J Appl Physiol, 2001 May, 90(5), 1788 - 97 Detergent inhibits 70-90% of responses to intravenous endotoxin in awake sheep; Staub NC Sr et al.; Sheep have reactive pulmonary intravascular macrophages, which are essential for the marked pulmonary vascular response to infusions of small quantities of endotoxin . In another species with reactive pulmonary intravascular macrophages, horses, our laboratory found that an intravenous biosafe detergent, tyloxapol, inhibited some systemic and pulmonary responses to endotoxin (Longworth KE, Smith BL, Staub NC, Steffey EP, and Serikov V . Am J Vet Res 57: 1063-1066, 1996) . We determined whether the same detergent would inhibit endotoxin responses in awake sheep . In 10 awake, instrumented sheep with chronic lung lymph fistulas, we did a control experiment by intravenously infusing 1 microg/kg Escherichia coli endotoxin . One week later, we gave 40 micromol/kg tyloxapol intravenously 1-4 h before infusing the same dose of endotoxin . In these paired studies, we compared pulmonary hemodynamics, lung lymph dynamics, body temperature, circulating leukocyte concentrations, and circulating tumor necrosis factor for 6 h . In all 10 sheep, tyloxapol blocked 80-90% of the pulmonary responses and 70-90% of the systemic responses . Tyloxapol is safe, inexpensive, easy to use, and effective immediately . It may be a clinically useful approach to contravening many of the effects of endotoxemia, in humans as well as animals. Lett Appl Microbiol, 2001 Apr, 32(4), 230 - 4 In vitro stability and expression of green fluorescent protein under high pressure conditions; Ehrmann MA et al.; AIMS: The objective of this work was to evaluate the use of wild-type GFP and mutant forms thereof as reporter for gene expression under high pressure conditions . METHODS AND RESULTS: The intensity of fluorescence after high pressure treatment was checked by subjecting cells, crude protein extracts containing GFPs and purified GFPs to pressures ranging from 100 MPa to 900 MPa . All tested GFP's retained fluorescence up to 600 MPa without loss of intensity . Expression of GFP under sublethal conditions was investigated in Escherichia coli with plasmid pQBI63, in which rsGFP is placed downstream of the T7 RNA polymerase binding site . T7 RNA polymerase is controlled in E . coli BL21 (DE3) pLysS by an IPTG inducible lacUV5 promoter . A pressure induced increase of GFP expression was monitored at 50 Mpa and 70 MPa . CONCLUSION: Fluorescence of GFPs is not influenced at pressures at which protein expression still occurs . We showed that the expression system used is inducible by pressurized conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: This study proved GFP to be a suitable reporter for gene expression studies capable to detect pressure induced gene expression. Immunology, 2001 Mar, 102(3), 344 - 51 Immunization onto bare skin with heat-labile enterotoxin of Escherichia coli enhances immune responses to coadministered protein and peptide antigens and protects mice against lethal toxin challenge; Beignon AS et al.; In this study, the potential of the bare skin as a non-invasive route for vaccination was examined . Following application of heat-labile enterotoxin (LT) of Escherichia coli onto bare skin of BALB/c mice, strong serum anti-LT antibody responses were observed, and mucosal immunoglobulin A (IgA) and IgG antibodies were measured in vagina washes . In addition, LT enhanced the serum and mucosal antibody and proliferative T-cell responses to the model protein antigen beta-galactosidase (beta-gal) when coadministered onto bare skin, highlighting its potential to exert an adjuvant effect . When a peptide representing a T-helper epitope (aa 307-319) from the haemagglutinin of influenza virus was applied onto bare skin with LT or cholera toxin (CT), it primed effectively peptide- and virus-specific T cells, as measured in vitro by the interleukin-2 (IL-2) secretion assay . LT was shown to be as immunogenic as CT . Binding activity to GM1 gangliosides was essential for effective induction of anti-CT serum and mucosal antibody responses . Finally, mice immunized onto bare skin with LT were protected against intraperitoneal challenge with a lethal dose of the homologous toxin . These findings give further support to a growing body of evidence on the potential of skin as a non-invasive route for vaccine delivery . This immunization strategy might be advantageous for vaccination programmes in Third World countries, because administration by this route is simple, painless and economical. Eur J Biochem, 2001 Apr, 268(8), 2421 - 9 GroEL-assisted refolding of adrenodoxin during chemical cluster insertion; Iametti S et al.; Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP . The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins . A possible scaffolding role for two bacterial sulfur transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates . The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH-cytochrome c reduction assay . These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times . The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio . GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL . A . vinelandii rhodanese, the E . coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution . The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein. Eur J Biochem, 2001 Apr, 268(8), 2408 - 15 Identification of catalytically important residues in the active site of Escherichia coli transaldolase; Schorken U et al.; The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis . The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior . X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes . Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates . Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis . Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity . The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate . The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate. Eur J Biochem, 2001 Apr, 268(8), 2362 - 8 A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin; Francky A et al.; Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine . This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen . Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p . To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7 . All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms . Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae . A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation . The apparently contradictory results for porcine pepsinogen A {Richter, C., Tanaka, T., Koseki, T . & Yada, R.Y . (1999) Eur . J . Biochem . 261, 746-752} can be reconciled with those for prochymosin . Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species . The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former. Eur J Biochem, 2001 Apr, 268(8), 2344 - 50 A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter-like activity; Sur R et al.; RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon . We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner . Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2 . We have located this P3 site by DNase I footprinting . A 63 base pair region centered around position - 100 with respect to galP1 is protected by polymerase . Interestingly, a Pribnow box TATAAT is present within this protected region (-103 to -108) . We have shown that transcription occurs from P3 in vitro . Primer extension analysis provides direct evidence that P3 is transcribed in vivo . The start site of transcription has been mapped at -96 position relative to galP1 . beta-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its -10 region result in a substantial reduction in the gal promoter activity. Eur J Biochem, 2001 Apr, 268(8), 2246 - 52 Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus; Ivens A et al.; Anthranilate phosphoribosyltransferase (TrpD; EC 2.4.2.18) from the hyperthermophilic archaeon Sulfolobus solfataricus (ssTrpD) was expressed in Escherichia coli, purified and crystallized . Analytical gel permeation chromatography revealed a homodimeric composition of the enzyme . The steady-state kinetic characteristics suggest tight binding of the substrate anthranilic acid and efficient catalysis at the physiological growth temperature of S . solfataricus . Crystals of ssTrpD diffract to better than 2.6 A resolution and preliminary X-ray characterization was carried out . The crystals are suitable for structure determination. Eur J Biochem, 2001 Apr, 268(8), 2239 - 45 Structural analysis of the O-antigen polysaccharide from the Shiga toxin-producing Escherichia coli O172; Landersjo C et al.; The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined . In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units . Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques . An O-acetyl group was present as 0.7 equivalent per repeating unit . Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized . Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E . coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1--> Cell Microbiol, 2001 May, 3(5), 341 - 57 Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli; Peiffer I et al.; Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells . We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV) . Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity . In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability . The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate . When the cells were infected with recombinant E . coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed . In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells . Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity. Cell Microbiol, 2001 Apr, 3(4), 213 - 22 EspA filament-mediated protein translocation into red blood cells; Shaw RK et al.; Type III secretion allows bacteria to inject effector proteins into host cells . In enteropathogenic Escherichia coli (EPEC), three type III secreted proteins, EspA, EspB and EspD, have been shown to be required for translocation of the Tir effector protein into host cells . EspB and EspD have been proposed to form a pore in the host cell membrane, whereas EspA, which forms a large filamentous structure bridging bacterial and host cell surfaces, is thought to provide a conduit for translocation of effector proteins between pores in the bacterial and host cell membranes . Type III secretion has been correlated with an ability to cause contact-dependent haemolysis of red blood cells (RBCs) in vitro . As EspA filaments link bacteria and the host cell, we predicted that intimate bacteria-RBC contact would not be required for EPEC-induced haemolysis and, therefore, in this study we investigated the interaction of EPEC with monolayers of RBCs attached to polylysine-coated cell culture dishes . EPEC caused total RBC haemolysis in the absence of centrifugation and osmoprotection studies were consistent with the insertion of a hydrophilic pore into the RBC membrane . Cell attachment and haemolysis involved interaction between EspA filaments and the RBC membrane and was dependent upon a functional type III secretion system and on EspD, whereas EPEC lacking EspB still caused some haemolysis . Following haemolysis, only EspD was consistently detected in the RBC membrane . This study shows that intimate bacteria-RBC membrane contact is not a requirement for EPEC-induced haemolysis; it also provides further evidence that EspA filaments are a conduit for protein translocation and that EspD may be the major component of a translocation pore in the host cell membrane. Cell Microbiol, 2001 Apr, 3(4), 197 - 211 Role of EspF in host cell death induced by enteropathogenic Escherichia coli; Crane JK et al.; Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries . Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island . One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function . EspF, like the other Esps, is a substrate for secretion by the type III secretory system . Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation . As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability . The espF mutant was deficient in host cell killing despite having normal adherence . The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death . The mode of cell death in cells transfected with espF appeared to be pure apoptosis . EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins. Bioelectromagnetics, 2001 May, 22(4), 260 - 6 Effects of high ELF magnetic fields on enzyme-catalyzed DNA and RNA synthesis in vitro and on a cell-free DNA mismatch repair; Harada S et al.; Environmental electromagnetic fields have been implicated in human cancers . We examined whether high extremely low frequency (ELF) AC magnetic fields could affect DNA synthesis, transcription or repair, using in vitro model systems with defined sequences . The rate and fidelity of DNA polymerase catalyzed DNA synthesis, as well as of RNA polymerase catalyzed RNA synthesis, were not statistically significantly affected by 60 Hz 0.25-0.5 Tesla magnetic fields . The efficiency of mutS dependent mismatch repair with human cell extracts was also not affected by the magnetic field exposure . The results suggest that the core processes related to the transmission of genetic information are stable under high ELF magnetic fields . Mol Microbiol, 2001 Apr, 40(1), 245 - 56 Positive regulation of motility and flhDC expression by the RNA-binding protein CsrA of Escherichia coli; Wei BL et al.; Many species of bacteria devote considerable metabolic resources and genetic information to the ability to sense the environment and move towards or away from specific stimuli using flagella . In Escherichia coli and related species, motility is regulated by several global regulatory circuits, which converge to modulate the overall expression of the master operon for flagellum biosynthesis, flhDC . We now show that the global regulator CsrA of E . coli K-12 is necessary for motility under a variety of growth conditions, as a result of its role as an activator of flhDC expression . A chromosomally encoded flhDC'-'lacZ translational fusion was expressed at three- to fourfold higher levels in csrA wild-type strains than in isogenic csrA mutants . Purified recombinant CsrA protein stimulated the coupled transcription-translation of flhDC'-' lacZ in S-30 extracts and bound to the 5' segment of flhDC mRNA in RNA mobility shift assays . The steady-state level of flhDC mRNA was higher and its half-life was approximately threefold greater in a csrA wild-type versus a csrA mutant strain . Thus, CsrA stimulates flhDC gene expression by a post-transcriptional mechanism reminiscent of its function in the repression of glycogen biosynthesis. Mol Microbiol, 2001 Apr, 40(1), 179 - 88 Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli; Xia B et al.; Escherichia coli contains a large CspA family, CspA to CspI . Here, we demonstrate that E . coli is highly protected against cold-shock stress, as these CspA homologues existed at approximately a total of two million molecules per cell at low temperature and growth defect was not observed until four csp genes (cspA, cspB, cspE and cspG) were deleted . The quadruple-deletion strain acquired cold sensitivity and formed filamentous cells at 15 degrees C although chromosomes were normally segregated . The cold-sensitivity and filamentation phenotypes were suppressed by all members of the CspA family except for CspD, which causes lethality upon overexpression . Interestingly, the cold sensitivity of the mutant was also suppressed by the S1 domain of polynucleotide phosphorylase (PNPase), which also folds into a beta-barrel structure similar to that of CspA . The present results show that cold-shock proteins and S1 domains share not only the tertiary structural similarity but also common functional properties, suggesting that these seemingly distinct protein categories may have evolved from a common primordial RNA-binding protein. Mol Microbiol, 2001 Apr, 40(1), 86 - 98 Site-directed mutagenesis of intimin alpha modulates intimin-mediated tissue tropism and host specificity; Reece S et al.; The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions . This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane . The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far . In addition to binding to Tir, intimin can also bind to a component encoded by the host . The consequence of latter intimin-binding activity may determine tissue tropism and host specificity . In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis . We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction . In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model) . We report that while an I237/897A substitution (positions numbered according to Int280alpha/whole intimin alpha) in intimin alpha did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo . In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model . This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity. Curr Opin Struct Biol, 2001 Apr, 11(2), 201 - 7 Recent advances in FRET: distance determination in protein-DNA complexes; Hillisch A et al.; Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A . Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling . Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology. Thromb Res, 2001 Mar 1, 101(5), 405 - 15 Domain specific monoclonal anti-factor VIII antibodies generated by inclusion body-renatured factor VIII peptides; Huang CC et al.; Production of monoclonal anti-factor VIII (FVIII) antibodies was hampered by the availability of FVIII proteins devoid of albumin and the von Willebrand factor (vWF) . We showed a successful way to generate domain specific anti-FVIII antibodies by using a series of Escherichia coli expressed FVIII fusion peptides . A total of eight fusion peptides were synthesized to cover almost the entire coding region of FVIII . All except one of the fusion peptides were insoluble and became aggregated as inclusion bodies . Purification and refolding of the peptides were accomplished by solublizing them with denaturants and dialyzing them in appropriate buffers, this being followed by chromatography of the refolded fractions on a metal-ion chelating column . These purified FVIII fusion peptides were used individually or as a pool to immunize mice and generate antibodies . Three monoclonal antibodies, D2, E6 and B12, were obtained . D2 recognizes a region (residues 1680-1703) of the light chain of FVIII, E6 recognizes a fragment (residues 744-1021) in the heavy chain, and B12, the A1 domain (residues 89-326) . Both D2 and B12 inhibited >80% FVIII function . The affinities (k(A)) of the antibodies for FVIII were 1.62x10(7) M(-1) for D2 and 2.2x10(8) M(-1) for E6 . Although B12 is inhibitory, it did not show a strong binding affinity with FVIII . The specificity of D2 and E6 for FVIII was demonstrated by immunoprecipitation of the FVIII protein in full-length recombinant FVIII (rFVIII) supplemented FVIII-deficient plasma, but not in FVIII-deficient plasma alone . An enzyme-linked immunosorbant assay (ELISA) using D2 or E6 was designed to detect plasma FVIII . The system may be useful in monitoring FVIII in cultured supernatants and in mouse models for gene therapy experiments. FEBS Lett, 2001 Apr 6, 494(1-2), 11 - 4 The structure and nucleotide occupancy of bovine mitochondrial F(1)-ATPase are not influenced by crystallisation at high concentrations of nucleotide; Menz RI et al.; Analysis of tryptophan mutants of F(1)-ATPase from Escherichia coli {Lobau et al . (1997) FEBS Lett . 404, 15-18} suggested that nucleotide concentrations used to grow crystals for the determination of the structure of bovine F(1)-ATPase {Abrahams et al . (1994) Nature 370, 621-628} would be sufficient to occupy only two catalytic sites, and that higher concentrations of nucleotide would result in all three sites being occupied . We have determined the structure of bovine F(1)-ATPase at 2.9 A resolution with crystals grown in the presence of 5 mM AMPPNP and 5 microM ADP . Similar to previous structures of bovine F(1)-ATPase determined with crystals grown in the presence of lower nucleotide concentrations, only two beta-subunits have bound nucleotide and the third subunit remains empty. J Gen Virol, 2001 May, 82(Pt 5), 1049 - 60 Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning; Arias A et al.; The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed . The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT--PCR products cloned in E . coli) . The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies . Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture . The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies . Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies . Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution . The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level. Protein Eng, 2001 Feb, 14(2), 135 - 40 Asn to Lys mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin; Narhi LO et al.; Erythropoietin (EPO) derived from Escherichia coli is unstable to elevated temperature and tends to aggregate with time, making it unsuitable for high-resolution structure analysis . The mammalian EPO contains about 40% carbohydrate, which makes this protein more stable and less prone to aggregate than non-glycosylated E.coli-derived EPO, but makes it unsuitable for high-resolution analysis owing to its size and flexibility . In an attempt to decrease the aggregation of E.coli-derived EPO, the three asparagine residues at positions 24, 38 and 83 were mutated to lysine residues . In the native protein, these residues are the sites of N-linked glycosylation, which suggests that they should be located on the surface of the protein and should not be involved in interactions in the hydrophobic protein core . Therefore, the substitution of basic amino acids for these neutral asparagine residues is not expected to affect the protein structure, but should increase the isoelectric point of the protein and its net positive charge, decreasing its tendency to aggregate at or below neutral pH due to electrostatic interactions . No apparent alterations in receptor binding, as determined by both cell-surface receptor competition assay and in vitro receptor dimerization experiments, were observed when these mutations were introduced into the EPO sequence . However, this mutant protein displayed a significant increase in stability to heat treatment and to storage, relative to the wild-type molecule . This resulted in a greater number of observable cross peaks in the mutant EPO in 2D NOESY experiments . However, the mutant was similar to the wild-type in stability when urea was used as a denaturant . This indicates that the introduced mutations resulted in a decrease in aggregation with heating or with prolonged incubation at ambient temperature, without changing the conformational stability or the receptor binding affinity of the mutant protein . This approach of placing charged residues at sites where N-glycosylation occurs in vivo could be applied to other systems as well. J Biol Chem, 2001 Jul 13, 276(28), 26269 - 75 Epub 2001 Apr 10. Cloning and expression of a novel human glutaredoxin (Grx2) with mitochondrial and nuclear isoforms; Lundberg M et al.; Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase . Today glutaredoxins are known as a multifunctional family of GSH-disulfide-oxidoreductases belonging to the thioredoxin fold superfamily . In contrast to Escherichia coli and yeast, a single human glutaredoxin is known . We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1 . The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area . The human Grx2 gene, located at chromosome 1q31.2--31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues . Two alternatively spliced Grx2 mRNA isoforms that differed in their 5' region were identified . These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively . The 125-residue Grx domain and the two full-length variants were expressed in E . coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities . Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria . We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal. J Biol Chem, 2001 May 4, 276(18), 15155 - 63 Epub 2001 Jan 31. Mutagenic and nonmutagenic bypass of DNA lesions by Drosophila DNA polymerases dpoleta and dpoliota; Ishikawa T et al.; cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B . Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpoletaDeltaC and dpoliotaDeltaC, respectively, exhibit DNA polymerase activity . dpoletaDeltaC and dpoliotaDeltaC efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner . dpoletaDeltaC shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in an error-prone manner . dpoliotaDeltaC scarcely bypasses these lesions . Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase . The human XPV gene product, hpoleta, bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6-4)PP, whereas Escherichia coli DNA polymerase V (UmuD'(2)C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J . S., O'Donnell, M., Woodgate, R., and Goodman, M . F . (2000) Nature 404, 1014-1018) . Both dpoletaDeltaC and DNA polymerase V preferentially incorporate GA opposite TT-(6-4)PP . The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases. Biochemistry, 2001 Mar 27, 40(12), 3730 - 6 Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center; Mulliez E et al.; In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide . The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx) . It has been proposed that a reductant serves to generate a reduced {4Fe-4S}(+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical . Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical . However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding . It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin . Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical . The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine. Biochemistry, 2001 Mar 27, 40(12), 3674 - 80 Evidence that SecB enhances the activity of SecA; Kim J et al.; In Escherichia coli, SecA is a critical component of the protein transport machinery which powers the translocation process by hydrolyzing ATP and recognizing signal peptides which are the earmark of secretory proteins . In contrast, SecB is utilized by only a subset of preproteins to prevent their premature folding and chaperone them to membrane-bound SecA . Using purified components and synthetic signal peptides, we have studied the interaction of SecB with SecA and with SecA-signal peptide complexes in vitro . Using a chemical cross-linking approach, we find that the formation of SecA-SecB complexes is accompanied by a decrease in the level of cross-linking of SecA dimers, suggesting that SecB induces a conformational change in SecA . Furthermore, functional signal peptides, but not dysfunctional ones, promote the formation of SecA-SecB complexes . SecB is also shown to directly enhance the ATPase activity of SecA in a concentration-dependent and saturable manner . To determine the biological consequence of this finding, the influence of SecB on the signal peptide-stimulated SecA/lipid ATPase was studied using synthetic peptides of varying hydrophobicity . Interestingly, the presence of SecB can sufficiently boost the response of signal peptides with moderate hydrophobicity such that it is comparable to the activity generated by a more hydrophobic peptide in the absence of SecB . The results suggest that SecB directly enhances the activity of SecA and provide a biochemical basis for the enhanced transport efficiency of preproteins in the presence of SecB in vivo. Biochemistry, 2001 Mar 27, 40(12), 3606 - 14 Protein kinase C phosphorylates nonmuscle myosin-II heavy chain from Drosophila but regulation of myosin function by this enzyme is not required for viability in flies; Su Z et al.; Conventional myosins (myosin-IIs) generate forces for cell shape change and cell motility . Myosin heavy chain phosphorylation regulates myosin function in simple eukaryotes and may also be important in metazoans . To investigate this regulation in a complex eukaryote, we purified the Drosophila myosin-II tail expressed in Escherichia coli and showed that it was phosphorylated in vitro by protein kinase C(PKC) at serines 1936 and 1944, which are located in the nonhelical globular tail piece . These sites are close to a conserved serine that is phosphorylated in vertebrate, nonmuscle myosin-IIs . If the two serines are mutagenized to alanine or aspartic acid, phosphorylation no longer occurs . Using a 341 amino acid tail fragment, we show that there is no difference in the salt-dependent assembly of wild-type phosphorylated and mutagenized polypeptides . Thus, the nonmuscle myosin heavy chain in Drosophila, which is encoded by the zipper gene, appears to be similar to rabbit nonmuscle myosin-IIA . In vivo, we generated transgenic flies that expressed the various myosin heavy chain variants in a zipper null or near-null genetic background . Like their wild-type counterparts, such variants are able to completely rescue the lethal phenotype due to severe zipper mutations . These results suggest that while the myosin-II heavy chain can be phosphorylated by PKC, regulation by this enzyme is not required for viability in Drosophila . Conservation during 530-1000 million years of evolution suggests that regulation by heavy chain phosphorylation may contribute to nonmuscle myosin-II function in some real, but minor, way. Biochemistry, 2001 Mar 27, 40(12), 3420 - 6 Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor; Chang AL et al.; The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis . This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG . Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme . Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity . O(2) regulation is due to deoxyheme being a better activator than oxyheme . AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain . The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors . The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold . The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher . Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger. Med Vet Entomol, 2001 Mar, 15(1), 58 - 63 Quantification of pyrethroid insecticides from treated bednets using a mosquito recombinant glutathione S-transferase; Enayati AA et al.; Recombinant glutathione S-transferase (agGST1-6) from the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) was expressed in Escherichia coli using a pET3a vector system . The expressed enzyme was biochemically active with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) . Activity of agGST1-6 with GSH and CDNB was inhibited to different degrees by both alpha-cyano and non-alpha-cyano pyrethroid insecticides . This inhibition was used to develop an assay for quantification of pyrethroids . Standard curves of insecticide concentration against percentage of enzyme inhibition or volume of iodine solution were established by spectrophotometry and iodine volumetric titration, respectively, for permethrin and deltamethrin . These assays allowed estimation of pyrethroid concentrations both spectrophotometrically and visually . For the residue assay of each insecticide, a cut-off point of 50% of the initial pyrethroid impregnation concentration was used, which should differentiate between biologically active and inactive treated bednets . The cross-reactivity of the primary permethrin photodegradants (3-phenoxyalcohol and 3-phenoxybenzoic acid) with the recombinant agGST1-6 was assayed in the same system . No agGST1-6 inhibition by the insecticide metabolites was observed, suggesting that the system is unaffected by primary permethrin metabolites and will accurately measure insecticide parent compound concentrations . The estimated pyrethroid insecticide concentrations, given spectrophotometrically and by iodine titration assay, were comparable to those obtained by direct HPLC quantification of residues extracted from bednets . Hence, it should be relatively easy to adapt this method to produce a test kit for residue quantification in the field. Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 4950 - 4 Epub 2001 Apr 10. Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells; Davis BM et al.; Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected . An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy . Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli . To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea . This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea . Of these, one was recovered significantly more frequently than the others . Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant. Proc Natl Acad Sci U S A, 2001 Apr 24, 98(9), 5007 - 12 Epub 2001 Apr 10. Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy; Henn A et al.; The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA . Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized . Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence . This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology . Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA . Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP . Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases . However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases. EMBO J, 2001 Apr 17, 20(8), 2041 - 50 The structural basis of acyl coenzyme A-dependent regulation of the transcription factor FadR; van Aalten DM et al.; FadR is an acyl-CoA-responsive transcription factor, regulating fatty acid biosynthetic and degradation genes in Escherichia coli . The apo-protein binds DNA as a homodimer, an interaction that is disrupted by binding of acyl-COA: The recently described structure of apo-FadR shows a DNA binding domain coupled to an acyl-CoA binding domain with a novel fold, but does not explain how binding of the acyl-CoA effector molecule > 30 A away from the DNA binding site affects transcriptional regulation . Here, we describe the structures of the FadR-operator and FadR- myristoyl-CoA binary complexes . The FadR-DNA complex reveals a novel winged helix-turn-helix protein-DNA interaction, involving sequence-specific contacts from the wing to the minor groove . Binding of acyl-CoA results in dramatic conformational changes throughout the protein, with backbone shifts up to 4.5 A . The net effect is a rearrangement of the DNA binding domains in the dimer, resulting in a change of 7.2 A in separation of the DNA recognition helices and the loss of DNA binding, revealing the molecular basis of acyl-CoA-responsive regulation. J Allergy Clin Immunol, 2001 Apr, 107(4), 724 - 31 Recombinant allergens Pru av 1 and Pru av 4 and a newly identified lipid transfer protein in the in vitro diagnosis of cherry allergy; Scheurer S et al.; BACKGROUND: In central and northern Europe food allergy to fruits of the Rosaceae family is strongly associated with birch pollinosis because of the existence of IgE cross-reactive homologous allergens in birch pollen and food . By contrast, in the Mediterranean population allergic reactions to these fruits frequently are not related to birch pollen allergy and are predominantly elicited by lipid transfer proteins (LTPs) . OBJECTIVE: We sought to determine the prevalence of IgE sensitization to the recombinant cherry allergens Pru av 1 and Pru av 4 in comparison with cherry extract within a representative group of patients who were allergic to cherries recruited in Germany and to compare the relevance of IgE to cherry LTPs in Italian patients . METHODS: Recombinant Pru av 1 and rPru av 4 were available from earlier studies . The cDNA of the cherry LTPs was obtained by using a PCR-cloning strategy . The protein was expressed in Escherichia coli and purified by means of metal chelate affinity chromatography . Sera from 101 German patients with birch pollinosis and oral allergy syndrome to cherry and sera from 7 Italian patients with cherry allergy were investigated by using enzyme allergosorbent tests for IgE reactivity with cherry extract, rPru av 1, rPru av 4, and the recombinant cherry LTP . Inhibition experiments were performed to compare the IgE reactivity of natural and recombinant cherry LTPs and to investigate potential cross-reactivity with birch pollen allergens . RESULTS: The LTP from cherry comprises 91 amino acids and a 26 amino acid signal peptide . The mature cherry LTP shows high amino acid sequence identity with allergenic LTPs from peach (Pru p 3, 88%), apricot (Pru ar 3, 86%), and maize (Zea m 14, 59%) and displays no IgE cross-reactivity with birch pollen . The IgE prevalences in the German patients were as follows: LTP, 3 of 101 (3%); rPru av 1, 97 of 101 (96.0%); rPru av 4, 16 of 101 (16.2%); and cherry extract, 98 of 101 (97%) . All 7 Italian patients had IgE against the cherry LTP . CONCLUSIONS: Recombinant allergens are useful tools for a more accurate in vitro IgE-based diagnosis of cherry allergy . Taken together, they mimic the allergenic activity of cherry extract, having slightly higher biologic activity . Sensitization to the cherry LTP is relevant for a minority of patients recruited in Germany, but our data indicate that it may be a major allergen in Italy. Biochim Biophys Acta, 2001 Apr 7, 1546(2), 412 - 21 Influence of ligand binding to human cytochrome P-450 1A2: conformational activation and stabilization by alpha-naphthoflavone; Cho US et al.; Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents . alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification . However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood . We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx . 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values . The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation . Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents . The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2 . The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content . The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx . 5 degrees C increase in thermal stability. Biochim Biophys Acta, 2001 Apr 7, 1546(2), 346 - 55 Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein; Feeney KA et al.; We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed . A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli . Activity of the synthetic genes in E . coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250 . However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC . The R4 and R5 peptides were not purified . The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts . Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits . We conclude that expression of perfect repeat peptides in E . coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins. Biochim Biophys Acta, 2001 Apr 7, 1546(2), 268 - 81 Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme; Nowicki C et al.; The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS . Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T . cruzi . Both recombinant and authentic T . cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds . Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors . Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated . TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate . The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T . cruzi TAT . Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T . cruzi's TAT activity. J Control Release, 2001 Apr 28, 71(3), 339 - 50 Oil components modulate physical characteristics and function of the natural oil emulsions as drug or gene delivery system; Chung H et al.; Oil-in-water (o/w) type lipid emulsions were formulated by using 18 different natural oils and egg phosphatidylcholine (egg PC) to investigate how emulsion particle size and stability change with different oils . Cottonseed, linseed and evening primrose oils formed emulsions with very large and unstable particles . Squalene, light mineral oil and jojoba bean oil formed stable emulsions with small particles . The remaining natural oils formed moderately stable emulsions . Emulsions with smaller initial particle size were more stable than those with larger particles . The correlation between emulsion size made with different oils and two physical properties of the oils was also investigated . The o/w interfacial tension and particle size of the emulsion were inversely proportional . The effect of viscosity was less pronounced . To study how the oil component in the emulsion modulates the in vitro release characteristics of lipophilic drugs, three different emulsions loaded with two different drugs were prepared . Squalene, soybean oil and linseed oil emulsions represented the most, medium and the least stable systems, respectively . For the lipophilic drugs, release was the slowest from the most stable squalene emulsion, followed by soybean oil and then by linseed oil emulsions . Cationic emulsions were also prepared with the above three different oils as gene carriers . In vitro transfection activity was the highest for the most stable squalene emulsion followed by soybean oil and then by linseed oil emulsions . Even though the in vitro transfection activity of emulsions were lower than the liposome in the absence of serum, the activity of squalene emulsion, for instance, was ca . 30 times higher than that of liposome in the presence of 80% (v/v) serum . In conclusion, the choice of oil component in o/w emulsion is important in formulating emulsion-based drug or gene delivery systems. J Microbiol Methods, 2001 May, 45(1), 31 - 9 Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner; Stender H et al.; A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described . The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E . coli . Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner . Results were provided as fluorescent spots representing E . coli microcolonies on the membrane filter surface . The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter . Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye. Mol Biochem Parasitol, 2001 Apr 6, 113(2), 289 - 301 Cloning and characterization of the subunits comprising the catalytic core of the Trypanosoma brucei mitochondrial ATP synthase; Brown B SV et al.; The Trypanosoma brucei mitochondrial F(1)-ATPase has been previously isolated and characterized . It is composed of five subunits of molecular weights 55000, 42000, 32000, 22000, and 17000 {1} . We have identified the alpha and beta subunits of the T . brucei F(1)-ATPase by N-terminal sequence determination together with analysis of cDNA and genomic clones . The genes for both subunits are homologous to the same subunits from other organisms . They contain the Walker A and B boxes of homology and a putative mitochondrial import sequence . The isolated T . brucei alpha subunit is unusually small at 42 kDa . The alpha cDNA clone encodes a protein of predicted size 59 kDa with a mitochondrial import presequence at the N-terminus . The predicted size was confirmed by expression of a 59 kDa protein from the cDNA clone in vitro . These results suggest that the alpha subunit may have an unusually large mitochondrial presequence of 159 amino acids . In contrast, the estimated size of the native beta subunit (55 kDa) correlates well with the size predicted from the cDNA clone, 57 kDa, from which a 21 amino acid presequence has been removed in vivo . The size of the beta subunit was confirmed by expression in an in vitro and an Escherichia coli expression system . The purified recombinant beta subunit, like the native F(1)-ATPase, can be labeled by the photoaffinity nucleotide analogue 8-azido ATP . Binding of the 8-azido ATP probe is best competed by the natural substrate ATP, and is significantly reduced by pretreatment with the inhibitor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazide as has been shown with beta subunits of other organisms . The differential binding of this photoaffinity analogue was used to resolve the identities of the alpha and beta subunits of the ATP synthase from T . brucei . These results are in contrast to results previously obtained for a related trypanosomatid Crithidia fasciculata. Mol Biochem Parasitol, 2001 Apr 6, 113(2), 241 - 9 Kinetic properties of dihydrofolate reductase from wild-type and mutant Plasmodium vivax expressed in Escherichia coli; Tahar R et al.; Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR) . In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR . Recently, mutations at homologous positions have been observed in the P . vivax gene . Since P . vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli . Induced expression yielded a protein product that precipitated as an inclusion body in E . coli . The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography . Kinetic properties of the recombinant P . vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH . Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected . The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance. J Biol Chem, 2001 Jun 15, 276(24), 21500 - 5 Epub 2001 Apr 09. Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase; Whittle E et al.; Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity . Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates . The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis . These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates . Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme . Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates . Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases . These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog. J Biol Chem, 2001 Jun 22, 276(25), 22844 - 9 Epub 2001 Apr 09. Minimal functional structure of Escherichia coli 4.5 S RNA required for binding to elongation factor G; Nakamura K et al.; Escherichia coli cells contain abundant amounts of metabolically stable 4.5 S RNA . Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA, and it plays an essential role in targeting proteins containing signal peptide to the secretory apparatus by forming an signal recognition-like particle with Ffh protein . It also binds independently to protein elongation factor G (EF-G) and functions in the translation process . This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges . We examined the binding activity of mutants with systematic deletions to define the minimal functional interaction domain of 4.5 S RNA that interacts with EF-G . This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A42, G48, and G49 did not affect binding . These data suggested that the 10 nucleotides in 4.5 S RNA, which are conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G binding . Based on the NMR-derived structure of mutant A47U, we further verified that substituting U at A47 causes striking structural changes and the loss of the symmetrical bulge . These results indicate the mechanism by which EF-G interacts with 4.5 S RNA and the importance of the bulge structure for EF-G binding. J Biol Chem, 2001 Jun 15, 276(24), 21343 - 50 Epub 2001 Apr 06. Functional expression, characterization, and purification of the catalytic domain of human 11-beta -hydroxysteroid dehydrogenase type 1; Walker EA et al.; 11-beta-hydroxysteroid dehydrogenase type 1 catalyzes the conversion of cortisone to hormonally active cortisol and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity . The enzyme is a glycosylated membrane-bound protein that has proved difficult to purify in an active state . Extracted enzyme typically loses the reductase properties seen in intact cells and shows principally dehydrogenase activity . The C-terminal catalytic domain is known to contain a disulfide bond and is located within the lumen of the endoplasmic reticulum, anchored to the membrane by a single N-terminal transmembrane domain . We report here the functional expression of the catalytic domain of the human enzyme, without the transmembrane domain and the extreme N terminus, in Escherichia coli . Moderate levels of soluble active protein were obtained using an N-terminal fusion with thioredoxin and a 6xHis tag . In contrast, the inclusion of a 6xHis tag at the C terminus adversely affected protein solubility and activity . However, the highest levels of active protein were obtained using a construct expressing the untagged catalytic domain . Nonreducing electrophoresis revealed the presence of both monomeric and dimeric disulfide bonded forms; however, mutation of a nonconserved cysteine residue resulted in a recombinant protein with no intermolecular disulfide bonds but full enzymatic activity . Using the optimal combination of plasmid construct and E . coli host strain, the recombinant protein was purified to apparent homogeneity by single step affinity chromatography . The purified protein possessed both dehydrogenase and reductase activities with a K(m) of 1.4 micrometer for cortisol and 9.5 micrometer for cortisone . This study indicates that glycosylation, the N-terminal region including the transmembrane helix, and intermolecular disulfide bonds are not essential for enzyme activity and that expression in bacteria can provide active recombinant protein for future structural and functional studies. Bioinformatics, 2001 Mar, 17(3), 237 - 48 Flexibility of the genetic code with respect to DNA structure; Baisnee PF et al.; MOTIVATION: The primary function of DNA is to carry genetic information through the genetic code . DNA, however, contains a variety of other signals related, for instance, to reading frame, codon bias, pairwise codon bias, splice sites and transcription regulation, nucleosome positioning and DNA structure . Here we study the relationship between the genetic code and DNA structure and address two questions . First, to which degree does the degeneracy of the genetic code and the acceptable amino acid substitution patterns allow for the superimposition of DNA structural signals to protein coding sequences? Second, is the origin or evolution of the genetic code likely to have been constrained by DNA structure? RESULTS: We develop an index for code flexibility with respect to DNA structure . Using five different di- or tri-nucleotide models of sequence-dependent DNA structure, we show that the standard genetic code provides a fair level of flexibility at the level of broad amino acid categories . Thus the code generally allows for the superimposition of any structural signal on any protein-coding sequence, through amino acid substitution . The flexibility observed at the level of single amino acids allows only for the superimposition of punctual and loosely positioned signals to conserved amino acid sequences . The degree of flexibility of the genetic code is low or average with respect to several classes of alternative codes . This result is consistent with the view that DNA structure is not likely to have played a significant role in the origin and evolution of the genetic code. Bioinformatics, 2001 Mar, 17(3), 226 - 36 Basic Gene Grammars and DNA-ChartParser for language processing of Escherichia coli promoter DNA sequences; Leung S et al.; MOTIVATION: The field of 'DNA linguistics' has emerged from pioneering work in computational linguistics and molecular biology . Most formal grammars in this field are expressed using Definite Clause Grammars but these have computational limitations which must be overcome . The present study provides a new DNA parsing system, comprising a logic grammar formalism called Basic Gene Grammars and a bidirectional chart parser DNA-ChartParser . RESULTS: The use of Basic Gene Grammars is demonstrated in representing many formulations of the knowledge of Escherichia coli promoters, including knowledge acquired from human experts, consensus sequences, statistics (weight matrices), symbolic learning, and neural network learning . The DNA-ChartParser provides bidirectional parsing facilities for BGGs in handling overlapping categories, gap categories, approximate pattern matching, and constraints . Basic Gene Grammars and the DNA-ChartParser allowed different sources of knowledge for recognizing E.coli promoters to be combined to achieve better accuracy as assessed by parsing these DNA sequences in real-world data sets. Biochemistry, 2001 Apr 17, 40(15), 4844 - 52 Properties of microtubules assembled from mammalian tubulin synthesized in Escherichia coli; Shah C et al.; When isolated from tissues, the alpha beta-dimeric protein tubulin consists of multiple isoforms which originate from the expression and subsequent posttranslational modification of multiple polypeptide sequences . Microtubules studied in vitro consist of mixtures of these isoforms . It is therefore not known whether dimers composed of single sequences of alpha- and beta-tubulin can polymerize to form microtubules, or whether posttranslational modifications may be necessary for microtubule assembly . To initiate investigation of these questions, rabbit reticulocyte lysate, which contains the cytoplasmic chaperonin CCT and its cofactors, was employed to prepare substantial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta-tubulins recombinantly synthesized in E . coli . This recombinant tubulin is composed of only a single alpha-chain and a single beta-chain . When analyzed after folding by isoelectric focusing, each chain yielded only one band, indicating that neither was detectably posttranslationally modified in the course of the folding reaction . When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein . Electron microscopy showed them to be of normal morphology . Analysis of their protein composition showed that they are composed nearly entirely of recombinant tubulin . These results demonstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules . They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro. Biochemistry, 2001 Apr 17, 40(15), 4738 - 44 Probing the active site of L-aspartate oxidase by site-directed mutagenesis: role of basic residues in fumarate reduction; Tedeschi G et al.; L-Aspartate oxidase is a very particular oxidase which behaves as a fumarate reductase in anaerobic conditions . Its primary and tertiary structures present remarkable similarity with the soluble fumarate reductase (FRD) from Shewanella frigidimarina and the flavin subunit of the membrane-bound fumarate reductase from Escherichia coli and Wolinella succinogenes . This and other extensive similarities are consistent with the idea that a common catalytic mechanism for the reduction of fumarate operates for all members of this enzyme group and that the key residues involved in the substrate binding and catalysis are conserved . This manuscript reports information about the role of these basic residues in L-aspartate oxidase: R290, R386, H244, and H351 . By means of site-directed mutagenesis, R290 and R386 are mutated to Leu and H351 and H244 are mutated both to Ala and Ser . H351, H244, and R386 mutants bind substrate analogues with higher dissociation constants and present lower k(cat)/K(m) values in the reduction of fumarate . Therefore, the results indicate that R386, H244, and H351 are important for the binding of the substrate fumarate and may play an important but not essential role in catalysis . R290, on the contrary, is mainly involved in catalysis and not in substrate binding since its mutation abolishes the catalytic activity without lowering the affinity of the enzyme for the substrate . The redox properties of all the mutants are identical to the wild-type . The findings are consistent with a model of L-aspartate oxidase active site based on the hypothesis proposed for the soluble FRD from S . fridimarina. Biochemistry, 2001 Apr 17, 40(15), 4714 - 21 Redox properties of the PutA protein from Escherichia coli and the influence of the flavin redox state on PutA-DNA interactions; Becker DF et al.; The PutA flavoprotein from Escherichia coli is both a transcriptional repressor and a membrane-associated proline dehydrogenase . PutA represses transcription of the putA and putP genes by binding to the control region DNA of the put regulon (put intergenic DNA) . Previous work has shown that FAD has a role in regulating the transcriptional repressor and membrane binding functions of the PutA protein . To test the influence of the FAD redox state on PutA--DNA interactions, we characterized the redox properties of the PutA flavoprotein from E . coli . At pH 7.5, an E(m)(E--FAD/E--FADH(2)) of --0.076 V for the two-electron reduction of PutA-bound FAD was determined by potentiometric titrations . Stabilization of semiquinone species was not observed during potentiometric measurements . Dithionite reduction of PutA, however, caused formation of red anionic semiquinone . The E(m) value for the proline/Delta(1)-pyrroline-5-carboxylate couple was determined to be --0.123 V, demonstrating the reduction of PutA by proline is favored by a potential difference (Delta E degrees ') of more than 0.045 V . Characterization of the PutA redox properties in the presence of put intergenic DNA revealed an E(m)(E(DNA)--FAD/E(DNA)--FADH(2)) of --0.086 V . The 10 mV negative shift in E(m) corresponds to just a 2.3-fold increase in the dissociation constant of PutA with the DNA upon reduction of FAD . Thus, it appears the FAD redox state has little influence on the overall PutA--DNA interactions. Biochemistry, 2001 Apr 17, 40(15), 4645 - 53 Ligand binding sites in Escherichia coli inorganic pyrophosphatase: effects of active site mutations; Hyytia T et al.; Type I soluble inorganic pyrophosphatases (PPases) are well characterized both structurally and mechanistically . Earlier we measured the effects of active site substitutions on pH--rate profiles for the type I PPases from both Escherichia coli (E-PPase) and Saccharomyces cerevisae (Y-PPase) . Here we extend these studies by measuring the effects of such substitutions on the more discrete steps of ligand binding to E-PPase, including (a) Mg(2+) and Mn(2+) binding in the absence of added ligand; (b) Mg(2+) binding in the presence of either P(i) or hydroxymethylbisphosphonate (HMBP), a competitive inhibitor of E-PPase; and (c) P(i) binding in the presence of Mn(2+) . The active site of a type I PPase has well-defined subsites for the binding of four divalent metal ions (M1--M4) and two phosphates (P1, P2) . Our results, considered in light of pertinent results from crystallographic studies on both E-PPase and Y-PPase and parallel functional studies on Y-PPase, allow us to conclude the following: (a) residues E20, D65, D70, and K142 play key roles in the functional organization of the active site; (b) the major structural differences between the product and substrate complexes of E-PPase are concentrated in the lower half of the active site; (c) the M1 subsite is functionally isolated from the rest of the active site; and (d) the M4 subsite is an especially unconstrained part of the active site. Biochemistry, 2001 Apr 17, 40(15), 4622 - 32 Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity; Edwards RA et al.; Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site . These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface . Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center . The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein . The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring . Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E . coli MnSOD) all lack the high pH transition of the wt enzyme . This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34 . Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding . These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis. Biochemistry, 2001 Apr 17, 40(15), 4569 - 82 An XAS investigation of product and inhibitor complexes of Ni-containing GlxI from Escherichia coli: mechanistic implications; Davidson G et al.; Escherichia coli glyoxalase I (GlxI) is a metalloisomerase that is maximally activated by Ni(2+), unlike other known GlxI enzymes which are active with Zn(2+) . The metal is coordinated by two aqua ligands, two histidines (5 and 74), and two glutamates (56 and 122) . The mechanism of E . coli Ni-GlxI was investigated by analyzing Ni K-edge X-ray absorption spectroscopic (XAS) data obtained from the enzyme and complexes formed with the product, S-D-lactoylglutathione, and various inhibitors . The analysis of X-ray absorption near edge structure (XANES) was used to determine the coordination number and geometry of the Ni site in the various Ni-GlxI complexes . Metric details of the Ni site structure were obtained from the analysis of extended X-ray absorption fine structure (EXAFS) . Interaction of S-D-lactoylglutathione (product) or octylglutathione with the enzyme did not change the structure of the Ni site . However, analysis of XAS data obtained from a complex formed with a peptide hydroxamate bound to Ni-GlxI is consistent with this inhibitor binding to the Ni center by displacement of both water molecules . XANES analysis of this complex is best fit with a five-coordinate metal and, given the fact that both histidine ligands are retained, suggests the loss of a glutamate ligand . The loss of a glutamate ligand would preserve the neutral charge on the Ni complex and is consistent with the lack of a significant shift in the Ni K-edge energy in this complex . These data are compared with data obtained from the E . coli Ni-GlxI selenomethionine-substituted enzyme . The replacement of three methionine residues in the native enzyme with selenomethionine does not affect the structure of the Ni site . However, addition of the peptide hydroxamate inhibitor leads to the formation of a complex whose structure as determined by XAS analysis is consistent with inhibitor binding via displacement of both water molecules but retention of both histidine and glutamate ligands . This leads to an anionic complex, which is consistent with an observed 1.7 eV decrease in the Ni K-edge energy . Plausible reaction mechanisms for Ni-GlxI are discussed in light of the structural information available. Biochemistry, 2001 Apr 17, 40(15), 4560 - 8 ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2; Zhu X et al.; Cholera toxin (CT) and the heat-labile enterotoxin (LT) from Escherichia coli are highly related in terms of structure and biochemical activities and are the causative agents of cholera and traveler's diarrhea, respectively . The pathophysiological action of these toxins requires their activity as ADP-ribosyltransferases, transferring the ADP-ribose moiety from NAD onto the stimulatory, regulatory component of adenylyl cyclase, Gs . This reaction is highly dependent on the protein cofactor, termed ADP-ribosylation factor (ARF), that is itself a 20 kDa regulatory GTPase . In this study, we define sites of interaction between LTA and human ARF3 . The residues identified as important to ARF binding include several of those previously shown to bind to the A2 subunit of the toxin and those important to the organization of two flexible loops, previously implicated as regulators of substrate entry . A model for how ARF acts to enhance the catalytic activity is proposed . A critical portion of the overlap between ARF and LTA(2) in binding LTA(1) includes a short region of sequence homology between LTA(2) and the switch II region of ARF . LTA(2) also interacted with ARF effectors in two-hybrid assays, and thus, we discuss the possibility that the LTA(2) subunit may function in cells as a partial ARF mimetic to compete for the binding of ARF to LTA(1) or regulate aspects of the toxin's transport from the cell surface to the ER. Genetica, 2000, 108(3), 229 - 37 The kinetics of transposable element autoregulation; Townsend JP et al.; Kinetic modeling of the self-regulatory mechanisms of transposable elements (TEs) involving interactions of one or a few gene products makes predictions that are often at odds with observed results . In particular, explanations of TE autorepression at high copy number that invoke a decrease in number of active monomers through dimerization, amyloidization, and protein-mRNA binding to create an inactive state are not supported by analysis of the corresponding kinetic models . This is also true for similar mRNA-mRNA binding models . Self-repression in mariner as well as other TEs can, however, be explained by a host-independent model in which inactive dimers compete with monomers for TE binding sites at the ends of the element . This model would also allow heterodimer poisoning to down-regulate transposition in the presence of divergent nonautonomous elements, since nondivergent monomers would be required at both TE ends for transposition. Can J Physiol Pharmacol, 2001 Mar, 79(3), 213 - 9 The role of K+ATP channels in the control of pre- and post-ischemic left ventricular developed pressure in septic rat hearts; Ismail JA et al.; Myocardial function is impaired 24 h after the induction of sepsis, however, recovery of left ventricular (LV) function after 35 min of global ischemia is complete . The mechanisms by which this protection occurs are unknown . Ischemic preconditioning, another form of myocardial protection from ischemia/reperfusion (I/R) injury, has been shown to be modulated by ATP-sensitive potassium (K+ATP) channels . To investigate the role of K+ATP channels in the regulation of coronary flow (CF) and protection from I/R injury in septic rat hearts, we assessed the effects of the K+ATP channel antagonist glibenclamide (GLIB) and the agonist cromakalim (CROM) on pre- and post-ischemic CF and left ventricular developed pressure (LVDP) . Although GLIB decreased pre-ischemic CF in both control and septic rat hearts, LVDP was unaffected . After I/R, CF was decreased in GLIB-treated control and septic rat hearts and LVDP was more severely depressed in control rat hearts than in septic rat hearts . CROM increased pre-ischemic CF in the septic group although LVDP was unaltered in both groups . After I/R, control rat heart CF was depressed but LVDP completely recovered . Post-ischemic CF in septic rat hearts was elevated compared with vehicle-treated septic rat hearts, but the recovery of LVDP was not improved . These results suggest that K+ATP channels modulate CF in septic rat hearts, but do not mediate cardioprotection as observed in control rat hearts. Epidemiol Infect, 2001 Feb, 126(1), 139 - 45 Selection bias in epidemiological studies of infectious disease using Escherichia coli and avian cellulitis as an example; Singer RS et al.; In epidemiological studies of infectious disease, researchers often rely on specific cues of the host, such as clinical signs, as surrogate indicators of pathogen presence . A selection bias would manifest if the specific visual cues used in sampling for the pathogen were not representative of the full range of signs caused by the strains of that pathogen . In our molecular epidemiological studies of Escherichia coli associated with avian cellulitis in broilers, we collect carcasses at the processing plant based on visual cues of lesion morphology . Therefore, the objectives of this study were to: (1) explore the potential impacts of selection bias in an application of infectious disease epidemiology, and (2) utilize a validation protocol to assess the potential for selection bias in our molecular epidemiological studies of E . coli and avian cellulitis . In two different trials, E . coli DNA fingerprints were compared between birds that our observers collected and the birds that the observers missed . Using Fisher's exact tests and simulation models, we determined that the isolates collected by the observers were not significantly different from the isolates missed by the observers (P > 0.60 in both trials) . Our method of selecting birds suspected of having cellulitis did not significantly bias our inferences about the population of E . coli associated with cellulitis in the flock . We encourage more investigators to critically assess the relationship of the sample to the target population in epidemiological studies of infectious disease. Acta Microbiol Pol, 2000, 49(3-4), 253 - 60 Prediction of a novel RNA 2'-O-ribose methyltransferase subfamily encoded by the Escherichia coli YgdE open reading frame and its orthologs; Bujnicki JM et al.; The amino acid sequence of the RNA 2'-O-ribose methyltranserase RrmJ was used as a probe for detecting putative homologs through iterative searches of genomic databases . We found a previously unannotated YgdE open reading frame (ORF) in the genome sequences of Escherichia coli and other gamma-Proteobacteria, which shares key features with RrmJ, despite the mutual sequence similarity of these proteins is relatively low . The predicted structural compatibility and the conservation of all functionally important residues between RrmJ and YgdE strongly suggests that the newly identified methyltranserase also modifies 2'-OH groups of ribose . The N-terminal region of YgdE, which has no counterpart in RrmJ, is predicted to form an independent domain, possibly involved in target recognition. J Biol Inorg Chem, 2001 Feb, 6(2), 189 - 95 Expression, purification, and characterization of NosL, a novel Cu(I) protein of the nitrous oxide reductase (nos) gene cluster; McGuirl MA et al.; NosL, one of the accessory proteins of the nos (nitrous oxide reductase) gene cluster, has been heterologously expressed, purified, and characterized . NosL is a monomeric protein of 18,540 MW that specifically and stoichiometrically binds Cu(I) . The copper ion in NosL is ligated by a Cys residue, and one Met and one His are thought to serve as the other ligands . While it is possible to oxidize Cu(I)-NosL with ferricyanide, the Cu(II) ion thus formed appears to dissociate from the protein . The function of Cu(I)NosL is not yet known, but the data indicate that NosL does not act as an electron transfer partner to nitrous oxide reductase . NosL is encoded on the same transcript as three other gene products (NosD, NosF, and NosY) . These have been shown to be required for assembly of the active site in nitrous oxide reductase, which is thought to be a copper cluster . Accordingly, it is possible that NosL is a copper chaperone involved in metallocenter assembly. Nucleic Acids Res, 2001 Apr 15, 29(8), 1733 - 40 A novel human hexameric DNA helicase: expression, purification and characterization; Biswas EE et al.; We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells . Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human . The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity . The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein . The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >/=5-fold by single-stranded DNA (ssDNA) . Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5'-->3' direction . The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein . We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis . Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent . The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis. Nucleic Acids Res, 2001 Apr 15, 29(8), 1695 - 702 The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2; Kondo E et al.; In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system . The hMLH1 protein forms three different heterodimers with one of the other MutL homologues . As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features . Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492-742 . In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1 . Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species . Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program . Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues . Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions. J Bacteriol, 2001 May, 183(9), 2952 - 6 Mapping of the Rsd contact site on the sigma 70 subunit of Escherichia coli RNA polymerase; Jishage M et al.; Rsd (regulator of sigma D) is an anti-sigma factor for the Escherichia coli RNA polymerase sigma(70) subunit . The contact site of Rsd on sigma(70) was analyzed after mapping of the contact-dependent cleavage sites by Rsd-tethered iron-p-bromoacetamidobenzyl EDTA and by analysis of the complex formation between Ala-substituted sigma(70) and Rsd . Results indicate that the Rsd contact site is located downstream of the promoter -35 recognition helix-turn-helix motif within region 4, overlapping with the regions involved in interaction with both core enzyme and sigma(70) contact transcription factors. J Bacteriol, 2001 May, 183(9), 2897 - 909 Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase; Sutton MD et al.; The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control . These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD' (which enables TLS) . In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD(2)C . We have previously reported that overexpression of the epsilon proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon with UmuD(2)C is important for the DNA damage checkpoint control function of the umuDC gene products . Here, we report that overexpression of the beta processivity clamp of the E . coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD'C gene products . Such a strain is not otherwise normally cold sensitive . To identify mutant beta proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD'C-overexpressing strain . In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of beta or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN . Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo . In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta . Taken together, these findings suggest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products . Four possible models for how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed. J Bacteriol, 2001 May, 183(9), 2866 - 73 Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70); Xu J et al.; We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter . We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations . The sequences selected under the two conditions differ from each other and from the consensus -10 sequence . The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro . Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint. J Bacteriol, 2001 May, 183(9), 2834 - 41 Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B; Cooper VS et al.; Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium . We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses . Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor . These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor . A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation . At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes) . The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon . The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence . To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150 . All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium . The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation. J Bacteriol, 2001 May, 183(9), 2823 - 33 Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli; Sanchez-SanMartin C et al.; To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin . The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes . With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit . We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions -204 and -157 was needed for the Ler-dependent activation of the tir operon . Sequence elements located between positions -204 and -97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions . Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein . However, its full activation was still repressed by growth in rich medium or at 25 degrees C, suggesting that negative regulation also occurs at or downstream of the promoter . Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene. J Bacteriol, 2001 May, 183(9), 2817 - 22 Interplay between the specific chaperone-like proteins HybG and HypC in maturation of hydrogenases 1, 2, and 3 from Escherichia coli; Blokesch M et al.; The hybG gene product from Escherichia coli has been identified as a chaperone-like protein acting in the maturation of hydrogenases 1 and 2 . It was shown that HybG forms a complex with the precursor of the large subunit of hydrogenase 2 . As with HypC, which is the chaperone-like protein involved in hydrogenase 3 maturation, the N-terminal cysteine residue is crucial for complex formation . Introduction of a deletion into hybG abolished the generation of active hydrogenase 2 but only quantitatively reduced hydrogenase 1 activity since HypC could replace HybG in this function . In contrast, HybG could not take over the role of HypC in a DeltahypC genetic background . Overproduction of HybG, especially of the variants with the replaced N-terminal cysteine residue, strongly interfered with hydrogenase 3 maturation, apparently by titrating some other component(s) of the maturation machinery . The results indicate that the three hydrogenase isoenzymes not only are interacting at the functional level but are also interconnected during the maturation process. J Bacteriol, 2001 May, 183(9), 2808 - 16 Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli; Yamanaka K et al.; Upon cold shock, Escherichia coli cell growth transiently stops . During this acclimation phase, specific cold shock proteins (CSPs) are highly induced . At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation . Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase . A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h . PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C . In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation. J Bacteriol, 2001 May, 183(9), 2779 - 84 Genes essential to iron transport in the cyanobacterium Synechocystis sp . strain PCC 6803; Katoh H et al.; Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp . strain PCC 6803 were identified . These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J . Bacteriol . 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake . Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium . All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation . The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding . The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively . These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters . Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport . This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium. J Bacteriol, 2001 May, 183(9), 2765 - 73 Escherichia coli ribosome-associated protein SRA, whose copy number increases during stationary phase; Izutsu K et al.; Protein D has previously been demonstrated to be associated with Escherichia coli ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis . In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the E . coli genetic map, between ompC and sfcA . The gene consists of 45 codons, coding for a protein of 5,096 Da . The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase . For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named sra . The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S) . In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of sra in the stationary phase. Infect Immun, 2001 May, 69(5), 3418 - 22 Escherichia coli CdtB mediates cytolethal distending toxin cell cycle arrest; Elwell C et al.; We previously reported that the CdtB polypeptide of Escherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases . In addition, the DNase-related residues of CdtB are required for cellular toxicity . Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E . coli extract containing CdtA and CdtC . CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action . In contrast to these findings, purified CdtB(H154A) lacked both DNA-nicking and cell cycle arrest activities . These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity. Infect Immun, 2001 May, 69(5), 3315 - 22 Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals; Goosney DL et al.; Enteropathogenic Escherichia coli (EPEC) is a human pathogen that attaches to intestinal epithelial cells and causes chronic watery diarrhea . A close relative, enterohemorrhagic E . coli (EHEC), causes severe bloody diarrhea and hemolytic-uremic syndrome . Both pathogens insert a protein, Tir, into the host cell plasma membrane where it binds intimin, the outer membrane ligand of EPEC and EHEC . This interaction triggers a cascade of signaling events within the host cell and ultimately leads to the formation of an actin-rich pedestal upon which the pathogen resides . Pedestal formation is critical in mediating EPEC- and EHEC-induced diarrhea, yet very little is known about its composition and organization . In EPEC, pedestal formation requires Tir tyrosine 474 phosphorylation . In EHEC Tir is not tyrosine phosphorylated, yet the pedestals appear similar . The composition of the EPEC and EHEC pedestals was analyzed by examining numerous cytoskeletal, signaling, and adapter proteins . Of the 25 proteins examined, only two, calpactin and CD44, were recruited to the site of bacterial attachment independently of Tir . Several others, including ezrin, talin, gelsolin, and tropomyosin, were recruited to the site of EPEC attachment independently of Tir tyrosine 474 phosphorylation but required Tir in the host membrane . The remaining proteins were recruited to the pedestal in a manner dependent on Tir tyrosine phosphorylation or were not recruited at all . Differences were also found between the EPEC and EHEC pedestals: the adapter proteins Grb2 and CrkII were recruited to the EPEC pedestal but were absent in the EHEC pedestal . These results demonstrate that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition. Infect Immun, 2001 May, 69(5), 2878 - 87 Recombinant urease and urease DNA of Coccidioides immitis elicit an immunoprotective response against coccidioidomycosis in mice; Li K et al.; Coccidioides immitis antigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis . Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) of C . immitis were expressed in Escherichia coli and tested as vaccine candidates in BALB/c mice . A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant . T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes . BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum of C . immitis arthroconidia demonstrated a significant reduction in the level of C . immitis infection compared to control animals . rHSP60 was much less effective as a protective antigen . Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 {IgG1} versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen . Urease was further evaluated by expression of the URE gene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route . In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p . infection, compared to only 10% of the mice immunized with the vector alone . In