Anal Biochem, 2002 Mar 1, 302(1), 123 - 30 Identification and characterization of hydrophobic Escherichia coli virulence proteins by liquid chromatography-electrospray ionization mass spectrometry; Hess S et al.; Virulence of enterotoxicogenic Escherichia coli is mediated by rodlike, rigid, highly hydrophobic proteins designated fimbriae or colonization factors (CFs) . More than 20 different colonization factors have been described so far using predominantly immunological and genetic methods . To characterize these hydrophobic proteins by liquid chromatography-mass spectrometry (LC-MS), different methodologies were explored . A novel LC-MS method was developed using hexafluoroisopropanol to maintain the hydrophobic proteins in solution . In addition, these proteins were digested with cyanogen bromide and peptide mapping by LC-MS was established . This technique was particularly useful in identification of closely related CFs . Both LC-MS and peptide mapping methodologies were found to be useful in characterizing highly hydrophobic CFs of E . coli . To search for molecular weights of mature proteins in the National Center for Biotechnology Information (NCBI) database, a new feature was developed and its applicability tested . The identification of a class of pathogenic virulence proteins, either intact or digested, is possible with molecular weight database searching. Int J Food Microbiol, 2002 Feb 5, 72(3), 225 - 31 The influence of temperature, salt and pH on the inhibitory effect of reuterin on Escherichia coli; Rasch M; The inhibitory effect of reuterin on Escherichia coli K12 was investigated at different conditions of temperature (10-30 degrees C), pH (4.5-6.5), and NaCl (0.5-3% (w/v)) . The maximum specific growth rate as a function of the different environmental conditions was modelled with a polynomial model . At increasing temperatures, there was an increasing effect of reuterin, whereas neither pH nor salt showed interactions with the effect of reuterin . The model was validated against new experimental data and proved to perform satisfactorily based on the bias and accuracy factors. Roum Arch Microbiol Immunol, 1999 Jul-Dec, 58(3-4), 223 - 39 Immune response to Escherichia coli antigens entrapped in liposomes . I . Immunopathological studies; Dima VF et al.; In this study, we have searched for an effective mucosal delivery system for a purified E . coli antigen which elicits anticolonization and anti-toxic immunity . E . coli colonization factor antigen (CFA/I) and heat-labile enterotoxin (LT) were encapsulated in liposomes . To determine the efficacies of soluble and liposome-encapsulated E . coli antigens young rabbits were mucosally treated with three oral doses of E . coli antigens given 7 days apart . Ten days after the last booster, rabbits were orally challenged with 5 x 10(9) bacterial cells (O78:H11 serotype) . The experimental results allow of making some remarks which can be correlated with the protection obtained in vaccinated animals: (a) immunization with E . coli antigens entrapped in liposomes ensured protection against ETEC strains; (b) lower protection against homologous and heterologous CFA/I +(LT- ST+) strains were noticed; (c) adhesion of labelled -3H-leucine-bacteria to the intestinal mucosa revealed a maximum distribution in duodenum-jejunum and minimum in the colonic mucosa; (d) it contributed to the release of inoculated virulent bacteria from intestinal tract; (e) humoral, cellular and histopathological findings confirm the afore mentioned observation . Summing up, these results suggest that liposomes are very good carriers for E . coli antigens and these findings highlight the potential use of LT and CFA/I antigens entrapped in liposomes as mucosal and humoral induction of immune response and make them a candidate for future use in prophylaxis of diarrhoea in man. J Bacteriol, 2002 Mar, 184(5), 1471 - 3 New class of IMP cyclohydrolases in Methanococcus jannaschii; Graupner M et al.; The enzyme responsible for observed IMP cyclohydrolase activity in Methanococcus jannaschii was purified and sequenced: its genetic locus was found to correspond to gene MJ0626 . The MJ0626 gene was cloned, and its protein product was expressed in Escherichia coli and shown to catalyze the cyclization of 5-formylamidoimidazole-4-carboxamide ribonucleotide to IMP . The enzyme has no sequence similarity to known enzymes, and its catalytic properties appear distinct from any characterized IMP cyclohydrolase . The purO gene for the enzyme is currently found only in the domain ARCHAEA: J Bacteriol, 2002 Mar, 184(5), 1438 - 43 Conserved low-affinity nickel-binding amino acids are essential for the function of the nickel permease NixA of Helicobacter pylori; Wolfram L et al.; Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori . The nickel permease NixA of H . pylori is a member of the single-component nickel-cobalt transporter family . To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis . This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities . The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity . Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody . Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E . coli system . The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid . The phenotype of the null mutants was independent of the culture medium . Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium . Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA . These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs. J Bacteriol, 2002 Mar, 184(5), 1417 - 22 Disruption of lolCDE, encoding an ATP-binding cassette transporter, is lethal for Escherichia coli and prevents release of lipoproteins from the inner membrane; Narita S et al.; ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli . Mutations resulting in defective LolD were previously shown to be lethal for E . coli . The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved . Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release . To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed . The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE . In addition to functional LolD, both LolC and LolE were required for growth . At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane . Furthermore, both LolC and LolE were essential for the release of lipoproteins . On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein . From these results, we conclude that the LolCDE complex is an essential ABC transporter for E . coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane. J Bacteriol, 2002 Mar, 184(5), 1407 - 16 Posttranscriptional activation of the transcriptional activator Rob by dipyridyl in Escherichia coli; Rosner JL et al.; The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function . Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly . We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially . A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed . However, dipyridyl enhanced Rob's activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional . Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity . Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl . In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD . In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct . Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro. J Bacteriol, 2002 Mar, 184(5), 1402 - 6 Quantitative assessment of oxygen availability: perceived aerobiosis and its effect on flux distribution in the respiratory chain of Escherichia coli; Alexeeva S et al.; Despite a large number of studies on the role of oxygen in cellular processes, there is no consensus as to how oxygen availability to the cell should be defined, let alone how it should be quantified . Here, a quantitative definition for oxygen availability (perceived aerobiosis) is presented; the definition is based on a calibration with reference to the minimal oxygen supply rate needed for fully oxidative catabolism (i.e., complete conversion of the energy source to CO(2) and water for glucose-limited conditions) . This quantitative method is used to show how steady-state electron fluxes through the alternative cytochrome oxidases of Escherichia coli are distributed as a function of the extent of aerobiosis of glucose-limited chemostat cultures . At low oxygen availability the electron flux is mainly via the high-affinity cytochrome bd oxidase, and, at higher oxygen availability, a similar phenomenon occurs but now via the low-affinity cytochrome bo oxidase . The main finding is that the catabolic activities of E . coli (and specifically its respiratory activity) are affected by the actual oxygen availability per unit of biomass rather than by the residual dissolved oxygen concentration of the culture. J Bacteriol, 2002 Mar, 184(5), 1359 - 69 Positive growth rate-dependent regulation of the pdxA, ksgA, and pdxB genes of Escherichia coli K-12; Pease AJ et al.; We found that transcription of the pdxA and pdxB genes, which mediate steps in the biosynthesis of the essential coenzyme pyridoxal 5"-phosphate, and the ksgA gene, which encodes an rRNA modification enzyme and is partly cotranscribed with pdxA, is subject to positive growth rate regulation in Escherichia coli K-12 . The amounts of the pdxA-ksgA cotranscript and pdxB- and ksgA-specific transcripts and expression from pdxA- and pdxB-lacZ fusions increased as the growth rate increased . The half-lives of ksgA- and pdxB-specific transcripts were not affected by the growth rate, whereas the half-life of the pdxA-ksgA cotranscript was too short to be measured accurately . A method of normalization was applied to determine the amount of mRNA synthesized per gene and the rate of protein accumulation per gene . Normalization removed an apparent anomaly at fast growth rates and demonstrated that positive regulation of pdxB occurs at the level of transcription initiation over the whole range of growth rates tested . RNA polymerase limitation and autoregulation could not account for the positive growth rate regulation of pdxA, pdxB, and ksgA transcription . On the other hand, growth rate regulation of the amount of the pdxA-ksgA cotranscript was abolished by a fis mutation, suggesting a role for the Fis protein . In contrast, the fis mutation had no effect on pdxB- or ksgA-specific transcript amounts . The amounts of the pdxA-ksgA cotranscript and ksgA-specific transcript were repressed in the presence of high intracellular concentrations of guanosine tetraphosphate; however, this effect was independent of relA function for the pdxA-ksgA cotranscript . Amounts of the pdxB-specific transcript remained unchanged during amino acid starvation in wild-type and relA mutant strains. J Bacteriol, 2002 Mar, 184(5), 1262 - 9 Reconstitution of the trimethylamine oxide reductase regulatory elements of Shewanella oneidensis in Escherichia coli; Gon S et al.; Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors . In Shewanella species, the TMAO reductase respiratory system is encoded by the torECAD operon . We showed that production of the TMAO reductase of S . oneidensis was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (torSTR) encoding a complex two-component regulatory system was present downstream of the torECAD operon . We introduced the torSTR gene cluster into Escherichia coli and showed that this regulatory gene cluster is involved in TMAO induction of the torE promoter but plays no role in the oxygen control . The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the tor structural operon . By deletion analysis, we confirmed that the TorR operator site is required for induction of the tor structural promoter . As the TMAO regulatory system of S . oneidensis is homologous to that of E . coli, we investigated a possible complementation between the TMAO regulatory components of the two bacteria . Interestingly, TorS(ec), the TMAO sensor of E . coli, was able to transphosphorylate TorR(so), the TMAO response regulator of S . oneidensis. J Bacteriol, 2002 Mar, 184(5), 1234 - 43 The flavoprotein MrsD catalyzes the oxidative decarboxylation reaction involved in formation of the peptidoglycan biosynthesis inhibitor mersacidin; Majer F et al.; The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II . Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA . We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue . MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family) . Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD . In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein . HFCD enzymes are characterized by a conserved His residue which is part of the active site . Exchange of this His residue for Asn led to inactivation of MrsD . The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities. Vet Microbiol, 2002 Mar 1, 85(2), 169 - 82 Prevalence of serogroups and virulence genes in Escherichia coli associated with postweaning diarrhoea and edema disease in pigs and a comparison of diagnostic approaches; Frydendahl K; Identification of Escherichia coli causing porcine postweaning diarrhoea (PWD) or edema disease (ED) requires knowledge regarding the prevalent pathotypes within a given region . This study was undertaken to determine the present distribution of serogroups, hemolytic activity and virulence factor gene profiles among porcine pathogenic E . coli isolates in Denmark and to compare detection of these characteristics as diagnostic approaches . Five hundred and sixty-three E . coli were serogrouped using E . coli O-antisera and investigated for hemolytic activity . Of these, 219 isolates were further characterized using a 5'-nuclease PCR assay detecting genes for adhesion factors, enterotoxins and verocytotoxin 2e (VT2e) . Forty-two different serogroups were found . The most prevalent serogroup was O149 accounting for 49.9% of all isolates, followed by O138 (14.9%), O139 (6.9%), O141 (4.1%) and O8 (3.7%) . Hemolytic activity was detected in 87.7% of all isolates . Virulence factor genes detected were F4 (44.7%), F18 (39.3%), intimin (1.4%), F6 (0.9%), STb (77.6%), EAST1 (65.8%), LT (61.6%), STa (26.5%) and VT2e (16.4%) . Six pathotypes accounted for 65.7% of all isolates investigated . Using possession of virulence factor genes as reference, O-serogrouping employing a selection of antisera representing common pig pathogenic serogroups and detection of hemolysis were evaluated as epidemiological markers for pathogenicity . Both criteria were associated with pathogenicity (P<0.001, for both), however, both methods also resulted in false classifications regarding pathogenicity for 11.9 and 13.2% of isolates, respectively . Detection of adhesion factor genes F4, F18 and intimin is suggested as an operational alternative when diagnosing PWD and ED. Vet Microbiol, 2002 Mar 1, 85(2), 125 - 32 O-serogroups, eae gene and EAF plasmid in Escherichia coli isolates from cases of bovine mastitis in Brazil; Correa MG et al.; Mastitis has been recognized for some time as the most costly disease in dairy herds . From March 1997 to August 1998, 2144 samples of bovine mastitic milk were collected, from which 182 Escherichia coli isolates were made, and from which 141 isolates had the somatic antigen (serogroup) determined . Twelve different serogroups were isolated from mastitic milk, and among them were O26, O55, O111 and O119, all of them classic enteropathogenic E . coli (EPEC) serogroups . These represented 40.0% of the isolates . The 20 of 57 isolates tested had plasmids and in dot blot hybridization, nine isolates were positive for an EaeA probe and an EPEC adherence factor (EAF) probe while two isolates were negative for EaeA probe but positive for the EAF probe . The nine isolates were characterized as attaching and effacing (A/E) E . coli (AEEC) isolates. Helicobacter, 2001 Dec, 6(4), 274 - 82 Immune response to a 26-kDa protein, alkyl hydroperoxide reductase, in Helicobacter pylori-infected Mongolian gerbil model; Yan J et al.; BACKGROUND: The host immune response is thought to play an important role in the outcome of Helicobacter pylori infection . The successful development of the H . pylori-infected Mongolian gerbil model that mimics human disease has enabled study of the antibody response against H . pylori antigens . MATERIALS AND METHODS: Serum samples from ulcer and carcinogenesis models of H . pylori-infected gerbils were used to screen for H . pylori antigens that cause a humoral immune response in the infected hosts . H . pylori alkyl hydroperoxide reductase (AhpC) is one such antigen on which we report here . The tsaA gene encoding AhpC was amplified by PCR from H . pylori ATCC 43504 strain, cloned into pMAL(TM)-c2 expression vector and expressed in Escherichia coli . Maltose-binding protein fusion protein (MBP-AhpC) was purified by a MBP affinity column . Using purified recombinant AhpC protein as an antigen, the antibody response and changes of antibody levels against AhpC in the gerbil models were studied by Western blotting and ELISA . RESULTS: Antibody against AhpC was negative in the early stages of infection, and became positive in the gerbils with the emergence of gastric diseases such as chronic active gastritis, gastric ulcer and gastric cancer . The antibody levels (ELISA) increased gradually over time and were higher in gerbils with gastric ulcer than that in gerbils without ulcers . CONCLUSIONS: Use of the gerbil model that mimics human H . pylori infection is likely to provide insights into the role of H . pylori-specific antigens possibly related to the subsequent development of gastric diseases. Mol Plant Microbe Interact, 2002 Jan, 15(1), 54 - 9 MucR and mucS activate exp genes transcription and galactoglucan production in Sinorhizobium meliloti EFB1; Lloret J et al.; When grown under standard conditions, Sinorhizobium meliloti EFB1 simultaneously produces two acidic exopolysaccharides, succinoglycan and galactoglucan, yielding very mucoid colonies . In this strain, MucR is essential for galactoglucan synthesis . A mutation in the mucS gene resulted in less mucoid colonies than in the wild-type EFB1 . This mucS- strain was complemented to the wild-type phenotype by the cloned mucS gene, indicating that mucS is necessary for a wild-type level of galactoglucan production . Reverse transcription-polymerase chain reaction analysis of exp genes, which encode the pathway for galactoglucan production, in EFB1 and in the mutants affected in mucS, mucR, and both genes simultaneously, showed that MucS is a transcriptional activator of the exp genes but does not affect its own transcription . Furthermore, MucR is necessary for mucS transcriptional activation . As introduction of a cloned mucS gene in a mucR- strain yielded colonies less mucoid than the wild type, MucR could also activate exp genes transcription through other pathways . Deletion analysis of the expE promoter showed a region important for transcription and MucS activation . This region, containing a palindrome, is present in the putative expA, expC, expD, and expE promoters but not in the mucS promoter, suggesting that it is the target for MucS . A mucR-mucS- mutant, which does not produce galactoglucan, was impaired in competitive nodulation of alfalfa in soil microcosms, indicating another possible role for this exopolysaccharide in symbiosis. Biol Chem, 2001 Dec, 382(12), 1707 - 14 Recombinant cryptic human fibronectinase cleaves actin and myosin: substrate specificity and possible role in muscular dystrophy; Schnepel J et al.; The N-terminal heparin/fibrin binding domain of human plasma fibronectin (pFN) contains a cryptic proteinase . The enzyme could be generated and activated in the presence of Ca2+ from the purified 70 kDa pFN fragment produced by cathepsin D digestion of pFN . In this work we cloned and expressed the serine proteinase, designated fibronectinase (Fnase), in E . coli . The recombinant pFN protein fragment was isolated from inclusion bodies, subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting fibronectin . Cleavage of pFN and the synthetic peptides Ac-I-E-G-K-pNA and Bz-I-E-G-R-pNA demonstrated identical specificity of the recombinant and the isolated fibronectinase . Further investigations of the substrate specificity revealed for the first time the muscle proteins actin and myosin as being substrates of fibronectinase . The enzyme can be inhibited by alpha1-proteinase inhibitor . In the context of induced cathepsin D release, e . g . from granulocytes under inflammatory conditions, these results indicate an increase in specific proteolytic potential against muscular proteins in dystrophic diseases by the release of cryptic fibronectinase. Biol Chem, 2001 Dec, 382(12), 1679 - 86 Structural and redox properties of the leaderless DsbE (CcmG) protein: both active-site cysteines of the reduced form are involved in its function in the Escherichia coli periplasm; Li Q et al.; The thiol/disulfide oxidoreductases play important roles in ensuring the correct formation of disulfide bonds, of which the DsbE protein, also called CcmG, is the one implicated in electron transfer for cytochrome c maturation in the periplasm of Escherichia coli . The soluble, N-terminally truncated DsbE was overexpressed and purified to homogeneity . Here we report the structural and redox properties of the leaderless form (DsbEL-) . During the redox reaction, the protein undergoes a structural transformation resulting in a more stable reduced form, but this form shows very low reactivity in thiol/ disulfide exchange of cysteine residues and low activity in accelerating the reduction of insulin . The standard redox potential (E'0) for the active thiol/ disulfide was determined to be -0.186 V; only one of the two cysteines (Cys80) was suggested to be the active residue in the redox reaction . From the aspect of biochemical properties, DsbE can be regarded as a weak reductant in the Escherichia coli periplasm . This implies that the function of DsbE in cytochrome c maturation can be ascribed to its active-site cysteines and the structure of the reduced form. J Periodontal Res, 2002 Feb, 37(1), 1 - 7 Inhibition of alveolar bone loss by matrix metalloproteinase inhibitors in experimental periodontal disease; Ramamurthy NS et al.; Periodontal disease is characterized by excessive host collagenase resulting in loss of gingival and periodontal ligament collagen and adjacent alveolar bone . Intragingival endotoxin injection induces a model of periodontal disease characterized by rapid bone loss with biochemical features similar to that of naturally occurring adult periodontitis . CH1766, a peptide with a zinc binding moeity which fits into the active site of the enzyme, and CH6631, a hydroxamic acid derivative with aryl-substituted sulphonamide residues, are inhibitors of matrix metalloproteinases (MMPIs) with differing inhibitory profiles as characterized by in vitro assays . In this study, endotoxin was injected into the gingivae of rats which were then treated orally with either 3 mg/kg or 30 mg/kg of one of the two inhibitory compounds . The gingival tissues were assessed for collagenase and gelatinase activity, plus three different pro-inflammatory cytokines . In addition, alveolar bone height in defleshed jaws was studied by computerized morphometric analysis and scanning electron microscopy . Both drugs reduced active and/or total MMP activity, in many cases to normal, and also partially normalized cytokine levels as well . A dose-response effect was seen with regard to amelioration of lipopolysaccharide-induced alveolar bone loss with both drugs . Other than studies with tetracyclines, this is the first report of beneficial effects of MMPIs in a model of periodontal disease, strongly suggesting that this class of agents could bring therapeutic benefit to patients with this disorder, and that periodontal disease can be used as a model to demonstrate in vivo efficacy of this class of drugs. Biopolymers, 2002, 67(1), 10 - 9 Changes in protein conformation and dynamics upon complex formation of brain-derived neurotrophic factor and its receptor: investigation by isotope-edited Fourier transform IR spectroscopy; Li T et al.; The interactions of brain-derived neurotrophic factor (BDNF) with the extracellular domain of its receptor (trkB) are investigated by employing isotope-edited Fourier transform IR (FTIR) spectroscopy . The protein secondary structures of individual BDNF and trkB in solutions are compared with those in their complex . The temperature dependence of the secondary structures of BDNF, trkB, and their complex is also investigated . Consistent with the crystal structure, we observe by FTIR spectroscopy that BDNF in solution contains predominantly beta strands (approximately 53%) and relatively low contents of other secondary structures including beta turns (approximately 16%), disordered structures (approximately 12%), and loops (approximately 18%) and is deficient in alpha helix . We also observe that trkB in solution contains mostly beta strands (52%) and little alpha helix . Conformational changes in both BDNF and trkB are observed upon complex formation . Specifically, upon binding of BDNF, the conformational changes in trkB appear to involve mostly beta turns and disordered structures while the majority of the beta-strand conformation remains unchanged . The IR data indicate that some of the disordered structures in the loop regions are likely converted to beta strands upon complex formation . The FTIR spectral data of BDNF, trkB, and their complex indicate that more amide NH groups of trkB undergo H-D exchange within the complex than those of the ligand-free receptor and that the thermal stability of trkB is decreased slightly upon binding of BDNF . The FT-Raman spectra of BDNF, trkB, and their complex show that the six intramolecular disulfide bonds of trkB undergo significant conformational changes upon binding of BDNF as a result of changes in the tertiary structure of trkB . Taken together, the FTIR and Raman data are consistent with the loosening of the tertiary structure of trkB upon binding of BDNF, which leads to more solvent exposure of the amide NH group and decreased thermal stability of trkB . This finding reveals an intriguing structural property of the neurotrophin ligand-receptor complex that is in contrast to other ligand-receptor complexes such as a cytokine-receptor complex that usually shows protection of the amide NH group and increased thermal stability upon complex formation . J Gen Virol, 2002 Mar, 83(Pt 3), 623 - 9 Six-helix bundle assembly and characterization of heptad repeat regions from the F protein of Newcastle disease virus; Yu M et al.; Paramyxoviruses may adopt a similar fusion mechanism to other enveloped viruses, in which an anti-parallel six-helix bundle structure is formed post-fusion in the heptad repeat (HR) regions of the envelope fusion protein . In order to understand the fusion mechanism and identify fusion inhibitors of Newcastle disease virus (NDV), a member of the Paramyxoviridae family, we have developed an E . coli system that separately expresses the F protein HR1 and HR2 regions as GST fusion proteins . The purified cleaved HR1 and HR2 have subsequently been assembled into a stable six-helix bundle heterotrimer complex . Furthermore, both the GST fusion protein and the cleaved HR2 show virus-cell fusion inhibition activity (IC(50) of 1.07-2.93 microM) . The solubility of the GST-HR2 fusion protein is much higher than that of the corresponding peptide . Hence this provides a plausible method for large-scale production of HR peptides as virus fusion inhibitors. J Gen Virol, 2002 Mar, 83(Pt 3), 581 - 93 Mutational analysis of the active centre of coronavirus 3C-like proteases; Hegyi A et al.; Formation of the coronavirus replication-transcription complex involves the synthesis of large polyprotein precursors that are extensively processed by virus-encoded cysteine proteases . In this study, the coding sequence of the feline infectious peritonitis virus (FIPV) main protease, 3CL(pro), was determined . Comparative sequence analyses revealed that FIPV 3CL(pro) and other coronavirus main proteases are related most closely to the 3C-like proteases of potyviruses . The predicted active centre of the coronavirus enzymes has accepted unique replacements that were probed by extensive mutational analysis . The wild-type FIPV 3CL(pro) domain and 25 mutants were expressed in Escherichia coli and tested for proteolytic activity in a peptide-based assay . The data strongly suggest that, first, the FIPV 3CL(pro) catalytic system employs His(41) and Cys(144) as the principal catalytic residues . Second, the amino acids Tyr(160) and His(162), which are part of the conserved sequence signature Tyr(160)-Met(161)-His(162) and are believed to be involved in substrate recognition, were found to be indispensable for proteolytic activity . Third, replacements of Gly(83) and Asn(64), which were candidates to occupy the position spatially equivalent to that of the catalytic Asp residue of chymotrypsin-like proteases, resulted in proteolytically active proteins . Surprisingly, some of the Asn(64) mutants even exhibited strongly increased activities . Similar results were obtained for human coronavirus (HCoV) 3CL(pro) mutants in which the equivalent Asn residue (HCoV 3CL(pro) Asn(64)) was substituted . These data lead us to conclude that both the catalytic systems and substrate-binding pockets of coronavirus main proteases differ from those of other RNA virus 3C and 3C-like proteases. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1905 - 9 Epub 2002 Feb 12. Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair; Minko IG et al.; DNA-protein crosslinks (DPCs) arise in biological systems as a result of exposure to a variety of chemical and physical agents, many of which are known or suspected carcinogens . The biochemical pathways for the recognition and repair of these lesions are not well understood in part because of methodological difficulties in creating site-specific DPCs . Here, a strategy for obtaining site-specific DPCs is presented, and in vitro interactions of the Escherichia coli nucleotide excision repair (NER) UvrABC nuclease at sites of DPCs are investigated . To create site-specific DPCs, the catalytic chemistry of the T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase (T4-pdg) has been exploited, namely, its ability to be covalently trapped to apurinic/apyrimidinic sites within duplex DNA under reducing conditions . Incubation of the DPCs with UvrABC proteins resulted in DNA incision at the 8th phosphate 5' and the 5th and 6th phosphates 3' to the protein-adducted site, generating as a major product of the reaction a 12-mer DNA fragment crosslinked with the protein . The incision occurred only in the presence of all three protein subunits, and no incisions were observed in the nondamaged complementary strand . The UvrABC nuclease incises DPCs with a moderate efficiency . The proper assembly and catalytic function of the NER complex on DNA containing a covalently attached 16-kDa protein suggest that the NER pathway may be involved in DPC repair and that at least some subset of DPCs can be removed by this mechanism without prior proteolytic degradation. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1954 - 9 Epub 2002 Feb 12. TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain; Frickel EM et al.; The lectin chaperone calreticulin (CRT) assists the folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER) . It interacts with ERp57, a thiol-disulfide oxidoreductase that promotes the formation of disulfide bonds in glycoproteins bound by CRT . Here, we investigated the interaction between CRT and ERp57 by using biochemical techniques and NMR spectroscopy . We found that ERp57 binds to the P-domain of calreticulin, an independently folding domain comprising residues 189-288 . Isothermal titration calorimetry showed that the dissociation constant of the CRT(189-288)/ERp57 complex is (9.1 +/- 3.0) x 10(-6) M at 8 degrees C . Transverse relaxation-optimized NMR spectroscopy provided data on the thermodynamics and kinetics of the complex formation and on the structure of this 66.5-kDa complex . The NMR measurements yielded a value of (18 +/- 5) x 10(-6) M at 20 degrees C for the dissociation constant and a lower limit for the first-order exchange rate constant of k(off) > 1,000 s(-1) at 20 degrees C . Chemical shift mapping showed that interactions with ERp57 occur exclusively through amino acid residues in the polypeptide segment 225-251 of CRT(189-288), which forms the tip of the hairpin structure of this domain . These results are analyzed with regard to the functional mechanism of the CRT/ERp57 chaperone system. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2439 - 44 Epub 2002 Feb 12. Sensitivity to Alternaria alternata toxin in citrus because of altered mitochondrial RNA processing; Ohtani K et al.; Specificity in the interaction between rough lemon (Citrus jambhiri Lush.) and the fungal pathogen Alternaria alternata rough lemon pathotype is determined by a host-selective toxin, ACR-toxin . Mitochondria from rough lemon are sensitive to ACR-toxin whereas mitochondria from resistant plants, including other citrus species, are resistant . We have identified a C . jambhiri mitochondrial DNA sequence, designated ACRS (ACR-toxin sensitivity gene), that confers toxin sensitivity to Escherichia coli . ACRS is located in the group II intron of the mitochondrial tRNA-Ala and is translated into a SDS-resistant oligomeric protein in C . jambhiri mitochondria but is not translated in the toxin-insensitive mitochondria . ACRS is present in the mitochondrial genome of both toxin-sensitive and -insensitive citrus . However, in mitochondria of toxin-insensitive plants, the transcripts from ACRS are shorter than those in mitochondria of sensitive plants . These results demonstrate that sensitivity to ACR-toxin and hence specificity of the interaction between A . alternata rough lemon pathotype and C . jambhiri is due to differential posttranscriptional processing of a mitochondrial gene. Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 1859 - 64 Epub 2002 Feb 12. Mechanism of action and NAD+-binding mode revealed by the crystal structure of L-histidinol dehydrogenase; Barbosa JA et al.; The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to l-histidine in 10 enzymatic reactions . This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway . l-histidinol dehydrogenase (HisD, EC ) catalyzes the last two steps in the biosynthesis of l-histidine: sequential NAD-dependent oxidations of l-histidinol to l-histidinaldehyde and then to l-histidine . HisD functions as a homodimer and requires the presence of one Zn(2+) cation per monomer . We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, Zn(2+), and NAD(+) (best resolution is 1.7 A) . Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping . Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication . Residues from both monomers form the active site . Zn(2+) plays a crucial role in substrate binding but is not directly involved in catalysis . The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule . NAD(+) binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold. Plant Physiol, 2002 Feb, 128(2), 661 - 8 Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli; Yeh CH et al.; Rice (Oryza sativa) class I low-molecular mass (LMM) heat shock protein (HSP), Oshsp16.9, has been shown to be able to confer thermotolerance in Escherichia coli . To define the regions for this intriguing property, deletion mutants of this hsp have been constructed and overexpressed in E . coli XL1-blue cells after isopropyl beta-D-thioglactopyranoside induction . The deletion of amino acid residues 30 through 36 (PATSDND) in the N-terminal domain or 73 through 78 (EEGNVL) in the consensus II domain of Oshsp16.9 led to the loss of chaperone activities and also rendered the E . coli incapable of surviving at 47.5 degrees C . To further investigate the function of these two domains, we determined the light scattering changes of Oshsp16.9 mutant proteins at 320 nm under heat treatment either by themselves or in the presence of a thermosensitive enzyme, citrate synthase . It was observed that regions of amino acid residues 30 through 36 and 73 through 78 were responsible for stability of Oshsp16.9 and its interactions with other unfolded protein substrates, such as citrate synthase . Studies of two-point mutants of Oshsp16.9, GST-N74E73K and GST-N74E74K, indicate that amino acid residues 73 and 74 are an important part of the substrate-binding site of Oshsp16.9 . Non-denaturing gel analysis of purified Oshsp16.9 revealed that oligomerization of Oshsp16.9 was necessary but not sufficient for its chaperone activity. Plant Physiol, 2002 Feb, 128(2), 578 - 90 FQR1, a novel primary auxin-response gene, encodes a flavin mononucleotide-binding quinone reductase; Laskowski MJ et al.; FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase . Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment . This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene . Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases . Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity . Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo . In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes . We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress. Plant Physiol, 2002 Feb, 128(2), 454 - 62 The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism; Hacham Y et al.; Cystathionine gamma-synthase (CGS) is a key enzyme of Met biosynthesis in bacteria and plants . Aligning the amino acid sequences revealed that the plant enzyme has an extended N-terminal region that is not found in the bacterial enzyme . However, this region is not essential for the catalytic activity of this enzyme, as deduced from the complementation test of an Escherichia coli CGS mutant . To determine the function of this N-terminal region, we overexpressed full-length Arabidopsis CGS and its truncated version that lacks the N-terminal region in transgenic tobacco (Nicotiana tabacum) plants . Transgenic plants expressing both types of CGS had a significant higher level of Met, S-methyl-Met, and Met content in their proteins . However, although plants expressing full-length CGS showed the same phenotype and developmental pattern as wild-type plants, those expressing the truncated CGS showed a severely abnormal phenotype . These abnormal plants also emitted high levels of Met catabolic products, dimethyl sulfide and carbon disulfide . The level of ethylene, the Met-derived hormone, was 40 times higher than in wild-type plants . Since the alien CGS was expressed at comparable levels in both types of transgenic plants, we further suggest that post-translational modification(s) occurs in this N-terminal region, which regulate CGS and/or Met metabolism . More specifically, since the absence of the N-terminal region leads to an impaired Met metabolism, the results further suggest that this region plays a role in protecting plants from a high level of Met catabolic products such as ethylene. Nucleic Acids Res, 2002 Feb 15, 30(4), 931 - 41 Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein; Kuhn JF et al.; Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs . Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described . Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein . Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein . Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D . Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction . These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region . The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea . L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis. Nucleic Acids Res, 2002 Feb 15, 30(4), 886 - 93 Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA; Cannon W et al.; The bacterial sigma54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein . Using DNA binding and transcription assays the ability of the enhancer-dependent sigma54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed . Different DNA regions important for stabilising sigma54 holoenzyme-promoter interactions, destabilizing binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking sigma54 Region I were identified . It appears that homoduplex structures are required for early events in sigma54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription . DNA opening from -5 to -1 appears important for stable engagement of the holoenzyme following activation . The regulatory Region I of sigma54 was shown to be involved in interactions with the sequences in the -5 to -1 area. J Biol Chem, 2002 May 10, 277(19), 17062 - 71 Epub 2002 Feb 12. The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin; Wilkinson SR et al.; Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione . Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin . The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract . Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro . The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin . Both tryparedoxin and TcGPXI operate by a ping-pong mechanism . Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides . TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle . Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol . The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites. Immunol Lett, 2002 Apr 1, 81(1), 31 - 40 Endomorphins delay constitutive apoptosis and alter the innate host defense functions of neutrophils; Azuma Y et al.; Recent studies have shown that opioid peptides are released from cells of the immune system during inflammation and stress, and are associated with altered immune responses . Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions . The aim of this study was to evaluate immunological effects of opioid peptides endomorphins 1 and 2 on constitutive apoptosis, superoxide anion production, hydrogen peroxide production, adhesion, phagocytosis, and chemotaxis of neutrophils . Neutrophils were isolated by peritoneal lavage from rats . Endomorphins 1 and 2 significantly delayed constitutive neutrophil apoptosis . The delay of neutrophil apoptosis was markedly attenuated by LY294002, a phosphoinositide 3-kinase inhibitor . Moreover, endomorphins 1 and 2 activated the phosphoinositide 3-kinase pathway as determined by phosphorylation of BAD . In contrast, endomorphins 1 and 2 blocked the production of superoxide anion and hydrogen peroxide by PMA-stimulated neutrophils . In addition, endomorphins 1 and 2 inhibited neutrophil adhesion to fibronectin . Moreover, endomorphins 1 and 2 potentiated neutrophil chemotaxis toward zymosan-activated serum and IL-8, respectively . However, endomorphins 1 and 2 did not alter phagocytosis of Escherichia coli by neutrophils . These results suggest that endomorphins 1 and 2 may act to delay neutrophil apoptosis and alter the natural immune functions of neutrophils. Immunol Lett, 2002 Apr 1, 81(1), 13 - 24 A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-gamma and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor; Kumar S et al.; We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein 1 (pMSP1(42)) from the 3D7 strain of Plasmodium falciparum (Pf3D7) . This plasmid expressed recombinant MSP1(42) after in vitro transfection in mouse VM92 cells . Rhesus monkeys immunized with pMSP1(42) produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1(19) (yMSP1(19)) . Immunization also induced antigen specific T cell responses as measured by interferon-gamma production, and by classical CTL chromium release assays . In addition, immunization with pMSP1(42) primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1(19) . We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1(42.) We tested both rhesus GM-CSF expressed from a DNA plasmid, and E . coli produced recombinant human GM-CSF . Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF . Co-immunization with a mixture of prhGM-CSF and pMSP1(42) induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses . In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose . However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1(19) . The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates. Carbohydr Res, 2002 Feb 18, 337(4), 327 - 33 Production of highly phosphorylated glycopolymers by expression of R1 in Escherichia coli; Vikso-Nielsen A et al.; The possible involvement of the starch bound R1 protein from potato (Solanum tuberosum L.) in the phosphorylation of starch was investigated by functional expression and characterisation of R1 in Escherichia coli . By expression of R1 in E . coli it is shown that it is possible to produce glycopolymers, e.g., glycogen, with an increased degree of phosphate substitution . The expression of R1 in E . coli resulted in a sixfold increase in glycogen bound phosphate and in an increased accumulation of glycogen leading to a glycogen excess (gex) phenotype . There was an overall shift in the unit-chain length of the isolated glycogen towards smaller degrees of polymerisation . The pleiotropic effects on the glycogen biosynthetic and amylolytic enzyme activities was investigated and showed an increase in ADPglucose pyrophosphorylase activity, as well as a decrease in exo-amylolytic activity . These results are discussed in relation to starch phosphorylation and a possible role of R1 in this respect. Biochem Pharmacol, 2002 Jan 15, 63(2), 305 - 20 The involvement of L-gamma-glutamyl-L-cysteinyl-glycine (glutathione/GSH) in the mechanism of redox signaling mediating MAPK(p38)-dependent regulation of pro-inflammatory cytokine production; Haddad JJ; Redox regulation of mitogen-activated protein kinase (MAPK(p38))-mediated pro-inflammatory cytokine production is not well characterized in the alveolar epithelium . It was hypothesized that the involvement of the MAPK(p38) pathway in regulating lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha and interleukin-6 secretion is redox-sensitive and affected by NAC, an antioxidant and a precursor of glutathione, and L-buthionine-(S,R)-sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis . Exposure of fetal alveolar type II epithelial cells to Escherichia coli-derived LPS induced, in a time-dependent manner, the phosphorylation/activation of MAPK(p38) (peak at 15min) . In addition, LPS up-regulated the phosphorylation of MAPK(p38) in a dose-dependent manner . The effect of LPS on the MAPK(p38) pathway was associated with the activation of MAPK-activated protein kinase, which phosphorylated the small 27kDa heat-shock protein (Hsp27) . LPS induced the phosphorylation of Hsp27 in a time- and dose-dependent manner . Selective blockage of the MAPK(p38) pathway by a pyridinyl-imidazole (SB-203580) abrogated LPS-induced release of TNF-alpha and IL-6 . Pre-treatment with NAC reduced LPS-mediated secretion of TNF-alpha and IL-6 . Incubation of cells with NAC induced intracellular accumulation of GSH, but reduced the concentration of GSSG . On the other hand, pre-treatment with BSO augmented LPS-mediated secretion of TNF-alpha and IL-6 . In addition, BSO induced intracellular accumulation of GSSG, but reduced the concentration of GSH . Whereas NAC blocked the phosphorylation/activation of MAPK(p38), BSO amplified the LPS-mediated effect on MAPK(p38) . These results indicated that intracellular redox signaling plays an important role in regulating LPS-induced activation of the MAPK(p38) pathway and MAPK(p38)-mediated regulation of LPS-dependent inflammatory cytokine production in the alveolar epithelium. Scand J Immunol, 2002 Jan, 55(1), 82 - 7 Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis; Harboe M et al.; The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis . The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M . bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein . Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase . In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide . Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau . After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate . In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II. J Am Chem Soc, 2002 Feb 20, 124(7), 1443 - 51 Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme; Mulder FA et al.; Recently developed carbon transverse relaxation dispersion experiments (Skrynnikov, N . R.; et al . J . Am . Chem . Soc . 2001, 123, 4556-4566) were applied to the study of millisecond to microsecond time scale motions in a cavity mutant of T4 lysozyme (L99A) using methyl groups as probes of dynamics . Protein expressed in E . coli cells with (13)CH(3)-pyruvate as the sole carbon source contained high levels of (13)C enrichment at a total of 80 Val gamma, Leu delta, Ile gamma (2), Ala beta, and Met epsilon methyl positions with little extraneous incorporation . Data for 72 methyl groups were available for analysis . Dispersion profiles with large amplitudes were measured for many of these residues and were well fit to a two-state exchange model . The interconversion rates and populations of the states, obtained from fitting relaxation dispersion profiles of each individual probe, were remarkably homogeneous and data for nearly all methyl groups in the protein could be collectively fit to a single cooperative conformational transition . The present study demonstrates the general applicability of methyl relaxation dispersion measurements for the investigation of millisecond time scale protein motions at a large number of side-chain positions . Potential artifacts associated with the experiments are described and methods to minimize their effects presented . These experiments should be particularly well suited for probing dynamics in high molecular weight systems due to the favorable NMR spectroscopic properties of methyl groups. Biochemistry, 2002 Feb 19, 41(7), 2452 - 8 Stopped-flow analysis on anion binding to blue-form halorhodopsin from Natronobacterium pharaonis: comparison with the anion-uptake process during the photocycle; Sato M et al.; Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells . The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column . Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore . Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)) . Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers . The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N . pharaonis membranes . Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing . Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes . The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol . These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously {Varo et al . (1995) Biochemistry 34, 14500-14507} . In addition, the Cl- concentration dependence on both rates was similar to each other . These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process. Biochemistry, 2002 Feb 19, 41(7), 2305 - 10 A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4; Pascal A et al.; Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors . This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids {Bassi, R., Croce, R., Cugini, D., and Sandona, D . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 10056-10061} . The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated . Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations . By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site . In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides. Biochemistry, 2002 Feb 19, 41(7), 2227 - 36 Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins; Markova SV et al.; A cDNA encoding the Ca2+-regulated photoprotein of the bioluminescent marine hydroid Obelia geniculata was cloned and sequenced . The cDNA is a 774 bp fragment containing two overlapping open reading frames, one of which contained 585 bp encoding a 195 amino acid polypeptide which obviously has the primary structure of the apoprotein of a calcium-regulated photoprotein . Many of the residues are identical to those in other Ca2+-regulated photoproteins: 86% compared with that from Obelia longissima, 76% with that from Clytia (Phialidium), 64% with that from Aequorea, and 64% with that from Mitrocoma(Halistaura) . The obelin from O . geniculata was overexpressed in Escherichia coli, refolded from inclusion bodies, and purified . The yield of highly purified recombinant protein was 55-80 mg/L of LB medium . O . geniculata obelin has absorption maxima at 280 and 460 nm and a shoulder at approximately 310 nm . The calcium-discharged protein loses visible absorption but exhibits a new absorption maximum at 343 nm . The bioluminescence of the obelin from O . geniculata is blue (lambda(max) = 495 nm) . In contrast, the fluorescence of the calcium-discharged protein is yellow-green (lambda(max) = 520 nm; excitation at 340 nm) . This is in sharp contrast to aequorin in which the bioluminescence and fluorescence emission spectra of the calcium-discharged protein are almost identical (lambda(max) = 465 nm) . The Ca2+ concentration-effect curve for O . geniculata obelin is similar to those of many other photoproteins: at {Ca2+} below approximately 10(-8) M, calcium-independent luminescence is observed, and at {Ca2+} approximately 10(-3) M, the luminescence reaches a maximum . Between these extremes, the curve spans a vertical range of almost 8 log units with a maximum slope on a log-log plot of about 2.5 . In the absence of Mg2+ the rate constant for the rise of bioluminescence determined by the stopped-flow technique is about 450 s(-1) . The effects of Mg2+ on the kinetics of bioluminescence are complicated, but at all concentrations studied they are relatively small compared to the corresponding effects on aequorin luminescence . At least with respect to speed and sensitivity to Mg2+, the obelins from both O . longissima and O . geniculata would appear to be more suitable than aequorin for use as intracellular Ca2+ indicators. Biochemistry, 2002 Feb 19, 41(7), 2158 - 65 Novel activity of Escherichia coli mismatch uracil-DNA glycosylase (Mug) excising 8-(hydroxymethyl)-3,N4-ethenocytosine, a potential product resulting from glycidaldehyde reaction; Hang B et al.; Glycidaldehyde is an industrial chemical which has been shown to be genotoxic in in vitro experiments and carcinogenic in rodent studies . It is a bifunctional alkylating agent capable of reacting with DNA to form exocyclic hydroxymethyl-substituted ethenobases . In this work, 8-(hydroxymethyl)-3,N4-etheno-2'-deoxycytidine (8-HM-epsilondC), a potential nucleoside derivative of glycidaldehyde, was synthesized using phosphoramidite chemistry and site-specifically incorporated into a defined 25-mer oligodeoxynucleotide . The 8-HM-epsilonC adduct is structurally related to 3,N4-ethenocytosine (epsilonC), a product of reaction with vinyl chloride or through lipid peroxidation . In Escherichia coli, epsilonC has been shown previously to be a primary substrate for the mismatch uracil-DNA glycosylase (Mug) . In this study, we report that the same glycosylase also acts on 8-HM-epsilonC in an oligonucleotide duplex . The enzyme binds to the 8-HM-epsilonC-oligonucleotide to a similar extent as the epsilonC-oligonucleotide . The Mug excision activity toward 8-HM-epsilonC is approximately 2.5-fold lower than that toward the epsilonC substrate . Both activities can be stimulated up to approximately 2-fold higher by the addition of E . coli endonuclease IV . These two adducts, when mispaired with normal bases, were all excised from DNA by Mug with similar efficiencies . Structural studies using molecular simulations showed similar adjustment and hydrogen bonding pattern for both 8-HM-epsilonC*G and epsilonC*G pairs in oligomer duplexes . We believe that these findings may have biological and structural implications in defining the role of 8-HM-epsilonC in glycosylase recognition/repair. Biochemistry, 2002 Feb 19, 41(7), 2140 - 8 Native state EX2 and EX1 hydrogen exchange of Escherichia coli CspA, a small beta-sheet protein; Rodriguez HM et al.; Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an apparent two-state mechanism . Our previous studies have shown that a large aromatic cluster on the surface of the protein participates in the rate-limiting step of folding and thus may be part of the folding nucleus of this protein . To obtain a more detailed picture of molecular events at the peptide backbone during unfolding and folding of CspA, we used native state hydrogen exchange and nuclear magnetic resonance spectroscopy (NMR) . The experiments with native CspA were performed over a range of pH values from low pH, where exchange is governed by a rapid equilibrium before chemical exchange (EX2 exchange), to high pH, where exchange is dictated by the rate of unfolding (EX1 exchange) . Rates of folding and unfolding were determined for 11 residues . The distribution of rates of folding within the structure of CspA suggests that hairpin turns, including one near the aromatic cluster, may nucleate the folding of CspA. Tsitologiia, 2001, 43(11), 1067 - 74 { Effect of gamma radiation, cis-diamminedichloroplatinum and its derivates on the Escherichia coli cell survival and potentiality for adaptive response}; Zhestianikov VD et al.; The sensitivity to the lethal effect of gamma-rays, cis- and trans-diamminedichloroplatinum (DDP), cis- and trans-iminoethers of DDP (IE) was compared in two groups of E . coli--K12 and B . In all experiments, cells of wild types appeared to be most resistant to these agents . gamma-Resistant and gamma-sensitivity/hypersensitive strains occupy an intermediate position according to their sensitivity to cis-DDP derivatives . In almost all the cases, both single and especially double mutants defective for the systems of nucleotide excision repair, recombination repair, and inducible SOS-repair are most sensitive to DDP derivatives . The data obtained show that in E . coli the repair of lethal lesions after cis-DDP action is more complicated than after gamma-irradiation . Of DDP derivatives cis-DDP is most effective, while trans-DDP is less effective, and cis- and trans-IE are considerably less effective, respectively . It is shown that the effects of ionizing radiation in low doses (more than 10 different regimes), or of treatment with cis-DDP in low concentrations do not change the survival of E . coli after their respective effects in high doses . In other words, under the effect of ionizing radiation and cis-DDP no adaptive response for the lethal action was found in E . coli. Trends Biochem Sci, 2002 Jan, 27(1), 11 - 8 Running rings around RNA: a superfamily of phosphate-dependent RNases; Symmons MF et al.; The exosome of Saccharomyces cerevisiae and the degradosome of Escherichia coli are multienzyme complexes involved in the degradation of mRNA . Both contain enzymes that are similar to the phosphate-dependent exoribonuclease RNase PH . These enzymes are phosphorylases that degrade RNA from the 3'-end . A recent X-ray crystallographic study of the polynucleotide phosphorylase (PNPase) from Streptomyces antibioticus reveals, for the first time, the atomic structure of a member of the RNase PH superfamily . Here, information from the structure of PNPase is used to address two related issues . First, the structure supports the idea that PNPase, which is a trimer of multidomain subunits, arose by duplication of a gene encoding an RNase PH-like enzyme . Second, the structure might explain how RNase PH-like enzymes associate into oligomeric rings that degrade RNA in a processive reaction. Int J Parasitol, 2002 Jan, 32(1), 81 - 9 Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination; Duraisingh MT et al.; The genome sequence of Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, rapidly approaches completion, but our ability to genetically manipulate this organism remains limited . Chromosomal integration has only been achieved following the prolonged maintenance of circularised episomal plasmids which selects for single crossover recombinants . It has not been possible to construct genetic deletions via double crossover recombination, presumably due to the low frequency of this event . We have used the Herpes simplex virus thymidine kinase gene and the Escherichia coli cytosine deaminase gene for negative selection of P . falciparum . Parasites were transformed with plasmids expressing the thymidine kinase and cytosine deaminase genes by positive selection for the human dihydrofolate reductase gene . Parasites expressing thymidine kinase are susceptible to the pro-drug ganciclovir while those expressing cytosine deaminase are sensitive to 5-fluorocytosine . Parental parasites were inherently resistant to these drugs . A significant 'bystander effect' was evident in cultures with either ganciclovir or 5-fluorocytosine . Positive and negative selection of the thymidine kinase transformants with both ganciclovir and WR99210 resulted in the selection of parasites containing a genetic deletion of the Pfrh3 gene, the first targeted double crossover deletions in P . falciparum . The use of negative selection for gene disruptions via double crossover recombination will dramatically improve our ability to analyse protein function and opens the possibility of using this strategy for a variety of gene deletion and modification experiments in the analysis of this important infectious agent. Arch Biochem Biophys, 2002 Jan 15, 397(2), 286 - 92 Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate; Hanashiro I et al.; Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P) . Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P . The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase . Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P . The effect of phosphorylation was studied in two ways . Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme . In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells . Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A . This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants . The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states . However, the role must differ subtly between the mammalian and the yeast enzymes . (c)2001 Elsevier Science. Arch Biochem Biophys, 2002 Jan 15, 397(2), 279 - 85 Truncation of the amino terminus of branching enzyme changes its chain transfer pattern; Binderup K et al.; Previous work has reported the production of an Escherichia coli branching enzyme with a 112-residue deletion at the amino terminal by limited proteolysis . Here, we study the chain transfer pattern of this enzyme . Gel-permeation chromatography of in vitro branched amylose shows that the truncated branching enzyme transfers fewer short chains (degree of polymerization {d.p.} <20) and a greater proportion of intermediate size chains (d.p . 30-90) than the native enzyme . High-performance anion-exchange chromatography (HPAEC) of the branching limited alpha-glucan product indicates that the truncated branching enzyme transfers a smaller proportion of chains with d.p . 4-11 and more chains longer than d.p . 12 . Also, the genes encoding native or truncated branching enzyme were individually expressed in a branching enzyme-deficient mutant, AC71 (glgB(-)) . By HPAEC analysis of the purified alpha-glucans we find that truncated branching enzyme transfers fewer chains of d.p . 5-11 and more chains longer than d.p . 12 relative to the full-length enzyme . These observations allow us to conclude that truncation of the amino-terminal domain has altered the branching pattern of the enzyme . Our results are consistent with the construction of hybrid branching enzymes from the maize isoforms . (c)2001 Elsevier Science. Arch Biochem Biophys, 2002 Jan 15, 397(2), 206 - 16 Degradation of L-glutamate dehydrogenase from Escherichia coli: allosteric regulation of enzyme stability; Maurizi MR et al.; L-glutamate dehydrogenase (GDH) is stable in exponentially growing Escherichia coli cells but is degraded at a rate of 20-30% per hour in cells starved for either nitrogen or carbon . GDH degradation is energy-dependent, and mutations in ATP-dependent proteases, ClpAP or Lon lead to partial stabilization . Degradation is inhibited by chloramphenicol and is completely blocked in relA mutant cells, suggesting that ribosome-mediated signaling may facilitate GDH degradation . Purified GDH has a single tight site for NADPH binding . Binding of NADPH in the absence of other ligands leads to destabilization of the enzyme . NADPH-induced instability and sensitivity to proteolysis is reversed by tri- and dicarboxylic acids or nucleoside di- and triphosphates . GTP and ppGpp bind to GDH at an allosteric site and reverse the destabilizing effects of NADPH . Native GDH is resistant to degradation by several purified ATP-dependent proteases: ClpAP, ClpXP, Lon, and ClpYQ, but denatured GDH is degraded by ClpAP . Our results suggest that, in vivo, GDH is sensitized to proteases by loss of a stabilizing ligand or interaction with an destabilizing metabolite that accumulates in starving cells, and that any of several ATP-dependent proteases degrade the sensitized protein . (c)2002 Elsevier Science. Arch Biochem Biophys, 2002 Jan 15, 397(2), 184 - 90 Human erythrocyte pyrimidine 5'-nucleotidase, PN-I; Amici A et al.; Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides . Pyrimidine 5'-nucleotidase, PN-I, has been purified and characterized . The molecular and enzymatic properties determined for the enzyme shows a 36-kDa and 5.1 pI monomeric protein with no disulfide bridges and no phosphate content . The activity is dependent on Mg(2+), while it is inactivated by heavy metals and by thiol-reactive reagents . PN-I is specific for pyrimidine nucleoside monophosphates, including the antineoplastic agents 5'-AZTMP and 5'-Ara-CMP . PN-I possess phosphotransferase activity able to exchange phosphate between pyrimidine nucleoside monophosphates and pyrimidine nucleosides, including AZT and Ara-Cyd . Amino acid sequence has been obtained from tryptic and CNBr peptides . PN-I cDNA sequence, coding for a 286-residue protein, has been retrieved from tag database, amplified by PCR, and expressed in Escherichia coli . The recombinant protein was fully active and showed identical properties with respect to PN-I . Substantial identity has been revealed with the partial sequences reported for p36, an alpha-interferon-induced protein . The significance of this identity is discussed . (c)2002 Elsevier Science. Proteomics, 2002 Feb, 2(2), 151 - 6 Protein purification by Off-Gel electrophoresis; Ros A et al.; A novel free-flow protein purification technique based on isoelectric electrophoresis is presented, where the proteins are purified in solution without the need of carrier ampholytes . The gist of the method is to flow protein solutions under an immobilised pH gradient gel (IPG) through which an electric field is applied perpendicular to the direction of the flow . Due to the buffering capacity of the IPG gel, proteins with an isoelectric point (pI) close to pH of the gel in contact with the flow chamber stay in solution because they are neutral and therefore not extracted by the electric field . Other proteins will be charged when approaching the IPG gel and are extracted into the gel by the electric field . Both a demonstration experiment with pI markers and a simulation of the electric field distribution are presented to highlight the principle of the system . In addition, an isoelectric fractionation of an Escherichia coli extract is shown to illustrate the possible applications. Leukemia, 2002 Jan, 16(1), 67 - 73 TRAIL (Apo2L) suppresses growth of primary human leukemia and myelodysplasia progenitors; Plasilova M et al.; Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, APO2L) has been shown to induce apoptosis in a number of tumor cell lines as well as in some primary tumors whereas cells from most normal tissues are highly resistant to TRAIL-induced apoptosis . We have studied the susceptibility of primary malignant and normal bone marrow hematopoietic progenitors to TRAIL-induced apoptosis . Extracellular domain of human TRAIL with N-terminal His(6) tag (His-TRAIL, amino acids 95-281) was produced in E . coli and its apoptosis-inducing ability was compared with the leucine-zipper containing TRAIL, LZ-TRAIL . Both variants of TRAIL had the same apoptosis-inducing ability . Clonogenic progenitor assays showed that His-TRAIL significantly reduced the number of myeloid colonies (CFU-GM) and clusters from patients with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS) . His-TRAIL had no negative effect on the number of CFU-GM colonies and clusters derived from bone marrow cells of AML patients in complete remission, and lymphoma patients without bone marrow involvement, as well as those derived from normal cord blood cells . Moreover, we found that normal human stem cells treated with high doses of His-TRAIL maintain a repopulating potential when transplanted into NOD/SCID mice . To conclude, our data document that TRAIL does not affect normal human hematopoiesis but suppresses the growth of early primary leukemia and myelodysplasia progenitors. J Biol Chem, 2002 Apr 26, 277(17), 14589 - 97 Epub 2002 Feb 11. Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics; Asagoshi K et al.; 2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species . In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro . The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context . mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency . The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site . Analysis of the nucleotide insertion (f(ins) = V(max)/K(m) for insertion) and extension (f(ext) = V(max)/K(m) for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis . When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) dTMP (2.4 x 10(-4)) approximately dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) mFapyG:A and mFapyG:G (extension not observed)) . Thus, mFapyG is a potentially lethal but not premutagenic lesion. J Biol Chem, 2002 May 3, 277(18), 15865 - 73 Epub 2002 Feb 11. Solution structure and dynamics of the lipoic acid-bearing domain of human mitochondrial branched-chain alpha-keto acid dehydrogenase complex; Chang CF et al.; The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain alpha-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex . We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain alpha-keto acid dehydrogenase complex (hbLBD) . Similar to LBD from other members of the alpha-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened beta-barrel formed by two four-stranded antiparallel beta-sheets . The lipoyl Lys(44) residue resides at the tip of a beta-hairpin comprising a sharp type I beta-turn and the two connecting beta-strands 4 and 5 . A prominent V-shaped groove formed by a surface loop, L1, connecting beta 1- and beta 2-strands and the lipoyl lysine beta-hairpin constitutes the functional pocket . We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from (15)N-T(1), (15)N-T(2), and ((1)H-(15)N) nuclear Overhauser effect data obtained at 600 MHz . The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys(44) beta-turn are highly flexible, whereas beta-sheet S1 appears to display a slow conformational exchange process. J Biol Chem, 2002 Apr 26, 277(17), 14986 - 95 Epub 2002 Feb 11. A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo; Nishida S et al.; The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro . When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro . This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle . We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected . The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA . When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner . Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication . These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication . Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides. Am J Physiol Lung Cell Mol Physiol, 2002 Mar, 282(3), L411 - 20 Intra-amniotic injection of IL-1 induces inflammation and maturation in fetal sheep lung; Willet KE et al.; Antenatal inflammation may be an important triggering event in the pathogenesis of bronchopulmonary dysplasia but may also accelerate fetal lung maturation . We examined the effects of intra-amniotic (IA) interleukin (IL)-1 alpha and IL-1 beta on maturation of the fetal sheep lung . These cytokine effects were compared with IA endotoxin, a potent proinflammatory stimulus that accelerated lung maturation . Date-bred ewes received 15 or 150 microg recombinant ovine IL-1 alpha or IL-1 beta or 10 mg Escherichia coli endotoxin by IA injection at 118 days gestation (term = 150 days), and fetuses were delivered at 125 days . IL-1 alpha and IL-1 beta improved lung function and increased alveolar saturated phosphatidylcholine (Sat PC) and surfactant protein mRNA expression at the higher dose . The maturation response to IL-1 alpha was greater than that to IL-1 beta, which was similar to endotoxin response . Inflammation was also more pronounced after IL-1 alpha treatment . Only endotoxin animals had residual inflammation of the fetal membranes at 7 days . Lung compliance, lung volume, and alveolar Sat PC were positively correlated with residual alveolar wash leukocyte numbers 7 days after IL-1 treatment, suggesting a link between lung inflammation and maturation. J Soc Gynecol Investig, 2002 Jan-Feb, 9(1), 22 - 6 Shiga toxin 1 and 2 induce apoptosis in the amniotic cell line WISH; Yoshimura K et al.; OBJECTIVE: The aim of this study was to evaluate the toxicity of Shiga toxin (Stx) 1 and 2 on amniotic cells in vitro . METHODS: WISH cells, which were derived from human amniotic cells, and Vero cells were cultured with or without Stxs . After 24 hours of culture, cell viability was measured by Cell Counting Kit-8, and extracted DNA was electrophoresed on a 1% agarose gel . The morphologic changes were observed by Papanicolaou staining, and the apoptotic index (percentage of apoptotic nuclei per total nuclei) was calculated . Quantification of apoptotic cells was also measured by an enzyme-linked immunosorbent assay . RESULTS: The viability of WISH cells decreased in proportion to the concentrations of Stxs . Cellular ladder formation was observed by DNA electrophoresis of Stx-treated WISH cells, and the typical morphologic changes were observed by Papanicolaou staining . The proportion of apoptotic cells increased in response to Stxs . CONCLUSIONS: Stxs injured WISH cells directly and induced apoptosis in vitro . WISH cells were as sensitive as Vero cells to Stxs and cell death occurred by apoptosis. Curr Opin Struct Biol, 2002 Feb, 12(1), 82 - 8 Poly(A) tail synthesis and regulation: recent structural insights; Hall TM; Polyadenylation at the 3' ends of mRNAs is critical to the translation and stability of the messages . Recently determined structures of poly(A) polymerase, U1A and domains of the poly(A)-binding protein provide a framework for understanding the synthesis and regulation of the poly(A) tail. Curr Opin Struct Biol, 2002 Feb, 12(1), 72 - 81 Towards the structure of the mammalian signal recognition particle; Wild K et al.; The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle involved in the co-translational targeting of proteins to membranes . Crystal structures are now available for three protein-RNA subcomplexes from the SRP, which give insights into fundamental aspects of protein-RNA recognition, the assembly of stable ribonucleoprotein particles and the mechanism of action of the SRP. J Reprod Immunol, 2002 Mar, 54(1-2), 93 - 115 Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides; Deng X et al.; Because of its location on the sperm surface and its multiple functions during fertilization, the PH-20 protein is a potential target for contraceptive vaccines . Cynomolgus macaques were immunized using four different adjuvants together with synthesized peptides or recombinant proteins representing selected regions of macaque PH-20 . The synthesized peptide (amino acids 387-412, designated Peptide 4) was used as a linear molecule in a 1:1 ratio with a peptide sequence of tetanus toxoid, as well as a multiple antigenic peptide (MAP) matrix held together by scaffolding lysine residues . In the MAP construct, the ratio of Peptide 4 to tetanus peptide was 4:1 . To circumvent the poor production of recombinant PH-20 in bacterial cells, two truncated forms of the molecule were expressed in Escherichia coli, G18 (encoding amino acids 143-510) and E10 (encoding amino acids 291-510) . The adjuvants were Montanide ISA 51, Titermax Gold, Syntex adjuvant formulation (SAF), and QS-21 . All of the antigen/adjuvant combinations produced significant immune responses as measured by ELISA . The circulating antibodies from immunized animals recognized macaque sperm surface PH-20 on Western blots and were shown by indirect immunofluorescence to bind to the surface of macaque sperm . Montanide and Titermax were associated with higher titers of anti-PH-20 antibodies than QS-21 and SAF adjuvants . Immunization with Titermax, however, resulted in sterile abscesses in 4 of 8 animals injected . We conclude that antigens derived from synthesized peptides and recombinant proteins representing selected regions of the PH-20 molecule can be used as vaccine components in combination with the adjuvant Montanide to elicit a significant sperm-directed antibody response in immunized macaques. Structure (Camb), 2002 Feb, 10(2), 195 - 204 Crystal structure of MJ1247 protein from M . jannaschii at 2.0 A resolution infers a molecular function of 3-hexulose-6-phosphate isomerase; Martinez-Cruz LA et al.; The crystal structure of the hypothetical protein MJ1247 from Methanococccus jannaschii at 2 A resolution, a detailed sequence analysis, and biochemical assays infer its molecular function to be 3-hexulose-6-phosphate isomerase (PHI) . In the dissimilatory ribulose monophosphate (RuMP) cycle, ribulose-5-phosphate is coupled to formaldehyde by the 3-hexulose-6-phosphate synthase (HPS), yielding hexulose-6-phosphate, which is then isomerized to fructose-6-phosphate by the enzyme 3-hexulose-6-phosphate isomerase . MJ1247 is an alpha/beta structure consisting of a five-stranded parallel beta sheet flanked on both sides by alpha helices, forming a three-layered alpha-beta-alpha sandwich . The fold represents the nucleotide binding motif of a flavodoxin type . MJ1247 is a tetramer in the crystal and in solution and each monomer has a folding similar to the isomerase domain of glucosamine-6-phosphate synthase (GlmS). Hybrid Hybridomics, 2001, 20(5-6), 369 - 75 Expression and detection of ScFvB9 and its mutant in recombinant phage antibody system; Yang EJ et al.; Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL) . In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E . coli . The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E . coli . The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS. Hybrid Hybridomics, 2001, 20(5-6), 351 - 60 Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma; Hamilton S et al.; Melanoma is among the few cancers with rising incidence . Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease . Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method . However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting . We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling . In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H) . Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies . Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo . Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments . When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies. Nat Prod Lett, 2001, 15(6), 393 - 9 Total synthesis of (+/-) tanikolide; Krauss J; The natural polyketide (+/-)-tanikolide (1) was prepared in eight steps starting from hex-5-enol . Key steps in this synthesis are a Sharpless dihydroxylation and a Grignard reaction between an alkyl halogenide and a ketone . The lactonization occurred spontaneously during the oxidation of the primary alcohol function to the carboxy group. Microbiol Immunol, 2001, 45(12), 829 - 40 Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells; Noguchi T et al.; The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN . To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2) . The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN . A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro . However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity . These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR . In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity. Yi Chuan Xue Bao, 2002 Jan, 29(1), 84 - 9 Cloning and high expression of hbFGF with a new strategy; Song ZL et al.; Computer program DNASIS v2.5 was used to help designing the site-directed mutations for optimizing the expression of hbFGF in E . coli . The secondary structure of the translation initiation region (TIR) is a determinant factor for translation initiation rate, meanwhile, codon preference plays an important role, too . According to the two principles, 4 sites in 5' end of hbFGF cDNA were definitely changed, and another 4 sites randomly changed . These mutations will lead to potential variation in the secondary structure of TIR . Then computer program DNASIS v2.5 was utilized to analyse the total 32 TIR sequences resulted from the combination of the 4 randomly mutated sites . Ten sequences with highest free formation energy (delta G0) were chosen for subsequent cloning . By PCR using synthetic primers containing the 8 changed sites described above, ten hbFGF cDNA were amplified and cloned to pET-3c respectively . E . coli strain BL21 (DE3) was transformed and induced to express recombinant hbFGF . Two high-expression clones were obtained by SDS-PAGE and MTT assay, indicating that computer program-aided design for optimizing expression of foreign genes in E . coli is useful. New Microbiol, 2002 Jan, 25(1), 75 - 82 Relations between hydrophobicity tested by three methods and surface chemical composition of Escherichia coli; Latrache H et al.; The cell surface hydrophobicity of three strains of Escherichia coli cultured in liquid medium and on solid medium was measured using various methods including adsorption to pxylene, partition of cells in a polyethylene glycol/dextran (PEG/DEX) two phase system and contact angle measurements . The percentage adsorbed to pxylene ranged from 1.6% to 67% and the percentage of cells in polyethylene glycol phase ranged from 19% to 64% . The contact angle data of less than 40 degrees C revealed a hydrophylic character of the E . coli strains studied here . No relations were found between paraxylene/water partitioning, PEG/DEX partioning and water contact angles . The linear correlation coefficients between the results of the three hydrophobicity assays and the elemental concentration ratios obtained by X-ray photoelectron spectroscopy (XPS) were calculated . A linear correlation was found between the contact angles and the O/C ratios (r=0.91) and the N/C ratios (0.67) . The adsorption to pxylene correlates better with N/C ratios (0.88) but does not correlate with O/C ratios (0.46) . However, this test correlates with N/P ratios (0.79) . No relation was obtained between partition in PEG/DEX system and any elemental concentration ratios . The surface composition determined by XPS was converted into a molecular composition in terms of proteins, polysaccharides, and hydrocarbon-like compounds . The proteins/polysaccharides and the hydrocarbons/polysaccharides seems to determine the contact angle of E . coli but not the adsorption to paraxylene or partition in the PEG/DEX system. New Microbiol, 2002 Jan, 25(1), 107 - 10 Expression of alkaline phosphatase induces rapid and artificial mineralization in specific transformed Escherichia coli; Ohara N et al.; Matrix vesicles (MV) having high alkaline phosphatase (ALP) activity act as initiators of biological mineralization . Although bacteria have similar membranous structures to MV, ALP mediated mineralization has not been studied in bacterial cells . Escherichia coli was transformed with a bacterial ALP gene in this study . Recombinant E . coli overproducing ALP induced mineralization through hydrolysis of calcium-glycerophosphate (Ca-GP) . Fourier transform infrared spectroscopy and electron microscopy combined with electron diffraction revealed newly formed hydroxyapatite mineral deposits . These findings suggest that hydrolysis of Ca-GP through ALP induced high Ca and Pi concentrations within bacterial cells followed by complete bacterial mineralization. Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2002 Feb, 19(1), 17 - 21 {Study on the construction of standard D12S391 allelic ladder and its genetic polymorphism in six populations}; Zhang L et al.; OBJECTIVE: To resolve the problem of the accuracy and standardization of short tandem repeat-polymerase chain reaction (STR-PCR) typing in forensic practice, the authors have designed a new method of producing standard D12S391 allelic ladder . METHODS: Nine different PCR amplified D12S391 allelic fragments were isolated from the gel, eluted into the distilled water and re-amplified by PCR . The purified allelic fragments were then blunt-end subcloned individually into the pUC plasmid vectors and transfected into competent E.coli DH5 alpha(TM) cells . The sequencing results confirmed that the size and the structure of the inserts were correct . The recombinant plasmids DNA with 9 inserts were then used as templates for PCR re-amplification to generate D12S391 standard ladder . RESULTS: With the ladder, the authors studied the genetic polymorphisms of D12S391 locus in six populations (German, Japanese and Chinese south-western Han, northern Han, Weiwu'er and Hui populations), and the respective primary data in the six populations were obtained . D12S391 locus showed high polymorphism in all six populations, and its exclusion power and discrimination power are 0.609-0.786 and 0.940-0.952 respectively . CONCLUSION: The results demonstrate that the standard ladder generated via this method is excellent, and D12S391 locus is robust for genetic research and forensic application. Oncol Rep, 2002 Mar-Apr, 9(2), 337 - 40 Suppression of tumor growth and pulmonary metastasis in murine osteosarcoma using gene therapy; Seto M et al.; We evaluated the effect of gene therapy in the murine osteosarcoma cell line, LM8, which preferentially metastasizes to the lungs . LM8 cells were transduced with the gene for a herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase (lacZ) . We investigated the cytotoxicity of LM8 cells bearing an HSV-tk gene after treatment with ganciclovir (GCV) . LM8 cells bearing an HSV-tk gene were more sensitive than non-transduced cells . The remarkable inhibition of tumor growth and pulmonary metastases was confirmed in vivo . Our findings indicated that GCV kills tumor cells transduced with HSV-tk in vitro and in vivo. J Virol, 2002 Mar, 76(5), 2449 - 59 Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice; Loiacono CM et al.; Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins . It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation . We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC) . Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated . Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins . The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus . Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice . The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age . beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody . Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1 . We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins . These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons. J Virol, 2002 Mar, 76(5), 2287 - 97 Baculovirus replication factor LEF-1 is a DNA primase; Mikhailov VS et al.; The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS(6) tag using recombinant baculoviruses . LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template . The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels . Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template . The LEF-1 primase was absolutely dependent on divalent cations (Mg(2+)), and optimal activity was supported by 10 mM MgCl(2) . An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory . Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished . Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer . Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1. J Biol Chem, 2002 May 3, 277(18), 15947 - 56 Epub 2002 Feb 08. Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress; Shen Y et al.; The activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family . Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates . DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding . Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins . By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4 . It is the first ER-localized type II DnaJ homologue to be reported . The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen . ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells . In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner . However, mutation of the hallmark tripeptide HPD (His --> Gln) in the J domain totally abolished this activation . ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta . We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation. Peptides, 2002 Mar, 23(3), 567 - 72 Preparation of an active recombinant peptide of crustacean androgenic gland hormone; Okuno A et al.; In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone . We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare . In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems . Neither recombinant precursors showed activity . Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide . These results indicate that the cDNA encodes the hormone. J Biochem Mol Toxicol, 2001, 15(6), 300 - 8 Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouse; Karoly ED et al.; The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E . consortium (I.M.A.G.E . CloneID 1432164) . Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5' flanking region, 1607 bases of coding region, and 309 bases of 3' flanking region . This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191 . Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively . The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli . FMO activity in particulate fractions isolated from isopropyl-beta-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45 degrees C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5 . The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E . coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate . Mol Reprod Dev, 2002 Mar, 61(3), 288 - 301 Golgi matrix protein gene, Golga3/Mea2, rearranged and re-expressed in pachytene spermatocytes restores spermatogenesis in the mouse; Banu Y et al.; In a transgenic mouse, Golga3/Mea2 gene (human homolog: GOLGA3/golgin-160) was disrupted by a translocation at the site of the transgene integration . Exons 8-24 of the disrupted gene remained intact and formed a fusion gene (DeltaMea2) with the antisense strand of E . coli-derived transgene by means of a cryptic splice signal in there . The protein product of DeltaMea2, virtually a form truncated to 2/3 of the normal size, localized to Golgi apparatus of pachytene spermatocytes and round spermatids . DeltaMea2 expression was specific to the testis, but varied among separate seminiferous tubules . It also showed variation among homozygous individuals from 0.5 to 4.3% of the wild type (wt) level . At the lowest levels, neither spermatids nor spermatozoa were present in the homozygous testes, but when the expression of DeltaMea2 increased to 4.3% of the wt