caetano-Anolles, WD Bauer [1988] 
Planta 175: 546-557). Mutants of R. meliloti deficient in exopolysaccharide
synthesis can induce the formation of organized nodular structures
(pseudonodules) on alfalfa roots but are defective in their ability to invade and
multiply within host tissues. The formation of empty pseudonodules by exo mutants
was found to elicit a feedback suppression of nodule formation similar to that
elicited by the wild-type bacteria. Inoculation of an exo mutant onto one side of
a split-root system 24 hours before inoculation of the second side with wild-type
cells suppressed wild-type nodule formation on the second side in proportion to
the extent of pseudonodule formation by the exo mutants. The formation of
pseudonodules is thus sufficient to elicit systemic feedback control of
nodulation in the host root system: infection thread development and internal
proliferation of the bacteria are not required for elicitation of feedback.
Pseudonodule formation by the exo mutants was found to be strongly suppressed in 
split-root systems by prior inoculation on the opposite side with the wild type. 
Thus, feedback control elicited by the wild-type inhibits Rhizobium-induced
redifferentiation of host root cells.

PMCID: PMC1062300
PMID: 16667284 [PubMed - as supplied by publisher]


674. J Gen Microbiol. 1990 Jan;136(1):105-13.

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii
USDA 191.

Ko YH, Gayda R.

Department of Microbiology, Louisiana State University, Baton Rouge.

Production of exopolysaccharides by Rhizobium has been linked with efficient
invasion and nodulation of leguminous plant roots by the bacteria.
Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were
isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo
mutants were investigated for exopolysaccharide synthesis and their ability to
nodulate soybeans. The exopolysaccharides produced by these mutants were analysed
for polysaccharide composition by column chromatography and thin-layer
chromatography. Two mutants designed exo-3 and exo-5 were deficient in both
neutral glucan and exopolysaccharide synthesis, but each induced some functional 
nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and
exo-4) synthesized neutral glucans at levels higher or lower than those in
wild-type and exhibited partial exopolysaccharide deficiencies. The data imply
that neither exopolysaccharides nor neutral glucans are essential for the
induction of determinate nodules by R. fredii.

PMID: 2351951 [PubMed - indexed for MEDLINE]


675. Biochem Cell Biol. 1990 Jan;68(1):387-92.

Endo-exonuclease of Aspergillus nidulans.

Koa H, Fraser MJ, Kafer E.

Department of Biochemistry, McGill University, Montreal, Que., Canada.

Endo-exonuclease (EE) has been found in both active and inactive, but
trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly 
in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in
the cytosol. The active form accounts for over 90% of the neutral
deoxyribonuclease activity extracted from mycelia. A single strand (ss)
DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially
purified and characterized. It was found to closely resemble, in size and
enzymological properties, the ss-DNA-binding EE previously purified from
Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically 
related to the N. crassa EE and, like that enzyme, was probably derived from a
polypeptide of 90 kDa or larger through proteolysis during extraction and
purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity 
on both DNA and RNA, which ultimately produced small 5'-P-terminated
oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with 
ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA.
Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then
linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322
DNA was extensively nicked internally under conditions where there was relatively
low exonuclease activity, but this nicking required that 5'-P-termini be present 
on the linear ds-DNA. The levels of active EE found in extracts of two
recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ
significantly from those in extracts of the wild type.

PMID: 2161674 [PubMed - indexed for MEDLINE]


676. Cell Biol Int Rep. 1989 Dec;13(12):1063-76.

The role of coated vesicles in recycling of synaptic vesicle membrane.

Heuser J.

Department of Cell Biology & Physiology, Washington University School of
Medicine, St. Louis, Missouri 63110.

The uptake of extracellular tracers into synaptic nerve terminals has been a
phenomenon of persistent interest. Uptake is into synaptic vesicles, hence
vesicles spend part of their life in continuity with the plasma membrane, as
expected if exocytosis underlies the quantal discharge of neurotransmitters.
However, exactly how or when synaptic vesicles acquire extracellular tracers has 
not been unambiguously determined. Two schools of thought have developed, one
holding that vesicles acquire tracers directly via a reversible exo/endocytotic
sequence in which they consistently maintain their biochemical identity during
their transient continuity with the plasma membrane, the other holding that
synaptic vesicles acquire tracers indirectly, via the formation of
clathrin-coated vesicles which are spatially and temporally separate from
exocytosis and reverse a temporary loss of the vesicles' individual identity upon
merger with the plasma membrane. Efforts to distinguish between these two
alternatives have generated an interesting diversity of electron microscopic
experiments, many of which are reviewed here. However, definitive determination
of which view is correct may ultimately require direct visualization of synaptic 
vesicle turnover in living nerve terminals. To this end, we here review the
results of visualizing endocytosis in tissue cultured cells, where light
microscopy can provide sufficient resolution to reveal membrane dynamics in
living cells. This has allowed visual discrimination of two different types of
endocytosis, one clathrin-mediated (coated vesicle formation) and the other
actin-mediated (macropinocytosis). Current work is also reviewed which aims at
determining experimental methods for inhibiting each type of endocytosis
selectively. Hypertonicity and severe cytoplasmic acidification turn out to
inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic
acidification selectively inhibit macropinocytosis. Applied to nerves, these
various treatments affect synaptic vesicle turnover in a manner that supports the
notion that synaptic vesicle membrane recycles via the "indirect" route of coated
vesicle formation.

PMID: 2576862 [PubMed - indexed for MEDLINE]


677. FEBS Lett. 1989 Oct 9;256(1-2):219-24.

Photoaffinity labeling of the mammalian dopamine transporter.

Sallee FR, Fogel EL, Schwartz E, Choi SM, Curran DP, Niznik HB.

Department of Psychiatry, Medical University of South Carolina, Charleston 29425.

A high affinity (1-2 nM) radioiodinated, photoaffinity probe for the dopamine
transporter, 1-(2-[bis-(4-fluorophenyl)-methoxylethyl)-4-(2-[4-azido-3-
[125I]iodophenyl]ethyl)piperazine ([125I]FAPP) has been synthesized. Upon
photolysis, [125I]FAPP incorporates into a striatal polypeptide of apparent Mr
62,000 as visualized by autoradiography following sodium dodecyl sulfate-PAGE.
Photoincorporation of [125I]FAPP into the Mr 62,000 polypeptide was
stereoselectively inhibited by various dopamine uptake agents with a potency
order typical of the dopamine transporter. The glycoprotein nature of the
apparent Mr 62,000 polypeptide was assessed following specific exo- and
endoglycosidase treatment. The dopamine transporter appears to be associated with
complex-type oligosaccharides as indexed by its susceptibility to neuraminidase
but not alpha-mannosidase digestion. Complete N-linked deglycosylation of the
neuronal dopamine transporter with the endoglycosidase, glycopeptidase-F,
increased the electrophoretic mobility of the 62 kDa polypeptide to apparent Mr
48,000. [125I]FAPP should prove to be a useful probe for the molecular
characterization of the dopamine uptake site in various tissues and under certain
pathophysiological states.

PMID: 2806548 [PubMed - indexed for MEDLINE]


678. Mol Pharmacol. 1989 Oct;36(4):566-74.

Glycoprotein nature of dopamine D1 receptors in the brain and parathyroid gland.

Jarvie KR, Booth G, Brown EM, Niznik HB.

Department of Pharmacology, University of Toronto, Ontario, Canada.

Dopamine D1 receptors can be covalently labeled with the photo-affinity ligand
(+-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrah yd
ro-1H-3-benzazepine ([125I]IMAB) and visualized following sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and autoradiography. In brain
membranes, [125I]IMAB labels a polypeptide of apparent Mr approximately equal to 
74,000 as the major ligand binding subunit of D1 receptors and two minor
polypeptides of Mr approximately equal to 64,000 and 52,000. In contrast,
[125I]IMAB labels a single polypeptide of apparent Mr approximately equal to
64,000 in bovine parathyroid glands. In this study, the carbohydrate nature of
dopamine D1 receptors from the brain and parathyroid gland were examined using
specific exo- and endoglycosidases and lectin affinity chromatography.
[125I]IMAB-labeled brain and parathyroid D1 receptors were sensitive to treatment
with the exoglycosidases neuraminidase or alpha-mannosidase, suggestive of the
existence of terminal sialic acid and oligomannose residues. Photolabeled D1
receptor polypeptides are not however, associated with distinct populations of
complex-type or high mannose-containing carbohydrate chains because 1) wheat germ
agglutinin and concanavalin A lectin chromatography of solubilized and
photolabeled neuronal D1 receptors followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and autoradiography revealed no
differences in the electrophoretic mobility of column pass-through and
specifically eluted [125I]IMAB-labeled polypeptides, and 2) [125I]IMAB-labeled D1
receptors specifically bound to and eluted from concanavalin A-Sepharose were
neuraminidase sensitive, indicative of the colocalization of oligomannose- and
complex-type glycans. Removal of these terminal glycan residues did not affect
the binding of [3H]SCH 23390 to dopamine D1 receptors. Complete N-linked
deglycosylation of photolabeled D1 receptors from both the brain and parathyroid 
with peptide N-glycosidase F resulted in the migration of a single major labeled 
polypeptide of apparent Mr approximately equal to 46,000. These data suggest
that, despite differences observed in the electrophoretic mobility and
glycosylation patterns of brain and parathyroid D1 receptor polypeptides, the
protein backbones of central and peripheral dopamine D1 receptors display similar
if not identical molecular weights.

PMID: 2682204 [PubMed - indexed for MEDLINE]


679. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7823-7.

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single
mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino 
acid in oxygen activation.

Imai M, Shimada H, Watanabe Y, Matsushima-Hibiya Y, Makino R, Koga H, Horiuchi T,
Ishimura Y.

Department of Biochemistry, School of Medicine, Keio University, Tokyo, Japan.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as
the terminal monooxygenase in the d-camphor monooxygenase system), in which
threonine-252 had been changed to alanine, valine, or serine, were employed to
study the role of the hydroxy amino acid in the monooxygenase reaction. The
mutant enzymes were expressed in Escherichia coli and were purified by a
conventional method. All the mutant enzymes in the presence of d-camphor
exhibited optical absorption spectra almost indistinguishable from those of the
wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide
ferrous forms. In a reconstituted system with putidaredoxin and its reductase,
the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to
that of the wild-type enzyme (1330 per min per heme), whereas the amount of
exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type
enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme
also exhibited an oxidase activity to yield H2O2 accompanied by a relative
decrease in the monooxygenase activity. On the other hand, the serine enzyme
exhibited essentially the same monooxygenase activity as that of the wild-type
enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was
produced by the substitution of an amino acid without a hydroxyl group. When
binding of O2 to the ferrous forms was examined, the alanine and valine enzymes
formed instantaneously an oxygenated form, which slowly decomposed to the ferric 
form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, 
respectively. Since these rates were too slow to account for the overall rates of
O2 consumption, the formation of H2O2 was considered to proceed not by way of
this route but through the decomposition of a peroxide complex formed by
reduction of the oxygenated form by reduced putidaredoxin. Based on these
findings, a possible mechanism for oxygen activation in this monooxygenase
reaction has been discussed.

PMCID: PMC298163
PMID: 2510153 [PubMed - indexed for MEDLINE]


680. Microbiologia. 1989 Sep;5(2):113-9.

The effect of Trichoderma viride C-1 UV mutagenization on cellulases activity.

Witkowska D, Bien M, Sobieszczanski J.

Department of Biotechnology and Food Microbiology, Academy of Agriculture,
Wroclaw, Poland.

Trichoderma viride C-1 strain was irradiated with UV until the survival level of 
0.03% was obtained. Sixteen mutants were isolated on the basis of the visible
clearance zone around the colonies on the media with cellulose and glycerol. Then
they were cultivated on a rotary shaker in the liquid Saunders medium
supplemented with microcrystalline cellulose and comminuted sugar beet pulp.
Exo-1,4-beta-glucanase, endo-1,4-beta-glucanase and beta-glucosidase were assayed
in the supernatants of postcultural liquids at different time intervals of
culture. The same mutants were characterized by higher biosynthesis level of
exoglucanase (1.2-5.0 times) endoglucanase (1.2-2.5 times) and beta-glucosidase
(1.5-1.7 times) when compared with the wild type strain.

PMID: 2629784 [PubMed - indexed for MEDLINE]


681. Arch Biochem Biophys. 1989 Aug 1;272(2):356-63.

New substrate specificity of modified porcine pancreatic alpha-amylase.

Ishikawa K, Hirata H.

National Chemical Laboratory for Industry, Ibaraki, Japan.

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA)
was studied using chemical modification of His residues. Diethyl pyrocarbonate
modified His residues in PPA and the activity of the modified PPA for the
hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides
decreased to less than 1% of that of the native enzyme. However, the activity for
the hydrolysis of the bond between p-nitrophenol and oligosaccharides in
p-nitrophenyl oligosaccharides was increased by chemical modification. When the
modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr
60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the
inhibitor. As a result, the remaining less than 1% hydrolytic activity of the
modified PPA for starch disappeared completely but that for p-nitrophenyl
oligosaccharides remained unaltered. The hydrolytic activity of the native PPA
for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that
between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides.
Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues)
were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in
the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex
hydrolyzed only the bond between p-nitrophenol and oligosaccharides in
p-nitrophenyl oligosaccharides. The above results reveal that, by chemical
modification with diethyl pyrocarbonate and biochemical modification with an
amylase inhibitor, amylase can be converted to a new exo-type enzyme which
hydrolyzes only the bond between p-nitrophenol and oligosaccharides in
p-nitrophenyl oligosaccharides.

PMID: 2473713 [PubMed - indexed for MEDLINE]


682. Biochim Biophys Acta. 1989 Jul 10;982(2):179-86.

Vesiculation induced by amphiphiles in erythrocytes.

Hagerstrand H, Isomaa B.

Department of Biology, Abo Akademi, Turku, Finland.

The ability of shape-transforming cationic, anionic, zwitterionic, and nonionic
amphiphiles to induce vesiculation in human erythrocytes was studied. At
concentrations where they exhibit maximum protection against hypotonic haemolysis
(CAHmax) echinocytogenic amphiphiles induced a rapid release of exovesicles.
Following 5 min of incubation, the vesicle release (acetylcholinesterase release)
amounted from 4% (sodium alkyl sulphates) to 13% (zwittergents) of the total
acetylcholinesterase activity of the erythrocytes. At concentrations
corresponding to CAH50 the vesicle release was less than 15% of that released at 
CAHmax. The size and the appearance of the vesicles varied with the type of
amphiphile. Stomatocytogenic amphiphiles which do not pass the erythrocytes
through echinocytic stages, did not induce release of exovesicles. Electron and
fluorescence microscopic observations of erythrocytes treated with
stomatocytogenic amphiphiles strongly indicated that an endovesiculation had
occurred. Amphiphiles which pass the erythrocytes through echinocytic stages
before stomatocytic shapes are attained, induced a release of both exo- and
endovesicles.

PMID: 2473779 [PubMed - indexed for MEDLINE]


683. J Mol Recognit. 1989 Jul;2(1):25-36.

The conformation of core oligosaccharides from Escherichia coli and Salmonella
typhimurium lipopolysaccharides as predicted by semi-empirical calculations.

Jansson PE, Wollin R, Bruse GW, Lindberg AA.

Department of Organic Chemistry, Arrhenius Laboratory, University of Stockholm,
Sweden.

The preferred conformation of the hexose and heptose regions of core saccharides 
from Enterobacteriaceae lipopolysaccharides was calculated. The Hard Sphere Exo
Anomeric (HSEA) approach was used and the minimum energy conformation of the
Salmonella typhimurium and Escherichia coli R1, R2, R3, R4 and K12 cores
calculated. The results indicate that most of the cores are sterically crowded,
with small degrees of freedom, and that the hexose and heptose parts form two
separate regions. The core structures exhibit a 'front'-side and a 'back'-side,
the former being similar for all the structures and the latter being
characteristic for each core type.

PMID: 2700070 [PubMed - indexed for MEDLINE]


684. J Biochem. 1989 Jul;106(1):17-22.

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the
porcine anterior pituitary.

Jarvie KR, Niznik HB.

Department of Pharmacology, University of Toronto, Ontario, Canada.

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were
visualized by autoradiography following photoaffinity labeling with
[125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel
electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine 
receptor migrated as two distinct bands of apparent Mr approximately equal to
150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from
porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding
subunits was investigated by the use of exo- and endo-glycosidase treatments and 
peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior
pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion 
as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate 
polyacrylamide gels, and suggests the presence of both complex type and terminal 
mannose carbohydrate residues. Moreover, the additive effects of sequential
treatment with these enzymes suggests that both types of carbohydrate chains are 
present on each receptor peptide. N-linked deglycosylation of pituitary D2
photolabeled receptors with glycopeptidase-F produced a further increase in the
mobility of the labeled protein to apparent Mr approximately equal to 44,000,
similar to that of deglycosylated D2 binding subunits of porcine and canine
brain. Peptide mapping experiments following limited proteolysis with
Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated
D2 dopamine receptors (Mr = 44,000), in different tissues and species, were
homologous. Taken together, these data suggest that despite the differences in
the overall molecular weight and tissue specific glycosylation pattern of
pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors 
appears to be conserved.

PMID: 2528540 [PubMed - indexed for MEDLINE]


685. J Histochem Cytochem. 1989 Jul;37(7):1115-24.

Histochemical analysis of the chemical structure of blood group-related
carbohydrate chains in serous cells of human submandibular glands using lectin
staining and glycosidase digestion.

Ito N, Nishi K, Nakajima M, Okamura Y, Hirota T.

Department of Legal Medicine, Nara Medical University, Japan.

Using lectin staining methods in combination with exo- and endo-glycosidase
digestion procedures, we analyzed the chemical structure of different types of
blood group-related substances in serous cells of formalin-fixed,
paraffin-embedded human submandibular glands. Serous cells produced only H
antigen; A and B antigens were not present, and the expression of H antigen is
dependent on the secretor status of the tissue donor. Although reactivity with
Ulex europaeus agglutinin I (UEA-I) was not markedly reduced by
alpha-L-fucosidase digestion, an affinity for peanut agglutinin (PNA) was seen
after fucosidase digestion in the cells from secretors. In those from
nonsecretors, no PNA reactivity appeared after enzyme digestion. On the other
hand, sialidase digestion elicited PNA reactivity in serous cells irrespective of
the donor's secretor status. PNA reactivity observed after fucosidase or
sialidase digestion was susceptible to endo-alpha-N-acetylgalactosaminidase
(endo-GalNAc-dase) digestion. SBA reactivity in UEA-I-negative cells from
secretors, or in cells from fetuses and newborn infants, was markedly reduced by 
beta-galactosidase digestion. After galactosidase digestion, reactivity with
Griffonia simplicifolia agglutinin II (GSA-II) appeared in the corresponding
cells. This GSA-II reactivity was almost completely eliminated by subsequent
beta-N-acetylhexosaminidase digestion. Whereas PNA reactivity in these cells was 
not reduced by beta-galactosidase treatment, it was significantly diminished by
endo-GalNAc-dase digestion. These results suggest that at least two kinds of
precursor disaccharides are produced in submandibular serous cells, i.e.,
SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and PNA-reactive
D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine
(O-glycosidically linked Type 3 chain or T antigen). Final fucosylation and
synthesis of these two types of precursor chain appear to be under the control of
the secretor gene.

PMID: 2499620 [PubMed - indexed for MEDLINE]


686. Int J Biol Macromol. 1989 Jun;11(3):177-85.

Conformational analysis and molecular modelling of the branching point of
amylopectin.

Imberty A, Perez S.

Centre de Recherches sur les Macromolecules Vegetales, CNRS, Grenoble, France.

The conformational properties of 6(2) alpha-D-glucosylmaltotriose have been
studied using energy calculations that include van der Waals interactions,
hydrogen bond stabilization, exo-anometric effect and torsional potential
contributions. The calculations focused mainly on the conformational properties
displayed at the alpha(1----6) linkage within the tetrasaccharide for which the
conformational space is reported. The tetrasaccharide molecule was then
considered as a model compound of the branching point in amylopectin. From
molecular modelling, some basic structural features associated with branching
were clearly established. It was found that, among the low energy arrangements,
the side chain would fold back onto the main backbone, thereby producing dense
three-dimensional structures in which a 'parallel' arrangement is achieved. The
branching between two strands of the double helix, as found in the crystalline
moiety of A and B starches, was further investigated. It was found that one
particular set of conformations about the glycosidic linkages in the two
different strands, could result in an arrangement such that strands could be
connected through an alpha(1----6) glycosidic linkage, with a minimum of
distortion. The three-dimensional features derived from the molecular modelling
agree with the physical properties and mode of biogenesis within the starch
granule; they are in accord with a 'cluster' type of structure.

PMID: 2489079 [PubMed - indexed for MEDLINE]


687. Biochemistry. 1989 May 30;28(11):4695-701.

Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to
kidney Na,K-ATPase.

Stewart JM, Jorgensen PL, Grisham CM.

Department of Chemistry, University of Virginia, Charlottesville 22901.

Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) 
have been used to determine the three-dimensional conformation of an ATP
analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous
studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to
MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21,
6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique
proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the
initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These
distances, taken together with previous 31P and 1H relaxation measurements with
paramagnetic probes, are consistent with a single nucleotide conformation at the 
active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a
glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring
is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in
this conformation are 100 degrees and 178 degrees, respectively. The nucleotide
adopts a bent configuration, in which the triphosphate chain lies nearly parallel
to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase
lies above and in the plane of the adenine ring. The distances from enzyme-bound 
Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are 
appropriate for second-sphere complexes involving, for example, intervening
hydrogen-bonded water molecules. The NMR data also indicate that the structure of
the bound ATP analogue is independent of the conformational state of the
enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2548590 [PubMed - indexed for MEDLINE]


688. Proc Natl Acad Sci U S A. 1989 May;86(9):3271-5.

Exonuclease III and the catalase hydroperoxidase II in Escherichia coli are both 
regulated by the katF gene product.

Sak BD, Eisenstark A, Touati D.

Division of Biological Sciences, University of Missouri, Columbia 65211.

The levels of both exonuclease III (exo III, product of xthA) and hydroperoxidase
II (HP-II, product of katE) activity in Escherichia coli were influenced by a
functional katF gene. The katF gene product is also necessary for synthesis of
HP-II. Mutations in either katF or xthA, but not katE, result in sensitivity to
H2O2 and near-UV (300-400 nm) radiation. Exo III, encoded by the xthA locus,
recognizes and removes nucleoside 5'-monophosphates near apurinic and
apyrimidinic sites in damaged DNA. Extracts of katF mutant strains had little
detectable exo III activity. When a katF+ plasmid was introduced into the katF
mutant, exo III activity exceeded wild-type levels. We propose that the katF gene
is a trans-acting positive regulator of exo III and HP-II enzymes, both of which 
are involved in cellular recovery from oxidative damage.

PMCID: PMC287112
PMID: 2541439 [PubMed - indexed for MEDLINE]


689. J Mol Biol. 1989 Apr 5;206(3):475-88.

Three-dimensional structure of ribonuclease T1 complexed with
guanylyl-2',5'-guanosine at 1.8 A resolution.

Koepke J, Maslowska M, Heinemann U, Saenger W.

Institut fur Kristallographie, Freie Universitat Berlin, F.R.G.

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was
cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG)
and the structure refined by the stereochemically restrained least-squares
refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 
sigma in the resolution range 6 to 1.8 A. The refined model consists of 781
protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a
minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end
of the refinement, the orientation of His, Asn and Gln side-chains was
reinterpreted on the basis of two-dimensional nuclear magnetic resonance data.
The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and
of the near-isomorphous RNase T1/2'-GMP complex are comparable. The
root-mean-square deviation is 0.73 A between equivalent protein atoms.
Differences in the unit cell dimensions are mainly due to the bound inhibitor.
The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in
the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center 
and the phosphate group are different and the 3'-terminal guanine forms no
hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold
disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from
differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for
the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy).
The orientation of the major site is stabilized through stacking interactions
between the 3'-terminal guanine and His92, an amino acid necessary for catalysis.
This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds
that of all other inhibitors of type 2',5'-GpN. On the basis of distance
criteria, one solvent peak in the electron density was identified as metal ion,
probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms 
and by six water molecules. The co-ordination polyhedron displays approximate 4m2
symmetry.

PMID: 2541256 [PubMed - indexed for MEDLINE]


690. Plant Physiol. 1989 Apr;89(4):1394-1400.

Cell Wall Metabolism in Ripening Fruit: IV. Characterization of the Pectic
Polysaccharides Solubilized during Softening of ;Bartlett' Pear Fruit.

Dick AJ, Labavitch JM.

Pomology Department, University of California, Davis, California 95616.

Fractionation of pectic polysaccharides from the juice of ripening ;Bartlett'
pears (Pyrus communis) gave two general types of polyuronides. The major type was
a homogalacturonan (HGA) whose molecular weight decreased upon ripening. The
other type comprised heteropolymers composed of various amounts of arabinose,
rhamnose, and galactose. Treatment of the major arabinose-containing
heteropolymeric fraction of high molecular weight (400,000) with a pear
exo-polygalacturonase to degrade contaminating HGA gave a polyuronide which was
inert to tomato endopolygalacturonase. Glycosyl-linkage analysis of this
arabinosyl-polyuronide gave results expected from a rhamnogalacturonan I-like
polysaccharide with large, highly branched araban side chains (RG-I). A linkage
between HGA and RG-I was not found. RG-I, in ripening pears, appeared to be
degraded with the initial loss of much of its arabinose.

PMCID: PMC1056027
PMID: 16666715 [PubMed - as supplied by publisher]


691. Biochemistry. 1989 Mar 21;28(6):2452-9.

Conformational analysis of d(C3G3), a B-family duplex in solution.

Wolk S, Thurmes WN, Ross WS, Hardin CC, Tinoco I Jr.

Department of Chemistry, University of California, Berkeley 94720.

NMR and circular dichroism studies of the duplex formed by the self-complementary
DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs
from standard B-form. An analysis of NMR coupling constants within the
deoxyribose moieties yields a 70% or greater contribution from pseudorotation
phase angles corresponding to the C3'-exo conformation, a conformation similar to
the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton
distances are consistent with a B-form structure, but some internucleotide
distances are intermediate between A- and B-form structures. Circular dichroism
spectra have B-form characteristics but also include an unusual negative band at 
282 nm. The solution spectroscopic results are in contrast with X-ray
crystallographic studies which find A-form structures for similar sequences.

PMID: 2730876 [PubMed - indexed for MEDLINE]


692. Biochemistry. 1989 Mar 7;28(5):2001-9.

Environmentally induced conformational changes in B-type DNA: comparison of the
conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its
crystalline complex with the restriction nuclease EcoRI.

Thomas GA, Kubasek WL, Peticolas WL, Greene P, Grable J, Rosenberg JM.

Department of Chemistry, University of Oregon, Eugene 97403.

Raman spectroscopic analysis of the secondary structure of the crystalline
restriction endonuclease EcoRI, the oligonucleotide d(TCGCGAATTCGCG) in solution,
and the corresponding crystalline EcoRI-oligonucleotide complex reveals
structural differences between the complexed and uncomplexed protein and
oligonucleotide components that appear to be linked to complex formation.
Structural differences that are spectroscopically identified include (1) an
increase in the population of furanose rings adopting the C3'-endo conformation
and (2) spectroscopically observed changes in base stacking which are probably
associated with the crystallographically observed distortion of the phosphate
backbone about positions C(3)-G(4) and C(9)-G(10) and unwinding between the
symmetry-related segments GAA-TTC which make up the central recognition core
(McClarin et al., 1986). Changes in base stacking due to distortions and
unwinding along the oligonucleotide result in differences in the base vibrational
region between the spectra of the complex and the oligonucleotide in solution.
The spectroscopic analysis indicates that the C2'-endo population is similar for 
the oligonucleotide in solution and in the complex. The additional C3'-endo
population in the complex appears to arise from the conversion of rings adopting 
alternative conformations such as C1'-exo and O1'-endo. Analysis of the
vibrational bands derived from guanine indicates that the population of guanine
residues associated with furanose rings in a C2'-endo conformation is similar for
the oligonucleotide in solution and in the crystalline complex. This implies that
the increase in C3'-endo population is not associated with guanine residues.
Large conformational distortions such as those observed in the crystal
distortions are not observed in either the crystal or the solution of the
oligomer d(CGCGAATTCGCG).(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2719943 [PubMed - indexed for MEDLINE]


693. Plasmid. 1989 Mar;21(2):142-6.

Plasmid localization and mapping of two Azospirillum brasilense loci that affect 
exopolysaccharide synthesis.

Michiels K, De Troch P, Onyeocha I, Van Gool A, Elmerich C, Vanderleyden J.

F. A. Janssens Memorial Laboratory for Genetics, Catholic University of Leuven,
Heverlee, Belgium.

Two Azospirillum brasilense loci that correct Rhizobium meliloti exoB and exoC
mutants for exopolysaccharide (EPS) synthesis have been identified previously (K.
W. Michiels, J. Vanderleyden, A. P. Van Gool, E. R. Signer, J. Bacteriol.,
1988b). A. brasilense exo mutants produce EPS of lower molecular weight than the 
wild type strain. Here, we show by hybridization that these exo loci are located 
on a 90-MDa plasmid in A. brasilense Sp7. In four other Azospirillum strains but 
not in A. lipoferum SpBr17, the loci are likewise located on a plasmid of
approximately the same size. Transposon Tn5 insertions in these loci were
isolated and mapped on the cloned DNA by restriction analysis. Hybridization of
restriction digests of purified 90-MDa plasmid DNA with probes containing the exo
loci confirmed their plasmid location. This is the first report on plasmid
localization of genes in Azospirillum.

PMID: 2544914 [PubMed - indexed for MEDLINE]


694. Nucleic Acids Res. 1989 Feb 25;17(4):1649-63.

Processing pathway of Escherichia coli 16S precursor rRNA.

Srivastava AK, Schlessinger D.

Department of Microbiology and Immunology, Washington University School of
Medicine, St Louis, MO 63110.

Immediate precursors of 16S rRNA are processed by endonucleolytic cleavage at
both 5' and 3' mature termini, with the concomitant release of precursor
fragments which are further metabolized by both exo- and endonucleases. In
wild-type cells rapid cleavages by RNase III in precursor-specific sequences
precede the subsequent formation of the mature ends; mature termini can, however,
be formed directly from pre-16S rRNA with no intermediate species. The direct
maturation is most evident in a strain deficient in RNase III, and the results in
whole cells are consistent with results from maturation reactions in vitro. Thus,
maturation does not require cleavages within the double-stranded stems that
enclose mature rRNA sequences in the pre-16S rRNA.

PMCID: PMC443564
PMID: 2646597 [PubMed - indexed for MEDLINE]


695. J Biochem. 1989 Jan;105(1):127-32.

Substrate specificities of exo- and endo-type cellulases in the hydrolysis of
beta-(1----3)- and beta-(1----4)-mixed D-glucans.

Kanda T, Yatomi H, Makishima S, Amano Y, Nisizawa K.

Department of Industrial Chemistry, Faculty of Engineering, Shinshu University,
Nagano.

An exo-type cellulase (Ex-1) was extracted from Irpex lacteus (Polyporus
tulipiferae) and purified essentially to homogeneity. This cellulase attacked
cellulosic substrates in an exo-wise fashion to produce almost exclusively
cellobiose. In contrast, Ex-1 was found to attack beta-glucans having
beta-(1----3)- and beta-(1----4)-mixed linkages in a way similar to an endo-type 
cellulase. The products formed from barley glucan by Ex-1 were
3(2)-O-beta-D-cellobiosyl-cellobiose much greater than
3(2)-O-beta-D-glucosyl-cellobiose greater than cellobiose much greater than or
equal to cellotriose much greater than glucose in the early stage, but no
laminaribiose was produced. An endo-type cellulase (En-1) obtained from the same 
fungus also hydrolyzed beta-glucans but in a typical endo-wise fashion and the
products from barley glucan were 3(2)-O-beta-D-glucosyl-cellobiose much greater
than 3(2)-O-beta-D-cellobiosyl-cellobiose greater than cellobiose much greater
than laminaribiose; no glucose or cellotriose was produced. Thus, it seems likely
that En-1 can attack any intramolecular linkage of beta-glucan, while Ex-1
requires the presence of at least cellobiosyl residues adjacent to a
beta-(1----3)-D-linked glucosyl residue. This finding, together with the mode of 
hydrolysis of cellulosic substrates by Ex-1, suggests that the stereochemical
structure of successive beta-(1----4)-cellobiosyl residues inserted by
beta-(1----3)-D-glucosidic linkage is permissible in the action of Ex-1, although
this enzyme prefers the beta-(1----4)-linked cellobiosyl sequence.

PMID: 2738039 [PubMed - indexed for MEDLINE]


696. Acta Neuropathol. 1989;79(3):262-70.

Susceptibility of brain cells to murine cytomegalovirus infection in the
developing mouse brain.

Tsutsui Y, Kashiwai A, Kawamura N, Nagahama M, Mizutani A, Naruse I.

Department of Pathology, Institute for Developmental Research, Aichi Prefectural 
Colony, Japan.

Mouse embryos were infected with murine cytomegalovirus (MCMV) by injecting the
virus into the cerebral ventricles in the late stage of gestation; the brains of 
the offspring were than analyzed using the histological and immunohistochemical
methods. Brains of the offspring, which were injected with relatively high titers
of MCMV [1 X 10(4) plaque-forming units (pfu)] on day 13 of gestation exo utero
or on day 15 of gestation in utero, showed massiv necrosis of the cerebral cortex
with gliomesodermal proliferation around 9 to 10 days after birth. In these
brains, viral antigen-positive cells were observed in zonal arrangement in the
lesion-free cortex and in the hippocampus. Immunohistochemical double staining
showed that some of the viral antigen-positive cells had also reacted with
antibody to neuron-specific enolase at the same time, but had hardly reacted with
antibodies to brain-type creatine kinase or glial fibrillary acidic protein.
Brains of the offspring, which were injected with relatively low titers of virus 
(1 x 10(3) pfu) on day 15 of gestation, showed zonal arrangement of viral
antigen-positive cells mainly in the cerebral cortex and in the hippocampus 7
days after birth, although the numbers of the positive cells were low. Fourteen
days after birth, some of these offspring showed atrophy of the cerebral cortex
and the hippocampus. These results suggest that some of the neuronal cells in the
cerebral cortex and the hippocampus have special susceptibility to MCMV
infection.

PMID: 2558485 [PubMed - indexed for MEDLINE]


697. Plant Cell. 1989 Jan;1(1):65-72.

A non-nodulating alfalfa mutant displays neither root hair curling nor early cell
division in response to Rhizobium meliloti.

Dudley ME, Long SR.

Department of Biological Sciences, Stanford University, California 94305-5020.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation 
of epidermal root hairs and the approximately concurrent stimulation of cell
dedifferentiation and cell division in the root inner cortex. These early steps
have been studied previously by analysis of R. meliloti mutants. Bacterial
strains mutated in nodABC, for example, fail to stimulate either root hair
curling or cell division events in the plant host, whereas exopolysaccharide
(exo) mutants of R. meliloti stimulate host cell division but the resulting
nodules are uninfected. As a further approach to understanding early symbiotic
interactions, we have investigated the phenotype of a non-nodulating alfalfa
mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating
plants were inoculated with wild-type R. meliloti and scored for root hair
curling and cell divisions. MN-1008 was found to be defective in both responses. 
Mutant plants inoculated with Exo- bacteria also showed no cell division
response. Therefore, the genetic function mutated in MN-1008 is required for both
root hair curling and cell division, as is true for the R. meliloti nodABC genes.
These observations support the model that the distinct cellular processes of root
hair curling and cell division are triggered by related mechanisms or components,
or are causally linked.

PMCID: PMC159737
PMID: 2535468 [PubMed - indexed for MEDLINE]


698. Histochemistry. 1989;92(4):307-12.

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens
in human pancreas using lectin staining and glycosidase digestion procedures.

Ito N, Nishi K, Nakajima M, Okamura Y, Hirota T.

Department of Legal Medicine, Nara Medical University, Japan.

Histochemical analyses of the chemical structures of sugar sequences with or
without blood group specificity were carried out by combined stepwise digestion
of tissue sections with exo- and endoglycosidases and subsequent lectin stainings
in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood
group A or AB secretor individuals, sequential digestion with
alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with
peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as
well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase
digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential
digestion with alpha-galactosidase and fucosidase likewise liberated the PNA
binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from
blood group B and AB secretors. Sialidase digestion liberated the PNA binding
sites not only in acinar cells but also intercalated duct cells, islet cells of
Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme
digestions was eliminted by endo-alpha-N-acetylgalactosaminidase
(endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from
non-secretors was also susceptible to endo-GalNAcdase treatment. Following the
endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA
reactivity in acinar cells from nonsecretors. These results show that ABH
determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta
1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in
pancreatic acinar cells and suggest that product coded by the secretor gene is
required for the complete conversion of type 3 precursor chains into H
determinants.

PMID: 2478505 [PubMed - indexed for MEDLINE]


699. J Biol Chem. 1988 Dec 15;263(35):18842-9.

The roles of active site hydrogen bonding in cytochrome P-450cam as revealed by
site-directed mutagenesis.

Atkins WM, Sligar SG.

Department of Biochemistry, University of Illinois, Urbana 61801.

The role of the active site hydrogen bond of cytochrome P-450cam has been studied
utilizing a combination of site-directed mutagenesis and substrate analogues with
altered hydrogen bonding capabilities. Cytochrome P-450cam normally catalyzes the
regiospecific hydroxylation of the monoterpene camphor. The x-ray crystal
structure of this soluble bacterial cytochrome P-450 (Poulos, T. L., Finzel, B.
C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260,
16122-16128) indicates a specific hydrogen bond between tyrosine 96 and the
carbonyl moiety of the camphor substrate. The site-directed mutant in which
tyrosine 96 has been changed to a phenylalanine and the substrate analogues
thiocamphor and camphane have been used to probe this interaction in several
aspects of catalysis. At room temperature, both the mutant enzyme with camphor
and the wild type enzyme with thiocamphor bound result in 59 and 65% high-spin
ferric enzyme as compared to the 95% high spin population obtained with native
enzyme and camphor as substrate. The equilibrium dissociation constant is
moderately increased, from 1.6 microM for the wild type protein to 3.0 and 3.3
microM for wild type-thiocamphor and mutant-camphor complexes, respectively.
Camphane bound to cytochrome P-450cam exhibits a larger decrease in high spin
fraction (45%) and a correspondingly larger KD (46 microM), suggesting that the
carbonyl moiety of camphor plays an important steric role in addition to its
interaction as a hydrogen bond acceptor. The absolute regioselectivity of the
mutant enzyme, and of the wild type enzyme with thiocamphor, is lost resulting in
production of several hydroxylated products in addition to the 5-exo-hydroxy
isomer. Based on rates of NADH oxidation, comparison of the substrate specificity
for these systems (kcat/KD) indicates a 5- and 7-fold decrease in specificity for
the mutant enzyme and thiocamphor-wild type complex, respectively. The
replacement of the cytochrome P-450cam active site tyrosine with phenylalanine
does not affect the branching ratio of monooxygenase versus oxidase chemistry or 
peroxygenase activity (Atkins, W.M., and Sligar, S.G. (1987) J. Am. Chem. Soc.
109, 3754-3760).

PMID: 3198602 [PubMed - indexed for MEDLINE]


700. Showa Shigakkai Zasshi. 1988 Dec;8(4):405-12.

Purification and characterization of intracellular dextranase of Bacteroides
oralis Ig4a.

Igarashi T, Yamamoto A.

Multiple forms of dextranase were detected in both intra- and extracellular
fractions of Bacteroides oralis Ig4a. The molecular weights of these enzymes
varied from 52,000 to 260,000 by sodium dodecyl sulfate-polyacrylamide-blue
dextran gel electrophoresis. The intracellular dextranases were fractionated by
chromatography and gel filtration steps, and the dextranases IV and V were
obtained. The former was only partially pure. The molecular weights of the
dextranases IV and V were estimated to be 120,000 and 105,000, respectively, by
SDS-PAGE. The dextranase V was further characterized and it was revealed that the
pH- and temperature optima were 5.0, and 55 degrees C, respectively. The Km value
was 6.7 x 10(-2) mM for dextran T-70. The enzyme did not exhibit any metal ion
requirements, but was inhibited by CoCl2 and HgCl2; lysine and alanine contents
were especially high; it hydrolyzed the alpha-1,6-glucan by an exo-type
mechanism, and was inactive toward glucans containing alpha-1,3-, alpha-1,4-, and
beta-1,4-linkages.

PMID: 3270938 [PubMed - indexed for MEDLINE]


701. J Bacteriol. 1988 Nov;170(11):5401-4.

Isolation and characterization of Azospirillum brasilense loci that correct
Rhizobium meliloti exoB and exoC mutations.

Michiels KW, Vanderleyden J, Van Gool AP, Signer ER.

F. A. Janssens Memorial Laboratory for Genetics, Catholic University of Leuven,
Heverlee, Belgium.

The occurrence in Azospirillum brasilense of genes that code for
exopolysaccharide (EPS) synthesis was investigated through complementation
studies of Rhizobium meliloti Exo- mutants. These mutants are deficient in the
synthesis of the major acidic EPS of Rhizobium species and form empty,
non-nitrogen-fixing root nodules on alfalfa (J. A. Leigh, E. R. Signer, and G. C.
Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We demonstrated that the 
exoC mutation of R. meliloti could be corrected for EPS production by several
cosmid clones of a clone bank of A. brasilense ATCC 29145. However, the EPS
produced differed in structure from the wild-type R. meliloti EPS, and the
symbiotic deficiency of the exoC mutation was not reversed by any of these cosmid
clones. The exoB mutation could be corrected not only for EPS production but also
for the ability to form nitrogen-fixing nodules on alfalfa by one particular
cosmid clone of A. brasilense. Tn5 insertions in the cloned DNA were isolated and
used to construct Azospirillum mutants with mutations in the corresponding loci
by marker exchange. It was found that these mutants failed to produce the
wild-type high-molecular-weight EPS, but instead produced EPSs of lower molecular
weight.

PMCID: PMC211624
PMID: 3182731 [PubMed - indexed for MEDLINE]


702. Nucleic Acids Res. 1988 Oct 11;16(19):9307-21.

Structural studies of O6-methyldeoxyguanosine and related compounds: a
promutagenic DNA lesion by methylating carcinogens.

Yamagata Y, Kohda K, Tomita K.

Faculty of Pharmaceutical Sciences, Osaka University, Japan.

O6-Methylation of guanine residues in DNA can induce mutations by formation of
base mispairing due to the deprotonation of N(1). The electronic, geometric and
conformational properties of three N(9)-Substituted O6-methylguanine derivatives,
O6-methyldeoxyguanosine (O6mdGuo), O6-methylguanosine (O6mGuo) and O6,
9-dimethylguanine (O6mdGua), were investigated by X-ray and/or NMR studies.
O6mdGuo crystallizes in the monoclinic space group P2(1) with cell parameters a =
5.267(1), b = 19.109(2), c = 12.330(2) A, beta = 92.45(1) degrees, V = 1239.8(3) 
A3, z = 4 (two nucleosides per asymmetric unit), and O6mGua in the monoclinic
space group P2(1)/n with cell parameters a = 10.729(2), b = 7.640(1) c =
10.216(1) A, beta = 92.17(2) degrees, V = 836.7(2) A3, z = 4. The geometry and
conformation of O6-methylguanine moieties observed in both crystals and are very 
similar. Furthermore, the molecular dimensions of the O6methylguanine residue
resemble more closely those of adenine than those of guanine. The methoxy group
is coplanar with the purine ring, the methyl group being cis to N(1). The
conformation of O6-methylguanine nucleosides is variable. The glycosidic
conformation of O6mdGuo is anti for molecule (a) and high-anti for molecule (b)
in the crystal, while that of O6mGuo is syn [Parthasarathy, R & Fridey, S. M.
(1986) Carcinogenesis 7, 221-227]. The sugar ring pucker of O6mdGuo is C(2')-endo
for molecule (a) and C(1')-exo for molecule (b). The C(4')-C(5') exocyclic bond
conformation in O6mdGuo is gauche- for molecule (a) but trans for molecule (b),
in contrast with gauche+ for O6mGuo. The hydrogen bonds exhibited by
O6-methylguanine derivatives differ from those in guanine derivatives; the amino 
N(2) and ring N(3) and N(7) atoms of O6-methylguanine residues are involved in
hydrogen bonding. 1H-NMR data for O6mdGuo and O6mdGuo reveal the predominance of 
a C(2')-endo type sugar puckering. In O6mdGuo, however, a contribution of a
C(1')-exo sugar puckering is significant. The NOE data also indicate that O6mdGuo
molecules exist with nearly equal population for anti (including high anti) and
syn glycosidic conformations. These observations and their biological
implications are discussed.

PMCID: PMC338708
PMID: 3174452 [PubMed - indexed for MEDLINE]


703. J Bacteriol. 1988 Sep;170(9):4239-48.

Genetic analysis of a cluster of genes required for synthesis of the
calcofluor-binding exopolysaccharide of Rhizobium meliloti.

Long S, Reed JW, Himawan J, Walker GC.

Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

Rhizobium meliloti produces an acidic, Calcofluor-binding exopolysaccharide which
plays a role in nodulation of alfalfa plants by this bacterium. We constructed
and mapped 102 transposon insertions in a 48-kilobase (kb) region previously
shown to contain several exo genes. Mutations affecting production of the
Calcofluor-binding exopolysaccharide were clustered in a 22-kb region and fell
into 12 complementation groups. Strains carrying mutations in seven of the
complementation groups (exoA, exoB, exoF, exoL, exoM, exoP, and exoQ) produced no
Calcofluor-binding exopolysaccharide and induced non-nitrogen-fixing nodules on
alfalfa. Mutants in an eighth complementation group, exoH (Leigh et al., Cell
51:579-587, 1987), produce an altered exopolysaccharide and also induce the
formation of non-nitrogen-fixing nodules. Mutants in the remaining four
complementation groups produced less Calcofluor-binding material than the wild
type. Mutants carrying mutations in two of these complementation groups (exoK and
exoN) formed apparently normal, nitrogen-fixing nodules, while mutants in the
other two groups (exoG and exoJ) formed normal nodules less efficiently than the 
wild type.

PMCID: PMC211433
PMID: 2842306 [PubMed - indexed for MEDLINE]


704. J Bacteriol. 1988 Aug;170(8):3327-32.

Characterization of polysaccharides of Rhizobium meliloti exo mutants that form
ineffective nodules.

Leigh JA, Lee CC.

Department of Microbiology, University of Washington, Seattle 98195.

Mutants of Rhizobium meliloti SU47 with defects in the production of the
Calcofluor-binding expolysaccharide succinoglycan failed to gain entry into
alfalfa root nodules. In order to define better the polysaccharide phenotypes of 
these exo mutants, we analyzed the periplasmic oligosaccharide cyclic
(1-2)-beta-D-glucan and lipopolysaccharide (LPS) in representative mutants. The
exoC mutant lacked the glucan and had abnormal LPS which appeared to lack a
substantial portion of the O side chain. The exoB mutant had a spectrum of LPS
species which differed from those of both the wild-type parental strain and the
exoC mutant. The presence of the glucan and normal LPS in the exoA, exoD, exoF,
and exoH mutants eliminated defects in these carbohydrates as explanations for
the nodule entry defects of these mutants. We also assayed for high- and
low-molecular-weight succinoglycans. All of the exo mutants except exoD and exoH 
completely lacked both forms. For the Calcofluor-dim exoD mutant, the
distribution of high- and low-molecular-weight forms depended on the growth
medium. The haloless exoH mutant produced high-molecular-weight and only a trace 
of low-molecular-weight succinoglycan; the succinyl modification was missing, as 
was expected from the results of previous studies. The implications of these
observations with regard to nodule entry are discussed.

PMCID: PMC211298
PMID: 3403505 [PubMed - indexed for MEDLINE]


705. Arch Biochem Biophys. 1988 Aug 1;264(2):607-17.

Production of hybrid glycoproteins and accumulation of oligosaccharides in the
brain of sheep and pigs administered swainsonine or locoweed.

Tulsiani DR, Broquist HP, James LF, Touster O.

Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee
37235.

Swainsonine and swainsonine-containing plants produce biochemical and
neurological changes in several mammalian species. The toxin is a potent
inhibitor of liver lysosomal alpha-D-mannosidase and Golgi mannosidase II. The
inhibition of the latter enzyme causes the production of abnormal glycoproteins
containing hybrid oligosaccharides instead of complex types in a variety of
cultured cells. In view of the widespread occurrence and biological importance of
N-linked glycoproteins in the central nervous system, we initiated studies to
determine the structure of oligosaccharides in glycoproteins prepared from the
brain of control, swainsonine-fed, and locoweed-fed animals. The results
presented here indicate that the feeding led to alteration in the structure of
brain glycoproteins. Over 25% of the glycoproteins which presumably contained
complex-type oligosaccharides were modified and now contained hybrid
oligosaccharides. The structure of the N-linked oligosaccharide (glycopeptide)
was established by (a) studying the binding properties of the glycopeptide to
immobilized lectins of known sugar specificity, and (b) comparing the size of the
glycopeptide before and after treatment with exo- and endoglycosidases. The
production of hybrid oligosaccharides occurred despite the apparent absence of
mannosidase II in brain. The relationships of the altered structure of brain
glycoproteins, accumulation of mannose-rich oligosaccharides in the brain, and
abnormal behavior of the animals administered swainsonine or locoweed are
discussed.

PMID: 3135781 [PubMed - indexed for MEDLINE]


706. Atherosclerosis. 1988 Aug;72(2-3):115-22.

The hypocholesterolemic action of the undigested fraction of soybean protein in
rats.

Sugano M, Yamada Y, Yoshida K, Hashimoto Y, Matsuo T, Kimoto M.

Laboratory of Nutrition Chemistry, Kyushu University School of Agriculture,
Fukuoka, Japan.

Erratum in:
    Atherosclerosis 1988 Nov;74(1-2):187.

Soybean protein was exhaustively digested with endo- and exo-type microbial
proteases and the effect of the digestible low molecular fraction (LMF) and the
undigested high molecular fraction (HMF) on the serum cholesterol level was
compared to that of the intact protein in rats given a cholesterol-enriched diet.
The HMF, peptides relatively abundant in hydrophobic amino acids, was found to be
substantially hypocholesterolemic when fed at the nitrogen level equivalent to
that of the 20% soybean protein diet, and not only serum but also liver
cholesterol levels were similar to those usually encountered in rats given diets 
free of cholesterol. There was a dose-dependent reduction of serum and liver
cholesterol when casein was replaced stepwise with HMF. The cholesterol-lowering 
action could be attributable to an increased fecal steroid excretion.

PMID: 3063266 [PubMed - indexed for MEDLINE]


707. Biochemistry. 1988 Jun 28;27(13):4840-8.

1H nuclear magnetic resonance studies of the conformation of an ATP analogue at
the active site of Na,K-ATPase from kidney medulla.

Stewart JM, Grisham CM.

Department of Chemistry, University of Virginia, Charlottesville 22901.

1H nuclear magnetic relaxation measurements have been used to determine the
three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active
site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is
a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C.,
& Grisham, C. M. (1982) Biochemistry 21, 6979; Gantzer, M. L., Klevickis, C., &
Grisham, C. M. (1982) Biochemistry 21, 4083] and that Mn2+ bound to a single,
high-affinity site on the ATPase can be an effective paramagnetic probe for
nuclear relaxation studies of the Na,K-ATPase [O'Connor, S. E., & Grisham, C. M. 
(1979) Biochemistry 18, 2315]. From the paramagnetic effect of Mn2+ bound to the 
ATPase on the longitudinal relaxation rates of the protons of Co(NH3)4ATP at the 
substrate site (at 300 and 361 MHz), Mn-H distances to seven protons on the bound
nucleotide were determined. Taken together with previous 31P nuclear relaxation
data, these measurements are consistent with a single nucleotide conformation at 
the active site. The nucleotide adopts a bent configuration, in which the
triphosphate chain lies nearly parallel to the adenine moiety. The glycosidic
torsion angle is 35 degrees, and the conformation of the ribose ring is slightly 
N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this
conformation are 100 degrees and 178 degrees, respectively. The bound Mn2+ lies
above and in the plane of the adenine ring. The distances from Mn2+ to N6 and N7 
are too large for first coordination sphere complexes but are appropriate for
second-sphere complexes involving, for example, intervening hydrogen-bonded water
molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2844241 [PubMed - indexed for MEDLINE]


708. Genes Dev. 1988 Jun;2(6):677-87.

Expression of nodule-specific genes in alfalfa root nodules blocked at an early
stage of development.

Dickstein R, Bisseling T, Reinhold VN, Ausubel FM.

Department of Genetics, Harvard Medical School, Boston, Massachusetts.

To help dissect the molecular basis of the Rhizobium-legume symbiosis, we used in
vitro translation and Northern blot analysis of nodule RNA to examine
alfalfa-specific genes (nodulins) expressed in two types of developmentally
defective root nodules elicited by Rhizobium meliloti. Fix- nodules were elicited
by R. meliloti nif mutants; these nodules were invaded by rhizobia and contained 
differentiated bacteroids. 'Empty' nodules were elicited by R. meliloti exo and
ndv mutants and by Agrobacterium tumefaciens strains carrying the R. meliloti nod
genes; these nodules contained a nodule meristem but lacked infection threads,
intracellular bacteria, and bacteroids. Fix- nodules contained a spectrum of
nodulins similar to wild-type nodules. In contrast, only two nodulins, Nms-30 and
a nodulin homologous to ENOD2 of soybean, were detected in empty nodules.
Although R. meliloti ndv and exo mutants elicited nodules with the same defective
phenotype, ndv and exo mutants (except for exoC mutants) had distinct biochemical
phenotypes. R. meliloti ndvA and ndvB mutants were deficient in cyclic glucan
production but not the acidic exopolysaccharide; the converse was true for exoA, 
exoB, and exoF mutants. exoC mutants were defective in both exopolysaccharide and
cyclic glucan biosynthesis. Our results support the model that the R. meliloti
nod genes produce a signal that results in nodule meristem induction. Both the
exopolysaccharide and cyclic glucan, however, appear to act at the next step in
the developmental process and are involved in the production of a signal (or
structure) that allows infection thread formation and invasion of the nodule.

PMID: 3417147 [PubMed - indexed for MEDLINE]


709. Mol Pharmacol. 1988 May;33(5):551-8.

Interaction of the antifolate antibiotic trimethoprim with phosphatidylcholine
membranes: a 13C and 31P nuclear magnetic resonance study.

Painter GR, Grunwald R, Roth B.

The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.

The interaction of the bacterial dihydrofolate reductase inhibitor trimethoprim
with small unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine vesicles
was studied using 13C and 31P NMR spectroscopy. In an effort to determine whether
trimethoprim passively permeates the vesicle membrane, an impermeant, anionic
complex of the paramagnetic ion Dy3+ was added to the extravesicular compartment.
Based on the downfield shift that the Dy3+ complex induces in the [2-13C]
resonance of trimethoprim in free solution, membrane permeation and movement of
the drug into the intravesicular space can in principle be established from
observation of the C2 chemical shift alone. In contrast to what is predicted by a
two-compartment model separated by a semipermeable barrier, the presence of
vesicles virtually reverses the effect of the shift reagent on the [2-13C] carbon
resonance. These results suggest that the majority of the trimethoprim might be
sequestered within the vesicle membrane. A saturable decrease in the spin-lattice
relaxation time and a saturable increase in the line width at half-height of the 
[2-13C] resonance as a function of vesicle concentration indicated that
trimethoprim does in fact bind to the phospholipid matrix of the membrane
bilayer. The KD for the interaction calculated from the relaxation data was 9.7
+/- 0.3 X 10(-4) M at a pH of 7.01 and an ionic strength of 0.015 M. The chemical
shift of the [2-13C] resonance is unaffected by interaction with the
electroneutral membrane, and the pKa increases by only 0.16 upon binding. These
results point to an interfacial location for the pyrimidine moiety. Using the
paramagnetic shift reagent Pr3+ and the 31P NMR signal from the phosphodiester
groups of the membrane lipids, trimethoprim was shown to displace Pr3+ ions from 
binding sites on the outer membrane surface as would be expected if the polar
pyrimidine ring were located at or near the membrane surface. The extent to which
trimethoprim and trimethoprim derivatives modified in the 3'- and 4'-positions
interact with the exo face of the membrane is strongly dependent on the type of
substituent and whether it is in the 3'- or 4'-position. Van der Waals
interactions between the 5-benzyl sidechain and the hydrophobic fatty acid region
of the membrane interior appear to be necessary for the polar portion of
trimethoprim to compete favorably for the membrane-binding site with the
polyvalent Pr3+ ion.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 3367902 [PubMed - indexed for MEDLINE]


710. Vutr Boles. 1988;27(4):29-32.

[The serum testosterone level of patients with bronchial asthma treated with
corticosteroids and untreated]

[Article in Bulgarian]

Mileva Zh, Maleeva A.

The testosterone basic level was followed up in 427 asthmatic male patients
treated and not treated with corticosteroids. In 172 of them (40.68%) basic low
blood testosterone was found, in 1/3 of the patients (32.32%) the morning
testosterone level was below 200 ng% and in 37 patients (8.67%) it was below 100 
ng%. Low blood testosterone was more frequent in the corticosteroid-treated
patients than in the patients who had never been treated with corticosteroids
(31.63%). Among the untreated patients low blood testosterone was more frequent
in the patients with nonatopic (40.82%) than in the patients with atopic (24.67%)
and mixed type (27.16%) of bronchial asthma. Low blood testosterone was found
mainly in the patients with severe (37.76%) and moderate (40.00%) form of the
disease and very rarely in patients with mild form of bronchial asthma (8.51%).
The basic testosterone level changes are probably due to the stress, hypoxia and 
corticosteroid treatment. The possibility of a direct suppressive action of exo- 
and endoallergens on the testes are discussed.

PMID: 3213021 [PubMed - indexed for MEDLINE]


711. Biochem J. 1987 Sep 15;246(3):651-8.

Nucleophilic re-activation of the PC1 beta-lactamase of Staphylococcus aureus and
of the DD-peptidase of Streptomyces R61 after their inactivation by
cephalosporins and cephamycins.

Faraci WS, Pratt RF.

Department of Chemistry, Wesleyan University, Middletown, CT 06457.

It has been shown previously [Faraci & Pratt (1985) Biochemistry 24, 903-910;
(1986) Biochemistry 25, 2934-2941; (1986) Biochem. J. 238, 309-312] that certain 
beta-lactam-processing enzymes form inert acyl-enzymes with cephems that possess 
good leaving groups at the C-3' position. These inert species arise by
elimination of the leaving group from the initially formed and more rapidly
hydrolysing acyl-enzyme, which has the 'normal' cephalosporoate structure. The
present paper shows that a strong nucleophile, thiophenoxide, can catalyse the
re-activation of three examples of these inert acyl-enzymes, generated on
reaction of cephalothin and cefoxitin with the PC1 beta-lactamase of
Staphylococcus aureus and of cephalothin with D-alanyl-D-alanine
transpeptidase/carboxypeptidase of Streptomyces R61. In view of the reversibility
of the elimination reaction, demonstrated in model systems [Pratt & Faraci (1986)
J. Am. Chem. Soc. 108, 5328-5333], this catalysis is proposed to arise through
nucleophilic addition to the exo-methylene carbon atom of the inert acyl-enzyme
to regenerate a more rapidly hydrolysing normal cephalosporoate. Strong support
for this scenario was obtained through comparison of the kinetics of the
catalysed re-activation reaction with those of turnover of the relevant
3'-thiophenoxycephems, thiophenoxycephalothin and thiophenoxycefoxitin. The
enzymes appear to stabilize the products of the elimination reaction with respect
to the normal cephalosporoate, but more strongly to destabilize the transition
states. The effects of other nucleophiles, including cysteine, glycine amide and 
imidazole, on the above enzymes and on other beta-lactamases can be understood in
terms of the model reaction kinetics and thermodynamics.

PMCID: PMC1148329
PMID: 3500712 [PubMed - indexed for MEDLINE]


712. Biotechnol Bioeng. 1987 Aug 5;30(2):251-7.

Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from
Trichoderma viride.

Beldman G, Voragen AG, Rombouts FM, Searle-van Leeuwen MF, Pilnik W.

Department of Food Science, Agricultural University, De Dreyen 12, 6703 BC
Wageningen.

Adsorption on crystalline cellulose of six endoglucanases (Endo I, II, III, IV, V
and VI; 1, 4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) and two exoglucanases
(Exo II and III; 1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.92), purified from
a commercial cellulase preparation of Trichoderma viride origin, was studied.
Endo I, III, and V adsorbed strongly on Avicel cellulose, while adsorption of
Endo II, IV, and VI was much lower. Also, the two exoglucanases could be divided 
into one enzyme (Exo III) that had a high adsorption affinity and another enzyme 
(Exo II) that adsorbed only moderately. Adsorption data fitted the Langmuir-type 
adsorption isotherm. However, adsorption was only partially reversible with
respect to dilution. No relation could be found between adsorption affinity and
degree of randomness in cellulose hydrolysis, measured as the diversity of
released hydrolytic products. Kinetic measurements indicated that only part of
the adsorbed enzyme molecules are hydrolytically active.

PMID: 18581306 [PubMed - in process]


713. Bioorg Khim. 1987 Aug;13(8):1093-101.

[Synthesis of capsular polysaccharide of Streptococcus pneumoniae type 14. 3.
Synthesis of a monomer for polycondensation]

[Article in Russian]

Nifant'ev NE, Bakinovskii LV, Kochetkov NK.

Synthesis of a tritylated tetrasaccharide 1,2-O-(1-cyano) ethylidene derivative
is described by glycosylation of 3,6-di-O-benzoyl-4-O-(2,4,6-tri-O-benzoyl-beta- 
D-galactopyranosyl)-1,2-O-[1-(exo-cyano)ethylidene]-alpha-D- glucopyranose with
6-O-acetyl-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoyl-beta-
D-galactopyranosyl)-2-deozy-2-phthalimido-D-glucopyranosyl. bromide followed by
selective deacetylation and tritylation.

PMID: 3675650 [PubMed - indexed for MEDLINE]


714. Appl Environ Microbiol. 1987 Apr;53(4):768-771.

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive
of Exo-beta-Amylase.

Nanmori T, Numata Y, Shinke R.

Department of Agricultural Chemistry, Faculty of Agriculture, Kobe University,
Rokkodai, Nada-ku, Kobe 657, Japan.

Starting with a strain of Bacillus cereus excreting about 40-fold more
beta-amylase than does the original wild-type strain, we isolated, after
mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a strain designated
BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase 
in beta-amylase activity. The amylase production of this strain was observed to
increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the
presence of 2% soluble starch in the culture medium. The enzyme produced by this 
strain was immunologically identical to the wild-type enzyme, suggesting that
either the copy number of the gene or the efficiency of enzyme synthesis from it,
or both, are altered in this strain.

PMCID: PMC203753
PMID: 16347320 [PubMed - as supplied by publisher]


715. Biochemistry. 1987 Mar 24;26(6):1645-55.

NMR studies of the AMP-binding site and mechanism of adenylate kinase.

Fry DC, Kuby SA, Mildvan AS.

NMR has previously been used to determine the conformation of enzyme-bound MgATP 
and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S.
A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the
conformation and location of the other substrate, AMP, distances have been
measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP,
to six protons and to the phosphorus atom of AMP on adenylate kinase, with the
paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs)
have been used to measure five interproton distances on enzyme-bound AMP. These
distances were used to determine the conformation of bound AMP in addition to its
position with respect to metal-ATP. Enzyme-bound AMP exhibits a high
anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose
pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the
C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20
degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A,
indicating a reaction coordinate distance of approximately 3 A, which is
consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten
intermolecular NOEs, from protons of the enzyme to those of AMP, were detected,
indicating the proximity of at least three hydrophobic amino acids to bound AMP. 
These constraints, together with the conformation of AMP and the intersubstrate
distances, were used to position AMP into the X-ray structure of adenylate
kinase. The AMP binding site is found to be near (less than or equal to 4 A from)
Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been 
found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A.,
Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, 
M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F.,
Maruyama, M., Tanizawa, Y., & Nakazawa, A. (1986) J. Biol. Chem. 261, 2942-2945].

PMID: 3036205 [PubMed - indexed for MEDLINE]


716. Ophthalmic Physiol Opt. 1987;7(1):37-41.

Fixation disparity in binocular stress.

Pickwell LD, Jenkins TC, Yetka AA.

School of Studies in Optometry, University of Bradford, U.K.

Fixation disparity has been taken as a sign of stress on binocular vision because
it is established that prism stress creates fixation disparity. This paper looks 
at the effect on fixation disparity of the stress caused by requiring subjects to
read in inadequate illumination. It is found that the reduction in illumination
does not in itself immediately change the magnitude of the fixation disparity.
There is, however, an increase in the mean slope of the central part of the
fixation disparity curve which suggests that when the effect of reduced
illumination is added to prism stress, fixation disparity is increased. The
stress created by asking subjects to read in reduced illumination for half an
hour resulted in the mean associated heterophoria being increased, and over half 
the subjects reported symptoms of stress. It is concluded that fixation disparity
is changed by this type of visual stress in some subjects, and in near vision is 
increased to a more marked degree of exo-disparity. Most of this increase occurs 
in the first ten minutes.

PMID: 3658422 [PubMed - indexed for MEDLINE]


717. Verh Dtsch Ges Pathol. 1987;71:231-6.

[Exo- and endocrine relations in pancreatic diseases]

[Article in German]

Domschke W.

PMID: 3439319 [PubMed - indexed for MEDLINE]


718. Hum Reprod. 1986 Dec;1(8):533-8.

Ultrastructure of the early human implantation in vitro.

Lindenberg S, Hyttel P, Lenz S, Holmes PV.

Four hatched human blastocysts obtained after in-vitro fertilization and
development were placed on monolayer cell cultures of human endometrial
epithelium, and subsequently examined by transmission electron microscopy. All
four blastocysts became adherent to the monolayer and three implanted and
exhibited outgrowth of their trophoblastic cells. During implantation the
blastocysts differentiated into mural and polar trophoblastic cells, and
embryonic cells including endodermal cells. The endometrial cells were displaced 
and stacked into a multilayer at the periphery of the implantation sites,
allowing the trophoblastic cells to come in contact with the culture dish. The
endometrial cells displayed local exo- or endo-cytosis where they contacted the
trophoblastic cells. The trophoblastic cells were not observed to be
phagocytosing endometrial cells. These observations suggest that human
blastocysts portray an intrusive type of implantation during the initial stages.

PMID: 3818911 [PubMed - indexed for MEDLINE]


719. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8221-5.

Conjugation rescue of an exocytosis-competent membrane microdomain in Tetrahymena
thermophila mutants.

Satir BH, Reichman M, Orias E.

Conjugation-rescue experiments with two Tetrahymena thermophila mutants (exo-)
incapable of exocytosis (SB255, SB258) have been used to dissect regulatory steps
in assembly of a functional membrane microdomain, the fusion rosette. "Rescue"
refers to the recovery of a secretory activity. Exo- mutants fail to secrete
mucus normally (form capsules) when stimulated by the secretagogue alcian blue
and are blocked before the assembly of a functional fusion rosette in the cell
membrane. Two criteria are used to assay recovery of the wild-type (exo+)
phenotype: the conjugant's ability to form capsules when stimulated and the
presence of assembled rosettes, which disperse upon stimulation. Conjugation of
exo+ X SB258 results in restoration of secretion in 60% of the mutant conjugants 
and reappearance of assembled rosettes. Secretory capacity is restored in the
SB258 cell within one-half hour of firm pair formation. This restoration is not
due to new gene expression or continued protein synthesis, since it occurs when
SB258 is crossed to a "star" strain (A*), which has defective micronuclei and
therefore cannot contribute wild-type genes, and restoration occurs in the
presence of cycloheximide during conjugation. Conjugation of exo+ X SB255 reveals
a real but inefficient restoration of exocytic capacity in the exo- conjugant and
a significant decrease of exocytic capacity in the exo+ conjugant. SB255 X SB258 
crosses also show a low but significant rescue of exocytic competence, indicating
that different components of the exocytic mechanism are affected in the two
mutants. This cross leads to restoration of rosette assembly and function in one 
of the partners, presumably SB258. These results provide data about some of the
steps necessary for rosette assembly and suggest that transferable factors that
promote and/or inhibit exocytosis are present in these cells.

PMCID: PMC386899
PMID: 3464949 [PubMed - indexed for MEDLINE]


720. J Biol Chem. 1986 Oct 5;261(28):12948-55.

Isolation and characterization of a 1,4-beta-D-glucan glucohydrolase from the
yeast, Torulopsis wickerhamii.

Himmel ME, Tucker MP, Lastick SM, Oh KK, Fox JW, Spindler DD, Grohmann K.

1,4-beta-D-Glucan glucohydrolase (exo-1,4-beta-D-glucosidase) (EC 3.2.1.74) was
isolated from growth supernatants of Torulopsis wickerhamii and was subjected to 
hydrodynamic, optical (CD), and kinetic analysis after purification to
homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion
exchange chromatography, and isopycnic banding centrifugation in cesium chloride.
The last step was found to separate the enzyme from strongly associating, high
molecular weight polysaccharide. Enzyme homogeneity was established by
isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical 
high performance size exclusion chromatography using dual detection. The native
exo-1,4-beta-D-glucosidase was found to be a dimer of 151,000 +/- 21,100 daltons 
by high performance size exclusion chromatography and 143,600 +/- 1,800 daltons
by sedimentation equilibrium. The enzyme has a 12% linked carbohydrate content
(mostly mannose) and no essential metal ions. Hydrolysis of
p-nitrophenyl-beta-D-glucopyranoside was found to be optimal at pH 4.25 and 50
degrees C. The enzyme was found to produce beta-D-glucose from cellodextrins
(indicating retention of anomeric configuration during hydrolysis) and
demonstrated depolymerization from the non-reducing polymer terminus. The enzyme 
followed competitive type inhibition with p-nitrophenyl-beta-D-glucopyranoside as
substrate and demonstrated high values of Ki for D-glucose and D-cellobiose
inhibition (190 and 230 mM, respectively). The exo-1,4-beta-D-glucosidase was
found to hydrolyze cellotetraose more rapidly than D-cellobiose and
aryl-beta-D-glycosides more rapidly than all other substrates. Low levels of
activity were found for the polymeric substrates beta-glucan (yeast cell walls), 
Avicel, and Walseth cellulose. Although this enzyme demonstrates broad
disaccharide substrate specificity, a characteristic common to
beta-D-glucosidases from many sources, the ability to hydrolyze higher
cellodextrins more rapidly than cellobiose renders this enzyme the first
exo-1,4-beta-D-glucosidase purified from yeast.

PMID: 3093475 [PubMed - indexed for MEDLINE]


721. J Biol Chem. 1986 Aug 25;261(24):11254-8.

Novel fucolipids of human adenocarcinoma: monoclonal antibody specific for
trifucosyl Ley (III3FucV3FucVI2FucnLc6) and a possible three-dimensional epitope 
structure.

Kaizu T, Levery SB, Nudelman E, Stenkamp RE, Hakomori S.

Immunization of mice with Ley-active trifucosylnonaosylceramide
(III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman,
E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261,
11247-11253) followed by selection of hybridoma by positive reaction with this
antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and 
V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM 
antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which
does not cross-react with Ley-active hexaosyl- or octaosylceramides
(III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution.
The three-dimensional structure of the trifucosylnonaosylceramide was simulated
based on previously published glycosidic torsion angles for fucosyl type 2 chain 
(Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere
exo-anomeric calculations (Thogersen, H., Lemieux, R. U., Bock, K., and Meyer, B.
(1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad
nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and
acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this
hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed
structure of Leb or Ley involved in their interactions with antibodies and
lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63,
2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could
be recognized by the antibody KH1, as shown in the model illustrated in the text.
Since the axis of ceramide, which is inserted in the lipid bilayer, is
perpendicular to the plane of type 2 chain, the epitope recognized by the
antibody KH1 is located at the external nonpolar surface of the carbohydrate
chain that is overlaid on the lipid bilayer.

PMID: 2426269 [PubMed - indexed for MEDLINE]


722. J Biol Chem. 1986 Aug 15;261(23):10839-43.

Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous
glycoprotein expression by different tissues.

Stiles GL.

Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently 
labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] 
iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis/autoradiography. The proteins comprising the A1 adenosine
receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this
study, the glycoproteins representing the radiolabeled A1 adenosine
receptor-binding subunit expressed in each of these tissues (brain and fat) were 
compared through the use of peptide mapping and exo- and endoglycosidase
treatments. Peptide mapping studies with several enzymes demonstrate that the
protein component of the radiolabeled A1 adenosine receptor-binding subunit is
conserved between different tissues. Both labeled receptor peptides demonstrate a
sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain 
complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a
lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled
receptor-binding subunits with endoglycosidase F results in a single labeled
polypeptide of Mr 32,000 for both systems. These data suggest that the A1
adenosine receptor-binding subunits expressed in the rat brain and fat are
similar glycoproteins as evidenced by similar overall molecular weights,
identical peptide maps, and equivalent responses to endo- and exoglycosidase
treatment.

PMID: 3015944 [PubMed - indexed for MEDLINE]


723. Biochemistry. 1986 May 20;25(10):2829-38.

Immunochemical studies on the interaction between synthetic glycoconjugates and
alpha-L-fucosyl binding lectins.

Petryniak J, Goldstein IJ.

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta
DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta
DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and
alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither 
precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta
DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of
Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues.
These results indicate that the minimal structural requirement for glycoprotein
precipitation or cell agglutination by the lectin includes any of the three
trisaccharides (fucosylated or nonfucosylated) derived from the blood group B
tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in
the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and
N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin 
in as much as removal of any one of these sugars reduces the inhibiting potency
of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta
DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H
type 2) were precipitated to the same extent. The E. europaea lectin neither
precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA,
Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor
agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal
D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or
alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding.
Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and
hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers
[Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can.
J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl
groups act to sterically hinder lectin binding to these oligosaccharides; these
observations also suggest that the lectin binds to the beta-side of these
oligosaccharides. These sides, on both blood group H type 1 and blood group H
type 2 oligosaccharides, provide a similar contour which can fully account for
their equal reactivity with E. europaea lectin. The only difference found between
Lotus and Ulex I lectins in precipitating ability was that only Lotus
precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta
DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 3718924 [PubMed - indexed for MEDLINE]


724. J Biol Chem. 1986 Apr 15;261(11):5145-53.

Structures of glycosphingolipids isolated from human embryonal carcinoma cells.
The presence of mono- and disialosyl glycolipids with blood group type 1
sequence.

Fukuda MN, Bothner B, Lloyd KO, Rettig WJ, Tiller PR, Dell A.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were
elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and 
exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide,
sialosylgangliotriaosylceramide, sialylated and nonsialylated
lacto-N-neotetraosylceramide, and the following glycolipids with a blood group
type 1 sequence: (formula; see text) The two former glycolipids,
lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with
monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present 
in many of the human embryonal carcinoma cells but not in a variety of other cell
lines, suggesting that sialylated but not fucosylated blood group type 1
sequences are characteristic markers for human embryonal carcinoma cells and
malignant teratocarcinomas.

PMID: 3514610 [PubMed - indexed for MEDLINE]


725. Virology. 1986 Apr 15;150(1):33-44.

Host and phage-coded functions required for coliphage N4 DNA replication.

Guinta D, Stambouly J, Falco SC, Rist JK, Rothman-Denes LB.

Escherichia coli strains containing mutations in various deoxyribonucleic acid
synthesis cistrons have been tested for their ability to support bacteriophage N4
growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of
the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep
genes. In contrast, N4 DNA replication requires the products of the dnaF,
(ribonucleotide reductase) and lig (DNA ligase) genes of E. coli. N4 DNA
replication, specifically processing of short DNA fragments requires the 5'-3'
exonuclease activity of the polA gene product. However, its DNA polymerizing
activity is not required. In addition, the sensitivity of N4 DNA synthesis to
inhibitors or temperature-sensitive mutants of E. coli DNA gyrase suggests that
this activity is required for N4 DNA synthesis. To date, we have found five N4
gene products required for N4 DNA replication: dbp (a single-stranded DNA binding
protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4
virion-associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease).

PMID: 3006344 [PubMed - indexed for MEDLINE]


726. Arkh Anat Gistol Embriol. 1986 Apr;90(4):48-54.

["Mixed" endocrine gland cells of the duodenal epithelium in various vertebrate
animals and man]

[Article in Russian]

Puzyrev AA, Ivanova VF.

Epithelium of the duodenal mucous membrane has been studied in frog, rat and man 
by means of electron microscopy. Together with typical endocrinocytes, incretory 
cells of "mixed" type are revealed in the epithelium. They are classified into
two types: 1. exo-endocrinic containing in their cytoplasm simultaneously: a)
mucous and EC-granules, b) mucous and S-granules, c) granules specific for
Paneth's cells and S-granules, d) granules specific for Paneth's cells and
L-granules; 2. endocrinocytes with incretory granules of various types: with D
and I, with D and EC, with D and L granules. Various ultramicroscopic
organization of the "mixed" exo-endocrinic cells is demonstrated. It can be
considered as various stages of their differentiation. In cytoplasm of the
"mixed" cells of the second type (endocrinic) a constant presence of D-granules
is revealed, that is, evidently, characteristic, within the limits of the
gastro-entero-pancreatic system, for the cells with two types of the endocrinic
granules.

PMID: 3718252 [PubMed - indexed for MEDLINE]


727. Neurochem Pathol. 1986 Apr;4(2):107-17.

Presence of glycoproteins containing the polylactosamine structure in brain and
liver of GM1 gangliosidosis patients. Comparative study between clinical types I 
and II, using endo-beta-galactosidase enzyme.

Berra B, De Gasperi R, Rapelli S, Okada S, Li SC, Li YT.

The material derived from defective degradation of glycoproteins, which
accumulates in brain and liver of a patient with GM1 gangliosidosis type I, was
investigated, and the structure of the main storage compounds determined. For
comparison, brain and liver of a patient with GM1 gangliosidosis type II were
also analyzed. Analysis of the glycopeptides obtained after pronase digestion of 
the defatted residue indicates the storage of glycoprotein-like material in type 
I, but not in type II. Treatment with endo-beta-galactosidase showed that the
stored material contained N-acetyllactosamine repeating units. Two major
oligosaccharides, OS I and OS II, were isolated after the enzyme treatment, whose
structures are: GlcNAc beta 1----3 Gal (OS I) and Gal beta l----4GlcNAc beta
1----3 Gal (OS II). Treatment with exo-beta-galactosidase transformed the
trisaccharide OS II into the disaccharide OS I, indicating that the deficiency of
beta-galactosidase in GM1 gangliosidosis type I, but not in type II, also affects
glycoprotein catabolism, leading to the accumulation of glycopeptides containing 
terminal beta-galactosyl residues and N-acetyllactosamine repeating units. These 
results indicate the severe impairment in the catabolism of glycoconjugates with 
beta-linked galactose in type I, although this impairment is not as pronounced in
type II.

PMID: 3088498 [PubMed - indexed for MEDLINE]


728. Biochemistry. 1986 Mar 11;25(5):1159-65.

Hydration of cellobial by exo- and endo-type cellulases: evidence for catalytic
flexibility of glycosylases.

Kanda T, Brewer CF, Okada G, Hehre EJ.

New insight has been obtained into the catalytic capabilities of cellulase.
Essentially homogeneous preparations of exo- (or Avicelase-) type and endo- (or
CMCase-) type cellulases from Irpex lacteus and Aspergillus niger, respectively, 
were shown to hydrate the enolic bond of cellobial to form 2-deoxycellobiose. The
A. niger enzyme also synthesized a small amount of a 2-deoxycellobiosyl-transfer 
product from cellobial. By use of digests conducted in deuterated buffer and 1H
NMR spectra for product analysis, both cellulases were found to protonate
(deuterate) the double bond of cellobial from below the si face of the D-glucal
moiety, i.e., from a direction opposite that assumed for protonation of the
beta-D-glycosidic linkages of cellulose and cellodextrins. The exo enzyme, which 
hydrolyzes the latter substrates primarily to cellobiose, rapidly catalyzed
cellobial hydration to produce the beta-anomer of
beta-D-glucopyranosyl(1----4)-2-deoxy-D-glucose-2(e)-d. The A. niger cellulase
produced the same 2-deoxycellobiose-d from cellobial, though too slowly for its
configuration to be determined. However, evidence was obtained for the formation 
of a beta-2-deoxycellobiosyl-d-D-glucose-transfer product by the enzyme. Thus, it
is likely that all of the observed reactions with cellobial represent trans
additions at the double bond. In any case, the anomeric configuration of products
is created de novo. Separate mechanisms are described for the reaction of
cellobial hydration and for the stereochemically different reaction of cellulose 
hydrolysis catalyzed by the present enzymes, assuming an arrangement of their
catalytic groups analogous to that found in lysozyme.(ABSTRACT TRUNCATED AT 250
WORDS)

PMID: 3964662 [PubMed - indexed for MEDLINE]


729. Gene. 1986;47(2-3):245-59.

Cloning of genes related to exo-beta-glucanase production in Saccharomyces
cerevisiae: characterization of an exo-beta-glucanase structural gene.

Nebreda AR, Villa TG, Villanueva JR, del Rey F.

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by
complementation of a mutant strain (exg1-2) with highly reduced extracellular
exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an
overlapping region of 5.2 kb were isolated from a genomic DNA library and
characterized by restriction mapping. The coding region was located by subcloning
the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg-
mutants transformed with yeast multicopy plasmids containing this DNA fragment
showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+
wild-type (wt) strains. The overproduced EXG had the same enzymic activity on
different substrates, and showed the same electrophoretic behaviour on
polyacrylamide gels and identical properties upon filtration through Sephacryl
S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed
Schizosaccharomyces pombe, yielding extracellular EXG activity which showed
cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only
a part of the EXG1 region was subcloned into the integrating vector YIp5, and the
resulting plasmid was used to transform an Exg+ strain. Genetic and Southern
analysis of several stable Exg- transformants showed that the fragment integrated
by homology with the EXG1 locus. The chromosomal DNA fragment into which the
plasmid integrated has a restriction pattern identical to that of the fragment on
which we had previously identified the putative EXG1 gene. Only one copy of the
EXG1 gene per genome was found in several strains tested by Southern analysis.
Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of 
about 4.1 kb, which partially complements the exg1-2 mutation but which shows no 
homology with the 2.7-kb fragment containing the EXG1 gene, were also identified 
in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic 
suppressor and could be related in some way to EXG production in S. cerevisiae.

PMID: 3104142 [PubMed - indexed for MEDLINE]


730. Gene. 1986;50(1-3):69-85.

The production of generalized transducing phage by bacteriophage lambda.

Sternberg N.

Generalized transduction has for about 30 years been a major tool in the genetic 
manipulation of bacterial chromosomes. However, throughout that time little
progress has been made in understanding how generalized transducing particles are
produced. The experiments presented in this paper use phage lambda to assess some
of the factors that affect that process. The results of those experiments
indicate: the production of generalized transducing particles by bacteriophage
lambda is inhibited by the phage lambda exonuclease (Exo). Also inhibited by
lambda Exo is the production of lambda docR particles, a class of particles whose
packaging is initiated in bacterial DNA and terminated at the normal phage
packaging site, cos. In contrast, the production of lambda docL particles, a
class of particles whose packaging is initiated at cos and terminated in
bacterial DNA, is unaffected by lambda Exo; lambda-generalized transducing
particles are not detected in induced lysis-defective (S-) lambda lysogens until 
about 60-90 min after prophage induction. Since wild-type lambda would normally
lyse cells by 60 min, the production of lambda-generalized transducing particles 
depends on the phage being lysis-defective; if transducing lysates are prepared
by phage infection then the frequency of generalized transduction for different
bacterial markers varies over a 10-20-fold range. In contrast, if transducing
lysates are prepared by the induction of a lambda lysogen containing an
excision-defective prophage, then the variation in transduction frequency is much
greater, and markers adjacent to, and on both sides of, the prophage are
transduced with much higher frequencies than are other markers; if the prophage
is replication-defective then the increased transduction of prophage-proximal
markers is eliminated; measurements of total DNA in induced lysogens indicate
that part of the increase in transduction frequency following prophage induction 
can be accounted for by an increase in the amount of prophage-proximal bacterial 
DNA in the cell. Measurements of DNA in transducing particles indicate that the
rest of the increase is probably due to the preferential packaging of the
prophage-proximal bacterial DNA. These results are most easily interpreted in
terms of a model for the initiation of bacterial DNA packaging by lambda, in
which the proteins involved (Ter) do not recognize any particular sequence in
bacterial DNA but rather recognize some feature of the DNA tht is sensitive to
lambda exonuclease, such as a nick or a double-stranded cut.(ABSTRACT TRUNCATED
AT 400 WORDS)

PMID: 3034738 [PubMed - indexed for MEDLINE]


731. Histol Histopathol. 1986 Jan;1(1):33-42.

Close association of centroacinar/ductular and insular cells in the rat pancreas.

Leeson TS, Leeson R.

Department of Anatomy, University of Alberta, Canada.

Close contacts between endocrine insular cells and exocrine acinar, centroacinar 
and ductular cells occur frequently in the rat pancreas as seen by both light and
electron microscopy. Islets of Langerhans are surrounded incompletely by a thin
connective tissue capsule or mantle but numerous exocrine-endocrine cell contacts
occur at the periphery, which is irregular with considerable "intermingling" of
the two cell types. Centroacinar and ductular cells are seen to be in contact
with all endocrine cell types but most commonly insulin-secreting B-cells. The
basal surface of centroacinar cells in the region of contact may be extensive,
sometimes with overlap of basal processes of these cells and their lateral
extension between acinar and insular cells. The areas of contact contain no
connective tissue or basal lamina and show no surface specializations. The
presence of both the "open" and "closed" type of enteroendocrine cells within
acini is confirmed, some also being in contact with centroacinar cells. The
functional significance of these exo-endocrine cell contacts is discussed in
terms of the endocrine-acinar portal system, possible direct paracrine secretion,
compartmentalization within the islet, and the known effects of islet hormones on
exocrine secretion. Also relevant is the developmental origin of islets from
ductal tissue and the cellular origin of some tumours, e.g., insulinomas, from
duct cells.

PMID: 2980100 [PubMed - indexed for MEDLINE]


732. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6231-5.

Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective
nodules.

Leigh JA, Signer ER, Walker GC.

By screening with the fluorescent stain Calcofluor, we have isolated 26
independent transposon Tn5 insertion mutants of Rhizobium meliloti that are
deficient in the production of a known extracellular polysaccharide (Exo-). The
mutants belonged to six distinct genetic groups based on the ability of their
Exo- phenotype to be complemented by different recombinant plasmids from a R.
meliloti clone bank. With few exceptions, all of the mutants formed ineffective
(non-nitrogen-fixing) nodules on alfalfa. For all but one group, the
complementing plasmids restored effective nodulation. These results establish a
firm and extensive correlation between the ability of Rhizobium to produce a
particular polysaccharide and symbiotic proficiency. The ineffective nodules
appeared to contain no bacteroids and to form without shepherds' crooks or
infection threads; this symbiotic phenotype matches that described for a set of
independently isolated mutants that belong phenotypically and genetically to the 
group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 
40, 869-877]. Apparently the exopolysaccharide, although not required for nodule 
formation, is involved in wild-type nodule invasion.

PMCID: PMC391026
PMID: 3862129 [PubMed - indexed for MEDLINE]


733. J Cell Biol. 1985 Sep;101(3):1144-52.

Characterization of the purified Chlamydomonas minus agglutinin.

Collin-Osdoby P, Adair WS.

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of
opposite mating-type (plus and minus) gametes during the first stages of mating. 
Purification and partial characterization of the plus agglutinin was previously
reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell,
33:183-193). Here we characterize the purified minus molecule. We show it to be a
high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3%
stacking region of an SDS-polyacrylamide gel and is absent from two
nonagglutinating minus mutants. Plus and minus agglutinins are remarkably
similar, although nonidentical, in amino acid composition, molecular morphology, 
and reactivity in vivo and in vitro with monoclonal antibodies raised against the
plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins,
when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate,
alkaline borohydride, reducing agents, or heat, but unaffected by exo- or
endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus 
molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes 
earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography,
and is sensitive to chymotrypsin digestion (unlike the plus agglutinin);
therefore, it differs from the plus agglutinin in apparent molecular weight, net 
charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our
results generally demonstrate a high degree of homology between these
complementary cell-cell recognition/adhesion molecules, which suggests that they 
are specified by genes that have a common evolutionary origin.

PMCID: PMC2113696
PMID: 2411736 [PubMed - indexed for MEDLINE]


734. Nucleic Acids Res. 1985 Aug 26;13(16):5949-63.

UV irradiation of nucleic acids: formation, purification and solution
conformational analysis of the '6-4 lesion' of dTpdT.

Rycyna RE, Alderfer JL.

Irradiation of dTpdT with 300 kJ/m2 of 254 nm produces numerous photo-products,
one of which labeled dT6pd4T[1] was purified by HPLC. dT6pd4T has a UV spectrum
(H20, pH 7) with lambda max = 326 nm and lambda min = 265 nm, and a P-31 NMR
resonance at -3.46 ppm (normal dTpdT occurs at -4.01 ppm; TMP, 30 degrees C). 2-D
COSY NMR spectra facilitated proton resonance assignments and 2-D NOESY spectra
aided analysis of spatial orientation. Carbon-13 and proton-coupled P-31 NMR
spectra of dT6pd4T were also obtained. These analyses indicate: C5=C6 of dT6p- is
saturated and the -pd4T base is more aromatic; the dT6p- base possesses a
configuration of 5R, 6S; dT6p- and -pd4T have anti-type glycosidic conformations;
furanose conformation of dT6p- is mainly C3'-endo and that of -pd4T exists in a
C3'-endo in equilibrium C3'-exo; exocyclic bonds gamma (C5'-C4'), beta (05'-C5') 
and epsilon (C3'-03') are non-classical rotamers; dihedral angle about epsilon
(C3'-03') is smaller relative to dTpdT.

PMCID: PMC321925
PMID: 4034399 [PubMed - indexed for MEDLINE]


735. Jpn J Pharmacol. 1985 Aug;38(4):403-9.

Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of
rat brain: influence of exo- and endo-glycosidases and glycopeptidase.

Tsuchihashi H, Nagatomo T.

The significance of carbohydrate moieties containing the beta-adrenoceptor
molecule in the rat brain was examined using radioligand binding assay methods.
Thus, this experiment was designed to assess the effects of exoglycosidase
(alpha-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and
endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor.
The main reason why five kinds of enzymes were used in the present study is that 
they can hydrolyze different carbohydrate molecules from cell membranes. Rat
brain was used and beta-adrenoceptor binding assay was carried out using
3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors
was sensitive to very low concentration of endoglycosidase H and glycopeptidase
A, thus indicating that the treatments with these enzymes of rat brain membrane
appear to decrease the number of beta-receptor binding sites. On the other hand, 
the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the 
membrane induced lower values of the dissociation constant (Kd) than those of the
control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of
the removal of hexose contents and total carbohydrate contents with these
treatments, respectively. These results imply that complex type N-linked acidic
carbohydrate chains containing neuraminic acid and high mannose type N-linked
carbohydrate chains, which are hydrolyzed with endoglycosidase H and
glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor
molecules play a crucial role in the drug-receptor interaction.

PMID: 2999487 [PubMed - indexed for MEDLINE]


736. Biochem J. 1985 Jul 15;229(2):441-51.

Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell
glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo
fibroblasts.

Hunt LA, Wright SE.

In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo
fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma
virus and from untransformed fibroblasts infected with a transformation-defective
derivative of Prague C Rous-sarcoma virus, we have detected
transformation-dependent alterations in both the acidic-type and the neutral-type
asparagine-linked oligosaccharides of cellular glycoproteins. Pronase-digested
glycopeptides were analysed by the combined techniques of gel filtration, exo-
and endo-glycosidase digestion and concanavalin A-agarose affinity
chromatography. The transformed cell glycoproteins contained more sialic acid and
were enriched for more highly branched (versus biantennary) acidic-type
structures compared with the untransformed cell glycoproteins, similarly to
previously reported transformation-dependent alterations. In addition, the
glycopeptides from the virus-transformed cells contained several neutral-type
structures that were apparently absent from the untransformed cells: small
neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to
endo-beta-N-acetylglucosaminidase D but resistant to
endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of
'truncated' precursor oligosaccharides (endoglycosidase-resistant,
alpha-mannosidase-sensitive). Endoglycosidase-released oligosaccharides with the 
properties of hybrid-type structures were derived from the glycoproteins of both 
transformed and untransformed cells.

PMCID: PMC1145076
PMID: 2994635 [PubMed - indexed for MEDLINE]


737. J Med Chem. 1985 Apr;28(4):461-6.

Analgesics of the 6,14-ethenomorphinan type. 6-deoxy-7 alpha-orvinols and
6-deoxy-8 alpha-orvinols.

Knipmeyer LL, Rapoport H.

6-Deoxythebaine (3) has been prepared as a precursor to C-6 alkyl substituted
orvinols 15 and 17. The C-6 methyl group was introduced by addition of
methyllithium to codeinone. Transformation of 6-methylcodeine to its 6-methyl
ether and 1,4-elimination of methanol with potassium tert-butoxide in Me2SO then 
gave 6-deoxythebaine (3) in 49% overall yield. Diels-Alder addition of methyl
vinyl ketone to this diene yielded four ketones: three regio- and stereoisomeric 
6,14-endo-ethenomorphinans and one exo adduct. The major ketone isomer provided
the set of C-19 diastereomeric orvinols 15 in which the pendant carbon has the 7 
alpha configuration. Regioisomeric ketone 8, in which the acetyl group is at C-8,
was formed in 3% yield and was similarly converted to the corresponding orvinols 
17. Orvinol (R)-15 (R at C-19) is an analgesic of very high potency, 2200 times
that of morphine; regioisomeric orvinols 17, in which the pendant tertiary
alcohols are on C-8, are much less potent. The higher activity of the C-6 methyl 
and methoxyl analogues (R)-15 and (R)-22 relative to hydrogen-substituted (R)-19 
indicates that C-6 alkyl substitution enhances analgesic potency.

PMID: 3981537 [PubMed - indexed for MEDLINE]


738. Eur J Biochem. 1985 Jan 15;146(2):301-8.

The cellulase of Trichoderma viride. Purification, characterization and
comparison of all detectable endoglucanases, exoglucanases and beta-glucosidases.

Beldman G, Searle-Van Leeuwen MF, Rombouts FM, Voragen FG.

Six endoglucanases (Endo I; II; III; IV; V; VI), three exoglucanases (Exo I; II; 
III) and a beta-glucosidase (beta-gluc I) were isolated from a commercial
cellulase preparation derived from Trichoderma viride, using gel filtration on
Bio-Gel, anion exchange on DEAE-Bio-Gel A, cation exchange on SE-Sephadex and
affinity chromatography on crystalline cellulose. Molecular masses were
determined by polyacrylamide gel electrophoresis. One group of endoglucanases
(Endo I, Endo II and Endo IV) with Mr of 50 000, 45 000 and 23 500 were more
random in their attack on carboxymethylcellulose than another group (Endo III,
Endo V and Endo VI) showing Mr of 58 000, 57 000 and 53 000 respectively. Endo
III was identified as a new type of endoglucanase with relatively high activity
on crystalline cellulose and moderate activity on carboxymethylcellulose. Exo II 
and Exo III with Mr of 60 500 and 62 000 respectively showed distinct adsorption 
affinities on a column of crystalline cellulose and could be eluted by a pH
gradient to alkaline regions. These enzymes were cellobiohydrolases as judged by 
high-pressure liquid chromatography of the products obtained from incubation with
H3PO4-swollen cellulose. It was concluded that these exoglucanases are primarily 
active on newly generated chain ends. Exo I was essentially another type of
exoglucanase which in the first instance was able to split off a cellobiose
molecule from a chain end and then hydrolyse this molecule in a second step to
two glucose units beta-Gluc I was a new type of aryl-beta-D-glucosidase which had
no activity on cellobiose. The enzyme had a Mr of 76 000 and was moderately
active on CM-cellulose, crystalline cellulose and xylan and highly active on
p-nitrophenyl-beta-D-glucose and p-nitrophenyl-beta-D-xylose.

PMID: 3917923 [PubMed - indexed for MEDLINE]


739. Biochim Biophys Acta. 1984 Nov 28;802(2):188-96.

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a 
Waldenstrom's macroglobulinemia immunoglobulin M.

Cahour A, Debeire P, Parente JP, Hartmann L, Montreuil J.

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ 
has been studied on oligosaccharides released by hydrazinolysis from the purified
glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains 
were separated by affinity chromatography on concanavalin A-Sepharose into
oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic
type were further fractionated by HPLC and those of the N-acetyllactosamine type 
by preparative high-voltage electrophoresis. The primary structure of the main
oligosaccharides was investigated on the basis of micro-methylation analysis,
mass spectrometry and sequential exo-glycosidase digestion. Glycans of the
oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2.
N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary 
and triantennary types. They presented a higher degree of heterogeneity due to
the presence of a variable number of NeuAc and fucose residues. The new
structures we report here were in addition to the major biantennary one we
previously described on the basis of methylation analysis and 500 MHz 1H-NMR
spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, 
H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha
2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta
1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.

PMID: 6437450 [PubMed - indexed for MEDLINE]


740. Hoppe Seylers Z Physiol Chem. 1984 Nov;365(11):1309-21.

[H-NMR spectroscopy. Specificity of microbial sialidases against complex
substrates]

[Article in German]

Friebolin H, Baumann W, Hauck M, Kurz D, Wajda R, Weisshaar G, Keilich G, Ziegler
D, Brossmer L, von Nicolai H.

The specificities of one viral and five bacterial sialidases were investigated by
1H-NMR-spectroscopy with substrates or substrate mixtures containing two sialic
acid residues of different linkage types. This technique allows - in contrast to 
the methods used before - the simultaneous determination of the rates of
hydrolysis of both NeuAc linkages in a single experiment. The substrate
specificities of the enzymes are discussed on the basis of the relation of the
rate constants k/k'. The data obtained are more exact and more informative than
those of separate experiments as reported previously. Among the enzymes
investigated, i.e. sialidases of fowl plague virus (FPV = VKH), Clostridium
perfringens (CP), Vibrio cholerae (VC), Bifidobacterium bifidum var.
pennsylvanicum (BBif), Bifidobacterium lactentis (BLac), and Arthrobacter
ureafaciens (AU), the activity of the viral sialidase VKH shows the highest, the 
activities of the Bifidobacterium sialidases the lowest dependence on the nature 
and on the linkage type of the different substrates. All sialidases
preferentially cleave the NeuAc alpha 2-3-Gal linkage with the exception of the
enzyme of Arthrobacter ureafaciens (AU) which shows a higher affinity to alpha
2-6 linkages. However, this does not apply to the side-arm-linked NeuAc alpha 2-6
structure in NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6)-GlcNAc beta 1-3Gal
beta 1-4Glc (Substrate B). This substrate in generally cleaved very slowly and is
hardly affected by the viral enzyme. After the alpha 2-3 linkage, the alpha 2-8
bond in NeuAc alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc(Substrate A) is most
susceptible for the sialidases VKH, CP and VC. An elongation of the carbohydrate 
chain (Substrate D) is accompanied by a reduction of the rate of cleavage for all
enzymes. The experiments with alpha 1-acid glycoprotein, fetuin, and with the
glycopeptides obtained by proteolytic degradation of the latter, revealed the
same specificity towards the alpha 2-3 and the alpha 2-6 linkages as the
oligosaccharides. Influenced by the chemical nature and the size of the
substrate, NeuAc is released from the native alpha 1-acid glycoprotein more
quickly than from the corresponding glycopeptide. All sialidases investigated so 
far are strictly exo-enzymes as could be demonstrated by the cleavage of NeuAc
alpha 2-8 NeuAc alpha 2-3 Gal beta 1-4Glc (Substrate A).

PMID: 6096253 [PubMed - indexed for MEDLINE]


741. Planta Med. 1984 Oct;50(5):438-440.

Serrulatoloside, a New Iridoid Glucoside from Penstemon serrulatus.

Junior P.

Institut fur Pharmazeutische Biologie der Philipps-Universitat Marburg,
Deutsch-hausstra?e 17S, D-3550 Marburg/L.

From leaves of PENSTEMON SERRULATUS Menz. a new iridoid glucoside of unusual type
in having an exo double bond at C-8 on the cyclopentane-ring has been isolated.
The structure of serrulatoloside has been established by spectroscopic methods.

PMID: 17340346 [PubMed - as supplied by publisher]


742. J Biomol Struct Dyn. 1984 Oct;2(2):333-44.

Structure of a novel drug-nucleic acid crystalline complex: 1,
10-phenanthroline-platinum (II) ethylenediamine--5'-phosphoryl-thymidylyl(3'-5') 
deoxyadenosine.

Vijay-Kumar S, Sakore TD, Sobell HM.

Department of Radiation Biology and Biophysics, University of Rochester School of
Medicine and Dentistry, New York 14642.

1,10-Phenanthroline-platinum (II) ethylenediamine (PEPt) forms a 1:2 crystalline 
complex with 5'-phosphorylthymidylyl (3'-5') deoxyadeno sine (d-pTpA). Crystals
are monoclinic, P2, with a = 10.204 A, b = 24.743 A, c = 21.064 A, Beta = 94.6
degrees. The structure has been determined by Patterson and Fourier methods, and 
refined by least squares to a residual of 0.128 on 2,367 observed reflections.
PEPt molecules form sandwich-like stacks with adenine-thymine hydrogen-bonded
pairs along the alpha axis. Intercalation in the classic sense is not observed in
this structure. Instead, d-pTpA molecules form an open chain structure in which
adenine-thymine residues hydrogenbond together with the reversed Hoogsteen type
base-pairing configuration. Deoxyadenosine residues exist in the syn conformation
and are C3' endo and C1' exo. Thymidine residues are in the high anti
conformation with C2' endo puckers. The structure is heavily hydrated, forming a 
channel-like water network along the alpha axis. Other features of the structure 
are described.

PMID: 6400939 [PubMed - indexed for MEDLINE]


743. Eur J Biochem. 1984 Aug 15;143(1):133-44.

Structural changes of carbohydrate chains of human thyroglobulin accompanying
malignant transformations of thyroid glands.

Yamamoto K, Tsuji T, Tarutani O, Osawa T.

Carbohydrate moieties were released from human thyroglobulins prepared from
normal and transformed, thyroid tissues by hydrazinolysis. The oligosaccharides
thus prepared were fractionated by DEAE-cellulose, Bio-Gel P-4,
concanavalin-A--Sepharose and Ricinus-communis-agglutinin--Sepharose columns and 
were characterized by exo-glycosidase and endo-glycosidase digestions,
methylation analysis and 400-MHz 1H-NMR spectroscopy. These studies showed that
the alterations accompanying malignancy occurred in complex-type carbohydrate
chains and exhibited the following structural features: (a) a complex-type
carbohydrate chain containing 1 phosphate in a diester linkage was present in all
the malignant thyroglobulins. (b) Asialo-carbohydrate chains with
biosynthetically incomplete structures were found in all of the malignant
thyroglobulins. (c) Carbohydrate chains of higher molecular mass, which have
repeating Gal-GlcNAc disaccharides and peripheral alpha-fucosyl residues were
present in metastatic papillary carcinoma.

PMID: 6468384 [PubMed - indexed for MEDLINE]


744. J Biol Chem. 1984 Jul 10;259(13):8655-63.

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing
high mannose or complex type carbohydrate chains.

Stiles GL, Benovic JL, Caron MG, Lefkowitz RJ.

Mammalian beta-adrenergic receptor binding peptides can be visualized by
covalently labeling them with the photoaffinity reagent
p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor
peptides migrate as broad bands of Mr approximately equal to 62,000. In the
present study, we examined the carbohydrate composition of the mammalian beta
receptor through the use of specific exo- and endoglycosidases and lectin
affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled
beta2-adrenergic receptors from hamster lung or rat erythrocyte with the
exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the
existence of both high mannose and complex type carbohydrate chains on beta
2-adrenergic receptors. The nonadditivity of the effect of sequential treatments 
with these enzymes suggested discrete populations of beta-adrenergic receptors
containing either complex or high mannose type chains. Deglycosylation of
receptor with endoglycosidase F results in a single labeled polypeptide at Mr =
49,000 for both systems. The same two populations of the beta receptors (high
mannose or complex type chain) could also be fractionated by lectin affinity
chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled
receptors. The high mannose-containing receptors could be absorbed to and
specifically eluted from concanavalin A-agarose. Those containing complex type
carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose.
Taken together, these data suggest that mammalian beta-adrenergic receptors
contain both complex and high mannose type carbohydrate chains and that
microheterogeneity of these chains likely explains the broad band pattern
typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

PMID: 6330118 [PubMed - indexed for MEDLINE]


745. Arch Biochem Biophys. 1984 May 1;230(2):668-75.

Studies on the mechanism of castanospermine inhibition of alpha- and
beta-glucosidases.

Saul R, Molyneux RJ, Elbein AD.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine
alkaloid that was isolated from the Australian plant, Castanospermum australe.
This alkaloid was found to be a potent inhibitor of lysosomal alpha- and
beta-glucosidases. In this report, the mechanism of inhibition of
amyloglucosidase (an exo-1,4-alpha-glucosidase) and almond emulsin
beta-glucosidase was examined. Castanospermine proved to be a competitive
inhibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the
p-nitrophenyl-alpha-D-glucoside. It was also a competitive inhibitor of almond
emulsin beta-glucosidase at pH 6.5, but in this case previous studies had shown
that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation
mixture had a marked effect on the inhibition. Thus, in all cases,
castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower
pH values. The pK for castanospermine was found to be 6.09, indicating that the
alkaloid was probably more active in the unprotonated form. This was also
suggested by the fact that the N-oxide of castanospermine, while still a
competitive inhibitor, was 50 to 100 times less active than was castanospermine, 
and its activity was not markedly altered by pH. These results probably explain
why castanospermine is a good inhibitor of the glycoprotein processing enzyme,
glucosidase I, since this is a neutral enzyme.

PMID: 6424575 [PubMed - indexed for MEDLINE]


746. Pharmacol Res Commun. 1984 Mar;16(3):281-94.

The effects of stereoisomers of 2-amino-6(7)- and
9-amino-6-trifluoromethylbenzonorbornenes on food intake, brain serotonin
concentration, and monoamine oxidase activity.

Gray NM, Lu MC, Bhargava HN.

To evaluate the importance of the conformation of fenfluramine in eliciting its
various central nervous system effects, the isomers of 2-amino-6(7)- and
9-amino-6-trifluoromethylbenzonorbornene were employed as conformationally
defined analogs of norfenfluramine. In this series of isomeric amines, the exo-2 
and anti-9 isomers resemble the fully extended conformation of fenfluramine,
whereas the endo-2 and syn-9 isomers resemble the folded conformation. The exo-2 
and anti-9 isomers were equi-effective in reducing food intake in the rat, but
were approximately seven times less potent than fenfluramine. The endo-2 and
syn-9 isomers had no effect on food intake up to a dose of 40 mg/kg. All of the
isomers were as effective as amphetamine in inhibiting brain monamine oxidase
type B. These isomers also inhibited monoamine oxidase type A to the same extent 
as type B, but were significantly less potent than amphetamine in inhibiting this
form of the enzyme. The effects at anorectic doses on brain serotonin (5-HT)
concentration were also studied. Although fenfluramine decreased brain 5-HT
concentration, the exo-2, syn-9 and anti-9 isomers had no significant effect. The
endo-2 isomers caused an 11% decrease in 5-HT concentration, but this effect was 
observed at higher doses of the compound. The data suggest that the fully
extended conformation of fenfluramine is preferred over the folded conformation
for eliciting its anorectic activity. However, no conclusion can be made for the 
conformational requirements for the other biological responses investigated in
this study.

PMID: 6718462 [PubMed - indexed for MEDLINE]


747. J Mol Biol. 1984 Feb 5;172(4):559-72.

An unusual conformation of NAD+ bound to sorbitol dehydrogenase? A time-dependent
transferred nuclear Overhauser effect study.

Gronenborn AM, Clore GM, Jeffery J.

The conformation of NAD+ in the sheep liver sorbitol dehydrogenase-NAD+ binary
complex has been investigated using time-dependent proton-proton transferred
nuclear Overhauser enhancement measurements to determine interproton distance
ratios and distances between bound NAD+ protons. The conformation about both the 
adenosine and nicotinamide riboside glycosidic bonds is anti, the conformations
of the adenosine and nicotinamide ribose rings are C3'-endo and C1'-exo,
respectively, and the conformations about the adenosine and nicotinamide riboside
C4'-C5' bonds are g+ and t, respectively, similar to those found in complexes of 
NAD+ with other A type dehydrogenases. In addition, however, the distance data
are indicative of an unusual overall conformation of NAD+ in the sorbitol
dehydrogenase-NAD+ binary complex, with the planes of the nicotinamide and
adenine rings separated by 6 to 8 A and at approximately 120 degrees to each
other. This overall conformation differs from the concensus extended conformation
found in the NAD+-dehydrogenase complexes crystallized to date, where the planes 
of the nicotinamide and adenine rings are 12 to 14 A apart and nearly
perpendicular to each other.

PMID: 6319720 [PubMed - indexed for MEDLINE]


748. Eur J Biochem. 1983 Dec 1;137(1-2):149-54.

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities.

Morrice LM, McLean MW, Long WF, Williamson FB.

Beta-Agarase I and II were characterised by their action on agar-type
polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was
specific for regions containing a minimum of one unsubstituted neoagarobiose unit
[3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at
the reducing side of this moiety. Yaphe demonstrated that agar was degraded by
this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a
predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993].
Beta-Agarase I slowly degraded neoagarohexaose but not the homologous
tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and
[1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to
methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were
invariably terminated at the reducing end by unsubstituted neoagarobiose. The
novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary
evidence indicated this enzyme was specific for sequences containing
neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to
neoagaro-oligosaccharides of which the disaccharide was limiting and predominant.
Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to 
the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the
alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol.
Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol.
Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24%
of the starting carbohydrate. This fraction was almost exclusively disaccharides 
(22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%).
Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by
Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. 
and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].

PMID: 6653550 [PubMed - indexed for MEDLINE]


749. Int J Pept Protein Res. 1983 Oct;22(4):410-21.

Cyclols, cyclodepsipeptides, and N-acyl-diketopiperazines from linear precursors.
Synthesis and crystal structure of 10-membered cyclodepsipeptides.

Zanotti G, Pinnen F, Lucente G, Cerrini S, Gavuzzo E, Mazza F.

In order to investigate the relative formation tendency of different tautomeric
ring systems (cyclols, cyclodepsipeptides and corresponding
N-acyl-diketopiperazines), two linear peptide precursors containing proline as
C-terminal residue, have been synthesized and subjected to cyclizing conditions. 
Boc-Ser-Phe-Pro-ONp (I) gave three isomeric cyclic compounds: Boc-Ser-Phe-Pro-
(IV), N-(Boc-Ser)-cyclo-(Phe-D-Pro) (III) and the corresponding aza-cyclol (II). 
Starting from Hyb-Phe-Pro-ONp (V) two epimeric
N(3-hydroxybutyryl)diketopiperazines (VI) and (VII) and the corresponding
10-membered cyclodepsipeptide (VIII) could be isolated. Crystal and molecular
structure of VIII is reported. Crystals of VIII are orthorhombic, P212121 with a 
= 9.684, b = 22.985, c = 7.841, z = 4. The two peptidic bonds are cis with omega 
values of 6.6 degrees and - 18.1 degrees, whereas the lactonic bond is of
transoid type. The pyrrolidine ring has C2-C gamma-exo conformation.

PMID: 6654588 [PubMed - indexed for MEDLINE]


750. Nucleic Acids Res. 1983 Sep 24;11(18):6475-86.

A novel guanine-guanine base pairing: crystal structure of a complex between
7-methylguanosine and its iodide.

Yamagata Y, Fukumoto S, Hamada K, Fujiwara T, Tomita K.

7-Methylguanosine, one of the biologically important minor nucleosides, could be 
crystallized as a complex of its zwitterionic form and its iodide, and the
crystal structure was determined by the X-ray diffraction method. The crystals
belong to the triclinic space group P1 with the unit cell dimensions: a =
7.678(1), b = 18.094(3), and c = 5.711(1) A, alpha = 79.32(1), beta = 80.14(1)
and gamma = 76.90(1) degrees. The structure was solved by the heavy atom method
and refined by the least-squares method to give a final R index of 0.075. The
novel reverse Watson-Crick type base pairing observed between a positively
charged molecule and a deprotonated one indicates that the deprotonation at the
N(1) position promoted by the alkylation at the N(7) position may interrupt the
formation of the normal Watson-Crick type GC base pair. The conformations about
the glycosidic bond and the sugar puckering are quite different between the two
molecules: the former has anti and C(4')-exo,C(3')-endo and the latter syn and
C(1')-exo-C(2')-endo.

PMCID: PMC326387
PMID: 6622257 [PubMed - indexed for MEDLINE]


751. Nucleic Acids Res. 1983 May 11;11(9):2801-14.

Molecular structure of deoxyadenylyl-3'-methylphosphonate-5'-thymidine dihydrate,
(d-ApT x 2H2O), a dinucleoside monophosphate with neutral phosphodiester
backbone. An X-ray crystal study.

Chacko KK, Lindner K, Saenger W, Miller PS.

dApT, a modified deoxyribose dinucleoside phosphate with an uncharged
methylphosphonate group, crystallizes as dihydrate in space group P2(1)2(1)2, a =
9.629(3), b = 20.884(6) and c = 14.173(4)A, Z = 4. The structure has been
determined using 2176 X-ray diffractometer reflections and refined to a final R
of 0.105. Torsion angles about P-O(5') and P-O(3') bonds are -91.8 degrees and
117.8 degrees. The former is in the normal (-)gauche range while the latter is
eclipsed. Bases are oriented anti, the sugar of adenosine is puckered 2T3
(C(2')endo) whereas that of thymidine displays puckering disorder with major and 
minor occupancy sites. Major site is a half-chair 2T (C(2')endo-C(1')exo) and
minor site an envelope 3T2 (C(3(1)endo). Adenine and thymine bases of symmetry
related molecules form reversed Hoogsteen type base pairs, water molecules are
disordered in the crystal lattice.

PMCID: PMC325924
PMID: 6574427 [PubMed - indexed for MEDLINE]


752. J Biol Chem. 1983 Mar 25;258(6):3576-82.

Cell-cell recognition in yeast. Characterization of the sexual agglutination
factors from Saccharomyces kluyveri.

Pierce M, Ballou CE.

The cell surface molecules responsible for sexual agglutination between haploid
cells of opposite mating type from Saccharomyces kluyveri have been purified and 
characterized. The 17-factor, released from 17-cells by beta-glucanase digestion 
(Zymolyase), is a glycoprotein of 6 X 10(4) Da. Its binding activity is heat- and
protease-labile, but it is stable to reducing agents and exo-alpha-mannosidase
digestion. The 16-factor, released from 16-cells by Zymolyase digestion, has a
molecular weight of 5 X 10(5) and is over 95% carbohydrate. An active binding
fragment can be released from 16-factor, from the factor purified from a mutant
of 16-cells (16(mnn1)-factor), and from the surfaces of the cells themselves by
dithiothreitol treatment. The 16(mnn1)-binding fragment has a molecular weight of
2 X 10(4) and is 30% carbohydrate. Its binding activity is stable to heat and
some proteases, but it is labile to pronase, carboxypeptidases A and Y,
alpha-mannosidases, and mild periodate treatment. 125I-16(mnn1)-binding fragment 
adheres specifically to 17-cells but does not bind to 16-cells or cells of other 
yeast strains. The binding of the labeled fragment to 17-cells is characterized
by a KA of 10(8) M-1, and 5 X 10(5) binding sites are present per cell. The
purified intact factors are monovalent and appear to interact in a lock and key
fashion to cause the specific agglutination of S. kluyveri 16- and 17-cells.

PMID: 6339487 [PubMed - indexed for MEDLINE]


753. J Biochem. 1983 Mar;93(3):787-94.

Transglycosylation activities of exo- and endo-type cellulases from Irpex lacteus
(Polyporus tulipiferae).

Kanda T, Noda I, Wakabayashi K, Nisizawa K.

Two highly purified cellulases, Ex-1 [exo-type, exo-cellobiohydrolase, EC
3.2.1.91] and En-1 [endo-type, EC 3.2.1.4] obtained from Driselase, a commercial 
enzyme preparation from Irpex lacteus (Polyporus tulipiferae), were used in this 
work. Both cellulases produced 14C-cellooligosaccharides such as 14C-G2 and
14C-G3 by transglycosylation when G3, G5, or beta-PNPC was used as a donor and
14C-G1 as an acceptor. However, the transglycosylation activity of Ex-1 was far
higher than that of En-1. When Ex-1 or En-1 was incubated with beta-PNPG only, no
p-nitrophenol was released, but it was readily released when G3 was added to the 
reaction mixture. In this reaction, the optimal donor (G3) concentration for Ex-1
was 1.0 mM, and the optimal pH values of Ex-1 were at 2.7 and 3.7 for beta-PNPG
and beta-PG as acceptors, respectively, these values being far lower than the
ordinary optimal pH values of the cellulase (4.0-5.0).

PMID: 6409895 [PubMed - indexed for MEDLINE]


754. Biochem J. 1983 Mar 1;209(3):659-67.

Glycosidase analysis of large acidic-type glycopeptides from viral and cellular
membrane glycoproteins. Evidence for a common oligomannosyl core with branch
sugar heterogeneity.

Hunt LA.

The asparagine-linked oligosaccharides of the complex acidic-type from
[3H]mannose-, [3H]glucosamine- or [3H]galactose-labelled membrane glycoproteins
of BHK21 cells and Rous-sarcoma virus were analysed by gel filtration combined
with extensive digestion with endo- and exo-glycosidases from bacterial and
eukaryotic sources. The neutral products from the digestion with a mixture of
exoglycosidases and endo-beta-N-acetylglucosaminidase D from Diplococcus
pneumoniae included a series of [3H]mannose- and [3H]glucosamine-labelled neutral
oligosaccharides that were all converted by digestion with eukaryotic
beta-N-acetylglucosaminidases into free N-acetylglucosamine and a small
oligomannosyl core containing two alpha-linked mannose residues and a third
mannose residue beta-linked to N-acetylglucosamine. These studies suggested that 
the complex acidic-type oligosaccharides from cellular and viral membrane
glycoproteins contained a common oligomannosyl core region (Man2 alpha leads to
Man beta leads to GlcNAc2), with heterogeneity in the number and/or linkage of
outer branch N-acetylglucosamine residues resulting in partial resistance to
beta-N-acetylglucosaminidase from a bacterial source.

PMCID: PMC1154143
PMID: 6307261 [PubMed - indexed for MEDLINE]


755. Eur J Cancer Clin Oncol. 1983 Feb;19(2):227-35.

Low- and high-risk malignant melanoma--I. Evaluation of clinical and histological
prognosticators in 585 cases.

Schmoeckel C, Bockelbrink A, Bockelbrink H, Koutsis J, Braun-Falco O.

In 585 cases with primary cutaneous stage I malignant melanoma (294 disease-free 
for at least 5 yr, 291 with later metastases) prognostic parameters were
examined. The most effective proved to be tumor thickness and mitotic activity,
particularly when combined as a prognostic index. Furthermore, vascular invasion,
ulceration in thick tumors (thickness greater than or equal to 3.0 mm), severe
cellular atypia, the small, lymphocytic-like cell type and the absence of an
inflammatory reaction were closely associated with a high rate of metastatic
cases. Less relevant prognostic factors were the level of invasion, sex, site,
tumor breadth, clinical diameters and infiltrative growth. Tumor type, age,
duration and an adjacent nevocellular nevus were not significantly associated
with the occurrence of later metastases. Furthermore, the growth-type (exo- or
endophytic) did not have a bearing on the prognosis.

PMID: 6681768 [PubMed - indexed for MEDLINE]


756. Biochem Int. 1983 Feb;6(2):141-8.

N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are
desulfated by the same enzyme.

Hopwood JJ, Elliott H.

We have prepared a series of oligosaccharides to assess the substrate specificity
of exo sulfatase activity in cultured human skin fibroblasts toward
N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and
heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from 
HS) were desulfated by a specific sulfatase that when deficient leads to the
accumulation of HS and the expression of mucopolysaccharidosis type IIID
(Sanfilippo D). Under the in vitro conditions studied there are two pathways for 
the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4
residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces 
GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4
residue is desulfated and then cleaved by the action of an
beta-N-acetylglucosaminidase activity. There was no detectable
beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to 
produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides.
There was approximately 10% of this normal beta-N-acetylglucosaminidase activity 
in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be
involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the
monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in
fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a
Sanfilippo D patient we propose that KS degradation in these patients proceeds by
the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4
which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 6236815 [PubMed - indexed for MEDLINE]


757. J Gen Virol. 1982 Dec;63(2):425-34.

Characterization of the oligosaccharides of Inkoo virus envelope glycoproteins.

Pesonen M, Ronnholm R, Kuismanen E, Pettersson RF.

Inkoo virus (a bunyavirus) was grown in BHK-21 cells and labelled with
[35S]methionine or [3H]mannose. [35S]Methionine labelled the two envelope
glycoproteins G1 (Mr = 125000) and G2 (Mr = 35000), as well as the nucleocapsid
protein N (Mr = 25000). Only G1 and G2 were labelled with the sugar precursor.
The [3H]mannose-labelled virus was solubilized with detergent and digested with
Pronase. The structure of the labelled glycopeptides originating from the mixture
of G1 and G2 was studied by degrading the glycans stepwise with specific exo- and
endoglycosidases, and by analysing the products by both gel and paper
chromatography, as well as lectin-affinity chromatography. Three classes of
N-glycosidic glycans were found: complex glycans with the monosaccharide sequence
(NeuNAc alpha Gal beta GlcNac beta) greater than or equal to 2 (Man)3 (GlcNAc)2
(occurrence of fucose was not studied), high mannose-type chains with the average
structure (Man)4-6 (GlcNAc)2, and endoglycosidase H-resistant small glycans which
were partly susceptible to mannosidase. These latter types of oligosaccharide
chains are a novel finding among virus glycoproteins. The relative ratio of the
three types of oligosaccharide chains was roughly 4 . 6:1:1 respectively. The G1 
glycoprotein carried most of the sugar chains, since it contained 85% of the
[3H]mannose label. The results are discussed in relation to the site of virus
maturation at smooth-surfaced vesicles in the Golgi region.

PMID: 7153764 [PubMed - indexed for MEDLINE]


758. Plant Physiol. 1982 Nov;70(5):1367-1372.

Characteristics and Activity Changes of Proteolytic Enzymes in Apple Leaves
during Autumnal Senescence.

Kang SM, Matsui H, Titus JS.

Department of Horticulture, University of Illinois, Urbana, Illinois 61801.

At least four different proteinases are present in senescing apple leaves (Malus 
domestica Borkh. cv. Golden Delicious) as determined by their pH optima,
substrate specificity, and their reactivity to proteinase inhibitors. An enzyme
active at pH 4.5 to 5.0 appears to be a sulfhydryl-dependent (iodoacetamide and
phenylmercuric acetate-sensitive) endoproteinase, and degradation of the large
subunit of ribulose bisphosphate carboxylase was observed only with this enzyme. 
It is tentatively concluded that this endoproteinase is responsible for the
breakdown of ribulose bisphosphate carboxylase in vivo. However, the presence of 
more than one endoproteinase in apple leaves is suggested by the broad range of
pH optima of the SH-dependent enzyme. Another enzyme active at pH 6.0 appears to 
be a carboxypeptidase, and was sensitive to phenylmethylsulfonylfluoride. This
enzyme showed a strong hydrolytic activity against
carbobenzoxyphenylalanylalanine. A sulfhydryl-dependent aminopeptidase and a
second hydroxyl-dependent carboxypeptidase were active at pH 7.5Total autolytic
activity (the sulfhydryl-dependent endoproteinase) as measured by the
disappearance of proteins decreased during the period of protein decline.
Evidence is presented that the measured proteinase activity can be dependent on
assay methods and substrates. While the disappearance of protein measures most of
endo-type activity, the ninhydrin assay appears to measure exo-type activity
preferentially.

PMCID: PMC1065889
PMID: 16662681 [PubMed - as supplied by publisher]


759. Nucleic Acids Res. 1982 Oct 11;10(19):6147-61.

Specific interaction of netropsin, distamycin-3 and analogs with LC duplexes:
reversion towards the B form of the 2'-deoxy-.2'-deoxy-2'-fluoro-hybrid duplexes 
upon specific interaction with netropsin, distamycin-3 and analogs.

Marck C, Kakiuchi N, Guschlbauer W.

Binding of the B-form specific ligands netropsin and distamycin-3, -4 and -5 has 
been used to monitor the presence and/or the inducibility of a B-type structure
in various poly-inosinic.poly-cytidilic double stranded polymers with
deoxyribose, ribose or 2'-deoxy-2'-fluororibose as sugar on either strand. The
efficiency of binding was followed by circular dichroism and further evaluated by
the increase in melting temperature of the complexes. The efficient binding of
netropsin and distamycins to the hybrid polymer (dIfl)n. (dC)n demonstrated that 
the fluorine carrying strand may undergo a A to B-type transition reflecting a
change of the 2'-deoxy-2'-fluororibose from the 3'-endo to the 1'-exo or 2'-endo 
pucker. The less efficient binding of the same ligands to the reverse hybrid
(dI)n.(dCfl)n showed that the geometry of the pyrimidine strand is the most
critical for the specific interaction. Taking into account the recent findings
about the regular hydration in the minor groove of the B-type dodecamer
dCGCGAATTCGCG in solid-state, the different binding modes observed between the
different polymers and antibiotics are explained by differences in their
possibilities of hydration. Binding of netropsin to a double stranded
deoxypolymer is interpreted as a local replacement of water molecules by
netropsin in the minor groove hydration network which is typical of the B-form.

PMCID: PMC320957
PMID: 6292869 [PubMed - indexed for MEDLINE]


760. Parasitology. 1982 Oct;85 (Pt 2):263-9.

Degenerating exo-erythrocytic forms of Plasmodium berghei in rat liver: an
ultrastructural and cytochemical study.

Jap PH, Meis JF, Verhave JP, Meuwissen JH.

Morphological and cytochemical aspects of the host response to almost mature
exo-erythrocytic forms (EEF) of Plasmodium berghei in rat hepatocytes were
studied by electron microscopy. Young stages (less than 47 h) never evoked a
local reaction. Two types of nearly mature EEF (47-51 h) could be distinguished, 
normal (EEF type I) and those that were the target of infiltrating cells (EEF
type II). The latter were in a stage of early or advanced degeneration and
generally exhibited increased electron density, especially in the contents of
their peripheral vacuoles. Neither type of EEF exhibited detectable enzyme
activity, although host cell enzymes, such as peroxidase and 5-nucleotidase, were
demonstrable. However, the infected liver cell appeared permeable to ruthenium
red whereas non-infected hepatocytes were not. When signs of degeneration were
present, as shown by the increasing density of the cytoplasm, loss of enzyme
activities occurred. The encompassing mononuclear cells were identified as true
monocytes, non-monocyte-derived and monocyte-derived macrophages by their
endogenous peroxidase activity. Macrophage filopodia penetrated and cleaved both 
hepatocyte and parasite cytoplasms. Subsequently, digestion and clearing of the
remnants took place. This study clearly demonstrated that a proportion of the
intra-hepatocytic EEF was destroyed by macrophages before they were able to
mature completely and release their merozoites.

PMID: 6755366 [PubMed - indexed for MEDLINE]


761. J Pharmacol Exp Ther. 1982 Apr;221(1):58-62.

The effects of stereoisomers of 2- and 9- aminobenzonorbornenes on food intake,
brain serotonin concentration and monoamine oxidase activity in the rat.

Gray NM, Lu MC, Bhargava HN.

To evaluate the importance of the conformation of amphetamine in eliciting its
various central nervous system effects, the conformationally defined analogs of
amphetamine. In this series of isomeric amines, the exo-2 and anti-9 isomers
resemble the fully extended conformation of amphetamine, whereas the endo-2 and
syn-9 isomers resemble the folded conformation. The exo-2 and anti-9 isomers were
equally as effective in reducing food intake in the rat, but were much less
potent than amphetamine. The endo-2 and syn-9 isomers had no effect on food
intake up to a dose of 40 mg/kg. These latter two isomers also caused very little
inhibition of monoamine oxidase in vitro. The exo-2 and anti-9 isomers, however, 
were nearly as effective as amphetamine in inhibiting monoamine oxidase type A,
but only amphetamine and the anti-9 isomer inhibited monoamine oxidase type B.
The effects at anorectic doses on brain serotonin (5-HT) concentration were also 
studied. Although amphetamine had no effect on the 5-HT concentration and
fenfluramine decreased its concentration, the exo-2 and anti-9 isomers caused a
significant increase in brain 5-HT. The anti-9 isomer caused a 14% increase in
5-HT, whereas the exo-2 isomer gave a 49% increase over controls. The data
suggest that the fully extended conformation of amphetamine is preferred over the
folded conformation for eliciting its biological responses, although the rigid
analogs were less potent than amphetamine.

PMID: 7062292 [PubMed - indexed for MEDLINE]


762. Hoppe Seylers Z Physiol Chem. 1982 Mar;363(3):279-93.

Enzymatic cleavage of the epsilon-peptide bond in alpha- and epsilon-substituted 
glycyl- and phenylalanyl-lysine peptides.

Plessing A, Siebert G, Wissler JH, Puigserver AJ, Pfaender P.

Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic
or non-classic routes. Some bacterial and mammalian enzymes, endo- and
exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for
their ability to split the epsilon-peptide bond in the above substrates. Kinetic 
constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney
and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas)
and hog intestinal aminopeptidase were additionally examined for their Ki values 
with the above substrates in comparison to the classic protease substrate leucine
p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be
efficient in cleaving epsilon-peptide bonds.

PMID: 6804347 [PubMed - indexed for MEDLINE]


763. J Virol. 1982 Feb;41(2):390-400.

Monosaccharide sequence of protein-bound glycans of Uukuniemi virus.

Pesonen M, Kuismanen E, Pettersson RF.

Uukuniemi virus, a member of the Bunyaviridae family, was grown in BHK-21 cells
in the presence of [(3)H]mannose. The purified virions were disrupted with sodium
dodecyl sulfate and digested with pronase. The [(3)H]mannose-labeled
glycopeptides of the mixture of the two envelope glycoproteins G1 and G2 were
characterized by degrading the glycans with specific exo-and endoglycosidases, by
chemical methods, and by analyzing the products with lectin affinity and gel
chromatography. The glycopeptides of Uukuniemi virus fell into three categories: 
complex, high-mannose type, and intermediate. The complex glycopeptides probably 
contained mainly two NeuNAc-Gal-GlcNAc branches attached to a core
(Man)(3)(GlcNAc)(2) peptide. The high-mannose-type glycans were estimated to
contain at least five mannose units attached to two N-acetylglucosamine residues.
Both glycan species appeared to be similar to the asparagine-linked
oligosaccharides found in many soluble and membrane glycoproteins. The results
suggested that the intermediate glycopeptides contained a mannosyl core. In about
half of the molecules, one branch appeared to be terminated in mannose, and one
appeared to be terminated in N-acetylglucosamine. Such glycans are a novel
finding in viral membrane proteins. They may represent intermediate species in
the biosynthetic pathway from high-mannose-type to complex glycans. Their
accumulation could be connected with the site of maturation of the members of the
Bunyaviridae family. Electron microscopic data suggest that the virions bud into 
smooth-surfaced cisternae in the Golgi region. The relative amounts of
[(3)H]mannose in the complex, high-mannose-type, and intermediate glycans were
25, 62, and 13%, respectively, which corresponded to the approximate relative
number of oligosaccharide chains of 2:2.8:1, respectively, in the roughly
equimolar mixture of G1 and G2. Endoglycosidase H digestion of isolated
[(35)S]methionine-labeled G1 and G2 proteins suggested that most of the complex
and intermediate chains were attached to G1 and that most of the
high-mannose-type chains were attached to G2.

PMCID: PMC256769
PMID: 7077748 [PubMed - indexed for MEDLINE]


764. Physiol Bohemoslov. 1982;31(3):213-6.

Ultrastructural changes in cortical synapses shortly after termination of a
seizure during kindling.

Langmeier M, Fischer J, Mares J.

Repeated electrical stimulation of the sensorimotor region of the rat cerebral
cortex at 10-min intervals led to progressive lengthening of the self-sustained
after-discharges (SSAD). 50-60 s after termination of the third SSAD we examined,
in the electron microscope, type I synapses (after Gray) in the second cortical
layer of the sensorimotor region of the contralateral hemisphere. In the
experimental animals we demonstrated swelling of both the pre- and post-synaptic 
elements, a decrease in the number of agranular synaptic vesicles and variability
of their shape and size. Frequent manifestations of exo- and endocytic activity
and a frequent incidence of complex vesicles have been described. Saccular
dilatation of the space between the outer and inner mitochondrial membrane in the
presynaptic terminal and dilatation of the terminal cisternae and extracellular
space occurred. Alteration of the spine apparatus was observed in the
postsynaptic elements. At the margin of the active zone we described simultaneous
invagination of the pre- and postsynaptic membrane up to the formation of rounded
structures with two concentric membranes. The changes in the synapses are
conceived as signs of exhaustion due to the previous epileptic seizure, which on 
the other hand, activated the restitution mechanisms of the structure of the
synaptic apparatus.

PMID: 6214805 [PubMed - indexed for MEDLINE]


765. J Gen Microbiol. 1981 Sep;126(1):55-61.

Autolysis of a division mutant of Escherichia coli.

Karibian D, Pellon G, Starka J.

The pleiotropic character of the envC chain-forming mutant of Escherichia coli
was found to include leakage of periplasmic enzymes and an abnormal tendency to
autolyse. Washed suspensions of envC cells released murein fragments into the
supernatant, and cell extracts from the mutant were richer than those of wild
type in exo-beta-N-acetylglucosaminidase (187% of the wild-type value) and in
soluble endopeptidase (256%) activities, but n-acetylmuramoylamidase,
D,D-carboxypeptidase, L,Dj-carboxypeptidase and transglycosylase were not
markedly different. When envC cells were grown in medium containing 0.58
M-sucrose, the chains broke up into rods, the L,D-carboxypeptidase activity
increased about sixfold and D,Dj-carboxypeptidase 1B about twofold. It is
suggested that L,D-carboxypeptidase is involved in septum splitting. The results 
suggest that the triggering of autolysis in E. coli envC depends on the
alteration of envelope constituents rather than on an enhanced activity of murein
hydrolases.

PMID: 6120994 [PubMed - indexed for MEDLINE]


766. Biochim Biophys Acta. 1981 Jul 27;654(2):242-8.

Synthesis and molecular conformation of
2',3'-O-isopropylidene-5'-deoxy-6(R),5'-cyclo-5,6-dihydrouridine.

Yamagata Y, Fujii S, Fujiwara T, Tomita K, Ueda T.

The title compound (hereafter abbreviated as 6(R),5'-cyclo-hUrd) is synthesized
from 2',3'-O-isopropylidene-5'-deoxy-5'-iodouridine and its molecular structure
has been determined by X-ray analysis. 6(R),5'-Cyclo-hUrd crystallizes in space
group C2 with Z = 4, and unit-cell dimensions a = 11.220 (2), b = 6.393 (1), c = 
18,963 (3) A and beta = 107.98 (1) degrees. The structure was solved by direct
interpretation of the three-dimensional Patterson function and refined to a final
R index of 0.063. In the crystal the glycosyl torsion angle chi CN is 60.7
degrees (anti conformation) and the dihydrouracil ring adopts a half-chair
conformation. The puckering of the ribose ring is an unusual O(1')-exo (P = 267
degrees, tau m = 47 degrees). The coupling constants of the 1H-NMR spectrum
measured in C2HCl3 solution indicate that the overall conformation of
6(R),5'-cyclo-hUrd found in crystal is also maintained in solution. The
theoretical calculations of coupling constants by the finite perturbation theory 
(FPT) intermediate neglect of differential overlap, self-consistent field
molecular orbital (INDO SCF-MO) method indicate that the deviation of the
observed coupling constants of sugar ring protons from those predicted by
applying modified Karplus-type formula to the X-ray structure could be due to the
strains around the sugar ring carbon atoms attached by protons.

PMID: 7284380 [PubMed - indexed for MEDLINE]


767. Farmakol Toksikol. 1981 Jul-Aug;44(4):453-9.

[Early triggering of the immune mechanism of chemical homeostasis in response to 
exo- and endogenous chemical compounds]

[Article in Russian]

Zaks AS, Bykova AA.

An early triggering of the immune mechanism of chemical homeostasis occurs in
response to intravenous, intraperitoneal or intramuscular injection of drugs
affecting the content of endogenous compounds of the mediator, enzymatic and
hormonal type. This response is accompanied by morphological and functional
changes in lymphoid cells of blood and organs as confirmed by immune
rosette-formation and blast transformation, by variation of intercellular
interactions that manifest in activation of immunocytoadhesion, in an increase in
the titers of antibodies to the drug administered and in those of autoantibodies 
to the endogenous compounds that realize the drug effect. It has been shown that 
lymphocyte receptors specific for the endo- and exogenous compounds participate
in the cell immune response to a chemical agent that might be pharmacologically
monitored by means of the action of antagonists of the endogenous mediators on
the receptor structure.

PMID: 7286206 [PubMed - indexed for MEDLINE]


768. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3920-3924.

Impaired induction and self-catabolite repression of extracellular pectate lyase 
in Erwinia chrysanthemi mutants deficient in oligogalacturonide lyase.

Collmer A, Bateman DF.

Department of Plant Pathology, Cornell University, Ithaca, New York 14853.

The pectate lyase (PL; EC 4.2.2.2) secreted by the plant pathogen Erwinia
chrysanthemi is induced and catabolite repressed by different concentrations of
its own product, digalacturonic acid 4,5-unsaturated at the nonreducing end
[u(GalUA)(2)]. Both activities of u(GalUA)(2) depend on its cleavage by
oligogalacturonide lyase (OGL; EC 4.2.2.6). This intracellular enzyme converts
u(GalUA)(2) to the deoxyketuronic acid 4-deoxy-L-threo-5-hexosulose uronic acid, 
which is then isomerized to 3-deoxy-D-glycero-2,5-hexodiulosonic acid. An
OGL-deficient mutant unable to grow on u(GalUA)(2) was poorly induced by
u(GalUA)(2) or by D-galacturonan but produced wild-type levels of PL when
supplied with 3-deoxy-D-glycero-2,5-hexodiulosonic acid. PL synthesis in the
mutant could also be stimulated by 4,5-unsaturated trigalacturonic acid, from
which deoxyketuronic acid is released by another intracellular enzyme. An
OGL-deficient mutant that grew slowly on u(GalUA)(2) in comparison with the
wild-type parent was hyperinduced by u(GalUA)(2) unless catabolite repression was
relieved by cyclic AMP or imposed by logarithmic growth on glycerol. PL synthesis
is also stimulated by saturated digalacturonic acid, which is released from
D-galacturonan by another extracellular enzyme,
exo-poly-alpha-D-galacturonosidase (EC 3.2.1.82). Because these dimers stimulate 
PL synthesis at concentrations (wt/vol) 1/1000th of the concentration required by
D-galacturonan, and because an OGL-deficient mutant uninducible by dimers was
also uninducible by D-galacturonan, we postulate that PL induction by pectic
polymers entails extracellular formation of dimers and subsequent intracellular
conversion to deoxyketuronic acids, the apparent inducers of PL.

PMCID: PMC319685
PMID: 16593039 [PubMed - as supplied by publisher]


769. Biochem J. 1981 Jun 1;195(3):701-13.

The structure of carbohydrate unit B of porcine thyroglobulin.

Yamamoto K, Tsuji T, Irimura T, Osawa T.

The oligosaccharide fraction was obtained from porcine thyroglobulin by
hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by
successive chromatographies on columns of DEAE-cellulose and concanavalin
A-Sepharose, and their structures were investigated by the combination of endo-
and exo-glycosidase digestions, methylation analysis and Smith degradation. From 
the results of these studies, the structures of the unit B oligosaccharides were 
proposed to be as follows: see formula in text. Thus the glycoprotein was found
to have triantennary and biantennary complex-type oligosaccharides as acidic
sugar chains. Concerning the triantennary oligosaccharides, the following
structural features were shown: (1) the sialic acid residues were not localized
on certain specific branches but distributed on all three branches; (2) however, 
alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the 
terminal of the branch arising from C-4 of the branching alpha-mannose residue,
whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of
the other branches; (3) the outer branching alpha-mannose residue was attached to
C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were
observed to exist.

PMCID: PMC1162943
PMID: 7316980 [PubMed - indexed for MEDLINE]


770. Biochem J. 1981 Jun 1;195(3):691-9.

Structure of carbohydrate unit A or porcine thyroglobulin.

Tsuji T, Yamamoto K, Irimura T, Osawa T.

The unit A-type glycopeptides were purified from porcine thyroglobulin by Pronase
digestion followed by chromatography on a DEAE-Sephadex A-25 column. These
glycopeptides were separated into five fractions (UA-I, -II, -IV and -V) by Dowex
50W (X2) column chromatography. Fractions UA-I, -II, -III, -IV and -V were found 
to have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn,
(Man)7(GlcNAc)2-Asn, (Man)6(GlcNAc)2-Asn and (Man)5(GlcNAc)2-Asn respectively.
The structures of these five fractions were investigated by the combination of
exo- and endo-glycosidase digestions, methylation analysis. Smith periodate
degradation and acetolysis. The results showed that fraction UA-V had the
simplest structure: see formula in text. The larger glycopeptides (fractions
UA-I, -II, -III and -IV) contained additional mannose residues alpha (1 leads to 
2)-linked to the terminal mannose residues in the above core structure. These
unit A-type glycopeptides appear to be biosynthetic intermediates that are to be 
processed to form complex-type glycopeptides (unit B-type sugar chains).

PMCID: PMC1162942
PMID: 7316979 [PubMed - indexed for MEDLINE]


771. Nucleic Acids Res. 1981 Feb 11;9(3):711-29.

The crystal and molecular structure of 2'-deoxy-2'-fluoroinosine monohydrate.

Hakoshima T, Omori H, Tomita K, Miki H, Ikehara M.

The structure of the hydrate of 2'-deoxy-2'-fluoroinosine has been determined by 
single-crystal x-ray diffraction. The nucleoside crystallizes in space group
P2(1)2(1)2(1) with unit cell dimensions, a = 33.291, b = 10. 871, c = 6.897A.
There are two nucleosides and two water molecules in the asymmetric unit. The
structure was solved by direct methods and refined to a residual R = 0.095. The
two independent nucleosides in the asymmetric unit show different conformations
about the glycosidic bond, while other structural details are similar. The base
orientation to the sugar is syn in molecule A, whereas anti in molecule B. The
exocyclic C(4')-C(5') bond conformation defined with respect to
C(3')-C(4')-C(5')-O(5') is gauche+ in both molecules A and B. The sugar ring
pucker defined by the pseudorotation phase angle P is a twisted conformation in
both, C(3')-endo-C(4')-exo with P = 29 degrees in molecule A and
C(4')-exo-C(3')-endo with P = 41 degrees in molecule B. It is shown by comparison
with x-ray results of other 2'-fluoronucleosides and unmodified nucleosides
including inosines that, in addition to a strong preference of the C(3')-endo
type pucker, twisted conformations involving C(4')-exo puckering may be one of
characteristic features of 2'-fluoronucleosides.

PMCID: PMC327232
PMID: 7220349 [PubMed - indexed for MEDLINE]


772. Biochemistry. 1981 Feb 3;20(3):560-6.

Structure of a complex-type sugar chain of human glycophorin A.

Irimura T, Tsuji T, Tagami S, Yamamoto K, Osawa T.

Tryptic glycopeptide T1 of human glycophorin A [Tomita, M., & Marchesi, V. T.
(1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968] was subjected to
hydrazinolysis. After N-acetylation, the complex-type oligosaccharides were
isolated by gel filtration. The major neutral oligosaccharide (2000 molecular
weight) was purified by a combination of ion-exchange and gel-permeation
chromatography. Treatments with endo- and exo-glycosidases, periodate oxidation, 
and methylation analysis indicated that the major neutral oligosaccharide has the
following structure: Gal beta 1 leads to -4GlcNAc beta 1 leads to 2Man alpha 1
leads to 6(GlcNAc beta 1 leads to 4)(Gal beta 1 leads to -4GlcNAc beta 1 leads to
2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4-(Fuc alpha 
1 leads to 6)GlcNAc. This oligosaccharide was retained by Ricinus communis
agglutinin-Sepharose and retarded by Sepharose 4B coupled with
erythroagglutinating E4 isolectin from Phaseolus vulgaris. Retention by
concanavalin A-Sepharose was observed only after treatment of the oligosaccharide
with beta-galactosidase.

PMID: 7213594 [PubMed - indexed for MEDLINE]


773. Ann Acad Med Singapore. 1981 Jan;10(1):99-106.

Chemotherapy of malaria.

Ponnampalam JT.

The chemotherapy of malaria is incomplete without taking into consideration the
widespread presence of chloroquine resistant falciparum malaria necessitating the
use of a combination of antimalarials in order to effect a radical cure.
Fortunately, there has been no evidence of chloroquine resistance thus far with P
vivax and P malariae. Chloroquine resistant falciparum strains are present in
about 85% of patients suffering from falciparum malaria but fortunately over 90
percent of the resistant cases only show a mild degree of resistance of the RI
(delayed) type which in the majority of cases responds to larger doses of
chloroquine over and above the amount administered in the standard three days
regime. A combination of antimalarials is necessary in order to effect a radical 
cure in chloroquine resistant falciparum malaria. Vivax malaria and malaria due
to P malariae best respond to sequential therapy with chloroquine and primaquine.

PIP: This review of the chemotherapy of malaria covers the following: history of 
malaria chemotherapy; the rationale of malaria chemotherapy (drug trials related 
to malariotherapy, drug trials utilizing human volunteers, drug trials on
hospital patients, field trials, extended field trials, and mass drug
administration); the malaria situation in Southeast Asia; drug resistant malaria;
the clinical features of malaria; diagnosis and treatment; and chemoprophylaxis. 
There is no single all purpose drug that is suitable for the treatment and
prevention of malaria. Single dose treatment is the method of choice. An urgent
need exists for a schizonticidal drug that would remain effective in the body for
3-6 months after a single dose, such a drug should be effective against both
trophozoites and gametocytes. Rather than the present 14-day course of primaquine
to eradicate exo-erythrocytic forms, a drug which is effective after a 3-day
course is urgently required. Malaria continues to be a major public health
problem in most of the countries of Southeast Asia. Prevalence of species of
malaria parasites show wide variation from country to country, but Plasmodium
falciparum is more prevalent than either P. vivax or P. malariae. Drug resistant 
malaria presents an added problem in Southeast Asia with P. falciparum showing
resistance to chloroquine and to other antimalarials. The degree of resistance
varies from country to country. Documented cases of resistance have been reported
from all the countries of the region, and dosage schedules of various
antimalarials have to be modified and combination of drugs used when treating
such cases. All that is needed to make a laboratory diagnosis of malaria is a
microscope, a few slides, and Field's stain. In view of the high incidence of
chloroquine resistant falciparum malaria in the countries of the Southeast Asia
region it is recommended that severe forms of falciparum infections be treated in
the hospital for the 1st week. About 15% of patients still respond to the
standard dose of chloroquine and remain parasite free for 28 days, i.e., the
strain of P. falciparum is said to be sensitive to chloroquine. Over 90% of the
resistant cases only show a mild degree of resistance of the RI (delayed) type
which in the majority of cases responds to larger doses of chloroquine over and
above the amount administered in the standard 3 day regimen. A combination of
antimalarials is required to effect a radical cure in chloroquine resistant
falciparum malaria. Vivax malaria and malaria due to P. malariae best respond to 
sequential therapy with chloroquine and primaquine.

PMID: 7025744 [PubMed - indexed for MEDLINE]


774. J Biol Chem. 1980 Dec 25;255(24):11737-42.

Purification and properties of alpha-N-acetylgalactosaminidase from Clostridium
perfringens.

Levy GN, Aminoff D.

Exo-alpha-N-acetylgalactosaminidase has been purified 8000-fold from Clostridium 
perfringens by gel filtration, ion exchange chromatography, isoelectric
precipitation, and negative adsorption on human O type erythrocytes. The
resulting enzyme is active at physiological pH and temperature. Phenyl
glycosides, oligosaccharides, mucins, glycolipids, and cell membranes are
substrates for this enzyme. The result of enzyme action on blood type A
erythrocytes is the loss of A activity and the simultaneous appearance of H
activity, characteristic of the O blood group type. Polyacrylamide gel
electrophoresis in sodium dodecyl sulfate demonstrates electrophoresis in sodium 
dodecyl sulfate demonstrates that the blood group A-destroying activity is
distinct from the other glycosidase activities found in C. perfringens.

PMID: 6254979 [PubMed - indexed for MEDLINE]


775. Prosthet Orthot Int. 1980 Dec;4(3):159-61.

Reproducibility of gait measurement using the Lamoreux goniometer.

Jansen E, Orbaek H.

The self-aligning goniometer system of the Lamoreux type mounted on an
exo-skeleton was applied ten times on the same normal person to measure hip and
knee flexion and extension movements. The standard deviation of full measurement 
was found to be approximately 5 per cent of the hip excursion and 10 per cent of 
the knee excursion. These figures were found throughout the test series as well
as within each single test run. The goniometer is considered a useful tool for
clinical gait analysis.

PMID: 7220248 [PubMed - indexed for MEDLINE]


776. J Exp Med. 1980 Oct 1;152(4):979-95.

Human monoclonal macroglobulins with specificity for Klebsiella K polysaccharides
that contain 3,4-pyruvylated-D-galactose and 4,6-pyruvylated-D-galactose.

Kabat EA, Liao J, Bretting H, Franklin EC, Geltner D, Frangione B, Koshland ME,
Shyong J, Osserman EF.

Two human IgM myeloma proteins, IgMWEA and IgMMAY, were found to react with agar 
and Klebsiella polysaccharides that contain pyruvylated D-galactose (DGal).
Quantitative precipitin data and precipitin inhibition studies with methyl alpha-
and beta-glycosides of 4,6-pyruvylated-D-galactose showed their combining sites
to be different, although each was directed against the pyruvylated-D-Gal, one
reacting most specifically with Klebsiella polysaccharides with terminal
nonreducing beta-linked 2,4 pyruvylated-D-Gal, whereas the other reacted equally 
well with Klebsiella polysaccharides that contain 3,4 beta-linked and 4,6
alpha-linked terminal nonreducing pyruvylated-DGal. Inhibition studies showed
that both sites are directed toward one of the two space isomers of 3,4- or
4,6-pyruvylated DGal, the form in which the methyl group of the pyruvate is
equatorial, or endo, and its carboxyl group axial, or exo, to the plane of the
acetal ring. Coprecipitation studies showed the combining site of IgMWEA to be
located on an (Fab')2 fragment and not on the (Fc)5mu fragment. The monoclonal
peak in the serum of IgMMAY was specifically precipitated by Klebsiella
polysaccharide. Myeloma proteins with specificities of this type may occur with
reasonable frequency in humans and may be a consequence of clonal expansion from 
inapparent infection, carrier states, or disease produced by various Klebsiella
organisms.

PMCID: PMC2185977
PMID: 6158553 [PubMed - indexed for MEDLINE]


777. J Biochem. 1980 Jun;87(6):1635-9.

Modes of action of exo- and endo-cellulases in the degradation of celluloses I
and II.

Kanda T, Wakabayashi K, Nisizawa K.

Cotton and Valonia-cellulose (cellulose I) were readily attacked by
endo-cellulase of a highly endowise-hydrolysis type, with a sharp decrease in the
degree of polymerization, while the simultaneous production of reducing sugar was
low. In contrast, viscose rayon and alkali-cellulose (cellulose II) showed little
lowering of the degree of polymerization by endo-cellulase, though the effect was
slightly greater than with an exo-cellulase, while the simultaneous production of
reducing sugar was very high. The synergistic effect of exo- and endo-cellulases 
on the hydrolysis of cellulose was much higher with cellulose I than with
cellulose II. These results can be explained in terms of differences in the
polarity of cellulose chains and in the crystallinity and/or ultrastructure of
the cellulose fibers.

PMID: 7400114 [PubMed - indexed for MEDLINE]


778. J Med Chem. 1980 Jun;23(6):698-702.

14-(Arylhydroxyamino)codeinones and derivatives as analgetics and antagonists.

Schwab LS.

Diels-Alder reaction of thebaine (1) and N-(cyclopropylmethyl)northebaine with
nitrosobenzene and p-fluoronitrosobenzene gave adducts
[6,14-exo-(phenyloxyamino)codeine 6-methyl esther and derivatives, 2a-d] which
yielded 14-(phenylhydroxyamino)codeinone and derivatives (3a-d) on acid
hydrolysis. Rearrangement of 3 (NaOMe) afforded
5,14-exo-(phenyloxyamino)thebainone and derivatives (4a-d); reduction of 3 led to
14-(phenylamino)dihydrocodeinone and derivatives (5a-d). Thebaine also reacted
with 1-halo-1-nitrosocyclohexane (halo = Cl, Br) and with benzohydroxamic acid
under oxidizing conditions to give 14-(hydroxyamino)codeinone (6). All compounds,
3-6, were evaluated as analgetics and antagonists by the tail-flick, writhing,
and Straub tail assays: compounds of types 3-5 were analgetics (N-Me), one-third 
to one-tenth as potent as morphine, or antagonists [N-(cyclopropylmethyl)], 50 to
100 times less potent than naloxone; 6 behaved as an antagonist in the tail-flick
test but as an agonist in the other assays. Opiate receptor binding studies
indicate that compounds of type 3 may be useful as opiate spin-label precursors.

PMID: 6248645 [PubMed - indexed for MEDLINE]


779. J Gen Virol. 1979 Nov;45(2):479-87.

Sequence analysis of lactosamine type glycans of individual membrane proteins of 
Semliki Forest virus.

Pesonen M.

3H-fucose and 14C-glucosamine labelled glycopeptides of the individual membrane
proteins E1, E2 and E3 of Semliki Forest virus could be sequentially digested
with alpha-neuraminidase, beta-galactosidase, N-acetyl-beta-glucosaminidase,
alpha- and beta-mannosidase, N-acetyl-beta-hexosaminidase and finally with
alpha-fucosidase. The degradations of the virus glycopeptides proceeded in the
same way as stepwise digestions of reference glycopeptides of the lactosamine
type obtained from IgG and alpha 1-acid glycoprotein. This suggests that all
three membrane glycoproteins of Semliki Forest virus contained glycans with a
monosaccharide sequence characteristic for lactosamine type oligosaccharides. The
number of both distal and proximal N-acetyl-glucosamine residues was estimated to
be usually two. According to exo- and endo-glycosidase digestions, fucose seemed 
to be attached to the innermost N-acetyl-glucosamine unit.

PMID: 541666 [PubMed - indexed for MEDLINE]


780. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2684-8.

Determination of enzyme mechanisms by radiationless energy transfer kinetics.

Lobb RR, Auld DS.

Rigorous definition of the elementary steps of an enzymatic reaction requires
visualization of transient enzyme-substrate (ES) complexes. Measurement of
radiationless energy transfer (RET) between enzyme tryptophan residues and a
fluorescent dansyl (5-dimethylaminonaphthalene-1-sulfonyl) substrate provides a
sensitive means to observe ES complexes directly. Analysis of the rate of
formation and breakdown of ES complexes by RET can serve as the basis of a rapid 
kinetic approach to enzyme mechanisms. Both pre-steady-state and steady-state
kinetics can be performed in the same RET experiment. Analysis at steady state
precisely determines k(cat) and K(m) values by multiple means. Analysis at
pre-steady state determines the number of intermediates, the type of reaction
mechanism, and all the individual binding and rate constants. Chymotrypsin was
chosen as a standard of reference for RET kinetics because extensive
investigations have established both the existence of transient intermediates in 
the course of its catalytic process and the range of values to be expected for
pertinent kinetic constants. As predicted, RET kinetics readily detects the two
known intermediates in the alpha-chymotrypsincatalyzed hydrolysis of specific
ester substrates. The results are both qualitatively and quantitatively in accord
with data derived for this enzyme from classical kinetics. Hence, this
experimental study both validates and demonstrates the theoretical advantages and
potential of RET kinetics. The generality of the approach has been investigated
by synthesizing a family of dansyl-labeled substrates designed to meet the
specificity requirements of a number of metallo- and nonmetallo- exo- and
endopeptidases. In all cases, the ES complex is observed readily at micromolar or
lower concentrations of enzyme under stopped-flow conditions. The success of the 
RET kinetic approach on proteolytic enzymes shows its broad utility.

PMCID: PMC383672
PMID: 288055 [PubMed - indexed for MEDLINE]


781. Hiroshima Daigaku Shigaku Zasshi. 1979;11(1):30-47.

[Studies on exo-type of beta-N-acetylglucosaminidase in rat parotid gland--the
variation of the basic and acidic forms of the enzyme in the subcellular
fractions (author's transl)]

[Article in Japanese]

Toda K.

PMID: 44881 [PubMed - indexed for MEDLINE]


782. J Biochem. 1978 Nov;84(5):1217-26.

Purification and properties of an exo-cellulase of Avicelase type from a
wood-rotting fungus, Irpex lacteus (Polyporus tulipiferae).

Kanda T, Nakakubo S, Wakabayashi K, Nisizawa K.

A cellulase component of Avicelase type was obtained from Driselase, a commercial
enzyme preparation from a wood-rotting fungus Irpex lacteus (Polyporus
tulipiferae). It showed a single band on SDS-polyacrylamide electrophoresis. The 
amino acid composition of this cellulase resembled those of cellulase components 
of endo-type from the same fungus. However, it produced exclusively cellobiose
from CMC as well as from water-insoluble celluloses such as Avicel or cotton at
earlier stages of hydrolysis. In addition, the hydrolysis of CMC practically
stopped after an initial rapid stage. The cellulase showed a strong synergistic
action with an endo-cellulase of higher randomness (typical CMCase-type) in the
hydrolysis of CMC as well as Avicel. In contrast to cellotriose and -tetraose,
cellopentaose and -hexaose were attacked very rapidly, and only cellobiose was
produced. These results suggest that the cellulase is an exo-type component.
However, it mutarotated the products from cellopentaitol in the same direction as
endo-cellulases. it represented a relatively large portion of the total cellulase
activity, and may play an important role in the degradation of native cellulose
in vivo.

PMID: 32175 [PubMed - indexed for MEDLINE]


783. Am J Optom Physiol Opt. 1978 Oct;55(10):670-6.

Association of symptoms with measures of oculomotor deficiencies.

Sheedy JE, Saladin JJ.

Phoria, vergence, and fixation-disparity were measured at near working distances 
for a nonclinical sample of 3rd-yr optometry students. A questionnaire divided
the sample into 33 symptomatic and 44 asymptomatic subjects. Discriminant
analysis was used to determine which clinical measures best predicted the group
to which each subject belonged. Sheard's criterion was a good discriminator for
exo deviations, and a variant of Percival's criterion was good for eso
deviations. Fixation-disparity variables proved to be valuable diagnostically.
The best fixation-disparity measures were the curve slope, curve type, and amount
of fixation disparity.

PMID: 747192 [PubMed - indexed for MEDLINE]


784. J Biochem. 1978 Jun;83(6):1625-30.

Mutarotation of hydrolysis products by different types of exo-cellulases from
Trichoderma viride.

Nisizawa K, Kanda T, Shikata S, Wakabayashi K.

Mutarotation of products from p-nitrophenyl beta-D-cellobioside and
cellopentaitol by two different types of exo-cellulases from Trichoderma viride
was investigated. It was found that an exo-cellulase of glucosidase type produced
from the former substrate D-glucose which was mutarotated in a downward
direction, while another exo-cellulase of Avicelase type produced from the latter
substrate cellobiose which was mutarotated in an upward direction.

PMID: 97279 [PubMed - indexed for MEDLINE]


785. Biotechnol Bioeng. 1978 May;20(5):637-63.

Kinetics of enzymatic hydrolysis of cellulose: analytical description of a
mechanistic model.

Okazaki M, Moo-Young M.

A generalized mechanistic model for the enzymatic hydrolysis of cellulose is
developed and expressed mathematically. The model is based on
Michaelis--Menten-type kinetics for concurrent random and endwise attack of the
substrate involving end-product inhibitions and three types of enzymes: an
endo-beta-1,4-glucanase, an exo-beta-1,4-glucanase, and beta-glucosidase. Basic
parameters of the model which can explain synergistic and other effects observed 
experimentally are quantified and discussed. It is shown that cellulose
degradation kinetics are expected to be strongly affected by the ratio of endo-
to exocellulases in the reaction mixture as indicated by previous experimental
data, and the substrate degree of polymerization, a factor not fully appreciated 
in previous studies, which appear to be overridingly important in many practical 
cases.

PMID: 417746 [PubMed - indexed for MEDLINE]


786. Biochim Biophys Acta. 1977 May 3;476(1):1-15.

Intramolecular hydrogen bonding and molecular conformations of nucleosides. N
(6)-dimethyl-2',3'-isopropylidene adenosine.

Plochocka D, Rabczenko A, Davies DB.

The physical properties of an adenosine derivative,
N(6)-dimethyl-2',3'-O-isopropylidene adenosine, Derivative 1, which is capable of
intramolecular hydrogen bond formation between base-ring and sugar exocyclic
hydroxymethyl group, have been studied in solution by infrared, circular dichroic
and nuclear magnetic resonance spectroscopy. Analysis of the 220 MHZ 1H NMR
spectrum of Derivative 1 in C2HCl2 solution indicated an overwhelming preference 
for the gg conformation for rotation about the C(4')--C5') bond and a predominant
conformation for rotation about the C(5')--O(5') bond in which OH(5') projects
towards the base ring. The purine base ring was shown to be in a predominant syn 
conformation with respect to the sugar ring by 100 MHZ 1H nuclear Overhauser
experiments, by analysis of 3J(13C,H1') magnitudes observed in proton-coupled 13C
NMR experiments and by CD measurements. Combination of each conformation feature 
of Derivative 1 in non-polar solvents is consistent with the overall molecular
conformation observed in the solid state in which intramolecular hydrogenbonding 
exists between purine N(3) and the sugar CH2OH group; the presence of a strong
intramolecular hydrogen bond was observed by infrared spectroscopy. The sugar
ring conformations of 2',3'-O-isopropylidene ribonucleosides were analysed in
terms of the pseudorotational properties of the ring; the N and S conformations
tend toward to C(2')-exo and C(3')-'exo conformations, respectively, compared to 
normal ribonucleosides (C(3')-endo and C(2')-endo, respectively). The presence of
the hydrogen bond in the derivative is sufficient to promote the S-type
conformations (approx. 80%--90%) compared to cases where such a strong hydrogen
bond is unlikely to be present approx. 40--50%).

PMID: 856281 [PubMed - indexed for MEDLINE]


787. Biochim Biophys Acta. 1976 Dec 2;455(2):510-25.

Serum glycoprotein-type sequence of monosaccharides in membrane glycoproteins of 
Semliki Forest virus.

Pesonen M, Renkonen O.

Semliki Forest virus was grown in BHK-21 cells and labelled in vivo with
radioactive monosaccharides. The virus was disrupted with sodium dodecyl sulphate
and the polypeptides were hydrolyzed with pronase. A mixture of type A
glycopeptides (for nomenclature, see Johnson and Clamp (1971) Biochem. J. 123,
739-745) of the membrane glycoproteins E1 and E3 was isolated by gel filtration
and subjected to sequential degradation with exo-glycosidases. The reduction in
the apparent molecular weight and the cleavage of radioactive monosaccharides
were monitored with gel filtration. The results suggest that the type A
oligosaccharides have similar average structures and contain at the non-reducing 
terminus 3.4 mol of alpha-D-sialic acid and 0.7 mol of alpha-L-focose, folloled
by 3.1 mol of beta-D-galactose, 4.2 mol of N-acetyl-beta-D-glucosamine, 0.7-1.5
mol of alpha-D-mannose, 0.5 mol of beta-D-mannose and 0.6-2.2 mol of
N-acetyl-beta-D-glucosamine attached to 1.0 mol of N-acetylglucosamine resistant 
to N-acetyl-beta-D-glucosaminidase. This innermost monosaccharide unit,
therefore, appears to be attached to the peptide. The peptides attached to this
N-acetyl-glucosamine had an apparent molecular weight of 720+/-100. We propose
the following average structure, compatible with most of our data, for the type A
glycopeptides of Semliki Forest virus:.

PMID: 999925 [PubMed - indexed for MEDLINE]


788. J Biol Chem. 1976 Oct 10;251(19):5904-10.

Structure of plant cell walls. Purification and characterization of a
beta-1,4-galactanase which degrades a structural component of the primary cell
walls of dicots.

Labavitch JM, Freeman LE, Albersheim P.

Wild type Bacillus subtilis, when grown on a soybean arabinan-galactan, secretes 
a beta-1,4-galactanase which has been purified more than 200-fold from the
culture fluid. Affinity chromatography was the most effective step in a
purification procedure which resulted in a preparation that contained only a
single 40,000 molecular weight protein band upon sodium dodecyl sulfate-disc gel 
electrophoresis. The purified galactanase digests a beta-1,4-galactan purified
from citrus pectin and digests partially the isolated cell walls of
suspension-cultured sycamore cells. The predominant product of the enzymic
degradation of the substrates tested is a 4-linked tetragalactose. Evidence is
presented to support the hypothesis that the galactanase attacks its substrates
in both an exo- and endo-manner. The products obtained upon galactanase digestion
of the soybean arabianin-galactan demonstrate that the earlier proposal
concerning the structure of this polysaccharide must be incorrect.

PMID: 823153 [PubMed - indexed for MEDLINE]


789. J Bacteriol. 1976 Jul;127(1):610-8.

Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells.

Lipke PN, Taylor A, Ballou CE.

Saccharomyces cerevisiae mating type a cells enlarged and elongated when exposed 
to alpha-factor, a sex pheromone produced by mating-type alpha cells. This
morphogensis required exogenous-D-glucose, nitrogen, and phosphate, and cells in 
exponential phase responded better than stationary-phase cells. Morphogenesis was
blocked by cycloheximide and by inhibitors of cell wall biosynthesis such as
2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-deoxy-2-fluoro-D-mannose,
but not by polyoxin D. One to two hours after addition of pheromone, a cells
became more susceptible to lysis by glucanases, a change that was dependendent on
the dose of alpha-factor and was blocked by drugs that block morphogenesis. On
the other hand, treatment with alpha-factor did not increase susceptibility to
attack by trypsin, subtilisin, or exo-alpha-mannanase. Radioactive label,
incorporated into cell wall polysaccharides during treatment with alpha-factor,
was not secreted into the medium during morphogenesis. Analysis of the labeled
wall polymers showed that alpha-factor-treated cells contain more glucan and less
mannan than control cells, and that the mannan of treated cells contains an
increased proportion of shorter side chains and unsubstituted backbone mannose
units. Thin-section electron microscopy of treated cells revealed that the cell
wall possesses a diffuse outer layer in the extension and is thinner at the tip.

PMCID: PMC233094
PMID: 776942 [PubMed - indexed for MEDLINE]


790. Bull Tokyo Med Dent Univ. 1976 Mar;23(1):23-6.

Dextran degrading activity of oral microbial flora.

Takamori K, Mizuno F, Matsuda Y, Takahashi N, Horikawa T.

Dextran degrading activity of the human oral microflora was detected by culturing
in TYD broth (Tryptose 1.0%, Yeast extract 0.3%, Dextran T-150 (Pharmacia, MW
150,000) 0.15%). All of the plaue and saliva samples collected from 10 subjects
showed a dextran degrading activity, both cultured aerobically and anaerobically,
while the anaerobic culture was more active than the aerobic. Furthermore, some
individual differences were observed in their activity. Crude enzyme(s) was
extracted from the supernatant of a mixed culture of plaque sample by adding
ammonium-sulfate to 0.6 and 0.8 saturation (called E-1 and E-2, respectively).
E-1 contained 2 dextran-degrading enzymes, one being thought to be an
endo-enzyme, with the optimal of H being 5.0 and the other an exo-enzyme with the
optimal pH being 7.0-7.5. On the other hand, E-2 contained 1 enzyme of the
endo-type. Thirteen strains producing dextranase were isolated from the plaque
and were identified as Bacteroides oralis-like organisms. Several other organisms
were thought to produce dextranase, although we failed to isolate them in this
experiment.

PMID: 1065508 [PubMed - indexed for MEDLINE]


791. Mol Gen Genet. 1975 Dec 30;143(1):13-23.

Restriction and modification in B. subtilis. Purification and general properties 
of a restriction endonuclease from strain R.

Bron S, Murray K, Trautner TA.

All Bacillus subtilis R-type strains showing the phenomena of restriction and
modification contain an endonuclease that inactivates in vitro the biological
activity of a variety of DNAs lacking R-specific modification, such as
transfecting SPPI, SPO2 and phi105 DNA, and transforming B. subtilis 168-type
DNA. The corresponding DNAs carrying R-specific modification are resistant to the
enzyme. The enzyme has been purified approximately 400-fold and is essentially
free from contaminating double strand-directed unspecific exo- or endonuclease
activity. Only Mg2+ is required as cofactor. The substrate DNAs are cleaved at
specific sites. The double-stranded fragments produced from SPP1 DNA (molecular
weight 2.5 x 10(7)) have an average molecular weight of about 3 x 10(5).

PMID: 2856 [PubMed - indexed for MEDLINE]


792. J Biochem. 1975 Sep;78(3):499-512.

Purification and properties of an exo-cellulase component of novel type from
Trichoderma miride.

Shikata S, Nsizawa K.

An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma
viride, was fractionated by Amberlite CG-50 column chromatography into three
cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both
beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase)
properties was obtained from peak III by extensive purification throuh
consecutive column chromatography. The enzyme was homogeneous on
ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and
molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this
enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose
residues one by one from the nonreducing end of higher cellooligosaccharides and 
CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and
beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore,
this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one 
by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin
apparently rich in amorphous structure.

PMID: 5409 [PubMed - indexed for MEDLINE]


793. C R Acad Sci Hebd Seances Acad Sci D. 1975 May 12;280(18):2125-8.

[The endocrine pancreas in Varanus niloticus: ultrastructural and cytochemical
electron microscopic study and X-ray microdiffraction]

[Article in French]

Theret C, Alliet J, Comlan G, Gourdier D.

Electron microscopy has confirmed that there are B and A1 insular cells in the
Varanidae; nevertheless, the majority of cells are A2. It is the transformation
of canalicular and acinar cells that is responsible for insular regeneration.
Mixed cell type can be found very frequently. X Ray microdiffraction has shown
that the beta granules contain high levels of zinc; similar concentrations are
found in peri-insular exocrine cells. Seasonal sexual activity intensifies the
process of exo-endocrine cellular transformation and increases both the zinc
level in B and pre-B cells and the sulphur level in A2 cells.

PMID: 807398 [PubMed - indexed for MEDLINE]


794. Arch Oral Biol. 1975 May-Jun;20(5-6):393-4.

Transglycosylation by the exo-type of beta-N-acetyglucosaminidase from human
parotid saliva.

Watanabe T, Nakamura R, Tsunemitsu A.

PMID: 1057392 [PubMed - indexed for MEDLINE]


795. Biochemistry. 1975 Feb 11;14(3):543-54.

Nuclear magnetic resonance studies of 2'- and 3'-ribonucleotide structures in
solution.

Davies DB, Danyluk SS.

A systematic 220-MHz proton nuclear magnetic resonance (nmr) study has been made 
of all common purine and pyrimidine 2'(3')-ribonucleotides in D2O solutions at 20
plus or minus 2 degrees. Spectra for the entire series were measured under
similar conditions of concentration, temperature, and ionic strength, thereby
facilitating intercomparisons of spectral properties. Spectral assignments were
accomplished with the aid of selected 31P-1H decoupling experiments and accurate 
values of nmr parameters were derived by simulation-iteration procedures. A
detailed analysis of the coupling constants and chemical shifts permitted a
determination of conformational properties for ribose rings, exocyclic carbinol
and phosphate groups, and the orientation of base-ribose rings. Following
procedures described elsewhere, an evaluation was made of ribose ring
pseudorotational parameters for each 2'(3')-nucleotide. The results show that
both the degree of pucker and pseudorotational angle vary only slightly
throughout the entire series of molecules, and lie within the ranges found in the
crystalline state. Furthermore, the ribose rings are in rapid equilibrium between
N type (C(3')-ENDO, C(2')-exo) and S type (C(2')-endo, C(3')-exo) conformers, N
forms and is formed by S, with an S type conformer favored in purine
2'(3')-ribonucleotides (-60:40) while pyrimidines exhibit approximately equal
compositions. Thus, the phosphate location on the ring has less of an effect on
ring properties than the nature of the base ring. An analysis is also reported of
rotamer equilibria about C(4')-C(5'), C(2')-O(2'), and C(3')-O(3') bonds. For the
former the nmr coupling constant data are consistent with a predominant gg
rotamer (-70%) with gt and tg rotamers populated to the extent of -20 and -10%,
respectively. No correlation of the type seen for 5'-nucleotides appears to exist
between C(4')-C(5') gg population and ribose ring equilibrium composition. For
2'-nucleotides the 31P-H(2') coupling data indicate a preferred C(3')g, C(1')t
conformer about C(2')-O(2') in agreement with 13C nmr results. A less definitive 
rotamer analysis follows from observed J31P,H(3') values, but when these results 
are combined with relevant chemical shift data for deoxynucleosides and
nucleotides the evidence strongly points to essentially free rotation and
approximately equal rotamer populations about C(3')-O(3'). Chemical shift
differences between purine and pyrimidine 2'(3')-ribonucleotides are
qualitatively accounted for by "in-plane" purine diamagnetic anisotropy effects. 
Also, the greater magnitude for purine deshieldings in 2'(3')-nucleotides
relative to 5'-nucleotides is explained by a more favored syn:anti ratio in the
former in line with recent nucle

PMID: 1111570 [PubMed - indexed for MEDLINE]


796. J Biol Chem. 1975 Feb 10;250(3):912-7.

Purification of Keratan Sulfate-endogalactosidase and its action on keratan
sulfates of different origin.

Nakazawa K, Suzuki S.

A glycosidase which attacks corneal keratan sulfate was purified from extracts of
Pseudomonas sp. IFO-13309. When corneal keratan sulfate was degraded by the
purified enzyme, Sephadex G-50 chromatography indicated the presence of a number 
of oligosaccharides differing in size and sulfate content. The characterization
of two major fractions of the oligosaccharides indicated that the point of enzyme
attack is limited to the endo-beta-D-galactoside bonds in which nonsulfated
D-galactose residues participate. The enzyme, unlike ordinary
exo-beta-D-galactosidases, did not catalyze the hydrolysis of phenyl
beta-D-galactoside. Moreover, beta-D-galactosyl-(1 leads to
3)-2-acetamido-2-deoxy-beta-D-glucosyl-(1 leads to 3)-beta-D-galactosyl-(1 leads 
to 4)-D-glucose ("lacto-N-tetraose") was completely refractory to the action of
this enzyme, suggesting that a structure of the type, X-(1 leads to
3)-beta-D-galactosyl-(1 leads to 4)-Y, is not the only specificity-determining
factor, i.e. neighboring sugars, X and Y, or even larger portions of substrate
molecule must have an important effect. Compared with corneal keratan sulfate,
keratan sulfates from human nucleus pulposus and shark cartilage were attacked at
lower rates with a resultant production of oligosaccharides of relatively large
size. The result is in agreement with the view that considerable variations exist
in the structure of keratan sulfates of different origin, and further suggests
that the enzyme may serve as a useful reagent in studying these variations.

PMID: 234443 [PubMed - indexed for MEDLINE]


797. Hiroshima Daigaku Shigaku Zasshi. 1975;7(2):134-45.

[Studies on exo-type of Beta-N-acetylglucosaminidase in human parotid saliva.
Purification and some properties of the enzyme having isoelectric point 7.2
(author's transl)]

[Article in Japanese]

Watanabe T.

PMID: 1074988 [PubMed - indexed for MEDLINE]


798. J Bacteriol. 1974 Dec;120(3):1219-22.

Differential thermolability of exonuclease and endonuclease activities of the
recBC nuclease isolated from thermosensitive recB and recC mutants.

Kushner SR.

The recBC nuclease (also called exonuclease V) has been partially purified from
Escherichia coli K-12 strains carrying the thermosensitive recB270, recC271, and 
recB270 recC271 mutations. Of the multiple activities associated with the enzyme,
only the adenosine 5'-triphosphate-dependent exonucleolytic hydrolysis of duplex 
deoxyribonucleic acid (DNA) is abnormally thermolabile. The exo- and
endonucleolytic degradation of single-stranded DNA is no more thermosensitive
than that catalyzed by the wild-type enzyme. These results suggest that the
defects in genetic recombination, DNA repair, and the maintenance of cell
viability observed in recBC mutants in vivo result primarily from the specific
loss of adenosine 5'-triphosphate-dependent exonuclease active on duplex DNA.

PMCID: PMC245903
PMID: 4612008 [PubMed - indexed for MEDLINE]


799. Arch Oral Biol. 1974 Apr;19(4):331-4.

Heterogeneity of exo-type of beta-N-acetylglucosaminidase in human parotid
saliva.

Watanabe T, Iwamoto Y, Yasutake A, Tsunemitsu A.

Department of Preventive Dentistry, Hiroshima University School of Dentistry,
Hiroshima 734, Japan.

PMID: 12692918 [PubMed - indexed for MEDLINE]


800. J Dent Res. 1973 Jul-Aug;52(4):846.

Hydrolysis of glycol chitin by an endo-and exo-type of
beta-N-acetylglucosaminidase from human parotid saliva.

Watanabe T, Iwamoto Y, Tsunemitsu A.

PMID: 4515872 [PubMed - indexed for MEDLINE]


801. Indian J Exp Biol. 1973 May;11(3):199-206.

Effect of interferon on tumor forming ability of Walker carcinosarcoma 256 cells 
and secretion of exo-toxin by the bacillus, Clostridium welchii (type C).

Babbar OP, Singh DP, Bajpai SK.

PMID: 4361113 [PubMed - indexed for MEDLINE]


802. J Biochem. 1973 Jan;73(1):55-60.

Studies on N-acetyl- -D-glucosaminidase of Aspergillus oryzae. V. Demonstration
of an exo-type of the enzyme and determination of partial binding energy of the
ES-complex.

Mega T, Ikenaka T, Arita H, Fukukawa K, Matsushima Y.

PMID: 4690234 [PubMed - indexed for MEDLINE]


803. Hiroshima Daigaku Shigaku Zasshi. 1973;5(1):41-5.

[The heterogeneity of exo-type beta-N-acetylglucosaminidase from human parotid
saliva]

[Article in Japanese]

Watanabe T, Iwamoto Y, Yasutake A, Tsunemitsu A.

PMID: 4516853 [PubMed - indexed for MEDLINE]


804. Biochem J. 1972 Nov;130(2):453-65.

The dimensions and shapes of the furanose rings in nucleic acids.

Arnott S, Hukins DW.

A survey was made of the geometry of furanose rings in beta-nucleotides and
beta-nucleosides (as monomers related to nucleic acids) for which structures have
been determined by X-ray crystallography. Mean values, and estimated standard
deviations from them, were calculated for bond-lengths, bond-angles and
conformation-angles. For parameters with values dependent on ring-puckering,
separate calculations were made for each ring type. (The rings are puckered in
one of three conformations: C-2- or C-3-endo or C-3-exo; C-2-exo has not been
observed.) The results were used to compute standard furanose rings with
C-2-endo, C-3-endo and C-3-exo conformations for use in nucleic acid molecular
model-building. The survey also showed that the only other conformation-angle in 
nucleotides dependent on the furanose ring conformation corresponds to the
relative orientation of the purine (or pyrimidine) base and the ring.

PMCID: PMC1174425
PMID: 4664573 [PubMed - indexed for MEDLINE]


805. J Bacteriol. 1972 Aug;111(2):443-6.

Cell wall alterations associated with the hyperproduction of extracellular
enzymes in Neurospora crassa.

Gratzner HG.

A pleiotropic mutation in Neurospora (exo-1), which confers derepression of
alpha-amylase, glucoamylase, beta-fructofuranosidase, and trehalase, appears to
also affect the composition of the cell wall. Segregants resulting from the
backcross of exo-1 to the wild-type strain from which it derived are altered in
the ratio of galactosamine to glucosamine in hydrolysates of isolated cell walls.
Conidial cell walls exhibit a marked decrease in the amount of galactosamine in
both exo-1 and exo-1(+) strains. Increased levels (approximately sevenfold) of
amylase are found in conidia of exo-1, as compared with those of exo-1(+).

PMCID: PMC251302
PMID: 4262302 [PubMed - indexed for MEDLINE]


806. Biochim Biophys Acta. 1972 Feb 28;258(2):541-7.

A new type of enzyme, and exo-splitting -1,3 glucanase from non-induced cultures 
of Aspergillus nidulans.

Zonneveld BJ.

PMID: 4622000 [PubMed - indexed for MEDLINE]


807. Infect Immun. 1970 Dec;2(6):691-697.

Monospecific Equine Antiserum Against Cholera Exo-Enterotoxin.

Finkelstein RA.

Department of Microbiology, The University of Texas (Southwestern) Medical
School, Dallas, Texas 75235.

An antiserum specific for Vibrio cholerae exo-enterotoxin was produced by
immunization of a horse with purified choleragenoid, a natural cholera toxoid.
The serum has a high titer against the toxin antigen in passive hemagglutination 
tests and a respectable antipermeability factor activity. It also passively
protected against choleragen-induced mouse foot edema. The serum was found to be 
useful for assaying toxin antigen in crude and refined products by in vitro tests
such as radial immunodiffusion, Lf, and quantitative precipitin titrations. Based
upon experimental observations, the serum was defined as containing 1,000
flocculating units of anticholera toxin antibody per ml. A flocculating dose, or 
Lf, of choleragen approximates 1 mug, and that of choleragenoid is 0.625 mug.
Formalin toxoids behaved like the parent toxin in these tests. The serum contains
approximately 2.2 mg of antibody protein per ml, which appears to be largely, if 
not entirely, of the Ig(T) type. It is suggested that this serum, which is
available in considerable supply, be considered for use as a reference cholera
antitoxin. The horse developed symptoms of anaphylactic shock during
immunization, suggesting the need for caution in projected studies on
toxoid-induced immunity in man.

PMCID: PMC416076
PMID: 16557901 [PubMed - as supplied by publisher]


808. FEBS Lett. 1970 Sep 24;10(2):81-84.

[No title available]

[Article in German]

Saenger W.

Max-Planck-Institut fur expetimentelle Medizin, Abteilung Chemie, 34,
Hermann-Rein-Str. 3, Gottingen, Germany

5'-Methylammonium-5'-deoxyadenosine is a poor subsrate for adenosine desaminase. 
The nucleoside was crystallized from aqueous methanol as its iodide monohydrate
in space group P2(1). The structure was solved from three dimensional X-ray data 
and refined to R = 4.1%. The structure of the nucleoside is determined by an
intramolecular hydrogen bond from N5' to N3 which implies that (1) the edenine
heterocycle is in syn position with respect to the sugar moiety; (2) the ribose
pucker is C2'-exo, C3'-endo and (3) the conformation about the C4'-C5' bond is
trans, gauche. The water of hydration is fourfold disordered and forms a linear
hydrophilic region which is surrounded by nucleoside molecules in a zigzag
arrangement. The adenine heterocycles are not stacked and in closer than Van der 
Waals distance with iodine ions suggesting a charge transfer type interaction.

PMID: 11945362 [PubMed - as supplied by publisher]


809. Infect Immun. 1970 May;1(5):464-467.

Antitoxic Immunity in Experimental Cholera: Observations with Purified Antigens
and the Ligated Ileal Loop Model.

Finkelstein RA.

Department of Microbiology, The University of Texas (Southwestern) Medical School
at Dallas, Dallas, Texas 75235.

Rabbits immunized with purified antigens, choleragen and choleragenoid (cholera
exo-enterotoxin and natural toxoid), were found to resist intra-intestinal
challenge with a variety of strains of cholera vibrios. The resistance occurred
in the absence of a significant antibacterial antibody response. In fact, the
antigens were incapable of protecting mice against the septicemic infection of
the standard mouse protection test used for bioassay of conventional cholera
vaccines. The immunized rabbits did, however, develop a significant antitoxin
titer demonstrable by passive hemagglutination, although the presence of
antitoxic coproantibody could not be established conclusively. The results
suggest the potential value of antitoxic immunity in cholera and are consistent
with the existence of only one serological type of cholera exo-enterotoxin.

PMCID: PMC415925
PMID: 16557759 [PubMed - as supplied by publisher]


810. J Med Chem. 1970 May;13(3):467-70.

Irreversible enzyme inibitiors. CLXXII. Proteolytic enzymes. XVI. Covalent
bonding of the sulfonyl fluoride group to serine outside the active site of
alpha-chymotrypsin by exo-type active-site directed irreversible inhibitors.

Cardinaud R, Baker BR.

PMID: 5460302 [PubMed - indexed for MEDLINE]


811. Proc Natl Acad Sci U S A. 1970 Apr;65(4):962-9.

A mechanism for penicillinasesecretion in Bacillus licheniformis.

Sargent MG, Lampen JO.

Cell-bound isozymes of penicillinase are distinguished from extracellular enzyme 
by their capacity to bind deoxycholate and to elute with an apparent molecular
weight of 45,000 on gel filtration in its presence. By methods that are unlikely 
to involve changes in primary structure, the cell-bound forms (both from the
plasma membrane and from the periplasmic vesicles) can be converted to forms that
are very similar if not identical to the exo-form (i.e., eluting with a molecular
weight of 24,000 in the presence and absence of deoxycholate). In the case of
plasma membrane penicillinase, addition of 25 per cent potassium phosphate at pH 
9.0 leads to a 65 per cent conversion in 20 minutes at 30 degrees . Vesicle
fraction penicillinase can be converted by pH 9.0 treatment alone. We suggest
that the conversion involves a change from a hydrophobic to a hydrophilic
conformational type, and that this is the crucial step for enzyme secretion in
microorganisms. A model is presented to account for existing data in which we
postulate that monomers of the newly synthesized penicillinase in an extended
hydrophobic conformation are inserted into the membrane at special growing points
where they may change to a hydrophilic exoform, or polymerize to the major plasma
membrane type of penicillinase.

PMCID: PMC283010
PMID: 5266165 [PubMed - indexed for MEDLINE]


812. J Med Chem. 1965 Jul;8(4):525-8.

exo-3-chloro-endo-6-cyano-2-norbornanone o-(methylcarbamoyl)oxime. A new type of 
methylcarbamate with acaricidal activity.

Payne LK Jr, Stansbury HA Jr, Weiden MH.

PMID: 5883719 [PubMed - indexed for MEDLINE]