Anal Chem, 2001 Sep 1, 73(17), 4063 - 70 Virtual 2-D gel electrophoresis: visualization and analysis of the E . coli proteome by mass spectrometry; Loo RR et al.; Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis . This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated . Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage . Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains . Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels . In the 5.7-6.0 pH range, E . coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels . Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E . coli proteins. Protein Sci, 2001 Oct, 10(10), 2037 - 49 Exploring the conformational equilibrium of E . coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy; van den Berg PA et al.; The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy . The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably . Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S . Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation . The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation . The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation . Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation . Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation . Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model . Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S . High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation. Protein Sci, 2001 Oct, 10(10), 2028 - 36 Examination of the folding of E . coli CspA through tryptophan substitutions; Vu DM et al.; Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism . However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated . The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site . To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein . The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing . Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants . These results indicate that the fast folding of CspA is driven by a concerted mechanism. Protein Sci, 2001 Oct, 10(10), 1989 - 2001 Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E . coli SSB cross-link to DNA; Steen H et al.; Protein-nucleic acid complexes are commonly studied by photochemical cross-linking . UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site . Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes . The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates . Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure . This study demonstrates the performance of four different MS-based strategies to characterize E . coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer . Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis . Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis . Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues . Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking . Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry. Vopr Med Khim, 2001 May-Jun, 47(3), 315 - 28 {Association of cytochrome P450 2B4 with molecular chaperones in heterological expression in E coli.}; Prozorovskii TV et al.; To produce a water-soluble form of microsomal P450 2B4, fusion proteins with glutathione-S-transpherase were genetically engineered . Specific proteolitic sites recognized by Factor Xa and Thrombin have been introduced into N-terminus of P450 2B4 (46-49), lacking signal anchor sequence (2-27) . It was supposed that proteolysis at this site could give the possibility to produce protein lacking hydrophobic N-terminus sequence (1-49) . However, it was shown that given region in P450 2B4 his resistant against specific proteinase action . Positive result has been obtained at specific proteolysis with IgA endoproteinase, recognizing the native sequence PPGP (31-34) in P450 2B4 . Thus, at first time truncated form of cytochrome 2B4, lacking its 33 N-terminal amino acid residues has been created . It was found that the expression of genetically engineered variants of GST-2B4 in Escherichia coli is accompanied by tight complex formation with molecular chaperones GroEL and DnaK . Dissociation of the complex occurred after proteolysis in: linker sequence (position 6-7) between C-terminal part of GST domain and N-terminal part of 2B4, and also before N-terminal methionine 2B4 and at position 33-34 (2B4) . These results suggest the possibility that interaction with a GroEL/DnaK molecular chaperones may be requirement for correct folding of eukaryotic cytochrome 2B4 during its biosynthesis in E . coli. Prog Nucleic Acid Res Mol Biol, 2001, 68, 349 - 64 Potential double-flipping mechanism by E . coli MutY; House PG et al.; To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted . MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation . However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency . Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A . The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY . Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy . The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase . Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix. Curr Biol, 2001 Sep 4, 11(17), 1369 - 73 Computational identification of noncoding RNAs in E . coli by comparative genomics; Rivas E et al.; Some genes produce noncoding transcripts that function directly as structural, regulatory, or even catalytic RNAs {1, 2} . Unlike protein-coding genes, which can be detected as open reading frames with distinctive statistical biases, noncoding RNA (ncRNA) gene sequences have no obvious inherent statistical biases {3} . Thus, genome sequence analyses reveal novel protein-coding genes, but any novel ncRNA genes remain invisible . Here, we describe a computational comparative genomic screen for ncRNA genes . The key idea is to distinguish conserved RNA secondary structures from a background of other conserved sequences using probabilistic models of expected mutational patterns in pairwise sequence alignments . We report the first whole-genome screen for ncRNA genes done with this method, in which we applied it to the "intergenic" spacers of Escherichia coli using comparative sequence data from four related bacteria . Starting from >23,000 conserved interspecies pairwise alignments, the screen predicted 275 candidate structural RNA loci . A sample of 49 candidate loci was assayed experimentally . At least 11 loci expressed small, apparently noncoding RNA transcripts of unknown function . Our computational approach may be used to discover structural ncRNA genes in any genome for which appropriate comparative genome sequence data are available. Gravit Space Biol Bull, 1997 Jun, 10(2), 43 - 7 A mechanosensitive channel protein and its gene in E . coli; Blount P et al.; Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are inferred from their functions but not yet characterized as isolated proteins or products of cloned genes . These receptors are often in low abundance and in animal and plant tissues that are inaccessible, thus making biochemical analysis difficult . Yet, the application of the sensitive patch-clamp technique to measure transmembrane currents has demonstrated the ubiquity of ion channels whose opening is favored by membrane stretch forces . We have discovered in E . coli the activity of a mechanosensitive ion channel of large conductance (MscL), and have successfully isolated the corresponding protein and gene (Sukharev et al . 1994a) . MscL channel appears to respond directly to stretch force in the lipid bilayer since it is active in artificial patches having only highly enriched MscL protein and lipids . Structurally, MscL is an integral membrane protein of only 136 amino-acid residues . Each channel pore is likely to be enclosed by six assembled MscL subunits . Hydropathy analysis suggests that the protein is largely hydrophobic with a more hydrophilic carboxyl tail . Targeted deletions and substitutions show that not all regions of the molecule contribute to channel function; however, strategic single amino-acid changes can alter channel kinetics and mechanosensitivity . MscL and its gene now form the first tangible system to study mechanosensing using a combination of genetic, biochemical, and biophysical techniques. Adv Space Res, 1994 Oct, 14(10), 203 - 6 Heavy ion induced DNA double strand breaks in cells of E . coli; Schafer M et al.; Vegetative cells of E . coli differing in their radiosensitivity have been used in heavy ion irradiation experiment . Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis . This method allows to separate linear DNA with length up to 8 Mio base pairs . After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation . This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA . The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences . From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined . These results will be discussed in comparison to the results for cell survival. Water Sci Technol, 1995, 31(5-6), 259 - 61 Comparative performance of Colisure (TM) and accepted methods in the detection of chlorine-injured total coliforms and E.coli; McFeters GA et al.; Studies were done to examine the comparability of Colisure (TM) and accepted reference methods to detect low numbers of total coliform bacteria and E.coli subjected to chlorine stress . Colisure (TM) is a medium designed to concurrently detect coliform bacteria and E.coli in drinking water by the specific action of beta-galactosidase (total coliforms) and beta-glucuronidase (E.coli) . The methods used to compare the performance of various media followed a protocol established by the USEPA . Samples (31) of sewage from six different regions of the US were treated with sufficient concentrations of chlorine (1.2-2.5mg/l) to reduce viability 1-3 logs (39% average injury) and diluted with drinking water to achieve ca . 3 viable coliforms/100ml . The mean log reductions in viable bacteria, determined with various media following disinfection of the 31 samples were: mEndo = 1.87 (TC), Colisure (TM) = 1.55 (TC), mTec = 3.63 (E.coli) and Colisure (TM) = 2.01 (E.coli) . When Colisure (TM) was compared with accepted methods to detect total coliforms in the dilute, disinfected samples, Colisure (TM) yielded results that were 1.6 times greater than LTB confirmed in BGLB at 28h . Colisure (TM) also detected 1.7 times greater levels of E.coli than LTB confirmed in EC/MUG at 28h . Sensitivity and specificity of Colisure (TM) were between 96 and 100% when positive and negative tests were verified . These findings indicate that Colisure (TM) is superior to certain accepted reference methods in the detection of chlorine-injured coliforms and E.coli under conditions that resemble contaminated drinking water. Water Sci Technol, 1993, 27(3-4), 261 - 5 Chlorine injury and the comparative performance of Colisure (TM), ColiLert (TM) and ColiQuik (TM) for the enumeration of coliform bacteria and E.coli in drinking water; McFeters GA et al.; Several factors have stimulated interest in recently developed substrate specific media for the detection of coliform bacteria in water . This study compared the performance of Colisure (TM) (Millipore), ColiLert (TM) (Environetics) and ColiQuick (TM) (Hach) with accepted membrane filtration and MPN methodologies for the enumeration of total coliforms and E . coli in chlorinated water . The performance of all three media was compared, in MPN configuration, with LTB/MPN (confirmed) using a variety of drinking and source water samples, both with and without chlorination . The Cochran-Mantel-Haenszel test yielded statistical correlations between results obtained with each of the three new enzyme detection media and accepted reference methods for the detection of low numbers of total coliforms . Another series of tests compared the performance of Colisure with accepted methods (LTB/MPN confirmed with BGLB and EC-MUG) in the detection of total coliforms and E . coli in sewage-spiked samples simulating contaminated drinking water, using an USEPA/AWWA test protocol . The results demonstrated that Colisure detected these indicator bacteria with greater sensitivity than the accepted methods and that this difference increased between 24 and 28 hours of incubation . The results of this study collectively support the validity of the new enzyme detection method for the detection of low levels of coliform bacteria and E . coli in source water and contaminated drinking water. Adv Space Res, 1992, 12(2-3), 65 - 8 Microdosimetric considerations of effects of heavy ions on E . coli K-12 mutants; Takahashi T et al.; The inactivation cross sections of E . coli K-12 recombination-deficient mutants, JC1553 (recA) and AB2470 (recB), for several MeV/u alpha-particles and N ions have been successfully analyzed by Katz's target theory in which radiosensitivity parameter E0 is assumed to be LET independent and equal to D37 for gamma-rays . For E . coli K-12 wild type, AB1157 (rec+, uvr+), however, it is impossible to interpret the inactivation cross section data by an LET-independent E0-value . In the latter case, as in the case of B . subtilis spore, it is necessary to assume that the radiosensitivity of the target for the core of a heavy ion is higher than that for delta-electrons . As well as Waligorski, Hamm and Katz's dose, the dose around the trajectory of an ion based on Tabata and Ito's energy deposition algorithm for electrons has been used in the course of analysis. Mol Gen Mikrobiol Virusol, 2001, (3), 15 - 8 {Modulation of the cat gene expression in E . coli cells by simple (AC)20 repeats}; Pisarchik AV et al.; Probable relationship between modulation of gene expression by simple repeating sequences and competition capacity of the host was studied . (AC)20 repeat can both stimulate and inhibit the expression of chloramphenicol acetyl transferase gene expression in E . coli cells depending on this gene's position . Modulation of the gene expression by simple (AC)20 repeat and competitive capacity of bacteria containing this plasmid are closely related. Nat Cell Biol, 2001 Sep, 3(9), 856 - 9 Enteropathogenic E . coli Tir binds Nck to initiate actin pedestal formation in host cells; Gruenheid S et al.; Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide . EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin . The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria . On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation . Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated . Here we show that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott-Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton . Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton . These results implicate Nck adaptors as host-cell determinants of EPEC virulence. Cell, 2001 Aug 24, 106(4), 429 - 41 Crystal structure of the processivity clamp loader gamma (gamma) complex of E . coli DNA polymerase III; Jeruzalmi D et al.; The gamma complex, an AAA+ ATPase, is the bacterial homolog of eukaryotic replication factor C (RFC) that loads the sliding clamp (beta, homologous to PCNA) onto DNA . The 2.7/3.0 A crystal structure of gamma complex reveals a pentameric arrangement of subunits, with stoichiometry delta':gamma(3):delta . The C-terminal domains of the subunits form a circular collar that supports an asymmetric arrangement of the N-terminal ATP binding domains of the gamma motor and the structurally related domains of the delta' stator and the delta wrench . The structure suggests a mechanism by which the gamma complex switches between a closed state, in which the beta-interacting element of delta is hidden by delta', and an open form similar to the crystal structure, in which delta is free to bind to beta. Cell, 2001 Aug 24, 106(4), 417 - 28 Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E . coli DNA polymerase III; Jeruzalmi D et al.; The dimeric ring-shaped sliding clamp of E . coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC) . The delta subunit of the gamma complex binds to the beta ring and opens it . The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized . Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench. Cell Stress Chaperones, 2001 Jan, 6(1), 29 - 37 DnaK/DnaJ chaperone system reactivates endogenous E . coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat shock proteins; Kedzierska S et al.; Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation . To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E . coli cells and the composition of the S fraction . We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing . A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction . Fda was purified from E . coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro . We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system . The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda . Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ . GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda . It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect. Biochemistry, 2001 Sep 4, 40(35), 10500 - 6 Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E . coli; Kol MA et al.; The mechanism by which phospholipids translocate (flop) across the E . coli inner membrane remains to be elucidated . We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane . As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E . coli phospholipids . Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane . Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C . The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation . In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop . For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG . In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE . A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity. Int J Biol Macromol, 2001 Aug 20, 29(2), 99 - 105 Unfolding and inactivation of monomeric superoxide dismutase from E . coli by SDS; Bozzi M et al.; The inactivation and the unfolding of the naturally monomeric Cu, Zn, superoxide dismutase from E . coli upon addition of sodium dodecylsulphate have been studied . In contrast to the bovine enzyme, CD, EPR, NMR spectroscopy and pulsed low resolution NMR measurements found an unfolding transition followed by inactivation of the enzyme . During this transition the active site becomes accessible to the bulk water . The unfolding is reversible and both, the tridimensional structure of the protein and the active site, can be restored upon dialysis . In addition, unfolding occurs without loss of metals in the solution. Biotechniques, 2001 Aug, 31(2), 322 - 3, 326-8 Novel vectors for co-expression of two proteins in E . coli; Kholod N et al.; Two new vectors, pAC28 and pEGST, for the co-expression of recombinant genes in E . coli were developed . This two-plasmid system allows for an efficient expression and purification of large amounts of protein-protein complexes formed in bacterial cells . We have utilized this new system to express and isolate a stable complex of two human proteins, hematopoietic cell tyrosine phosphatase (HePTP) and mitogen-activated proteins kinase Erk2 . This approach is useful for biochemical and structural studies of protein-protein interactions. Zhonghua Gan Zang Bing Za Zhi, 2001 Jul, 9 Suppl, 69 - 72 {The expression of HCV RNA polymerase in E.coli and the study of its solubility and antigenicity}; Hou L et al.; OBJECTIVE: To express HCV RNA polymerase and study its soluble condition and antigenicity . METHODS: We constructed expression vectors pQE-5B-Fl and pQE-5B-C21 and expressed them in E.coli (M15) . We analyzed their solubility in different conditions and purified soluble pQE-5B-C21 protein by Ni-NTA column, then detected its antigenicity by ELISA and western blot . RESULTS: We obtained the purified soluble pQE-5B-C21 protein in the induction conditions of 18 degrees C and the protein was proved to be of good antigenicity by ELISA and western blot . CONCLUSIONS: The RNA polymerase of HCV expressed in E.coli has good solubility and antigenicity. Mutat Res, 2001 Sep 1, 480-481, 77 - 84 Contribution of E . coli AlkA, TagA glycosylases and UvrABC-excinuclease in MMS mutagenesis; Grzesiuk E et al.; MMS, an S(N)2 alkylating agent, is a moderate inducer of SOS mutagenesis and adaptive response . Our previous studies have shown that transient starvation of Escherichia coli AB1157argE3 strain causes a decrease of MMS-induced argE3-->Arg(+) reversions and this decrease is accompanied by the disappearance of the Fpg protein sensitive sites on plasmids isolated from MMS-treated and subsequently starved bacteria . This suggests that in such cells the mutation frequency decline (MFD) repair takes place . Here, we study the relation between MMS-induced mutagenesis as well as mutation frequency decline during starvation, and the repair of alkylated bases and AP-sites by base and nucleotide excision repair systems . In the AB1157alkA(-) strain, MMS-induced mutagenesis was over five-fold higher than in the wild type strain and no MFD repair occurred during starvation . Surprisingly, the lack of TagA glycosylase diminished MMS mutagenesis and accelerated the MFD effect . However, in double tagA(-)alkA(-) mutant, the frequency of Arg(+) reversions increased over 10-fold during 60 min of aminoacid starvation after MMS-treatment . Lack of the uvrA gene function did not affect the MMS-induced mutation rate and MFD in AB1157alkA(+)tagA(+) . Starvation of MMS treated AB1157tagAalkAuvrA triple mutant caused a decrease of mutation frequency almost to the level of spontaneous mutation rate . Examination of the repair of 3-MeAde, 7-MeGua and AP sites during starvation using repair glycosylases and plasmids isolated from MMS-treated and starved bacteria revealed that in E . coli uvr(+) but tagAalkA strain, neither 3-MeAde nor 7-MeGua were repaired during 60 min starvation and these persistent lesions could be responsible for the induction of the SOS system and an increase in mutation rate during starvation . In the triple tagAalkAuvrA mutant the repair of 3-MeAde, 7-MeGua and AP sites was carried out effectively and this could explain the observed decrease in the mutation rate during starvation . These results suggest that only in the absence of the "first choice" repair enzymes TagA, AlkA glycosylases and UvrABC excinuclease, a third error-free repair system of alkylated bases is activated . In the absence of only TagA and AlkA glycosylases, UvrABC excinuclease mediates activation of the SOS response, and this results in an increase of mutagenesis induced by the presence of alkylated bases in DNA. Mutat Res, 2001 Sep 1, 480-481, 71 - 5 The antimutator phenotype of E . coli mud is only apparent and results from delayed appearance of mutants; Schaaper RM et al.; Antimutator strains are strains that have a lower mutation rate than the wild-type strain . We have reexamined the properties of one reported antimutator strain of Escherichia coli, termed mud {Mol . Gen . Genet . 153 (1977) 87} . This strain contains a temperature-sensitive mutation in the purB gene, leading to adenine-dependent growth at higher temperature . When grown at permissive or semi-permissive temperature in the absence of adenine it displays large reductions in the number of both spontaneous and mutagen-induced mutants (e.g . several hundred-fold for valine-resistant mutants) . However, our studies show that strains containing the purB allele generate mutations at the same level as the wild-type strain, and that the apparent antimutator effect is the consequence of the delayed appearance of mutants on the selective plates . This delay likely results from the combined stress exerted by the adenine deficiency and the presence of the selective agent (i.e . valine). Biochemistry, 2001 Aug 21, 40(33), 9968 - 76 Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E . coli transhydrogenase; Althage M et al.; The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport . In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction . Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed . Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266 . A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge . The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions . Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values . It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III. J Mol Biol, 2001 May 25, 309(1), 255 - 66 E . coli 5'-nucleotidase undergoes a hinge-bending domain rotation resembling a ball-and-socket motion; Knofel T et al.; Structures of nine independent conformers of E . coli 5'-nucleotidase (5'-NT) have been analyzed using four different crystal forms . These data show that the two-domain protein undergoes an unusual 96 degrees hinge-bending domain rotation . Structures of the open and closed forms with substrates and inhibitors reveal that the substrate moves by approximately 25 A with the large domain rotation into the catalytic site . The domain motions derived from a comparison of the nine conformations agree well with motions obtained from a normal mode analysis in that all independent domain rotations are around axes that are roughly located in the plane which includes the domain centers and the hinge . Two residues, Lys355 and Gly356, form the core of the hinge region and undergo a large change of the main-chain torsion angles . The hinge-bending movement observed for 5'-nucleotidase differs markedly from a classical hinge-bending closure motion which involves an opening of the substrate or ligand-binding cleft between two domains . In contrast, the movement observed in 5'-nucleotidase resembles that of a ball-and-socket joint . The smaller C-terminal domain rotates approximately around its center such that the residues at the domain interface move in a sliding motion along the interface . Few direct interdomain contacts and a layer of water molecules between the two domains facilitate the sliding motion. Cell Microbiol, 2001 Aug, 3(8), 499 - 510 The enterohaemorrhagic Escherichia coli (serotype O157:H7) Tir molecule is not functionally interchangeable for its enteropathogenic E . coli (serotype O127:H6) homologue; Kenny B; A major virulence determinant of enteropathogenic Escherichia coli (EPEC) is the Tir molecule that is translocated into the plasma membrane where it orchestrates cytoskeletal rearrangements . Tir undergoes several phosphorylation events within host cells, with modification on a tyrosine essential for its actin-nucleating function . The EHEC (serotype O157:H7) Tir homologue is not tyrosine phosphorylated implying that it uses an alternative mechanism to nucleate actin . This is supported in this study by the demonstration that EHEC Tir is unable to functionally substitute for its EPEC homologue . Like EPEC, the EHEC Tir molecule is phosphorylated within host cells, with the actin-nucleating dysfunction correlated to an altered modification profile . In contrast to EHEC Tir, the EPEC Tir molecule mediated actin nucleation whether delivered into host cells by either strain . Thus, it would appear that EHEC encodes specific factor(s) that facilitate the correct modification of its Tir molecule within host cells . Domain-swapping experiments revealed that the N-terminal, alpha-actinin binding, Tir domains were functionally interchangeable, with both the actin-nucleating dysfunction and altered modification profiles linked to the EHEC C-terminal Tir domain . This tyrosine-independent modification process presumably confers an advantage to EHEC O157:H7 and may contribute to the prevalence of this strain in EHEC disease . The presented data are also consistent with EPEC and EHEC sharing non-phosphotyrosine phosphorylation event(s), with an important role for such modifications in Tir function . An EHEC-induced phosphotyrosine dephosphorylation activity is also identified. Water Res, 2001 Sep, 35(13), 3085 - 8 The occurrence of E . coli O157:H7 in South African water sources intended for direct and indirect human consumption; Muller EE et al.; The occurrence of Escherichia coli O157:H7 in selected water samples in South Africa was investigated . The chromogenic Rainbow agar O157 medium designed for the rapid identification of E . coli O157:H7 was used for the detection of these organisms in various river-water samples in the Vaal Barrage Reservoir drainage basin of South Africa . A total of 204 samples were obtained from 15 sites where water was used for direct and indirect human consumption . Samples were filtered through Gelman filter-units and incubated on Rainbow agar O157 which produced different colours according to the bacterial chromogenic properties . Six hundred and sixty-three suspected E . coli O157:H7 colonies, with colours ranging between dark blue, grey and black, were subcultured onto sorbitol-MacConkey agar and screened for different virulence factors specific for E . coli O157:H7 and agglutination with anti-E . coli O157 antiserum . The results indicated that none of the suspected colonies contained all of the virulence factors necessary to classify them as E . coli O157:H7 . None of these organisms agglutinated with antisera against E . coli O157 . The probability of being infected with E . coli O157:H7 from direct or indirect consumption of these river water sources is therefore low . Some samples did, however, contain enterohaemorrhagic E . coli virulence properties, such as Stx1, Stx2 and enterohaemolysin, which might impose a health risk if ingested. Biotechnol Prog, 2001 Jul-Aug, 17(4), 624 - 8 Cell growth and by-product formation in a pyruvate kinase mutant of E . coli; Zhu T et al.; In this paper, we report on the analysis of acid formation in an E . coli pyk mutant . The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations . However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type . This substantial reduction in acids is accompanied by an increase in CO(2) production . Moreover, unlike the B . subtilis pyk mutant, the E . coli pyk mutant did not show a substantial increase in the PEP pool. Neurosci Lett, 2001 Aug 10, 308(3), 149 - 52 Impaired retrograde axonal transport of adenovirus-mediated E . coli LacZ gene in the mice carrying mutant SOD1 gene; Murakami T et al.; A replication-defective recombinant adenoviral vector containing E . coli lacZ gene was injected into the gastrocnemius muscles of transgenic mice carrying mutant Cu/Zn superoxide dismutase (SOD1) gene and non-transgenic wild-type mice at 40 weeks of age . After 60 and 90 h of the injection, lacZ staining was observed at the distal ends of the sciatic nerves in both mice groups, with the number and the distances greatly reduced in the transgenic mice . Mean velocities of retrograde transport for lacZ was estimated to be 2.1 and 0.05 mm/24 h in non-transgenic and transgenic mice, respectively . These results indicate that the retrograde axonal transport of foreign gene product is impaired in the mice model for familial amyotrophic lateral sclerosis. Science, 2001 Jul 27, 293(5530), 705 - 8 Role of inorganic polyphosphate in promoting ribosomal protein degradation by the Lon protease in E . coli; Kuroda A et al.; Inorganic polyphosphate (polyP), a polymer of hundreds of phosphate (Pi) residues, accumulates in Escherichia coli in response to stresses, including amino acid starvation . Here we show that the adenosine 5'-triphosphate-dependent protease Lon formed a complex with polyP and degraded most of the ribosomal proteins, including S2, L9, and L13 . Purified S2 also bound to polyP and formed a complex with Lon in the presence of polyP . Thus, polyP may promote ribosomal protein degradation by the Lon protease, thereby supplying the amino acids needed to respond to starvation. J Photochem Photobiol B, 2001 Jul, 60(2-3), 136 - 42 Induction of phr gene expression in E . coli strain KY706/pPL-1 by He-Ne laser (632.8 nm) irradiation; Kohli R et al.; We have observed that He-Ne laser irradiation of E . coli strain KY706/pPL-1 leads to induction of photolyase gene, phr . The magnitude of induction was found to depend on the He-Ne laser fluence, fluence rate and post-irradiation incubation period in the nutrient medium . The optimum values for fluence and fluence rate were 7x10(3) J/m(2) and 100 W/m(2), respectively, and the induction of phr gene was observed to saturate beyond an incubation period of approximately 2 h . Experiments carried out with singlet oxygen quenchers and with D(2)O suggest that the effect is mediated via singlet oxygen . Photoreactivation studies carried out after UVC exposure of both the He-Ne laser-exposed as well as unexposed cells showed a larger surviving fraction in the He-Ne laser pre-irradiated cells . This can be attributed to He-Ne laser irradiation-induced induction of phr expression . However, since even without photoreactivating light He-Ne laser pre-irradiated cells show higher survival against UVC radiation it appears that He-Ne laser irradiation induces both light-dependent as well as dark DNA repair processes. Mol Genet Genomics, 2001 Jun, 265(4), 683 - 6 The RadB protein from Pyrococcus does not complement E . coli recA mutations in vivo; Inwood W et al.; A previous publication claimed that the radB gene called Pk-REC from Pyrococcus furiosus complemented an E . coli recA mutation . We found that a sequencing error had led to the test of a mutant form of Pk-REC . The wild-type radB gene from P . furiosus cloned in a similar expression vector to the mutant Pk-REC also appeared to complement an E . coli recA mutation . However, the cloned P . furiosus gdh (glutamate dehydrogenase) gene showed the same activity . We therefore concluded that overexpression of any protein can produce an artificial growth inhibition or stationary phase in recA mutant cells, which allows cells to recover from UV damage due to the action of repair systems that do not require RecA-like activity. Bioinformatics, 2001 Jul, 17(7), 622 - 33 Conformational model for binding site recognition by the E.coli MetJ transcription factor; Liu R et al.; MOTIVATION: Current methods for identifying sequence specific binding sites in DNA sequence using position specific weight matrices are limited in both sensitivity and specificity . Double strand DNA helix exhibits sequence dependent variations in conformation . Interactions between macromolecules result from complementarity of the two tertiary structures . We hypothesize that this conformational variation plays a role in transcription factor binding site recognition, and that the use of this structure information will improve the predictive power of transcription factor binding site models . RESULTS: Conformation models for the sequence dependence of DNA helix distortion have been developed . Using our conformational models, we defined a tertiary structure template for the met operon repressor MetJ binding site . Both naturally occurring sites and precursor binding sites identified through in vitro selection were used as the basis for template definition . The conformational model appears to recognize features of protein binding sites that are distinct from the features recognized by primary sequence based profiles . Combining the conformational model and primary sequence profile yields a hybrid model with improved discriminatory power compared with either the conformational model or sequence profile alone . Using our hybrid model, we searched the E.coli genome . We are able to identify the documented MetJ sites in the promoter regions of metA, metB, metC, metR and metF . In addition, we find several novel loci with characteristics suggesting that they are functional MetJ repressor binding sites . Novel MetJ binding sites are found upstream of the metK gene, as well as upstream of a gene, abc, a gene that encodes for a component of a multifunction transporter which may transport amino acids across the membrane . The false positive rate is significantly lower than the sequence profile method . AVAILABILITY: The programs of implementation of this algorithm are available upon request . The list of crystal structures used for compiling the mean base step parameters of DNA is available by anonymous ftp at http://stateslab.wustl.edu/pub/helix/StructureList. Cell, 2001 Jun 15, 105(6), 733 - 43 Structural basis of the interaction of the pyelonephritic E . coli adhesin to its human kidney receptor; Dodson KW et al.; PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney . The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide . Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin . The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis . These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E . coli for the human kidney and is critical to the pathogenesis of pyelonephritis. Mol Cell, 2001 Jun, 7(6), 1337 - 43 Topological regulation of cell division in E . coli . spatiotemporal oscillation of MinD requires stimulation of its ATPase by MinE and phospholipid; Hu Z et al.; Topological regulation of cell division in E . coli requires positioning a cell division inhibitor, MinC, at the poles of the cell, thus restricting the potential for division to midcell . This positioning is achieved through a rapid oscillation of MinC from pole to pole, a process requiring MinD and MinE . However, the mechanistic basis for this oscillation is not known . Here we report that MinE stimulates MinD ATPase activity, but only in the presence of phospholipid vesicles . Analysis of MinE mutants demonstrates that this stimulation is required for MinD oscillation and suggests that the level of stimulation determines the period of the oscillation . A model is presented in which the requirements for the MinD ATPase contribute spatial and temporal inputs that provide the mechanistic basis for the oscillation. J Mol Biol, 2001 Jul 6, 310(2), 485 - 98 High enzymatic activity and chaperone function are mechanistically related features of the dimeric E . coli peptidyl-prolyl-isomerase FkpA; Ramm K et al.; We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity . We have now investigated the molecular mechanism of these two activities in vitro in greater detail . The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate . This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site . The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis . Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site . Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520 . Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain . The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity . Mutat Res, 2001 Jul 1, 478(1-2), 191 - 7 Bisulfite-induced cytosine deamination rates in E . coli SSB:DNA complexes; Lough J et al.; E . coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA) . We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex . When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon . Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone . After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events . The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations . The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations. Curr Microbiol, 2001 Aug, 43(2), 89 - 92 An E . coli 5S rRNA deletion mutant useful for the study of 5S rRNA structure/function relationships; Ammons D et al.; We report on the construction of a novel strain of E . coli that can be useful for studies on the structure/function relationship of 5S rRNAs . The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA . The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function . We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence. Biochemistry, 2001 Jun 12, 40(23), 6713 - 9 Activation of class III ribonucleotide reductase from E . coli . The electron transfer from the iron-sulfur center to S-adenosylmethionine; Padovani D et al.; The anaerobic ribonucleotide reductase (ARR) from E . coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals . ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a {4Fe-4S}-containing activating enzyme (beta) and AdoMet under reducing conditions . Here we show that the EPR-active S = 1/2 reduced {4Fe-4S}+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical . These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet . Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT . However, in that case, 2 equiv of methionine per reduced cluster was formed . This unusual stoichiometry and combined EPR and Mossbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active {3Fe-4S} intermediate clusters. Neurol Sci, 2000, 21(5 Suppl), S983 - 4 A novel mtDNA mutation in the ATPase6 gene studied by E . coli modeling; Carrozzo R et al.; This study aimed to understand the pathogenesis of a new mtDNA-related etiology of Leigh syndrome . We identified the T9176G mutation as the molecular basis of Leigh syndrome in a child and looked for alterations in cellular ATP production . We then modeled the new mtDNA mutation in E . coli and analyzed ATP synthesis, hydrolysis, and the ability of the mutated enzyme to pump protons . Our results suggest that the T9176G change results in a novel, fully assembled enzyme which inhibits the holoenzyme probably by blocking the proton pathway. Mutat Res, 2001 May 31, 492(1-2), 91 - 7 Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E . coli; Ohta T et al.; Ethidium bromide (EtBr) and SYBR Green I are nucleic acid gel stains and used commonly in combination with UV-illumination . EtBr preferentially induces frameshift mutations but only in the presence of an exogenous metabolic activation system, while SYBR Green I is a very weak mutagen that induces frameshift mutations . We found that EtBr and SYBR Green I, without an added metabolic activation system, strongly potentiated the base-substitution mutations induced by UV-irradiation in E . coli B/r WP2 cells . Each DNA stain alone showed no mutagenicity to the strain . Base-substitutions induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and 4-nitroquinoline-1-oxide were similarly potentiated by EtBr and SYBR Green I . SYBR Green I had a much greater effect . No enhancing effects were observed on mutations induced by mitomycin C, cisplatin, transplatin, cumene hydroperoxide, base analogs, and alkylating agents . Another DNA stain, acridine orange, showed similar enhancing effects on UV- and MX-mutagenicity, but no effect was observed for 4',6-diamidino-2-phenylindole (DAPI) . UV- and MX-induced mutations in E . coli WP2s (uvrA), which is defective in nucleotide excision repair activity, were not potentiated by the addition of EtBr, SYBR Green I, or acridine orange . Those nucleic acid stains might inhibit the nucleotide excision repair of DNA damaged by UV and MX treatment. Structure (Camb), 2001 May 9, 9(5), 439 - 50 The crystal structure of E . coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily; von Delft F et al.; BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis . It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction . RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E . coli . The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution . The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid . CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily . This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex. Arch Biochem Biophys, 2001 Jan 15, 385(2), 311 - 21 The kinetic and spectral characterization of the E . coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate; Loughran PA et al.; The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins . CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E . coli by adding the first 10 codons of CYP17alpha producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies . CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min(-1) and corresponding Km values of 74.5, 27, and 16.7 microM, respectively, in the presence of cytochrome b5 . In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min(-1) and corresponding Km values of 3.9 and 33 microM, respectively . Arachidonate was not metabolized in the absence of cytochrome b5 . Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min(-1) and Km values of 74 and 25 microM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role . Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 microM, respectively) . In stopped-flow kinetics experiments, the flavin reduction (approximately 90 s(-1)) and heme reduction (approximately 9 s(-1)) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5 . Estimations of the rate of CPR (0.3 s(-1)) or cytochrome b5 (9.1 s(-1)) binding with CYP4A7 were also determined. Arzneimittelforschung, 2001, 51(4), 332 - 8 Double-blind randomised placebo-controlled phase III study of an E . coli extract plus 5-fluorouracil versus 5-fluorouracil in patients with advanced colorectal cancer; Unger C et al.; The primary aim of this study was to evaluate the toxicity (mucositis, diarrhea and leucopenia) of a therapy with 5-fluorouracil (CAS 51-21-8; 5-FU) plus an E . coli extract (LC-Extract, Laves coli extract, Colibiogen inject, cell-free soluble fraction from lysed E . coli, Laves strain) in comparison with 5-FU plus placebo . Secondary endpoints included general toxicity, response rate according to WHO, survival time and quality of life . 164 patients with advanced colorectal cancer were enrolled in this randomised, placebo-controlled, double-blind, multicenter phase III study . The treatment consisted of 0.167 ml/kg/d LC-Extract or placebo followed by 500-750 mg/m2/d 5-FU on five consecutive days, repeated every three weeks for up to six treatment cycles . 158 (77 verum, 81 placebo) patients were evaluable for toxicity, 144 (72 verum, 72 placebo) evaluable for response . The therapy with LC-Extract was well tolerated . Adverse events that occurred during the study were mainly judged as 5-FU- or tumor-related . Toxicity from treatment with 600 mg/m2/d 5-FU in both treatment groups was very low . After treatment with 750 mg/m2/d 5-FU patients in the placebo-group experienced a higher CTC toxicity than in the LC-Extract groups . Remission rate and survival time showed a slight trend in favour of LC-Extract . These results suggest a positive benefit-risk ratio of the additional application of LC-Extract to 5-FU in the treatment of advanced colorectal cancer especially for administration of high doses of 5-FU. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1998 Aug, 20(4), 279 - 84 {The expression study of human stem cell factor (hSCF) in E . coli}; Mao H et al.; OBJECTIVE: To improve the expression level of recombinant human stem cell factor (rhSCF) . METHODS: With PCR techniques and synthesized oligonucleotide as primers, the number of nucleotides between the SD sequence and the starting codon ATG of human stem cell factor cDNA was altered, and the preferable codons of E . coli in the N-terminal amino acid sequence were selected . RESULTS: SDS-PAGE electrophoresis indicated that the ratio of expressed rhSCF to total E . coli proteins increased from 12% to 40% by thermal inducing . With DNA sequencing, the reading frame of the gene was proved to be correct . The sequence of 17 N-terminal amino acids of purified recombinant hSCF has been proved to be identical to the natural hSCF, except the first amino acid-Met . CONCLUSIONS: These studies suggest that the 5'-flanking region of hSCF cDNA plays an important role in its gene expression. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 385 - 93 Regulation of E . coli glycogen phosphorylase activity by HPr; Seok YJ et al.; Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients . One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS) . Many proteins interact with and are regulated by PTS components in bacteria . Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS . HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown . The evolution of the allosteric regulation of E . coli GP by HPr is compared to that of other phosphorylases. J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 347 - 54 Three-dimensional structures of protein-protein complexes in the E . coli PTS; Peterkofsky A et al.; The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport . The soluble proteins of the glucose transport pathway also function as regulators of diverse systems . The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy . The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated . An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions . Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase . Thus, there is a common motif for the protein-protein interactions characteristic of the PTS. Lung, 2000 Nov-Dec, 178(6), 331 - 40 Attenuation of live E . coli-induced acute lung injury by X-ray irradiation in guinea pigs; Ishizaka A et al.; Irradiation is suspected to injure inflammatory cells, such as neutrophils and mononuclear phagocytes, cells known to contribute to the development of acute lung injury (ALI) . This study examined whether preexposure to x-ray irradiation modifies ALI induced by E . coli injected intravenously in guinea pig . Thirty animals were divided into two control and two irradiated subgroups: the first control group received saline only (n = 8), and the second control group received E . coli, 2 x 10(9)/kg body weight, suspended in saline (n = 6), IV . The first irradiated group received a single 12-Gy dose + saline (n = 6), and the second irradiated group received a single 12-Gy dose + E . coli (n = 10) . The lung wet-to-dry-weight ratio (W/D) and 125I-albumin lung tissue/plasma ratio (T/P) were measured as markers of lung injury . W/D was significantly higher in the control E . coli group than in the other groups . T/P in the control E . coli group was also increased compared with T/P measured in the other groups . In the control E . coli group, a marked increase in bronchoalveolar lavage (BAL) neutrophils was observed compared with the control saline group . However, no significant difference in BAL neutrophil counts was observed between the control and irradiated E . coli groups . In contrast, BAL macrophages were significantly reduced in the irradiated E . coli groups compared with the control E . coli group . These findings suggest that x-ray irradiation attenuates E . coli-induced ALI in guinea pigs, an effect explained, at least in part, by a reduction in the number of alveolar macrophages. Chin Med Sci J, 1997 Dec, 12(4), 229 - 31 The development of monoclonal antibody against rhTNF and its curative effects on E . coli infected mice; Guo X et al.; Fifteen hybridoma cell lines producing monoclonal antibodies (McAb) against recombinant human tumor necrosis factor alpha (rhTNFa) have been established by fusing SP 2/0 cells with spleen cells from a BALB/c mouse immunized with rhTNFa . Following J . M . Davis's Works, semi-solid medium was used for initial cloning . Five of them were studied further . Their main chromosome numbers range were 96 to 105, all of them were IgG1 subclass . The affinities of these McAbs were estimated to be 1.25 x 10(8) mol/L, 1.12 x 10(8) mol/L, 2.34 x 10(8) mol/L, 8.55 x 10(7) mol/L, 1.04 x 10(8) mol/L, respectively . Two groups of mice challenging with E . Coli (10(7) organisms), one group treated with 2 mg/kg anti-TNF monoclonal antibody, the other did not . There was a higher survival rate in treated group, the serum TNF level was significantly lower too, and the untreated mice had severe pathologic changes in viscera. FEMS Microbiol Lett, 2001 May 15, 199(1), 61 - 6 A subtractive hybridisation analysis of genomic differences between the uropathogenic E . coli strain 536 and the E . coli K-12 strain MG1655; Janke B et al.; Suppression subtractive hybridisation (SSH) was performed to identify genomic differences between the uropathogenic Escherichia coli strain 536 and the non-pathogenic E . coli K-12 strain MG1655 . In total, 22 DNA fragments were isolated which were specific for strain 536 . Five of these fragments showed homology to known virulence determinants and four fragments matched genes for lipopolysaccharide (LPS) or capsule biosynthesis and a siderophore receptor . Seven fragments did not show any homology to known genes . These fragments may represent parts of putative pathogenicity islands (PAIs) . Whereas two fragments were highly specific for uropathogenic E . coli (UPEC), the other fragments could also be detected among the other tested wild-type strains. Mutat Res, 2001 Jun 5, 486(1), 11 - 9 Expression of E . coli RecA targeted to mitochondria of human cells; Paul R et al.; Mitochondrial DNA integrity is ensured by several nuclear-encoded proteins in vertebrates, and a number of mtDNA alterations in human diseases, including deletions and duplications, have been suspected to result from errors in the mitochondrial recombination pathway . However, the presence of the latter system is still a matter of controversy as RecA proteins display various functions in vitro . In Escherichia coli, RecA plays a central role in homologous recombination by pairing and transferring a single strand to a homologous duplex DNA . To address indirectly the issue of a mitochondrial recombination pathway in vivo, we have constructed a chimeric gene containing an N terminal mitochondrial targeting sequence and the E . coli RecA gene . Cells were transfected by the recombinant plasmid, then tested for their mtDNA repair upon bleomycin treatment . We found an increased repair rate of the mitochondrial DNA in cells expressing RecA as compared to control cells . These results indicate that the transfected cells display an improved mtDNA repair replication pathway due to the exogenous RecA, likely in synergy with an endogenous rate-limiting mitochondrial recombination pathway. Vet J, 2001 May, 161(3), 269 - 79 E . coli nitroreductase/CB1954: in vitro studies into a potential system for feline cancer gene therapy; Blackwood L et al.; Investigations were carried out to identify a suitable prodrug activating system for feline gene therapy with the eventual aim of treating feline thyroid disease and feline neoplasia . The E . coli nitroreductase (NTR)/CB1954 prodrug activating system was evaluated in vitro in feline cells by transient transfection with a nitroreductase expressing construct and subsequent treatment with the prodrug CB1954 . The feline cells successfully expressed E . coli nitroreductase, which was able to activate the prodrug CB1954 resulting in cytotoxicity to both transformed and adjacent cells (a bystander effect) in vitro . In the absence of nitroreductase, CB1954 was non-toxic to feline cells . In addition, the nitroreductase gene was expressed in rat thyroid cells under the control of the cell type specific feline thyroglobulin promoter . This paper demonstrates that the E . coli nitroreductase/CB1954 system may be suitable for in vivo feline gene therapy, and further investigations are warranted. Biochemistry, 2001 Feb 20, 40(7), 2075 - 9 pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E . coli in planar lipid bilayers; Das S et al.; Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH . This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations . Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity . PHB/polyP complexes, isolated from E . coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH . When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5 . However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure . Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH . Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH . Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH . The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel. Mol Cell Endocrinol, 2001 Apr 25, 175(1-2), 41 - 56 Ovine caveolin-1: cDNA cloning, E . coli expression, and association with endothelial nitric oxide synthase; Chen D et al.; Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of endothelial nitric oxide (NO) synthase (eNOS) . We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy . To test this hypothesis in the sheep model, we isolated the full-length cDNA of ovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells . Thirty-two positive oCav-1 clones were recognized by a partial oCav-1 cDNA from this library, of which eight were sequenced . Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from approximately 2.1 to 2.7 kb . Northern analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of approximately 2.1 and 2.7 kb, respectively . Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC . Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1 protein, full-length oCav-1 cDNAs apparently possess a approximately 1.6-2.1 kb 3'-untranslated region . Database searches with oCav-1 cDNA revealed that the coding region of mammalian Cav-1 genes is highly conserved . We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a {His}6-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1 . Incubation of exogenous {His}6-tagged oCav-1 with resting UAEC extracts led to the formation of a {His}6-tagged oCav-1-eNOS complex . In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, {His}6-tagged oCav-1 associated eNOS was dose (0-10 microM)-dependently inhibited . eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes . Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation. J Ocul Pharmacol Ther, 2001 Feb, 17(1), 93 - 9 Bovine eye 1-Cys peroxiredoxin: expression in E . coli and antioxidant properties; Peshenko IV et al.; Peroxiredoxins constitute a molecular family of novel antioxidant proteins and are distributed broadly in non-mammalian and mammalian tissues, including the eye . In this study, a recombinant bovine eye 1-Cys peroxiredoxin (BRPrx) was expressed in Escherichia coli (E . coli) . The recombinant protein protected glutamine synthetase from oxidative damage caused by a metal ion-catalyzed oxidation system (ascorbate/Fe3+/O2) in the presence of dithiothreitol as an electron donor . The protector activity of BRPrx is attributed to its peroxidase activity exhibited in the presence of dithiothreitol . Both hydrogen peroxide and short chain hydroperoxides are substrates for the protein . Glutathione could not support antioxidant properties of the recombinant protein . The antioxidant activity of BRPrx in the glutamine synthetase protection assay was as high as the activity of catalase and about one order of magnitude lower than that of selenium glutathione peroxidase . These results support the premise that Prx is an important component of the antioxidant defense system in eye tissues. Biotechnol Bioeng, 2001 Jun 5, 73(5), 406 - 11 Integrated flow-injection processing for on-line quantification of plasmid DNA during cultivation of E . coli; Nandakumar MP et al.; An integrated flow-injection processing (FIP) system for the quantification of plasmids during cultivation is described . The system performs on-line sampling, cell lysis, and quantification of plasmids in an integrated manner during cultivation of E . coli . The system was operated by using a miniaturized expanded-bed column which can be used for handling samples containing cells and cell debris without interfering with the binding analysis . Two types of detectors (one measuring UV absorbance at 254 nm and a fluorometer) are used for on-line plasmid detection . The system was developed using standard solutions and it was successfully applied in monitoring plasmid contents during a cultivation of E . coli . Genes Cells, 2001 Apr, 6(4), 279 - 90 Induction of CspA, an E . coli major cold-shock protein, upon nutritional upshift at 37 degrees C; Yamanaka K et al.; BACKGROUND: The synthesis of CspA, the major cold-shock protein of Escherichia coli, is dramatically induced upon cold shock . It was recently reported that there is massive presence of CspA under nonstress conditions, and it is thus claimed that CspA as the cold-shock protein is a misnomer . RESULTS: Here, we re-examined and confirmed that CspA is induced upon culture dilution at 37 degrees C . However, its induction level is one-sixth of the cold-shock-induced level, clearly indicating that the major stress that induces CspA is cold shock . It was further found that CspA induction can be achieved not only by culture dilution but also by the simple addition of nutrients, and that it was almost completely abolished in the presence of rifampicin or nalidixic acid . Nutritional upshift causes the induction of only CspA but not other cold-shock-inducible CspA homologues . The amount of cspA mRNA rapidly and transiently increased by culture dilution, but its stability was not significantly changed . CONCLUSIONS: These results suggest that CspA is a nutritional-upshift stress protein as well as a cold-shock stress protein, and that CspA induction following nutritional upshift may be due to transcriptional activation. J Protein Chem, 2000 Nov, 19(8), 685 - 92 The proteolytic activity of the recombinant cryptic human fibronectin type IV collagenase from E . coli expression; Schnepel J et al.; Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain . The enzyme could be generated and activated in the presence of Ca2+ from the purified 70-kDa pFN fragment produced by cathepsin D digestion . In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E . coli . The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, alpha- and beta-casein, insulin b-chain, and a synthetic Mca-peptide . In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates . Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2. Clin Nephrol, 2000 May, 53(5), 319 - 24 Elevated tissue factor circulating levels in children with hemolytic uremic syndrome caused by verotoxin-producing E . coli; Kamitsuji H et al.; BACKGROUND: Microvascular thrombosis in the kidney plays an important role in the pathogenesis of hemolytic uremic syndrome (HUS) . Tissue factor (TF), present on the vascular surface of endothelial cells, binds factor VIIa . The complex initiates the coagulating cascade by activating factors X and IX . PATIENTS AND METHODS: In cases of HUS associated with verotoxin-producing E . coli (VTEC) infection, VTEC gastroenteritis without HUS and normal controls, we measured plasma concentrations of TF and tissue factor pathway inhibitor (TFPI) to evaluate their clinical significance . In children with non-HUS chronic renal failure (CRF), the TF levels were also measured as another control group . RESULTS: In the acute phase of HUS, plasma levels of TF and TFPI were significantly elevated, then returned to normal range in the recovery phase . The TF levels were closely correlated with the thrombin antithrombin-III complex, a marker of thrombin activity in circulating blood, and with creatinine clearance (Ccr) . Furthermore, a positive correlation was noted between plasma TF levels and plasma soluble thrombomodulin (sTM) levels, which is a marker of endothelial cell injury . The influence of decreased excretion from damaged kidneys should be considered since a definite lot correlation was observed between plasma TF levels and Ccr in children with non-HUS CRF . CONCLUSION: From these findings, we concluded that elevated TF circulating levels may also play an important role in blood-clotting activation observed in VTEC-HUS patients, and may also be a useful marker for renal damage. Pediatr Infect Dis J, 2001 Mar, 20(3), 316 - 8 Hemolytic-uremic syndrome surveillance to monitor trends in infection with Escherichia coli O157 and non-O157 enterohemorrhagic E . coli in Austria; Fischer H et al.; Austrian data underline that relying on the number of enterohemorrhagic Escherichia coli (EHEC) O157 strains isolated from clinical specimens does not allow assessment of the actual incidence of EHEC infections . A hospital-based system for identification of hemolytic-uremic syndrome cases based on voluntary cooperation was established in 1995 and provides information needed to monitor trends in the incidence of O157 and non-O157 EHEC infections. Fresenius J Anal Chem, 2001 Feb, 369(3-4), 295 - 301 Detection of E . coli using a microfluidics-based antibody biochip detection system; Stokes DL et al.; This work demonstrates the detection of E . coli using a 2-dimensional photosensor array biochip which is efficiently equipped with a microfluidics sample/reagent delivery system for on-chip monitoring of bioassays . The biochip features a 4 x 4 array of independently operating photodiodes that are integrated along with amplifiers, discriminators and logic circuitry on a single platform . The microfluidics system includes a single 0.4 mL reaction chamber which houses a sampling platform that selectively captures detection probes from a sample through the use of immobilized bioreceptors . The independently operating photodiodes allow simultaneous monitoring of multiple samples . In this study the sampling platform is a cellulosic membrane that is exposed to E . coli organisms and subsequently analyzed using a sandwich immunoassay involving a Cy5-labeled antibody probe . The combined effectiveness of the integrated circuit (IC) biochip and the immunoassay is evaluated for assays performed both by conventional laboratory means followed by detection with the IC biochip, and through the use of the microfluidics system for on-chip detection . Highlights of the studies show that the biochip has a linear dynamic range of three orders of magnitude observed for conventional assays, and can detect 20 E . coli organisms . Selective detection of E . coli in a complex medium, milk diluent, is also reported for both off-chip and on-chip assays. Yonsei Med J, 2001 Feb, 42(1), 114 - 9 Increase in rat plasma antioxidant activity after E . coli lipopolysaccharide administration; Lee KY et al.; It is well recognized that the sensitivity of animals to lipopolysaccharide (LPS) endotoxin varies tremendously . And, it has been recently observed that Sprague-Dawley rats dramatically increase the activity of hepatic endogenous antioxidative enzyme systems after LPS administration . This finding suggests that the relative resistance of rats to LPS may be related to a concomitant increase in the activities of the hepatic antioxidant systems . This study was designed to examine if the above reported hepatic change in rats given LPS could be observed at the systemic level . Male Sprague-Dawley or Wistar rats, weighing 250 - 350 g, were given increasing doses (10 - 100 mg/kg) of LPS i.p . under 1.0% isoflurane anesthesia . Antioxidant capacity (AOC), blood gas analysis, and the cardiovascular parameters of the arterial blood of animals were determined over a 4 hour period following LPS administration . In addition, we studied the effect of pretreatment with the non-specific nitric oxide synthase inhibitor, L-N(G)-Nitroarginine methyl ester hydrochloride (L-NAME), given 50 mg/kg s.c . one and 24 hours before the administration of 20 mg/kg LPS i.p . in Sprague-Dawley rats . Rats given sufficiently high doses of E . coli LPS to produce behavioral effects also showed increased plasma AOCs in the early period after the administration of LPS . Similar changes were noted in Sprague-Dawley and Wistar rat strains, but at different doses that reflect their differential sensitivities to the LPS induced inflammatory response . Also, the resistance of the Sprague-Dawley strain of rats to LPS was not altered by the prior administration of L-NAME, nor was the plasma AOC altered . In conclusion, our study suggests that the rat strains are relatively resistant to develop the toxic signs of LPS in the early period after the administration of LPS, especially in Sprague-Dawley rats . Moreover, endotoxin-induced increases in plasma AOC may contribute to the rats' resistance to LPS intoxication. J Immunol Methods, 2001 May 1, 251(1-2), 187 - 93 Protein affinity maturation in vivo using E . coli mutator cells; Coia G et al.; This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E . coli mutator cells . The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences . Selection conditions can be modified for antibody fragments with increased production levels . Growth conditions in E . coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment. Biochemistry, 2001 Apr 10, 40(14), 4261 - 71 Structural basis for the functional switch of the E . coli Ada protein; Lin Y et al.; The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center . The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes . Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage . Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer . To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated . Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada . The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA . This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group. Virology, 2001 Mar 15, 281(2), 272 - 80 A new strategy in design of +RNA virus infectious clones enabling their stable propagation in E . coli; Yamshchikov V et al.; Infectious clone methodology is a valuable tool of modern experimental virology . However, its use is often constrained by the instability of infectious clone constructs during propagation in E . coli . To circumvent this problem, we have devised a strategy that could be suitable for design of +RNA virus molecular clones in general . An infectious clone is assembled as "infectious DNA," and expression of problem regions present in the viral cDNA is prevented during propagation in E . coli by insertion of short introns . To demonstrate the feasibility of this approach, a highly unstable Japanese encephalitis flavivirus infectious clone has been successfully converted into a remarkably stable infectious DNA construct with the specific infectivity of 10(6) pfu/microg in cell culture . The proposed strategy may be useful in the design of self-amplifying gene therapy vectors and development of new immunization methodologies, and could facilitate creation of molecular repositories of existing viral vaccines . Genes Dev, 2001 Mar 15, 15(6), 748 - 61 Topoisomerase IV, alone, unknots DNA in E . coli; Deibler RW et al.; Knotted DNA has potentially devastating effects on cells . By using two site-specific recombination systems, we tied all biologically significant simple DNA knots in Escherichia coli . When topoisomerase IV activity was blocked, either with a drug or in a temperature-sensitive mutant, the knotted recombination intermediates accumulated whether or not gyrase was active . In contrast to its decatenation activity, which is strongly affected by DNA supercoiling, topoisomerase IV unknotted DNA independently of supercoiling . This differential supercoiling effect held true regardless of the relative sizes of the catenanes and knots . Finally, topoisomerase IV unknotted DNA equally well when DNA replication was blocked with hydroxyurea . We conclude that topoisomerase IV, not gyrase, unknots DNA and that it is able to access DNA in the cell freely . With these results, it is now possible to assign completely the topological roles of the topoisomerases in E . coli . It is clear that the topoisomerases in the cell have distinct and nonoverlapping roles . Consequently, our results suggest limitations in assigning a physiological function to a protein based upon sequence similarity or even upon in vitro biochemical activity. Protein Sci, 2001 Jan, 10(1), 116 - 28 Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E . coli: fragment complementation and circular permutation reveal stable, alternatively folded forms; Smith VF et al.; The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods . Cleavage of the backbone after position 86 resulted in two fragments, (1--86) and (87--159) each of which are poorly structured and enzymatically inactive . When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K(a) = 2.6 x 10(7) M(-1)) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations . The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart . In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein . After addition of ligand or the tight-binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native-like secondary and tertiary structures . These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR. Biochemistry, 2001 Mar 13, 40(10), 3117 - 26 Determination of an optimal potential window for catalysis by E . coli dimethyl sulfoxide reductase and hypothesis on the role of Mo(V) in the reaction pathway; Heffron K et al.; Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential . The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)) . At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0) . Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance . The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state. Biochemistry, 2001 Mar 13, 40(10), 3047 - 55 The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E . coli and its relationship with that found in eukaryotic protein kinases; Oudot C et al.; The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E . coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue . As such, it belongs to the serine/threonine protein kinase family . However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure . In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases . In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403 . Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues) . Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases . Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification . Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain . In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme . However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme . On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected . Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family. Mol Biotechnol, 2000 Nov, 16(3), 253 - 60 Application of the E . coli trp promoter; Bass SH et al.; The Escherichia coli tryptophan (trp) promoter has been used extensively for the high level production of proteins on a small and large scale . This regulated promoter is readily available, relatively easy to turn on, and can be used in essentially any E . coli host background . This article gives a detailed use of the trp promoter including the design of expression vectors, subsequent culture conditions for promoter induction, and, finally, a protocol for the most common way of detecting the newly synthesized protein of interest . Its successful use for heterologous protein expression, however, sometimes requires consideration of parameters other than transcription such as translation initiation, translation elongation, and proteolysis . In this respect we offer guidance in getting through these post-transcriptional problems, which can occur with the use of any promoter. Int J Food Microbiol, 2001 Feb 15, 63(3), 217 - 23 Characterization of Shiga toxin producing E . coli and O157 serotype E . coli isolated in France from healthy domestic cattle; Rogerie F et al.; A study was carried out in France in collaboration with the meat industry to investigate the occurrence and characteristics of Shiga toxin-producing E . coli (STEC) and O157 E . coli in a population of healthy bovines representative of French livestock . A total of 851 animals belonging to three bovine classes (106 young bulls, 374 dairy cows and 371 meat cows) were included in the study . Samples of feces and of the corresponding carcasses were collected from March 97 to August 97 in seven abattoirs spread throughout the national territory . STEC cultures from the 1702 samples were screened using PCR for the presence of stx genes . Positive samples were further subjected to colony blot hybridization and to O157-specific immunomagnetic separation . Probe-positive colonies and O157 colonies were then analyzed for the presence of virulence genes and phenotypic characters (serotype, Stx production) . In 154 (18.1%) feces and 91 (10.7%) carcass samples stx genes were detected . Two hundred and twenty-two STEC colonies were isolated from 67 (7.9%) feces and 16 (1.9%) carcass samples, with 183 STEC isolated from feces and 39 from carcasses . Only eight O157 isolates were collected from feces samples . None of these O157 E . coli isolates presented stx genes and thus could not be considered as pathogenic regarding hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) . In 3.2% of STEC isolated from feces and in 10.2% of STEC from carcasses eae genes were detected . In 17% of STEC from feces and in 30.7% from carcasses ehx genes were detected . Using these data, the 222 STEC colonies could be classified in 11 different 'virulence patterns' (presence/absence of stx1, stx2, eae and ehx genes), showing that more than 77% of isolates presented only one virulence factor . Only three STEC on 222 colonies (1.3%) presented the three virulence factors stx, eae and ehx in association, none of them reacting with antisera specific for enterohemorrhagic E . coli . (EHEC) . These data, together with the fact that only five isolates on the 222 (2.2%) reacted with such antisera (three O111 and two O26 isolates) demonstrated that the natural bacterial populations isolated during this study were clearly distinct from EHEC. J Vet Diagn Invest, 2001 Jan, 13(1), 26 - 9 Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and enterotoxin genes in E . coli isolated from diarrheic piglets; Choi C et al.; A total of 720 Escherichia coli strains isolated from diarrheic piglets on 756 swine farms were screened for the presence of the enteroaggregative E . coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR) . Escherichia coli strains that carried EAST1 genes were also tested by PCR for the presence of 4 fimbriae (F4, F5, F6, F41), 2 heat-stable enterotoxins (STa and STb), and 1 heat-labile enterotoxin (LT) gene . One hundred sixty-four (22.7%) of the 720 E . coli isolates carried genes for EAST1 . Of these 164 isolates, 62 (37.8%) carried EAST1 genes only, 11 (6.7%) carried genes for at least 1 of the fimbrial adhesins, 51 (31.1%) carried genes for at least 1 of the enterotoxins, and 40 (23.8%) carried genes for at least 1 of the fimbrial adhesins and enterotoxins . Forty-six percent of strains that carried EAST1 genes carried STa genes, and 16% of strains that carried EAST1 genes carried F4 . The isolation rate of enterotoxigenic E . coli strains carrying genes for EAST1 gene was 63% . The 6 major genotypes observed in this study (in decreasing order) were EAST1+, EAST1+STa+, EAST1+STa+STb+, EAST1+STa+F5+, EAST1+STa+F4+, and EAST1+STb+F4+ . EAST1 is widely prevalent among diarrheagenic strains of E . coli and may represent an important virulence determinant in the pathogenesis of enteric colibacillosis of preweaned pigs. Chin Med Sci J, 1997 Mar, 12(1), 37 - 40 Expression of HBV Pre S1 peptide in E . coli and product characterization; Mi Z et al.; HBV Pre S1 sequence is supposed to play an important role in the infection of HBV . Presence of Pre S1/anti-Pre S1 in serum has valuable clinical implications . In order to improve the study of Pre S1, Pre S1 sequence was overexpressed in E . coli as a fusion protein with MBP (Maltose-binding protein), and anti-Pre S1 antiserum was elicited in rabbits by Pre S1-MBP purified by affinity chromatography . The recombinant plasmid constructed from pMAL-cRI expressed the 106aa Pre S1 sequence at the C terminal of MBP by tac promoter . The resulting protein is about 54 kD in size . Western-blot analysis confirmed its reactivity with antiserum derived from synthetic Pre S1 peptide and serum from patients with acute hepatitis B (AHB) . ELISA showed that Pre S1-MBP and Dane particles purified from AHB patient's serum reacted with antiserum against synthetic Pre S1 peptide, and this reaction was specifically inhibited by synthetic Pre S1 peptide . ELISA also demonstrated that antiserum against Pre S1-MBP reacted with synthetic Pre S1 peptide, but not with synthetic HCV peptide or HEV peptide. J Microbiol Methods, 2001 Apr, 44(3), 225 - 33 Optimization of a fluorescent-based phosphor imaging dot blot DNA hybridization assay to assess E . coli virulence gene profiles; Zhang L et al.; To increase the efficiency and consistency in screening Escherichia coli for virulence genes, a Phosphor Imager was adopted for signal detection in Dot Blot DNA hybridization replacing X-ray film read by eye . We assessed not only the reliability of the instrument-based procedure, but the impact of going from an outcome measured by visualization on a semi-quantitative scale to a digitized readout on an interval scale . We analyzed technical and biological variability of the assay and the factors contributing to the variability . In spite of high variability both within and between membranes in the Phosphor Imager readings, we were able to define classification rules for gene presence that were remarkably consistent . Using the X-ray film signal detection procedure with Southern confirmation as a gold standard, we obtained a sensitivity and specificity of 87-99% for a rule requiring no retesting for all but one gene probe. Genes Dev, 2001 Mar 1, 15(5), 491 - 506 Fine structure of E . coli RNA polymerase-promoter interactions: alpha subunit binding to the UP element minor groove; Ross W et al.; The alpha subunit of E . coli RNAP plays an important role in the recognition of many promoters by binding to the A+T-rich UP element, a DNA sequence located upstream of the recognition elements for the sigma subunit, the -35 and -10 hexamers . We examined DNA-RNAP interactions using high resolution interference and protection footprinting methods and using the minor groove-binding drug distamycin . Our results suggest that alpha interacts with bases in the DNA minor groove and with the DNA backbone along the minor groove, but that UP element major groove surfaces do not make a significant contribution to alpha binding . On the basis of these and previous results, we propose a model in which alpha contacts UP element DNA through amino acid residues located in a pair of helix-hairpin-helix motifs . Furthermore, our experiments extend existing information about recognition of the core promoter by sigma(70) by identifying functional groups in the major grooves of the -35 and -10 hexamers in which modifications interfere with RNAP binding . These studies greatly improve the resolution of our picture of the promoter-RNAP interaction. J Mol Biol, 2001 Feb 16, 306(2), 363 - 73 Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E . coli; Fribourg S et al.; Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes . We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis . Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains . Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation. J Mol Biol, 2001 Feb 16, 306(2), 213 - 25 Interaction of the C-terminal domain of the E . coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface; Yasuno K et al.; The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA . Previously, we determined the solution structure of alphaCTD . Here, we investigated the interaction between alphaCTD and UP element DNA by NMR . DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA . Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding . There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay . We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending . Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA . The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction . Based on these observations, we propose a model for the UP element-alphaCTD complex. Virus Res, 2001 Apr, 74(1-2), 59 - 73 Expression of deletion mutants of the hepatitis B virus protein HBx in E . coli and characterization of their RNA binding activities; Rui E et al.; The hepatitis B virus protein HBx has been implicated in the development of liver cancer . It has been shown that the HBx protein is able to bind to single-stranded DNA in a specific manner . This DNA binding activity might be relevant for HBx oncogene character . To study the HBx interaction with nucleic acids in more detail we expressed full-length HBx as well as several N- and C-terminally truncated HBx proteins as 6xHis and GST-fusions in E . coli . Using a gel shift assay, we were able to demonstrate that all of the truncated HBx proteins have the ability to bind to an AU-rich RNA . The affinity of GST-HBx #3 (residues 80-142) was an order of magnitude higher than that of GST-HBx #2 (residues 5-79), indicating that a high affinity RNA binding site is located in HBx C-terminal half . AUF1 is the protein ligand that binds to AU-rich RNA regions present in certain proto-oncogene mRNAs and causes their rapid degradation . By a competitive binding experiment of AUF1 and HBx to the AU-rich RNA oligonucleotide, we show that HBx is able to displace AUF1 from its binding site on the RNA oligonucleotide . This new aspect of HBx function is discussed in the context of cellular transformation. Bioorg Khim, 2000 Oct, 26(10), 728 - 34 {Cleavage of RNA in hybrid duplexes by ribonuclease H from E . coli . I . Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides}; Vorob'ev PE et al.; We studied the E . coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem) . It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments . On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed . Thus, the initial rates of accumulation of the 17- and 8-mer were close . Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20 . These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation . This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem . Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness. J Protein Chem, 2000 Jul, 19(5), 389 - 97 Characterization of glyoxalase I (E . coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis; Stokvis E et al.; Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis . An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid . The E . coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I . Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I . Two specific binding sites were observed per dimer . The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-(2-{3-(hexyloxy)benzoyl}-vinyl)glutathione . Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction . This study shows that selective inhibitors may exist for the E . coli homologue of the glyoxalase I enzyme. Bioorg Med Chem, 2001 Jan, 9(1), 51 - 6 Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E . coli tRNA(Asp); Persson T et al.; A 77-mer RNA with the sequence of Eschlerichia coli tRNA(Asp) has been chemically synthesised using standard automated phosphoramidite chemistry with the coupling reagent 4,5-dicyanoimidazole (DCI) . The synthesis was carried out on a 1000 A CPG-column and . after deprotection and gel purification, a yield of about 7 mmol with a purity of > 95% was reproducibly obtained . By comparing automated synthesis of the 77-mer RNA using 1H-tetrazole and DCI as activator, DCI is advantageous in producing longer RNAs . However, for shorter RNAs ( <40 mer) no difference could be observed . In addition to the all-ribo tRNA(Asp) carrying the wild-type sequence, two variants were synthesised, one with a single C to G48 mutation and the second with a 2'-deoxy modification at C48 . The three tRNAs were tested for their aminoacylation efficiency and high affinity binding to E . coli RNase P RNA . The results demonstrate that chemically synthesised 77-mer oligoribonucleotides can be successfully used for structure function studies. Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 423 - 8 {Cloning and expression of the E . coli serine hydroxymethyltransferase gene (glyA)}; Shen T et al.; The E . coli K12 glyA gene(13 kb), encoding serine hydroxymethyltransferase (SHMT), has been cloned in the plasmid vector pBR329 using insertion inactivation and complementation test . Subcloning of segments of the original insert (13 kb) into plasmids pBR322, pBR329 and pSMY901 established that a 2.6 kb PstI-EcoR fragment carries the glyA gene . The 12 strains of transforments containing recombined plasmid . were obtained . SHMT and glyA gene product level in strains carrying glyA plasmids were different . No SHMT activity was observed in host strains . The glyA gene products for JM109(pSM13), K12(pSM13), K12(pSM14) and K12(pSM15) account for 15.7%, 15.4%, 11.8%, and 9.48% of the total dissoluble cell protein, respectively. Am J Physiol Gastrointest Liver Physiol, 2001 Feb, 280(2), G216 - 21 Effect of E . coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice; Charney AN et al.; We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice . Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions . Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net