Escherichia coli

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 level, high sperm production was restored and a sporadic (1/22) fertile homozygous male was obtained . The earliest apoptotic degeneration of pachytene spermatocytes evidenced at 17 dpp in homozygous testes in some discrete seminiferous tubules was preceded by DeltaMea2 expression in a variegated fashion at 16 dpp . These results consistently indicated that in homozygous testes, the pachytene spermatocytes which failed to express DeltaMea2 may undergo apoptotic degeneration . Golga3/Mea2, and DeltaMea2 in homozygotes, in a certain excessive amount may be important for survival of pachytene spermatocytes in the mouse .

Proteins, 2002 Feb 15, 46(3), 308 - 20
Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: thermal stability of protein domains and unfolding pathway; Sham YY et al.; Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations . The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure . The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding . This region, which is made up of part of the N terminus domain and part of the large domain of the E . coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains . The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway . This observation is compared with the recently proposed hierarchical folding model . Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E . coli dihydrofolate reductase involves sequential formation of the substrate binding sites .

Proteins, 2002 Feb 15, 46(3), 278 - 86
3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism; Richter W; 3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II . The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs . Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme . The present study shows by computational analysis that the enzyme encoded by the E . coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases . They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH{E/D}-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature . Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis . In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases .

Proteins, 2001, Suppl 5, 98 - 118
Assessment of novel fold targets in CASP4: predictions of three-dimensional structures, secondary structures, and interresidue contacts; Lesk AM et al.; In the Novel Fold category, three types of predictions were assessed: three-dimensional structures, secondary structures, and residue-residue contacts . For predictions of three-dimensional models, CASP4 targets included 5 domains or structures with novel folds, and 13 on the borderline between Novel Fold and Fold Recognition categories . These elicited 1863 predictions of these and other targets by methods more general than comparative modeling or fold recognition techniques . The group of Bonneau, Tsai, Ruczinski, and Baker stood out as performing well with the greatest consistency . In many cases, several groups were able to predict fragments of the target correctly-often at a level somewhat larger than standard supersecondary structures-but were not able to assemble fragments into a correct global topology . The methods of Bonneau, Tsai, Ruczinski, and Baker have been successful in addressing the fragment assembly problem for many but not all the target structures .

Proteins, 2001, Suppl 5, 86 - 91
What is the value added by human intervention in protein structure prediction?
Karplus K, Karchin R, Barrett C, Tu S, Cline M, Diekhans M, Grate L, Casper J, Hughey R.
This article presents results of blind predictions submitted to the CASP4 protein structure prediction experiment . We made two sets of predictions: one using the fully automated SAM-T99 server and one using the improved SAM-T2K method with human intervention . Both methods use iterative hidden Markov model-based methods for constructing protein family profiles, using only sequence information . Although the SAM-T99 method is purely sequence based, the SAM-T2K method uses the predicted secondary structure of the target sequence and the known secondary structure of the templates to improve fold recognition and alignment . In this article, we try to determine what aspects of the SAM-T2K method were responsible for its significantly better performance in the CASP4 experiment in the hopes of producing a better automatic prediction server . The use of secondary structure prediction seems to be the most valuable single improvement, though the combined total of various human interventions is probably at least as important .

Proteins, 2001, Suppl 5, 76 - 85
CASP2 knowledge-based approach to distant homology recognition and fold prediction in CASP4; Murzin AG et al.; In 1996, in CASP2, we presented a semimanual approach to the prediction of protein structure that was aimed at the recognition of probable distant homology, where it existed, between a given target protein and a protein of known structure (Murzin and Bateman, Proteins 1997; Suppl 1:105-112) . Central to our method was the knowledge of all known structural and probable evolutionary relationships among proteins of known structure classified in the SCOP database (Murzin et al., J Mol Biol 1995;247:536-540) . It was demonstrated that a knowledge-based approach could compete successfully with the best computational methods of the time in the correct recognition of the target protein fold . Four years later, in CASP4, we have applied essentially the same knowledge-based approach to distant homology recognition, concentrating our effort on the improvement of the completeness and alignment accuracy of our models . The manifold increase of available sequence and structure data was to our advantage, as well as was the experience and expertise obtained through the classification of these data . In particular, we were able to model most of our predictions from several distantly related structures rather than from a single parent structure, and we could use more superfamily characteristic features for the refinement of our alignments . Our predictions for each of the attempted distant homology recognition targets ranked among the few top predictions for each of these targets, with the predictions for the hypothetical protein HI0065 (T0104) and the C-terminal domain of the ABC transporter MalK (T0121C) being particularly successful . We also have attempted the prediction of protein folds of some of the targets tentatively assigned to new superfamilies . The average quality of our fold predictions was far less than the quality of our distant homology recognition models, but for the two targets, chorismate lyase (T0086) and Appr>p cyclic phosphodiesterase (T0094), our predictions achieved the top ranking .

Bioessays, 2002 Feb, 24(2), 141 - 8
The "tale" of UmuD and its role in SOS mutagenesis; Gonzalez M et al.; Recently, the Escherichia coli umuD and umuC genes have been shown to encode E . coli's fifth DNA polymerase, pol V (consisting of a heterotrimer of UmuD'(2)C) . The main function of pol V appears to be the bypass of DNA lesions that would otherwise block replication by pols I-IV . This process is error-prone and leads to a striking increase in mutations at sites of DNA damage . While the enzymatic properties of pol V are now only beginning to be fully appreciated, a great deal is known about how E . coli regulates the intracellular levels of the Umu proteins so that the lesion-bypassing activity of pol V is available to help cells survive the deleterious consequences of DNA damage, yet keeps any unwarranted activity on undamaged templates to a minimum . Our review summarizes the multiple restrictions imposed upon pol V, so as to limit its activity in vivo and, in particular, highlights the pivotal role that the N-terminal tail of UmuD plays in regulating SOS mutagenesis . Published 2002 Wiley Periodicals, Inc.

Biotechnol Bioeng, 2002 Mar 30, 77(7), 776 - 85
Direct recovery of glutathione S-transferase by expanded bed adsorption: anion exchange as an alternative to metal affinity fusions; Clemmitt RH et al.; The use of expanded beds of ion-exchange adsorbents for the direct recovery of a recombinant intracellular protein, glutathione S-transferase (GST), from unclarified Escherichia coli homogenates is described . The results form the basis for a comparison between this approach for purifying GST and a chelating fusion strategy and highlight the need to consider the additional costs entailed by these more-complicated approaches . The separation performance was investigated with respect to choice of anion or cation exchanger, adsorption pH, load volume, sample preparation, and stepwise elution protocol . Anion exchange was found to be more appropriate than cation exchange, as the low pHs involved in the latter caused a loss of activity . The optimal pH for adsorption was found to be 9 with a dynamic capacity from clarified homogenate in packed mode of 112 U mL(-1) (11.2 mg GST mL(-1)) . As increasing volumes of unclarified homogenate were applied to the expanded bed, the yield of GST in the eluate decreased, and the purification factor was found to increase and then decrease . This was due to the displacement of weakly bound proteins by GST and then its displacement by even more strongly binding proteins . The dynamic capacity of the anion exchanger, STREAMLINE DEAE, from unclarified homogenate in expanded mode decreased slightly to 85 U mL(-1) (8.5 mg GST mL(-1)) . The elution protocol for GST from the anion exchanger was then adjusted to try to maximize the degree of purification . Anion exchange expanded bed adsorption of GST from unclarified E . coli homogenate gave an eluted yield of 95.7% and 1.64-fold purification . Interestingly, a decrease in the expression level of GST in the feedstream from 23 down to 13% caused a decrease in the dynamic capacity from 85 to 14.5 U mL(-1) whereas the degree of purification remained similar .

Biotechnol Bioeng, 2002 Mar 30, 77(7), 764 - 75
Modeling of the biotransformation of crotonobetaine into L-(-)-carnitine by Escherichia coli strains; Canovas M et al.; A simple unstructured model, which includes carbon source as the limiting and essential substrate and oxygen as an enhancing substrate for cell growth, has been implemented to depict cell population evolution of two Escherichia coli strains and the expression of their trimethylammonium metabolism in batch and continuous reactors . Although the model is applied to represent the trans-crotonobetaine to L-(-)-carnitine biotransformation, it is also useful for understanding the complete metabolic flow of trimethylammonium compounds in E . coli . Cell growth and biotransformation were studied in both anaerobic and aerobic conditions . For this reason we derived equations to modify the specific growth rate, mu, and the cell yield on the carbon source (glycerol), Y(xg), as oxygen increased the rate of growth . Inhibition functions representing an excess of the glycerol and oxygen were included to depict cell evolution during extreme conditions . As a result, the model fitted experimental data for various growth conditions, including different carbon source concentrations, initial oxygen levels, and the existence of a certain degree of cell death . Moreover, the production of enzymes involved within the E . coli trimethylammonium metabolism and related to trans-crotonobetaine biotransformation was also modeled as a function of both the cell and oxygen concentrations within the system . The model describes all the activities of the different enzymes within the transformed and wild strains, able to produce L-(-)-carnitine from trans-crotonobetaine under both anaerobic and aerobic conditions . Crotonobetaine reductase inhibition by either oxygen or the addition of fumarate as well as its non-reversible catalytic action was taken into consideration . The proposed model was useful for describing the whole set of variables under both growing and resting conditions . Both E . coli strains within membrane high-density reactors were well represented by the model as results matched the experimental data .

J Biol Chem, 2002 Apr 19, 277(16), 13449 - 54 Epub 2002 Feb 07.
Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli; Ollagnier-de Choudens S et al.; Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin . Reconstituted BioB from Escherichia coli contains a {4Fe-4S}(2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction . Using EPR and Mossbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 {4Fe-4S}(1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 {4Fe-4S}(2+) cluster . Dethiobiotin is not required for the reaction . Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the {4Fe-4S}(2+/1+) cluster.

J Biol Chem, 2002 Apr 19, 277(16), 14315 - 20 Epub 2002 Feb 07.
Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex; Kurumizaka H et al.; The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates . The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development . In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity . The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs . An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA . In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.

Am J Physiol Heart Circ Physiol, 2002 Mar, 282(3), H1111 - 7
Nitric oxide modulates endotoxin-induced platelet-endothelial cell adhesion in intestinal venules; Cerwinka WH et al.; Although platelets have been implicated in the pathogenesis of vascular diseases, little is known about factors that regulate interactions between platelets and the vessel wall under physiological conditions . The objectives of this study were to 1) define the contribution of nitric oxide (NO) to endotoxin (lipopolysaccharide, LPS)-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules and 2) determine whether the antiadhesive action of NO is mediated by soluble guanylate cyclase (sGC) . Adhesive interactions between platelets and endothelial cells were monitored by intravital microscopy . LPS administration into control wild-type mice (WT) resulted in a >15-fold increase in P/E adhesion . Similar responses were observed using endothelial NO synthase (eNOS)-deficient platelets . However, treatment with the NO donor diethylenetriamine-nitric oxide (DETA-NO) attenuated the P/E adhesion response to LPS, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester or eNOS deficiency resulted in an exacerbation . P/E adhesion response did not differ between LPS-treated WT and inducible NOS-deficient mice . Inhibition of sGC abolished the attenuating effects of DETA-NO, whereas the sGC activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) reduced LPS-induced P/E adhesion . These findings indicate that 1) eNOS-derived NO attenuates endotoxin-induced P/E adhesion and 2) sGC is responsible for the antiadhesive action of NO.

Allergol Immunopathol (Madr), 2001 Nov-Dec, 29(6), 229 - 37
Inhibition of enteropathogenic Escherichia coli (EPEC) adherence to HEp-2 cells by bovine colostrum and milk; Palmeira P et al.; BACKGROUND: enteropathogenic Escherichia coli (EPEC) is the main etiological agent of infantile diarrhea in Brazil and other developing countries . Human milk IgA protects newborn intestinal mucosa by inhibiting bacterial adhesion to epithelial cells and this effect is shown by in vitro assays of EPEC adhesion to HEp-2 cultured cells . Bovine milk, if effective in promoting this protection, could be an useful tool in the absence of the natural breastfeeding, in high-risk nurseries or in hospital infections . METHODS: the effect of colostrum, milk, and serum from dairy cows on the adherence to EPEC to HEp-2 cells was investigated . Colostrum from immunized and control animals and industrialized milk formulas were fractionated through a membrane device with a molecular weight cut off 10 kDa . The high molecular weight fraction (HMWF) of bovine colostrum was depleted of IgG through an affinity column and absorbed with an EPEC adherent strain . Antibodies were searched by ELISA and immunoblotting (IB) . RESULTS: colostrum and milk from EPEC-immunized animals showed and inhibitory activity on adherence similar to that of control non-immunized animals . The inhibitory effect on adhesion was related to the HMWF . IgG-depleted colostrum partially retained the inhibitory effect, whereas IgG-rich eluate lost this property . The EPEC-absorbed fraction retained the inhibitory property . Industrialized milk formulas and respective HMWF also inhibited bacterial adherence . In IB assays, colostrum and milk samples from immunized animals recognized proteins of 30-40 kDa and 94 kDa, a molecular weight consistent with the adhesin intimin, in EPEC extracts . CONCLUSIONS: the inhibitory effect of EPEC adherence may be mediated by HMWF components, and IgG was not the only component responsible for this phenomenon.

Clin Ther, 2002 Jan, 24(1), 100 - 11
An open-label comparison of the immunogenicity and tolerability of intranasal and intramuscular formulations of virosomal influenza vaccine in healthy adults; de B et al.; BACKGROUND: Many intramuscular inactivated influenza vaccines achieve suboptimal results in the prevention of respiratory disease and influenza complications . This has led to the current interest in developing effective oral or intranasal preparations . OBJECTIVE: This study compared the immunogenicity and tolerability of intranasal and intramuscular formulations of virosomal subunit influenza vaccine in healthy adults . It also assessed the immunogenicity and tolerability of 3 different production lots of the intranasal vaccine containing Escherichia coli heat-labile toxin adjuvant . METHODS: This was a multicenter, Phase I, randomized, open-label pilot study in which the primary end point was immunogenicity (hemagglutination-inhibition {HI} antibody assay on days 1 and 29) . The secondary end point was the frequency of adverse events (AEs) . Subjects were assigned to 4 vaccination groups: groups AI, AII, and AIII received intranasal influenza vaccine from batches that differed in the hemagglutinin and neuraminidase strains used, and group B received intramuscular virosomal subunit vaccine . Assessments of health status, hematology, biochemistry, body temperature, heart rate, blood pressure, and incidence of AEs were made on days 1, 8, and 29, and serology was assessed on days 1 and 29 . RESULTS: The study enrolled 88 subjects . All 3 production lots of intranasal vaccine induced an immune response to most of the viral strains administered (A/Singapore, A/Texas, A/Wuhan, B/Beijing), with no notable immunogenic differences between lots . After intranasal vaccination, geometric mean titers (GMT) increased 2.7-fold against A/Singapore (group AI); 1.8- and 3.1-fold against A/Texas (groups AII and AIII, respectively); 1.9- to 2.4-fold against A/Wuhan; and 1.5- to 1.7-fold against B/Beijing . After intramuscular vaccination . GMT increased 11.3-, 6.3-, and 2.7-fold against A/Texas, A/Wuhan, and B/Beijing, respectively . Seroprotection (HI antibody titers > or = 1:40 in > 70% of those vaccinated) was achieved against all strains in the group that received intramuscular vaccination, against A/Wuhan in all groups that received intranasal vaccination, and against A/Texas in group AII . Both vaccine formulations were well tolerated . Intranasal vaccination was associated with a low incidence ( < 20%) of nasal AEs . CONCLUSIONS: Both the intranasal and intramuscular vaccinations elicited a systemic immune response and were well tolerated . The different batches of intranasal vaccine showed a similar immunogenic profile . Intranasal administration may be preferred to intramuscular administration by some patients.

Nat Prod Lett, 2001, 15(4), 237 - 41
N2,N2,7-trimethylguanine, a new trimethylated guanine natural product from the New Zealand ascidian, Lissoclinum notti; Pearce AN et al.; From the New Zealand ascidian, Lissoclinum notti a new natural product, N2,N2,7-trimethylguanine (1) has been isolated . The structure of 1 was elucidated by analysis of spectroscopic data.

World J Gastroenterol, 2002 Feb, 8(1), 124 - 7
Lipopolysaccharide induced synthesis of CD14 proteins and its gene expression in hepatocytes during endotoxemia; Li SW et al.; AIM: To observe synthesis of CD14 protein and expression of CD14 mRNA in hepatic tissue and hepatocytes of rats during endotoxemia . METHODS: The endotoxemia model of Wistar rat was established by injection of a dose of lipopolysaccharide (LPS) (5mg x kg(-1), Escherichia coli O111:B4) via the tail vein, then the rats were sacrificed after 3, 6, 12 and 24 h in batches respectively . Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique and were collected to measure the expression of CD14 mRNA and synthesis of CD14 protein by reverse transcript-polymerase chain reaction (RT-PCR) or Western blot analysis . The binding of fluorescein isothiocyanate (FITC)-CD14 polyclonal antibody to isolated hepatocytes was also assessed by flow cytometric analysis (FCM) . RESULTS: In the rats with endotoxemia, the expressions of CD14 mRNA in hepatic tissue and isolated hepatocytes were stronger at 3, 6, and 12 h than that in control rats (3.48+/-0.15, 5.89+/-0.62, 4.33+/-0.18, vs 1.35+/-0.14 in hepatic tissue, P<0.01; 4.12+/-0.17, 6.24+/-0.64, 4.35+/-0.18, vs 1.87+/-0.15 in hepatocytoes, P<0.01).The synthesis of CD14 protein in hepatic tissue and isolated hepatocytes increases also obviously in 6 and 12 h when compared to that in control rats (13.27+/-1.27, 17.32+/-1.35, 11.42+/-1.20, vs 7.34+/-0.72 in hepatic tissue, P<0.01; 14.68+/-1.30, 17.95+/-1.34,11.65+/-1.19, vs 7.91+/-0.70 in hepatocytes, P<0.01) . FCM showed that mean fluorescence intensity (MFI) and numbers of FITC-CD14 positive cells in the rats with endotoxemia increased obviously at 3,6,12 and 24h when compared with normal control group (43.4%, 70.2%, 91.4%, 32.6% vs 4.5%, P<0.01) . CONCLUSION: LPS can markedly promote the synthesis of CD14 protein and up-regulate the expression of CD14 mRNA in isolated hepatocytes and hepatic tissue . Liver might be a main source for soluble CD14 production during endotoxemia.

World J Gastroenterol, 2002 Feb, 8(1), 99 - 102
Expression and bioactivity identification of soluble MG7 scFv; Yu ZC et al.; AIM: To examine the molecular mass and identify the bioactivity of MG7 scFv for its application as a targeting mediator in gene therapy of gastric cancer . METHODS: Two strongly positive recombinant phage clones screened from MG7 recombinant phage antibody library were separately transfected into E.coli TG1 . Plasmid was isolated from the transfected E.coli TG1 and digested by EcoR I and Hind III to examine the length of exogenous scFv gene . Then, the positive recombinant phage clones were individually transfected into E.coli HB2151.The transfectant was cultured and induced by IPTG . Perplasmic extracts was prepared from the induced transfectant by osmotic shock . ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv . Immunodotting assay was adopted to evaluate the yield of soluble MG7 scFv produced by transfected E.coli HB2151 . Western blot was used to examine the molecular mass of MG7 scFv . Finally, the nucleotide sequence of MG7 scFv was examined by DNA sequencing . RESULTS: Two positive recombinant phage clones were found to contain the exogenous scFv gene . ELISA showed that MG7 scFv had strong antigen-binding affinity . Immuodotting assay showed that transfected E.coli HB2151 could successfully produce the soluble MG7 scFv with high yield via induction by IPTG . The molecular mass of MG7 scFv was 30 kDa by western blot . DNA sequencing demonstrated that the VH and VL genes of MG7 scFv were 363 bp and 321 bp,respectively . CONCLUSION: We have successfully developed the soluble MG7 scFv which possessed strong antigen-binding affinity.

Nature, 2002 Feb 7, 415(6872), 662 - 6
RanGAP mediates GTP hydrolysis without an arginine finger; Seewald MJ et al.; GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude . Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket . This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis . Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells . Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP . Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic . The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.

Microbiology, 2002 Feb, 148(Pt 2), 391 - 404
Streptomyces spp . contain class Ia and class II ribonucleotide reductases: expression analysis of the genes in vegetative growth; Borovok I et al.; Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces . One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of approximately 108 kDa . The activity of this protein depended on the presence of 5'-deoxyadenosylcobalamine (coenzyme B12), establishing it as a class II RNR . The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa . Molecular phylogenetic analysis showed that the S . clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs . Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR . Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp . The S . coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR . It was shown, using quantitative real-time PCR, that the S . clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth . The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3-5x10(5) copies per 400 ng total RNA after reverse transcription) . In contrast, the copy number of the class Ia nrdAB transcripts was some 10- to 20-fold less than that of nrdJ in the early-exponential growth phase (2.8x10(4) copies), and decreased markedly at the mid-exponential (4x10(3) copies) and late-exponential phases (1.1x10(3) copies) of growth . A possible role for the involvement of two RNRs during vegetative growth is discussed.

Microbiology, 2002 Feb, 148(Pt 2), 353 - 60
Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore; Gonzalez Carrero MI et al.; Brucella abortus is known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions . In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5 in the virulent B . abortus strain 2308 . This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA . Production of catecholic compounds by both the Brucella mutant and parental strains under iron-deprivation conditions was assayed by TLC . Two catecholic substances were identified in the supernatant of the parental strain 2308 . The faster migrating spot showed the same retention factor (R(f)) as that of purified 2,3-DHBA . The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol . This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308 . The genomic region containing Tn5 in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing . The sequence revealed that the insertion had occurred at a gene with homology to Escherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin . Intracellular survival and growth rates of the B . abortus wild-type and entF mutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection . It is concluded that B . abortus synthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.

Zhonghua Wai Ke Za Zhi, 2000 Jun, 38(6), 462 - 4
{The effect of pentoxifylline on endotoxin-induced biopterin formation in rabbits}; Yao Y et al.; OBJECTIVE: To observe the potential effect of pentoxifylline on endotoxin-mediated biopterin (tetrahydrobiopterin and more oxidized species) formation and systemic hemodynamics . METHODS: Rabbits were subjected to endotoxic shock induced by a bolus intravenous injection of E . coli O26B6 lipopolysaccharide (400 microg/kg) . 28 animals were divided into sham-operation group (n = 8), endotoxic shock group (n = 10), and pentoxifylline-treated group (n = 10) . Plasma biopterin and tumor necrosis factor-alpha (TNF) levels, and guanosine triphosphate cyclohydrolase I (GTP-CHI) activity in tissues were determined at various intervals . RESULTS: Treatment with pentoxifylline significantly decreased plasma biopterin and TNF levels at 2 to 8 hours after endotoxin challenge (P < 0.05, P < 0.01), and inhibited GTP-CHI activities in the liver, lung, and myocardial tissues (P < 0.05) . Meanwhile, systemic hemodynamic parameters, including mean arterial pressure, cardiac output, and total peripheral resistance, in the treatment group were much higher than those in the endotoxic shock group . CONCLUSIONS: These data suggest that early treatment with pentoxifylline can effectively inhibit endotoxin-induced biopterin synthesis and release, and markedly improve systemic hemodynamics during septic shock.

J Eukaryot Microbiol, 2001 Nov-Dec, 48(6), 676 - 82
Isolation of a unique membrane protein from Naegleria fowleri; Reveiller FL et al.; Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system . In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N . fowleri RNA . A specific protein was found to be expressed from a cDNA clone designated Mp2CL5 . Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N . fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba . Western blot analysis using anti-N . fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein . The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E . coli . A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein . This antibody was used to further characterize the Mp2CL5 native protein expressed by N . fowleri . Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N . fowleri trophozoites . The native N . fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth . Studies are in progress to examine further its role as a virulence factor.

Virology, 1995 Apr 1, 208(1), 349 - 53
The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus; Hammond RW et al.; The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA . The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs) . ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da . ORF 2 corresponds to the coat protein gene . Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum . Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein . Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation . ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids . The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate . These relationships are also reflected at the nucleotide sequence level . These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Virology, 1995 Apr 1, 208(1), 328 - 35
Passage of Autographa californica nuclear polyhedrosis virus through the midgut epithelium of Spodoptera exigua larvae; Flipsen JT et al.; A special recombinant of Autographa californica multicapsid nuclear polyhedrosis virus (AcNPV) was designed to study the early histopathological events of baculovirus infection in Spodoptera exigua larvae . This recombinant contained a Drosophila melanogaster heat shock 70 promoter driving an Escherichia coli beta-galactosidase (Lac-Z) reporter gene to monitor the presence of early viral gene expression and a second reporter gene, the E . coli beta-glucuronidase (GUS) gene, under control of the very late AcNPV p10 promoter to monitor viral replication . In S . exigua larvae, permissive Spodoptera spp . cultured cells, and nonpermissive D . melanogaster cultured cells early viral gene expression was indicated by the appearance of Lac-Z as early as 3 hr p.i . Late viral gene expression was indicated by the appearance of GUS and occurred only in the permissive cultured cells and larvae . Early and late viral gene expression could be detected simultaneously using differential enzyme histochemistry . Analysis of infected S . exigua larvae revealed that midgut columnar cells and, at a low frequency, midgut regenerative cells were the primary sites of infection . Parental nucleocapsids were apparently transported through columnar cells to underlaying regenerative cells before virus replication and progeny production . Infection of tissues beside the midgut epithelium was not detected prior to viral replication within the midgut, suggesting that infection of the midgut is an important prelude to systemic infection.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1303 - 8
Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor; Nakamura GR et al.; Recently we described a family of peptides, unrelated in sequence to IgE, that form stable beta-hairpins in solution and inhibit IgE activity in the microM range {Nakamura, G . R., Starovasnik, M . A., Reynolds, M . E . & Lowman, H . B . (2001) Biochemistry 40, 9828-9835} . Using an expanded set of peptide-phage libraries, we found a simpler motif, X(2)CPX(2)CYX, for binding to the high-affinity IgE receptor . In solution, one of these peptides spontaneously formed a covalent antiparallel dimer . We subsequently linked these monomers in a single-chain construct on phage and optimized receptor binding . Ultimately, peptides with 30 nM affinity were produced . NMR studies showed that the peptide adopts a stable fold consisting of two "zeta" (zeta)-shaped moieties . Structure-activity analyses reveal a single binding site created by the zeta-dimer, with two tyrosine residues important for structural stability and two proline residues important for Fc epsilon RI binding . The peptides inhibit histamine release from cultured cells and are extremely stable in biological fluids . The zeta peptides appear to act as competitive IgE inhibitors and suggest possibilities for design of novel IgE antagonists.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1170 - 5
Product dependence and bifunctionality compromise the ultrasensitivity of signal transduction cascades; Ortega F et al.; Covalent modification cycles are ubiquitous . Theoretical studies have suggested that they serve to increase sensitivity . However, this suggestion has not been corroborated experimentally in vivo . Here, we demonstrate that the assumptions of the theoretical studies, i.e., irreversibility and absence of product inhibition, were not trivial: when the conversion reactions are close to equilibrium or saturated by their product, "zero-order" ultrasensitivity disappears . For high sensitivities to arise, not only substrate saturation (zero-order) but also high equilibrium constants and low product saturation are required . Many covalent modification cycles are catalyzed by one bifunctional 'ambiguous' enzyme rather than by two independent proteins . This makes high substrate concentration and low product concentration for both reactions of the cycle inconsistent; such modification cycles cannot have high responses . Defining signal strength as ratios of modified (e.g., phosphorylated) over unmodified protein, signal-to-signal response sensitivity equals 1: signal strength should remain constant along a cascade of ambiguous modification cycles . We also show that the total concentration of a signalling effector protein cannot affect the signal emanating from a modification cycle catalyzed by an ambiguous enzyme if the ratio of the two forms of the effector protein is not altered . This finding may explain the experimental result that the pivotal signal transduction protein PII plus its paralogue GlnK do not control steady-state N-signal transduction in Escherichia coli . It also rationalizes the absence of strong phenotypes for many signal-transduction proteins . Emphasis on extent of modification of these proteins is perhaps more urgent than transcriptome analysis.

Proc Natl Acad Sci U S A, 2002 Feb 19, 99(4), 2164 - 9 Epub 2002 Feb 05.
Amplification-mutagenesis: evidence that "directed" adaptive mutation and general hypermutability result from growth with a selected gene amplification; Hendrickson H et al.; When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation) . This observation suggested that growth limitation stimulates mutability . Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps . First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac(+) revertant allele within the amplified array . Finally, it favors loss of mutant copies until a stable haploid lac(+) revertant arises and overgrows the colony . By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone . This sequence of events appears to direct mutations to useful sites . General mutagenesis is a side-effect of growth with an amplification (SOS induction) . The F' plasmid, which carries lac, contributes by stimulating gene duplication and amplification . Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose . The sequence of events--amplification, mutation, segregation--may help to explain both the origins of some cancers and the evolution of new genes under selection.

Proc Natl Acad Sci U S A, 2002 Mar 5, 99(5), 2977 - 82 Epub 2002 Feb 05.
Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA; Tieng V et al.; MICA are distant homologs of MHC class I molecules expressed in the normal intestinal epithelium . They are ligands of the NKG2D activating receptor expressed on most gammadelta T cells, CD8+ alphabeta T cells, and natural killer cells and therefore play a critical role in innate immune responses . We investigated MICA cell-surface expression on infection of epithelial cell lines by enteric bacteria and show here that MICA expression can be markedly increased by bacteria of the diffusely adherent Escherichia coli diarrheagenic group . This effect is mediated by the specific interaction between bacterial adhesin AfaE and its cellular receptor, CD55, or decay-accelerating factor . It is extremely rapid after AfaE binding, consistent with a stress-induced signal . MICA induction on epithelial cells triggered IFN-gamma release by the NKG2D expressing natural killer cell line NKL . This host-bacteria interaction pathway could play a role in the pathogenesis of inflammatory bowel disease, a condition that implicates a bacterial trigger in genetically susceptible individuals . This was supported by the increased MICA expression at the surface of epithelial cells in colonic biopsies from Crohn's disease-affected patients compared with controls.

Cancer Res, 2002 Feb 1, 62(3), 773 - 80
Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas; Miller CR et al.; 5-Fluorouracil (5-FU) is a potent antimetabolite used for chemotherapy of gastrointestinal (GI), breast, and head and neck malignancies . Although clinical trials have been conducted, the poor therapeutic index of 5-FU has precluded its clinical use for a number of other tumor types . It is unclear whether this lack of utility is due to problems with drug delivery or inherent insensitivity . Adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy has the potential to overcome pharmacokinetic issues associated with systemic 5-FU and is particularly well suited to use with tumors in which local control is paramount, such as recurrent, localized prostate cancer and malignant gliomas . In this study, the in vitro response by a panel of human tumor cell lines derived from both GI (colon, pancreas) and non-GI (prostate, glioma) tumors to 5-FU and to AdCMVCD (an Ad encoding Escherichia coli CD)/5-FC was examined . Whereas the sensitivity (IC(50)) of individual cell lines to these agents varied, no significant difference in median IC(50) for either 5-FU or AdCMVCD/5-FC was evident for the four tumor types tested (P > 0.1) . The relevant contributions of Ad gene transfer efficiency and inherent 5-FU sensitivity in determining response to AdCMVCD/5-FC were then assessed . Multiple linear regression analysis revealed that whereas both factors significantly contribute to the response, inherent 5-FU sensitivity was substantially more important (beta= 0.78 versus 0.48; P < 0.001) . Finally, the therapeutic efficacy of a single intratumoral injection of AdCMVCD followed by systemic 5-FC was assessed in three intracranial C.B17 severe combined immunodeficient mouse models of human glioma . AdCMVCD/5-FC efficacy was specific, virus dose-dependent, and closely paralleled in vitro 5-FU and CD/5-FC sensitivity in two of three models tested . These results reveal that glioma cells are as sensitive as GI tumor cells to the antineoplastic effects of 5-FU, identify inherent 5-FU sensitivity as an important factor in determining CD/5-FC efficacy, and confirm previous findings in rat models that demonstrate the potential clinical utility of AdCMVCD/5-FC gene therapy for gliomas.

Zhonghua Wai Ke Za Zhi, 1999 Jul, 37(7), 391 - 4
{Transfection of articular chondrocytes with PcNDA3-hBMP3 and its stable expression}; Han Y et al.; OBJECTIVE: To explore the possibility of stable expression of PcDNA3-hBMP3 in cultured articular chondrocytes of rabbit . METHODS: PcDNA3-hBMP3 was constructed using gene clone technique and recombined DNA technique . With the help of profectamine, the cultured articular chondrocytes were transfected with PcDNA3-hBMP3, and the evidence of successfully stable transfection in these cells could be obtained by positive northern blot . RESULTS: The cultured articular chondrocytes of rabbits seemed to be polygonal, and its logarithmic growth phase was 2 - 4 days after cell inoculation . The two fragments cut from PcDNA3-hBMP3 by EcoR I and Xba I represented 5.4 kb and 1.4 kb by electrophoresis, which were confirmed to be the carrier and the fragment inserted originally, indicating that the construction of PcDNA3-hBMP3 was successful . The RNA extracted from cultured chondrocytes was screened for 4 weeks by G418 hibrided with the fragment cut from hBMP3 positively . CONCLUSIONS: With the help of profectamine, the cultured articular chondrocytes can be transfected by recombined gene of PcDNA3-hBMP3 successfully, and their stable expression at 4 weeks after transfection is obtained.

Biochem J, 2002 Feb 15, 362(Pt 1), 89 - 96
Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis; Ward RJ et al.; Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A(2) variant present in the venom of Bothrops jararacussu, in which the Asp(49) residue is replaced with a lysine, which damages artificial membranes by a Ca(2+)-independent mechanism . Wild-type BthTx-I and the mutants Lys(49)-->Asp, His(48)-->Gln and Lys(122)-->Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom . The Ca(2+)-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His(48)Gln and Lys(49)Asp mutants were similar; however, the Lys(122)Ala mutant demonstrated reduced levels of both activities . Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants . In the case of the Lys(49)Asp mutant, this demonstrates that the absence of catalytic activity in Lys(49)-PLA(2) is not a consequence of the single Asp(49)-->Lys replacement . Furthermore, these results provide unambiguous evidence that the Ca(2+)-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis . The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.

J Agric Food Chem, 2002 Feb 13, 50(4), 784 - 9
Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability; Ken CF et al.; A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach . Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues . The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species . Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences . To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3) . The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow . Dimer was the major form of the enzyme in equilibrium . The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0) . The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C . The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole . The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.

Mol Ther, 2002 Feb, 5(2), 204 - 10
Generation of helper-dependent adenoviral vectors by homologous recombination; Toietta G et al.; Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo . The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb) . The ligation to produce the viral plasmid is generally very inefficient . Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183 . Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb . To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter . The viruses were rescued and tested in vitro and for in vivo expression in mice . The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E . coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector.

J Mol Biol, 2002 Feb 8, 316(1), 79 - 88
Structure of 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase involved in mevalonate-independent biosynthesis of isoprenoids; Steinbacher S et al.; Isoprenoids are biosynthesized from isopentenyl diphosphate and the isomeric dimethylallyl diphosphate via the mevalonate pathway or a mevalonate-independent pathway that was identified during the last decade . The non-mevalonate pathway is present in many bacteria, some algae and in certain protozoa such as the malaria parasite Plasmodium falciparum and in the plastids of higher plants, but not in mammals and archaea . Therefore, these enzymes have been recognised as promising drug targets . We report the crystal structure of Escherichia coli 2C- methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), which converts 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate and CMP in a Mg-dependent reaction . The protein forms homotrimers that tightly bind one zinc ion per subunit at the active site, which helps to position the substrate for direct attack of the 2-phosphate group on the beta-phosphate .

J Theor Biol, 2001 Oct 7, 212(3), 391 - 8
Synchronous cultures from the baby machine: anatomy of a model; Grover NB et al.; The baby-machine system, which produces newborn Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth . In the present article, we describe in some detail a model designed to analyse such cells with a view to characterizing the nature and quality of the synchrony in a quantitative manner; it can also serve to evaluate the methodology itself, its potential and its limitations . The model consists of five elements, giving rise to five adjustable parameters (and a proportionality constant): a major, essentially synchronous group of cells with ages distributed normally about zero; a minor, random component from a steady-state population on the membrane that had undergone only very little age selection during the elution process; a fixed background count, to account for the signals recorded by the electronic particle counter produced by debris and electronic noise; a time-shift, to allow for differences between collection time and sampling time; and the coefficient of variation of the interdivision-time distribution, taken to be a Pearson type III . The model is fitted by nonlinear least-squares to data from cells grown in glucose minimal medium . The standard errors of the parameters are quite small, making their estimates all highly significant; the quality of the fit is striking . We also provide a simple yet rigorous procedure for correcting cell counts obtained in an electronic particle counter for the effect of coincidence . An example using real data produces an excellent fit.

Vet Rec, 2002 Jan 12, 150(2), 35 - 7
Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea; Kwon D et al.; A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STb), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease . Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins . Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively . Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively . Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.

J Refract Surg, 2002 Jan-Feb, 18(1), 51 - 7
Endotoxin levels in steam and reservoirs of table-top steam sterilizers; Whitby JL et al.; PURPOSE: To document endotoxin levels in "Statim" cassette sterilizer reservoirs and in steam delivered to the cassette in the unwrapped instrument cycle . To document endotoxin levels in sterilizer reservoir water using different management protocols . METHODS: Endotoxin levels were determined using the Limulus Amebocyte Lysate test . Endotoxin preparations were from Escherichia coli and Ralstonia pickettii . All samples were collected in depyrogenated glassware and stored at -20 degrees C until assayed . RESULTS: The majority of water samples contained < 1.0 Endotoxin Unit (EU)/ml . The highest level found in sterilizers in clinical use was 5.3 EU/ml . Endotoxin was not detected in steam condensate within the limits of the assay . When the endotoxin level in the reservoir water was experimentally enhanced to 200 EU/ml, cassette steam condensate endotoxin levels were from 0.5% to 5% of the reservoir level . Daily and weekly emptying of the cassette reservoir consistently yielded low endotoxin levels as did monthly emptying, but with the latter there was a trend toward higher levels that favors weekly emptying as a precautionary measure . CONCLUSIONS: Endotoxin levels in the reservoirs of 23 sterilizers involving 240 samplings were never high enough to yield detectable endotoxin levels in steam in the sterilizer cassette . Regular weekly emptying of sterilizer reservoirs would eliminate the risk of endotoxin transfer during steam sterilization.

J Surg Res, 2002 Feb, 102(2), 150 - 5
Construction and preliminary screening of a human phage single-chain antibody library associated with gastric cancer; He J et al.; BACKGROUND: The aim of this study was to construct a phage library of human single-chain antibodies associated with gastric cancer and screen such a library for CEA binding scFv . MATERIALS AND METHODS: The cDNA library of antibody variable regions was constructed using mRNA from metastatic lymph nodes or spleen of patients with stomach cancer by RT-PCR . These cDNA were assembled into a single-chain format and cloned into phagemid pCANTAB-5 and then transformed into Escherichia coli TG1 . The scFv gene library was rescued by M13KO7 helper phage . CEA and the viable CEA-positive gastric cancer cell line MKN-28 were used to screen the phage antibody library . Indirect and tumor cell ELISA was used to determine the specificity of phage antibody . Fixed cell immunofluorescence and live cell FACS analysis were used to further characterize the binding of phage scFv . RESULTS: After transformation into E . coli TG1, 2.5 x 10(7) cfu/microg ampicillin-resistant clones grew . Sequences of those positive insert clones showed that the V(H) genes were derived from the V(H) III subgroup, while the V(L) genes belonged to the V(kappa) III subgroup . After four rounds of panning, the titer of eluted binding phage increased 135- to 158-fold and ELISA results showed that 20/95 clones can bind CEA and 47/95 clones can bind fixed tumor cells . Immunofluorescence and FACS analysis results showed that these phage scFv fragments could bind CEA-positive cells . CONCLUSIONS: We successfully constructed a human phage antibody library from lymph nodes of stomach cancer patients . Such kinds of library prove useful for generating tumor-antigen-specific human antibody fragments . (c)2001 Elsevier Science.

Chembiochem, 2001 Aug 3, 2(7-8), 517 - 23
Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket; Muller BH et al.; We describe a strategy that allowed us to confer on a bacterial (E . coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability . First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation . We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside . Second, the gene encoding the inactive mutant was submitted to random mutagenesis . Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket . Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium . As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP . Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C) . Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1) . Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two . Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability . Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported.

Chembiochem, 2001 May 4, 2(5), 343 - 54
Inhibition studies of porphobilinogen synthase from Escherichia coli differentiating between the two recognition sites; Stauffer F et al.; Porphobilinogen synthase condenses two molecules of 5-aminolevulinate in an asymmetric way . This unusual transformation requires a selective recognition and differentiation between the substrates ending up in the A site or in the P site of porphobilinogen synthase . Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites . The P site, whose structure is known from X-ray crystallographic studies, tolerates ester functions well . The A site interacts very strongly with nitro groups, but is not very tolerant to ester functions . This differentiation is a central factor in the asymmetric handling of the two identical substrates . Finally, it could be shown that the keto group of the substrate bound at the A site is not only essential for the recognition, but that an increase in electrophilicity of the carbon atom also increases the inhibition potency considerably . This has important consequences for the recognition process at the A site, whose exact structure is not yet known.

Chembiochem, 2001 Mar 2, 2(3), 171 - 9
Peptoid - peptide hybrids that bind Syk SH2 domains involved in signal transduction; Ruijtenbeek R et al.; Peptoid-peptide hybrids are oligomeric peptidomimetics that contain one or more N-substituted glycine residues . In these hybrids, the side chains of one or several amino acids are "shifted" from the alpha-carbon atom to the amide nitrogen atom . A library of phosphorylated peptoid-peptide hybrids derived from the sequence pTyr-Glu-Thr-Leu was synthesized and tested for binding to the tandem SH2 domain of the protein tyrosine kinase Syk . A considerable influence of the side chain position was observed . Compounds 19-21, 24, and 25 comprising a peptoid NpTyr and/or NGlu residue did not show any binding . Compounds 22, 23, and 26 containing an NhThr (hThr=homothreonine) and/or NLeu peptoid residue showed binding with IC(50) values that were only five to eight times higher than that of the tetrapeptide lead compound 18 . These data show that side chain shifting is possible with retention of binding capacity, but only at the two C-terminal residues of the tetramer . This method of a peptoid scan using peptoid-peptide hybrids appears to be very useful to explore to what extent a peptide sequence can be transformed into a peptoid while retaining its affinity.

Nat Struct Biol, 2002 Mar, 9(3), 225 - 30
A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits; Schmeing TM et al.; The large ribosomal subunit catalyzes peptide bond formation during protein synthesis . Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol . Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active . Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed . When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed . The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451) . Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

Plant Cell Physiol, 2002 Jan, 43(1), 27 - 34
GA(3)-induced expression of a new functional AAA-ATPase (FsA1) is correlated with the onset of germination in Fagus sylvatica L . seeds (beechnuts); Lorenzo O et al.; A full-length cDNA clone, named FsA1, has been isolated from a cDNA library constructed using mRNA from Fagus sylvatica L . dormant seeds (beechnuts) . This clone shows high identity with members of the AAA superfamily, for ATPases Associated with a variety of cellular Activities, encoding subunit 8 of the 26S proteasome or Tat binding proteins (TBPs) . Direct biochemical evidence supporting Mg(2+)-dependent ATPase activity has been obtained by expressing FsA1 in Escherichia coli as histidine tag fusion protein and using the recombinant protein in the stimulation of ATP hydrolysis . Analysis of the expression of FsA1 transcripts during stratification shows an increase in the presence of gibberellic acid (GA(3)), a treatment that proved to be efficient in breaking dormancy and increasing germination percentages of these seeds, while the addition of paclobutrazol, a well-known GA biosynthesis inhibitor, greatly reduces the expression of the clone . A low level of expression was maintained in the stratification control in H(2)O, where dormancy is slowly released . These results show that this new member of the AAA-ATPase family is up-regulated by GAs and its expression correlated with the germination arise in Fagus sylvatica seeds . The possible function of this protein during the transition from dormancy to germination is discussed.

J Biol Chem, 2002 Apr 19, 277(16), 13771 - 7 Epub 2002 Feb 04.
Molecular characterization of a specific thiamine triphosphatase widely expressed in mammalian tissues; Lakaye B et al.; Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins . In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis . In this report we purified a soluble thiamine triphosphatase (ThTPase; EC ) from calf brain . The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity . Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank data base . A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652 . The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced . The human open reading frame was expressed in E . coli as a GST fusion protein . Transformed bacteria had a high isopropyl-beta-d-thiogalactopyranoside-inducible ThTPase activity . The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP . The mRNA was expressed in most human tissues but at relatively low levels . This is the first report of a molecular characterization of a specific ThTPase.

J Biol Chem, 2002 Apr 26, 277(17), 14910 - 5 Epub 2002 Feb 04.
Folding of the Plasmodium falciparum cysteine protease falcipain-2 is mediated by a chaperone-like peptide and not the prodomain; Sijwali PS et al.; Papain-family cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are hemoglobinases and potential drug targets . Available data suggest that papain-family proteases require prodomains for correct folding into functional conformations . However, in prior studies of falcipain-2, an Escherichia coli-expressed construct containing only a small portion of the prodomain refolded efficiently, suggesting that this enzyme differs in this regard from other papain-family enzymes . To better characterize the determinants of folding for falcipain-2, we expressed multiple pro- and mature constructs of the enzyme in E . coli and assessed their abilities to refold . Mature falcipain-2 refolded into active protease with very similar properties to those of proteins resulting from the refolding of proenzyme constructs . Deletion of a 17-amino acid amino-terminal segment of the mature protease yielded a construct incapable of correct folding, but inclusion of this segment in trans allowed folding to active falcipain-2 . The prodomain was a potent, competitive, and reversible inhibitor of mature falcipain-2 (K(i) 10(-10) m) . Our results identify a chaperone-like function of an amino-terminal segment of mature falcipain-2 and suggest that protease inhibition, but not the mediation of folding, is a principal function of the falcipain-2 prodomain.

Mutat Res, 2002 Feb 20, 499(2), 197 - 211
Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro; Martomo SA et al.; Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool . We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells . By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations . However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation . During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated . Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low {dGTP} . An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency . We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP) . This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.

Altern Lab Anim, 2002 Jan-Feb, 30(1), 87 - 92
Use of the SOS-chromotest spot assay as a screening system for detecting genotoxic compounds in crude plant extracts; Baez DA et al.; An SOS-chromotest spot assay was used to detect genotoxic compounds in crude plant extracts . The method allows simultaneous testing of extracts from different species in either a liquid or a solid crystalline form . Extracts from two species of the genus Senna, native to the state of Morelos, Mexico, were assayed . Four genotoxic compounds were isolated, and were identified as quercetin and rutin from S . wislizeni, and 5,7-di- O-methylrutin and 5,7-di-O-methylquercetin from S . skinneri . The SOS-chromotest spot assay proved to be useful for activity- guided fractionation at the beginning of screening for genotoxic compounds in crude plant extracts.

Biochemistry, 2002 Feb 12, 41(6), 2022 - 7
Testing of the portal hypothesis: analysis of a V32G, F57G, K58G mutant of the fatty acid binding protein of the murine adipocyte; Jenkins AE et al.; The portal region of fatty acid binding proteins is hypothesized to function as a dynamic aperture, controlling accessibility of external ligands to the internal fatty acid binding cavity . To test this hypothesis, a triple mutant of the murine FABP4 has been developed (V32G, F57G, K58G, referred to as the portal mutant) that is predicted to constitutively enlarge the opening due to a reduction in the molecular dimensions of the side chains of key portal amino acids . The portal mutant was purified from expressing Escherichia coli, its stability was evaluated, and the thermodynamics and kinetics of ligand binding were compared to that of wild-type protein . Introduction of the three amino acid substitutions caused no significant change in the stability of the protein with a free energy of unfolding of 13.7 kJ/mol as compared to 14.0 kJ/mol for the wild-type protein . The portal mutant exhibited a modest decrease (4-fold) in ligand binding affinity using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as a surrogate ligand . 1,8-ANS displacement assays revealed that the binding affinity for oleate increased from a K0.5 of 196 +/- 15 nM for the wild-type protein to 165 +/- 8 for the portal mutant, while that for arachidonate decreased from the wild type of 186 +/- 11 nM to 418 +/- 26 nM for the portal mutant . To evaluate cavity accessibility, rate of 1,8-ANS binding was assessed between the portal and wild-type protein . Using equimolar amounts of ligand and protein at 4 degrees, 1,8-ANS bound within the cavity to 95% saturation (t0.95) in 750 ms, while the mutant protein was fully modified in less than 1.4 ms . To independently evaluate cavity accessibility, modification of the sole protein cysteine residue, C117 residing within the cavity near C2-C4 of the bound ligand, was monitored using 5,5'-dithio-bis(2-nitrobenzoic acid) modification . The half time for modification (t0.5) for the wild-type protein was approximately 20 s, while that for V32G F57G K58G occurred in less than a second . As such, enlargement of the portal region of FABP4 markedly increased the accessibility of ligands to the cavity while having only modest effects on ligand affinity . Taken together, these data provide support for the portal region hypothesis and suggest dynamic fluctuations in this region regulate cavity access, but not ligand affinity or selectivity.

Biochemistry, 2002 Feb 12, 41(6), 1869 - 76
Conformational heterogeneity is revealed in the dissociation of the oligomeric chaperonin GroEL by high hydrostatic pressure; Panda M et al.; We investigated the dissociation of tetradecameric GroEL by high hydrostatic pressure in the range of 1-2.5 kbar . Kinetics of the dissociation of GroEL in the absence and presence of Mg(2+) and/or KCl were monitored using light scattering . All of the kinetics were biphasic in nature . At any given pressure, only monomers and 14mers were produced, and below 2.5 kbar, the 14mers only partially dissociated to monomers, which did not significantly reassemble on depressurization . Under identical reaction conditions, the observed dissociation rates decreased by only 2-fold when the concentration of GroEL was increased by 20-fold . At 2.5 kbar the observed rates decreased exponentially with the increase in {KCl} and reached a minimum at approximately 75mM . Similarly, the rates decreased with the increase in {Mg(2+)} and reached a minimum at approximately 3 mM Mg(2+) . In the presence of saturating amounts of Mg(2+) (10 mM) and KCl (100 mM), the rates were much faster than with 10 mM Mg(2+) alone . The results could be rationalized in terms of the presence of GroEL heterogeneity, which could not be assessed easily by common techniques such as sedimentation velocity, HPLC, gel electrophoresis, and dissociation by chaotropes . This heterogeneity is evidence of subpopulations of GroEL that dissociate at different pressures . At low pressures, the oligomer without added Mg(2+) only partially dissociates to monomers, leading to an apparent plateau in the kinetics, whereas in the presence of Mg(2+) the species are converted to a tighter Mg(2+)-bound species, leading to a much slower dissociation process . The presence of KCl in the sample also leads to similar heterogeneity.

Biochemistry, 2002 Feb 12, 41(6), 1861 - 8
Identification of glutamic acid 479 as the gluzincin coordinator of zinc in FtsH (HflB); Saikawa N et al.; Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent metalloprotease . Its cytoplasmic domain contains a zinc-binding motif, H(417)EXXH, whose histidine residues have been shown to be functionally important . Although they are believed to be involved directly in zinc coordination, nothing is known about the third zinc ligand of this protease . Sequence alignment indicates that glutamic acid residues are conserved among the FtsH homologues at positions corresponding to Glu(479) and Glu(585) of E . coli FtsH . We replaced each of them by Gln, Asp, Lys, or Val . Mutations at position 479 compromised the proteolytic functions of FtsH in vivo . In vitro proteolytic activities of the E479Q, E479V, and E479D mutant enzymes were much lower than that of the wild-type protein and were significantly stimulated by a high concentration of zinc ion . These mutant proteins retained the wild-type levels of ATPase activities, and their trypsin susceptibilities as well as CD spectra were essentially indistinguishable from those of the wild-type protein, indicating that the mutations did not cause gross conformational changes in FtsH . They exhibited reduced zinc contents upon purification . From these results, we conclude that Glu(479) is a zinc-coordinating residue.

J Mol Biol, 2002 Feb 1, 315(5), 1179 - 87
Low resolution solution structure of the Apo form of Escherichia coli haemoglobin protease Hbp; Scott DJ et al.; We have studied the solution properties of the apo form of the haemoglobin protease or "haemoglobinase", Hbp, a principal component of an important iron acquisition system in pathogenic Escherichia coli . Experimental determination of secondary structure content from circular dichroism (CD) spectroscopy, obtained using synchrotron light, showed that the protein contains predominately beta-sheets in agreement with secondary structure prediction from the primary sequence . Next, the size and shape of the protein were probed using analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) . These showed that Hbp is a monomer, with an extended conformation . Using ab initio reconstruction methods we have produced a model of Hbp, which shows that the protein adopts an extended crescent-shaped conformation . Analysis of the resulting model gives hydrodynamic parameters in good agreement with those observed experimentally . Thus we are able to construct a hydrodynamically rigorous model of apo-Hbp in solution, not only giving a greater level of confidence to the results of the SAXS reconstruction methods, but providing the first three-dimensional view of this intriguing molecule .

J Mol Biol, 2002 Feb 1, 315(5), 1027 - 37
Concerted binding and bending of DNA by Escherichia coli integration host factor; Dhavan GM et al.; Integration host factor (IHF) is a heterodimeric Escherichia coli protein that plays essential roles in a variety of cellular processes including site-specific recombination, transcription, and DNA replication . The IHF-DNA interface extends over three helical turns and includes sequential minor groove contacts that present strong, sequence specific protection patterns against hydroxyl radical cleavage . Synchrotron X-ray footprinting has been used to follow the kinetics of formation of DNA-protein contacts in the IHF-DNA complex with single base-pair spatial, and millisecond time, resolution . The three sites of IHF protection on the DNA develop with similar time-dependence, indicating that sequence specific binding and bending occur concertedly . Two distinct phases are observed in the association process . The first "burst" phase is characterized by a rate that is greater than diffusion limited (>10(10) s(-1) M(-1)) and the second phase is on the order of diffusion controlled (approximately 10(8) M(-1) s(-1)) . The overall kinetics of association become faster with increasing IHF concentration showing that complex formation is second-order with protein . The rate of association is maximal between 100 and 200 mM KCl decreasing at higher and lower concentrations . The rate of IHF dissociation from site-specifically bound DNA increases monotonically as KCl concentration is increased . The dissociation progress curves are biphasic with the amplitude of the first phase dependent upon competitor DNA concentration . These results are the first analysis by synchrotron footprinting of the fast kinetics of a protein-DNA interaction and suggest that IHF binds its specific site through a multiple-step mechanism in which the first step is facilitated diffusion along the length of the duplex followed by subsequent binding and bending of the DNA in a concerted manner .

Genomics, 2002 Jan, 79(1), 124 - 36
Sequence analysis of LRPPRC and its SEC1 domain interaction partners suggests roles in cytoskeletal organization, vesicular trafficking, nucleocytosolic shuttling, and chromosome activity; Liu L et al.; LRPPRC (originally called LRP130) is an intracellular, 130-kD, leucine-rich protein that copurifies with the fibroblast growth factor receptor from liver cell extracts and has been detected in diverse multiprotein complexes from the cell membrane, cytoskeleton, and nucleus . Here we report results of a sequence homology analysis of LRPPRC and its SEC1 domain interactive partners . We found that 23 copies of tandem repeats that are similar to pentatricopeptide, tetratricopeptide, and huntingtin-elongation A subunit-TOR repeats characterize the LRPPRC sequence . The amino terminus exhibits multiple copies of leucine-rich nuclear transport signals followed by ENTH, DUF28, and SEC1 homology domains . We used the SEC1 domain to trap interactive partners expressed from a human liver cDNA library . Interactive C19ORF5 (XP_038600) exhibited a strong homology to microtubule-associated proteins and a potential arginine-rich mRNA binding motif . UXT (XP_033860) exhibited alpha-helical properties homologous to the actin-associated spectrin repeat and L/I heptad repeats in mobile transcription factors . C6ORF34 (XP_004305) was homologous to the non-DNA-binding carboxy terminus of the Escherichia coli Rob transcription factor . CECR2 (AAK15343) exhibited a transcription factor AT-hook motif next to two bromodomains and a homology to guanylatebinding protein-1 . Together these features suggest a regulatory role of LRPPRC and its SEC1 domain-interactive partners in integration of cytoskeletal networks with vesicular trafficking, nucleocytosolic shuttling, transcription, chromosome remodeling, and cytokinesis.

Med Microbiol Immunol (Berl), 2001 Dec, 190(3), 145 - 9
Binding inhibition of type 1 fimbriae to human granulocytes: a flow cytometric inhibition assay using trivalent cluster mannosides; Horst AK et al.; The binding of type 1 fimbriae from Escherichia coli to vital human neutrophilic granulocytes was inhibited by synthetic trivalent cluster mannosides . Binding of type 1 fimbriae was measured in a flow cytometric assay . Based on the molarity of mannosyl residues, the clusters exceed the inhibitory potency of methyl alpha-D-mannoside by a factor of more than 1,000 and the inhibitory potency of p-nitrophenyl alpha-D-mannoside by a factor of more than 10 . The inhibition studies indicate that the trivalent cluster mannosides are very potent inhibitors of the binding of type 1 fimbriae to human neutrophilic granulocytes . Based on their defined structure, cluster mannosides are well suited for characterizing the molecular interactions of mannose-sensitive fimbriae with their cell membrane receptors.

Mol Cell Biochem, 2001 Nov, 227(1-2), 31 - 6
A surface plasmon resonance study of the interactions between the component subunits of protein kinase CK2 and two protein substrates, casein and calmodulin; Benitez MJ et al.; Surface plasmon resonance has been used to study the interaction between the subunits composing protein kinase CK2 (two catalytic, alpha-subunits, and two regulatory, beta-subunits), as well as the interaction of each subunit with two types of protein substrates, casein, the phosphorylation of which is activated by the regulatory subunit, and calmodulin, which belongs to the kind of substrates on which the catalytic subunit is downregulated by the regulatory subunit . The interaction of casein with the catalytic subunit differs from the interaction with the holoenzyme . Similarly to the interaction with the regulatory subunit, the catalytic subunit interacts with the protein substrate forming a very stable, irreversible complex . The reconstituted holoenzyme, however, binds casein reversibly, displaying a binding mode similar to that displayed by the regulatory subunit . The interaction of calmodulin with the catalytic subunit gives place, like in the case of casein, to an irreversible complex . The interactions with the regulatory subunit and with the holoenzyme were practically negligible, and the interaction with the regulatory subunit disappeared upon increasing the temperature value to close to 30 degrees C . The presence of polylysine induced a high increase in the extent of calmodulin binding to the holoenzyme . The results obtained suggest that CK2beta subunit and protein substrates share a common, or at least an overlapping, site of interaction on the catalytic subunit . The interaction between both subunits would prevent substrates from binding irreversibly to alpha subunit, and, at the same time, it would generate a new and milder site of interaction between the whole holoenzyme and the protein substrate . The main difference between casein and calmodulin would consist in the lower affinity display by the last for the new site generated upon the binding of the regulatory subunit, in the absence of polycations like polylysine.

Mol Cell Biochem, 2001 Nov, 227(1-2), 129 - 35
Cloning and characterization of the cDNA coding for the catalytic alpha subunit of CK2 from tobacco; Salinas P et al.; We have previously reported the participation of the protein kinase CK2 in the mechanism by which salicylic acid activates transcription of genes, such as those coding for glutathion S-transferases, in tobacco . With the purpose of further studying the participation of CK2 in this signal transduction pathway, we isolated and sequenced the cDNA from the NtCK2A gene, coding for the catalytic alpha subunit of CK2 from tobacco . The NtCK2A cDNA was isolated by screening of a tobacco cDNA library with a heterologous probe from Arabidopsis thaliana, followed by 3' RACE to obtain the 3' region . Sequence analysis of the NtCK2A cDNA showed a high level of identity between this CK2alpha protein sequence and the corresponding sequences of other plant species such as Arabidopsis and maize (92-95% identity), or those of animal species such as human and Xenopus laevis (75% identity) . The expression of the NtCK2A gene in different tissues from tobacco plants was analyzed by Northern blot . High levels of expression of this gene were observed in proliferating tissues such as shoot and root apical meristems . A recombinant CK2alpha protein was obtained after expression of the NtCK2A cDNA in Escherichia coli . The ability of this recombinant CK2alpha subunit to phosphorylate casein was inhibited by heparin and stimulated by the CK2beta subunit from Xenopus laevis.

Biosci Biotechnol Biochem, 2001 Dec, 65(12), 2741 - 8
A gene encoding phosphatidylethanolamine N-methyltransferase from Acetobacter aceti and some properties of its disruptant; Hanada T et al.; Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter . To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid . The pmt gene of A . aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced . One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A . aceti . The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein . The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation . When the pmt gene was expressed in E . coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated . A . aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed . The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses . The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG) . The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate . These results suggest that the lack of PC in the A . aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A . aceti membrane is related to its acetic acid tolerance.

Arch Biochem Biophys, 2002 Jan 15, 397(2), 336 - 41
Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages; Lazarus M et al.; We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli . The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1) . Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1 . The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages . Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide . Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages . Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide . (c)2001 Elsevier Science.

Arch Biochem Biophys, 2002 Jan 15, 397(2), 324 - 35
Tryparedoxin peroxidase of Leishmania donovani: molecular cloning, heterologous expression, specificity, and catalytic mechanism; Flohe L et al.; Tryparedoxin peroxidase (TXNPx) of Trypanosomatidae is the terminal peroxidase of a complex redox cascade that detoxifies hydroperoxides by NADPH (Nogoceke et al., Biol . Chem . 378, 827-836, 1997) . A gene putatively coding for a peroxiredoxin-type TXNPx was identified in L . donovani and expressed in Escherichia coli to yield an N-terminally His-tagged protein (LdH6TXNPx) . LdH6TXNPx proved to be an active peroxidase with tryparedoxin (TXN) 1 and 2 of Crithidia fasciculata as cosubstrates . LdH6TXNPx efficiently reduces H2O2, is moderately active with t-butyl and cumene hydroperoxide, but only marginally with linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide . The enzyme displays ping-pong kinetics with a k(cat) of 11.2 s(-1) and limiting K(m) values for t-butyl hydroperoxide and CfTXN1 of 50 and 3.6 microM, respectively . Site-directed mutagenesis confirmed that C52 and C173, as in related peroxiredoxins, are involved in catalysis . Exchanges of R128 against D and T49 against S and V, supported by molecular modelling, further disclose that the SH group of C52 builds the center of a novel catalytic triad . By hydrogen bonding with the OH of T49 and by the positive charge of R128 the solvent-exposed thiol of C52 becomes deprotonated to react with ROOH . Molecular models of oxidized TXNPx show C52 disulfide-bridged with C173' that can be attacked by C41 of TXN2 . By homology, the deduced mechanism may apply to most peroxiredoxins and complements current views of peroxiredoxin catalysis . (c)2002 Elsevier Science.

Plant Cell, 2002 Jan, 14(1), 119 - 31
A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion; Elumalai RP et al.; Potassium ions (K(+)) are the most abundant cations in plants and are necessary for cell growth . Arabidopsis shy3-1 mutant plants have a short hypocotyl, small leaves, and a short flowering stem, and these defects result from decreased cell expansion . The semidominant shy3-1 mutation changes an amino acid in KT2/KUP2, a K(+) transporter related to the Escherichia coli Kup protein . Second mutations in the KT2/KUP2/SHY3 gene, including presumed null mutations, suppress the shy3-1 phenotypes . Plants with these intragenic suppressor mutations appear similar to wild-type plants, suggesting that KT2/KUP2/SHY3 acts redundantly with other genes . Expression of the shy3-1 mutant version of KT2/KUP2/SHY3 in wild-type plants confers shy3-1-like phenotypes, indicating that shy3-1 probably either causes a gain of function or creates an interfering protein . The shy3-1 mutation does not eliminate the ability of the KT2/KUP2 cDNA to rescue the growth of a potassium transport-deficient E . coli mutant . A P(SHY3)::GUS fusion is expressed in growing portions of the plant . These results suggest that KT2/KUP2/SHY3 mediates K(+)-dependent cell expansion in growing tissues.

J Clin Microbiol, 2002 Feb, 40(2), 694 - 7
Type IV longus pilus of enterotoxigenic Escherichia coli: occurrence and association with toxin types and colonization factors among strains isolated in Argentina; Pichel MG et al.; The longus type IV pilus structural gene (lngA) was sought among 217 clinical enterotoxigenic Escherichia coli (ETEC) strains isolated in Argentina . lngA was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors . The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.

J Clin Microbiol, 2002 Feb, 40(2), 645 - 8
Diffusely adherent Escherichia coli as a cause of acute diarrhea in young children in Northeast Brazil: a case-control study; Scaletsky IC et al.; In a prospective study carried out in two urban centers in northeastern Brazil, 195 HEp-2-adherent Escherichia coli strains were isolated; 110 were identified as the only pathogen in stools of children with diarrhea, and 85 were from controls . Enteropathogenic E . coli isolates were identified in 21 children with diarrhea (8.9%) and 7 children without diarrhea (3.0%), and they were significantly associated with diarrhea (P < 0.01) . Enteroaggregative E . coli strains were isolated from 40 children with diarrhea (16.9%) and 38 children without diarrhea (16.4%) and showed no correlation with diarrhea (P > 0.5) . In 49 children with diarrhea (20.7%) and 40 children without diarrhea (17.3%), diffusely adherent E . coli (DAEC) isolates were detected and were not found to be associated with diarrhea (P = 0.41) . However, after stratification, for children older than 12 months of age a significant correlation between DAEC infection and diarrhea was detected (P = 0.01) . These results suggest that DAEC isolates should be considered potential pathogens in northeastern Brazil and also confirm the association of DAEC with age-dependent diarrhea.

J Biol Chem, 2002 Apr 19, 277(16), 14077 - 84 Epub 2002 Feb 01.
Hinge-bending motion of D-allose-binding protein from Escherichia coli: three open conformations; Magnusson U et al.; Conformational changes of periplasmic binding proteins are essential for their function in chemotaxis and transport . The allose-binding protein from Escherichia coli is, like other receptors in its family, composed of two alpha/beta domains joined by a three-stranded hinge . In the previously determined structure of the closed, ligand-bound form (Chaudhuri, B . N., Ko, J., Park, C., Jones, T . A., and Mowbray, S . L . (1999) J . Mol . Biol . 286, 1519-1531), the ligand-binding site is buried between the two domains . We report here the structures of three distinct open, ligand-free forms of this receptor, one solved at 3.1-A resolution and two others at 1.7-A resolution . Together, these allow a description of the conformational changes associated with ligand binding . A few large, coupled torsional changes in the hinge strands are sufficient to generate the overall bending motion, with only minor disruption of the individual domains . Integral water molecules appear to act as structural "ball bearings" in this process . The conformational changes of the related ribose-binding protein follow a distinct pattern . The observed differences between the two proteins can be interpreted in the context of changes in sequence and in crystal packing and provide new insights into the nature of hinge bending motion in this class of periplasmic binding proteins.

J Biol Chem, 2002 Apr 19, 277(16), 13739 - 44 Epub 2002 Feb 01.
Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p; Tanida I et al.; Autophagy is a process of bulk degradation of cytoplasmic components by the lysosome/vacuole and has a significant relationship to several neurodegenerative disorders and myopathies in mammals . One of APG gene products essential for autophagy in yeast, Apg3p, is a protein-conjugating enzyme for Apg8p lipidation (Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and Ohsumi, Y . (2000) Nature 408, 488-492) . In this study, the cloning of a human Apg3p homologue (hApg3p) as an E2 enzyme essential for human Apg8p homologues (i.e . GATE-16, GABARAP, and MAP-LC3) is shown, and its unique characteristics are described . The predicted amino acid sequence of the isolated clone shows 34.1% identity and 48.1% similarity to yeast Apg3p . Site-directed mutagenesis revealed that Cys(264) of hApg3p is an authentic active-site cysteine residue essential for the formation of hApg3p small middle dothApg8p homologue intermediates . Overexpression of hApg7p enhances the formation of a stable E2-substrate complex between hApg3p(C264S) and each of the hApg8p homologues, and MAP-LC3 is preferred as the substrate over the other two Apg8p homologues . These results indicate that hApg3p is an E2-like enzyme essential for three human Apg8p homologues . Co-immunoprecipitation of hApg7p with hApg3p indicates that hApg3p forms an E1.E2 complex with hApg7p as in the case of yeast Apg3p and Apg7p . Furthermore, hApg3p coimmunoprecipitates with hApg12p, and the overexpression of hApg3p facilitates the formation of the GFPhApg12p.thApg5p conjugate, suggesting that hApg3p cross-talks with the hApg12p conjugation system.

J Biol Chem, 2002 Apr 19, 277(16), 13724 - 31 Epub 2002 Feb 01.
Allosteric communication between signal peptides and the SecA protein DEAD motor ATPase domain; Baud C et al.; SecA, the preprotein translocase ATPase is built of an amino-terminal DEAD helicase motor domain bound to a regulatory C-domain . SecA recognizes mature and signal peptide preprotein regions . We now demonstrate that the amino-terminal 263 residues of the ATPase subdomain of the DEAD motor are necessary and sufficient for high affinity signal peptide binding . Binding is abrogated by deletion of residues 219-244 that lie within SSD, a novel substrate specificity element of the ATPase subdomain . SSD is essential for protein translocation, is unique to SecA, and is absent from other DEAD proteins . Signal peptide binding to the DEAD motor is controlled in trans by the C-terminal intramolecular regulator of ATPase (IRA1) switch . IRA1 mutations that activate the DEAD motor ATPase also enhance signal peptide affinity . This mechanism coordinates signal peptide binding with ATPase activation . Signal peptide binding causes widespread conformational changes to the ATPase subdomain and inhibits the DEAD motor ATPase . This involves an allosteric mechanism, since binding occurs at sites that are distinct from the catalytic ATPase determinants . Our data reveal the physical determinants and sophisticated intramolecular regulation that allow signal peptides to act as allosteric effectors of the SecA motor.

J Biol Chem, 2002 Apr 12, 277(15), 12868 - 73 Epub 2002 Feb 01.
Repair of nitric oxide-modified ferredoxin {2Fe-2S} cluster by cysteine desulfurase (IscS); Yang W et al.; Iron-sulfur proteins are among the sensitive targets of the nitric oxide cytotoxicity . When Escherichia coli cells are exposed to nitric oxide, iron-sulfur clusters are modified forming protein-bound dinitrosyl iron complexes . Such modified protein dinitrosyl iron complexes are stable in vitro but are efficiently repaired in aerobically growing E . coli cells even without any new protein synthesis . Here we show that cysteine desulfurase encoded by the gene iscS of E . coli can directly convert the ferredoxin dinitrosyl iron complex to the ferredoxin {2Fe-2S} cluster in the presence of L-cysteine in vitro . A reassembly of the {2Fe-2S} cluster in the ferredoxin dinitrosyl iron complex does not require any addition of iron or other protein components . Furthermore, a complete removal of the dinitrosyl iron complex from ferredoxin prevents reassembly of the {2Fe-2S} cluster in the protein . The results suggest that cysteine desulfurase (IscS) together with L-cysteine can efficiently repair the nitric oxide-modified ferredoxin {2Fe-2S} cluster and that the iron center in the dinitrosyl iron complex may be recycled for the reassembly of iron-sulfur clusters in proteins.

J Biol Chem, 2002 Apr 19, 277(16), 14206 - 10 Epub 2002 Feb 01.
Tandem function of nucleotide binding domains confers competence to sulfonylurea receptor in gating ATP-sensitive K+ channels; Zingman LV et al.; Fundamental to the metabolic sensor function of ATP-sensitive K(+) (K(ATP)) channels is the sulfonylurea receptor . This ATP-binding cassette protein, which contains nucleotide binding domains (NBD1 and NBD2) with conserved Walker motifs, regulates the ATP sensitivity of the pore-forming Kir6.2 subunit . Although NBD2 hydrolyzes ATP, a property essential in K(ATP) channel gating, the role of NBD1, which has limited catalytic activity, if at all, remains less understood . Here, we provide functional evidence that cooperative interaction, rather than the independent contribution of each NBD, is critical for K(ATP) channel regulation . Gating of cardiac K(ATP) channels by distinct conformations in the NBD2 ATPase cycle, induced by gamma-phosphate analogs, was disrupted by point mutation not only of the Walker motif in NBD2 but also in NBD1 . Cooling membrane patches to decelerate the intrinsic ATPase activity counteracted ATP-induced K(ATP) channel inhibition, an effect that mimicked stabilization of the MgADP-bound posthydrolytic state at NBD2 by the gamma-phosphate analog orthovanadate . Temperature-induced channel activation was abolished by mutations that either prevent stabilization of MgADP at NBD2 or ATP at NBD1 . These findings provide a paradigm of K(ATP) channel gating based on integration of both NBDs into a functional unit within the multimeric channel complex.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 191 - 8
Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II; Fujimoto K et al.; Sepiapterin reductase (SPR) catalyzes the last step in the pathway of tetrahydrobiopterin biosynthesis in tissues . SPR is phosphorylated by Ca2+-dependent protein kinases, which indicates that Ca2+-activated protein kinases may play a role in the regulation of SPR in vivo . Phosphorylation sites of rat sepiapterin reductase (rSPR) by Ca2+/calmodulin-dependent protein kinase II were determined in the present study . Using specific monoclonal anti-phospho-Ser and -Thr antibodies, we found that only Ser residues of rSPR were phosphorylated . We constructed several point mutants of SPR by systematically replacing the three Ser residues by Ala ones . These mutants showed that all three Ser residues, i.e . S46, S196, and S214, of rSPR were phosphorylated . We also recognized that only Ser-213 of human SPR was phosphorylated . Each of these serine residues in SPR was found in the consensus sequence (Arg-X-X-Ser/Thr) of the phosphorylation site.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 160 - 7
Chorismate lyase: kinetics and engineering for stability; Holden MJ et al.; By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway . We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB) . Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity . CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods . The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient . We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site . The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 127 - 35
Cloning, expression and characterisation of a human Nudix hydrolase specific for adenosine 5'-diphosphoribose (ADP-ribose); Lin S et al.; The human NUDT9 gene has been mapped to 4q22 and shown to give rise to two alternatively spliced mRNAs, NUDT9alpha and NUDT9beta, that encode a member of the Nudix hydrolase family . Both transcripts were readily detected in heart and skeletal muscle and also in liver, kidney and pancreas . NUDT9alpha protein was expressed in Escherichia coli and shown specifically to hydrolyse ADP-ribose and IDP-ribose to the corresponding nucleoside 5'-monophosphates and ribose 5-phosphate . No other nucleotide substrates were hydrolysed significantly . NUDT9alpha was inhibited by fluoride and by N-acetyl-p-benzoquinoneimine and had K(m) and kcat values of 180 microM and 8 s(-1) respectively with ADP-ribose as substrate . The full-length 39.1 kDa NUDT9alpha has a potential mitochondrial leader sequence, which would give rise to a mature 34.2 kDa mitochondrial protein . Apart from the high K(m) value, the properties of NUDT9alpha are close to those of the known mammalian 40 kDa cytoplasmic ADPRibase-1 and 35 kDa mitochondrial ADPRibase-m . However, any relationship between the NUDT9 species and the previously reported ADPRibases remains to be established.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 74 - 83
A site-directed mutagenesis analysis of tNOX functional domains; Chueh PJ et al.; Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min . A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min . The tNOX cDNA has been cloned and expressed . These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface . In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length . Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains . The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity . The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive . The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min . The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 64 - 73
Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis; Pan JJ et al.; Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone . Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity . Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton {12} . In order to understand the mechanism of enzyme activation for E . coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s(-1)) was 100 times larger than the steady-state rate (0.02 s(-1)) . The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s(-1)) but insufficient to cause full activation (100-fold) . A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 17 - 26
Temperature-dependent binding of monoclonal antibodies to C hordein; Brett GM et al.; The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614 . The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA) . The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding . Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide . Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations . Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures . It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.

Biochim Biophys Acta, 2002 Jan 31, 1594(1), 6 - 16
Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase; Kelley-Loughnane N et al.; Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50% . Contradictory kinetic properties of the partially purified E . coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate . In this investigation, a new purification for E . coli FBPase has been devised yielding enzyme with purity levels as high as 98% . This highly purified E . coli FBPase was characterized and the data compared to that for the pig kidney enzyme . Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme . The kcat of the E . coli FBPase was 14.6 s(-1) as compared to 21 s(-1) for the pig kidney enzyme, while the K(m) of the E . coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme . The concentration of Mg2+ required to bring E . coli FBPase to half maximal activity was estimated to be 0.62 mM Mg2+, which is twice that required for the pig kidney enzyme . Unlike the pig kidney enzyme, the Mg2+ activation of the E . coli FBPase is not cooperative . AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E . coli FBPase displays no cooperativity . Although cooperativity is not observed, the E . coli and pig kidney enzymes show similar AMP affinity . The quaternary structure of the E . coli enzyme is tetrameric, although higher molecular mass aggregates were also observed . The homology model of the E . coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme . The homology model of the E . coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.

Zhonghua Wai Ke Za Zhi, 1998 Dec, 36(12), 759 - 62
{Dietary fiber protects intestinal structure and barrier function}; Deng G et al.; OBJECTIVE: To evaluate the effect of dietary fiber on intestinal structure and barrier function of 5-fluorouracil (5-Fu) challenged rat . METHOD: Thirty Wistar rats were divided randomly into three groups (10 each group): Chow, enteric nutrition (EN), and EN + Fiber group . Both EN and EN + Fiber group were isonitrogenic and isocaloric . The rats kept their diet respectively for 8 days . 5-Fu was injected intraperitoneally on day 4 postoperatively . Intestinal permeability (L/M) was measured respectively on day 3 and day 7 . On day 8, the bacterial translation, wet weight and mucous thickness of both small intestine and colon and the villus height of small intestine were measured . The rats were weighted before and after experiment respectively . RESULT: The body weight loss of the EN + Fiber group (-3.1 +/- 3.4 g) was less than that of the EN group (-6.6 +/- 5.2 g) (P < 0.05), whereas the Chow group gained body weight (4.9 +/- 4.3 g) (P < 0.01 when compared with EN and EN + Fiber group) . The parameters of intestinal structures of the EN + Fiber group was superior to the EN group (P < 0.05) . The L/M of both EN + Fiber (from 0.0265 +/- 0.0073 to 0.0274 +/- 0.0068) and the Chow group (from 0.0268 +/- 0.0039 to 0.0281 +/- 0.0044) was unchanged (P > 0.05 for both) after 5-Fu challenged, whereas that of the EN group increased (from 0.0289 +/- 0.0070 to 0.0331 +/- 0.0084) (P < 0.01) . The incidence of bacterial translocation to MLN of both EN + Fiber group and the Chow group (20%) was lower than that of the EN group (70%) (P < 0.05) . CONCLUSION: The dietary fiber protects the intestinal structure and barrier function of 5-Fu challenged rats.

Inorg Chem, 2002 Feb 11, 41(3), 521 - 31
Sterically hindered carboxylate ligands support water-bridged dimetallic centers that model features of metallohydrolase active sites; Lee D et al.; The synthesis and characterization of carboxylate-bridged dimetallic complexes are described . By using m-terphenyl-derived carboxylate ligands, a series of dicobalt(II), dicobalt(III), dinickel(II), and dizinc(II) complexes were synthesized . The compounds are {Co(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)} (1), {Co(2)(mu-OH(2))(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)} (2a-c), {Co(2)(mu-OH)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)} (3), {Ni(2)(mu-O(2)CAr(Tol))(4)L(2)} (4), {Ni(2)(mu-HO...H)(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)} (5), and {Zn(2)(mu-O(2)CAr(Tol))(2)(O(2)CAr(Tol))(2)L(2)} (6), where Ar(Tol)CO(2)H = 2,6-di(p-tolyl)benzoic acid and L = pyridine, THF, or N,N-dibenzylethylenediamine . Structural analysis of these complexes revealed that additional bridging ligands can be readily accommodated within the {M(2)(mu-O(2)CAr(Tol))(2)}(2+) core, allowing a wide distribution of M...M distances from 2.5745(6) to 4.0169(9) A . Unprecedented bridging units {M(2)(mu-OH(2))(2)(mu-O(2)CR)(2)}(n+) and {M(2)(mu-HO...H)(2)(mu-O(2)CR)(2)}(n+) were identified in 2a-c and 5, respectively, in which strong hydrogen bonding accommodates shifts of protons from bridging water molecules toward the dangling oxygen atoms of terminal monodentate carboxylate groups . Such a proton shift along the O...H...O coordinate attenuates the donor ability of the anionic carboxylate ligand, which can translate into increased Lewis acidity at the metal centers . Such double activation of bridging water molecules by a Lewis acidic metal center and a metal-bound general base may facilitate the reactivity of metallohydrolases such as methionine aminopeptidase (MAP).

Biol Pharm Bull, 2002 Jan, 25(1), 115 - 7
Expression of a synthetic gene encoding the Asp-hemolysin from Aspergillus fumigatus in Escherichia coli; Kumagai T et al.; Asp-hemolysin is a hemolytic toxin from Aspergillus fumigatus and is a specific binding protein with high affinity for oxidized low density lipoprotein (Ox-LDL) . As a first step in clarifying the structure-function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli (E . coli) as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography on an amylose resin . The apparent molecular size of the protein produced by E . coli was approximately 57 kDa, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . This is consistent with the predicted molecular size of 56.9 kDa for a fusion protein which includes 14.2 kDa of Asp-hemolysin and 42.7 kDa from MBP . The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody as revealed by western blot analysis . Furthermore, in dot blot analysis, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin.

Endothelium, 2001, 8(4), 261 - 8
Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells; Ohmi K et al.; Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome . The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder . Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs) . The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1 . However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1 . NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs . The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

Science, 2002 Feb 1, 295(5556), 855 - 7
A role for interaction of the RNA polymerase flap domain with the sigma subunit in promoter recognition; Kuznedelov K et al.; In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme . The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme . We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit . The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element . Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.

Science, 2002 Feb 1, 295(5556), 851 - 5
Role of Escherichia coli curli operons in directing amyloid fiber formation; Chapman MR et al.; Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases . Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid . The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli . In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively . Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.

J Biol Chem, 2002 Apr 12, 277(15), 12861 - 7 Epub 2002 Jan 31.
Dye-linked D-proline dehydrogenase from hyperthermophilic archaeon Pyrobaculum islandicum is a novel FAD-dependent amino acid dehydrogenase; Satomura T et al.; The activity of dye-linked d-proline dehydrogenase was found in the crude extract of a hyperthermophilic archaeon, Pyrobaculum islandicum JCM 9189 . The dye-linked d-proline dehydrogenase was a membrane associated enzyme and was solubilized from the membrane fractions by treatment with Tween 20 . The solubilized enzyme was purified 34-fold in the presence of 0.1% Tween 20 by four sequential chromatographies . The enzyme has a molecular mass of about 145 kDa and consisted of homotetrameric subunits with a molecular mass of about 42 kDa . The N-terminal amino acid sequence of the subunit was MKVAIVGGGIIGLFTAYHLRQQGADVVI . The enzyme retained its full activity both after incubation at 80 degrees C for 10 min and after incubation in the range of pH 4.0-10.0 at 50 degrees C for 10 min . The enzyme-catalyzed dehydrogenation of several d-amino acids was carried out using 2,6-dichloroindophenol as an electron acceptor, and d-proline was the most preferred substrate among the d-amino acids . The Michaelis constants for d-proline and 2,6-dichloroindophenol were determined to be 4.2 and 0.14 mm, respectively . Delta(1)-Pyrroline-2-carboxylate was identified as the reaction product from d-proline by thin layer chromatography . The prosthetic group of the enzyme was identified to be FAD by high-performance liquid chromatography . The gene encoding the enzyme was cloned and expressed in Escherichia coli . The nucleotide sequence of the dye-linked d-proline dehydrogenase gene was determined and encoded a peptide of 363 amino acids with a calculated molecular weight of 40,341 . The amino acid sequence of the Pb . islandicum enzyme showed the highest similarity (38%) with that of the probable oxidoreductase in Sulfolobus solfataricus, but low similarity with those of d-alanine dehydrogenases from the mesophiles so far reported . This shows that the membrane-bound d-proline dehydrogenase from Pb . islandicum is a novel FAD-dependent amino acid dehydrogenase.

J Biol Chem, 2002 Apr 12, 277(15), 12572 - 8 Epub 2002 Jan 31.
A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase; Jung BG et al.; A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library . PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)) . The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor . In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides . Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx . Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx . Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).

EMBO J, 2002 Feb 1, 21(3), 387 - 97
Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins; Lutzmann M et al.; Now that it is likely that all yeast nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its approximately 30 individual components . Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex . We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five nucleoporins in a single Escherichia coli cell . During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex . Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an approximately 40 nm long stalk . Taken together, our data document that the Nup84p-Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.

Appl Environ Microbiol, 2002 Feb, 68(2), 981 - 4
Effect of oxidizing disinfectants (chlorine, monochloramine, and ozone) on Helicobacter pylori; Baker KH et al.; The susceptibility of Helicobacter pylori to disinfectants was compared to that of Escherichia coli . H . pylori is more resistant than E . coli to chlorine and ozone but not monochloramine . H . pylori may be able to tolerate disinfectants in distribution systems and, therefore, may be transmitted by a waterborne route.

Appl Environ Microbiol, 2002 Feb, 68(2), 464 - 9
FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli; Ideno A et al.; The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii . We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli . The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C . Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro . In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C . When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E . coli, whose expressed product tended to form an inactive aggregate in E . coli, it improved the yield of the soluble Fab fragments with antibody specificity . PhFKBP29 exerted protein folding and aggregation suppression in E . coli cells.

Exp Hematol, 2002 Feb, 30(2), 108 - 15
Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy; Bagnis C et al.; OBJECTIVE: The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene . The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas . PATIENTS AND METHODS: Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy . CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E . coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector . Cells were reinfused to the patients after myeloablation, without prior ex vivo selection . RESULTS: Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion . One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft . Patients experienced side effects that are often observed after high-dose chemotherapy . CONCLUSIONS: Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study . Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.

Parasitol Res, 2002 Jan, 88(1), 69 - 72
Potential of beta-galactosidase-expressing Toxoplasma gondii for in situ localization and observation of rare stages of the parasite life cycle; Dao A et al.; A cyst-forming strain of Toxoplasma gondii was transfected with the Escherichia coli LacZ gene and expressed beta-galactosidase constitutively . This strain has been used to localize and analyze the early stages of development and reactivation of T . gondii in mice . The chromogenic detection of the enzyme allows an easy detection of the parasites after light fixation and therefore allows a submacroscopic analysis of tissue distribution within the organism . Also, it allows further embedding and retrieval of rare stages for electron microscopic observation . that detect the presence of the parasite and initiate the response, and (2) the early stages of reactivation, when the cysts are supposed to break open and release the infectious bradyzoites . We have taken advantage of the possibility of detecting the enzymatic activity of beta-galactosidase (beta-gal) in transfected parasites to show that one could perform a semi-macroscopic detection and that this was compatible with further analysis by histological or electron microscopic techniques, being therefore able to detect the rare events and then to analyze them further with more refined morphological techniques.

Curr Microbiol, 2002 Mar, 44(3), 224 - 8
MutS2 family protein from Pyrococcus furiosus; Vijayvargia R et al.; MutS2 protein of Pyrococcus furiosus has been cloned and over-expressed . Initial characterization reveals that PfuMutS2 possesses a thermostable ATPase activity and a thermostable, nonspecific DNA binding activity . However, PfuMutS2 does not have any detectable mismatch-specific DNA binding activity . It is the first in vitro characterization of an MutS2 family protein.

Curr Microbiol, 2002 Mar, 44(3), 178 - 83
Relationship between the persistence of mer operon sequences in Escherichia coli and their resistance to mercury; Murtaza I et al.; Studies related to geographic distribution of E . coli carrying mer operon sequences were carried out on the Indian subcontinent . Out of the 80 E . coli isolates, collected from five geographically distinct regions of India, 68 were found to be resistant to one or the other heavy metal used in the study . Among these isolates, 36 were found to be resistant to the inorganic form (HgCl2) and only 5 to resist both the inorganic and organic forms of mercury . Colony hybridization studies revealed 35 isolates out of 68 to hybridize with the probe . Interestingly, some of the mercury-sensitive isolates (Hgs), especially from the Dal Lake, were found positive in hybridization studies . These findings, supported by mercury volatilization studies, indicate the presence of nonfunctional/vestigial mer sequences in the isolates collected from different environments . On the other hand, few of the mercury-resistant isolates (Hgr) from the Yamuna River did not show any sign of hybridization . Further, volatilization studies also indicated an alternate mode of resistance mechanism operating in them . The studies demonstrate that the mer operon sequences share very high homology among the E . coli isolates collected from different geographical locations, and this metal resistance may be a genetic character that arose from a common ancestral background.

Nat Biotechnol, 2002 Feb, 20(2), 177 - 82
An unnatural base pair for incorporating amino acid analogs into proteins; Hirao I et al.; An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code . The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase . This transcription was coupled with translation in an Escherichia coli cell-free system . The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine . Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products . This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

J Biol Chem, 2002 Apr 12, 277(15), 12718 - 23 Epub 2002 Jan 30.
Targeting, insertion, and localization of Escherichia coli YidC; Urbanus ML et al.; YidC was recently shown to play an important role in the assembly of inner membrane proteins (IMPs) both in conjunction with and separate from the Sec-translocon . Little is known about the biogenesis and structural and functional properties of YidC, itself a polytopic IMP . Here we analyze the targeting and membrane integration of YidC using in vivo and in vitro approaches . The combined data indicate that YidC is targeted by the signal recognition particle and inserts at the SecAYEG-YidC translocon early during biogenesis, unlike its mitochondrial homologue Oxa1p . In addition, YidC is shown to be relatively abundant compared with other components involved in IMP assembly and is predominantly localized at the poles of the cell.

J Biol Chem, 2002 Apr 5, 277(14), 12118 - 27 Epub 2002 Jan 30.
Physical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sulfolobus solfataricus; Carpentieri F et al.; Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya . Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM) was shown to possess a robust ATP-dependent 3'-5' DNA helicase activity in vitro . Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM) . Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers . The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5'-tail and is stimulated by the single-stranded DNA binding protein from S . solfataricus (Sso SSB) . In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein.protein complexes . These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways.

J Biol Chem, 2002 Apr 5, 277(14), 12089 - 98 Epub 2002 Jan 30.
Presteady-state analysis of avian sarcoma virus integrase . I . A splicing activity and structure-function implications for cognate site recognition; Bao KK et al.; Integrase catalyzes insertion of a retroviral genome into the host chromosome . After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner . In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends . This splicing reaction showed an initial exponential phase (k(splicing) = 0.02 s(-1)) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred . With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products . Furthermore, the splicing reaction allowed characterization of substrate binding and specificity . Although integrase requires only a 3' hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile . These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.

J Biol Chem, 2002 Apr 5, 277(14), 11756 - 64 Epub 2002 Jan 30.
Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts; Odersky A et al.; In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair . Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells . The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes . We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB . The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site . We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence . The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.

Diagn Microbiol Infect Dis, 2002 Jan, 42(1), 1 - 8
Evaluation of the VTEC-Screen "Seiken" test for detection of different types of Shiga toxin (verotoxin)-producing Escherichia coli (STEC) in human stool samples; Beutin L et al.; An immunoassay for in vitro detection of Shiga (Vero) toxins Stx1 and Stx2 (VTEC-Screen "Seiken") was compared with the verocell toxicity test (VCA) and an stx-gene specific PCR for detection of Shiga toxin-producing E . coli (STEC) from 234 human stool samples selectively enriched on sorbitol-MacConkey (SMAC) agar . Culturable STEC were isolated from 59 (25.2%) of the 234 stool specimens and were found to be distributed over 20 different O-serogroups . Fifty-three (89.8%) of the 59 STEC-positive samples were identified with the VTEC-Screen compared to 55 (93.2%) with the PCR and 58 (98.3%) with the VCA . A possible false positive reaction with the VTEC-Screen was obtained with one sample and five samples showed aspecific reactions with both the test- and the control latex . The VTEC-Screen detected all samples which contained Stx1 producing strains (77.9% of STEC-positive samples) but was negative with six samples (10.2%) which contained Stx2 and/or Stx2 variant producers, although secondary enrichment of on brain-heart infusion agar detected three of these to improve the detection rate to 94.9% . Examination of reference strains encoding different genotypes of stx(1) and stx(2) indicated that certain variants of Stx2 reacted poorly (Stx2d-Ount, Stx2e and Stx2ev) or not at all with the VTEC-Screen . Overall, however, the test was found to be accurate, rapid and easy to perform, thus being suitable for the routine screening of clinical stool specimens for STEC.

FEBS Lett, 2002 Jan 30, 511(1-3), 118 - 22
Carbonyl formation on a copper-bound prion protein fragment, PrP23-98, associated with its dopamine oxidase activity; Shiraishi N et al.; The amino-terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion . This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline . In this study, we have investigated copper-catalyzed oxidation of an N-terminal part of human PrP, PrP23-98, that was prepared by the recombinant DNA technique . Carbonyl formations on copper-bound PrP23-98 induced by dopamine and L-ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23-98-bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.

FEBS Lett, 2002 Jan 30, 511(1-3), 97 - 101
Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production; Revilla-Nuin B et al.; N-Acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) are the essential precursors of N-acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant . Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate-dependent phosphotransferase system that shows a broad range of specificity . Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino-acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively . The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro . These findings account for the low in vivo PA production observed when E . coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

FEBS Lett, 2002 Jan 30, 511(1-3), 51 - 8
Components required for membrane assembly of newly synthesized K+ channel KcsA; van Dalen A et al.; An Escherichia coli in vitro transcription-translation system was used to study the components involved in the biogenesis of the homotetrameric potassium channel KcsA . We show that a functional signal recognition particle pathway is essential for tetramer formation, probably to direct correct monomer insertion in the membrane . In the absence of YidC or at reduced SecYEG levels, KcsA assembly occurs with lower efficiency . Strikingly, the highest efficiency of tetramerization was observed when transcription-translation was carried out in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal membrane requirement to form the KcsA tetramer . It is concluded that SecYEG and YidC are not required for the formation of tetrameric KcsA in vitro.

FEBS Lett, 2002 Jan 30, 511(1-3), 6 - 10
Mechanism of oligomerization of Escherichia coli carbamoyl phosphate synthetase and modulation by the allosteric effectors . A site-directed mutagenesis study; Mora P et al.; We use site-directed mutagenesis to clarify the role of effector-mediated oligomerization changes on the modulation of the activity of Escherichia coli carbamoyl phosphate synthetase (CPS) by its allosteric activator ornithine and its inhibitor UMP . The regulatory domain mutations H975L, L990A and N992A abolished, and N987V decreased CPS oligomerization . The oligomerization domain mutation L421E prevented tetramer but not dimer formation . None of the mutations had drastic effects on enzyme activity or changed the sensitivity or apparent affinity of CPS for ornithine and UMP . Our findings exclude the involvement of oligomerization changes in the control of CPS activity, and show that CPS dimers are formed by the interactions across regulatory domains, and tetramers by the interactions of two dimers across the oligomerization domains . A mechanism for effector-mediated changes of the oligomerization state is proposed.

FEBS Lett, 2002 Jan 30, 511(1-3), 1 - 5
The role of 2-methyl-6-phytylbenzoquinone methyltransferase in determining tocopherol composition in Synechocystis sp . PCC6803; Shintani DK et al.; A putative 2-methyl-6-phytylbenzoquinone (MPBQ) methyltransferase gene, SLL0418, was identified from the Synechocystis PCC6803 genome based on its homology to previously characterized gamma-tocopherol methyltransferases . Genetic and biochemical evidence confirmed open reading frame (ORF) SLL0418 encodes a MPBQ methyltransferase . An SLL0418 partial knockout mutant accumulated beta-tocopherol with no effect in the overall tocopherol content of the cell . In vitro assays of the SLL0418 gene expressed in Escherichia coli showed the enzyme efficiently catalyzes methylation of ring carbon 3 of MPBQ . In addition, the enzyme also catalyzes the methylation of ring carbon 3 of 2-methyl-6-solanylbenzoquinol in the terminal step of plastoquinone biosynthesis.

J Biochem (Tokyo), 2002 Feb, 131(2), 241 - 6
Molecular cloning and expression of the mannose/glucose specific lectin from Castanea crenata cotyledons; Nakamura S et al.; cDNA clones encoding a mannose/glucose specific lectin, CCA, from Castanea crenata cotyledons have been isolated and sequenced . The cloned CCA cDNA had an open reading frame of 927 bp encoding 309 amino acid residues . Compared with the amino acid sequence determined for the protein chemically, it was clarified that CCA has no signal peptide and undergoes no proteolytic cleavage as do other mannose specific Jacalin-related lectins . The coding region of CCA was introduced into an expression vector, pET-22b(+), and then transferred into Escherichia coli BL21(DE3) . Although recombinant CCA (rCCA) accumulated as inclusion bodies, refolded rCCA exhibited a similar CD spectrum to nCCA and regained the hemagglutination activity . In addition, a hapten inhibition assay revealed that nCCA and rCCA showed the same specificities toward sugars and glycoproteins . On measurement by GPC-MALLS in the native state, the absolute molecular mass of nCCA was found to be 332 7 kDa, which indicated that nCCA is a decamer of identical subunits having a molecular mass of 33 kDa . The same as the natural molecule, rCCA showed a molecular mass of 320 +/- 5 kDa and was judged to also be a decamer . These results indicate that the rCCA obtained in this study is equivalent to nCCA.

J Biochem (Tokyo), 2002 Feb, 131(2), 225 - 31
Genomic clones encoding two isoforms of pokeweed antiviral protein in seeds (PAP-S1 and S2) and the N-glycosidase activities of their recombinant proteins on ribosomes and DNA in comparison with other isoforms; Honjo E et al.; Pokeweed antiviral proteins (PAPs) are single-chain ribosome-inactivating proteins (RIPs) isolated from several organs of Phytolacca americana (Pokeweed) that are characterized by their ability to depurinate not only ribosomes but also various nucleic acids . PAP-S is one of the isoforms found in seeds . In this study, we obtained three different genomic clones encoding two forms of PAP-S (here designated as PAP-S1 and PAP-S2) and alpha-PAP after PCR using a pair of degenerated primers based on the known N- and C-terminal amino acid sequences of PAP-S . The nucleotide sequences of the genomic clones contained no introns . The deduced amino acid sequences of PAP-S1 and PAP-S2, which showed 83% identity to each other, were found to correspond to sequences reported independently for PAP-S protein and cDNA, respectively, demonstrating that at least two forms of PAP-S actually exist in seeds of the same plant . The recombinant PAP-S1, PAP-S2, alpha-PAP, and PAP I (a form appearing in spring leaves) exhibit the same level of depurinating activity on rat ribosomes, while their efficiencies on Escherichia coli ribosomes and salmon sperm DNA differ substantially from one another in the order of PAP I > alpha-PAP > PAP-S1 > PAP-S2 and alpha-PAP > PAP I > PAP-S1 > PAP-S2 . Structural comparisons suggest that the large difference in ribosome recognition between PAP-S1 (or S2) and PAP I is caused by the alteration of residues adjacent to the adenine-binding site.

J Biochem (Tokyo), 2002 Feb, 131(2), 219 - 24
Phospholipids promote dissociation of ADP from the Mycobacterium avium DnaA protein; Yamamoto K et al.; The biochemical aspects of the initiation of DNA replication in Mycobacterium avium are unknown . As a first step towards understanding this process, M . avium DnaA protein, the counterpart of Escherichia coli replication initiator protein, was overproduced in E . coli with an N-terminal histidine tag and purified to homogeneity on a nickel affinity column . The recombinant DnaA protein bound both ATP and ADP with high affinity and showed a weak ATPase activity . ADP, following the hydrolysis of ATP, remained bound to the protein strongly and the exchange of ATP for bound ADP was found to be weak . Acidic phospholipids such as phosphatidylinositol, phosphatidylglycerol, and cardiolipin, promoted the dissociation of ADP from the DnaA protein, whereas the neutral phospholipid, phosphatidylethanolamine, did not . The phospholipid promoted dissociation of ADP from DnaA protein was stimulated in the presence of the M . avium origin of replication . We suggest that the initiation of DNA replication in M . avium involves an interplay among DnaA, adenine nucleotides and phospholipids.

Org Lett, 2002 Feb 7, 4(3), 355 - 7
Molecular design and biological potential of galacto-type trehalose as a nonnatural ligand of shiga toxins; Dohi H et al.; Galacto-type trehalose, a "C-4 epimer of trehalose", possesses a stereochemical structure around the alpha(1-1)-linkage analogous to that of the globobiosyl alpha(1-4)-linkage in Gb(2) and Gb(3) ceramides, which are known as the ligands of Shiga toxins produced by pathogenic E . coli . This paper presents evidence supporting the new idea of using a trehalosyl alpha(1-1)-linkage as a substitute for the galactobiosyl alpha(1-4)-linkage.

J Chromatogr A, 2002 Jan 11, 943(1), 77 - 90
Impact of the physical and topographical characteristics of adsorbent solid-phases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates; Thwaites E et al.; A comparison is made of the performance of two types of adsorbent solid phases (commercially sourced Streamline composites and custom-assembled Zirblast pelliculates), derivatised with similar anion exchange chemistries and applied to the recovery of plasmid DNA from Escherichia coli extracts prepared by chemical lysis and coarse filtration . Streamline and Zirblast adsorbents were characterised by average particle diameters of 200 and 95 microm, densities of 1.16 and 3.85 g/m2, and small ion capacities of 170 and 8 micromol/ml settled adsorbent, respectively . Detailed analysis of products and impurities in a full operational cycle of adsorption, washing, pre-elution, elution and regeneration processes was enabled by the harnessing of a battery of analyses for nucleic acid and organic solute content of feedstocks and bed effluents exploiting ultra-violet spectrophotometry, agarose gel electrophoresis and specific reactions with the fluorescent probe PicoGreen . In comparative tests operated under near identical conditions, Streamline and Zirblast adsorbents exhibited plasmid recoveries of 76 and 90% of bound product characterised by purity ratios (relative PicoGreen and A254 estimates of mass) of 9 and 32, respectively . Conclusions are drawn regarding the specific impact of the physical and topographical characteristics of solid-phase geometry upon product throughput, achievable product purity, process time-scales and operational economics for the manufacture of plasmid DNA.

Nat Genet, 2002 Feb, 30(2), 227 - 32 Epub 2002 Jan 30.
Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors; Al-Tassan N et al.; Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A . We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis . Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis . Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp . These mutations affect residues that are conserved in mutY of E . coli (Tyr82 and Gly253) . Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity . Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly . Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1467 - 72 Epub 2002 Jan 29.
Cho, a second endonuclease involved in Escherichia coli nucleotide excision repair; Moolenaar GF et al.; Nucleotide excision repair removes damages from the DNA by incising the damaged strand on the 3' and 5' sides of the lesion . In Escherichia coli, the two incisions are made by the UvrC protein, which consists of two functional halves . The N-terminal half contains the catalytic site for 3' incision and the C-terminal half contains the residues involved in 5' incision . The genome of E . coli contains an SOS-inducible gene (ydjQ) encoding a protein that is homologous to the N-terminal half of UvrC . In this paper we show that this protein, which we refer to as Cho (UvrC homologue), can incise the DNA at the 3' side of a lesion during nucleotide excision repair . The incision site of Cho is located 4 nt further away from the damage compared with the 3' incision site of UvrC . Cho and UvrC bind to different domains of UvrB, which is probably the reason of the shift in incision position . Some damaged substrates that are poorly incised by UvrC are very efficiently incised by Cho . We propose that E . coli uses Cho for repair of such damages in vivo . Initially, most of the lesions in the cell will be repaired by the action of UvrC alone . Remaining damages, that for structural reasons obstruct the 3' incision by UvrC, will be repaired by the combined action of Cho (for 3' incision) and UvrC (for 5' incision).

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1212 - 7 Epub 2002 Jan 29.
Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP; Maita N et al.; In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin . The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers . Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller . Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins . The complex structure suggests that phenylalanine-induced GTPCHI x GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state.

Proc Natl Acad Sci U S A, 2002 Feb 5, 99(3), 1176 - 81 Epub 2002 Jan 29.
The mechanism of catalysis of the chorismate to prephenate reaction by the Escherichia coli mutase enzyme; Hur S et al.; Molecular dynamics studies of the Escherichia coli chorismate mutase (EcCM), containing at the active site chorismate and in turn the transition state (TS), have been performed . The simulations show that TS is not bound any tighter than chorismate . Comparison of average polar interactions show they are virtually identical for interactions of EcCM with chorismate and the TS, whereas hydrophobic interactions with TS are much weaker than with chorismate . Interactions and the mechanism of catalysis of chorismate --> prephenate by the EcCM enzyme are discussed.

Am J Respir Crit Care Med, 2002 Feb 1, 165(3), 372 - 7
Activation of poly(ADP-Ribose) polymerase-1 is a central mechanism of lipopolysaccharide-induced acute lung inflammation; Liaudet L et al.; Recent studies demonstrated that activation of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) by oxidant-mediated DNA damage is an important pathway of tissue injury in conditions associated with oxidative stress . Using a dual approach of PARP-1 suppression, by genetic deletion or pharmacological inhibition with the phenanthridinone PARP inhibitor PJ-34, we now demonstrate an essential role of PARP-1 in the development of pulmonary inflammation induced by lipopolysaccharide (LPS) . PARP-1+/+ and PARP-1-/- mice received an intratracheal instillation of LPS (50 microg), followed after 24 h by bronchoalveolar lavage to measure the cytokines TNF-alpha, IL-1beta, and IL-6, the chemokines MIP-1alpha and MIP-2, leukocyte counts and myeloperoxidase activity (neutrophil accumulation), protein content (high permeability edema), and nitrite/ nitrate (nitric oxide production) . Malondialdehyde (an index of lipid peroxidation) was measured in lung tissue . Similar experiments were conducted in BALB/c mice treated with PJ-34 or vehicle . The absence of functional PARP-1 reduced LPS-induced increases of cytokines and chemokines, alveolar neutrophil accumulation, lung hyperpermeability, NO production, and lipid peroxidation . Histological analysis revealed attenuated lung damage after PARP inhibition . Our findings support a mechanistic role of PARP-1 in the regulation of LPS-induced lung inflammation . Pharmacological inhibition of PARP may be useful in clinical conditions associated with overwhelming lung inflammation.

Psychoneuroendocrinology, 2002 Apr, 27(3), 353 - 65
Prior stressor exposure primes the HPA axis; Johnson JD et al.; Exposure to stressors often alters the subsequent responsiveness of many systems . The present study tested whether prior exposure to inescapable tailshock (IS) alters the corticosterone (CORT) or adrenocorticotropin hormone (ACTH) response to either an injection of bacterial endotoxin (lipopolysaccharide; LPS) or subsequent placement on a pedestal . Rats were exposed to IS or remained as home cage controls (HCC) . 1, 4, 10, or 21 days later animals were injected i.p . with either 10 microg/kg LPS or equivolume sterile saline . Prior IS significantly increased plasma CORT 1 h, but not 2 or 5 h after LPS, compared to controls 1, 4, and 10 days, but not 21 days after IS . Exposure to IS 24 h earlier also significantly increased plasma ACTH 1 h after LPS . Additional animals were placed on a pedestal 24 h after IS, and plasma CORT was measured 15, 30, and 60 min later . IS significantly increased plasma CORT 15 min after pedestal exposure, but not after 30 or 60 min . These results suggest that exposure to IS sensitizes the CORT and ACTH response to subsequent HPA activation.

Indian J Gastroenterol, 2001 Nov-Dec, 20(6), 246 - 7
Ruptured splenic abscess presenting as pneumoperitoneum; Rege SA et al.; Spontaneous pneumoperitoneum follows perforation of hollow viscus; rarely, it may arise from pulmonary interstitial emphysema or intestinal inflammatory disease . We report a 30-year-old man with ruptured splenic abscess who presented with acute abdomen and had pneumoperitoneum . He was treated with splenectomy and is asymptomatic 2 months later.

Genes Genet Syst, 2001 Oct, 76(5), 327 - 34
Isolation and characterization of a cDNA from soybean and its homolog from Escherichia coli, which both complement the light sensitivity of Escherichia coli hemH mutant strain VS101; Kanjo N et al.; Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated XWH-1) from a X phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101 . In VS101 bacteria lysogenized with lambdaWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria . On the other hand, in the wild-type E . coli strains lysogenized with lambdaWH-1, significant accumulation of delta-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed . The growth of the wild-type bacteria in which the insert cDNA from deltaWH-1 had been introduced via a plasmid vector was markedly inhibited . By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway . ORF296 showed about 30% identity with the E . coli hypothetical protein YicL . By cloning and examining the gene for YicL in E . coli, we found that YicL shows the same effect as that of the soybean cDNA . From these findings, we concluded that the clone from soybean and yicL from E . coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB) . Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.

Antonie Van Leeuwenhoek, 2001 Sep, 79(3-4), 277 - 84
Putative lmbI and lmbH genes form a single lmbIH ORF in Streptomyces lincolnensis type strain ATCC 25466; Janata J et al.; The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al . 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified . Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e . conversion of N-demethyllincomycin (NDL) to lincomycin . In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes . The product, LmbIH, is a protein of an unknown function and is homologous with the T1dD protein family . Escherichia coli T1dD protein was previously shown to be involved in the control of DNA gyrase by LetD protein . Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression . This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

Mol Gen Mikrobiol Virusol, 2001, (4), 18 - 22
{The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product}; Dobrynina OIu et al.; Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12 . A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes . FruB protein possessing a pseudo-HPr activity replaces the HPr . It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid . The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated . The fruS mutations were located in the fru operon . Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose . Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated . They were mapped using the P1vir transduction . The fruS mutations were found in the structural gene of the fructose operon . Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.

Int Arch Allergy Immunol, 2001 Dec, 126(4), 286 - 93
cDNA cloning and expression of Blo t 11, the Blomia tropicalis allergen homologous to paramyosin; Ramos JD et al.; Blomia tropicalis is an important mite species in many parts of the world and the most predominant mite species in tropical countries . The prevalence of sensitization to this species has probably been underestimated because commercial extracts are largely unavailable . Identification and characterization of B . tropicalis allergens is an important step toward understanding the role of this species in allergic sensitization and could provide appropriate reagents for diagnostic and therapeutic procedures . This paper describes the isolation, sequence analysis, expression and allergenicity of a cDNA gene coding for a B . tropicalis allergen with homology to paramyosin, a high-molecular-weight allergen previously identified in Dermatophagoides farinae . The full-length Blo t 11 cDNA gene was isolated by cDNA library screening, 5'-rapid amplification of cDNA ends and long-distance PCR . Sequence analysis was performed with a combination of CLUSTAL W, CGC and BLAST program packages . The cDNA gene was expressed as a GST fusion protein in Escherichia coli and purified by affinity chromatography using the glutathione Sepharose column . Allergenicity of the rBlo t 11 was tested by human IgE dot blot immunoassay . Blo t 11 is a 3,111-bp cDNA gene with a 2,625-bp open reading frame coding for an 875-amino acid protein, exhibiting significant homology with different invertebrate paramyosins . The human IgE dot blot immunoassay showed that the rBlo t 11 reacted positively to 52% (33/63) of sera from asthmatic patients . Blo t 11 is the homolog of Der f 11 exhibiting potentially important allergenic activity .

J Biol Chem, 2002 Apr 12, 277(15), 13175 - 83 Epub 2002 Jan 28.
Global expression profiling of acetate-grown Escherichia coli; Oh MK et al.; This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides . Glucose-grown cultures were used as a reference . At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were down-regulated compared with the glucose-grown culture . Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated . It appears that E . coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source . The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes . It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate . The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations . In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.

J Antimicrob Chemother, 2002 Feb, 49(2), 383 - 6
Nucleotide sequence and organization of plasmid pMVSCS1 from Mannheimia varigena: identification of a multiresistance gene cluster; Kehrenberg C et al.; OBJECTIVES: A small resistance plasmid of Mannheimia varigena was analysed with regard to its gene organization and the development of a multiresistance gene cluster . MATERIALS AND METHODS: The 5621 bp plasmid pMVSCS1 was transformed into Escherichia coli JM107, cloned and sequenced completely . RESULTS: Three intact resistance genes, sulII, catAIII and strA, a truncated strB gene and a novel replication gene were identified . A potential recombination site for the integration of catAIII in the spacer region between sulII and strA was identified . CONCLUSION: The physical linkage of the plasmid-borne resistance genes organized in a cluster would facilitate the spread of the different resistance genes by co-selection, even in the absence of a direct selective pressure.

Front Biosci, 2002 Feb 01, 7, a9 - 14
Protein denitration/modification by Escherichia coli nitrate reductase and mammalian cytochrome P-450 reductase; Kuo WN et al.; The incubation of peroxynitrite (PN)-pretreated histone III-S (NH) with Escherichia coli nitrate reductase (cytochrome, NADPH/GSH-independent) and that of NADPH-treated NH (NHNADPH) with liver cytochrome P-450 reductase (NADPH-dependent) resulted in decreased 3-nitrotyrosine immunoreactivity found in Western blot analysis . Additionally, increased nitrate was noted as an end product of these reactions . These findings imply that varied enzymatic denitration/modification of NO/PN-reacted protein, either with or without a reductant, may be important in regulating related signal transduction cascade(s) and relieving oxidative stress.

Bioorg Med Chem, 2002 Mar, 10(3), 545 - 50
Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase; Griffiths JS et al.; The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli . The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1) . During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature . A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C . The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates . Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist . A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.

Bioorg Med Chem, 2002 Mar, 10(3), 525 - 30
Synthesis and enzymatic evaluation of pyridinium-substituted uracil derivatives as novel inhibitors of thymidine phosphorylase; Murray PE et al.; A series of water soluble N(1)- and C(6)-substituted uracil pyridinium compounds were prepared as potential inhibitors of thymidine phosphorylase (TP) . The C(6)-uracil substituted derivatives were the most active . 1-{(5-Chloro-2,4-dihydroxypyrimidin-6-yl)methyl}pyridinium chloride, was identified as the best inhibitor being 5-fold more potent than the known inhibitor, 6-amino-5-bromouracil.

Gene, 2002 Jan 9, 282(1-2), 169 - 77
An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis; Lew BM et al.; Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide . The ability of inteins to promote protein splicing even when inserted into a foreign context has facilitated the study of the modulation of protein splicing . In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis . It involves the activation of the cytotoxic CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing is blocked . The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZ alpha-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by protein splicing . Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion protein . To induce large amounts of CcdB fusion proteins, the fusion protein is expressed from a high-copy-number plasmid . Such a screening system detects even low levels of protein splicing and we have used it to show that protein splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction . In order to search for protein splicing inhibitors, which may attenuate protein splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when splicing of the expressed CcdB fusion protein is only moderately suppressed . We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of protein splicing.

FEMS Microbiol Lett, 2002 Jan 10, 206(2), 169 - 74
Antiserum raised against gyrase A of Acholeplasma laidlawii reacts with phytoplasma proteins; Koui T et al.; Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector . The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli . The fusion protein was purified and used as an antigen for rabbit immunization . Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A . laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants . The antiserum could also detect phytoplasmas in infected plant sap . These results demonstrate that the gyrase A protein (GyrA) of A . laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.

Mol Biochem Parasitol, 2002 Feb, 119(2), 191 - 201
Molecular cloning, recombinant expression and partial characterization of the aspartate transcarbamoylase from Toxoplasma gondii; Mejias-Torres IA et al.; A cDNA coding for a monofunctional aspartate transcarbamoylase (ATCase) was isolated from a Toxoplasma gondii tachyzoite cDNA library using a complementation method . The calculated molecular mass of the deduced amino acid sequence was 46.8 kDa, with a predicted pI of 7.1 . Size exclusion chromatography/laser-light scattering showed a single, monodisperse peak with molecular mass of 144 kDa . Amino acid sequence alignments revealed that active site residues of the Escherichia coli ATCase catalytic chain were conserved in the T . gondii sequence, and the latter shared 26-33% overall sequence identity with other ATCases . A recombinant enzyme was overexpressed in E . coli, and was purified with a yield of approximately 0.8 mg l(-1) culture . The temperature dependence of the recombinant enzyme was similar to that of native ATCase in T . gondii extracts . The K(m)'s for aspartate and carbamoyl phosphate were 7.82 mM, and 67.6 microM, respectively . The V(max) was 23900 micromol h(-1) mg(-1) . Pyrimidine nucleotides had no significant effect on the enzyme's activity . N-phosphonoacetyl-L-aspartate (PALA) inhibited the enzyme with K(i)=0.38 microM . The T . gondii ATCases contained two additional sequences of approximately 24 residues each, which are not found in other ATCases . One of these sequences was susceptible to proteolysis by elastase.

Clin Chim Acta, 2002 Mar, 317(1-2), 159 - 69
Development of the diagnostic immunoassay to detect anti-PreS1(21-47aa) antibody--a marker suggesting the health improvement of hepatitis B patients; Wei J et al.; BACKGROUND: A new immunoassay has been developed for the detection of the anti-PreS1(21-47aa) antibody in sera of hepatitis B virus (HBV)-infected patients . Anti-PreS1(21-47aa) antibody involves virus neutralization and is a new marker for diagnosing acute and chronic B hepatitis . METHODS: The expression plasmids pGEXS I and pGEXS II, which expressed glutathione S-transferase (GST) fusion proteins containing a copy of PreS1(21-47aa) peptide and two orderly joined copies of PreS1(21-47aa) peptide, were constructed . The soluble expression products were purified by affinity chromatography . RESULTS: The two PreS1(21-47aa) fusion proteins were both successfully applied in the immunoassay based on biotin-protein A and streptavidin-HRP, and could detect the anti-PreS1(21-47aa) antibody with high sensitivity in sera from hepatitis B patients . The anti-PreS1(21-27aa) antibody was detected during the recovery phase of acute hepatitis B patients, but it was found only in few of the chronic carriers by the established conventional system . CONCLUSIONS: The follow-up study suggested that the presence of the anti-PreS1(21-27aa) antibody correlated well with the recovery of patients from hepatitis and the improvement in health.

Biochemistry, 2002 Feb 5, 41(5), 1654 - 62
Interactions between heme d and heme b595 in quinol oxidase bd from Escherichia coli: a photoselection study using femtosecond spectroscopy; Borisov VB et al.; Femtosecond spectroscopy was performed on CO-liganded (fully reduced and mixed-valence states) and O(2)-liganded quinol oxidase bd from Escherichia coli . Substantial polarization effects, unprecedented for optical studies of heme proteins, were observed in the CO photodissociation spectra, implying interactions between heme d (the chlorin ligand binding site) and the close-lying heme b(595) on the picosecond time scale; this general result is fully consistent with previous work {Vos, M . H., Borisov, V . B., Liebl, U., Martin, J.-L., and Konstantinov, A . A . (2000) Proc . Natl . Acad . Sci . U.S.A . 97, 1554-1559} . Analysis of the data obtained under isotropic and anisotropic polarization conditions and additional flash photolysis nanosecond experiments on a mutant of cytochrome bd mostly lacking heme b(595) allow to attribute the features in the well-known but unusual CO dissociation spectrum of cytochrome bd to individual heme d and heme b(595) transitions . This renders it possible to compare the spectra of CO dissociation from reduced and mixed-valence cytochrome bd under static conditions and on a picosecond time scale in much more detail than previously possible . CO binding/dissociation from heme d is shown to perturb ferrous heme b(595), causing induction/loss of an absorption band centered at 435 nm . In addition, the CO photodissociation-induced absorption changes at 50 ps reveal a bathochromic shift of ferrous heme b(595) relative to the static spectrum . No evidence for transient binding of CO to heme b(595) after dissociation from heme d is found in the picosecond time range . The yield of CO photodissociation from heme d on a time scale of < 15 ps is found to be diminished more than 3-fold when heme b(595) is oxidized rather than reduced . In contrast to other known heme proteins, molecular oxygen cannot be photodissociated from the mixed-valence cytochrome bd at all, indicating a unique structural and electronic configuration of the diheme active site in the enzyme.

Biochemistry, 2002 Feb 5, 41(5), 1520 - 8
Stopped-flow kinetic studies of the interaction between Escherichia coli Fpg protein and DNA substrates; Fedorova OS et al.; Formamidopyrimidine-DNA-glycosylase of Escherichia coli (Fpg protein) repairs oxidative DNA damage by removing formamidopyrimidine lesions and 8-oxoguanine residues from DNA . This enzyme possesses three types of activities resulting in the excision of oxidized residue from DNA: hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP-lyase), and delta-elimination . In our work, the kinetic mechanism for 8-oxoguanine excision from DNA substrate with Fpg protein has been determined from stopped-flow measurements of changes in the tryptophan fluorescence . The 12-nucleotide duplex d(CTCTC(oxo)GCCTTCC)*d(GGAAGGCGAGAG) containing the 8-oxoG nucleotide in the sixth position of one strand was used as the specific substrate . Four distinct phases in the time traces were detected . These four-phase transition changes in the Fpg protein fluorescence curves were analyzed by global fitting to determine the intrinsic rate constants . We propose that the first two phases represent the equilibrium steps . The first of them describes the bimolecular binding step and the second, formation of the apurinic site . The third, irreversible step is believed to describe the beta-elimination process . The fourth step reflects the delta-elimination and decomposition of complex between enzyme and the product of 8-oxoG nucleotide excision . The results obtained provide direct evidence of conformational transitions of the Fpg protein during the catalytic process . The significance of these results for the functioning of Fpg protein is discussed.

Biochemistry, 2002 Feb 5, 41(5), 1421 - 7
Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site; Szafranska AE et al.; The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA . During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme . The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His(6)) fusion protein in high yield . The His(6)-MCAT was purified to homogeneity using standard techniques, and kinetic analysis of the malonylation of S . coelicolorFAS holo-ACP, catalyzed by His(6)-MCAT, gave K(infinity) (M) values of 73 (ACP) and 60 microM (malonyl CoA) . A catalytic constant k (infinity) (M) of 450 s(-1) and specificity constants k (infinity) (M)/K (infinity) (M) of 6.2 (ACP) and 7.5 microM(-1) s(-1) (malonyl CoA) were measured . Malonyl transfer to the E . coli FAS holo-ACP, catalyzed by His(6)-MCAT, was less efficient (k (infinity) (M)/K (infinity) (M) was 10% of that of the S . coelicolor ACP) . Incubation of MCAT with the serine specific agent PMSF caused inhibition of malonyl transfer to FAS ACPs, and an S97A MCAT mutant was incapable of catalyzing malonyl transfer . Our results show that in the reaction with FAS holo-ACPs the S . coelicolor MCAT is very similar to the E . coli MCAT paradigm in terms of its kinetic mechanism and active site residues . These results indicate that no other active site nucleophile is involved in catalysis as has been suggested to explain recently reported observations.

Parasitology, 2001 Dec, 123(Pt 6), 631 - 9
A Cooperia punctata gene family encoding 14 kDa excretory-secretory antigens conserved for trichostrongyloid nematodes; Yatsuda AP et al.; A polymorphic set of 14 kDa excretory-secretory (E-S) antigen-encoding cDNAs, with similarity to a previously characterized 15 kDa E-S antigen of Haemonchus contortus, was cloned from Cooperia punctata . Five cDNAs encoding predicted proteins of 70-80% identity were sequenced . Genomic analyses of individuals proved the existence of three 14 kDa E-S antigen-encoding genes, excluding that the differences reflected polymorphisms between individuals in a population . Southern blots indicated the presence of additional members of this gene family . Thus, despite the fact that heterologously expressed C . punctata 14 kDa E-S products are shown to be recognized by immune sera, potential pitfalls in the development of a recombinant vaccine are presented by this genetic diversity . Vaccine design could be further rationalized by knowledge of the function, and possible redundancy in function, of the E-S products which is presently lacking . The limitations encountered in assigning a function to the 14/15 kDa family of E-S proteins that is thus far unique to the trichostrongyloid nematodes are discussed.

J Mass Spectrom, 2002 Jan, 37(1), 99 - 107
Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level; Martinovic S et al.; The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification . We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae . Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added . The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR) . The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum . The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein . Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues . The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g . His, Arg, Ile, Phe, Lys) . A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously . Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications .

Environ Mol Mutagen, 2002, 39(1), 55 - 68
Characterization of mutant spectra generated by a forward mutational assay for gene A of Phi X174 from ENU-treated transgenic mouse embryonic cell line PX-2; Valentine CR et al.; The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing . A forward mutational assay exists for Phi X174 that requires an altered, functional Phi X174 protein and therefore should have fewer targets (sense, base-pair substitutions) than forward assays that inactivate a protein . We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N-ethyl-N-nitrosourea (ENU) . We identified 25 target sites and 33 different mutations in Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 am3, cs70 (line 54) . All six types of base-pair substitution were represented among both the spontaneous and ENU-treated mutant spectra . The mutant spectra from cells treated with 200 and 400 microg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other . The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose . The spontaneous mutant frequency was 1.9 x 10(-5) and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations . Thirteen mutations at 12 targets were identified only from ENU-treated cells . Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases . The results suggest that the Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

J Cell Biochem, 2002, 84(3), 590 - 600
Identification and mapping of nuclear matrix-attachment regions in a one megabase locus of human chromosome 19q13.12: long-range correlation of S/MARs and gene positions; Chernov IP et al.; The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA . However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes . One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold . We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene-rich D19S208-COX7A1 locus of human chromosome 19 . A library of S/MAR elements from the locus was prepared and shown to contain -20 independent S/MARs . Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus . A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains . These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb . The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs . The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed . The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements .

Nucleic Acids Res . 2001 Dec 15;29(24):E122.
Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein-DNA footprinting; Henn A et al.; Nucleic acid fragmentation (footprinting) by *OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions . This method has proven valuable because it provides structural information with single base pair resolution . Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair fragmentation of nucleic acid in tens of milliseconds . We developed a complementary method that utilizes X-rays generated from a conventional rotating anode machine in which nucleic acid footprints can be generated by X-ray exposures as short as 100-300 ms . Our theoretical and experimental studies indicate that efficient cleavage of nucleic acids by X-rays depends upon sample preparation, energy of the X-ray source and the beam intensity . In addition, using this experimental set up, we demonstrated the feasibility of conducting X-ray footprinting to produce protein-DNA protection portraits at sub-second timescales.

Nucleic Acids Res, 2001 Dec 15, 29(24), 5129 - 39
NMR structure of a ribosomal RNA hairpin containing a conserved CUCAA pentaloop; Nagaswamy U et al.; The structure of a 23 nt RNA sequence, rGGACCCGGGCUCAACCUGGGUCC, was elucidated using homonuclear NMR, distance geometry and restrained molecular dynamics . This RNA is analogous to residues 612-628 of the Escherichia coli 16S rRNA . The structure of the RNA reveals the presence of a pentaloop closed by a duplex stem in typical A-form conformation . The loop does not form a U-turn motif, as previously predicted . A non-planar A.C.A triple base interaction (hydrogen bonds A13 NH6-C10 O2 and C10 N3-A14 NH6) stabilizing the loop structure is inferred from structure calculations . The CUCAA loop structure is asymmetrical, characterized by a reversal of the phosphodiester backbone at the UC step (hydrogen bond C12 NH4-C10 O2') and 3'-stacking within the CAA segment . Loop base U11 is oriented towards the major groove and the consecutive adenosines on the 3'-end of the loop are well stacked, exposing their reactive functional groups in the minor groove defined by the duplex stem . The solution structure of the loop resembles that seen in the 3.3 A X-ray structure of the entire 30S subunit, where the analogous loop interacts with a ribosomal protein and a receptor RNA helix.

Nucleic Acids Res, 2001 Dec 15, 29(24), 5107 - 14
An architectural role of the Escherichia coli chromatin protein FIS in organising DNA; Schneider R et al.; The Escherichia coli chromatin protein FIS modulates the topology of DNA in a growth phase-dependent manner . In this study we have investigated the global effect of FIS binding on DNA architecture in vitro . We show that in supercoiled DNA molecules FIS binds at multiple sites in a non-random fashion and increases DNA branching . This global DNA reshaping effect is independent of the helical phasing of FIS binding sites . We propose, in addition to the previously inferred stabilisation of tightly bent DNA microloops in the upstream regions of certain promoters, that FIS may perform the distinct architectural function of organising branched plectonemes in the E.coli nucleoid.

Nucleic Acids Res, 2001 Dec 15, 29(24), 5067 - 70
A protonated base pair participating in rRNA tertiary structural interactions; Kubarenko AV et al.; In the recently published X-ray crystallographic structure for the 50S subunit of Haloarcula marismortui ribosomes, residue U2546 of the 23S rRNA forms a non-Watson-Crick base pair with U2610 . The corresponding residues in the secondary structure of the Escherichia coli 23S molecule are U2511 and C2575, and it follows that the latter base (C2575) should be protonated in order to form a base pair that is isostructural with its counterpart in H.marismortui . This prediction was demonstrated experimentally by reduction with sodium borohydride followed by primer extension analysis; borohydride is able to reduce positively charged bases, yielding products which block reverse transcription . In the course of the analysis a further charged base pair (AH(+)1528-G1543) was identified in the E.coli 23S molecule . Both charged pairs (U2511-CH(+)2575 and AH(+)1528-G1543) were only observed in the context of the intact ribosomal subunit and were not seen in deproteinized rRNA.

Nucleic Acids Res, 2001 Dec 15, 29(24), 5044 - 51
Directed evolution of a recombinase for improved genomic integration at a native human sequence; Sclimenti CR et al.; We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes . The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site . Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells . Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.

Nucleic Acids Res, 2001 Dec 15, 29(24), 4930 - 4
A second NAD(+)-dependent DNA ligase (LigB) in Escherichia coli; Sriskanda V et al.; Escherichia coli DNA ligase (LigA) is the prototype of the NAD(+)-dependent class of DNA ligases found in all bacteria . Here we report the characterization of E.coli LigB, a second NAD(+)-dependent DNA ligase identified by virtue of its sequence similarity to LigA . LigB differs from LigA in that it lacks the BRCA1 C-terminus domain (BRCT) and two of the four Zn-binding cysteines that are present in LigA and all other bacterial NAD(+) ligases . We found that recombinant LigB catalyzed strand joining on a singly-nicked DNA in the presence of a divalent cation and NAD(+), and that LigB reacted with NAD(+) to form a covalent ligase-adenylate intermediate . Alanine substitution for the motif I lysine ((126)KxDG) abolished nick joining and ligase-adenylate formation by LigB, thus confirming that the ligase and adenylyltransferase activities are intrinsic to the LigB protein.

Nucleic Acids Res, 2001 Dec 15, 29(24), 4909 - 19
Mode of DNA-protein interaction between the C-terminal domain of Escherichia coli RNA polymerase alpha subunit and T7D promoter UP element; Ozoline ON et al.; The C-terminal domain (CTD) downstream from residue 235 of Escherichia coli RNA polymerase alpha subunit is involved in recognition of the promoter UP element . Here we have demonstrated, by DNase I and hydroxyl radical mapping, the presence of two UP element subsites on the promoter D of phage T7, each located half and one-and-a-half helix turns, respectively, upstream from the promoter -35 element . This non-typical UP element retained its alphaCTD-binding capability when transferred into the genetic environment of the rrnBP1 basic promoter, leading to transcription stimulation as high as the typical rrnBP1 UP element . Chemical protease FeBABE conjugated to alphaCTD S309C efficiently attacked the T7D UP element but not the rrnBP1 UP element . After alanine scanning, most of the amino acid residues that were involved in rrnBP1 interaction were also found to be involved in T7D UP element recognition, but alanine substitution at three residues had the opposite effect on the transcription activation between rrnBP1 and T7D promoters . Mutation E286A stimulated T7D transcription but inhibited rrnBP1 RNA synthesis, while L290A and K304A stimulated transcription from rrnBP1 but not the T7D promoter . Taken together, we conclude that although the overall sets of amino acid residues responsible for interaction with the two UP elements overlap, the mode of alphaCTD interaction with T7D UP element is different from that with rrnBP1 UP element, involving different residues on helices III and IV.

J Biol Chem, 2002 Apr 12, 277(15), 12525 - 31 Epub 2002 Jan 25.
Rap-specific GTPase activating protein follows an alternative mechanism; Brinkmann T et al.; Rap1 is a small GTPase that is involved in signal transduction cascades . It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs) . To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies . The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C . The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site . However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction . We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect . Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61 . The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP . As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant . This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.

Zhonghua Jie He He Hu Xi Za Zhi, 1999 Mar, 22(3), 138 - 41
{The 38,000 protein of Mycobacterium tuberculosis is overexpressed in Escherichia coli}; He X et al.; OBJECTIVE: To obtain recombinant 38,000 protein in large quantities and to study its immunologic characteristics by stable expression of the gene encoding for 38,000 antigen of Mycobacterium tuberculosis in E . coli . METHODS: Expression plasmid was constructed with DNA recombinant technique . Positive clones were screened using double digestion and polymerase chain reaction . Recombinant plasmid was transformed into E . coli . Then E . coli carrying recombinant plasmid were induced . The expression of 38,000 antigen was identified by SDS-polyacrylamid gel electrophoresis (PAGE) and immunoblotting . Stained gel was scanned to detect expression level of recombinant antigen . RESULTS: Gel stained with coomassie blue G-250 showed that the induced E . coli carrying recombinant plasmid can produce 38,000 protein at high level . Gel scan showed that 38,000 antigen expression in E . coli was about 36% - 40% of total cellular protein . The recombinant 38,000 antigen existed mostly in inclusion bodies . The recombinant antigen can react with antibodies: serum of pulmonary tuberculosis patients and the goats immuned with Mycobacterium tuberculosis . CONCLUSIONS: Constructed recombinant E . coli can overproduce 38,000 antigen of Mycobacterium tuberculosis . Inclusion bodies are easy to purify and can protect the recombinant antigen from protease, on the other hand, it has not biological activity unless the denatured protein is accurately folded.

Protein Expr Purif, 2002 Feb, 24(1), 163 - 70
Low concentrations of free hydrophobic amino acids disrupt the Escherichia coli RNA polymerase core-sigma(70) protein-protein interaction; Maitra A et al.; Studies of the Escherichia coli RNA polymerase subunit sigma-70 employing limited proteolytic digestion and binding by monoclonal antibodies indicate that conserved region 3 is solvent accessible in the free protein and in the RNA polymerase holoenzyme . Conversely, when sigma-70 binds to core RNA polymerase, proteolytic cleavage of region 3 is dramatically reduced . The former set of results seems to indicate the physical presence of region 3 on or near the surface of the holoenzyme while the latter of these results suggest that region 3 is sequestered in a direct protein-protein contact within the RNA holoenzyme which alters its protease sensitivity . To further investigate these possibilities we inserted an internal histidine-tag within region 3 of sigma(70) (sigma(70)-R3-His6) between amino acids 508 and 509 . Confirmation that the internal His-tag insertion does not disrupt normal sigma(70) function was verified by genetic complementation . His-tagged protein was immobilized on nickel-agarose and core RNAP was tethered via the sigma-core interaction . Our results are consistent with the localization of region 3 on or near the surface both of free sigma(70) and of RNA polymerase holoenzyme . Furthermore, we find that the sigma(70)-core interaction is resistant to high ionic conditions but is completely disrupted by the presence of the low-molecular-weight hydrophobic amino acids phenylalanine and leucine free in solution . These results demonstrate the general usefulness of this approach to the disruption of protein-protein interactions and its potential application for protein purification .

Protein Expr Purif, 2002 Feb, 24(1), 131 - 7
Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica; Hellberg A et al.; Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity . To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form . The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography . Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme . Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C . Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E . histolytica lysates . Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein . Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA . The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors . In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity .

Protein Expr Purif, 2002 Feb, 24(1), 105 - 10
Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana; Paris S et al.; In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH) . In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana) . The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol of NADPH formed min(-1) mg(-1) of protein, respectively . These values are 200-fold higher than those reported previously for partially purified plant enzymes . AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, respectively . Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors . In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa . Addition of the effector converted the enzyme into a tetrameric form of 320 kDa .

Protein Expr Purif, 2002 Feb, 24(1), 61 - 70
Processive degradation of nascent polypeptides, triggered by tandem AGA codons, limits the accumulation of recombinant tobacco etch virus protease in Escherichia coli BL21(DE3); Kapust RB et al.; Due to its high degree of sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV protease) is a useful reagent for cleaving genetically engineered fusion proteins . However, the overproduction of TEV protease in Escherichia coli has been hampered in the past by low yield and poor solubility . Here we demonstrate that the low yield can be attributed to the presence of arginine codons in the TEV protease coding sequence that are rarely used in E . coli and specifically to a tandem pair of AGA codons . The yield of protease can be improved by replacing these rare arginine codons with synonymous ones or by increasing the supply of cognate tRNA that is available to the cell . Furthermore, we show that when ribosomes become stalled at rare arginine codons in the TEV protease mRNA, the nascent polypeptides are targeted for proteolytic degradation in BL21(DE3) cells by a mechanism that does not involve tmRNA-mediated peptide tagging .

Protein Expr Purif, 2002 Feb, 24(1), 56 - 60
Overexpression of Escherichia coli genes encoding nucleoside phosphorylases in the pET/Bl21(DE3) system yields active recombinant enzymes; Esipov RS et al.; The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E . coli BL21(DE3) strains producing each of these enzymes . Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found . The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation . The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel .

Protein Expr Purif, 2002 Feb, 24(1), 51 - 5
The purification of polyphenol oxidase from tobacco; Shi C et al.; A new polyphenol oxidase (PPO) named PPO II was purified from tobacco (Nicotiana tobacum) by using acetone powder, ammonium sulfate precipitation, and column chromatography on DEAE-Sephadex A-50, Sephadex G-75, and CM-Sephadex C-50 . It has an active site of a pair of type 3 coppers bridged to phenolate oxygen, which represents a new catalytic mechanism for polyphenol oxidase . PAGE, SDS-PAGE, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry of the purified enzyme demonstrated that the enzyme is a single band with a molecular mass 35,600 Da . Biochemical characteristics include the optimum pH at 6.0, optimum temperature at 40 degrees C, and K(m) of 1.2 mM for catechol as substrate (pH 6.5, 30 degrees C) . Substrate specificity studies indicate that the enzyme is of the catechol oxidase family . PPO II inhibits cultures of Escherichia coli and it accumulates on the wounded sites of tobacco leaves indicating that it may act as a defense role in plant defense systems .

Protein Expr Purif, 2002 Feb, 24(1), 33 - 42
Heterologous expression, purification, and characterization of human triacylglycerol hydrolase; Alam M et al.; Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver . A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library . The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence . The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family . The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide . However, the expressed protein was insoluble and inactive . Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein . Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme . N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide . The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen . The results suggest that hTGH expressed in insect cells is properly folded . Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship .

Protein Expr Purif, 2002 Feb, 24(1), 25 - 32
Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases; Savopoulos JW et al.; The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines . Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy . Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE . In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2 . We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays .

Protein Expr Purif, 2002 Feb, 24(1), 13 - 7
High-level expression of human extracellular superoxide dismutase in Escherichia coli and insect cells; He HJ et al.; Much is known about the physical properties of the Cu,Zn- and Mn-superoxide dismutases (SODs) . However, the biochemical characteristics and pharmacological properties of extracellular (EC)-SOD have been severely limited due to difficulties in obtaining and purifying the enzyme . The EC-SOD cDNA was inserted into the Escherichia coli expression plasmid pET-28a(+) which contains the T7 promoter and transformed into the E . coli BL21(DE3) . After induction with 1 mmol/L isopropyl beta-D-thiogalactoside, the recombinant human EC-SOD was highly expressed as inclusion bodies . SDS-PAGE analysis revealed that recombinant EC-SOD accumulated up to 26% of the total soluble protein of E . coli cells . The expression product was purified by a Ni(2+)-IDA-Sepharose 6B column . After the denaturing and refolding processes, the recombinant human EC-SOD retains the specific enzymatic activity of 920 U/mg of the purified product . The gene encoding human EC-SOD mature peptide was also inserted into the donor plasmid pFastBacHTb . After transposition, transfection, and amplification were performed, the recombinant baculoviruses infected the Tn-5B1-4 cells and EC-SOD was highly expressed in Tn-5B1-4 cells . SDS-PAGE and Western blot analysis revealed that the subunit molecular weight of the expression product is 28 kDa . Furthermore, recombinant human EC-SOD retains the enzymatic specific activity of 200 U/mg of the Tn-5B1-4 cell lysates .

J Mol Biol, 2002 Jan 25, 315(4), 831 - 43
The ATPase domain of SecA can form a tetramer in solution; Dempsey BR et al.; Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG . SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex . SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization . We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure . The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)={monomer}(4)/{tetramer}) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution . Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure . Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore .

J Mol Biol, 2002 Jan 25, 315(4), 787 - 98
Stimulation of the weak ATPase activity of human hsp90 by a client protein; McLaughlin SH et al.; Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways . In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23 . The role of these proteins in Hsp90-mediated assembly processes is poorly understood . It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro.Here we show, for the first time, that human Hsp90 has ATPase activity in vitro . The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli . Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C . Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold . This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity . In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity.We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90 . In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate . Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further . In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis.Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding . We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo . We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur .

J Mol Biol, 2002 Jan 25, 315(4), 573 - 86
Engineered RNase P ribozymes inhibit gene expression and growth of cytomegalovirus by increasing rate of cleavage and substrate binding; Trang P et al.; We have previously employed an in vitro (genetic) selection procedure to select RNase P ribozyme variants for their activity in cleaving a mRNA substrate from a pool of ribozymes containing randomized sequences . In this study, one of the variants was used to target the overlapping region of the mRNAs encoding the major transcription regulatory proteins, IE1 and IE2, of human cytomegalovirus (HCMV) . The ribozyme variant exhibited an enhanced substrate binding and rate of chemical cleavage, and was at least 25 times more efficient in cleaving the target mRNA in vitro than the ribozyme derived from the wild-type sequence . Our results provide the first direct evidence that a point mutation at nucleotide 86 of RNase P catalytic RNA from Escherichia coli (A(86)-->C(86)) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G(205)-->C(205)) enhances substrate binding of the ribozyme . Moreover, the variant was also more effective in inhibiting IE1 and IE2 expression and HCMV growth in cultured cells . A reduction of more than 97% in IE1 and IE2 expression and a reduction of 3000-fold in viral growth were observed in cells expressing the variant . Thus, RNase P ribozyme variant is highly effective in inhibiting HCMV gene expression and growth . Our results provide the direct evidence that increasing the rate of chemical cleavage and substrate-binding affinity of the ribozymes should lead to an improvement of their anti-HCMV efficacy . Moreover, our data also suggest that highly effective anti-HCMV ribozyme variants can be developed using genetic engineering approaches including in vitro selection .

J Mol Biol, 2002 Jan 25, 315(4), 561 - 71
Manipulating conformational equilibria in the lactose permease of Escherichia coli; Weinglass AB et al.; A mechanism proposed for lactose/H(+) symport by the lactose permease of Escherichia coli indicates that lactose permease is protonated prior to ligand binding . Moreover, in the ground state, the symported H(+) is shared between His322 (helix X) and Glu269 (helix VIII), while Glu325 (helix X) is charge-paired with Arg302 (helix IX) . Substrate binding at the outer surface between helices IV (Glu126) and V (Arg144, Cys148) induces a conformational change that leads to transfer of the H(+) to Glu325 and reorientation of the binding site to the inner surface . After release of substrate, Glu325 is deprotonated on the inside due to re-juxtapositioning with Arg302 . The conservative mutation Glu269-->Asp causes a 50-100-fold decrease in substrate binding affinity and markedly reduced active lactose transport, as well as decreased rates of equilibrium exchange and efflux . Gly-scanning mutagenesis of helix VIII was employed systematically with mutant Glu269-->Asp in an attempt to rescue function, and two mutants with increased activity are identified and characterized . Mutant Thr266-->Gly/Met267-->Gly/Glu269-->Asp binds ligand with increased affinity and catalyzes active lactose transport with a marked increase in rate; however, little improvement in efflux or equilibrium exchange is observed . In contrast, mutant Gly262-->Ala/Glu269-->Asp exhibits no improvement in ligand binding but a small increase in the rate of active transport; however, an increase in the steady-state level of accumulation, as well as efflux and equilibrium exchange is observed . Remarkably, when the two sets of mutations are combined, all translocation reactions are rescued to levels approximating those of wild-type permease . The findings support the contention that Glu269 plays a pivotal role in the mechanism of lactose/H(+) symport . Moreover, the results suggest that the two classes of mutants rescue activity by altering the equilibrium between outwardly and inwardly facing conformations of the permease such that impaired protonation and/or H(+) transfer is enhanced from one side of the membrane or the other . When the two sets of mutants are combined, the equilibrium between outwardly and inwardly facing conformations and thus protonation and H(+) transfer are restored .

J Mol Biol, 2002 Jan 25, 315(4), 497 - 511
A phosphorylation site mutant of OmpR reveals different binding conformations at ompF and ompC; Mattison K et al.; In Escherichia coli, the two-component regulatory system that controls the expression of outer membrane porins in response to environmental osmolarity consists of the sensor kinase EnvZ and the response regulator OmpR . Phosphorylated OmpR activates expression of the OmpF porin at low osmolarity, and at high osmolarity represses ompF transcription and activates expression of OmpC . We have characterized a substitution in the amino-terminal phosphorylation domain of OmpR, T83I, its phenotype is OmpF(-) OmpC(-) . The mutant protein is not phosphorylated by small molecule phosphodonors such as acetyl phosphate and phosphoramidate, but it is phosphorylated by the cognate kinase EnvZ . Interestingly, the active site T83I substitution alters the DNA binding properties of the carboxyl-terminal effector domain . DNase I protection assays indicate that DNA binding by the mutant protein is similar to wild-type OmpR at the ompF promoter, but at ompC, the pattern of protection is different from OmpR . Our results indicate that all three of the OmpR binding sites at the ompC promoter must be filled in order to activate gene expression . Furthermore, it appears that OmpR-phosphate must adopt different conformations when bound at ompF and ompC . A model is presented to account for the reciprocal regulation of OmpF and OmpC porin expression .

J Mol Biol, 2002 Jan 25, 315(4), 487 - 95
The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism; Izard T; Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) in the presence of magnesium . To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant . Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis . The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism . The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified . Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state .

Biochem Biophys Res Commun, 2002 Feb 1, 290(4), 1328 - 35
A rapid method to capture and screen for transcription factors by SELDI mass spectrometry; Forde CE et al.; A novel method to screen for transcription factors binding to promoter DNA sequences has been developed using DNA chip surfaces and mass spectrometry . This technique was demonstrated with Escherichia coli lac repressor, LacI . The consensus promoter binding sequence for LacI and a scrambled version of the same DNA sequence were prepared on two affinity chip surfaces . Total E . coli protein lysate was applied to the two surfaces . A 38.2 kDa protein, as detected by SELDI-MS, was captured on the chip surface containing the binding sequence for LacI but not on the surface containing the scrambled sequence . The protein was identified following one-step, small-scale affinity capture and peptide mapping . Subsequent database searches identified the 38.2 kDa protein as the lac repressor of E . coli . We discuss application of DNA chip affinity capture to characterize transcription factors and to screen for differences in cellular regulatory networks . (c)2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 Feb 1, 290(4), 1206 - 13
Expression in Escherichia coli, purification and kinetic characterization of human heparan sulfate 3-O-sulfotransferase-1; Myette JR et al.; Heparan sulfate (HS) glycosaminoglycans are a structurally diverse class of complex biomolecules that modulate many important events at the cell surface and within the extracellular matrix and whose structural heterogeneity derives largely from the sequence-specific N- and O-sulfations catalyzed by an extensive repertoire of sulfating enzymes . We have expressed the human heparan sulfate 3-OST-1 isoform in Escherichia coli and subsequently purified a soluble, active enzyme . To assess its functionality, we determined the kinetic parameters for the recombinant 3-O-sulfotransferase-1 using a radiochemical assay that directly measures the 3-O-sulfation of unlabeled bovine kidney heparan sulfate in vitro using {(35)S}PAPS as the sulfate donor . The apparent K(m) values measured were in the low micromolar range (K(HS)(m) = 4.3 microM; K(PAPS)(m) = 38.6 microM); V(max) values of 18 and 21 pmol sulfate/min/pmol of enzyme for HS and PAPS, respectively . These values were compared with kinetic parameters likewise measured for recombinant 3-OST-1 purified from baculovirus-infected sf9 cells . The two enzymes appear to modify heparan sulfate in vitro to roughly the same extent and with comparable specificities . The expression of 3-OST-1 in E . coli represents an important step in subsequent structure-function studies . (c)2002 Elsevier Science (USA).

Biochem Biophys Res Commun, 2002 Feb 1, 290(4), 1183 - 7
Alteration of stringent response of the Escherichia coli rnpB promoter by mutations in the -35 region; Park JW et al.; It is well known that the GC-rich discriminator region between the -10 region and the transcription start site is important for the stringent control of the transcription . However, the discriminator activity is influenced by flanking regions, in particular in conjunction with the promoter -35 and -10 sequences . In this study, we changed the sequence in the -35 region of the rnpB P-1 promoter to see how such changes affect the stringent control . The sequence variation in the -35 region changed the stringent signal . The change to the consensus TTGACA sequence caused the most prominent relieving effect on stringent repression of the rnpB transcription . The spacing between the -35 and -10 regions is also significant because the relieving effect of the TTGACA was offset by the change of the spacing from 17 to 16 bp . The nucleotide just upstream of the -35 region contributes toward generating stringent signals as well . (c)2002 Elsevier Science (USA).

Life Sci, 2001 Dec 21, 70(5), 523 - 34
Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols; Autore G et al.; In order to validate the use of Crescentia alata (Bignoniaceae) in the traditional medicine of Guatemala as an antiinflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for antiinflammatory activity on carrageenin paw edema in rats and in vitro on Escherichia coli lipopolysaccharide- (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774.A1 macrophage cell line . This extract exerted in vivo a significant anti-inflammatory activity at the highest dose tested . The same extract showed in vitro an inhibitory activity on inducible nitric oxide synthase expression and on NO formation in LPS-primed J774.A1 cells . Subsequent fractionation and analysis of the extract has led to the isolation and characterization as major constituents of two flavonol glycosides: quercetin 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (rutin) 1, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (kaempferol 3-O-rutinoside) 2, and flavonol aglycone, kaempferol 3 . Their structures were elucidated by spectral methods . The bioassay-directed analysis of flavonols 1-3 indicated that kaempferol (3) was the most active compound contained in the MeOH extract because it reduced in vitro both NO production and iNOS expression in LPS-primed J774.A1 cells, whereas rutin (1) and kaempferol 3-O-rutinoside (2) showed no significant activity . The MeOH extract and all of flavonoids tested did not show in vitro significant cytotoxic effect in J774.A1 macrophage cell line.

Free Radic Res, 2001 Dec, 35(6), 907 - 16
Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress; Victor VM et al.; Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock . The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg) . The intake by cells of this antioxidant present in vitro at different concentrations was also studied . The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection . The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min . The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection . The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h . The AA levels decreased after this time, in agreement with changes in the AA hepatic levels . The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection . Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro . This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection . The incorporation decreased at these times of endotoxic shock, a few hours before death . In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration . These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.

Free Radic Res, 2001 Dec, 35(6), 867 - 72
Glycerol metabolism in superoxide dismutase-deficient Escherichia coli; Benov L et al.; Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various phenotypic deficits if grown aerobically . Here we report that sodAsodB E . coli cannot use glycerol under aerobic conditions . The reason is low activity of glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism . Superoxide does not inactivate GK, but makes it susceptible to inactivation by a heat-labile factor present in the cell-free extracts . This factor seems to be part of a proteolytic system, which recognizes and degrades oxidatively modified proteins.

Anal Chem, 2002 Jan 15, 74(2), 347 - 54
Toward electrochemical resolution of two genes on one electrode: using 7-deaza analogues of guanine and adenine to prepare PCR products with differential redox activity; Yang IV et al.; The 7-deaza analogues of guanine and adenine were incorporated into polymerase chain reaction (PCR) products by substitution of the appropriate nucleotide triphosphates into the reaction . These PCR products can be immobilized on ITO electrodes and detected by catalytic cyclic voltammetry with ruthenium polypyridyl complexes . Immobilization on indium tin oxide (ITO) electrodes of 330- and 1200-base pair (bp) PCR amplicons from the E . coli dacA gene containing one or both of the 7-deazapurines was effected by precipitation from a 9:1 DMF/acetate solution . Amplicons containing the 7-deazaguanine base were detected by observing current enhancement in the cyclic voltammogram of Ru(dmb)3(3)+/2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine) due to the selective oxidation of the modified base by this mediator . Oxidation of incorporated 7-deazaadenine bases in addition to native guanines gives rise to a higher current enhancement in the cyclic voltammogram of Ru(bpy)3(3)+/2+ (bpy = 2,2'-bipyridine) compared to the enhancement observed in the presence of guanine only . This strategy was employed to simultaneously detect the 330-bp sequence containing 7-deazaadenine and the 1200-bp sequence containing 7-deazaguanine on the same ITO electrode . Such a strategy may provide a means for detecting multiple genes on a single microlocation and may thereby lead to more highly multiplexed gene assays.

J Infect Chemother, 2001 Sep, 7(3), 180 - 5
Analysis of the serological response to Chlamydia pneumoniae in patients with ischemic heart disease by recombinant MOMP-ELISA; Kido Y et al.; To investigate the humoral immune response to the major outer membrane protein (MOMP) of Chlamydia pneumoniae, a fusion protein, thioredoxin-(His)6-MOMP (rMOMP) was produced in Escherichia coli and purified; this served as an antigen to establish an enzyme-linked immunosorbent assay (ELISA) . Specific IgG and IgA antibodies against rMOMP were determined in sera from patients with ischemic heart disease . The findings were compared with those obtained by ELISA using the outer membrane protein complex (Hitazyme) . The positivity rates for IgG antibody by rMOMP-ELISA were low (28%) compared with those by Hitazyme (72%) . However, the positivity rates of IgA antibody by rMOMP-ELISA were similar to those by Hitazyme (76%) . Interestingly, antigen positivity by immunohistochemical staining in the atherosclerotic lesions of coronary arteries was high in the groups with a high IgA titer of rMOMP-ELISA.

J Infect Chemother, 2000 Mar, 6(1), 30 - 4
Medium compositions and culture conditions for the assay of fosfomycin susceptibility by Etest; Horii T et al.; The Etest (AB Biodisk, Solna, Sweden), a susceptibility testing method, was used for fosfomycin and was evaluated by comparison with the agar dilution method for 73 clinical isolates of Escherichia coli, including two resistant strains, and an optimal Etest method for fosfomycin was determined . Media and culture conditions greatly affected the minimum inhibitory concentrations (MICs) determined by the Etest for fosfomycin, as shown for the agar dilution methods . Our results showed that the most favorable conditions for the Etest for fosfomycin were with Mueller-Hinton agar (Becton-Dickinson Japan, Tokyo, Japan) under aerobic conditions . However, the MICs for the resistant strains were much higher than those determined by agar dilution methods, using Nutrient agar (Becton-Dickinson Japan) under anaerobic conditions . The addition of glucose-6-phosphate did not significantly affect the Etest results.

J Infect Chemother, 2000 Mar, 6(1), 21 - 5
Effect of a soluble pseudo-receptor on verotoxin 2-induced toxicity; Takenaga M et al.; To neutralize the toxicity of verotoxin (VT) produced by Escherichia coli type O-157, a soluble pseudo-receptor (Lyso Gb3) was synthesized with the deacylated form of the natural receptor, globotrisylceramide (Galalpha 1-4Galbeta1-4-glucosylceramide; Gb3) . In this study, we evaluated the characteristics and pharmacological effects of Lyso Gb3, using VT2 . It was confirmed that Lyso Gb3 specifically recognized VT2 . Lyso Gb3 itself had little influence on the in-vitro growth of Vero cells, but markedly augmented VT2-induced cytotoxicity . In addition, the VT2-induced killing of mice was not decreased, but was, rather, increased by Lyso Gb3 . These results indicate that the soluble pseudo-receptor Lyso Gb3 recognized VT2 . However, it did not reduce, but, rather, enhanced, VT2-induced toxicity in the presence of the natural receptor, although Lyso Gb3 alone had no toxicity.

Mol Genet Genomics, 2002 Jan, 266(5), 873 - 81 Epub 2001 Nov 21.
Characterisation of the allelic variation in the rpoS gene in thirteen K12 and six other non-pathogenic Escherichia coli strains; Atlung T et al.; The nucleotide sequence of rpoS, the gene for the stress sigma factor, was determined in 13 different K12 strains of Escherichia coli . The results indicate that the original K12 isolate carried an amber mutation at codon 33, which in 50% of the derivatives is mutated by a single base substitution to a coding triplet, in most cases to CAG encoding glutamine . The six non-K12 strains examined here had GAG, encoding glutamate, in position 33 . The two most divergent strains had three and seven neutral substitutions in rpoS and carried insertions of 2100 and 2900 bp, respectively, just downstream of the gene . The genetic variations in rpoS were compared with the variation in RpoS-related phenotypes, by measuring catalase (KatE) activity, glycogen accumulation and acid phosphatase levels, and a katEp-gfp fusion was used to visualise katE gene transcription . The RpoS phenotypes of the six rpoS(33E) strains varied significantly more than that of the K12 rpoS(33Q) strains, especially with respect to acid phosphatase levels . This was due to the absence of the gene for the transcriptional activator AppY from four of the rpoS(33E) strains, while all the K12 derivatives carried this gene . When cloned into a LacI-controlled vector and compared in a rpoS::Tn 10 background, the RpoS(33Q) and RpoS(33E) variants showed the same activity.

Mol Genet Genomics, 2002 Jan, 266(5), 865 - 72 Epub 2001 Nov 21.
Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements; Izu H et al.; The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome . In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively . The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions . In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd . In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd . In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression . Similar results were obtained with IHF-defective strains . Thus, the expression of the two genes is regulated in a mutually exclusive manner . Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.

Mol Genet Genomics, 2002 Jan, 266(5), 827 - 31 Epub 2001 Nov 01.
Screening for stabilization of proteins with a trans-translation signature in Escherichia coli selects for inactivation of the ClpXP protease; Bohn C et al.; Trans-translation is a process that adds a hydrophobic peptide tag to the C-terminus of polypeptides, which causes them to become unstable . We designed a genetic screen to identify factors involved in the degradation of trans-translated products, using the green fluorescent protein (GFP) fused to the trans-translation tag as a reporter . Two screens were devised to identify insertional mutants that stabilize such substrates . Only disruption of the clpX or clpP gene resulted in stabilization of the tagged substrates . The sspB gene product was recently shown to be a specificity-enhancing factor for the ClpXP degradation machine . In the wild-type background, targeted inactivation of the sspB gene failed to stabilize the tagged substrate . These results indicate that the ATP-dependent ClpXP protease is probably the only main component involved in the degradation of cytoplasmic trans-translated proteins in Escherichia coli that can be completely inactivated.

Mol Genet Genomics, 2002 Jan, 266(5), 806 - 12 Epub 2001 Oct 20.
Isolation and characterization of novel F-plasmid sopB mutants defective in gene silencing; Kubo Y et al.; The product of the sopB gene on the Escherichia coli F-plasmid has been shown to silence genes in the vicinity of its binding region, sopC, when overexpressed . We searched for mutants defective in SopB-dependent silencing by screening for a plasmid incompatibility phenotype, in order to examine the relationship between gene silencing and the intracellular localization of SopB, as revealed by a green fluorescent protein (GFP)-SopB fusion . Nine new mutants were isolated . One of them, in which leucine 92 is replaced by proline, was completely compatible with a sopC-carrying plasmid and was defective in other silencing activities . When expressed as a GFP fusion protein, the L92P mutant was found to be uniformly distributed in the cell . This implies a link between silencing and SopB localization, supporting the view that a high local concentration of SopB drives non-specific DNA binding in segments of the plasmid adjacent to sopC . Despite the lack of apparent localization of GFP fluorescence, the mutant protein, like the wild-type SopB, was found mostly in the inner membrane fraction, indicating that the association with the inner membrane was retained.

Mol Genet Genomics, 2002 Jan, 266(5), 768 - 77 Epub 2001 Nov 09.
Genes for mevalonate biosynthesis in Phycomyces; Ruiz-Albert J et al.; Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids . The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and its conversion to mevalonate . We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus . Single copies of these genes are present in the Phycomyces genome . The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain . The hydrophilic domain suffices for catalytic activity, as shown by expressing it in Escherichia coli . Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing . Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMG-CoA.

Mol Genet Genomics, 2002 Jan, 266(5), 747 - 57 Epub 2001 Nov 16.
Transcriptional regulation of the Antifungal Protein in Aspergillus giganteus; Meyer V et al.; Regulation of the expression of the afp gene that codes for the Antifungal Protein of Aspergillus giganteus was investigated using a reporter system . For this purpose, the E . coli reporter gene uidA encoding beta-glucuronidase (GUS) was placed under the control of the afp promoter . No homologous integration of the reporter construct into the afp site was observed among 156 transformants tested . In one of the transformants carrying a single, ectopically integrated, copy of the construct, GUS and AFP both displayed exactly the same temporal expression patterns under various cultivation conditions, as assayed by Northern and protein analyses . Thus, this transformant was used to identify factors that are involved in the transcriptional regulation of afp expression . Expression is only detectable in the vegetative mycelium, whereas no expression occurs in aerial hyphae or conidia, indicating that afp expression is developmentally regulated . Transcription of afp is regulated by ambient pH, being suppressed under acidic conditions and strongly induced under alkaline conditions . This observation suggests that PacC regulates the afp gene, which is consistent with the presence of two putative PacC binding sites within the 5' upstream region . Transcription is not subject to carbon catabolite repression or nitrogen metabolite repression . The expression of afp is up-regulated by heat shock, upon growth in the presence of excess NaCl and ethanol, and under conditions of carbon starvation . In contrast, expression decreases slightly in the presence of hydrogen peroxide and under nitrogen starvation . These data are compatible with the presence of a putative heat shock element (NTTCNNGANTTCN) and five putative C(4)T stress-responsive elements within the afp promoter.

Mol Genet Genomics, 2001 Dec, 266(4), 546 - 55 Epub 2001 Aug 30.
Enhanced homologous recombination caused by the non-transcribed spacer of the rDNA in Arabidopsis; Urawa H et al.; The problem of the low frequency of homologous recombination observed in higher plants has been approached in several ways . Here, we report a new strategy to enhance homologous recombination in Arabidopsis . In Escherichia coli and Saccharomyces cerevisiae, hotspots that enhance homologous recombination nearby have been identified in regions close to sites associated with the blocking of DNA replication forks or with intensive transcriptional activity . In yeast, a recombination hotspot (HOT1) was found in a region spanning two non-transcribed spacers (NTS) between ribosomal RNA genes (rDNA), which contains both a replication fork barrier ( RFB) and the promoter for transcription of the 35S rRNA gene . Since rDNA has a common structure among eukaryotes, we analyzed the effect of the endogenous NTS on homologous mitotic recombination in a higher plant . We constructed transgenic lines of Arabidopsis containing this NTS and a recombination substrate, in which two 3'- and 5'-deleted uidA (beta-glucuronidase) genes with partially overlapping sequences are separated by a Hyg(r) gene . Reconstitution of functional uidA genes by homologous recombination was monitored by histochemical GUS staining . We found that recombination occurred more frequently in all organs tested in F (Fork block) lines transgenic for the NTS than in C (Control) lines without the NTS . The average number of GUS+ spots on leaves in F lines was more than nine-fold higher than in C lines . Furthermore, by genomic Southern analysis, post-recombinational molecules were detected in a transgenic line, F43, which had an extremely high number of GUS+ blue spots . These results strongly suggest that NTS-dependent enhancement of homologous recombination may be a common feature of higher plants as well as yeast.

Mol Genet Genomics, 2001 Dec, 266(4), 537 - 45 Epub 2001 Oct 12.
The foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL; Derkx PM et al.; Here we report the isolation and characterization of the cypB gene from Aspergillus niger . The cypB gene encodes a protein with a predicted molecular weight of 20.7 kDa, which shows a high degree of identity to the cyclophilin family of peptidyl prolyl cis-trans isomerases (PPIases) from other eukaryotes . The 5' untranslated region of cypB includes three sequences resembling UPREs (unfolded protein response elements) . The expression of cypB is upregulated by tunicamycin and DTT, suggesting that at least one UPRE is functional . The CYPB protein also has a 23-amino acid sequence which serves to target the protein to the endoplasmic reticulum (ER), and the ER retention sequence HEEL . CYPB-(His)(6) was expressed in Escherichia coli; the purified protein is capable of isomerizing a substrate peptide in vitro . This is also the first report to show that C-terminal addition of the sequence HEEL is sufficient to ensure retention of the green fluorescent protein (GFP) within the ER.

Protein Eng, 2001 Dec, 14(12), 1043 - 52
Selection of catalytically active biotin ligase and trypsin mutants by phage display; Heinis C et al.; Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it . However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes . We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide . Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction . In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes . These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII . Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors . In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls . Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls . In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants . Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated . The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Protein Eng, 2001 Dec, 14(12), 1035 - 41
Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion; Thompson J et al.; Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models . Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance . After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin . This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv) . Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone . Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S) . In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv . The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon . T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct . The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

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