Escherichia coli

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 addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens . These data support the need for further evaluation of the C . immitis urease as a candidate vaccine against coccidioidomycosis.

Am J Physiol Gastrointest Liver Physiol, 2001 May, 280(5), G858 - 65
Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model; Beno DW et al.; Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG) . Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG . Despite significant elevations in mean peak serum tumor necrosis factor-alpha (TNF-alpha) concentrations (90.9 +/- 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 microg/kg endotoxin at 6 or 24 h . Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 microg/kg endotoxin (mean peak TNF-alpha 129.6 +/- 24.4 ng/ml) . Furthermore, when rats were treated with up to 16 microg/kg of recombinant TNF-alpha, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values . These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-alpha.

J Immunol Methods, 2001 May 1, 251(1-2), 161 - 76
An improved procedure for the generation of recombinant single-chain Fv antibody fragments reacting with human CD13 on intact cells; Peipp M et al.; A procedure was developed to generate recombinant single chain Fv (scFv) antibody fragments reacting with the extracellular domain of human cell surface antigen CD13 (hCD13; aminopeptidase N) on intact cells . Membrane fractions prepared from a stably transfected hCD13-positive murine NIH/3T3 cell line were used to immunize BALB/c mice, with the intention that hCD13 would be the major immunogenic molecule recognized by the immune system . Spleen RNA from the immunized mice served to generate a combinatorial scFv phage display library . The library was adsorbed against non-transfected NIH/3T3 or Sf21 insect cells to eliminate nonrelevant binders . The supernatant was then used for panning with either hCD13-transfected Sf21 insect cells or a hCD13-expressing human leukemia-derived cell line . Therefore, the key concepts of the procedure were the presentation of hCD13 as the sole human antigen on murine NIH/3T3 cells and a screening strategy where hCD13 was the major common antigen of the material used for immunization and panning . Two different hCD13-reactive phages were isolated and the soluble scFvs were expressed in E . coli and purified . The two scFvs, anti-hCD13-1 and anti-hCD13-3, differed at four amino acid positions in their V(H) regions and both had high affinities for hCD13 as determined by surface plasmon resonance (K(D)=7 and 33x10(-10) M, respectively) . Both efficiently recognized hCD13 on intact cells . Therefore, the procedure allowed the production of high affinity scFvs reacting with a desired antigen in its native conformation without requiring extensive purification of the antigen and should be useful for the preparation of scFvs against other conformation-sensitive cell-surface antigens.

J Immunol Methods, 2001 May 1, 251(1-2), 151 - 9
Development of a quantitative assay for residual host cell proteins in a recombinant subunit vaccine against human respiratory syncytial virus; Dagouassat N et al.; We have developed and validated a process-specific immunoligand assay based on the Threshold system for the quantification of residual host cell proteins (HCPs) in a recombinant subunit vaccine candidate against the human respiratory syncytial virus (hRSV) . The industrial process of this vaccine produced in Escherichia coli, involved five chromatography steps for the production of clinical-grade batches . The clearance of non-product-related protein throughout the purification process was documented by the evaluation of the HCP content in the chromatographic fractions at each step of the downstream processing . The assay had a detection limit of 0.5 ng/ml of HCP equivalent to 10 parts per million (ppm) . The quantification limit was 1.3 ng/ml of HCP, giving a sensitivity range of the assay of 10 to 30 ppm . To our knowledge, this is the first sensitive HCP assay reported for a vaccine.

J Immunol Methods, 2001 May 1, 251(1-2), 137 - 49
Altering the fine specificity of an anti-Legionella single chain antibody by a single amino acid insertion; McCarthy BJ et al.; Antibody engineering provides the potential to clone and manipulate antibody genes to produce fragments with altered specificity . We have produced an anti- Legionella single chain fragment with broader specificity towards Legionella serotypes than the parent monoclonal antibody . Using this relationship between the parent monoclonal and the recombinant antibody derived from it as a model, we attempted to identify those residues responsible for this change in fine specificity . Sequence analysis of this recombinant antibody revealed the deletion of a conserved residue, Asp101, in the CDR-H3 region . Using site-directed mutagenesis, we have created a mutant form of this single chain fragment with an aspartic acid insertion mutation at position 101 of the antibody heavy chain . This mutant scFv demonstrates improved specificity compared to the wild-type recombinant antibody, indicating an important role for Asp101.

Nitric Oxide, 2001 Apr, 5(2), 208 - 11
Time course of inducible nitric oxide synthase activity following endotoxin administration in dogs; Preiser JC et al.; An increased production of nitric oxide (NO) via the inducible isoform of NO synthase (iNOS) has been incriminated in the pathogenesis of septic shock . Since the time course of iNOS activity is not known during endotoxic shock in dogs, we measured iNOS activity, estimated by the rate of conversion of (14)C-arginine to (14)C-citrulline in the absence of calcium, in the heart, lung, liver, kidney, and gut at 1, 2, 3, 4, and 6 h after a bolus of Escherichia coli endotoxin (2 mg/kg, iv), in the dog . This model, including generous fluid administration, is associated with typical features of human septic shock, including low systemic vascular resistance, altered myocardial function and limited oxygen extraction capability . An increase in iNOS activity was observed at 4 h in the liver (0.24 vs 0.04 mU/mg/min) and at 6 h in the heart (0.26 vs 0.09 mU/mg/min) . These findings may contribute to a better delineation of the involvement of NO in endotoxic shock, and to the evaluation of the therapeutic effects of NO inhibitors .

J Mol Biol, 2001 Apr 13, 307(5), 1341 - 9
Direct localization by cryo-electron microscopy of secondary structural elements in Escherichia coli 23 S rRNA which differ from the corresponding regions in Haloarcula marismortui; Matadeen R et al.; Insertions were introduced by a two-step mutagenesis procedure into each of five double-helical regions of Escherichia coli 23 S rRNA, so as to extend the helix concerned by 17 bp . The helices chosen were at sites within the 23 S molecule (h9, h25, h45, h63 and h98) where significant length variations between different species are known to occur . At each of these positions, with the exception of h45, there are also significant differences between the 23 S rRNAs of E . coli and Haloarcula marismortui . Plasmids carrying the insertions were introduced into an E . coli strain lacking all seven rrn operons . In four of the five cases the cells were viable and 50 S subunits could be isolated; only the insertion in h63 was lethal . The modified subunits were examined by cryo-electron microscopy (cryo-EM), with a view to locating extra electron density corresponding to the insertion elements . The results were compared both with the recently determined atomic structure of H . marismortui 23 S rRNA in the 50 S subunit, and with previous 23 S rRNA modelling studies based on cryo-EM reconstructions of E . coli ribosomes . The insertion element in h45 was located by cryo-EM at a position corresponding precisely to that of the equivalent helix in H . marismortui . The insertion in h98 (which is entirely absent in H . marismortui) was similarly located at a position corresponding precisely to that predicted from the E . coli modelling studies . In the region of h9, the difference between the E . coli and H . marismortui secondary structures is ambiguous, and the extra electron density corresponding to the insertion was seen at a location intermediate between the position of the nearest helix in the atomic structure and that in the modelled structure . In the case of h25 (which is about 50 nucleotides longer in H . marismortui), no clear extra cryo-EM density corresponding to the insertion could be observed .

J Mol Biol, 2001 Apr 13, 307(5), 1207 - 21
Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament; Solinger JA et al.; RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae . Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function . Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity . In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair . In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity . Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation . This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA . Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation . Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation . We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments .

J Mol Biol, 2001 Apr 13, 307(5), 1195 - 206
Directionality of DNA replication fork movement strongly affects the generation of spontaneous mutations in Escherichia coli; Yoshiyama K et al.; Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error . All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement . It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations . A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis . It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation . Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations . These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR . Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms .

Plant Mol Biol, 2001 Feb, 45(3), 353 - 63
Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh; Hibino T et al.; Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants . Among four mangrove species, glycinebetaine could be detected only in Avicennia marina . Pinitol was the main osmoprotectant in the other three species . The level of glycinebetaine in A . marina increased under high salinity . Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected . A cDNA library was constructed from the leaves of A . marina . Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus . The BADH transcription levels of the former were higher than those of the latter . The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity . BADHs were expressed in Escherichia coli and purified . Two kinds of A . marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH . A . marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of omega-aminoaldehydes and were more stable at high temperature than the spinach BADH.

Life Sci, 2001 Mar 9, 68(16), 1931 - 7
Inhibition of Shiga toxin-induced tumor necrosis factor-alpha production and gene expression in human monocytic cells by CV6209; Zhang HM et al.; Cytokines, in particular TNF-alpha, appear to be necessary to develop the pathological process of Shiga toxin-producing Escherichia coli (STEC) infection . In this study, we investigated whether CV6209, a PAF antagonist, could modulate Shiga toxin (Stx)-induced TNF-alpha production in human monocytic cells . Cells were stimulated by Stx1 or Stx2 (5 ng/ml) with or without CV6209 addition (12-100 microg/ml) for various periods of time . CV6209 significantly suppressed Stx-induced TNF-alpha production in a dose- and time-dependent manner . Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that CV6209 suppressed Stx-mediated TNF-alpha mRNA expression . Our results indicated that CV6209 had an important regulatory effect on Stx-induced TNF-alpha production and gene expression.

J Clin Lab Anal, 2001, 15(2), 100 - 3
Identification of human enterovirulent Escherichia coli strains by multiplex PCR; Rich C et al.; Some strains of Escherichia coli are involved in enteric infections in both adults and children . However the classical diagnostic methods can not differentiate pathogenic from nonpathogenic E . coli, because of the lack of phenotypic differences . In this study, we developed multiplex PCR in order to amplify fragments of specific virulence genes of the five main E . coli pathotypes . Fragments of the expected size were obtained using previously or newly designed primers and allowed identification of 10 virulence genes in only 5 reactions . This method was applied to the detection of pathogenic E . coli isolated from 90 patients' stools specimens during an 18-month survey . Patients were suffering from diarrhea or hemolytic uremic syndrome and in 13 cases (14.4%), an enterovirulent E . coli strain was detected . This diagnostic method could therefore represent an important technique in clinical laboratories which lack standard tests for these pathogens.

Biotechnol Bioeng, 2000-2001, 71(4), 286 - 306
Combining pathway analysis with flux balance analysis for the comprehensive study of metabolic systems; Schilling CH et al.; The elucidation of organism-scale metabolic networks necessitates the development of integrative methods to analyze and interpret the systemic properties of cellular metabolism . A shift in emphasis from single metabolic reactions to systemically defined pathways is one consequence of such an integrative analysis of metabolic systems . The constraints of systemic stoichiometry, and limited thermodynamics have led to the definition of the flux space within the context of convex analysis . The flux space of the metabolic system, containing all allowable flux distributions, is constrained to a convex polyhedral cone in a high-dimensional space . From metabolic pathway analysis, the edges of the high-dimensional flux cone are vectors that correspond to systemically defined "extreme pathways" spanning the capabilities of the system . The addition of maximum flux capacities of individual metabolic reactions serves to further constrain the flux space and has led to the development of flux balance analysis using linear optimization to calculate optimal flux distributions . Here we provide the precise theoretical connections between pathway analysis and flux balance analysis allowing for their combined application to study integrated metabolic function . Shifts in metabolic behavior are calculated using linear optimization and are then interpreted using the extreme pathways to demonstrate the concept of pathway utilization . Changes to the reaction network, such as the removal of a reaction, can lead to the generation of suboptimal phenotypes that can be directly attributed to the loss of pathway function and capabilities . Optimal growth phenotypes are calculated as a function of environmental variables, such as the availability of substrate and oxygen, leading to the definition of phenotypic phase planes . It is illustrated how optimality properties of the computed flux distributions can be interpreted in terms of the extreme pathways . Together these developments are applied to an example network and to core metabolism of Escherichia coli demonstrating the connections between the extreme pathways, optimal flux distributions, and phenotypic phase planes . The consequences of changing environmental and internal conditions of the network are examined for growth on glucose and succinate in the face of a variety of gene deletions . The convergence of the calculation of optimal phenotypes through linear programming and the definition of extreme pathways establishes a different perspective for the understanding of how a defined metabolic network is best used under different environmental and internal conditions or, in other words, a pathway basis for the interpretation of the metabolic reaction norm.

J Biol Chem, 2001 Jun 22, 276(25), 22024 - 31 Epub 2001 Apr 04.
A sulfurtransferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z in Escherichia coli; Leimkuhler S et al.; It has been shown that conversion of precursor Z to molybdopterin (MPT) by Escherichia coli MPT synthase entails the transfer of the sulfur atom of the C-terminal thiocarboxylate from the small subunit of the synthase to generate the dithiolene group of MPT and that the moeB mutant of E . coli contains inactive MPT synthase devoid of the thiocarboxylate . The data presented here demonstrate that l-cysteine can serve as the source of the sulfur for the biosynthesis of MPT in vitro but only in the presence of a persulfide-containing sulfurtransferase such as IscS, cysteine sulfinate desulfinase (CSD), or CsdB . A fully defined in vitro system has been developed in which an inactive form of MPT synthase can be activated by incubation with MoeB, Mg-ATP, l-cysteine, and one of the NifS-like sulfurtransferases, and the addition of precursor Z to the in vitro system gives rise to MPT formation . The use of radiolabeled l-{(35)S}cysteine has demonstrated that both sulfurs of the dithiolene group of MPT originate from l-cysteine . It was found that MPT can be produced from precursor Z in an E . coli iscS mutant strain, indicating that IscS is not required for the in vivo sulfuration of MPT synthase . A comparison of the ability of the three sulfurtransferases to provide the sulfur for MPT formation showed the highest activity for CSD in the in vitro system.

J Biol Chem, 2001 Jun 15, 276(24), 21618 - 26 Epub 2001 Apr 04.
Glutaredoxin protects cerebellar granule neurons from dopamine-induced apoptosis by dual activation of the ras-phosphoinositide 3-kinase and jun n-terminal kinase pathways; Daily D et al.; Glutaredoxin 2 (Grx2) from Escherichia coli protects cerebellar neurons from dopamine-induced apoptosis via nuclear factor kappa B (NF-kappaB) activation, which is mediated by the expression of redox factor-1 (Ref-1) . An analysis of the mechanisms underlying Grx2 protective activity revealed dual activation of signal transduction pathways . Grx2 significantly activated the Ras/phosphoinositide 3-kinase/Akt/NF-kappaB cascade in parallel to the Jun N-terminal kinase (JNK)/AP1 cascade . Dopamine, in comparison, down-regulated both pathways . Treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate Akt and AP-1 but had no effect on the phosphorylation of JNK1/2, suggesting that Akt/NF-kappaB and AP-1 are regulated by Ref-1 . Exposure of the neurons to JNK1/2 antisense oligonucleotide in the presence of Grx2 significantly reduced AP-1 and NF-kappaB DNA binding activities and abolished Grx2 protection . These results demonstrate that dual activation of Ras/phosphoinositide 3-kinase and AP-1 cascades, which are mediated by Ref-1, is an essential component of the Grx2 mechanism of action.

Genetics, 2001 Apr, 157(4), 1413 - 23
Localized remodeling of the Escherichia coli chromosome: the patchwork of segments refractory and tolerant to inversion near the replication terminus; Guijo MI et al.; The behavior of chromosomal inversions in Escherichia coli depends upon the region they affect . Regions flanking the replication terminus have been termed nondivisible zones (NDZ) because inversions ending in the region were either deleterious or not feasible . This regional phenomenon is further analyzed here . Thirty segments distributed between 23 and 29 min on the chromosome map have been submitted to an inversion test . Twenty-five segments either became deleterious when inverted or were noninvertible, but five segments tolerated inversion . The involvement of polar replication pause sites in this distribution was investigated . The results suggest that the Tus/pause site system may forbid some inversion events, but that other constraints to inversion, unrelated to this system, exist . Our current model for deleterious inversions is that the segments involved carry polar sequences acting in concert with other polar sequences located outside the segments . The observed patchwork of refractory and tolerant segments supports the existence of several NDZs in the 23- to 29-min region . Microscopic observations revealed that deleterious inversions are associated with high frequencies of abnormal nucleoid structure and distribution . Combined with other information, the data suggest that NDZs participate in the organization of the terminal domain of the nucleoid.

Gene, 2001 Mar 21, 266(1-2), 25 - 34
Characterization of a heavy metal ATPase from the apicomplexan Cryptosporidium parvum; LaGier MJ et al.; P1-ATPases are transporters which pump heavy metals across membranes, either to provide enzymes with essential cofactors or to remove excess, toxic metal cations from the cytosol . The first protist P1-ATPase (CpATPase2) has been isolated from the apicomplexan Cryptosporidium parvum, an opportunistic pathogen of AIDS patients . This single copy gene encodes 1260 amino acids (aa), predicting a protein of 144.7 kDa . Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis confirmed CpATPase2 expression . Immunofluorescence microscopy of C . parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase (GST) fusion protein suggests that CpATPase2 is associated with the plasma- and cytoplasmic membranes . The protein shares greatest overall sequence similarity to previously characterized copper P1-ATPases . Expression and subsequent biochemical analyses of the N-terminal heavy metal binding domain (HMBD, GMxCxxC) of CpATPase2 as a maltose-binding protein (MBP) in Escherichia coli reveals that the protein specifically binds reduced copper, Cu(I), in vitro and in vivo, and that the cysteine residues of HMBD are responsible for heavy metal coordination . Overall, these data show that the apicomplexan C . parvum possesses a heavy metal P-ATPase transporter with a specificity for reduced copper . Since this discovery represents the first time a heavy metal P-ATPase has been identified and characterized from a protist, further molecular and biochemical studies are needed to understand the roles heavy metal P-ATPases play in heavy metal metabolism and potential virulence for this and other apicomplexa.

Gene, 2001 Mar 21, 266(1-2), 1 - 13
Cloning and characterization of a novel serine/threonine protein phosphatase type 5 from Trypanosoma brucei; Chaudhuri M; Reversible protein phosphorylation is essential for the regulation of numerous cellular functions and differentiation . The haemo-flagellated parasitic protozoan Trypanosoma brucei, the causative agent for African trypanosomiasis undergoes various stages of cellular differentiation during its digenetic life cycle . A complete cDNA of a unique serine/threonine phosphatase type five (TbPP5) was cloned and characterized from T . brucei . TbPP5 contains an open reading frame of 1416 bp that encodes a protein of about 53 kDa and exists as a single copy gene in the T . brucei genome . The deduced amino acid sequence showed 45-48% overall identity and 60-65% similarity with protein phosphatase 5's (PP5) from different species . Analysis of the primary sequence revealed that TbPP5 contains three TPR motifs at the N-terminal region (amino acid residues 7-107) while the phosphatase catalytic domain occurs in the C-terminal region (amino acid residues 210-410) . A TbPP5 cDNA hybridized with a transcript of 2.5 kb which is present at similar levels in the procyclic and the bloodstream forms . However, the level of expression of the TbPP5 protein (52 kDa) detected by an antibody developed against a recombinant protein produced in E . coli was about 2-fold higher in the procyclic than the bloodstream form . The TbPP5 transcript level gradually decreased in cells grown in the logarithmic phase to the stationary phase in culture . Moreover, 18 h serum starvation of the procyclic forms decreased the level of the specific transcript about 3-fold suggesting that this protein may play a role during the active growth phase of the organism . The recombinant protein showed phosphatase activity which was stimulated about 2.6-fold by arachidonic acid with half-maximal activity at 75 microM . Indirect immuno-fluorescence of permeabilized cells revealed that the protein is localized in the cytosol and the nucleus This is the first report for the identification of a type 5 serine/threonine protein phosphatase in an ancient eukaryote such as T . brucei.

Metab Eng, 2001 Apr, 3(2), 138 - 50
The organization of metabolic reaction networks . II . Signal processing in hierarchical structured functional units; Kremling A et al.; Based on the analysis of molecular interactions of proteins with DNA binding sites, a new approach to developing mathematical models describing gene expression is introduced . Detection of hierarchical structures in metabolic networks can be used to decompose complex reaction schemes . This will be achieved by assigning each regulator protein to one level in the hierarchy . Signals are then transduced from the top level to the lower level, but not vice versa . The method is shown by a simple example with two interacting proteins . A comparison of simulation results shows good agreement between a model taking all interactions into account and a model developed with the new approach . Finally, the method is applied to the crpA modulon in Escherichia coli, which controls uptake and metabolism for a number of carbohydrates . Here, RNA polymerase represents the top level, CrpA the second level, and the lactose-specific repressor LacI the lowest level, respectively . Besides the lactose operon, the method is applied to the adenylate cyclase gene and the gene for the regulator CrpA .

Metab Eng, 2001 Apr, 3(2), 124 - 37
A MILP-based flux alternative generation and NMR experimental design strategy for metabolic engineering; Phalakornkule C et al.; A mixed-integer linear program (MILP) is described that can enumerate all the ways fluxes can distribute in a metabolic network while still satisfying the same constraints and objective function . The multiple solutions can be used to (1) generate alternative flux scenarios that can account for limited experimental observations, (2) forecast the potential responses to mutation (e.g., new reaction pathways may be used), and (3) (as illustrated) design (13)C NMR experiments such that different potential flux patterns in a mutant can be distinguished . The experimental design is enabled by using the MILP results as an input to an isotopomer mapping matrices (IMM)-based program, which accounts for the network circulation of (13)C from a precursor such as glucose . The IMM-based program can interface to common plotting programs with the result that the user is provided with predicted NMR spectra that are complete with splittings and Lorentzian line-shape features . The example considered is the trafficking of carbon in an Escherichia coli mutant, which has pyruvate kinase activity deleted for the purpose of eliminating acetate production . Similar yields and extracellular measurements would be manifested by the flux alternatives . The MILP-IMM results suggest how NMR experiments can be designed such that the spectra of glutamate for two flux distribution scenarios differ significantly .

Planta, 2001 Feb, 212(3), 460 - 5
Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin; Wallaart TE et al.; The sesquiterpenoid artemisinin, isolated these from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs . A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene . Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase . The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A . annua . When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate . Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight.

J Orthop Sci, 2001, 6(1), 75 - 81
Cationic polymer-mediated genetic transduction into cultured human chondrosarcoma-derived HCS-2/8 cells; Ohashi S et al.; The usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent; Oiagen), linear polyethylenimine (PEI; ExGen 500; Euromedex), and branched PEI in gene delivery into chondrocytes was examined comparatively . A plasmid vector containing the Escherichia coli LacZ (pSES.beta) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8 . Gene expression was evaluated by an O-nitrophenyl beta-D-galactopyranoside (ONPG) assay and by staining with 0.05% 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal; Nacalai Tesque) . The ONPG assay showed that the highest delivery rate was achieved when 2microg of pSES.beta was combined with either 21 microg of dendrimer, 1.7microg of linear PEI, or 2.0microg of branched PEI . At the same DNA/polymer ratios, the proportions of X-gal-stained cells were also the highest (31.3 +/- 7.5%, 30.3 +/- 9.0%, and 8.3 +/- 3.1%, respectively) . LacZ expression reached the highest level 3 days after the dendrimer-mediated transduction, and gradually declined, returning to the background level on day 14 . Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations . Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer . The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful nonviral vector for gene delivery to cells of chondrocytes.

Plant Mol Biol, 2001 Jan, 45(2), 133 - 44
Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp . PCC 6803; Hirani TA et al.; The gene products of sll0337 and slr0081 in Synechocystis sp . PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively . Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions . A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin . In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting . Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon . Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp . PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation . This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp . PCC 7942 {Ray, J.M., Bhaya, D., Block, M.A . and Grossman, A.R . (1991) J . Bact . 173: 4297-4309} . An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp . PCC 7942.

Cancer Res, 2001 Mar 15, 61(6), 2632 - 40
Single nucleotide instability without microsatellite instability in rat mammary carcinomas; Watanabe N et al.; Mutation frequencies (MnFs) of the lacI transgene and mutation rates (MRs) of the endogenous hprt gene were analyzed in two mammary carcinoma cell lines that we established from mammary carcinomas that had been induced by 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) in female lacI-transgenic rats . Using the lacI transgene, corrected MnF, which is the number of independent lacI mutations that occurred while 102 cells expanded into 10(7) cells and which reflect the dynamic increase of point mutations, was measured . The corrected MnFs in the two mammary carcinoma cell lines (59 x 10(-6) and 72 x 10(-6) mutations) were significantly higher than that in the primary culture of normal mammary epithelium (4.7 x 10(-6)) . MRs of the hprt gene in the two mammary carcinoma cell lines (8.2 x 10(-7) and 11 x 10(-7) mutations/hprt/cell division) were also higher than the same control (1.4 x 10(-7)) . A:T to C:G transversion was observed at significantly higher frequencies in the two cell lines (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt) than in the control (0 of 6 for lacI; 0 of 4 for hprt) . Taking advantage of the lacI transgene, high frequencies of A:T to C:G transversion (6 of 38 and 8 of 33, respectively) was also confirmed in the primary carcinomas of the two cell lines, which indicated the presence of a common abnormality in the cell lines and in the primary carcinomas . Both the established cell lines and their primary carcinomas were negative for microsatellite instability, which is known to be caused mainly by mismatch repair insufficiency and to increase point mutations, and for p53 mutations . These findings showed that the two cell lines, and possibly their primary carcinomas, had increases in the MRs of point mutations attributable to a mechanism(s) different from mismatch repair insufficiency, and we would suggest that such a state be designated as single nucleotide instability (SNI).

Eur Biophys J, 2001, 29(8), 559 - 68
Structure of the Fe-heme in the hemodimeric hemoglobin from Scapharca inaequivalvis and in the T721 mutant: an X-ray absorption spectroscopic study at low temperature; Della Longa S et al.; The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K . According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO . The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form . XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K . The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI . This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.

J Nat Toxins, 2001 Feb, 10(1), 43 - 55
Identification of key residues responsible for enzymatic and platelet-aggregation-inhibiting activities of acidic phospholipase A2S from Agkistrodon halys Pallas; Liu X et al.; Site-directed mutagenesis was used to probe the structural and functional relationship of acidic phospholipase A2 from Agkistrodon halys Pallas . The mutants are AP-E6R (E6R), AP-D115K (D115K), AP-6R115K (E6R, D115K), AP-Y118M (Y118M), and AP-W119T (W119T) . All mutants were inserted into a bacterial expression vector and effectively expressed in E . coli RR1 . The purified recombinant enzymes were used to assay for enzymatic and inhibiting platelet aggregation activities . The enzymatic activities of AP-D115K, AP-Y118M and AP-W119T are close to that of denatured-refolded acidic phospholipase A2, while the enzymatic activities of AP-E6R, AP-6R1 15K are lower than that of denatured-refolded acidic phospholipase A2 (AP-WT) . In these five mutants, AP-Y118M showed strongest inhibiting effect on platelet aggregation, which is the same as that of AP-WT, AP-W119T showed only modest activity and AP-E6R, AP-D115K, AP-6R115K showed little activity . To study the structural and functional relationships among these five mutants, molecular modeling of these five mutants was done . The roles of various amino acid residues in the enzymatic activity and pharmacological activity of acidic phospholipase A2 are discussed.

J Nat Toxins, 2001 Feb, 10(1), 17 - 25
Expression, purification and biochemical characterization of a recombinant phospholipase A2, with anticoagulant activity from Agkistrodon halys Pallas; Zhong X et al.; A cloned cDNA encoding a PLA2 from Agkistrodon halys Pallas was found to have conservative residues Glu53 and Trp70 but with Lys56 and Lys67 substituted by Thr56 and Asp67, respectively, when compared with sequences of other class II PLA2 with anticoagulant activity . It was inserted into a temperature-sensitive bacterial expression vector and effectively expressed in Escherichia coli RR1 . The protein was produced as insoluble inclusion bodies and recovered by centrifugation after enzyme digestion . By washing to partial purification, the expression product was refolded and was purified by FPLC superose 12 to appear as a single band in SDS-PAGE . The recombinant protein proved to have obvious enzymatic, anticoagulant and hemolytic activities, which were removed after modification by p-BPB . These findings suggest that the pharmacological activities of this recombinant PLA2 may be related to its catalytic activity and warrant further research on the structure-function relationships of the pharmacological site of the PLA2 from Agkistrodon halys Pallas.

Microsurgery, 2001, 21(2), 58 - 62
Effects of liposome-mediated gene transfer of VEGF in ischemic rat gracilis muscle; Neumeister MW et al.; The purpose of the current study was to determine the effects of vascular endothelial growth factor (VEGF) on muscle flap survival and vascularity in a rat gracilis ischemia-reperfusion model . A total of 12 adult male Wistar rats were divided into two groups (n = 6) . The experimental group received the plasmid encoding VEGF(165) cDNA plus lipofectamine (cationic liposome) injected directly to the gracilis muscle following 4 h of ischemia . The control group received lipofectamine only . The viability and vascularity of the flaps were evaluated after 7 days of reperfusion . The data demonstrated that the VEGF plasmid- and lipofectamine-treated muscle flaps had significantly greater total survival and capillary count 7 days after reperfusion compared with the flaps treated only with lipofectamine . These results indicate that VEGF exerts a protective effect on ischemic skeletal muscle flaps .

Nature, 2001 Apr 5, 410(6829), 715 - 8
Functional proteins from a random-sequence library; Keefe AD et al.; Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences . Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3' end of its encoding mRNA, to sample a large number of distinct random sequences . Starting from a library of 6 x 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP . This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins . The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries.

Pharmacology, 2001, 62(3), 181 - 7
Antidiarrheal activity of wood creosote: inhibition of muscle contraction and enterotoxin-induced fluid secretion in rabbit small intestine; Ogata N et al.; Wood creosote has long been used as an antidiarrheal agent, but its mechanism of action is not well understood . To elucidate the mechanism of its antidiarrheal activity, we have addressed questions whether it inhibits fluid secretion induced by Escherichia coli heat-stable enterotoxin (STa) in rabbit jejunum in vivo, and whether it inhibits muscle contraction of isolated rabbit ileum ex vivo . Wood creosote (10-100 mg/l) instilled in a ligated loop of jejunum inhibited STa-induced fluid secretion (p < 0.05) . It also inhibited the spontaneous phasic, acetylcholine-induced tonic and Ba2+-induced tonic contractions of longitudinal and circular muscles of ileum dose-dependently with IC(50) values of 130-530 mg/l . These data provide further evidence that the antidiarrheal activity of wood creosote is attributable to its antisecretory and antimotility effects .

J Biomed Sci, 2001 Mar-Apr, 8(2), 170 - 5
Construction of a tagging system for subcellular localization of proteins encoded by open reading frames; Chuang CH et al.; We have previously characterized a monoclonal antibody (SC1D7) that is directed to maltose-binding protein (MBP) of Escherichia coli and other closely related enteric bacteria . SC1D7 does not cross-react with proteins in eucaryotes and appears to be a highly specific tool in immunochemical analyses . To better map the epitope, we took advantage of an available plasmid, pMAL-c2, that encodes the E . coli MBP-coding sequence and constructed plasmids to express MBP fragments . A construct containing the N-terminal portion of MBP does not react with SC1D7, whereas a second construct expressing glutathione S-transferase fused with the C-terminal half of MBP does react with SC1D7 . To precisely define the epitope, random peptides displayed on M13 were used to react with SC1D7 . Sequences of reactive peptides were aligned, and a consensus sequence of XDXRIPX was deduced . This sequence matches MBP with an amino acid stretch of KDPRIAA . To consolidate the mapping result, a sequence encoding this epitope was inserted into an expression vector and the resulting recombinant protein did react with SC1D7 . Thereafter, this epitope was incorporated into a eucaryotic expression plasmid containing a previously defined hepatitis delta virus epitope for protein tagging . This two-epitope-tagging vector is useful in various molecular analyses . We demonstrate its usage for localization of a bacterial virulence factor in host cells . This vector should be applicable for high-throughput characterization of new open reading frames found in genome sequencing .

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4788 - 93 Epub 2001 Apr 03.
Jasmonic acid carboxyl methyltransferase: a key enzyme for jasmonate-regulated plant responses; Seo HS et al.; Methyl jasmonate is a plant volatile that acts as an important cellular regulator mediating diverse developmental processes and defense responses . We have cloned the novel gene JMT encoding an S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Arabidopsis thaliana . Recombinant JMT protein expressed in Escherichia coli catalyzed the formation of methyl jasmonate from jasmonic acid with K(m) value of 38.5 microM . JMT RNA was not detected in young seedlings but was detected in rosettes, cauline leaves, and developing flowers . In addition, expression of the gene was induced both locally and systemically by wounding or methyl jasmonate treatment . This result suggests that JMT can perceive and respond to local and systemic signals generated by external stimuli, and that the signals may include methyl jasmonate itself . Transgenic Arabidopsis overexpressing JMT had a 3-fold elevated level of endogenous methyl jasmonate without altering jasmonic acid content . The transgenic plants exhibited constitutive expression of jasmonate-responsive genes, including VSP and PDF1.2 . Furthermore, the transgenic plants showed enhanced level of resistance against the virulent fungus Botrytis cinerea . Thus, our data suggest that the jasmonic acid carboxyl methyltransferase is a key enzyme for jasmonate-regulated plant responses . Activation of JMT expression leads to production of methyl jasmonate that could act as an intracellular regulator, a diffusible intercellular signal transducer, and an airborne signal mediating intra- and interplant communications.

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4373 - 8 Epub 2001 Apr 03.
Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium; Rivera SB et al.; Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units . Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family . In C . cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides . CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced . A C . cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein . The first 50 aa of the ORF constitute a putative plastidial targeting sequence . Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography . Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP . The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure . The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.

Proc Natl Acad Sci U S A, 2001 Apr 10, 98(8), 4403 - 8 Epub 2001 Apr 03.
Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry; Miyashita M et al.; Edman degradation remains the primary method for determining the sequence of proteins . In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of (14)C . The transgenic transferase was labeled by growing transformed Escherichia coli on {(14)C}glucose and purified by microaffinity chromatography . An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier . This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.

J Biol Chem, 2001 Jun 15, 276(24), 21242 - 9 Epub 2001 Apr 03.
Stimulation of human endonuclease III by Y box-binding protein 1 (DNA-binding protein B) . Interaction between a base excision repair enzyme and a transcription factor; Marenstein DR et al.; Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation . Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family . This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs . A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA-binding protein B (DbpB) . The in vitro addition of His(6)YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity . Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon-amino group of the hNth1 active site lysine . This equilibrium may be a checkpoint in modulating hNth1 activity.

J Biol Chem, 2001 Jun 29, 276(26), 23221 - 9 Epub 2001 Apr 03.
Isolation and characterization of major glycoproteins of pigeon egg white: ubiquitous presence of unique N-glycans containing Galalpha1-4Gal; Suzuki N et al.; Ovotransferrin (POT), two ovalbumins (POA(hi) and POA(lo)), and ovomucoid (POM) were isolated from pigeon egg white (PEW) . Unlike their chicken egg white counterparts, PEW glycoproteins contain terminal Galalpha1-4Gal, as evidenced by GS-I lectin (specific for terminal alpha-Gal), anti-P(1) (Galalpha1-4Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) monoclonal antibody, and P fimbriae on uropathogenic Escherichia coli (specific for Galalpha1-4Gal) . Galalpha1-4Gal on PEW glycoproteins were found in N-glycans releasable by treatment with glycoamidase F . The respective contents of N-glycans in each glycoprotein were 3.5%, POT; 17%, POA(hi); and 31-37%, POM . POA(hi) has four N-glycosylation sites, in contrast to chicken ovalbumin, which has only one . High performance liquid chromatography analysis showed that N-glycans on POA(hi) were highly heterogeneous . Mass spectrometric analysis revealed that the major N-glycans were monosialylated tri-, tetra-, and penta-antennary oligosaccharides containing terminal Galalpha1-4Gal with or without bisecting N-acetylglucosamine . Oligosaccharide chains terminating in Galalpha1-4Gal are rare among N-glycans from the mammals and avians that have been studied, and our finding is the first predominant presence of (Galalpha1-4Gal)-terminated N-glycans.

Am J Physiol Endocrinol Metab, 2001 May, 280(5), E703 - 11
Fructose augments infection-impaired net hepatic glucose uptake during TPN administration; Donmoyer CM et al.; During chronic total parenteral nutrition (TPN), net hepatic glucose uptake (NHGU) and net hepatic lactate release (NHLR) are markedly reduced (downward arrow approximately 45 and approximately 65%, respectively) with infection . Because small quantities of fructose are known to augment hepatic glucose uptake and lactate release in normal fasted animals, the aim of this work was to determine whether acute fructose infusion with TPN could correct the impairments in NHGU and NHLR during infection . Chronically catheterized conscious dogs received TPN for 5 days via the inferior vena cava at a rate designed to match daily basal energy requirements . On the third day of TPN administration, a sterile (SHAM, n = 12) or Escherichia coli-containing (INF, n = 11) fibrin clot was implanted in the peritoneal cavity . Forty-two hours later, somatostatin was infused with intraportal replacement of insulin (12 +/- 2 vs . 24 +/- 2 microU/ml, SHAM vs . INF, respectively) and glucagon (24 +/- 4 vs . 92 +/- 5 pg/ml) to match concentrations previously observed in sham and infected animals . After a 120-min basal period, animals received either saline (Sham+S, n = 6; Inf+S, n = 6) or intraportal fructose (0.7 mg x kg(-1) x min(-1); Sham+F, n = 6; Inf+F, n = 5) infusion for 180 min . Isoglycemia of 120 mg/dl was maintained with a variable glucose infusion . Combined tracer and arteriovenous difference techniques were used to assess hepatic glucose metabolism . Acute fructose infusion with TPN augmented NHGU by 2.9 +/- 0.4 and 2.5 +/- 0.3 mg x kg(-1) x min(-1) in Sham+F and Inf+F, respectively . The majority of liver glucose uptake was stored as glycogen, and NHLR did not increase substantially . Therefore, despite an infection-induced impairment in NHGU and different hormonal environments, small amounts of fructose enhanced NHGU similarly in sham and infected animals . Glycogen storage, not lactate release, was the preferential fate of the fructose-induced increase in hepatic glucose disposal in animals adapted to TPN.

Curr Opin Biotechnol, 2001 Apr, 12(2), 195 - 201
Advances in Escherichia coli production of therapeutic proteins; Swartz JR; Escherichia coli offers a means for the rapid and economical production of recombinant proteins . These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically sensitive products as insulin and bovine growth hormone . Although significant progress has been made in transcription, translation and secretion, one of the major challenges is obtaining the product in a soluble and bioactive form . Recent progress in oxidative cytoplasmic folding and cell-free protein synthesis offers attractive alternatives to standard expression methods.

J Nutr Biochem, 2001 Apr, 12(4), 235 - 241
Fructose triggers DNA modification and damage in an Escherichia coli plasmid; Levi B et al.; The nonenzymatic reaction between reducing sugars and amino groups of long-lived macromolecules results in an array of chemical modifications that may account for several physiological complications . The characteristics of the reaction are directly related to the type of the reducing sugars involved, whether aldoses or ketoses, phosphorylated or non-phosphorylated, and these in turn determine the consequences of the induced modifications . So far, most studies have been focused on the nonenzymatic reaction between glucose and proteins, while the reaction with fructose, a faster glycating agent, attracted only a minor attention . We have recently demonstrated that long-term fructose consumption induces age-related changes in collagen from skin and cortical bones faster than glucose . In the present study we provide evidence that fructose and its phosphate metabolites can modify DNA faster than glucose and its phosphate metabolites under in vitro conditions . Incubating the plasmid pBR322 with fructose and glucose phosphate metabolites induced DNA modifications and damage that were verified by gel electrophoresis and transformation capacity of the plasmid into an Escherichia coli host . The intensity of the tested sugars to modified and damage DNA after incubation for 15 days increased significantly in the following order: glucose 1-phosphate < glucose < glucose 6-phosphate < fructose 1-phosphate < fructose < fructose 6-phosphate . The data suggest that fructose should deserve more attention as a factor that may influence glycation and induce physiological complications.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 91 - 7
FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions; Pruss BM et al.; The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes . Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized . These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 79 - 84
Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome; Salvadori MR et al.; Recently, a novel verocytotoxin named VT2y was described which belongs to the STx family and is produced by Escherichia coli isolated from domestic poultry with swollen head syndrome (SHS) . The VT2y toxin induced apoptosis in Vero, HeLa, CHO, CEF (primary chicken embryo fibroblast) and PCK (primary chicken kidney) cell lines . Morphological evidence (nuclear shrinkage, chromatin condensation and blebbing of the plasma membrane) of apoptosis could be distinguished in 15 min and was maximal at 1 h after treatment with VT2y . This was confirmed by the terminal dUTP nick-end-labeling (TUNEL) method.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 53 - 8
Molecular cloning, nucleotide sequence and structural analysis of the Streptomyces galbus DSM40480 fda gene: the S . galbus fructose-1,6-bisphosphate aldolase is a member of the class II aldolases; Wehmeier UF; The fda gene of Streptomyces galbus DSM40480 encoding the fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) was cloned, sequenced and characterised . The fda gene encodes a protein of 341 amino acids with a molecular mass of 36.5 kDa and belongs to the class II aldolases . When the S . galbus fda gene was expressed in the Escherichia coli fda(ts) mutant NP315, the growth defect of the strain was complemented at temperatures >35 degrees C . In Northern hybridisations, we identified an fda transcript of 1200 bp length . The transcript length indicates that the fda gene is transcribed from its own promoter . Attempts to isolate fda knock out mutants were not successful . Streptomyces lividans strains with a second copy of the fda gene were constructed and analysed.

FEBS Lett, 2001 Mar 30, 493(2-3), 139 - 43
Directed evolution of beta-galactosidase from Escherichia coli by mutator strains defective in the 3'-->5' exonuclease activity of DNA polymerase III; Stefan A et al.; Directed evolution of Escherichia coli beta-galactosidase into variants featuring beta-glucosidase activity was challenged . To this end, mutagenesis of lacZ was performed by replication in E . coli CC954, a mutator strain containing a DNA polymerase III defective in 3'-->5' exonuclease activity . beta-Galactosidase variants can be isolated upon mutagenesis of lacZ hosted into the self-transmissible episome F'128 . Optimal evolution of lacZ can be achieved by propagation of E . coli CC954/F'128 cultures for 15 generations; further growth of mutator cultures for 37 or 55 generations imposes a high mutational load on lacZ and hinders the selection of efficiently evolved clones.

FEBS Lett, 2001 Mar 30, 493(2-3), 101 - 5
Functional coupling with Galpha(q) and Galpha(i1) protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli; Grisshammer R et al.; To analyze the coupling of Galpha subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Galpha subunits covalently linked to the receptor C-terminus . The presence of Galpha(q) or Galpha(i/q), in which the six C-terminal residues of Galpha(i1) were replaced with those from Galpha(q), increased the percentage of receptors in the agonist high-affinity state . This effect was less pronounced for wild-type Galpha(i1) and not observed for Galpha(i/s) . Functional coupling of neurotensin receptor to Galpha was demonstrated by neurotensin-induced {(35)S}GTPgammaS binding for the Galpha(q), Galpha(i/q) and Galpha(i1) subunits, but not for Galpha(i/s) . Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.

FEBS Lett, 2001 Mar 30, 493(2-3), 85 - 90
Depletion of phosphatidylethanolamine affects secretion of Escherichia coli alkaline phosphatase and its transcriptional expression; Mikhaleva NI et al.; In this report we demonstrate that depletion of the major phospholipid phosphatidylethanolamine, a single non-bilayer forming phospholipid of Escherichia coli, significantly reduces the secretion efficiency of alkaline phosphatase in vivo . Secretion, however, is correlated with the content in membranes of cardiolipin, which in combination with selected divalent cations has a strong tendency to adopt a non-bilayer state indicating the possible involvement of lipid polymorphism in efficient protein secretion . Depletion of this zwitterionic phospholipid also inhibits expression of the protein controlled by the endogenous P(PHO) promoter but not the P(BAD) promoter, which is suggested to be due to the effect of unbalanced phospholipid composition on the orthophosphate signal transduction system (Pho regulon) through an effect on its membrane bound sensor.

Structure (Camb), 2001 Mar 7, 9(3), 245 - 53
Structural basis for the function of pyridoxine 5'-phosphate synthase; Franco MG et al.; BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism . In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer . PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate . RESULTS: The crystal structure of E . coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A . The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold . Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry . In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer . Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function . CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E . coli PNP synthase suggest essentials of the enzymatic mechanism . The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state . All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture . Thus, a common mechanism can be anticipated.

Structure (Camb), 2001 Mar 7, 9(3), 233 - 43
Structures of beta-ketoacyl-acyl carrier protein synthase I complexed with fatty acids elucidate its catalytic machinery; Olsen JG et al.; BACKGROUND: beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E . coli membrane lipids . The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction . KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes . RESULTS: Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively . The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer . A detailed model is proposed for the catalysis of KAS I . Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group . CONCLUSIONS: The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad . Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group . The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.

J Mol Biol, 2001 Apr 6, 307(4), 1103 - 12
The importance of hinge sequence for loop function and catalytic activity in the reaction catalyzed by triosephosphate isomerase; Xiang J et al.; We have determined the sequence requirements for the N-terminal protein hinge of the active-site lid of triosephosphate isomerase . The codons for the hinge (PVW) were replaced with a genetic library of all possible 8000 amino acid combinations . The most active of these 8000 mutants were selected using in vivo complementation of a triosephosphate isomerase-deficient strain of Escherichia coli, DF502 . Approximately 0.3 % of the mutants complement DF502 with an activity that is between 10 and 70 % of wild-type activity . They all contain Pro at the first position . Furthermore, the sequences of these hinge mutants reveal that hydrophobic packing is very important for efficient formation of the enediol intermediate . However, the reduced catalytic activities observed are not due to increased rates of loop opening . To explore the relationship between the N-terminal and C-terminal hinges, three semi-active mutants from the N-terminal hinge selection experiment (PLH, PHS and PTF), and six active C-terminal hinge mutants from previous work (NSS, LWA, YSL, KTK, NPN, KVA) were combined to form 18 "double-hinge" mutants . The activities of these mutants suggest that the N-terminal and C-terminal hinge structures affect one another . It appears that specific side-chain interactions are important for forming a catalytically active enzyme, but not for preventing release of the unstable enediol intermediate from the active site of the enzyme . The independence of intermediate release on amino acid sequence is consistent with the absence of a "universal" hinge sequence in structurally related enzymes .

J Mol Biol, 2001 Apr 6, 307(4), 1075 - 90
Backbone dynamics of Escherichia coli thioesterase/protease I: evidence of a flexible active-site environment for a serine protease; Huang YT et al.; Escherichia coli thioesterase/protease I (TEP-I) is a member of a novel subclass of the lipolytic enzymes with a distinctive GDSLS motif . In addition to possessing thioesterase and protease activities, TEP-I also exhibits arylesterase activity . We have determined the (15)N nuclear magnetic spin relaxation rates, R(1) and R(2), and the steady state (1)H-(15)N heteronuclear Overhauser effect, measured at both 11.74 T and 14.09 T, of (u-(15)N) TEP-I . These data were analyzed using model-free formalism (with axially symmetric rotational diffusion anisotropy) to extract the backbone dynamics of TEP-I . The results reveal that the core structure of the central beta-sheet and the long alpha-helices are rigid, while the binding pocket appears to be rather flexible . The rigid core serves as a scaffold to anchor the essential loops, which form the binding pocket . The most flexible residues display large amplitude fast (ps/ns time-scale) motion and lie on one stripe whose orientation is presumed to be the ligand-binding orientation . We also detected the presence of several residues displaying slow (microseconds/ms time-scale) conformational exchanging processes . These residues lie around the binding pocket and are oriented perpendicularly to the orientation of the flexible stripe . Two of the putative catalytic triads, Ser10 and His157, and their neighbors show motion on the microseconds/ms time-scale, suggesting that their slow motion may have a role in catalysis, in addition to their possible roles in ligand binding . The presence of a flexible substrate-binding pocket may also facilitate binding to a wide range of substrates and confer the versatile functional property of this protein .

J Mol Biol, 2001 Apr 6, 307(4), 1001 - 9
Mapping arm-DNA-binding domain interactions in AraC; Wu M et al.; AraC protein, the regulator of the l-arabinose operon in Escherichia coli has been postulated to function by a light switch mechanism . According to this mechanism, it should be possible to find mutations in the DNA-binding domain of AraC that result in weaker arm-DNA-binding domain interactions and which make the protein constitutive, that is, it no longer requires arabinose to activate transcription . We isolated such mutations by randomizing three contiguous leucine residues in the DNA-binding domain, and then by systematically scanning surface residues of the DNA-binding domain with alanine and glutamic acid . As a result, a total of 20 constitutive mutations were found at ten different positions . They form a contiguous trail on the DNA-distal face of the DNA-binding domain, and likely define the region where the N-terminal arm that extends from the N-terminal dimerization domain contacts the C-terminal DNA-binding domain .

Clin Immunol, 2001 Apr, 99(1), 65 - 74
Incidence of GM-CSF antibodies in cancer patients receiving GM-CSF for immunostimulation; Ullenhag G et al.; We have assessed the immunogenicity profile of GM-CSF in patients with either colorectal carcinoma (CRC) at different stages of disease or with multiple myeloma who were given recombinant human GM-CSF (Escherichia coli-derived) combination therapy . Metastatic CRC patients received a colon carcinoma-reactive antibody and high doses of GM-CSF (425--500 microg/day for 10 days), while other CRC patients and those with myeloma received low doses of GM-CSF (75--80 microg/day for 4 days) as an adjuvant along with appropriate tumor antigens . We found that 55% of the patients (11/20) given high doses of GM-CSF developed GM-CSF-reactive antibodies in comparison with an incidence of only 16% (4/25) in patients given low doses of GM-CSF . None of the patients developed neutralizing antibodies and so the biological effects of GM-CSF were not compromised . A majority of patients (80%) (36/45) also developed antibodies to E . coli proteins that were present as trace contaminants in the GM-CSF product . Treatment with recombinant GM-CSF products, therefore, may induce antibodies against this cytokine depending on the regimen and the amounts used . In this study, multiple immunizations with low doses of GM-CSF was associated with a low incidence of GM-CSF antibodies, which did not neutralize the effect of the cytokine . This therapeutic strategy was effective in inducing adjuvant-type effects and needs to be explored in further clinical trials with this cytokine .

J Med Virol, 2001 May, 64(1), 51 - 7
Use of bacterially expressed EBNA-1 protein cloned from a nasopharyngeal carcinoma (NPC) biopsy as a screening test for NPC patients; Chen MR et al.; EBV serological tests have been used for many years as accessory diagnostic predictors of nasopharyngeal carcinoma (NPC) . To increase the sensitivity and specificity of the NPC detection rate, a novel enzyme-linked immunosorbent assay (ELISA) was established using a bacterially-expressed GST-EBNA-1 protein, containing the EBNA-1 sequence cloned from an NPC patient . Serum samples were collected from age- and gender-matched patients with NPC, community control subjects and hospital control patients and tested using this ELISA . The positivity rates were 78.7% (247/314) in NPC, 11.5% (28/244) in hospital controls and 3.8% (10/263) in the community control group . These serum samples were also tested for IgA anti-VCA antibodies and their ability to neutralize EBV DNase and the sensitivities of the anti-VCA antibody and DNase-neutralization tests also were analyzed . The optimum combination is VCA plus EBNA-1, which can identify 92.5% (287/310) of NPC patients, and shows a specificity of 92.7% (242/261) for normal individuals .

Braz J Med Biol Res, 2001 Apr, 34(4), 463 - 70
Further biochemical characterization of Mycobacterium leprae laminin-binding proteins; Marques MA et al.; It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells . Searching for M . leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp) and ribosomal proteins S4 and S5 as potential adhesins involved in M . leprae-Schwann cell interaction . Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells . In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5 . The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays . However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins . The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells . Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

EMBO J, 2001 Apr 2, 20(7), 1563 - 72
Dynamic localization cycle of the cell division regulator MinE in Escherichia coli; Hale CA et al.; The MinC protein directs placement of the division septum to the middle of Escherichia coli cells by blocking assembly of the division apparatus at other sites . MinD and MinE regulate MinC activity by modulating its cellular location in a unique fashion . MinD recruits MinC to the membrane, and MinE induces MinC/MinD to oscillate rapidly between the membrane of opposite cell halves . Using fixed cells, we previously found that a MinE-green fluorescent protein fusion accumulated in an annular structure at or near the midcell, as well as along the membrane on only one side of the ring . Here we show that in living cells, MinE undergoes a rapid localization cycle that appears coupled to MinD oscillation . The results show that MinE is not a fixed marker for septal ring assembly . Rather, they support a model in which MinE stimulates the removal of MinD from the membrane in a wave-like fashion . These waves run from a midcell position towards the poles in an alternating sequence such that the time-averaged concentration of division inhibitor is lowest at midcell.

EMBO J, 2001 Apr 2, 20(7), 1530 - 7
Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase; Durbecq V et al.; Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway . This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP . DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature . The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism . The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site . Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site . The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere . W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

EMBO J, 2001 Apr 2, 20(7), 1508 - 18
Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili; Hung DL et al.; P pili are important virulence factors in uropathogenic Escherichia coli . The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm . We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis . P pili are assembled via the chaperone/usher pathway . Subunits that go 'OFF-pathway' during pilus biogenesis generate a signal . This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm . In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors . This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.

Biochemistry, 2001 Apr 10, 40(14), 4512 - 20
A novel PCNA-binding motif identified by the panning of a random peptide display library; Xu H et al.; Proliferating cell nuclear antigen (PCNA) has recently been identified as a target for the binding of proteins involved in DNA replication, DNA repair, and cell cycle control . The interactions between PCNA and a number of these proteins are known to be mediated by a conserved peptide motif . In this study, a random peptide library in which peptide sequences are displayed on the E . coli bacterial flagellin protein was screened for PCNA-binding sequences . Analysis of the retrieved peptide sequences verified the presence of the known PCNA-binding motif . In addition, a second, larger group of peptides containing a different consensus sequence for PCNA binding was discovered . This sequence was found to be present on DNA polymerase delta, and a peptide conforming to this sequence was demonstrated to bind to PCNA . Database search and analysis show that many proteins contain the second consensus sequence . These include proteins that are involved in DNA replication, repair, and cell cycle control . The demonstration of this second PCNA-binding motif may provide a basis for identifying and experimentally testing specific proteins for the structural basis for PCNA binding.

Biochemistry, 2001 Apr 10, 40(14), 4478 - 83
Editing by a tRNA synthetase: DNA aptamer-induced translocation and hydrolysis of a misactivated amino acid; Farrow MA et al.; Aminoacyl-tRNA synthetases establish the rules of the genetic code by aminoacylation reactions . Occasional activation of the wrong amino acid can lead to errors of protein synthesis . For isoleucyl-tRNA synthetase, these errors are reduced by tRNA-dependent hydrolytic editing reactions that occur at a site 25 A from the active site . These reactions require that the misactivated amino acid be translocated from the active site to the center for editing . One mechanism describes translocation as requiring the mischarging of tRNA followed by a conformational change in the tRNA that moves the amino acid from one site to the other . Here a specific DNA aptamer is investigated . The aptamer can stimulate amino acid-specific editing but cannot be aminoacylated . Although the aptamer could in principle stimulate hydrolysis of a misactivated amino acid by an idiosyncratic mechanism, the aptamer is shown here to induce translocation and hydrolysis of misactivated aminoacyl adenylate at the same site as that seen with the tRNA cofactor . Thus, translocation to the site for editing does not require joining of the amino acid to the nucleic acid . Further experiments demonstrated that aptamer-induced editing is sensitive to aptamer sequence and that the aptamer is directed to a site other than the active site or tRNA binding site of the enzyme.

Biochemistry, 2001 Apr 10, 40(14), 4381 - 90
Insight into the chemistry of flavin reduction and oxidation in Escherichia coli dihydroorotate dehydrogenase obtained by rapid reaction studies; Palfey BA et al.; Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis . The enzyme from Escherichia coli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate . The chemistry of the reduction of the flavin in DHOD from E . coli by the substrate dihydroorotate (DHO) was studied at 4 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values . A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 1 ms) upon mixing with DHO . The flavin of the intermediate was reduced by DHO, forming a reduced flavin-orotate charge-transfer complex . The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s(-1) at pH 6.5 to approximately 360 s(-1) at pH values greater than an observed pK(a) of 9.5 which was ascribed to Ser175, the active-site base . At all pH values, the reduced flavin-orotate charge-transfer complex dissociated too slowly to be catalytically relevant . Therefore, the oxidizing quinone substrate must bind to the reduced enzyme-orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies {Bjornberg, O., Gruner, A.-C., Roepstorff, P., and Jensen, K . F . (1999) Biochemistry 38, 2899-2908} . Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD . The reduced enzyme-orotate complex reacted rapidly with menadione (180 s(-1)), demonstrating that the reduced enzyme-orotate complex is a catalytically competent intermediate.

Biochemistry, 2001 Apr 10, 40(14), 4234 - 41
Structural basis for a change in substrate specificity: crystal structure of S113E isocitrate dehydrogenase in a complex with isopropylmalate, Mg2+, and NADP; Doyle SA et al.; Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate and has negligible activity toward other (R)-malate-type substrates . The S113E mutant of IDH significantly improves its ability to utilize isopropylmalate as a substrate and switches the substrate specificity (k(cat)/K(M)) from isocitrate to isopropylmalate . To understand the structural basis for this switch in substrate specificity, we have determined the crystal structure of IDH S113E in a complex with isopropylmalate, NADP, and Mg(2+) to 2.0 A resolution . On the basis of a comparison with previously determined structures, we identify distinct changes caused by the amino acid substitution and by the binding of substrates . The S113E complex exhibits alterations in global and active site conformations compared with other IDH structures that include loop and helix conformational changes near the active site . In addition, the angle of the hinge that relates the two domains was altered in this structure, which suggests that the S113E substitution and the binding of substrates act together to promote catalysis of isopropylmalate . Ligand binding results in reorientation of the active site helix that contains residues 113 through 116 . E113 exhibits new interactions, including van der Waals contacts with the isopropyl group of isopropylmalate and a hydrogen bond with N115, which in turn forms a hydrogen bond with NADP . In addition, the loop and helix regions that bind NADP are altered, as is the loop that connects the NADP binding region to the active site helix, changing the relationship between substrates and enzyme . In combination, these interactions appear to provide the basis for the switch in substrate specificity.

Naunyn Schmiedebergs Arch Pharmacol, 2001 Mar, 363(3), 276 - 80
Endotoxin inhibits gastric emptying in rats via a capsaicin-sensitive afferent pathway; Calatayud S et al.; The effects of endotoxin on gastric emptying of a solid nutrient meal and the neural mechanisms involved in such a response were investigated in conscious rats . The intraperitoneal (i.p.) administration of E . coli endotoxin (40 microg/kg) significantly reduced the 4-h rate of gastric emptying of a standard solid nutrient meal . Ablation of primary afferent neurons by systemic administration of high doses of capsaicin (20+30+50 mg/kg s.c.) to adult rats did not modify the rate of gastric emptying in control animals but prevented the delay in gastric transit induced by endotoxin . Local application of capsaicin to the vagus nerve rather than application of capsaicin to the celiac ganglion significantly repressed endotoxin-induced delay in gastric emptying . Neither treatment modified the rate of gastric emptying in vehicle-treated animals . Blockade of CGRP receptors (CGRP 8-37, 100 microg/kg i.v.) did not alter gastric emptying in control animals but significantly prevented endotoxin-induced inhibition of gastric emptying . In contrast, a tachykinin receptor antagonist ({D-Pro2, D-Trp7.9}-substance P, 2 mg/kg i.p.) significantly reduced the rate of gastric emptying in control animals and did not modify the inhibitory effects of endotoxin . Adrenergic blockade with phentolamine (3 mg/kg i.p.) +/- propranolol (5 mg/kg i.p.) or muscarinic antagonism with atropine (0.1 mg/kg i.p.) failed to reverse the delay in gastric emptying induced by endotoxin . These observations indicate that endotoxin-induced delay in gastric emptying of a solid nutrient meal is mediated by capsaicin-sensitive afferent neurons.

Naunyn Schmiedebergs Arch Pharmacol, 2001 Mar, 363(3), 267 - 75
Protective effects of yangambin on cardiovascular hyporeactivity to catecholamines in rats with endotoxin-induced shock; Araujo CV et al.; The protective effects of a new, selective, plant-derived platelet-activating factor (PAF) antagonist, yangambin, on the cardiovascular alterations and mortality due to endotoxic shock were investigated in anaesthetized rats . We also studied the involvement of PAF in the induction of the vascular and cardiac hyporesponsiveness to adrenergic stimulation observed during endotoxaemia . The animals were sensitized to the lethal effects of Escherichia coli lipopolysaccharide (LPS) with D(+)-galactosamine (50 mg/kg, i.v.) 15 min before LPS injection . LPS (3 mg/kg, i.v.) induced a progressive and marked decrease in mean arterial blood pressure from 85+/-4 to 30+/-3 mmHg and a reduction of cardiac output (CO) from 180+/-7 to 37+/-3 ml/min (120 min) accompanied by a maintenance of systemic vascular resistance, suggesting that cardiovascular collapse resulted mainly from myocardial depression . The maximum pressor responses to noradrenaline (0.3-3.0 microg/kg, i.v.) fell from 72+/-9 (control) to 5+/-1 mmHg (LPS) while the CO responses decreased from 81+/-5 to 8+/-3 ml/min . Pre-treatment with yangambin (30 mg/kg, i.v.) or with WEB 2086 (5 mg/kg, i.v.), a reference PAF receptor antagonist, completely prevented the LPS-induced cardiovascular collapse and abolished the sharp reductions of the arterial blood pressure and CO responses to noradrenaline observed during endotoxaemia . Post-treatment with yangambin 90 min after LPS administration did not reverse the arterial hypotension, cardiac failure or cardiovascular hyporesponsiveness to catecholamines . Finally, the acute (150 min) survival rates of endotoxic shock increased from 0% (LPS group) to 100% in the groups pretreated with either yangambin or WEB 2086 . The long-term (7-day) survival also increased from 0% (LPS group) to 85% (yangambin pre-treatment group) . In conclusion, these data suggest a role for PAF in the pathogenesis of endotoxin-induced vascular and cardiac hyporesponsiveness to catecholamines and confirm its involvement in the complex cascade of multiple mediators released during endotoxic/septic shock . Yangambin proved to be an effective pharmacological agent against cardiovascular collapse and mortality in endotoxin shock.

Actas Urol Esp, 2001 Jan, 25(1), 67 - 8
{Pneumocysts, unusual infective complication in diabetics}; Anglada Curado FJ et al.; We report on a case of fully gas-filled bladder with no evidence of intramural gas, fistula between bladder and gastrointestinal tract or instrumentation . The patient is diagnosed of a diabetic neurogenic bladder . We comment the causes of this rare finding and its relation with emphysematous cystitis.

Ann Trop Paediatr, 2001 Mar, 21(1), 91 - 3
Necrotizing fasciitis of the scalp in a neonate; Ameh EA et al.; We report an 11-day-old baby who presented with necrotizing fasciitis of the scalp from which Escherichia coli was cultured . Treatment consisted of administration of parenteral broad-spectrum antibiotics and debridement . Skin grafting of the resulting scalp defect was not permitted by the parents . The wound healed with scar tissue over 3 months.

Plant Cell, 2001 Apr, 13(4), 965 - 78
Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms; Frankenberg N et al.; Phytobilins are linear tetrapyrrole precursors of the light-harvesting prosthetic groups of the phytochrome photoreceptors of plants and the phycobiliprotein photosynthetic antennae of cyanobacteria, red algae, and cryptomonads . Previous biochemical studies have established that phytobilins are synthesized from heme via the intermediacy of biliverdin IX alpha (BV), which is reduced subsequently by ferredoxin-dependent bilin reductases with different double-bond specificities . By exploiting the sequence of phytochromobilin synthase (HY2) of Arabidopsis, an enzyme that catalyzes the ferredoxin-dependent conversion of BV to the phytochrome chromophore precursor phytochromobilin, genes encoding putative bilin reductases were identified in the genomes of various cyanobacteria, oxyphotobacteria, and plants . Phylogenetic analyses resolved four classes of HY2-related genes, one of which encodes red chlorophyll catabolite reductases, which are bilin reductases involved in chlorophyll catabolism in plants . To test the catalytic activities of these putative enzymes, representative HY2-related genes from each class were amplified by the polymerase chain reaction and expressed in Escherichia coli . Using a coupled apophytochrome assembly assay and HPLC analysis, we examined the ability of the recombinant proteins to catalyze the ferredoxin-dependent reduction of BV to phytobilins . These investigations defined three new classes of bilin reductases with distinct substrate/product specificities that are involved in the biosynthesis of the phycobiliprotein chromophore precursors phycoerythrobilin and phycocyanobilin . Implications of these results are discussed with regard to the pathways of phytobilin biosynthesis and their evolution.

Microbiology, 2001 Apr, 147(Pt 4), 1069 - 77
The gene yghK linked to the glc operon of Escherichia coli encodes a permease for glycolate that is structurally and functionally similar to L-lactate permease; Nunez MF et al.; In Escherichia coli the glc operon involved in glycolate utilization is located at 67.3 min and formed by genes encoding the enzymes glycolate oxidase (glcDEF) and malate synthase G (glcB) . Their expression from a single promoter upstream of glcD is induced by growth on glycolate and regulated by the activator encoded by the divergently transcribed gene glcC . Gene yghK, located 350 bp downstream of glcB, encodes a hydrophobic protein highly similar to the L-lactate permease encoded by lldP . Expression studies have shown that the yghK gene (proposed name glcA) is transcribed from the same promoter as the other glc structural genes and thus belongs to the glc operon . Characterization of a glcA::cat mutant showed that GlcA acts as glycolate permease and that glycolate can also enter the cell through another transport system . Evidence is presented of the involvement of L-lactate permease in glycolate uptake . Growth on this compound was abolished in a double mutant of the paralogous genes glcA and lldP, and restored with plasmids expressing either GlcA or LldP . Characterization of the putative substrates for these two related permeases showed, in both cases, specificity for the 2-hydroxymonocarboxylates glycolate, L-lactate and D-lactate . Although both GlcA and LldP recognize D-lactate, mutant analysis proved that L-lactate permease is mainly responsible for its uptake.

Microbiology, 2001 Apr, 147(Pt 4), 965 - 72
The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions; Franke S et al.; Transcription of the ybcZ--ylcA ylcBCD--ybdE region of the Escherichia coli K38 chromosome was analysed by Northern RNA--DNA hybridization, RT-PCR and primer extension . Transcription of a dicistronic ybcZ--ylcA mRNA and a tetracistronic ylcBCD--ybdE mRNA was induced by silver and was initiated from the sigma-70 promoters ylcAp and ylcBp . Expression of beta-galactosidase activity from a Phi(ylcBp--lacZ) operon fusion was also induced by Ag(+) and Cu(2+), but not by Zn(2+) . In-frame deletion of ybdE from the chromosome yielded a silver-sensitive E . coli mutant strain which did not differ in its copper resistance from its wild-type strain . On the other hand, deletion of the copA gene for the copper-exporting P-type ATPase CopA resulted in copper sensitivity, but not in silver sensitivity . A Delta ybdE Delta copA double mutant strain behaved towards copper as the Delta copA strain and towards silver as the Delta ybdE strain . Thus, in E . coli, the YlcBCD--YbdE system may be involved in silver- but not in copper resistance, and CopA may be involved in copper- but not in silver resistance.

Mol Cell Biol, 2001 Apr, 21(8), 2858 - 66
Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs; Takata M et al.; The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells . Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs) . We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant . The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange . Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays . Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51 . We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

J Exp Bot, 2001 Feb, 52(355), 215 - 21
Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells; Davletova S et al.; A calmodulin like domain protein kinase (CPK) homologue was identified in alfalfa and termed MsCPK3 . The full-length sequence of cDNA encoded a 535 amino acid polypeptide with a molecular weight of 60.2 kDa . The deduced amino acid sequence showed all the conserved motifs that define other members of this kinase family, such as serine-threonine kinase domain, a junction region and four potential Ca2+ -binding EF sites . The recombinant MsCPK3 protein purified from E . coli was activated by Ca2+ and inhibited by calmodulin antagonist (W-7) in in vitro phosphorylation assays . The expression of MsCPK3 gene increased in the early phase of the 2,4-D induced alfalfa somatic embryogenesis . Heat shock also activated this gene while kinetin, ABA and NaCl treatment did not result in MsCPK3 mRNA accumulation . The data presented suggest that the new alfalfa CPK differs in stress responses from the previously described homologues and in its potential involvement in hormone and stress-activated reprogramming of developmental pathways during somatic embryogenesis.

J Biol Chem, 2001 Jun 22, 276(25), 23135 - 43 Epub 2001 Mar 29.
Functional characterization of arginine 30, lysine 40, and arginine 62 in Tn5 transposase; Twining SS et al.; Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules . Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E . coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo . In vitro analysis revealed these mutants were inactive, because both Arg(30) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone . These results suggest Arg(30) and Arg(62) play critical roles in DNA binding and/or synaptic complex formation . Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E . coli proteinases . This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro . These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.

Int Immunol, 2001 Apr, 13(4), 541 - 51
Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia coli enterotoxin B subunit (EtxB) to CD4(+) T cells; Nashar TO et al.; Successful antigen presentation by antigen-presenting cells is governed by a number of factors including the efficiency of antigen capture by cell-surface receptors, targeting to compartments of antigen processing, surface expression of MHC II-peptide complexes and presence of co-stimulatory signals . Ganglioside GM1 is an important component of membrane glycosphingolipids, and has been implicated in cell differentiation, apoptosis and signal transduction pathways . Using the B subunit of Escherichia coli enterotoxin (EtxB), a potent immunogen that binds GM1 with high affinity, and a non-binding mutant of EtxB, EtxB(G33D), we demonstrate that GM1 is intimately involved in several aspects of antigen presentation . Thus, GM1-mediated presentation of EtxB by B cells and CD11c(+) dendritic cells (DC) significantly enhanced the proliferation and cytokine expression of EtxB-specific CD4(+) T cells . Investigation regarding potential mechanisms revealed that EtxB binding directly augments the expression of MHC class II on B cells, and fractionation of B cells demonstrated that EtxB binding to GM1 results in rapid internalization and targeting to class II-rich compartments . GM1-mediated uptake of antigens and access to class II compartments in B cells can be exploited to significantly enhance the presentation of ovalbumin-conjugated to EtxB . These results demonstrate that GM1 can play an important role in antigen presentation via the MHC II pathway.

Int Immunol, 2001 Apr, 13(4), 421 - 9
Chondrocyte antigen expression, immune response and susceptibility to arthritis; Chan VS et al.; The association of HLA-B27 with certain forms of arthritis implies a role for MHC class I-restricted T cells in the arthritic process . Our aim was to study CD8(+) T cell responses towards specific antigens localized in joint tissue . Known determinants were introduced into chondrocytes of transgenic (TG) mice, under the control of the cis-regulatory sequences of the human type II collagen gene (COL2A1) . Two Escherichia coli beta-galactosidase (beta-gal)-expressing lines were derived (CIIL73 and CIIL64) as well as two lines (CIINP) expressing influenza A virus nucleoprotein (NP) . Expression of the antigens could be demonstrated in cartilaginous tissues . The TG lines showed variable degrees of responsiveness towards the transgene-introduced antigens; whilst 75% of CIIL73 mice had an impaired cytotoxic T lymphocyte (CTL) response towards beta-gal, the response in CIIL64 mice was essentially normal . However, both lines displayed normal proliferative and antibody responses to beta-gal . A reduced CTL response was seen to NP in the CIINP lines in approximately 65% of the animals . In spite of the persistence of T cell responses to the transgene antigens in these lines, induction of CTL responses alone has so far failed to induce clinical signs of arthritis . Interestingly, some animals expressing beta-gal were susceptible to arthritis following challenge with type II collagen alone, whilst their non-TG littermates and TG mice from other lines remained unaffected . As beta-gal is expressed by E . coli, a component of the normal gut flora, this suggests a possible role for gut-derived immune responses . We believe these lines could form the basis of a model for studying links between intestinal inflammation and arthritis.

Am J Respir Crit Care Med, 2001 Mar, 163(4), 977 - 82
Lipopolysaccharide-induced diaphragmatic contractile dysfunction and sarcolemmal injury in mice lacking the neuronal nitric oxide synthase; Comtois AS et al.; In this study we evaluated the role of the neuronal nitric oxide synthase (nNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction and sarcolemmal injury . Wild-type (WT) mice or mice deficient in the nNOS gene (nNOS(-/-)) were injected with either saline (control) or Escherichia coli LPS (LPS groups) and sacrificed 12 h later . The diaphragm was then examined for NOS expression, NOS activity, and in-vitro contractility . We also assessed sarcolemmal injury in isolated muscle strips under resting condition and after 3 min of artificial stimulations . In WT mice, LPS injection reduced maximum force to about 75% of that of control animals and raised total NOS activity significantly due to the induction of the iNOS isoform . Although muscle fiber injury was minimal under resting condition, the percentage of injured fibers in control and LPS-injected mice approached 27% and 40% of total fibers, respectively, in response to artificial stimulation . By comparison, LPS injection in nNOS(-/-) mice elicited a worsening of muscle contractility (maximum force < 60% of control animals) but elicited degrees of sarcolemmal injury similar to those observed in the WT animals . In addition, muscle NOS activity and iNOS protein level in nNOS(-/-) mice injected with LPS reached about 10% and 60% of that of WT animals, respectively (p < 0.05 compared with WT animals) . Protein level of endothelial NOS isoform in the diaphragm was not altered by LPS injection in either WT or nNOS(-/-) animals . We conclude that nNOS plays a protective role in attenuating the negative influence of sepsis on diaphragmatic contractility but is not involved in the pathogenesis of sepsis-induced sarcolemmal injury.

Am J Respir Crit Care Med, 2001 Mar, 163(4), 958 - 64
Mechanical ventilation-induced air-space enlargement during experimental pneumonia in piglets; Goldstein I et al.; Mechanical ventilation-induced air-space enlargement was investigated in a porcine model of multifocal pneumonia . Following the intrabronchial inoculation of Escherichia coli, 9 piglets (22 +/- 2 kg) were ventilated with a tidal volume (VT) of 15 ml/kg for 43 +/- 15 h . Five noninoculated piglets ventilated for 60 h with the same VT served as control animals . Following death, the lungs were fixed and lung morphometry was assessed . In inoculated animals, unventilated infected and normally ventilated noninfected pulmonary lobules coexisted . In normally ventilated lung regions (1) emphysema-like lesions were present, (2) mean alveolar area and mean linear intercept were significantly greater in inoculated than in control animals, and (3) the degree of alveolar distension correlated with the decrease in respiratory compliance . In unventilated lung areas (1) pseudocysts were frequent, (2) alveolar edema was rare, (3) bronchiolectasis was frequent, (4) mean bronchiolar area was greater in inoculated than in control animals, and (5) the degree of bronchiolar distension correlated with the increase in inspiratory plateau pressure . In conclusion, in piglets with severe bronchopneumonia, air-space enlargement rather than pulmonary edema was the major feature of mechanical ventilation-induced lung barotrauma and resembled lesions previously reported in critically ill patients ventilated using high inspiratory pressures.

Appl Environ Microbiol, 2001 Apr, 67(4), 1964 - 9
Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli; Drewlo S et al.; On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods . A 1,251-bp open reading frame (bec) was cloned and sequenced . The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.

Appl Environ Microbiol, 2001 Apr, 67(4), 1601 - 6
Purification and characterization of the recombinant Thermus sp . strain T2 alpha-galactosidase expressed in Escherichia coli; Ishiguro M et al.; The nucleotide sequence of the Thermus sp . strain T2 DNA coding for a thermostable alpha-galactosidase was determined . The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M(r), 53,514) . The observed homology between the deduced amino acid sequences of the enzyme and alpha-galactosidase from Thermus brockianus was over 70% . Thermus sp . strain T2 alpha-galactosidase was expressed in its active form in Escherichia coli and purified . Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric . The enzyme was most active at 75 degrees C for p-nitrophenyl-alpha-D-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70 degrees C . The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40 degrees C for 1 h . The enzyme acted on the terminal alpha-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea alpha-galactosidase I . The enzyme has only one Cys residue in the molecule . para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

Front Biosci, 2001 Apr 01, 6, A1 - A12
Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules; Torshin IY; Structural classes of protein domains correlate with their amino acid compositions . Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance . This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition . Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents . Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified . The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources . About 90% of the proteins of the core are intracellular proteins . 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins . At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins . The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges . The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins . As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules . The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

Curr Opin Microbiol, 2001 Apr, 4(2), 178 - 85
Transcriptional responses to DNA damage; Volkert MR et al.; In Escherichia coli, DNA repair and protective responses are regulated at the transcriptional level . Regulatory mechanisms have evolved that allow cells to respond to DNA damage by mounting the appropriate responses . The regulatory proteins controlling these responses are activated when they recognize the presence of a specific DNA damaging agent, the production of specific DNA lesions, or the production of damage intermediates resulting from replication of lesions containing DNA . Transcription of the responses to DNA damage are induced when the activated regulatory proteins stimulate transcription of the genes they control by a variety of complex and unique molecular mechanisms.

Curr Opin Microbiol, 2001 Apr, 4(2), 132 - 7
The AraC transcriptional activators; Martin RG et al.; The AraC family of bacterial transcriptional activators regulate diverse genetic systems . Recent X-ray diffraction studies show that the monomeric MarA and Rob activators bind to their asymmetric degenerate DNA sites via two different helix-turn-helix elements . Activation by MarA, SoxS or Rob requires a particular orientation of the asymmetric binding sequence (and hence the activator), depending on its distance from the -10 RNAP signal . Genetic studies are beginning to clarify how the activators interact with RNAP . Growing evidence suggests that for the sugar metabolism activators, multiple binding sites upstream of the promoter anchor the activator in a repressing or nonactivating configuration . By interaction with the sugar and/or CRP, the activator is allosterically altered so it can bind a new set of sites that enable it to activate the promoter . Surprisingly, the virulence activator, Rns, must bind to both upstream and downstream sites in order to activate the rns promoter.

Curr Opin Microbiol, 2001 Apr, 4(2), 126 - 31
How sigma docks to RNA polymerase and what sigma does; Burgess RR et al.; It is clear that multiple sites of interaction exist between sigmas and core subunits, likely reflecting the changing pattern of interactions that occur sequentially during the complex process of holoenzyme formation, open promoter formation, and initiation of transcription . Recent studies have revealed that a major site of interaction of Escherichia coli sigma factors is the amino acid 260-309 coiled-coil region of the beta' subunit of core RNA polymerase . This region of beta' interacts with region 2.1-2.2 of sigma(70) . Binding of this region of beta' to sigma(70) triggers a conformational change in sigma that allows it to bind to a -10 nontemplate promoter DNA strand oligonucleotide.

J Biochem Biophys Methods, 2001 Mar 28, 48(1), 77 - 84
A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody; Dansithong W et al.; A DNA fragment for a heavy chain variable domain (VH) was prepared from a hybridoma that produces a monoclonal antibody against human midkine (MK) . The antibody fragment was produced in Escherichia coli and its affinity for chemically synthesized full length MK or recombinant midkine c-terminus (MKc-half) protein was confirmed by ELISA . An Escherichia coli cell lysate expressing MKc-half was applied to a VH fragment-coupled Sepharose 4B column and eluted with a buffer containing 0.5 M NaCl . SDS-PAGE analysis revealed a high degree of purity of the MKc-half protein in the eluent, showing the utility of a recombinant VH fragment in purification of proteins by affinity chromatography.

Vaccine, 2001 Apr 6, 19(20-22), 2898 - 907
Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA- or IgG-mediated protective mucosal immunity; Haan L et al.; Local mucosal IgA antibodies play a central role in protection of the respiratory tract against influenza virus infection . Therefore, new-generation influenza vaccines should aim at stimulating not only systemic, but also local antibody responses . Previously, we demonstrated that the recombinant B subunit of the Escherichia coli heat-labile toxin (LTB) is a potent adjuvant towards nasally administered influenza subunit antigen . Here, we investigated the protection conferred by LTB-supplemented influenza subunit antigen given intranasally (i.n.) or intramuscularly (i.m.) to mice . Both i.n . and i.m . immunization with subunit antigen and LTB completely protected the animals against viral infection . Protection upon i.n . immunization was associated with the induction of antigen-specific serum IgG and mucosal IgA, whereas protection upon i.m . immunization correlated with strong serum and mucosal IgG, but not IgA responses . We conclude that LTB-supplemented influenza subunit antigen, given either i.n . or i.m, induces protective antibody-mediated mucosal immunity and thus represents a promising novel flu vaccine candidate.

Protein Expr Purif, 2001 Apr, 21(3), 485 - 91
Expression of cloned cDNA for the human mitochondrial RNA polymerase in Escherichia coli and purification; Nam SC et al.; A full-length cDNA for the mature, mitochondrial form of human mitochondrial RNA polymerase was cloned and expressed under the control of T5 or tac promoter in Escherichia coli . The cDNA was efficiently expressed at 37 degrees C, but virtually all the polymerase produced was insoluble, and renaturation of the inclusion bodies was unsuccessful . When the cells were grown at 25 degrees C, however, a portion of approximately 10% was soluble and active . The protein was purified 100-fold from the soluble lysates to homogeneity by two-step chromatography using Ni-nitrilotriacetic acid-Sepharose and heparin-agarose columns, as an N-terminal histidine tag attached and as the tag cleaved away . The purified polymerases with and without the histidine tag were both active in RNA polymerization in vitro as measured with poly(dA-dT) template, and specific activity was 140,000 units/mg . The purified enzyme has the same biochemical properties as the polymerase fraction partially purified from the human mitochondria, except for the promoter-specific activity that was not observed with the purified polymerase in the presence of mitochondrial transcription factor A . Additional factor(s) and/or mammalian-specific or regulatory modification(s) of the polymerase should be necessary for promoter-specific transcription .

Protein Expr Purif, 2001 Apr, 21(3), 462 - 9
The cochaperone murine stress-inducible protein 1: overexpression, purification, and characterization; van der Spuy J et al.; Murine stress-inducible protein 1 (mSTI1) is a cochaperone that is homologous with the human heat shock cognate protein 70 (Hsc70)/heat shock protein 90 (Hsp90)-organizing protein (Hop) . To analyze the biochemical properties of mSTI1 and the stoichiometry of the Hsc70.mSTI1.Hsp90 association, recombinant mSTI1 was produced in untagged, histidine (His)-tagged, and glutathione S-transferase (GST)-tagged forms . His-mSTI1 was detected either as a dimer during size-exclusion-high-performance liquid chromatography (SE-HPLC) or as a monomer during Superdex 200 gel filtration chromatography . SE-HPLC on GST-mSTI1 and untagged mSTI1 suggested that mSTI1 existed as a monomer . Cross-linking of His-mSTI1 detected a compact monomeric species and a dimeric species . Gel filtration on the association of bovine STI1 or His-mSTI1 with Hsc70 detected species of molecular mass consistent with a dimeric STI1 species or a 1:1 complex of STI1 and Hsc70 . Our data and that of others suggest that mSTI1 and its homologues exist as either a monomer or a dimer and that this facilitates its proposed function as an Hsc70/Hsp90 organizing protein .

Protein Expr Purif, 2001 Apr, 21(3), 412 - 6
Expression, purification, and crystallization of the RGS-like domain from the Rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach; Garrard SM et al.; Lsc-homology domains are found in several eukaryotic nucleotide exchange factors which act on Rho-family GTPases . They show limited amino acid sequence similarity to RGS proteins, which down-regulate the cellular signaling by the alpha-subunits of trimeric G-proteins and have been shown to interact with Galpha12 and Galpha13 . It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases . We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF . To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues . Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source . The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit .

Protein Expr Purif, 2001 Apr, 21(3), 401 - 11
Expression, purification, and biophysical characterization of the BRCT domain of human DNA ligase IIIalpha; Thornton KH et al.; The C-terminal regions of several DNA repair and cell cycle checkpoint proteins are homologous to the breast-cancer-associated BRCA-1 protein C-terminal region . These regions, known as BRCT domains, have been found to mediate important protein-protein interactions . We produced the BRCT domain of DNA ligase IIIalpha (L3{86}) for biophysical and structural characterization . A glutathione S-transferase (GST) fusion with the L3{86} domain (residues 837-922 of ligase IIIalpha) was expressed in Escherichia coli and purified by glutathione affinity chromatography . The GST fusion protein was removed by thrombin digestion and further purification steps . Using this method, (15)N-labeled and (13)C/(15)N-double-labeled L3{86} proteins were prepared to enable a full determination of structure and dynamics using heteronuclear NMR spectroscopy . To obtain evidence of binding activity to the distal BRCT of the repair protein XRCC1 (X1BRCTb), as well as to provide insight into the interaction between these two BRCT binding partners, the corresponding BRCT heterocomplexes were also prepared and studied . Changes in the secondary structures (amount of helix and sheet components) of the two constituents were not observed upon complex formation . However, the melting temperature of the complex was significantly higher relative to the values obtained for the L3{86} or X1BRCTb proteins alone . This increased thermostability imparted by the interaction between the two BRCT domains may explain why cells require XRCC1 to maintain ligase IIIalpha activity .

Protein Expr Purif, 2001 Apr, 21(3), 378 - 85
Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p17 of B- and C-subtypes; Gupta S et al.; A simple and efficient method for expression in Escherichia coli and purification of matrix protein, p17, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described . DNA sequences encoding p17 of B- and C-subtype were cloned from respective gag sequences . The gag sequences were obtained by PCR amplification using DNA extracted from peripheral blood lymphocytes of an HIV-1 infected patient from India . A T7-promoter-based expression system was optimized for expression of p17 in soluble form . p17 (B- and C-subtype) was purified to near homogeneity using conventional chromatographic techniques . Purification of p17 (C-subtype) is described for the first time with yield of 7.7 mg from a 1-liter culture . The yield of p17 (B-subtype) is 14.7 mg from a 1-liter culture, which is severalfold better than that reported earlier . N-terminal sequencing and CD spectra of the purified proteins, p17B and p17C, show that the proteins are properly processed and well-folded . The immunoreactivity of both types of p17 to sera from HIV-infected individuals is comparable .

Protein Expr Purif, 2001 Apr, 21(3), 367 - 77
Solution studies of recombinant human stromal-cell-derived factor-1; Holmes WD et al.; Stromal-cell-derived factor-1 (SDF-1alpha) is an 8-kDa chemokine that is constitutively expressed in bone-marrow-derived stromal cells and has been identified as a ligand for the CXCR4 receptor . We produced the chemokine recombinantly as methionine-SDF-1alpha in Escherichia coli without the leader peptide sequence . The protein was denatured, refolded, and further purified by reversed-phase HPLC . SDF-1alpha was shown to be >95% pure as judged by SDS-PAGE . The final yield of purified and refolded SDF-1alpha was 1-2 mg per gram of wet cell paste . The refolded protein is a ligand for the CXCR4 receptor and has been used to block HIV-mediated cell fusion and downmodulates the CXCR4 receptor . Our ability to purify hundreds of milligrams of refolded protein allowed us to conduct detailed studies of the biophysical properties of the protein . We have used a combination of biophysical techniques to study the solution properties of SDF-1alpha . The average mass of SDF-1alpha, as determined by static light scattering, gave us the first indications that the chemokine may self-associate . Further investigation with sedimentation velocity ultracentrifugation confirmed the existence of two species . The measured s(20, W) values defined two masses corresponding to monomer and dimer . Finally, sedimentation equilibrium ultracentrifugation and dynamic light scattering yielded a composite value of 150 +/- 30 microM for the dimerization constant . We conclude that SDF-1alpha exists in a monomer-dimer equilibrium .

J Am Soc Mass Spectrom, 2001 Mar, 12(3), 329 - 36
Metastable ion formation and disparate charge separation in the gas-phase dissection of protein assemblies studied by orthogonal time-of-flight mass spectrometry; Versluis C et al.; The dissection of specific and nonspecific protein complexes in the gas phase is studied by collisionally activated decomposition . In particular, the gas phase dissection of multiple protonated homodimeric Human Galectin I, E . Coli Glyoxalase I, horse heart cytochrome c, and Hen egg Lysozyme have been investigated . Both the Human Galectin I and E . Coli Glyoxalase I enzymes are biologically active as a dimer, exhibiting molecular weights of approximately 30 kDa . Cytochrome c and Lysozyme are monomers, but may aggregate to some extent at high protein concentrations . The gas phase dissociation of these multiple protonated dimer assemblies does lead to the formation of monomers . The charge distribution over the two concomitant monomers following the dissociation of these multiple protonated dimers is found to be highly dissimilar . There is no evident correlation between the solution phase stability of the dimeric proteins and their gas-phase dissociation pattern . Additionally, in the collisionally activated decomposition spectra diffuse ion signals are observed, which are attributed to monomer ions formed via slow decay of the collisionally activated dimer ions inside the reflectron time-of-flight . Although, the formation of these diffuse metastable ions may complicate the interpretation of collisionally activated decomposition mass spectra, especially when studying noncovalent protein complexes, a simple mathematical equation may be used to reveal their origin and pathway of formation.

Res Microbiol, 2001 Jan-Feb, 152(1), 75 - 81
eae-negative attaching and effacing Escherichia coli from piglets with diarrhea; Penteado AS et al.; One hundred and ninety strains of Escherichia coli that were isolated from pigs with diarrhea in the state of Sao Paulo, Brazil, and that were negative for enterotoxins and cytotoxins were investigated . Strains which adhered to HeLa cells were examined for fluorescence actin staining (FAS), the ability to induce attaching and effacing (A/E) lesions on HEp-2 cells detectable by transmission electron microscopy and the presence of eae gene sequences detected by PCR . Intimin production was detected by western blot and serogrouping was performed . Forty-seven isolates adhered to HeLa cells in several patterns, but none adhered in a localized adherence pattern . However, seven of the 47 adherent strains were positive for the FAS reaction, although the reactions were usually weak or atypical . One FAS-negative and three FAS-positive strains, which were examined for their ability to induce A/E lesions, were all positive . Subsequently, testing of these strains for the eae gene showed that they all lacked this gene . These findings, along with earlier reports of eae-negative A/E E . coli, suggest that higher quantities of E . coli in this category might be detected if more reliance were placed on phenotypic tests rather than on gene detection tests alone.

Free Radic Biol Med, 2000 Jan 15, 28(2), 167 - 73
Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells; Su Y et al.; The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood . Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP) . Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site . Therefore, we hypothesized that PAO may have a direct effect on eNOS activity . To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli . High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions . PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity . PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS . The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent . These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.

Photochem Photobiol, 2001 Mar, 73(3), 304 - 11
Photoinduced inactivation of T7 phage sensitized by symmetrically and asymmetrically substituted tetraphenyl porphyrin: comparison of efficiency and mechanism of action; Gabor F et al.; We investigated the efficiency and the mechanism of action of two--one symmetrically and one asymmetrically substituted--glycoconjugated tetraphenyl porphyrins in their photoreaction with T7 phage as a model of nucleoprotein (NP) complexes . A correlation was found between the dark inactivation of T7 and the binding of porphyrins determined by fluorescence spectroscopy . Both types of porphyrin sensitized the photoinactivation of T7, but the slopes of inactivation kinetics were markedly different . There was no correlation between the dark binding and the photosensitizing efficacy of the two derivatives . Inactivation was moderated by 1,3-diphenylisobenzofuran and 1,3-dimethyl-2-thiourea; however, neither of them inhibited T7 inactivation completely . This result suggests that both Type-I and Type-II reactions play a role in the virus inactivation . Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated NP complex . Polymerase chain reaction analysis of a 555 bp segment of gene 1 and a 3826 bp segment of genes 3 and 4 failed to demonstrate any DNA damage.

Yi Chuan Xue Bao, 2001, 28(3), 278 - 84
{Effects of Pro229-->Ser and Glu243-->Gly on the characters of thermostable catechol 2, 3-dioxygenase}; Jiang T et al.; In order to investigate the effects of amino acid replacement on the characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis . The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E . coli TG1 and purified . The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed . The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60 degrees C) and the Kcat/Km value of Pro229Ser and Glu243Gly (4.89 +/- 0.01 x 10(6) mol-1 s-1 and 5.88 +/- 0.01 x 10(6) mol-1 s-1, respectively) were reduced compared with the wild type enzyme (6.97 +/- 0.01 x 10(6) mol-1 s-1) . However, the thermostability of Pro229Ser extremely decreased 10.2 degrees C and the thermostability of Glu243Gly slightly increased 1.5 degrees C . It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.

Intensive Care Med, 2001 Jan, 27(1), 258 - 63
Reduction in intestinal leukocyte adherence in rat experimental endotoxemia by treatment with the 21-aminosteroid U-74389G; Lehmann C et al.; OBJECTIVES: To investigate leukocyte adherence in intestinal venules in experimental endotoxemia after treatment with the 21-aminosteroid U-74389G . DESIGN AND SETTING: Prospective, randomized, controlled animal study in an experimental laboratory . SUBJECTS: Twenty-one male Wistar rats weighing 190 +/- 40 g . INTERVENTIONS: The rats were divided equally into three groups: (a) control group, (b) endotoxemia (5 mg/kg lipopolysacharide from Escherichia coli O55:B5), and (c) endotoxemia and U-74389G administration 30 min before (3 mg/kg) and 60 min after endotoxin challenge (1.5 mg/ kg) . MEASUREMENTS AND MAIN RESULTS: The distal small intestine of the animals was examined using intravital fluorescence videomicroscopy 2 h after endotoxin challenge . Leukocytes were stained in vivo by means of rhodamine 6G . In the endotoxemic animals we observed a fourfold increase in the count of firmly adherent leukocytes in submucosal post-capillary and collecting venules . Treatment with the 21-aminosteroid U-74389G significantly attenuated the count of sticking leukocytes in the collecting venules (control, 61 +/- 10 cells/mm2; lipopolysaccharide, 237 +/- 42 cells/mm2; U-74389G 125 +/- 9 cells/mm2; p < 0.05) . In these venules leukocyte rolling behavior was comparable to that in the control group without endotoxin challenge . CONCLUSIONS: Administration of U-74389G, which has radical scavenging properties, attenuates leukocyte adherence in selected populations of intestinal venules which is found increased during endotoxemia . Thus, 21-aminosteroids may have an impact in the treatment of endotoxin-induced intestinal injury.

Intensive Care Med, 2001 Jan, 27(1), 251 - 7
Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin; Holzmann A et al.; OBJECTIVES: To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin/lipopolysaccharide (LPS) . DESIGN AND SETTING: Prospective, controlled animal study in the animal research facility of a university hospital . INTERVENTIONS: Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg/ kg E . coli 0111:B4 LPS administration, lungs were isolated and perfused, and the thromboxane mimetic U46619 was employed to increase the mean pulmonary artery pressure by 5-7 mmHg . The lungs were then ventilated with or without 0.4 ppm NO, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; PDE type 2 inhibitor), milrinone (PDE type 3 inhibitor), or zaprinast (inhibitor of PDE types 5 and 9) were added to the perfusate . MEASUREMENTS AND RESULTS: In the presence of EHNA (12.5, 25, 50 microM) the vasodilator response to inhaled NO was not greater than in its absence (0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg vs . 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg, respectively) . In the presence of milrinone (125, 250, 500 nM), the vasodilator response to inhaled NO was also not improved . In contrast, zaprinast (3.7, 7.4, 14.8 microM) augmented the pulmonary vasodilatory effect of inhaled NO in lungs from LPS-pretreated rats from 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg to 0.75 +/- 0.25, 1.5 +/- 0.5, 1.75 +/- 0.75 mmHg, respectively (p < 0.05) . CONCLUSIONS: Our results demonstrate that inhibition of pulmonary PDE enzyme activity with zaprinast increases vasodilator responsiveness to inhaled NO in lungs obtained from rats 16 h after LPS challenge.

Indian J Med Res, 2001 Jan, 113, 5 - 10
Escherichia coli heat stable enterotoxin receptors & guanylyl cyclase activity in the intestinal brush border membrane of hamsters & guinea pigs; Bhattacharya J et al.; BACKGROUND & OBJECTIVES: Although Escherichia coli heat stable enterotoxin (STa) causes diarrhoea in laboratory animals, no studies were done to find out the species specific variation of distribution of the STa receptors in laboratory animals . The present investigation evaluates the density of STa receptors and the guanylyl cyclase (GC) activity in the small intestinal epithelial cells of hamsters and guinea pigs . METHODS: Brush border membrane (BBM) was prepared from the small intestines of hamsters and guinea pigs . Receptor binding assay, GC assay and autoradiography were performed to determine the density of STa receptors, the GC activity and molecular weights of the STa binding proteins respectively . RESULTS: The receptor densities, per mg BBM protein at equilibrium, were found to be 4.1 x 10(9) and 1.5 x 10(12) in hamsters and guinea pigs respectively . The GC activity was found to be lower in STa treated hamster BBM compared to that of guinea pig . Scatchard analysis of the stoichiometric data showed a linear plot, and STa bound with association constants of 0.31 x 10(12) M-1 and 1.04 x 10(12) M-1 in hamsters and guinea pigs respectively . Autoradiographic analysis of the SDS-PAGE, revealed that 125I-STa bound apparently to a 45 kDa membrane protein in hamster and a 115 kDa membrane protein in guinea pig . INTERPRETATION & CONCLUSIONS: It appears that a lower density of STa receptor exists in hamsters compared to that in guinea pigs . STa binds with a single population of STa receptors in each species with different ligand binding affinities . Also, the molecular weights of the STa binding proteins differ in these species . Moreover, the GC activity was found to be lower in hamsters than in guinea pigs.

J Biol Chem, 2001 Jun 8, 276(23), 20213 - 9 Epub 2001 Mar 28.
TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli; Bolhuis A et al.; In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway . In the present study, we have purified the Tat complex from E . coli, and we show that it contains only TatA, TatB, and TatC . Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association . This has been confirmed by expression of a translational fusion between TatB and TatC . This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms . The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association . The molecular mass of the complex is approximately 600 kDa, demonstrating the presence of multiple copies of TatA, B, and C . Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.

J Biol Chem, 2001 May 18, 276(20), 17106 - 10 Epub 2001 Mar 06.
Transcription factor Rho does not require a free end to act as an RNA-DNA helicase on an RNA; Burgess BR et al.; Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript . Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5' terminus . To determine whether Rho's activity is 5'-end-dependent, its ability to bind to and function on a circular derivative of lambda cro mRNA was investigated . The circular derivative was made in vitro by action of RNA ligase on a derivative of lambda cro RNA containing an extra 10-nucleotide sequence near the 5'-end that was complementary to a sequence located near the 3'-end . Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript . Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction . Thus, the action of Rho on a transcript does not depend on the availability of a free 5' terminus.

J Biol Chem, 2001 May 18, 276(20), 17111 - 6 Epub 2001 Mar 13.
Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax duffy-binding protein; Singh S et al.; Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions . Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes . The P . vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein . The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins . Here, we have expressed P . vivax region II (PvRII), the P . vivax Duffy binding domain, in Escherichia coli . Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies . We have developed methods to refold and purify recombinant PvRII in its functional conformation . Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity . Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P . vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P . vivax malaria . Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.

J Biol Chem, 2001 May 11, 276(19), 16425 - 31 Epub 2001 Feb 13.
Crystal structure of activated CheY . Comparison with other activated receiver domains; Lee SY et al.; The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution . BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively . The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules . All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r) . Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group . The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4 . The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89) . This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop . Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop . This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM . The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r) . A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride . This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

J Biol Chem, 2001 May 25, 276(21), 18597 - 604 Epub 2001 Feb 27.
Pkd1 unusual DNA conformations are recognized by nucleotide excision repair; Bacolla A et al.; The 2.5-kilobase pair poly(purine.pyrimidine) (poly(R.Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene . To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3) . The full-length poly(R.Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response . The extent of cell loss was correlated with the length of the poly(R.Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci . We conclude that the unusual DNA conformations formed by the PKD1 poly(R.Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid . These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.

J Biol Chem, 2001 May 11, 276(19), 15810 - 5 Epub 2001 Feb 15.
"Weak toxin" from Naja kaouthia is a nontoxic antagonist of alpha 7 and muscle-type nicotinic acetylcholine receptors; Utkin YN et al.; A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with {(125)I}alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm . In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N . kaouthia, representing short-type and long-type alpha-neurotoxins, respectively . WTX and alpha-cobratoxin displaced {(125)I}alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm . Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm) . Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin . It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.

J Biol Chem, 2001 May 11, 276(19), 16070 - 5 Epub 2001 Mar 08.
Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2; Chen Z et al.; Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) . In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways . Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2 . Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7 . Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38 . Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6 . Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.

J Biol Chem, 2001 May 25, 276(21), 17878 - 86 Epub 2001 Feb 23.
CRP modulates fis transcription by alternate formation of activating and repressing nucleoprotein complexes; Nasser W et al.; The DNA architectural proteins FIS and CRP are global regulators of transcription in Escherichia coli involved in the adjustment of cellular metabolism to varying growth conditions . We have previously demonstrated that FIS modulates the expression of the crp gene by functioning as its transcriptional repressor . Here we show that in turn, CRP is required to maintain the growth phase pattern of fis expression . We demonstrate the existence of a divergent promoter in the fis regulatory region, which reduces transcription of the fis promoter . In the absence of FIS, CRP activates fis transcription, thereby displacing the polymerase from the divergent promoter, whereas together FIS and CRP synergistically repress fis gene expression . These results provide evidence for a direct cross-talk between global regulators of cellular transcription during the growth phase . This cross-talk is manifested in alternate formation of functional nucleoprotein complexes exerting either activating or repressing effects on transcription.

J Biol Chem, 2001 May 18, 276(20), 17022 - 9 Epub 2001 Mar 06.
Excision of 3' termini by the Trex1 and TREX2 3'-->5' exonucleases . Characterization of the recombinant proteins; Mazur DJ et al.; The excision of nucleotides from DNA 3' termini is an important step in DNA replication, repair, and recombination pathways to generate correctly base paired termini for subsequent processing . The mammalian TREX1 and TREX2 proteins contain potent 3'-->5' exonucleases capable of functioning in this capacity . To study the activities of these exonucleases we have developed strategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical characterization . The Trex1 and TREX2 proteins are homodimers that exhibit robust 3' excision activities with very similar preferred reaction conditions and preferences for specific DNA substrates . In a steady-state kinetic analysis, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2 . The Michaelis constants derived from these data indicate similar apparent kcat values of 22 s(-1) for Trex1 and 16 s(-1) for TREX2 using single-stranded oligonucleotides . The apparent KM values of 19 nm for Trex1 and 190 nm for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2 . An exonuclease competition assay was designed using heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2 . The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for producing correctly base paired 3' termini.

J Biol Chem, 2001 Jun 1, 276(22), 19182 - 9 Epub 2001 Mar 06.
Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme; Stewart J et al.; The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome . The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers . This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA . delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta . Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta . Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer . We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer . These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

J Biol Chem, 2001 Jun 15, 276(24), 21724 - 36 Epub 2001 Feb 21.
Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome . Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria; Suzuki T et al.; The mammalian mitochondrial ribosome (mitoribosome) is a highly protein-rich particle in which almost half of the rRNA contained in the bacterial ribosome is replaced with proteins . It is known that mitochondrial translation factors can function on both mitochondrial and Escherichia coli ribosomes, indicating that protein components in the mitoribosome compensate the reduced rRNA chain to make a bacteria-type ribosome . To elucidate the molecular basis of this compensation, we analyzed bovine mitoribosomal large subunit proteins; 31 proteins were identified including 15 newly identified proteins with their cDNA sequences from human and mouse . The results showed that the proteins with binding sites on rRNA shortened or lost in the mitoribosome were enlarged when compared with the E . coli counterparts; this suggests the structural compensation of the rRNA deficit by the enlarged proteins in the mitoribosome.

J Biol Chem, 2001 May 18, 276(20), 16992 - 7 Epub 2001 Feb 27.
Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases; Handa P et al.; Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase . Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway . We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E . coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG . In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E . coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs . The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex . However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG . Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG . The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG . The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.

J Biol Chem, 2001 May 11, 276(19), 16439 - 46 Epub 2001 Feb 08.
Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence; Kamath-Loeb AS et al.; Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability . The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities . WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions . Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions . We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon . The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products . The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN . Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta . Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures.

J Biol Chem, 2001 May 4, 276(18), 15131 - 6 Epub 2001 Jan 26.
Human UDP-galactose 4-epimerase . Accommodation of UDP-N-acetylglucosamine within the active site; Thoden JB et al.; UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism . One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket . Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined . Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa . Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc . To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199 . Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E . coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase . This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E . coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.

J Biol Chem, 2001 May 18, 276(20), 17373 - 9 Epub 2001 Feb 13.
The FadR.DNA complex . Transcriptional control of fatty acid metabolism in Escherichia coli; Xu Y et al.; In Escherichia coli, the expression of fatty acid metabolic genes is controlled by the transcription factor, FadR . The affinity of FadR for DNA is controlled by long chain acyl-CoA molecules, which bind to the protein and modulate gene expression . The crystal structure of FadR reveals a two domain dimeric molecule where the N-terminal domains bind DNA, and the C-terminal domains bind acyl-CoA . The DNA binding domain has a winged-helix motif, and the C-terminal domain resembles the sensor domain of the Tet repressor . The FadR.DNA complex reveals how the protein interacts with DNA and specifically recognizes a palindromic sequence . Structural and functional similarities to the Tet repressor and the BmrR transcription factors suggest how the binding of the acyl-CoA effector molecule to the C-terminal domain may affect the DNA binding affinity of the N-terminal domain . We suggest that the binding of acyl-CoA disrupts a buried network of charged and polar residues in the C-terminal domain, and the resulting conformational change is transmitted to the N-terminal domain via a domain-spanning alpha-helix.

J Biol Chem, 2001 Jun 1, 276(22), 19648 - 55 Epub 2001 Mar 06.
Functional signal peptides bind a soluble N-terminal fragment of SecA and inhibit its ATPase activity; Triplett TL et al.; The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA . The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex . Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG . A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation . Several recent reports show that functions of SecA can be studied using separated domains . Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA . We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA . Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant . Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.

J Biol Chem, 2001 Apr 27, 276(17), 14385 - 92 Epub 2001 Jan 22.
Tumor targeting of mono-, di-, and tetravalent anti-p185(HER-2) miniantibodies multimerized by self-associating peptides; Willuda J et al.; Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule . Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185(HER-2) single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus . Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli . We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts . Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185(HER-2) and on SK-OV-3 cells . The (99m)technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h . The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g . The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight . These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.

J Biol Chem, 2001 May 11, 276(19), 16548 - 54 Epub 2001 Feb 13.
Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus; Iwai H et al.; A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain . Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales . There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins . Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 m guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cis-proline) had disappeared . Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.

J Biol Chem, 2001 May 11, 276(19), 16365 - 73 Epub 2001 Feb 08.
Ca2+-independent smooth muscle contraction . a novel function for integrin-linked kinase; Deng JT et al.; Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19) . Several agonists acting via G protein-coupled receptors elicit a contraction without a change in {Ca(2+)}(i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation . We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase . In this study, we identify this kinase as integrin-linked kinase . Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E . coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18) . Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner . These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.

J Biol Chem, 2001 May 11, 276(19), 16207 - 15 Epub 2001 Jan 17.
DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular and intermolecular protein.protein interactions; Greenall A et al.; The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism . Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues . Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3 . DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain . A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3 . By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3 . PEA3 and USF-1 form DNA complexes in a cooperative manner . Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module . Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.

J Biol Chem, 2001 May 4, 276(18), 14916 - 23 Epub 2001 Feb 13.
A novel osteoblast-derived C-type lectin that inhibits osteoclast formation; Zhou H et al.; We have cloned and expressed murine osteoclast inhibitory lectin (mOCIL), a 207-amino acid type II transmembrane C-type lectin . In osteoclast formation assays of primary murine calvarial osteoblasts with bone marrow cells, antisense oligonucleotides for mOCIL increased tartrate-resistant acid phosphatase-positive mononucleate cell formation by 3-5-fold, whereas control oligonucleotides had no effect . The extracellular domain of mOCIL, expressed as a recombinant protein in Escherichia coli, dose-dependently inhibited multinucleate osteoclast formation in murine osteoblast and spleen cell co-cultures as well as in spleen cell cultures treated with RANKL and macrophage colony-stimulating factor . Furthermore, mOCIL acted directly on macrophage/monocyte cells as evidenced by its inhibitory action on adherent spleen cell cultures, which were depleted of stromal and lymphocytic cells . mOCIL completely inhibited osteoclast formation during the proliferative phase of osteoclast formation and resulted in 70% inhibition during the differentiation phase . Osteoblast OCIL mRNA expression was enhanced by parathyroid hormone, calcitriol, interleukin-1alpha and -11, and retinoic acid . In rodent tissues, Northern blotting, in situ hybridization, and immunohistochemistry demonstrated OCIL expression in osteoblasts and chondrocytes as well as in a variety of extraskeletal tissues . The overlapping tissue distribution of OCIL mRNA and protein with that of RANKL strongly suggests an interaction between these molecules in the skeleton and in extraskeletal tissues.

J Biol Chem, 2001 Apr 27, 276(17), 14204 - 11 Epub 2001 Jan 19.
Structural characterization of protein kinase A as a function of nucleotide binding . Hydrogen-deuterium exchange studies using matrix-assisted laser desorption ionization-time of flight mass spectrometry detection; Andersen MD et al.; Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J . A . (1999) Biochemistry 38, 12072-12079) . To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP . Four regions of the protein are protected from exchange in the presence of ADP . Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site . Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure . One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases . The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site . The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.

J Biol Chem, 2001 Jun 1, 276(22), 18836 - 42 Epub 2001 Mar 12.
The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable; Shinkai A et al.; Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases . Motif A in the polymerase active site has a required role in catalysis and is highly conserved . To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection . We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity . Hence, the primary structure of motif A is plastic . We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive . In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence . As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.

J Biol Chem, 2001 May 25, 276(21), 18478 - 84 Epub 2001 Feb 27.
Alteration of a nonconserved active site residue in the chemotaxis response regulator CheY affects phosphorylation and interaction with CheZ; Silversmith RE et al.; CheY is a response regulator in the well studied two-component system that mediates bacterial chemotaxis . Phosphorylation of CheY at Asp(57) enhances its interaction with the flagellar motor . Asn(59) is located near the phosphorylation site, and possible roles this residue may play in CheY function were explored by mutagenesis . Cells containing CheY59NR or CheY59NH exhibited hyperactive phenotypes (clockwise flagellar rotation), and CheY59NR was characterized biochemically . A continuous enzyme-linked spectroscopic assay that monitors P(i) concentration was the primary method for kinetic analysis of phosphorylation and dephosphorylation . CheY59NR autodephosphorylated at the same rate as wild-type CheY and phosphorylated similarly to wild type with acetyl phosphate and faster (4-14x) with phosphoramidate and monophosphoimidazole . CheY59NR was extremely resistant to CheZ, requiring at least 250 times more CheZ than wild-type CheY to achieve the same dephosphorylation rate enhancement, whereas CheY59NA was CheZ-sensitive . However, several independent approaches demonstrated that CheY59NR bound tightly to CheZ . A submicromolar K(d) for CheZ binding to CheY59NR-P or CheY.BeF(3)(-) was inferred from fluorescence anisotropy measurements of fluoresceinated-CheZ . A complex between CheY59NR-P and CheZ was isolated by analytical gel filtration, and the elution position from the column was indistinguishable from that of the CheZ dimer . Therefore, we were not able to detect large CheY-P.CheZ complexes that have been inferred using other methods . Possible structural explanations for the specific inhibition of CheZ activity as a result of the arginyl substitution at CheY position 59 are discussed.

J Biol Chem, 2001 Apr 27, 276(17), 13622 - 7 Epub 2001 Jan 17.
Delta 3,5,delta 2,4-dienoyl-CoA isomerase is a multifunctional isomerase . A structural and mechanistic study; Zhang D et al.; Delta(3,5),Delta(2,4)-Dienoyl-CoA isomerase (DI), an auxiliary enzyme of unsaturated fatty acid beta-oxidation, was purified from rat mitochondria and peroxisomes and subjected to N-terminal sequencing to facilitate a mechanistic study of this enzyme . The mature mitochondrial DI from rat heart was lacking its 34 N-terminal amino acid residues that have the properties of a mitochondrial targeting sequence . The peroxisomal isomerase was identified as a product of the same gene with a truncated and ragged N terminus . Expression of the cDNA coding for the mature mitochondrial DI in Escherichia coli yielded an enzyme preparation that was as active as the native DI . Because the recombinant DI also exhibited Delta(3,5,7),Delta(2,4,6)-trienoyl-CoA isomerase (TI) activity, both isomerases reside on the same protein . Mutations of any of the 3 acidic amino acid residues located at the active site (Modis, Y., Filppula, S . A., Novikov, D . K., Norledge, B., Hiltunen, J . K., and Wierenga, R . K . (1998) Structure 6, 957-970) caused activity losses . In contrast to only a 10-fold decrease in activity upon replacement of Asp(176) by Ala, substitutions of Asp(204) by Asn and of Glu(196) by Gln resulted in 10(5)-fold lower activities . Such activity losses are consistent with the direct involvement of these latter two residues in the proposed proton transfers at carbons 2 and 6 or 8 of the substrates . Probing of the wild-type and mutants forms of the enzyme with 2,5-octadienoyl-CoA as substrate revealed low Delta(2),Delta(3)-enoyl-CoA isomerase and Delta(5),Delta(4)-enoyl-CoA isomerase activities catalyzed by Glu(196) and Asp(204), respectively . Altogether, these data reveal that positional isomerizations of the diene and triene are facilitated by simultaneous proton transfers involving Glu(196) and Asp(204), whereas each residue alone can catalyze, albeit less efficiently, a monoene isomerization.

J Biol Chem, 2001 Jun 1, 276(22), 18804 - 11 Epub 2001 Mar 05.
The early interaction of the outer membrane protein phoe with the periplasmic chaperone Skp occurs at the cytoplasmic membrane; Harms N et al.; Spheroplasts were used to study the early interactions of newly synthesized outer membrane protein PhoE with periplasmic proteins employing a protein cross-linking approach . Newly translocated PhoE protein could be cross-linked to the periplasmic chaperone Skp at the periplasmic side of the inner membrane . To study the timing of this interaction, a PhoE-dihydrofolate reductase hybrid protein was constructed that formed translocation intermediates, which had the PhoE moiety present in the periplasm and the dihydrofolate reductase moiety tightly folded in the cytoplasm . The hybrid protein was found to cross-link to Skp, indicating that PhoE closely interacts with the chaperone when the protein is still in a transmembrane orientation in the translocase . Removal of N-terminal parts of PhoE protein affected Skp binding in a cumulative manner, consistent with the presence of two Skp-binding sites in that region . In contrast, deletion of C-terminal parts resulted in variable interactions with Skp, suggesting that interaction of Skp with the N-terminal region is influenced by parts of the C terminus of PhoE protein . Both the soluble as well as the membrane-associated Skp protein were found to interact with PhoE . The latter form is proposed to be involved in the initial interaction with the N-terminal regions of the outer membrane protein.

J Biol Chem, 2001 Jun 1, 276(22), 19327 - 31 Epub 2001 Feb 21.
The cost of exposing a hydrophobic loop and implications for the functional role of 4.5 S RNA in the Escherichia coli signal recognition particle; Cleverley RM et al.; The signal recognition particle (SRP) is an RNA-protein complex that directs ribosomes to the rough endoplasmic reticulum membrane by binding to targeting signals found on the nascent chain of proteins destined for export to the endoplasmic reticulum . We found evidence from studies with fragments of the protein component of the Escherichia coli SRP that a long hydrophobic loop (the so-called "finger loop") is detrimental to the stability of its signal peptide-binding domain, the M domain . This hydrophobic loop is highly conserved and thus may have a critical role in the function of the SRP . Given our previously reported evidence that 4.5 S RNA stabilizes the tertiary fold of the M domain (Zheng, N., and Gierasch, L . M . (1997) Mol . Cell 1, 79-87), we now propose that the functional requirement for 4.5 S RNA resides in its ability to counteract the destabilizing influence of the finger loop.

J Biol Chem, 2001 Jun 1, 276(22), 18695 - 701 Epub 2001 Mar 13.
Cloning, expression, and characterization of a human inosine triphosphate pyrophosphatase encoded by the itpa gene; Lin S et al.; ITP and dITP exist in all cells . dITP is potentially mutagenic, and the levels of these nucleotides are controlled by inosine triphosphate pyrophosphatase (EC ) . Here we report the cloning, expression, and characterization of a 21.5-kDa human inosine triphosphate pyrophosphatase (hITPase), an enzyme whose activity has been reported in many animal tissues and studied in populations but whose protein sequence has not been determined before . At the optimal pH of 10.0, recombinant hITPase hydrolyzed ITP, dITP, and xanthosine 5'-triphosphate to their respective monophosphates whereas activity with other nucleoside triphosphates was low . K(m) values for ITP, dITP, and xanthosine 5'-triphosphate were 0.51, 0.31, and 0.57 mm, respectively, and k(cat) values were 580, 360, and 640 s(-1), respectively . A divalent cation was absolutely required for activity . The gene encoding the hITPase cDNA sequence was localized by radiation hybrid mapping to chromosome 20p in the interval D20S113-D20S97, the same interval in which the ITPA inosine triphosphatase gene was previously localized . A BLAST search revealed the existence of many similar sequences in organisms ranging from bacteria to mammals . The function of this ubiquitous protein family is proposed to be the elimination of minor potentially mutagenic or clastogenic purine nucleoside triphosphates from the cell.

J Biol Chem, 2001 May 4, 276(18), 15453 - 7 Epub 2001 Feb 13.
Expression of Semliki Forest virus E1 protein in Escherichia coli . Low pH-induced pore formation; Nyfeler S et al.; Exposure of Semliki Forest virus 1 to mildly acidic conditions results in conformational changes of the viral spike proteins, which in turn leads to a pore formation across its membrane . The ability to form a pore has been ascribed to the ectodomain of the Semliki Forest virus (SFV) E1 spike protein . To elucidate whether the E1 protein per se is sufficient for low pH-dependent pore formation, we expressed E1 in Escherichia coli in an inducible manner using the pET11c expression system . The data obtained clearly showed that the E1 protein was expressed in the bacterial cell membrane and that exposure of E . coli expressing the SFV E1 protein to low pH (<6.2) resulted in a permeability change of the membrane . Thus, we conclude that the E1 protein of SFV per se is sufficient to promote pore formation under mildly acidic conditions.

J Biol Chem, 2001 May 11, 276(19), 16289 - 95 Epub 2001 Jan 30.
Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase sigma 28 subunit; Yoshida M et al.; The effects of polyamines on the synthesis of various final sigma subunits of RNA polymerase were studied using Western blot analysis . Synthesis of final sigma(28) was stimulated 4.0-fold and that of final sigma(38) was stimulated 2.3-fold by polyamines, whereas synthesis of other final sigma subunits was not influenced by polyamines . Stimulation of final sigma(28) synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation . Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation . Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgarno sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.

J Biol Chem, 2001 Apr 27, 276(17), 13902 - 10 Epub 2001 Jan 23.
The kinetic pathway of RNA binding to the Escherichia coli transcription termination factor Rho; Kim DE et al.; The Escherichia coli transcription termination factor Rho is structurally and functionally homologous to hexameric helicases that assemble into ring structures . Using stopped-flow fluorescence and presteady-state ATPase kinetics, we have determined the kinetic pathway of poly(C) RNA binding to Rho hexamer, both in the presence and in absence of ATP . These studies indicate a four-step sequential mechanism of RNA binding and reveal the respective roles of the primary and secondary RNA binding sites in initiation and ATPase activation of Rho . The primary RNA binding sites of Rho hexamer interact with poly(C) RNA at a diffusion-limited rate constant close to 8 x 10(8) m(-1) s(-1), resulting in the Rho-RNA species PR1, which subsequently isomerizes to PR2 with a rate constant 21 s(-1) . The PR2 isomerizes to PR3 with a rate constant of 32 s(-1) in the presence of ATP, and the formation of PR4 from PR3 results in a species that is fully competent in hydrolyzing ATP at the RNA-stimulated rate . The PR3 to PR4 isomerization occurs at a relatively slow rate of 4.1 s(-1); thus, the presteady-state ATPase kinetics show a distinct lag due to the slow initiation step . The interactions of the RNA with the primary sites trigger ring opening, and we propose that during the last two steps, the RNA migrates into the central channel and interacts with the secondary sites, resulting in the activation of the ATPase activity . The primary RNA binding sites, in addition to promoting sequence specific initiation, kinetically facilitate loading of the RNA into the secondary sites, which are relatively inaccessible, since they are present in the central channel . These studies reveal common features used by hexameric helicases to bind nucleic acids in an efficient and specific manner.

J Biol Chem, 2001 Apr 20, 276(16), 13308 - 13 Epub 2001 Jan 22.
The beta ' subunit of Escherichia coli RNA polymerase is not required for interaction with initiating nucleotide but is necessary for interaction with rifampicin; Naryshkina T et al.; Using a modification of a highly selective affinity labeling protocol, we demonstrated that the alpha(2)beta subassembly of Escherichia coli RNA polymerase efficiently and specifically interacts with the initiating purine nucleotide . Isolated beta is also active in this reaction . In contrast, neither beta nor alpha(2)beta is able to interact with a chimeric molecule composed of rifampicin attached to an initiation substrate . Based on these results, we conclude that the RNA polymerase initiation site, specific for purine nucleotides, which ultimately become the 5'-end of the transcript, is essentially complete in the absence of the largest subunit, beta' . However, the rifampicin binding center is formed only in the alpha(2)betabeta' core enzyme . We interpret our results in light of the high resolution structure of core RNA polymerase from Thermus aquaticus.

J Biol Chem, 2001 Apr 27, 276(17), 14083 - 91 Epub 2001 Jan 29.
Energetics of target peptide binding by calmodulin reveals different modes of binding; Brokx RD et al.; Thermodynamic parameters of interactions of calcium-saturated calmodulin (Ca(2+)-CaM) with melittin, C-terminal fragment of melittin, or peptides derived from the CaM binding regions of constitutive (cerebellar) nitric-oxide synthase, cyclic nucleotide phosphodiesterase, calmodulin-dependent protein kinase I, and caldesmon (CaD-A, CaD-A*) have been measured using isothermal titration calorimetry . The peptides could be separated into two groups according to the change in heat capacity upon complex formation, DeltaC(p) . The calmodulin-dependent protein kinase I, constitutive (cerebellar) nitric-oxide synthase, and melittin peptides have DeltaC(p) values clustered around -3.2 kJ.mol(-1).K(-1), consistent with the formation of a globular CaM-peptide complex in the canonical fashion . In contrast, phosphodiesterase, the C-terminal fragment of melittin, CaD-A, and CaD-A* have DeltaC(p) values clustered around -1.6 kJ.mol(-1).K(-1), indicative of interactions between the peptide and mostly one lobe of CaM, probably the C-terminal lobe . It is also shown that the interactions for different peptides with Ca(2+)-CaM can be either enthalpically or entropically driven . The difference in the energetics of peptide/Ca(2+)-CaM complex formation appears to be due to the coupling of peptide/Ca(2+)-CaM complex formation to the coil-helix transition of the peptide . The binding of a helical peptide to Ca(2+)-CaM is dominated by favorable entropic effects, which are probably mostly due to hydrophobic interactions between nonpolar groups of the peptide and Ca(2+)-CaM . Applications of these findings to the design of potential CaM inhibitors are discussed.

J Biol Chem, 2001 Apr 20, 276(16), 12744 - 8 Epub 2001 Jan 19.
A single carboxyl mutant of the multidrug transporter EmrE is fully functional; Yerushalmi H et al.; EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons . Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein . This residue was shown to be an essential part of the binding site, common to protons and substrate . EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type . This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity . The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls . Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner . Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities . This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.

J Biol Chem, 2001 Apr 20, 276(16), 13178 - 85 Epub 2001 Jan 26.
An active site tyrosine influences the ability of the dimethyl sulfoxide reductase family of molybdopterin enzymes to reduce S-oxides; Johnson KE et al.; Dimethyl sulfoxide reductase (DMSOR), trimethylamine-N-oxide reductase (TMAOR), and biotin sulfoxide reductase (BSOR) are members of a class of bacterial oxotransferases that contain the bis(molybdopterin guanine dinucleotide)molybdenum cofactor . The presence of a Tyr residue in the active site of DMSOR and BSOR that is missing in TMAOR has been implicated in the inability of TMAOR, unlike DMSOR and BSOR, to utilize S-oxides . To test this hypothesis, Escherichia coli TMAOR was cloned and expressed at high levels, and site-directed mutagenesis was utilized to generate the Tyr-114 --> Ala and Phe variants of Rhodobacter sphaeroides DMSOR and insert a Tyr residue into the equivalent position in TMAOR . Although all of the mutants turn over in a manner similar to their respective wild-type enzymes, mutation of Tyr-114 in DMSOR results in a decreased specificity for S-oxides and an increased specificity for trimethylamine-N-oxide (Me(3)NO), with a greater change observed for DMSOR-Y114A . Insertion of a Tyr into TMAOR results in a decreased preference for Me(3)NO relative to dimethyl sulfoxide . Kinetic analysis and UV-visible absorption spectra indicate that the ability of DMSOR to be reduced by dimethyl sulfide is lost upon mutation of Tyr-114 and that TMAOR does not exhibit this activity even in the Tyr insertion mutant.

J Biol Chem, 2001 Apr 20, 276(16), 12827 - 31 Epub 2001 Jan 25.
A possible role of Ku in mediating sequential repair of closely opposed lesions; Hashimoto M et al.; One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions . We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA . DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate . It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU . However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme . The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku . Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present . Similar results were also observed with Escherichia coli endonuclease III . These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex . In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.

J Biol Chem, 2001 May 4, 276(18), 14829 - 34 Epub 2001 Feb 05.
A role for the Ppz Ser/Thr protein phosphatases in the regulation of translation elongation factor 1Balpha; de Nadal E et al.; In vivo 32P-labeled yeast proteins from wild type and ppz1 ppz2 phosphatase mutants were resolved by bidimensional electrophoresis . A prominent phosphoprotein, which in ppz mutants showed a marked shift to acidic regions, was identified by mixed peptide sequencing as the translation elongation factor 1Balpha (formerly eEF1beta) . An equivalent shift was detected in cells overexpressing HAL3, a inhibitory regulatory subunit of Ppz1 . Subsequent analysis identified the conserved Ser-86 as the in vivo phosphorylatable residue and showed that its phosphorylation was increased in ppz cells . Pull-down experiments using a glutathione S-transferase (GST)-EF1Balpha fusion version allowed to identify Ppz1 as an in vivo interacting protein . Cells lacking Ppz display a higher tolerance to known translation inhibitors, such as hygromycin and paromomycin, and enhanced readthrough at all three nonsense codons, suggesting that translational fidelity might be affected . Overexpression of a GST-EF1Balpha fusion counteracted the growth defect associated to high levels of Ppz1 and this effect was essentially lost when the phosphorylatable Ser-86 is replaced by Ala . Therefore, the Ppz phosphatases appear to regulate the phosphorylation state of EF1Balpha in yeast, and this may result in modification of the translational accuracy.

J Biol Chem, 2001 Jun 8, 276(23), 20516 - 22 Epub 2001 Mar 08.
Crystal structure of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III; Scarsdale JN et al.; Mycolic acids (alpha-alkyl-beta-hydroxy long chain fatty acids) cover the surface of mycobacteria, and inhibition of their biosynthesis is an established mechanism of action for several key front-line anti-tuberculosis drugs . In mycobacteria, long chain acyl-CoA products (C(14)-C(26)) generated by a type I fatty-acid synthase can be used directly for the alpha-branch of mycolic acid or can be extended by a type II fatty-acid synthase to make the meromycolic acid (C(50)-C(56)))-derived component . An unusual Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein (ACP) synthase III (mtFabH) has been identified, purified, and shown to catalyze a Claisen-type condensation between long chain acyl-CoA substrates such as myristoyl-CoA (C(14)) and malonyl-ACP . This enzyme, presumed to play a key role in initiating meromycolic acid biosynthesis, was crystallized, and its structure was determined at 2.1-A resolution . The mtFabH homodimer is closely similar in topology and active-site structure to Escherichia coli FabH (ecFabH), with a CoA/malonyl-ACP-binding channel leading from the enzyme surface to the buried active-site cysteine residue . Unlike ecFabH, mtFabH contains a second hydrophobic channel leading from the active site . In the ecFabH structure, this channel is blocked by a phenylalanine residue, which constrains specificity to acetyl-CoA, whereas in mtFabH, this residue is a threonine, which permits binding of longer acyl chains . This same channel in mtFabH is capped by an alpha-helix formed adjacent to a 4-amino acid sequence insertion, which limits bound acyl chain length to 16 carbons . These observations offer a molecular basis for understanding the unusual substrate specificity of mtFabH and its probable role in regulating the biosynthesis of the two different length acyl chains required for generation of mycolic acids . This mtFabH presents a new target for structure-based design of novel antimycobacterial agents.

J Biol Chem, 2001 Apr 6, 276(14), 10929 - 34 Epub 2001 Jan 17.
Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation; Salim M et al.; Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin . BVR is unique in having dual pH/dual cofactor requirements . Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity . An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein . Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated . Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent . In addition, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower K(d) at pH 8.7 than at pH 7.4 (15.5 versus 28.0 micrometer) . Mn(2+) was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60 degrees C for 10 min . The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase . Potato acid phosphatase treatment reversibly inactivated the enzyme . The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition . Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate . The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H(2)O(2) . The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.

J Biol Chem, 2001 Apr 20, 276(16), 12756 - 63 Epub 2001 Jan 17.
Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease; van Montfort BA et al.; Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain . The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis . Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit . The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124) . The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry . Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124) . The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length . The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.

J Biol Chem, 2001 May 25, 276(21), 17968 - 75 Epub 2001 Mar 12.
Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64; Sakai D et al.; Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus . The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases . The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified . Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5 . By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer . To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs . Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings . Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag . The purified mutant PilQ proteins contained various levels of reduced ATPase activity . Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable . PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not . These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.

J Biol Chem, 2001 May 25, 276(21), 18038 - 45 Epub 2001 Feb 27.
A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage; Betti M et al.; Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain . The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions . The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates . Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E . coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks . ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity . ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.

J Biol Chem, 2001 Apr 20, 276(16), 12879 - 84 Epub 2001 Jan 22.
In vivo interaction between dynamitin and MacMARCKS detected by the fluorescent resonance energy transfer method; Jin T et al.; Dynamitin is a subunit of the dynactin complex regulating microtubule-dependent motor functions, and MacMARCKS (Macrophage-enriched myristoylated alanine-rich protein kinase C substrate) is a major protein kinase C substrate regulating integrin activation . The interaction between dynamitin and MacMARCKS has been implicated in integrin-dependent cell spreading . However, the in vivo interaction of these two proteins in living cells has not been demonstrated . Spatial and temporal information about the interaction is also lacking . In this study, we used the fluorescent resonance energy transfer method to demonstrate in vivo interaction between MacMARCKS and dynamitin with cyan fluorescent protein (CFP)-conjugated dynamitin as the donor fluorophore and yellow fluorescent protein (YFP)-conjugated MacMARCKS as the acceptor fluorophore . The interaction of these two fusion proteins was studied both in vitro and in vivo, and typical fluorescent resonance energy transfer was observed; the CFP emission peak increased while the YFP emission peak decreased when protein interaction was abolished . Spatial and temporal information was obtained in RAW macrophage cells . In resting macrophage cells, dynamitin-MacMARCKS interaction is concentrated at the cell periphery, although the majority of dynamitin is distributed at the perinuclear region of the cells . When cells were treated with phorbol 12-myristate 13-acetate, both proteins concentrated to perinuclear regions of the cells, and yet the interaction disappeared as the cell spread . Similar events were also observed in 293 cells . Thus, we conclude that dynamitin and MacMARCKS indeed interact in living cells.

J Biol Chem, 2001 May 25, 276(21), 17851 - 6 Epub 2001 Feb 27.
Class II recombinant phosphoribosyl diphosphate synthase from spinach . Phosphate independence and diphosphoryl donor specificity; Krath BN et al.; A recombinant form of spinach (Spinacia oleracea) phosphoribosyl diphosphate (PRPP) synthase isozyme 3 resembling the presumed mature enzyme has been synthesized in an Escherichia coli strain in which the endogenous PRPP synthase gene was deleted, and has been purified to near homogeneity . Contrary to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds . In addition spinach PRPP synthase isozyme 3 shows an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP . The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x mg of protein)(-1) . The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6.

J Biol Chem, 2001 Jun 8, 276(23), 19812 - 9 Epub 2001 Mar 01.
The Escherichia coli RNA polymerase.anti-sigma 70 AsiA complex utilizes alpha-carboxyl-terminal domain upstream promoter contacts to transcribe from a -10/-35 promoter; Orsini G et al.; During infection of Escherichia coli, the phage T4 early protein AsiA inhibits open complex formation by the RNA polymerase holoenzyme Efinal sigma(70) at -10/-35 bacterial promoters through binding to region 4.2 of the final sigma(70) subunit . We used the -10/-35 lacUV5 promoter to study the properties of the Efinal sigma(70).AsiA complex in the presence of the glutamate anion . Under these experimental conditions, inhibition by AsiA was significantly decreased . KMnO(4) probing showed that the observed residual transcriptional activity was due to the slow transformation of the ternary complex Efinal sigma(70).AsiA.lacUV5 into an open complex . In agreement with this observation, affinity of the enzyme for the promoter was 10-fold lower in the ternary complex than in the binary complex Efinal sigma(70).lacUV5 . A tau plot analysis of abortive transcription reactions showed that AsiA binding to Efinal sigma(70) resulted in a 120-fold decrease in the second-order on-rate constant of the reaction of Efinal sigma(70) with lacUV5 and a 55-fold decrease in the rate constant of the isomerization step leading to the open complex . This ternary complex still responded to activation by the cAMP.catabolite activator protein complex . We show that compensatory Efinal sigma(70)/promoter upstream contacts involving the C-terminal domains of alpha subunits in Efinal sigma(70) become essential for the binding of Efinal sigma(70).AsiA to the lacUV5 promoter.

J Biol Chem, 2001 Jun 1, 276(22), 19580 - 7 Epub 2001 Mar 14.
Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions; Wang QJ et al.; The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity . Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction . {(3)H}Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity . Lipid dependence was determined as a function of lipid concentration and composition . The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity . As expected, Leu-20 and Leu-24 strongly influenced binding . Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine . Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions . Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect . Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu . In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity . More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.

J Biol Chem, 2001 May 25, 276(21), 17844 - 50 Epub 2001 Feb 20.
Amino acid residue mutations uncouple cooperative effects in Escherichia coli D-3-phosphoglycerate dehydrogenase; Grant GA et al.; d-3-Phosphoglycerate dehydrogenase from Escherichia coli contains two Gly-Gly sequences that occur at junctions between domains . A previous study (Grant, G . A., Xu, X . L., and Hu, Z . (2000) Biochemistry 39, 7316-7319) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains . Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the K(m) and k(cat) . Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domains, which form the active site cleft, have a significant effect on the k(cat) of the enzyme without substantially altering the enzyme's sensitivity to serine . In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition . Interestingly, even though cooperativity of inhibition can be reduced significantly, there is little apparent effect on the cooperativity of serine binding itself . An additional mutant, G336V,G337V, also reduces the cooperativity of inhibition, but in this case serine binding also is reduced to the point at which it cannot be measured by equilibrium dialysis . The double mutant G294V,G336V demonstrates that strain imposed by mutation at one hinge can be relieved partially by mutation at the other hinge, demonstrating linkage between the two hinge regions . These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled . Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme . A comparison of these values with the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme, which will allow modeling of the dynamics of domain movement.

J Biol Chem, 2001 May 11, 276(19), 15712 - 9 Epub 2001 Feb 07.
Inhibition of cytosolic phospholipase A2 by annexin I . Specific interaction model and mapping of the interaction site; Kim SW et al.; Annexins (ANXs) display regulatory functions in diverse cellular processes, including inflammation, immune suppression, and membrane fusion . However, the exact biological functions of ANXs still remain obscure . Inhibition of phospholipase A(2) (PLA(2)) by ANX-I, a 346-amino acid protein, has been observed in studies with various forms of PLA(2) . "Substrate depletion" and "specific interaction" have been proposed for the mechanism of PLA(2) inhibition by ANX-I . Previously, we proposed a specific interaction model for inhibition of a 100-kDa porcine spleen cytosolic form of PLA(2) (cPLA(2)) by ANX-I (Kim, K . M., Kim, D . K., Park, Y . M., and Na, D . S . (1994) FEBS Lett . 343, 251-255) . Herein, we present an analysis of the inhibition mechanism of cPLA(2) by ANX-I in detail using ANX-I and its deletion mutants . Deletion mutants were produced in Escherichia coli, and inhibition of cPLA(2) activity was determined . The deletion mutant ANX-I-(1-274), containing the N terminus to amino acid 274, exhibited no cPLA(2) inhibitory activity, whereas the deletion mutant ANX-I-(275-346), containing amino acid 275 to the C terminus, retained full activity . The protein-protein interaction between cPLA(2) and ANX-I was examined using the deletion mutants by immunoprecipitation and mammalian two-hybrid methods . Full-length ANX-I and ANX-I-(275-346) interacted with the calcium-dependent lipid-binding domain of cPLA(2) . ANX-I-(1-274) did not interact with cPLA(2) . Immunoprecipitation of A549 cell lysate with anti-ANX-I antibody resulted in coprecipitation of cPLA(2) . These results are consistent with the specific interaction mechanism rather than the substrate depletion model . ANX-I may function as a negative regulator of cPLA(2) in cellular signal transduction.

J Biol Chem, 2001 Apr 13, 276(15), 11980 - 7 Epub 2001 Jan 16.
Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system . Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA; Haney SA et al.; The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli . We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site . A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence . In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact . Two mutant proteins examined restored this interaction significantly . In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion . We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA . However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein . Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

J Biol Chem, 2001 Apr 27, 276(17), 13982 - 8 Epub 2001 Jan 25.
Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition; Merithew E et al.; Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes . We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J . J., Zhu, Z., Connolly, J . L., and Lambright, D . G . (1999) Structure 7, 413-423) . A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A . T . (1999) Cell 96, 363-374) . Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase . As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions . However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A . This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central beta sheet and the alpha2 helix . These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies . Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.

J Biol Chem, 2001 Jun 1, 276(22), 19301 - 9 Epub 2001 Feb 27.
The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1; Gabus C et al.; Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP) . Although PrP is conserved in vertebrates, its function remains to be identified . In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP . Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome . NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT) . In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes . This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA . Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT . The NC-like properties of huPrP map to the N-terminal region of huPrP . Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells . These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

J Biol Chem, 2001 Apr 20, 276(16), 13256 - 63 Epub 2001 Jan 26.
Trax (translin-associated factor X), a primarily cytoplasmic protein, inhibits the binding of TB-RBP (translin) to RNA; Chennathukuzhi VM et al.; Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays . Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions . The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific single stranded DNA sequences . The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the yeast two-hybrid system . Cell fractionation and confocal microscope studies reveal that Trax is predominantly cytoplasmic . In contrast, TB-RBP is present in both the nuclei and cytoplasm of transfected cells and uses a highly conserved nuclear export signal to exit nuclei . In addition to a leucine zipper, two basic domains in TB-RBP are essential for RNA binding, but only one of these domains is needed for DNA binding . Trax restores DNA binding to TB-RBP containing an altered form of this domain . These data suggest that Trax-TB.RBP interactions modulate the DNA- and RNA-binding activity of TB-RBP.

J Biol Chem, 2001 May 18, 276(20), 17221 - 8 Epub 2001 Feb 20.
Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters; Kim DK et al.; A cDNA was isolated from rat small intestine by expression cloning which encodes a novel Na+-independent transporter for aromatic amino acids . When expressed in Xenopus oocytes, the encoded protein designated as TAT1 (T-type amino acid transporter 1) exhibited Na+-independent and low-affinity transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine (Km values: approximately 5 mm), consistent with the properties of classical amino acid transport system T . TAT1 accepted some variations of aromatic side chains because it interacted with amino acid-related compounds such as l-DOPA and 3-O-methyl-DOPA . Because TAT1 accepted N-methyl- and N-acetyl-derivatives of aromatic amino acids but did not accept their methylesters, it is proposed that TAT1 recognizes amino acid substrates as anions . Consistent with this, TAT1 exhibited sequence similarity (approximately 30% identity at the amino acid level) to H+/monocarboxylate transporters . Distinct from H+/monocarboxylate transporters, however, TAT1 was not coupled with the H+ transport but it mediated an electroneutral facilitated diffusion . TAT1 mRNA was strongly expressed in intestine, placenta, and liver . In rat small intestine TAT1 immunoreactivity was detected in the basolateral membrane of the epithelial cells suggesting its role in the transepithelial transport of aromatic amino acids . The identification of the amino acid transporter with distinct structural and functional characteristics will not only facilitate the expansion of amino acid transporter families but also provide new insights into the mechanisms of substrate recognition of organic solute transporters.

J Biol Chem, 2001 Apr 20, 276(16), 13490 - 8 Epub 2001 Jan 23.
Cloning and characterization of adenylate kinase from Chlamydia pneumoniae; Miura K et al.; Chlamydiae proliferate only within the infected host cells and are thought to be "energy parasites," because they take up ATP from the host cell as an energy source . In the present study, we isolated from Chlamydia pneumoniae the gene encoding adenylate kinase (AK) . Using the enzyme produced in Escherichia coli, its properties were characterized . K(m) values for AMP and for ADP of the purified C . pneumoniae AK (AKcpn) were each 330 microm, which is significantly higher than the reported values of other AKs, whereas K(m) for ATP was 24 microm, which was rather lower than others . AKcpn contains 1 g atom of zinc/mol of 24,000-dalton protein . Mass spectrometric analysis of AKcpn and analysis of properties of mutated AKcpn strongly suggested that zinc is associated with four cysteine residues in the LID domain of the enzyme . The apo-AKcpn that lost zinc retained AK activity, although K(m) for AMP of apo-AKcpn increased about 2-fold and V(max) decreased about one-half from that of holo-AKcpn . The apo-AKcpn was more thermolabile and sensitive to trypsin digestion than the holo-AKcpn . Moreover, the recovery in vitro of the AK activity during the renaturation process of the denatured apo-AKcpn was dependent on zinc . A mutated protein in which cysteine residues in the LID domain were substituted by other amino acids lost both zinc and enzyme activity . The mutated protein was more sensitive to protease than the apo-AKcpn . These results indicate that zinc in AKcpn, although not essential for the catalysis, stabilizes the enzyme and probably plays a crucial role in proper folding of the protein . Furthermore, the catalytic properties of AKcpn suggest a distinctive regulatory mechanism in the metabolism compared with AKs in other organisms.

J Biol Chem, 2001 May 4, 276(18), 15225 - 31 Epub 2001 Jan 22.
Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1; Bergeron A et al.; Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH) . Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients . Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH . Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV-1 cells . Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins . Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme . The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme . Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.

J Biol Chem, 2001 May 25, 276(21), 17994 - 9 Epub 2001 Feb 22.
An essential role of Glu-243 and His-239 in the phosphotransfer reaction catalyzed by pyruvate dehydrogenase kinase; Tuganova A et al.; This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239 . We used site-directed mutagenesis to convert Glu-243 to Ala, Asp, or Gln and His-239 to Ala . The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase . The Glu-243 to Asp mutant had approximately 2% residual activity, whereas the Glu-243 to Ala or Gln mutants exhibited less than 0.5 and 0.1% residual activity, respectively . Activity of the His-239 to Ala mutant was decreased by approximately 90% . Active-site titration with {alpha-(32)P}ATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding . All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex . Furthermore, neither of the mutations affected the inter-subunit interactions . Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity . Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the gamma phosphate . The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).

J Biol Chem, 2001 Apr 13, 276(15), 11524 - 30 Epub 2001 Jan 18.
Riboflavin synthase of Escherichia coli . Effect of single amino acid substitutions on reaction rate and ligand binding properties; Illarionov B et al.; Conserved amino acid residues of riboflavin synthase from Escherichia coli were modified by site-directed mutagenesis . Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins . S41A and H102Q mutants had substantially reduced reaction velocities . Replacements of various other conserved polar residues had little impact on catalytic activity . (19)F NMR protein perturbation experiments using a fluorinated intermediate analog suggest that the N-terminal sequence motif MFTG is part of one of the substrate-binding sites of the protein.

J Biol Chem, 2001 May 18, 276(20), 16674 - 82 Epub 2001 Feb 21.
Inhibition of human endogenous retrovirus-K10 protease in cell-free and cell-based assays; Kuhelj R et al.; A full-length and C-terminally truncated version of human endogenous retrovirus (HERV)-K10 protease were expressed in Escherichia coli and purified to homogeneity . Both versions of the protease efficiently processed HERV-K10 Gag polyprotein substrate . HERV-K10 Gag was also cleaved by human immunodeficiency virus, type 1 (HIV-1) protease, although at different sites . To identify compounds that could inhibit protein processing dependent on the HERV-K10 protease, a series of cyclic ureas that had previously been shown to inhibit HIV-1 protease was tested . Several symmetric bisamides acted as very potent inhibitors of both the truncated and full-length form of HERV-K10 protease, in subnanomolar or nanomolar range, respectively . One of the cyclic ureas, SD146, can inhibit the processing of in vitro translated HERV-K10 Gag polyprotein substrate by HERV-K10 protease . In addition, in virus-like particles isolated from the teratocarcinoma cell line NCCIT, there is significant accumulation of Gag and Gag-Pol precursors upon treatment with SD146, suggesting the compound efficiently blocks HERV-K Gag processing in cells . This is the first report of an inhibitor able to block cell-associated processing of Gag polypeptides of an endogenous retrovirus.

J Biol Chem, 2001 Jun 8, 276(23), 20357 - 63 Epub 2001 Mar 13.
Divergent N-terminal sequences of a deubiquitinating enzyme modulate substrate specificity; Lin H et al.; Ubiquitin-specific processing proteases (UBPs) are characterized by a conserved core domain with surrounding divergent sequences, particularly at the N-terminal end . We previously cloned two isoforms of a testis UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini that target the two isoforms to different subcellular locations (Lin, H., Keriel, A., Morales, C . R., Bedard, N., Zhao, Q., Hingamp, P., Lefrancois, S., Combaret, L., and Wing, S . S . (2000) Mol . Cell . Biol . 20, 6568-6578) . To determine whether the N termini also influence the biochemical functions of the UBP, we expressed UBP-t1, UBP-t2, and the common core domain, UBP core, in Escherichia coli . The three isoforms cleaved branched triubiquitin at >20-fold faster rates than linear diubiquitin, suggesting that UBP-testis functions as an isopeptidase . Both N-terminal extensions inhibited the ability of UBP-core to generate free ubiquitin when linked in a peptide bond with itself, another peptide, or to small adducts . The N-terminal extension of UBP-t2 increased the ability of UBP-core to cleave branched triubiquitin . UBP-core removed ubiquitin from testis ubiquitinated proteins more rapidly than UBP-t2 and UBP-t1 . Thus, UBP enzymes appear to contain a catalytic core domain, the activities and specificities of which can be modulated by N-terminal extensions . These divergent N termini can alter localization and confer multiple functions to the various members of the large UBP family.

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