J Biol Chem, 2002 May 31, 277(22), 19811 - 6 Epub 2002 Mar 23. Structure of formamidopyrimidine-DNA glycosylase covalently complexed to DNA; Gilboa R et al.; Formamidopyrimidine-DNA glycosylase (Fpg) is a DNA repair enzyme that excises oxidized purines from damaged DNA . The Schiff base intermediate formed during this reaction between Escherichia coli Fpg and DNA was trapped by reduction with sodium borohydride, and the structure of the resulting covalently cross-linked complex was determined at a 2.1-A resolution . Fpg is a bilobal protein with a wide, positively charged DNA-binding groove . It possesses a conserved zinc finger and a helix-two turn-helix motif that participate in DNA binding . The absolutely conserved residues Lys-56, His-70, Asn-168, and Arg-258 form hydrogen bonds to the phosphodiester backbone of DNA, which is sharply kinked at the lesion site . Residues Met-73, Arg-109, and Phe-110 are inserted into the DNA helix, filling the void created by nucleotide eversion . A deep hydrophobic pocket in the active site is positioned to accommodate an everted base . Structural analysis of the Fpg-DNA complex reveals essential features of damage recognition and the catalytic mechanism of Fpg. Cancer Res, 2002 Mar 15, 62(6), 1624 - 31 Characterization of human UMP/CMP kinase and its phosphorylation of D- and L-form deoxycytidine analogue monophosphates; Liou JY et al.; Pyrimidine nucleoside monophosphate kinase {UMP/CMP kinase (UMP/CMPK);EC 2.7.4.14} plays a crucial role in the formation of UDP, CDP, and dCDP, which are required for cellular nucleic acid synthesis . Several cytidine and deoxycytidine analogues are important anticancer and antiviral drugs . These drugs require stepwise phosphorylation to their triphosphate forms to exert their therapeutic effects . The role of UMP/CMPK for the phosphorylation of nucleoside analogues has been indicated . Thus, we cloned the human UMP/CMPK gene, expressed it in Escherichia coli, and purified it to homogeneity . Its kinetic properties were determined . UMP and CMP proved to be far better substrates than dCMP . UMP/CMPK used all of the nucleoside triphosphates as phosphate donors, with ATP and dATP being the best donors and CTP being the poorest . Furthermore, UMP/CMPK was able to phosphorylate all of the deoxycytidine analogue monophosphates that we tested . The relative efficiency was as follows: arabinofuranosyl-CMP > dCMP > beta-L-2',3'-dideoxy-3'-thia-CMP > Gemcitabine monophosphate > beta-D-2',3'-dideoxy-CMP; beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CMP; beta-L-2',3'-dideoxy-5-fluoro-3'-thia-CMP > beta-L-2',3'-dideoxy-CMP > beta-L-dioxolane-CMP . By comparing the relative V(max)/K(m) values of D- and L-form dideoxy-CMP, we showed that this kinase lacked stereoselectivity . Reducing agents, such as DTT, 2-mercaptoethanol, and thioredoxin, were able to activate this enzyme, suggesting that its activity may be regulated by redox potential in vivo . UMP/CMPK localized predominantly to the cytoplasm . In addition, 196-amino acid UMP/CMPK was the actual form of UMP/CMPK, rather than the 228-amino acid form as suggested before. Am J Vet Res, 2002 Mar, 63(3), 448 - 53 Apoptosis of bovine neutrophils during mastitis experimentally induced with Escherichia coli or endotoxin; Van Oostveldt K et al.; OBJECTIVE: To determine whether apoptosis of neutrophils was accelerated during mastits experimentally induced by use of Escherichia coli or E coli endotoxin and whether differences were apparent in the response to E coli or endotoxin . ANIMALS: 11 healthy lactating Holstein cows . PROCEDURE: Blood samples were collected from cows at various intervals after intramammary inoculation with E coli or endotoxin . Percentage of apoptotic neutrophils detected after in vitro incubation for 3 hours was determined . Fluorescein isothiocyanate-labeled annexin-V in combination with propidium iodide was used to distinguish apoptosis and necrosis of neutrophils . Total and differential circulating leukocyte counts and rectal temperature were determined at the time of collection of blood samples . Milk yield and milk somatic cell counts were determined at the time of milking . RESULTS: Inoculation of endotoxin did not accelerate in vitro induction of neutrophil apoptosis . However, inoculation of E coli increased the percentage of apoptotic neutrophils . At 18 hours after inoculation, 20% of the neutrophils were apoptotic, compared with 5% before inoculation . Milk somatic cell count and rectal temperature increased, milk production and total leukocyte count decreased, and percentage of immature neutrophils increased after inoculation with E coli or endotoxin . However, kinetics of the responses were more rapid, more severe, and of shorter duration during endotoxin-induced mastitis . CONCLUSIONS AND CLINICAL RELEVANCE: In vitro induction of apoptosis of neutrophils was accelerated only during E coli-induced mastitis and not during endotoxin-induced mastitis . Endotoxin inoculation as a model for studying coliform mastitis in dairy cows should be viewed with caution. Mol Cells, 2002 Feb 28, 13(1), 154 - 6 Electron paramagnetic resonance study reveals a putative iron-sulfur cluster in human rpS3 protein; Lee CH et al.; The human ribosomal protein S3 (rpS3) functions as a component of the 40S subunit and as a UV DNA repair endonuclease . This enzyme has an endonuclease activity for UV-irradiated and oxidatively damaged DNAs . DNA repair endonucleases recognize a variety of UV and oxidative base damages in DNA from E . coli to human cells . E . coli endonuclease III is especially known to have an iron-sulfur cluster as a co-factor . Here, we tried an electron paramagnetic resonance (EPR) method for the first time to observe a known iron-sulfur cluster signal from E . coli endonuclease III that was previously reported . We compared it to the human rpS3 in order to find out whether or not the human protein contains an iron-sulfur cluster . As a result, we succeeded in observing a Fe EPR signal that is apparently from an iron-sulfur cluster in the human rpS3 endonuclease . The EPR signal from the human enzyme, consisting of three major parts, is similar to that from the E . coli enzyme, but it has a distinct extra peak. Mol Cells, 2002 Feb 28, 13(1), 118 - 24 Refolding of the catalytic and hinge domains of human MT1-mMP expressed in Escherichia coli and its characterization; Koo HM et al.; The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli . The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea . During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form . By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290 . These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding . Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay . In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2 . These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP. Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 703 - 5 {Isolation, purification and renaturation of recombinant-DNA-derived porcine somatotropin}; Li Z et al.; Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described . The r-pGH is found in genetic engineering E . coli as the form of inclusion bodies . Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected . Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction . Inclusion bodies were dissolved using 6 mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl . The Guanidine/HCl protein mixture were diluted by renaturation solution . Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained . Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained. Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 678 - 82 {Bone-inducing activity of human bone morphogenetic protein-2 102 peptide}; Zhang B et al.; To analyze the bone-inducing activity of C terminal of hBMP-2 and get a new recombinant product of hBMP-2, the gene encoding 102 aa of hBMP-2 mature peptide C terminal was cloned and expressed in E . coli and the first Cys was mutated with Ser . The fragments encoding the target peptide were amplified and cloned into heat-inducible expression vector pDH and transformed into E . coli DH5 alpha . After induction, a new protein bond appeared on the SDS-PAGE . The expressed products amounted to 30% of the total bacterial protein, which existed in the form of inclusion body . The products of bacterial lysates were purified through the ion-exchange chromatography . The denatured proteins were dialysed and diluted directly into the refolding buffer . The renatured products were implanted into mouse thigh muscles to analyze their bone-inducing activity respectively . The results of histological assay showed that the 102 peptide of hBMP-2 could ectopically induce formation of bone, while the mutated 102 peptide of hBMP-2 could not . It suggested hBMP-2 102 peptide still had bone-inducing activity . The first Cys of hBMP-2 mature peptide might be necessary for integrity of three pairs of disulfide bond, and also essential for bone-inducing activity of hBMP-2. Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 631 - 4 {Cloning and expression of heptad repeat regions of the Newcastle disease virus fusion protein}; Liu YF et al.; Two heptad repeat regions (HR1, HR2) from F clone of the virulent and avirulent NDV were cloned, expressed with vector pGEX-6p-1 in E . coli BL21 (DE3), and purified the fusion protein by Glutathiones Sepharose 4B Column . After cleaved by precission protease, the desired protein was purified by Glutathione Sepharose 4B Column and high temperature . Purified HR1 and HR2 were analyzed by Mass spectrum, the results shows that the Molecular Weights of HR1 and HR2 of virulent NDV are 7103 and 6301, and the Molecular Weights of HR1 and HR2 of avirulent NDV is 7107 and 6309. Sheng Wu Gong Cheng Xue Bao, 2001 Nov, 17(6), 608 - 12 {Advances in in vitro refolding of inclusion body proteins}; Fang M et al.; Overexpression of recombinant proteins in E . coli often results in formation of insoluble, inactive inclusion bodies . These inclusion bodies, which contain the recombinant proteins in a highly enriched form, can be isolated by solid/liquid separation . After solubilization, active proteins can be generated through an appropriate refolding process . Within the last decade, specific strategies and methods have been developed for preparing active recombinant proteins from inclusion bodies . Recent developments in renaturation procedure include the inhibition of aggregation during refolding by the application of low molecular weight additives and matrix-bound renaturation techniques. Curr Microbiol, 2002 Apr, 44(4), 291 - 6 HtpG plays a role in cold acclimation in cyanobacteria; Hossain MM et al.; Abstracts.The heat shock protein HtpG is homologous to members of the Hsp90 protein family of eukaryotes and is essential for basal and acquired thermotolerances in cyanobacteria . In this study we have examined the role of HtpG in the cyanobacterium, Synechococcus sp . PCC 7942, in the acclimation to low temperatures . The inactivation of the htpG gene resulted in severe inhibition of cell growth and of the photosynthetic activity when the htpG mutant was shifted to 16 degrees C from 30 degrees C . Wild-type cells were able to resume growth without a lag period when shifted to 30 degrees C after 5 days at 16 degrees C, while the mutant displayed a detectable lag . The HtpG protein was induced in the wild-type cells at 16 degrees C . Electrophoresis in the absence of sodium dodecyl sulfate (SDS) showed that a novel, high-molecular-weight complex containing GroEL and DnaK accumulated at 16 degrees C, but the accumulation was strongly inhibited in the htpG mutant . Our results demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures. Curr Microbiol, 2002 Apr, 44(4), 273 - 9 Construction of double-copy glucose isomerase gene engineering strain of Streptomyces diastaticus by homologous recombination; Zhang Y et al.; The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance ( tsr(R)) gene into the E . coli plasmid pUB1-GI1 . Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI . After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr(R) transformants were obtained, further based on enzyme assays . These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination . This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI ( GIG138P, GI1) gene . Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively . The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome . Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. World J Surg, 2002 Apr, 26(4), 448 - 50 Epub 2002 Feb 04. Effect of bile acid replacement on endotoxin-induced tumor necrosis factor-alpha production in obstructive jaundice; Sheen-Chen SM et al.; There is a high incidence of perioperative morbidity and mortality in patients with obstructive jaundice due to sepsis . Tumor necrosis factor-a (TNF-alpha) is considered a crucial mediator in inducing and processing the inflammatory cascade . We hypothesize that obstructive jaundice leads to an increased endotoxin-induced TNF-alpha production and that intestinal bile acid replacement can prevent this phenomenon . Sprague-Dawley rats were randomized to three groups of 12 animals each . Group 1 underwent common bile duct ligation (CBDL) with oral intestinal bile acid (deoxycholic acid 5 mg/100 g body weight/3 times daily) replacement (CBDL + bile acid); group 2 underwent common bile duct ligation with the same amount of normal saline replacement orally (CBDL + saline); and group 3 underwent a sham operation (sham control) . After 2 days, endotoxin was given to the animals, and after 90 minutes, tissues (liver and lung) and blood were collected for checking the TNF-alpha levels and biochemical analyses . Comparisons among these three groups were performed and recorded . While serum and tissue (liver and lung) TNF-alpha levels of group 2 (CBDL + saline) were significantly increased after endotoxin challenge, these elevations were reduced to control levels (sham control) following oral replacement of intestinal bile acid (CBDL + bile acid) . Obstructive jaundice leads to an increased endotoxin-induced TNF-alpha production and intestinal bile acid replacement can inhibit this phenomenon. Protein Sci, 2002 Apr, 11(4), 924 - 32 Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI); Buczek O et al.; A major goal of this paper was to estimate a dynamic range of equilibrium constant for the opening of a single peptide bond in a model protein, bovine pancreatic trypsin inhibitor (BPTI) . Ten mutants of BPTI containing a single Xaa-->Met substitution introduced in different parts of the molecule were expressed in Escherichia coli . The mutants were folded, purified to homogeneity, and cleaved with cyanogen bromide to respective cleaved forms . Conformation of the intact mutants was similar to the wildtype, as judged from their circular dichroism spectra . Substantial conformational changes were observed on the chemical cleavage of three single peptide bonds--Met46-Ser, Met49-Cys, and Met53-Thr--located within the C-terminal helix . Cleavage of those peptide bonds caused a significant destabilization of the molecule, with a drop of the denaturation temperature by 56.4 degrees C to 68 degrees C at pH 4.3 . Opening of the remaining seven peptide bonds was related to a 10.8 degrees C to 39.4 degrees C decrease in T(den) . Free energies of the opening of 10 single peptide bonds in native mutants (Delta G(op,N)) were estimated from the thermodynamic cycle that links denaturation and cleavage free energies . To calculate those values, we assumed that the free energy of opening of a single peptide bond in the denatured state (Delta G(op,D)) was equal to -2.7 kcal/mole, as reported previously . Calculated Delta G(op,N) values in BPTI were in the range from 0.2 to 10 kcal/mole, which was equivalent to a >1 million-fold difference in equilibrium constants . The values of Delta G(op,N) were the largest for peptide bonds located in the C-terminal helix and significantly lower for peptide bonds in the beta-structure or loop regions . It appears that opening constants for single peptide bonds in various proteins span across 33 orders of magnitude . Typical equilibrium values for a single peptide bond opening in a protein containing secondary structure elements fall into negligibly low values, from 10(-3) to 10(-8), and are efficient to ensure stability against proteolysis. Protein Sci, 2002 Apr, 11(4), 883 - 93 Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site; Korndorfer IP et al.; The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns . The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution . We report here the crystal structures of the streptavidin mutants '1' and '2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II . Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site . The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 A . Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket--comprising residues 45 through 52--adopts an 'open' conformation in all four subunits within the asymmetric unit . The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin . Our findings suggest that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide . Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism. Protein Sci, 2002 Apr, 11(4), 875 - 82 Complex behavior in solution of homodimeric SecA; Woodbury RL et al.; SecA, a homodimeric protein involved in protein export in Escherichia coli, exists in the cell both associated with the membrane translocation apparatus and free in the cytosol . SecA is a multifunctional protein involved in protein localization and regulation of its own expression . To carry out these functions, SecA interacts with a variety of proteins, phospholipids, nucleotides, and nucleic acid and shows two enzymic activities . It is an ATPase and a helicase . Its role during protein localization involves interaction with the precursor polypeptides to be exported, the cytosolic chaperone SecB, and the SecY subunit of the membrane-associated translocase, as well as with acidic phospholipids . At the membrane, SecA undergoes a cycle of binding and hydrolysis of ATP coupled to conformational changes that result in translocation of precursors through the cytoplasmic membrane . The helicase activity of SecA and its affinity for its mRNA are involved in regulation of its own expression . SecA has been reported to exist in at least two conformational states during its functional cycle . Here we have used analytical centrifugation, as well as column chromatography coupled with multi-angle light scatter, to show that in solution SecA undergoes at least two monomer-dimer equilibrium reactions that are sensitive to temperature and to concentration of salt. Protein Sci, 2002 Apr, 11(4), 806 - 19 Analysis of serine proteinase-inhibitor interaction by alanine shaving; Buczek O et al.; We analyzed the energetic importance of residues surrounding the hot spot (the P(1) position) of bovine pancreatic trypsin inhibitor (BPTI) in interaction with two proteinases, trypsin and chymotrypsin, by a procedure called molecular shaving . One to eight residues of the structural epitope, composed of two extended and exposed loops, were mutated to alanine(s) . Although truncation of the side chains of residues surrounding the P(1) position to methyl groups caused a decrease in Delta G(den) values up to 6.4 kcal mole(-1), it did not influence the overall conformation of the inhibitor . We found that the replacement of up to six residues with alanines was fully additive at the level of protein stability . To analyze the influence of the structural epitope on the association energy, we determined association constants for BPTI variants and both enzymes and applied the additivity analysis . Shaving of two binding loops led to a progressive drop in the association energy, more pronounced for trypsin (decrease up to 9.6 kcal mole(-1)) than chymotrypsin (decrease up to 3.5 kcal mole(-1)) . In the case of extensively mutated variants interacting with chymotrypsin, the association energies agreed very well with the values calculated from single mutational effects . However, when P(1)-neighboring residues were shaved to alanine(s), their contribution to the association energy was not fully removed because of the presence of methyl groups and main chain-main chain intermolecular hydrogen bonds . Moreover, the hot spot had a different contribution to the complex stability in the fully shaved BPTI variant compared with the wild type, which was caused by perturbations of the P(1)-S(1) electrostatic interaction. Protein Sci, 2002 Apr, 11(4), 778 - 94 Fine-tuning function: correlation of hinge domain interactions with functional distinctions between LacI and PurR; Swint-Kruse L et al.; LacI and PurR are highly homologous proteins . Their functional units are homodimers, with an N-terminal DNA binding domain that comprises the helix-turn-helix (HTH), N-linker, and hinge regions from both monomers . Hinge structural changes are known to occur upon DNA dissociation but are difficult to monitor experimentally . The initial steps of hinge unfolding were therefore examined using molecular dynamics simulations, utilizing a truncated, chimeric protein comprising the LacI HTH/N-linker and PurR hinge . A terminal Gly-Cys-Gly was added to allow "dimerization" through disulfide bond formation . Simulations indicate that differences in LacI and PurR hinge primary sequence affect the quaternary structure of the hinge x hinge' interface . However, these alternate hinge orientations would be sterically restricted by the core domain . These results prompted detailed comparison of recently available DNA-bound structures for LacI and truncated LacI(1-62) with the PurR structure . Examination revealed that different N-linker and hinge contacts to the core domain of the partner monomer (which binds effector molecule) affect the juxtapositions of the HTH, N-linker, and hinge regions in the DNA binding domain . In addition, the two full-length repressors exhibit significant differences in the interactions between the core and the C-linker connection to the DNA binding domain . Both linkers and the hinge have been implicated in the allosteric response of these repressors . Intriguingly, one functional difference between these two proteins is that they exhibit opposite allosteric response to effector . Simulations and observed structural distinctions are correlated with mutational analysis and sequence information from the LacI/GalR family to formulate a mechanism for fine-tuning individual repressor function. J Biol Chem, 2002 Jun 21, 277(25), 22648 - 55 Epub 2002 Mar 21. Catalytic properties of rice alpha-oxygenase . A comparison with mammalian prostaglandin H synthases; Koeduka T et al.; Long-chain fatty acids can be metabolized to C(n)(-1) aldehydes by alpha-oxidation in plants . The reaction mechanism of the enzyme has not been elucidated . In this study, a complete nucleotide sequence of fatty acid alpha-oxygenase gene in rice plants (Oryza sativa) was isolated . The deduced amino acid sequence showed some similarity with those of mammalian prostaglandin H synthases (PGHSs) . The gene was expressed in Escherichia coli and purified to apparently homogeneous state . It showed the highest activity with linoleic acid and predominantly formed 2-hydroperoxide of the fatty acid (C(n)), which is then spontaneously decarboxylated to form corresponding C(n)(-1) aldehyde . With linoleic or linoleic acids as a substrate, rice alpha-oxygenase formed no product having a lambda(max) at approximately 234 nm, which indicated that the enzyme could not oxygenize the pentadiene system in the substrate . The spectroscopic feature of the purified enzyme in its ferrous state is similar to that of mammalian PGHS, whereas that of dithionite-reduced state showed significant difference . Site-directed mutagenesis revealed that His-158, Tyr-380, and Ser-558 were essential for the alpha-oxygenase activity . These residues are conserved in PGHS and known as a heme ligand, a source of a radical species to initiate oxygenation reaction and a residue involved in substrate binding, respectively . This finding suggested that the initial step of the oxygenation reaction in alpha-oxygenase has a high similarity with that of PGHS . The rice alpha-oxygenase activity was inhibited by imidazole but hardly inhibited by nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen, and flurbiprofen, which are known as typical PGHS inhibitors . In addition, peroxidase activity could not be detected with alpha-oxygenase when palmitic acid 2-hydroperoxide was used as a substrate . From these findings, the catalytic resemblance between alpha-oxygenase and PGHS seems to be evident, although there still are differences in their substrate recognitions and peroxidation activities. Mutat Res, 2002 Mar 25, 515(1-2), 73 - 83 Dinitropyrenes induce gene mutations in multiple organs of the lambda/lacZ transgenic mouse (Muta Mouse); Kohara A et al.; Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust . DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations . To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human . For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs . A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks . Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis . The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E . coli . In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E . coli . Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis . Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene . Three-four-fold increases were observed in stomach for both genes . A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene . The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice . Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups . The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%) . The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule) . The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo. Curr Biol, 2002 Mar 19, 12(6), 439 - 45 Chromatin motion is constrained by association with nuclear compartments in human cells; Chubb JR et al.; BACKGROUND: In comparison with many nuclear proteins, the movement of chromatin in nuclei appears to be generally constrained . These restrictions on motion are proposed to reflect the attachment of chromatin to immobile nuclear substructures . RESULTS: To gain insight into the regulation of chromosome dynamics by nuclear architecture, we have followed the movements of different sites in the human genome in living cells . Here, we show that loci at nucleoli or the nuclear periphery are significantly less mobile than other, more nucleoplasmic loci . Disruption of nucleoli increases the mobility of nucleolar-associated loci . CONCLUSIONS: This is the first report of distinct nuclear substructures constraining the movements of chromatin . These constraints reflect the physical attachment of chromatin to nuclear compartments or steric impairment caused by local ultrastructure . Our data suggest a role for the nucleolus and nuclear periphery in maintaining the three-dimensional organization of chromatin in the human nucleus. J Nat Prod, 2002 Mar, 65(3), 377 - 8 Tunichrome sp-1: new pentapeptide tunichrome from the hemocytes of Styela plicata; Tincu JA et al.; A modified pentapeptide has been isolated from the hemocytes of the ascidian Styela plicata . The structure of the peptide was determined by Edman sequence analysis, mass spectrometry, and NMR spectroscopy with the stereochemistry assigned by acid hydrolysis followed by both (a) GC-MS of the volatile amino acid derivatives on a chiral column and (b) ultrasensitive detection of fluorescent diasteromeric derivatives of the component amino acids after reversed-phase HPLC . The peptide L-DOPA-L-DOPA-Gly-L-Pro-dcdeltaDOPA (where DOPA = 3,4-dihydroxyphenylalanine and dcdeltaDOPA = decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine) we designate as tunichrome Sp-1. J Nat Prod, 2002 Mar, 65(3), 306 - 13 Rare sesquiterpenes from the algicolous fungus Drechslera dematioidea; Osterhage C et al.; From the inner tissue of the marine red alga Liagora viscida the fungus Drechslera dematioidea was isolated . After mass cultivation, the fungus was investigated for its secondary metabolite content, and 10 new sesquiterpenoids {isosativenetriol (1), drechslerines A (2) and B (3), 9-hydroxyhelminthosporol (5), drechslerines C-G (6-10), and sativene epoxide (12)} were isolated . Compounds 8 and 10 exhibited antiplasmodial activity against Plasmodium falciparum strains K1 and NF54 . The known compounds helminthosporol (4), cis-sativenediol (11), isocochlioquinone A (14), isocochlioquinone C (15), and cochlioquinone B (16) were also isolated . All structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR and MS. Eur Biophys J, 2002 Feb, 30(8), 559 - 70 A model for the thermal unfolding of amicyanin; La RC et al.; In the present study the thermal unfolding of amicyanin has been addressed using differential scanning calorimetry, fluorescence emission, optical density, circular dichroism and electron paramagnetic resonance . The combined use of these techniques has allowed us to assess, during unfolding of the protein, its global conformational changes in relationship to the local structural modifications occurring in the copper environment and close to the fluorescent chromophore Trp46 of the protein . The thermal transition from the native to the denatured state is on the whole irreversible and occurs in the temperature range between 65 and 72 degrees C, depending on the scan rate and technique used . Amicyanin as a whole shows a complex unfolding pathway, which has been described in terms of a three-step model: N <--> U --> F1 --> F2 . According to this model, in the first step the native state of the protein (N) goes reversibly to the unfolded state (U), in the second one U goes irreversibly to F1 and, finally, the state F2 is irreversibly reached in the third step . Kinetic factors prevent the experimental separation of these steps . Nevertheless, the comparison of the data obtained with the different experimental techniques testifies the presence, within the unfolding pathway, of some intermediate states, although not sufficiently long-lived to allow a detailed characterization . A first intermediate transient state has been identified around 68 degrees C, whereas a second one can be related to conformational changes that involve the copper environment . Finally, an exothermal phenomenon, caused by irreversible rearrangements of the melted polypeptide chains, is evidenced . In addition, according to the EPR findings, the type 1 copper ion, which is four-fold coordinated by two N and two S atoms in a distorted tetrahedron in the native state of the protein, shows type 2 features after denaturation . A mathematical model simulating the unfolding Cp(exc) profile has been also developed. Tohoku J Exp Med, 2001 Dec, 195(4), 237 - 43 Different binding property of verotoxin-1 and verotoxin-2 against their glycolipid receptor, globotriaosylceramide; Itoh K et al.; We determined the binding of verotoxin-1 (VT1) and verotoxin-2 (VT2) against globotriaosylceramide (Gb3) by a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) . Ethanolic solution of Gb3 containing cholesterol and phosphatidylcholine was passively adsorbed onto the wells of microtiter plate, and Gb3-bound VT1 and VT2 were detected by anti-VT1 and anti-VT2 mAbs, respectively . Although both VT1 and VT2 reacted with Gb3 in a concentration dependent manner, terminal galactose requirement for Gb3 binding was also different from each other . Pretreatment of VT1 showed the inhibitory effect on the binding of VT2 to Gb3, while the VT2-pretreatment showed no inhibitory effect on VT1 binding to Gb3 . This was not due to the replacement of Gb3-bound VT2 with post-treated VT1 . These results suggest that the binding sites of VT1 and VT2 on Gb3 are not identical to each other. J Am Soc Mass Spectrom, 2002 Mar, 13(3), 221 - 31 Visualization and analysis of molecular scanner peptide mass spectra; Muller M et al.; The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner . Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions . The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid . First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise . Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints . Then, the correlation between neighboring spectra is used to recalibrate the peptide masses . Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented . This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel. Autoimmunity, 2001, 34(3), 177 - 85 A peptide fragment of beta cardiac myosin heavy chain (beta-CMHC) can provoke autoimmune myocarditis as well as the corresponding alpha cardiac myosin heavy chain (alpha-CMHC) fragment; Kohno K et al.; The validity of the general belief that alpha cardiac myosin heavy chain (alpha-CMHC) is primarily responsible for causing experimental autoimmune myocarditis because of the more profound tolerance induction to beta-CMHC due to its expression during the embryonic stage has been examined . In order to completely avoid cross-contamination among components of the two myosin heavy chains, recombinant myosin fragments were synthesized in Escherichia coli using cDNA fragments of rat alpha- and beta-CMHC cloned by reverse transcription polymerase chain reaction (RT-PCR) . Two fragments corresponding to amino acid residues 1107-1164 derived from alpha- and beta-heavy chains were equally capable of provoking severe myocarditis in Lewis rats when immunized in complete Freund's adjuvant . No significant differences in the severity, as judged from histological scoring, were observed between the diseases induced by the two different peptide fragments, indicating conclusively that beta-CMHC is as pathogenic as alpha-CMHC. Arch Microbiol, 2002 Mar, 177(3), 209 - 16 Epub 2002 Jan 11. Signal peptides of secreted proteins of the archaeon Sulfolobus solfataricus: a genomic survey; Albers SV et al.; Analysis of the recently completed genome sequence of the thermoacidophilic archaeon Sulfolobus solfataricus reveals that about 4.2% of its proteome consists of putative secretory proteins with signal peptides . This includes members of the four major classes of signal peptides: secretory signal peptides, twin-arginine signal peptides, possible lipoprotein precursors, and type IV pilin signal peptides . The latter group is surprisingly large compared to the size of the groups in other organisms and seems to be used predominately for a subset of extracellular substrate-binding proteins. Nature, 2002 Mar 21, 416(6878), 323 - 6 Reduced adaptation of a non-recombining neo-Y chromosome; Bachtrog D et al.; Sex chromosomes are generally believed to have descended from a pair of homologous autosomes . Suppression of recombination between the ancestral sex chromosomes led to the genetic degeneration of the Y chromosome . In response, the X chromosome may become dosage-compensated . Most proposed mechanisms for the degeneration of Y chromosomes involve the rapid fixation of deleterious mutations on the Y . Alternatively, Y-chromosome degeneration might be a response to a slower rate of adaptive evolution, caused by its lack of recombination . Here we report patterns of DNA polymorphism and divergence at four genes located on the neo-sex chromosomes of Drosophila miranda . We show that a higher rate of protein sequence evolution of the neo-X-linked copy of Cyclin B relative to the neo-Y copy is driven by positive selection, which is consistent with the adaptive hypothesis for the evolution of the Y chromosome . In contrast, the neo-Y-linked copies of even-skipped and roundabout show an elevated rate of protein evolution relative to their neo-X homologues, probably reflecting the reduced effectiveness of selection against deleterious mutations in a non-recombining genome . Our results provide evidence for the importance of sexual recombination for increasing and maintaining the level of adaptation of a population. J Virol, 2002 Apr, 76(8), 3678 - 87 Herpes simplex virus vectors elicit durable immune responses in the presence of preexisting host immunity; Brockman MA et al.; Herpes simplex virus (HSV) recombinants are being developed as vaccine vectors for the expression of heterologous antigens . There is concern, however, that preexisting HSV immunity may decrease their effectiveness . We have addressed this issue in an animal model . Immunized mice were inoculated with a replication-defective HSV-1 vector that expressed the Escherichia coli beta-galactosidase protein as a model antigen . We assessed vector efficacy by analyzing the immunoglobulin G (IgG) antibody response and cellular proliferative response directed against beta-galactosidase . We report that the ability of the vector to induce antibody or proliferative responses was not diminished by preexisting immunity to HSV . Of further note, the anti-HSV and anti-beta-galactosidase IgG responses following vector administration were extremely durable in both immunized and naive mice . These results indicate that the ability of a replication-defective HSV-derived vaccine vector to elicit long-lived immune responses in mice is not impaired by prior HSV exposure. J Virol, 2002 Apr, 76(8), 3670 - 7 Rapid construction of adenoviral vectors by lambda phage genetics; McVey D et al.; Continued improvements of adenoviral vectors require the investigation of novel genome configurations . Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production . In this way failure to generate a virus is not associated with an inability to generate the desired genome . We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli . The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability . Furthermore, the process is independent of any restriction endonucleases . The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification{s} and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E . coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid . Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule . This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions . The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations. J Virol, 2002 Apr, 76(8), 3605 - 14 Replication fidelity of the supF gene integrated in the thymidine kinase locus of herpes simplex virus type 1; Hwang YT et al.; Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1 . These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene . A mutation frequency of approximately 10(-4) was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene . However, recombinants derived from the PAA(r)5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three- to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y . T . Hwang, B.-Y . Liu, C.-Y . Hong, E . J . Shillitoe, and C . B . C . Hwang, J . Virol . 73:5326-5332, 1999) . Thus, the PAA(r)5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain . The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA . Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication . Furthermore, the replication mode of the target gene also affected the mutational spectrum. J Pharmacol Exp Ther, 2002 Apr, 301(1), 160 - 7 Mechanism-based inactivation of cytochrome P450 3A4 by 17 alpha-ethynylestradiol: evidence for heme destruction and covalent binding to protein; Lin HL et al.; 17 alpha-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6 beta-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner . The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH . The values for the K(I) and k(inact) were 18 microM and 0.04 min(-1), respectively, and the t(1/2) was 16 min . Incubation of 50 microM EE with P450 3A4 at 37 degrees C for 30 min resulted in a 67% loss of testosterone 6 beta-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex . The inactivation of P450 3A4 by EE was irreversible . Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation . The partition ratio was approximately 50 . The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated . SDS-polyacrylamide gel electrophoresis analysis demonstrated that {(3)H}EE was irreversibly bound to the P450 3A4 apoprotein . After extensive dialysis of the {(3)H}EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein . Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M - H)(-) of 479 Da . HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites . In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site. J Biol Chem, 2002 May 31, 277(22), 19424 - 32 Epub 2002 Mar 20. Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism; Wong I et al.; Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site . A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping . However, the order of these two steps during catalysis has not been conclusively established . We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair . Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis . Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping . Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision . The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix . The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping. J Biol Chem, 2002 May 24, 277(21), 18373 - 82 Epub 2002 Mar 20. Functional characterization of betaine/proline transporters in betaine-accumulating mangrove; Waditee R et al.; Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter from tomato, a betaine-nonaccumulating plant . In this study, two full-length and one partial transporter genes were isolated from betaine-accumulating mangrove Avicennia marina . Their homologies to betaine transporters from bacteria and betaine/4-aminobutyrate transporters from mammalian cells were low but were high to proline transporters from Arabidopsis and tomato . Two full-length transporters could complement the Na(+)-sensitive phenotype of the Escherichia coli mutant deficient in betT, putPA, proP, and proU . Both transporters could efficiently take up betaine and proline with similar affinities (K(m), 0.32-0.43 mm) and maximum velocities (1.9-3.6 nmol/min/mg of protein) . The uptakes of betaine and proline were significantly inhibited by mono- and dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-aminobutyrate . Sodium and potassium chloride markedly enhanced betaine uptake rates with optimum concentrations at 0.5 m, whereas sucrose showed only modest activation . The change of amino acids Thr(290)-Thr-Ser(292) in a putative periplasmic loop to Arg(290)-Gly-Arg(292) yielded the active transporter independent of salts, suggesting the positive charge induced a conformational change to the active form . These data clearly indicate that the betaine-accumulating mangrove contains betaine/proline transporters whose properties are distinct from betaine transporters of bacteria and mammalian cells. Vaccine, 2002 Mar 15, 20(13-14), 1733 - 40 The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the coadministered adjuvant; Choi AH et al.; Intranasal (i.n.) immunization of BALB/c mice with chimeric murine rotavirus EDIM (epizootic diarrhea of infant mice) VP6 and attenuated E . coli heat-labile toxin (LT), LT(R192G), stimulated >99% protection against rotavirus shedding after EDIM challenge . Here, we evaluated other potential adjuvants with chimeric VP6 administered by two mucosal routes: i.n . and oral . Besides LT(R192G), the adjuvants examined included Adjumer, CpG oligodeoxynucleotides (CpG ODN), chimeric A1 subunit of cholera toxin (CTA1)-DD, and QS-21 . All except QS-21 significantly (P<0.05) increased VP6-specific serum IgG responses after i.n . immunization, but none significantly increased these responses when administered orally . The i.n . delivery of chimeric VP6 alone induced both rotavirus IgG1 and IgG2a whose relative titers suggested a skewed Th2-like response . Inclusion of Adjumer greatly increased Th2-like responses, while CpG ODN shifted the response to a less Th2-like response . The adjuvants CTA1-DD, LT(R192G), QS-21 had no significant effect on ratios of IgG1/IgG2a titers . Following EDIM challenge of mice immunized i.n . with chimeric VP6 and either LT(R192G), CTA1-DD, Adjumer or CpG ODN, shedding was reduced >99, 95, 80, 74, respectively, relative to that found in unimmunized mice (P<0.05) . QS-21 induced less protection (43%, not significant (N.S.)) while immunization with chimeric VP6 alone reduced shedding by only 16% (N.S.) . Oral immunization with chimeric VP6 and all selected adjuvants except QS-21 was less effective than after i.n . immunization, with protection levels of 94 (P<0.05), 71 (P<0.05), 55, 35 and 28% for LT(R192G), QS-21, CpG ODN, CTA1-DD, and Adjumer, respectively, while immunization with chimeric VP6 alone gave no protection . Thus, different adjuvants induced different degrees of protection and oral immunization was generally less effective then the i.n . route. J Virol Methods, 2002 May, 103(1), 67 - 74 Overexpression and purification of the hepatitis B e antigen precursor; Laine S et al.; Circumstantial evidence suggests that the secreted hepatitis B virus (HBV) e antigen (HBeAg) and/or its 22 kDa precursor (P22) have an essential role in the establishment of persistent infection . In order to identify cellular proteins that could interact with P22, large amounts of this protein are required to perform pull-down assays . A plasmid was constructed encoding a recombinant P22 with a Histidine-tag at its N-terminal extremity (P22r) . The initial attempts to overexpress P22r in a conventional Escherichia coli strain failed, most likely due to the presence of rare AGA/AGG codon clusters in the 3' part of the gene . To overcome this difficulty, P22r was overexpressed in the Epicurian coli BL21-codonplus (DE3)-RIL strain, which possesses extra copies of the ArgU gene that encodes the tRNA(AGA/AGG) . In this strain, P22r was overexpressed successfully and then purified in milligram quantities by metal affinity chromatography on Ni2+-chelated His-Bind resin . The purified recombinant protein P22r was able to interact with a cellular protein (P32), which had previously been shown to co-immunoprecipitate with native P22, indicating that at least some of the P22r molecules were folded correctly. J Med Chem, 2002 Mar 28, 45(7), 1466 - 76 Carbonic anhydrase inhibitors . A general approach for the preparation of water-soluble sulfonamides incorporating polyamino-polycarboxylate tails and of their metal complexes possessing long-lasting, topical intraocular pressure-lowering properties; Scozzafava A et al.; Reaction of polyamino-polycarboxylic acids or their dianhydrides with aromatic/heterocyclic sulfonamides possessing a free amino/imino/hydrazino/hydroxy group afforded mono- and bis-sulfonamides containing polyamino-polycarboxylic acid moieties in their molecule . The acids/anhydrides used in synthesis included IDA, NTA, EDDA, EDTA and EDTA dianhydride, DTPA and DTPA dianhydride, EGTA and EGTA dianhydride, and EDDHA, among others . All the newly prepared derivatives showed strong affinity toward isozymes I, II, and IV of carbonic anhydrase (CA) . Metal complexes of the new compounds have also been prepared . Metal ions used in such preparations included di- and trivalent main-group and transition cations, such as Zn(II), Cu(II), Al(III), etc . Some of the new sulfonamides/disulfonamides obtained in this way, as well as their metal complexes, behaved as nanomolar CA inhibitors against isozymes II and IV, being slightly less effective in inhibiting isozyme I . Some of these sulfonamides as well as their metal complexes strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 1-2% water solutions/suspensions . The good water solubility of these sulfonamide CA inhibitors, correlated with the neutral pH of their water solutions used in the ophthalmologic applications and the long duration of action of the IOP-lowering effect, makes them interesting candidates for developing novel types of antiglaucoma drugs devoid of serious topical side effects. J Med Chem, 2002 Mar 28, 45(7), 1439 - 46 Structural basis for the glucocorticoid response in a mutant human androgen receptor (AR(ccr)) derived from an androgen-independent prostate cancer; Matias PM et al.; The crystal structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) in complex with the agonist 9alpha-fluorocortisol has been determined at 1.95 A resolution . This mutant AR contains two mutations (L701H and T877A) and was previously reported as a high-affinity cortisol/cortisone responsive AR (AR(ccr)) isolated from the androgen-independent human prostate cancer cell lines MDA PCa 2a and 2b (Zhao et al . Nature Med . 2000, 6, 703-6) . The three-dimensional structure of the AR(ccr) LBD complexed with 9alpha-fluorocortisol shows the typical conformation of an agonist-bound nuclear receptor in which helix 12 is precisely positioned as a "lid" for the ligand-binding pocket . Binding of 9alpha-fluorocortisol to the AR(ccr) involves favorable hydrogen bond patterns on the C17 and C21 substituents of the ligand due to the mutations at 701 and 877 in the AR(ccr) . Our studies provide the first structural explanation for the glucocorticoid activation of AR(ccr), which is important for the development of new therapeutic treatments for androgen-independent prostate cancer. Bioconjug Chem, 2002 Mar-Apr, 13(2), 370 - 7 Effects of coligand variation on the in vivo characteristics of Tc-99m-labeled interleukin-8 in detection of infection; Rennen HJ et al.; In our previous studies, interleukin-8 (IL-8) was labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) as bifunctional coupling agent and tricine as coligand . This preparation showed excellent characteristics for imaging of infection in a rabbit model of soft-tissue infection . In the present study, the propylaldehyde hydrazone formulation of HYNIC was introduced to stabilize HYNIC-IL-8 . (99m)Tc-HYNIC-IL-8 was prepared using 5 different coligand formulations . The effect of these coligand formulations on the in vitro characteristics and in vivo behavior of (99m)Tc-HYNIC-IL-8 was investigated . HYNIC-conjugated IL-8 was labeled with (99m)Tc in the presence of either (A) tricine, (B) ethylenediaminediacetic acid (EDDA), (C) tricine and trisodium triphenylphosphinetrisulfonate (TPPTS), (D) tricine and nicotinic acid (NIC), or (E) tricine and isonicotinic acid (ISONIC) . These preparations were characterized in vitro by RP-HPLC, determination of the octanol/water partition coefficient, stability studies, and receptor binding assays . The in vivo biodistribution of the radiolabel in rabbits with E . coli-induced soft-tissue infection was determined both by gamma-camera imaging as well as by tissue counting at 6 h pi . Specific activity (MBq/microg) was highest for (ISO)NIC (up to 80) > TPPTS (40) > tricine (15) > EDDA (7) . RP-HPLC and octanol/water partition coefficients showed a shift toward higher lipophilicity for the TPPTS preparation . The leukocyte receptor binding fractions were around 40-55% for all preparations except for TPPTS, which showed predominantly nonspecific binding . All preparations were stabilized in serum, but the stability in PBS was highest for NIC and TPPTS > EDDA > ISONIC > tricine . The in vivo biodistribution showed highest abscess/muscle for NIC and ISONIC (>200) > EDDA and tricine (approximately 100) > TPPTS (<40) . Gamma camera imaging rapidly visualized the abscess from 2 h pi onward for all formulations . The abscess/background (A/B) at 6 h pi for ISONIC was significantly higher (P < 0.05) than that of tricine and the A/B of TPPTS was significantly lower (P < 0.05) . IL-8 can be rapidly and easily labeled with (99m)Tc using HYNIC as a chelator in combination with various coligands . The most optimal infection imaging characteristics were found for formulations using nicotinic acid/tricine as coligand system combining a high specific activity and high in vitro stability with high abscess/muscle ratios (>200) and high abscess/background ratios (>20) . Protein doses to be administered were as low as 70 ng/kg bodyweight . At these low protein doses, side effects are not to be expected in the human system . This paves the way for infection imaging studies in patients. Bioconjug Chem, 2002 Mar-Apr, 13(2), 309 - 16 Synthesis of new modified DNAs by hyperthermophilic DNA polymerase: substrate and template specificity of functionalized thymidine analogues bearing an sp3-hybridized carbon at the C5 alpha-position for several DNA polymerases; Sawai H et al.; Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 alpha-position with amino-linker arms, a methyl ester, or a carboxyl group at the C5 sidearm, were good substrates for primer-extension reactions by DNA polymerase from Pyrococcus kodakaraensis (KOD Dash DNA polymerase), yielding exclusively full-length products . The resulting modified DNA was further allowed to react with a functional molecule such as fluorescein isothiocyanate . By contrast, only truncated products were formed from the thymidine analogue substrate bearing the amino-linker arm or the negatively charged carboxyl group using Taq, Tth DNA polymerase, or DNA polymerase I from E . coli (Klenow fragment) . The results indicate either that the thymidine analogue was not accepted by the enzymes, or that the polymerases could not extend the products, once the analogue had been incorporated, depending on the type of the analogue . A conventional thymidine analogue bearing an aminopropenyl group at the C5-position was accepted by all enzymes, among which KOD Dash DNA polymerase showed the highest activity for the polymerization with this analogue . Templates bearing the thymidine analogues in place of one thymidine residue were read by KOD Dash, Taq, Tth DNA polymerases, and the Klenow fragment giving the full-length product . KOD Dash DNA polymerase could expand structural diversities of substrates that can be used to prepare modified DNAs. Biochem Biophys Res Commun, 2002 Mar 29, 292(2), 396 - 401 The function of OmpA in Escherichia coli; Wang Y; Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane . In this study, the function of OmpA in E . coli stress survival was examined . An E . coli K1 ompA-deletion mutant was significantly more sensitive than that of its parent strain to sodium dodecyl sulfate (SDS), cholate, acidic environment, high osmolarity, and pooled human serum . A number of amino acid changes at the extracellular loops of OmpA did not affect the viability of E . coli, while short peptide insertions in the periplasmic turns of the OmpA beta-barrel decreased E . coli resistance to environmental stresses . Moreover, ompA mutants were found to survive much better within brain microvascular endothelial cells than the wild-type strain, supporting that OmpA is a major target in mammalian host cell defense . These results indicated that OmpA plays a vital structural role in E . coli, and suggested that a perfect beta-barrel structure of OmpA is important for outer membrane stability . Based on these results and the published OmpA structural analyses, I propose that OmpA is composed of three functional domains including a hydrophilic extracellular mass, a beta-barrel transmembrane structure, and a peptidoglycan binding domain . (c)2002 Elsevier Science (USA). Plant Mol Biol, 2002 Mar, 48(4), 361 - 8 Molecular and biochemical characterization of an Arabidopsis thaliana arogenate dehydrogenase with two highly similar and active protein domains; Rippert P et al.; The present study reports the first molecular characterization of a plant arogenate dehydrogenase, the enzyme that catalyses the transformation of arogenate into tyrosine . The structure of the Arabidopsis thaliana tyrA gene is very peculiar since it encodes two highly similar, and putatively active, protein domains . PCR analyses confirmed the existence of a transcript encoding the two protein domains . The complete coding sequence and sequences corresponding to the two separate domains were independently cloned into Escherichia coli mutant AT 2471 lacking prephenate dehydrogenase activity . Our results revealed that the three recombinant enzymes are active . They all exhibit a high specificity toward arogenate and NADP, and have very similar kinetic properties. Plant Mol Biol, 2002 Mar, 48(4), 339 - 50 Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara; Huang T et al.; The gene for a thermal hysteresis (antifreeze) protein (sthp-64) from the bittersweet nightshade, Solanum dulcamara, was cloned and characterized . An expression cDNA library prepared from November S . dulcamara was screened using a polyclonal antibody generated against a previously purified 67 kDa thermal hysteresis protein, and positive clones were identified and sequenced . The full-length thermal hysteresis protein gene was cloned into an Escherichia coli expression vector and expressed as a fusion protein . The putative thermal hysteresis protein (STHP-64) contains two conserved regions 56 and 57 amino acids in length which have the C-X4-C-X22-23-H-X1-H zinc finger motif which is present in WRKY proteins, a family of transcription factors which play a role in regulating expression of pathogenesis-related proteins in plants . Additional features of transcription factors, such as an acidic domain between the two zinc-fingers and a glutamine-rich region upstream of the first zinc-finger are also present in STHP-64 . A DNA binding assay showed that the expressed STHP-64 fusion protein has specific DNA-binding ability . A unique feature of STHP-64 is that the C-terminus contains 10 consecutive 13-mer repeats . Such repeats are a common feature of animal antifreeze proteins . The expressed STHP-64 fusion protein had low levels of thermal hysteresis activity, but this activity was considerably increased by addition of citrate, which is known as an enhancer of certain insect antifreeze proteins . Northern blots demonstrated that the STHP-64 transcript was not present in leaves until November and December, suggesting that cold acclimation induces STHP-64 production. Nippon Rinsho, 2002 Mar, 60(3), 545 - 50 {Shiga toxin producing Escherichia coli infection}; Nakao H; Shiga toxin producing Escherichia coli(STEC) has been recognized as an emerging food-borne pathogen that causes bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome(HUS), especially in developed countries . As the specific therapy for STEC infection has not been developed, currently available medical therapy is inadequate to prevent life-threatening complications . Here are described the possibilities and problems of using and developing therapies such as antibiotics, Synsorb-Pk and humanized anti-Stx monoclonal antibody therapy . In conclusion, the prevention of primary infection is thought to be the best way to prevent the life-threatening complications caused by STEC, and second way is identification as early as possible. Nippon Rinsho, 2002 Mar, 60(3), 473 - 9 {Anti Shiga-like toxin II(SLT-II) humanized monoclonal antibody}; Matsumoto Y; Anti-Shiga-Like Toxin II(SLT-II) Humanized Monoclonal Antibody(TMA-15) was constructed from Mouse Monoclonal Antibody(MuVTm1.1) recognizing the same antigen using recombinant and CDR grafting technology . TMA-15 had almost the same affinity to SLT-II as MuVTm1.1 and showed the good protective activity of mice challenged either with SLT-II or with SLT-II secreting Shiga-like Toxin producing E . coli(STEC) . TMA-15 showed no acute toxicity to monkeys and no cross-reactivity to human tissues in pre-clinical safety studies . From the preliminary results of Phase 1 clinical trial using healthy adult volunteers, doses up to planned maximum dose were well tolerated and TMA-15 showed long half life in blood almost comparable to gamma globulin preparations . Therefore, TMA-15 is expected to show clinical efficacy in coming clinical trial using pediatric STEC patients. Mol Gen Mikrobiol Virusol, 2002, (1), 31 - 6 {Possibility of using fusion proteins for detection of nonsense mutations and frame-shift mutations in BRCA1 gene}; Gutkina NI et al.; The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase" . The ability/inability of this newly constructed ORF to cause alpha-complementation in E . coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment . A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E . coli delta M15gal . Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid . Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E . coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E . coli delta M15gal . We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence . Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation . On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E . coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence . We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence. Am J Obstet Gynecol, 2002 Mar, 186(3), 523 - 30 Bacterially induced preterm labor in the mouse does not require maternal interleukin-1 signaling; Hirsch E et al.; OBJECTIVE: We tested the hypothesis that intrauterine bacterial inoculation induces labor via expression of the inflammatory cytokine interleukin-1 (IL-1) in a murine model . STUDY DESIGN: Pregnant mice on day 14.5 of a 19-20 day gestation were inoculated with killed Escherichia coli or sterile media into either (a) the right uterine horn, (b) the right uterine horn following its surgical isolation from the contralateral horn and cervix, or (c) the kidney . Cytokine levels in gestational tissues and maternal serum were determined by use of enzyme-linked immunosorbent assay (ELISA) . In a separate experiment, bacterially induced preterm delivery was compared between mice lacking a functional IL-1 receptor and wild-type control litter mates . RESULTS: Killed E coli induced delivery within 48 hours with similar dose-response curves regardless of inoculation site (intact uterine horn, isolated uterine horn, or kidney) . Bacterial inoculation of an isolated right horn caused dramatic increases in local expression of IL-1 and IL-6 . However, delivery occurred from the uninjected horn without corresponding upregulation of cytokines, with the exception of a modest rise within fetal membranes . Mice lacking a functional IL-1 receptor were no different from wild-type mice in their susceptibility to bacterially induced delivery . CONCLUSION: Bacterially induced labor in the murine model does not require IL-1 signaling. J Cardiovasc Pharmacol, 2002 Apr, 39(4), 544 - 51 Dexamethasone inhibits the inducible bioconversion of glyceryl trinitrate to nitric oxide; Mollace V et al.; The aim of this study was to assess the effects of dexamethasone (DEX) on the inducible bioconversion of glyceryl trinitrate (GTN) into nitric oxide in cultured smooth muscle cells, endothelial cells, and the J774 macrophage cell line as well as in vivo and ex vivo in rats either untreated or pretreated with Escherichia coli lipopolysaccharide . In vitro, an increased bioconversion of GTN to nitrite and an elevation of cyclosine guanosine 3,5;-monophosphate (cGMP) levels occurred after treatment with lipopolysaccharide (LPS) (0.5 microg/ml, 18 h) . This effect was ablated by co-incubation with DEX (10 microM, 18 h) . Rats treated with an intraperitoneal (IP) injection of LPS (4 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (1 mg/kg, intravenously {IV}) and this was prevented when DEX (4 mg/kg, IP) was given together with LPS . Progesterone (50 mg/kg, IP) had no effect on GTN-induced hypotensive response . Conversely, exposure of rat aortic strips obtained from animals pretreated with LPS produced an enhanced vasorelaxant response in LPS-treated rats . Also, this effect was inhibited by pretreatment with DEX . Thus, the induction of the pathway leading to the formation of nitric oxide from GTN is blocked by DEX both in vitro and in vivo, and this may represent a useful tool in the assessment of the enhanced bioconversion of organic nitrates into nitric oxide occurring via inflammatory mechanisms. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3523 - 8 Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA; Hazra TK et al.; 8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging . Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes . One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1 . E . coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site . Here, we report the presence of two human orthologs of E . coli mutM nei genes in the human genome database, and characterize one of their products . Based on the substrate preference, we have named it NEH1 (Nei homolog) . The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E . coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides . Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site . The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases. Proc Natl Acad Sci U S A, 2002 Mar 19, 99(6), 3475 - 80 Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking; Guan L et al.; Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix . Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-A-long cross-linking agent containing bis-methane thiosulfonate reactive groups {position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)} . The results are consistent with a current helix-packing model of the permease . Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends . The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol . Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics . Furthermore, cross-linking is markedly decreased at 0 degrees C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking . The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer. J Biol Chem, 2002 May 24, 277(21), 19064 - 70 Epub 2002 Mar 19. The Escherichia coli cyclic AMP receptor protein forms a 2:2 complex with RNA polymerase holoenzyme, in vitro; Dyckman D et al.; Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and RNA polymerase holoenzyme associate to form a 2:2 complex in vitro . No complexes of lower stoichiometry (1:1, 2:1, 1:2) were detected over a wide range of CAP and RNA polymerase concentrations, suggesting that the interaction is highly cooperative . The absence of higher stoichiometry complexes, even in the limit of high {protein}, suggests that the 2:2 species represents binding saturation for this system . The 2:2 pattern of complex formation is robust . A lower-limit estimate of the formation constant in our standard buffer (40 mm Tris (pH 7.9), 10 mm MgCl(2), 0.1 mm dithiothreitol, 5% glycerol, 100 mm KCl) is 2 x 10(20) m(-3) . The qualitative pattern of association is unchanged over the temperature range 4 degrees C < or = T < or = 20 degrees C, by substitution of glutamate for chloride as the dominant anion, or on addition of 20 microm cAMP to the reaction mix . These results limit the possible mechanisms of CAP-polymerase association . In addition, they support the idea that CAP binding may influence the availability of the monomeric form of RNA polymerase that mediates transcription at many promoters. J Biol Chem, 2002 May 10, 277(19), 16351 - 4 Epub 2002 Mar 19. A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins; Kranz JK et al.; Ca(2+)-saturated calmodulin (CaM) directly associates with and activates CaM-dependent protein kinase I (CaMKI) through interactions with a short sequence in its regulatory domain . Using heteronuclear NMR (13)C-(15)N-(1)H correlation experiments, the backbone assignments were determined for CaM bound to a peptide (CaMKIp) corresponding to the CaM-binding sequence of CaMKI . A comparison of chemical shifts for free CaM with those of the CaM.CaMKIp complex indicate large differences throughout the CaM sequence . Using NMR techniques optimized for large proteins, backbone resonance assignments were also determined for CaM bound to the intact CaMKI enzyme . NMR spectra of CaM bound to either the CaMKI enzyme or peptide are virtually identical, indicating that calmodulin is structurally indistinguishable when complexed to the intact kinase or the peptide CaM-binding domain . Chemical shifts of CaM bound to a peptide (smMLCKp) corresponding to the calmodulin-binding domain of smooth muscle myosin light chain kinase are also compared with the CaM.CaMKI complexes . Chemical shifts can differentiate one complex from another, as well as bound versus free states of CaM . In this context, the observed similarity between CaM.CaMKI enzyme and peptide complexes is striking, indicating that the peptide is an excellent mimetic for interaction of calmodulin with the CaMKI enzyme. Biochim Biophys Acta, 2002 Feb 11, 1594(2), 297 - 306 Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate (KDO-8-P) synthase is a zinc-metalloenzyme; Krosky DJ et al.; 3-Deoxy-D-manno-2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D-arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate . All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli, were believed not to require a metal cofactor for catalytic activity . However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme . Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes . One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori . In order to test this model, we have cloned the kdsa gene encoding H . pylori KDO-8-P synthase, and overexpressed and purified the protein . This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity . The Zn(2+) in the enzyme could be quantitatively replaced by Cd(2+), which increased the observed k(cat) by approximately 2-fold, and decreased the apparent K(m)(A-5-P) by approximately 6.5-fold . Furthermore, removal of the Zn(2+) from the enzyme did not greatly perturb its circular dichroism spectra . Thus, the divalent metal most likely serves as cofactor directly involved in catalysis. Biochim Biophys Acta, 2002 Feb 11, 1594(2), 286 - 96 Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe; Behling Agree AK et al.; Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells . The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I . To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C . These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods . The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I . However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties . From fluorescence energy transfer data, average distances between the N-terminal region containing Trp residues and the various AC locations were obtained . The current results, together with previously published observations, led to the construction of a three-dimensional model for the folding of lipid-free apoA-I. FEBS Lett, 2002 Mar 6, 514(1), 102 - 5 A bilayer cell-free protein synthesis system for high-throughput screening of gene products; Sawasaki T et al.; A high-throughput cell-free protein synthesis method has been described . The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture . With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction . Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses . This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era. FEBS Lett, 2002 Mar 6, 514(1), 78 - 83 Functional evidence for D- and T-loop interactions in tmRNA; Barends S et al.; During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features . The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs . Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence . Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342) . Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB. FEBS Lett, 2002 Mar 6, 514(1), 74 - 7 The role of SmpB protein in trans-translation; Shimizu Y et al.; The function of SmpB protein in the trans-translation system was evaluated using the well-defined cell-free translation system consisting of purified ribosome, alanyl-tRNA synthetase and elongation factors . The analysis showed that SmpB protein enhances alanine-accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the trans-translation process in the presence of translational components . Moreover, SmpB is indispensable in the addition of tag-peptide onto ribosomes by tmRNA . In particular, the A-site binding of tmRNA is inhibited in the absence of SmpB. FEBS Lett, 2002 Mar 6, 514(1), 60 - 6 Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3' end of tRNA to ribosomal proteins at the P and E sites; Kirillov SV et al.; Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at their 3' termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome . When bound to the P site of poly(U)-programmed ribosomes, deacylated tRNA(Phe), Phe-tRNA(Phe) and N-acetyl-Phe-tRNA(Phe) probes labeled protein L27 and two main sites within domain V of the 23S RNA . In contrast, deacylated tRNA(Phe) bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA . In the absence of poly(U), the deacylated tRNA(Phe) probe also labeled protein L1 . Cross-linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27 . In the course of this process, tRNA passes through the intermediate P/E binding state . These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order . Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker . The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit. FEBS Lett, 2002 Mar 6, 514(1), 55 - 9 How does tmRNA move through the ribosome? Ivanov PV, Zvereva MI, Shpanchenko OV, Dontsova OA, Bogdanov AA, Aglyamova GV, Lim VI, Teraoka Y, Nierhaus KH. To test the structure of tmRNA in solution, cross-linking experiments were performed which showed two sets of cross-links in two different domains of tmRNA . Site-directed mutagenesis was used to search for tmRNA nucleotide bases that might form a functional analogue of a codon-anticodon duplex to be recognized by the ribosomal A-site . We demonstrate that nucleotide residues U85 and A86 from tmRNA are significant for tmRNA function and propose that they are involved in formation of a tmRNA element playing a central role in A-site recognition . These data are discussed in the frame of a hypothetical model that suggests a general scheme for the interaction of tmRNA with the ribosome and explains how it moves through the ribosome. FEBS Lett, 2002 Mar 6, 514(1), 44 - 8 Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli; Brunelli CA et al.; 4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum . To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA . Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only . These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA. Chin J Traumatol, 2002 Apr, 5(2), 97 - 102 Construction of recombinant adenoviral vector Ad-CMV-hTGFbeta1 for reversion of intervertebral disc degeneration by gene transfer; Tan J et al.; OBJECTIVE: To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration . METHODS: A newly developed recombinant adenoviral vector construction system was used in the study . The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV . The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli . BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1 . Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis . Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells . Recombinant adenoviruses were generated within 2 weeks . RESULTS: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis . The infected 293 cells showed evident cytopathic effect (CPE) . The productions of PCR confirmed the presence of recombinant adenovirus . The expression of hTGFbeta1 was verified by immunohistochemical staining . CONCLUSIONS: The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy. Physiol Plant, 2002 Feb, 114(2), 165 - 171 Isolation and characterization of an extended thioredoxin h from poplar; Gelhaye E et al.; A cDNA coding for a thioredoxin h has been isolated from a xylem/phloem poplar cDNA library by RACE-PCR . The nucleotide sequence called popTrx-h2 is homologous to other thioredoxins h isolated from plants but differs from the other thioredoxins h by presenting a 30 amino acid long N-terminus extension . A variant of this cDNA lacking the N-terminal extension was also generated by PCR . Both cDNAs have been introduced into an expression plasmid (pET-3d) and the recombinant proteins have been expressed to a high level and purified from Escherichia coli cells . Protein sequencing showed that a part of the N-terminal extension was cleaved in the E . coli cells, with the first 19 amino acids missing, suggesting the presence of a putative cleavage site in the N-terminal extension of popTrx-h2 . Both recombinant proteins display unusual catalytic properties compared to other thioredoxins h characterized so far, i.e . a weak reduction by Arabidopsis thaliana NADPH-dependent thioredoxin reductase, and a weak activation of the chloroplastic NADP-malate dehydrogenase, a non-physiological target enzyme . Northern blot experiments indicate that the transcripts of popTrx-h2 are present in leaves and roots, albeit at a lower level compared to the earlier characterized popTrx-h1. J Vet Pharmacol Ther, 2001 Dec, 24(6), 379 - 83 A pharmacokinetic study of amoxycillin in febrile beagle dogs following repeated administrations of endotoxin; Marier JF et al.; The pharmacokinetics of amoxycillin was studied in nine male beagle dogs under healthy and febrile conditions . In Period 1, dogs received 20 mg/kg of an oral suspension of amoxycillin . Intravenous doses of saline, 2 and 20 microg/kg of endotoxin (LPS from Escherichia coli serotype) were administered to dogs (three per group) prior to administration of 20 mg/kg of amoxycillin in Period 2 . Rectal temperature and behavioral changes were recorded and blood samples were collected over 12 h for pharmacokinetic analysis . Amoxycillin was assessed in plasma using liquid chromatography coupled with mass spectrometry . Plasma concentrations were analysed using a one-compartment model with lag-time for absorption using an iterative two-stage method . As compared with control groups, amoxycillin clearance decreased significantly with preliminary treatments of 2 microg/kg endotoxin (0.209 vs . 0.140 L/h kg, P < 0.05) and 20 microg/kg endotoxin (0.214 vs . 0.075 L/h kg, P < 0.05) . As a result of this, the area under curve for the 2 and 20 microg/kg endotoxin groups increased significantly 100.4 vs . 149.4 microg h/mL (P < 0.05) and 99.2 vs . 277.7 microg h/mL (P < 0.05), respectively . Other drugs currently used for the treatment of fever and septic shock should be re-evaluated using a febrile animal model to avoid improper dose administration. Biochem J, 2002 Apr 1, 363(Pt 1), 81 - 7 Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations; Fang J et al.; Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix . In mammals, this enzyme is highly regulated by allosteric effectors . The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion . The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition . In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A) . The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively) . Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site . Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation . The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site . These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide insight into the structural mechanisms involved in positive and negative allosteric control and in inter-subunit co-operativity of human GDH. J Mol Biol, 2002 Mar 22, 317(2), 309 - 23 Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate; Scott JW et al.; We have expressed a truncated form of the alpha1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD) . A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies . We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/alpha (ACC1) as a GST fusion . This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain . We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases . The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations . The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues) . The features of the interaction can be summarised as follows: (i) an amphipathic helix from P-16 to P-5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P-6 and P-4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P+3 interacts with D56 on the small lobe; (iv) the side-chain of P+4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position . These interactions position the serine residue to be phosphorylated in close proximity to the gamma-phosphate group of ATP . Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date . Mol Microbiol, 1997 Jul, 25(1), 53 - 64 Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor; Valent QA et al.; The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system . In this study, we use an E . coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment . Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E . coli SRP . Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP . In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E . coli ribosome. Nat Prod Rep, 2002 Feb, 19(1), 60 - 9 From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase; Teplyakov A et al.; This review summarizes the state of knowledge on D-glucosamine-6P synthesis catalyzed by glucosamine-6P synthase . The mechanisms of L-glutamine hydrolysis, ammonia transfer and fructose-6P conversion into D-glucosamine-6P are analyzed with the E . coli enzyme in light of recent X-ray structures . With 92 references this paper covers the literature up to June 2001 and emphasizes the potential implication of the mammalian glucosamine-6P synthase in type 2 diabetes. Poult Sci, 2002 Mar, 81(3), 302 - 8 Genetic and phenotypic correlations between antibody responses to Escherichia coli, infectious bursa disease virus (IBDV), and Newcastle disease virus (NDV), in broiler lines selected on antibody response to Escherichia coli; Yunis R et al.; The genetic control of antibody (Ab) response to Escherichia coli (EC), infectious bursa disease virus, and Newcastle disease virus and the genetic and phenotypic correlation between these Ab responses, were evaluated under farm conditions in which chicks were simultaneously exposed to these antigens . The experimental population comprised five groups: two lines divergently selected for high (HH) or low (LL) Ab response to EC vaccination; a commercial broiler dam-line (CC), from which HH and LL had been derived; and the HH x CC and LL x CC hybrid groups (HC and LC, respectively) . Lines LL and HH expressed similar symmetric divergence to all three antigens . The ranking of the LL, LC, CC, HC, and HH genetic groups according to their mean Ab responses and their very high linear correlation with the LL vs . HH genomic scale clearly indicate the additive nature of the genetic divergence between these lines . Several estimates of correlation were calculated between Ab responses of each pair of antigens and between BW and Ab to each antigen . The high correlation between group means, the near-zero within-group correlation, and the low phenotypic correlation indicate the strongly positive genetic correlation between Ab responses and no correlation with BW . The results of this study suggest that overall immunocompetence of commercial broilers can be improved by selection for high Ab response of young chicks to controlled immunization with a single antigen, without counteracting further selection for high BW. Crit Care Med, 2002 Jan, 30(1), 190 - 4 Thioredoxin is associated with endotoxin tolerance in mice; Sano H et al.; OBJECTIVE: Oxidative stress and subsequent lipid peroxidation appear to be central to the lethal effect of lipopolysaccharide . We hypothesized that induction of an antioxidant protein, thioredoxin, would play an important role in the development of endotoxin tolerance and reduce mortality rates in lipopolysaccharide-treated mice . DESIGN: Prospective, randomized, controlled study . SETTING: University research laboratory . SUBJECTS: Adult, male, ddy mice . INTERVENTIONS: In survival-curve experiments, mice were pre-treated intravenously with a low dose of Escherichia coli lipopolysaccharide (20 microg/mouse, pretreatment group) or saline (control group) . A large dose of lipopolysaccharide (200 microg/mouse) subsequently was injected into the tail vein 16 hrs after pretreatment . In experiments to measure the expression of thioredoxin after lipopolysaccharide challenge, mice were injected intravenously with different concentrations of lipopolysaccharide (between 20 and 200 microg/mouse, designated the lipopolysaccharide group) or with saline (control group) . MEASUREMENTS AND MAIN RESULTS: The survival rate during the 72-hr observation period was significantly higher in the pretreatment group (82%) than in the control group (30%; p = .025 by Mantel-Cox log rank analysis) . After lipopolysaccharide challenge, thioredoxin concentrations in the lung, heart, and liver of mice in the pretreated group were about 1.5- to 2-fold higher than those in the control group . Enhancement in these organs became apparent about 6 hrs after lipopolysaccharide challenge and lasted until at least 24 hrs . The levels of accumulation of 4-hydroxy-2-nonenal modified proteins after a large dose of lipopolysaccharide challenge were lower in the pretreatment group than in the control group . CONCLUSIONS: These results demonstrate that mice with an increased concentration of thioredoxin, induced by pretreatment with a low dose of lipopolysaccharide, had increased survival when given a subsequent high-dose challenge of lipopolysaccharide . Thus, thioredoxin is associated with the development of endotoxin tolerance in mice. J Trauma, 2002 Mar, 52(3), 449 - 52 Free hemoglobin enhances tumor necrosis factor-alpha production in isolated human monocytes; Carrillo EH et al.; BACKGROUND: A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively . Many feel this response is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity . Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR . Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb) . We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR . METHODS: Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours . Treatment groups included saline controls, lipopolysaccharide ({LPS}, 10 ng/mL, from Escherichia coli), human Hb (25 microg/mL), and Hb + LPS . Supernatants were analyzed by enzyme-linked immunosorbent assay . Student's t test with Mann-Whitney posttest was used for statistical analysis with p < or = 0.05 considered significant . RESULTS: Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02) . LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls . CONCLUSION: These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha . These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively. Genetics, 2002 Mar, 160(3), 851 - 9 Crossing over between regions of limited homology in Escherichia coli . RecA-dependent and RecA-independent pathways; Lovett ST et al.; We have developed an assay for intermolecular crossing over between circular plasmids carrying variable amounts of homology . Screens of Escherichia coli mutants demonstrated that known recombination functions can only partially account for the observed recombination . Recombination rates increased three to four orders of magnitude as homology rose from 25 to 411 bp . Loss of recA blocked most recombination; however, RecA-independent crossing over predominated at 25 bp and could be detected at all homology lengths . Products of recA-independent recombination were reciprocal in nature . This suggests that RecA-independent recombination may involve a true break-and-join mechanism, but the genetic basis for this mechanism remains unknown . RecA-dependent crossing over occurred primarily by the RecF pathway but considerable recombination occurred independent of both RecF and RecBCD . In many respects, the genetic dependence of RecA-dependent crossing over resembled that reported for single-strand gap repair . Surprisingly, ruvC mutants, in both recA(+) and recA mutant backgrounds, scored as hyperrecombinational . This may occur because RuvC preferentially resolves Holliday junction intermediates, critical to both RecA-dependent and RecA-independent mechanisms, to the noncrossover configuration . Levels of crossing over were increased by defects in DnaB helicase and by oxidative damage, showing that damaged DNA or stalled replication can initiate genetic recombination. Domest Anim Endocrinol, 2002 Mar, 22(1), 37 - 50 Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils; Van Oostveldt K et al.; Polymorphonuclear neutrophil leukocytes (PMN) play an important role in intramammary defense against infections by Escherichia coli . During mastitis, PMN are confronted with various inflammatory mediators that can modulate their function . In severely diseased cows, increased concentrations of lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha (TNF-alpha) are detected in plasma . Binding of LPS to membrane bound CD14 molecules on monocytes cause release of inflammatory mediators such as TNF-alpha . Because apoptosis of PMN promotes resolution of inflammation and because the LPS and TNF-alpha response in milk and blood is related to the severity of E . coli mastitis, the effect on apoptosis of bovine PMN of increased concentrations LPS and TNF-alpha was studied together with the functionality of apoptotic PMN.Bovine PMN apoptosis, as determined with annexin-V, was induced with high concentrations of either LPS (1000 and 10,000ng/mL) or TNF-alpha (10,000ng/mL) in whole blood following a 6h incubation at 37 degrees C . The apoptosis inducing effect of LPS on PMN was not inhibited following coculture with either anti-bovine TNF-alpha or anti-ovine CD14 monoclonal antibodies . When compared to controls, apoptotic PMN had a similar level of CD18 expression but lacked phagocytic and respiratory burst activity . This is the first study reporting the effects of apoptosis on bovine PMN function . These functional impairments in apoptotic PMN could be important in contributing to the establishment of intramammary infection . Well functioning PMN could finally determine the severity of mastitis following an invasion of bacteria in the mammary gland. Biochimie, 2002 Jan, 84(1), 27 - 47 DNA mismatch repair defects: role in colorectal carcinogenesis; Jacob S et al.; The inactivation of the DNA mismah repair (MMR) system, which is associated with the predisposition to the hereditary non-polyposis colorectal cancer (HNPCC), has also been documented in nearly 20% of the sporadic colorectal cancers . These tumors are characterized by a high frequency of microsatellite instability (MSI(+) phenotype), resulting from the accumulation of small insertions or deletions that frequently arise during replication of these short repeated sequences . A germline mutation of one of the two major MMR genes (hMSH2 or hMLH1) is found in half to two-thirds of the patients with HNPCC, whereas in sporadic cases hypermethylation of the hMLH1 promoter is the major cause of the MMR defect . Germline mutations in hMSH6 are rare and rather confer predisposition to late-onset familial colorectal cancer, and frequent extracolonic tumors . Yet, the genetic background of a number of HNPCC patients remains unexplained, indicating that other genes participate in MMR and play important roles in cancer susceptibility . The tumor-suppressor genes that are potential targets for the MSI-driven mutations because they contain hypermutable repeated sequences are likely to contribute to the etiology and tissue specificity of the MSI-associated carcinogenesis . Because the prognosis and the chemosensitivity of the MSI(+) colorectal tumors differ from those without instability, the determination of the MSI phenotype is expected to improve the clinical management of patients . This review gives an overview of various aspects of the biochemistry and genetics of the DNA mismah repair system, with particular emphasis in its role in colorectal carcinogenesis. Eur J Med Chem, 2002 Mar, 37(3), 207 - 17 Reactions of purines-containing butenolides with L-cysteine or N-acetyl-L-cysteine as model biological nucleophiles: a potent mechanism-based inhibitor of ribonucleotide reductase caused apoptosis in breast carcinoma MCF7 cells; Hakimelahi GH et al.; Thiols are the most reactive nucleophilic reagents among the biological models investigated . The reactivity of butenolides 1a-c, 2-4, and 6-8 toward L-cysteine, a model biological nucleophile, was studied spectrophotometrically . The rates of the reactions were measured and correlated with antitumour activity of these molecules . N-Acetylcysteine addition product 5, resulting from the treatment of butenolide 4 with glutathione precursor, N-acetyl-L-cysteine, was isolated . Unlike purine-containing gamma-(Z)-ethylidene-2,3-dimethoxybutenolides 1a-c, 4, 6, and 7, adduct 5 and butenolides 10-12 did not exhibit inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines . As such, the biological activity of purine-containing butenolides can be attributed to their adenine moiety as a recognition site as well as their reactivity towards the cysteine residues of functional proteins forming covalent bond via reverse Michael type addition . Adenine-containing phosphonothioanhydride derivative 8 was also synthesised . Its reaction with N-acetyl-L-cysteine produced N,S-diacetylcysteine and thiophosphonate 9 . Compound 9 did not exhibit anticancer activity; yet its precursor 8 displayed the most pronounced inhibition on all the examined malignant tumour cell lines . In the presence of L-cysteine, cytotoxicity of 4 and 8 was decreased, whereas glutathione addition more influenced on the cytotoxicity of 8 . It was found that adenine-containing phosphonothioanhydride 8 functions as a significant irreversible inactivator of the Escherichia coli ribonucleoside diphosphate reductase . After treatment of MCF7 cells with compound 8, fluorescence microscopy demonstrated the presence of nucleus shrinkage or segmentation . This apoptotic morphology, however, was not pronounced in the presence of glutathione or dithiotheritol.