Protein Expr Purif, 1997 Oct, 11(1), 47 - 52 Stable expression and rapid purification of Escherichia coli GroEL and GroES chaperonins; Kamireddi M et al.; An Escherichia coli expression vector pRE (P . Reddy, A . Peterkofsky, and K . McKenney, 1989, Nucleic Acids Res . 17, 10473-10488), originally developed for the cloning and expression of lethal genes, was used for cloning and hyperexpression of GroEL and GroES genes . Regulated gene expression is achieved in the pRE vector under the tight control of the lambda PL promoter . Upon induction of the promoter, stable expression of GroEL to about 60% of the total cell protein was observed . Similarly, stable expression of GroES to about 40% of the total cell protein was achieved . GroES was found to be a heat-stable protein while GroEL was not . Both GroE chaperonins were purified in a single chromatographic step with a yield of about 100 mg GroEL and 25 mg GroES per liter of E . coli culture . GroE chaperonins purified by the protocols described here were active in the renaturation of urea-denatured rhodanese. Protein Expr Purif, 1997 Oct, 11(1), 41 - 6 Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli; Martinez-Barajas E et al.; Fructokinase (FK; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) cloned from a tomato fruit cDNA library has been expressed in Escherichia coli . The recombinant protein was purified 159-fold to greater than 99% purity, based on SDS-PAGE analysis . The subunit molecular mass is estimated to be 35 kDa and the nondissociated molecular mass is 72.4 kDa, indicating that the functional form is a dimer . Two-dimensional IEF/SDS-PAGE analyses combined with immunodetection show that both native and recombinant proteins exhibit the same pattern of six closely grouped peptides with pI values ranging from 5.66 to 6.17 . Biochemical characterization of the purified recombinant enzyme shows properties essentially identical to those of the native fructokinase purified from young tomato fruit: the pH optimum is 8.0, the K(m) for fructose is 0.22 mM, and severe substrate inhibition is observed when fructose concentration is greater than 0.5 mM (Ki = 3.0 mM) . ATP is the preferred phosphate donor (K(m) = 0.13 mM and Vmax/K(m) = 212), followed by GTP (K(m) = 0.45 mM and Vmax/K(m) = 76) and UTP (K(m) = 1.68 mM and Vmax/K(m) = 20), but Vmax values are slightly greater with GTP and UTP . Product inhibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration {Ki (ADP) = 0.41 mM with 0.5 mM fructose and decreased to 0.12 mM with 3 mM fructose} . Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fructose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK. Protein Expr Purif, 1997 Oct, 11(1), 26 - 34 Expression of three functional domains of connexin 32 as thioredoxin fusion proteins in Escherichia coli and generation of antibodies; Mambetisaeva ET et al.; Gap junctions, intercellular channels that allow cells to communicate directly, are constructed from connexin protein subunits . Connexins traverse the membrane four times and have two extracellular loops and one intracellular loop; the amino and carboxyl tails are located at the intracellular aspect of the plasma membrane . The first extracellular domain (EL1; residues 42-75), the intracellular domain (IL; 94-130), the carboxyl-terminus (CT; 208-283), and the full-length rat connexin 32 (Cx32) gene were subcloned and expressed in Escherichia coli as fusion proteins to the thioredoxin protein (Trx) . Under optimal conditions, the expression levels of the various Cx32 domains differed . The Trx-EL1 and Trx-IL fusions were expressed at high levels in G1768 cells and represented 40-60% of total soluble cellular protein 4 h after induction with tryptophan . The Trx-CT fusion protein was produced less efficiently (20% of total soluble cellular protein) . However, Trx-Cx32 (full length) was not expressed in E . coli . Linkage of full-length Cx32 to thioredoxin caused a dramatic decrease in the growth of the cultures after induction . Expressed Trx-EL1, Trx-IL, and Trx-CT fusion products were affinity purified over a Thio-Bond resin and used as antigens for generation of polyclonal antibodies to rat connexin32 in rabbits . Antibodies generated to thioredoxin-CT fusion and an antibody produced against a short synthetic peptide (GAP9) corresponding in sequence to an intracellular carboxyl-terminal tail of Cx32 (residues 264-283) identified the same proteins on Western blots . The antibodies to the Trx-CT fusion protein were of higher titer than those generated previously to synthetic peptides . Immunofluorescent staining of rat liver sections with Trx-IL and Trx-CT antibodies demonstrated Cx32 immunoreactivity in punctate areas of the cell surface corresponding to the expected positions of gap junctions. Protein Expr Purif, 1997 Oct, 11(1), 17 - 25 Effects of codon usage and vector-host combinations on the expression of spinach plastocyanin in Escherichia coli; Ejdeback M et al.; Spinach plastocyanin has been expressed in Escherichia coli and exported to the periplasmic space . The effects of codon usage, expression system, growth length, and temperature on expression levels in LB medium were investigated . A stretch of codons, rare in E . coli, was identified and replaced with highly expressed codons, increasing the yield by at least 20% . Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter . Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction . The optimized expression system produced 38 mg holoprotein per liter culture . In this system it was also possible to express plastocyanin in minimal medium, at a yield of 10 mg per liter . N-terminal sequencing and mass spectrometry showed that plastocyanin was correctly processed . The expressed plastocyanin was purified to homogeneity, as shown by an A278/A597 ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorption coefficients for epsilon 278 and for epsilon 597 were determined to be 4700 M-1 cm-1. J Mol Biol, 1997 Sep 26, 272(3), 327 - 35 An A to U transversion at position 1067 of 23 S rRNA from Escherichia coli impairs EF-Tu and EF-G function; Saarma U et al.; Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (kcat/KM) for both EF-Tu and EF-G binding . In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are significantly increased by the A to U transversion . The U1067 mutation impairs EF-Tu function more than EF-G function . The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu.GDP release from the mutated ribosomes . There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition . With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu . J Mol Biol, 1997 Sep 26, 272(3), 293 - 300 Repression and activation of promoter-bound RNA polymerase activity by Gal repressor; Choy HE et al.; By binding to the DNA site OE at position -60.5 in the gal operon, the GalR protein activates transcription from the P2 promoter located on the opposite face of DNA (position -5) and represses transcription from the P1 promoter located on the same face (position +1) . GalR increases RNA polymerase binding at P2 and inhibits isomerization at P1 by forming a GalR-DNA-RNA polymerase ternary complex in each case . The specific effect of GalR at one promoter is independent of the presence of the other promoter . The enhancement or repression is also not the intrinsic property of a promoter; the regulation can be reversed by switching the angular orientation of the promoters relative to OE . Both enhancement and repression appear to require the same interaction between RNA polymerase alpha-subunit and GalR and/or the same interaction between RNA polymerase alpha-subunit and DNA in the ternary complexes . We have discussed how GalR might exert opposite effects in the steps involved in the formation of the open complex from free RNA polymerase and DNA . Copyright Academic Press Limited. Genomics, 1997 Sep 15, 44(3), 253 - 65 Functional characterization of human nucleosome assembly protein-2 (NAP1L4) suggests a role as a histone chaperone; Rodriguez P et al.; Histones are thought to play a key role in regulating gene expression at the level of DNA packaging . Recent evidence suggests that transcriptional activation requires competition of transcription factors with histones for binding to regulatory regions and that there may be several mechanisms by which this is achieved . We have characterized a human nucleosome assembly protein, NAP-2, previously identified by positional cloning at 11p15.5, a region implicated in several disease processes including Wilms tumor (WT) etiology . The deduced amino acid sequence of NAP-2 indicates that it encodes a protein with a potential nuclear localization motif and two clusters of highly acidic residues . Functional analysis of recombinant NAP-2 protein purified from Escherichia coli demonstrates that this protein can interact with both core and linker histones . We demonstrate that recombinant NAP-2 can transfer histones onto naked DNA templates . Deletion mutagenesis of NAP-2 demonstrates that both NH3- and COOH-terminal domains are required for histone transfer activity . Subcellular localization studies of NAP-2 indicate that it can shuttle between the cytoplasm and the nucleus, suggesting a role as a histone chaperone . Given the potential role of the human NAP-2 gene (HGMW-approved symbol NAP1L4) in WT etiology, we have elucidated the exon/intron structure of this gene and have analyzed the mutational status of NAP-2 in sporadic WTs . Our results, coupled with tumor suppression assays in G401 WT cells, do not support a role for NAP-2 in the etiology of WT . A putative role for NAP-2 in regulating cellular differentiation is discussed. Anal Biochem, 1997 Oct 1, 252(1), 143 - 52 Palindromic oligonucleotide-directed enzymatic determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate in human cells; Zhang H et al.; A new method is presented for the determination of 2'-deoxythymidine 5'-triphosphate and 2'-deoxycytidine 5'-triphosphate concentrations within human cells based on a DNA polymerase reaction directed by a palindromic oligonucleotide precursor . Two 19-mer oligonucleotide precursors are employed that contain a common 8-mer palindromic sequence followed by a sequence-specific insertion site and a 5'-oligodeoxythymidylate tail . To conduct a measurement, two molecules of the 19-mer oligonucleotide precursor are first annealed to form a pair of symmetrical template-primer addition sites at their 3'-termini that are coded for the analyte of interest, present in limiting amounts . The Klenow fragment of Escherichia coli DNA polymerase I then elongates the template-primer by the addition of two molecules of the complementary deoxyribonucleotide analyte . Following the addition of the analyte molecules, the template-primer is extended with a 10-mer oligo(dA) tail in the presence of excess dATP and the Klenow fragment . The result is a 30-mer palindromic oligonucleotide that can be separated from any remaining 19-mer precursor and quantified by paired-ion HPLC using UV detection . Since the molar extinction coefficient of the 30-mer palindromic oligonucleotide is much larger than that of the nucleotide analyte alone, the UV signal is markedly enhanced, thereby increasing sensitivity . Details describing this method and the application of it to measure these analytes in as few as 2.5 x 10(6) human cells are presented. Magn Reson Med, 1997 Oct, 38(4), 565 - 8 Determination and characterization of nitric oxide generation in mice by in vivo L-Band EPR spectroscopy; Fujii H et al.; The authors have shown direct, real-time, in vivo measurement of nitric oxide (NO) in mice by using the water soluble metal chelator complex, N-methyl-D-glucamine dithiocarbamate (MGD), and Fe(II) as monitored by EPR at L-band . The three-line EPR spectrum from the product {(MGD)2-Fe(II)-NO} was observed noninvasively in lipopolysaccharide (LPS)-treated mice . The spectrum was markedly suppressed by the administration, before LPS injection, of phenyl N-tert-butyl nitrone (PBN), an inhibitor of the expression of induced nitric oxide synthase (iNOS) . When 15N-arginine was administered to LPS-treated mice, a diagnostic EPR spectrum was observed, consisting of both three- and two-line EPR signals, due to (MGD)2-Fe(II)-14NO and (MGD)2-Fe(II)-15NO, respectively . The results strongly suggested that the NO detected in these experiments was synthesized by iNOS . In vivo EPR measurements of {(MGD)2-Fe(II)-NO} at several regions in the body (from the head to the tail) indicated that the NO was generated mostly in the upper abdomen near the liver . These observations were confirmed by ex vivo EPR measurements on isolated organs where higher NO levels were detected in vivo in the liver and kidney . The spectroscopic results, combined with the pharmacokinetic data, support the model that NO detected in LPS-treated mice was produced mainly in the liver, and that it did not reflect NO-adduct complex accumulated in the liver via the blood circulation. Pediatr Nephrol, 1997 Oct, 11(5), 556 - 9 Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome . British Association for Paediatric Nephrology; Inward CD et al.; Autopsy material was examined from British children dying early in the course of haemolytic uraemic syndrome (HUS) . These presented after 1983, the period in which verocytotoxin-producing Escherichia coli (VTEC) infection was confirmed as the leading cause of diarrhoea-associated (D+HUS) in the United Kingdom . Of 18 cases referred for this study, 3 were found on review to have no history of a diarrhoeal prodrome (D-HUS) . In the D+ patients, the median duration from onset of diarrhoea to death was 8 days (range 4-42 days) . VTEC infection was confirmed in 6 cases . All had neutrophilia at presentation (median 21, range 15-49.8 x 10(9)/l) . The 15 cases had uniform pathological features, consisting of glomerular thromboses and congested rather than ischaemic glomeruli . Arteriolar thromboses were common at the hilum of glomeruli and were sometimes also seen proximally, including in interlobular arteries . There were cortical infarcts in 5 cases with extensive thrombosis . Cases were demonstrated to have significantly greater numbers of neutrophils expressed per 100 glomeruli than controls, when counted using immunohistological stains to neutrophil elastase and CD15 . This study showed uniformity of the renal changes in D+HUS and gave further evidence of the importance of neutrophils in the pathogenesis of the disease. Plant Physiol, 1997 Sep, 115(1), 181 - 90 The Arabidopsis TCH4 xyloglucan endotransglycosylase . Substrate specificity, pH optimum, and cold tolerance; Purugganan MM et al.; Xyloglucan endotransglycosylases (XETs) modify a major component of the plant cell wall and therefore may play critical roles in generating tissue properties and influencing morphogenesis . An XET-related gene family exists in Arabidopsis thaliana, the members of which show differential regulation of expression . TCH4 expression is rapidly regulated by mechanical stimuli, temperature shifts, light, and hormones . As a first step in determining whether Arabidopsis XET-related proteins have distinct properties, we produced recombinant TCH4 protein in bacteria and determined its enzymatic characteristics . TCH4 specifically transglycosylates only xyloglucan . The enzyme prefers to transfer a portion of a donor polymer onto another xyloglucan polymer (acceptor); TCH4 will also utilize xyloglucan-derived oligosaccharides as acceptors but discriminates between differentially fucosylated oligosaccharides . TCH4 is most active at pH 6.0 to 6.5 and is surprisingly cold-tolerant with an optimum of 12 to 18 degrees C . TCH4 activity is enhanced by urea and bovine serum albumin, but nor cations, reducing agents, or carboxymethylcellulose . These studies indicate that TCH4 is specific for xyloglucan, but that the molecular mass and the fucosyl content of the substrates influence enzymatic reaction rates . TCH4 is unlikely to play a role in acid-induced wall loosening but may function in cold acclimation or cold-tolerant growth. J Physiol, 1997 Sep 1, 503 ( Pt 2), 347 - 52 Tetrahydrobiopterin regulates cyclic GMP-dependent electrogenic Cl- secretion in mouse ileum in vitro; Rolfe VE et al.; 1 . Basal electrogenic Cl- secretion, measured as the short-circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)-deficient hph-1 mice and wild-type controls in vitro, although values were not significantly different . 2 . The basal nitrite release and mucosal cyclic guanosine 3',5'-monophosphate (cyclic GMP) production were similar in control and BH4-deficient ileum . 3 . Mucosally added Escherichia coli heat-stable toxin (STa, 55 ng ml-1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4-deficient ileum . 4 . L-Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4-deficient ileum . 5 . Serosal carbachol (1 mM) stimulated maximum short-circuit currents of similar magnitude in both control and BH4-deficient ileum, whilst nitrite release and cyclic GMP production were minimal . 6 . E . coli STa and L-arginine increased electrogenic Cl- secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP . The inhibition of these processes in the hph-1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut. Environ Mol Mutagen, 1997, 30(2), 139 - 46 Comet assay in human biomonitoring studies: reliability, validation, and applications; Collins A et al.; The comet assay (single-cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies . For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion-specific endonuclease . Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra- and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers . We applied the assay in investigations of human disease and occupational exposure of factory workers. Indian J Biochem Biophys, 1997 Feb-Apr, 34(1-2), 97 - 104 Cloning and expression of SAT-3 involved in SA-Le(x) biosynthesis: inhibition studies with polyclonal antibody against GST-SAT-3 fusion protein; Basu SS et al.; The SAT-3 activity (CMP-NeuAc:Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4Glc-ceramide alpha 2-3 sialytransferase) involved in the biosynthesis of sialy Le(x) has been characterized in human colon carcinoma cells and embryonic chicken brains . Using RT-PCR-based strategy, we have isolated partial cDNA clones of SAT-3 from ECB and Colo-205 mRNAs . Suitable primers from sialylmotif and N-terminal sequence of human placenta SAT-3 (HP-SAT-3) were used . The 800 bp cDNA fragment encoding a region (90%) of alpha 2-3 sialyltransferase (SAT-3) catalytic domain from ECB has been expressed as a glutathione S-transferase (GST) soluble fusion protein (62 kDa) in E . coli and purified over glutathione-agarose affinity matrix . Polyclonal antibody has been produced against affinity-purified catalytic domain of SAT-3 (GST-SAT-3 fusion protein) . A concentration-dependent polydonal antibody binding to native SAT-3 has also been demonstrated by measuring the residual SAT-3 activity in the enzyme fractions from Colo-205 . The marked inhibition (> 80%) of SAT-3 activity and relatively less inhibition (< 20%) of SAT-4 activity (CMP-NeuAc:GgOse4Cer alpha 2-3sialyl transferase) suggests strongly the existence of two different gene products (SAT-3 and SAT-4) in human colon carcinoma Colo-205 cells and in embryonic chicken brains (ECB). Indian J Biochem Biophys, 1997 Feb-Apr, 34(1-2), 61 - 71 Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors; Wu AM et al.; Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface . Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain . A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins . (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins . (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL) . (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL) . (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4) . (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E . coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a. Chin J Biotechnol, 1997, 13(2), 63 - 70 Temperature-induced expression of human-mouse chimeric Fab; Zan H et al.; The temperature-induced expression vector pHZ01 with lambda PRPL promoter for the efficient expression of human-mouse chimeric Fab was constructed . Three kinds of chimeric Fab were expressed in E . coli: anti-prostate specific antigen (PSA) chimeric Fab, anti-lysozyme (HEL) chimeric Fab, and anti-tetanus toxoid (TT) chimeric Fab . All the soluble chimeric Fabs expressed showed specific antigen-binding activities. J Mol Biol, 1997 Oct 3, 272(4), 633 - 41 Helix-helix packing in a membrane-like environment; Mingarro I et al.; The unique ability of the glycophorin A transmembrane helix to dimerize in SDS has previously been exploited in studies of the sequence specificity of helix-helix packing in a micellar environment . Here, we have made different insertion mutants in the critical helix-helix interface segment, and find that efficient dimerization can be mediated by a wider range of sequence motifs than suggested by the earlier studies . We also show that certain mutants that are unable to dimerize can nevertheless form relatively high amounts of tetramers, and that specific tetramerization can be induced by duplication of the critical interface motif on the lipid-exposed side of the transmembrane helix . J Mol Biol, 1997 Oct 3, 272(4), 541 - 52 Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles; Sastri M et al.; The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector . The recombinant protein was found to self assemble into capsids in vivo . The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(+/-2) nm . In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed . The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T=3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis . The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA . The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids . Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable . These results demonstrate that the N-terminal 26 amino acid residues are not essential for T=3 capsid assembly in PhMV . In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles . J Mol Biol, 1997 Oct 3, 272(4), 484 - 92 The active domain of the herpes simplex virus protein ICP47: a potent inhibitor of the transporter associated with antigen processing; Neumann L et al.; The herpes simplex virus type 1 (HSV-1) protein ICP47 binds specifically to the transporter associated with antigen processing (TAP), thereby blocking peptide-binding and translocation by TAP and subsequent loading of peptides onto MHC class I molecules in the endoplasmic reticulum . In consequence, HSV-infected cells are masked for immune recognition by cytotoxic T-lymphocytes . To investigate the molecular details of this, so far, unique transporter-inhibitor interaction, the active domain and critical amino acid residues were identified by using short overlapping fragments and systematic deletions of the viral inhibitor . A fragment of 32 amino acid residues, ICP47(3-34), was found to be the minimal region harboring an activity to inhibit peptide-binding to TAP comparable to the action of the full-length protein and therefore representing the active domain . Further N or C-terminal truncations cause an abrupt loss in activity . Within the identified active domain, various mutants and chimeras of ICP47 derived from HSV-1 and HSV-2 helped to identify amino acid residues critical for TAP inhibition . On the basis of these results, therapeutic drugs could be designed that are applicable in treatment of allograft rejection or in novel vaccination strategies against HSV, restoring the ability of the immune system to recognize HSV-infected cells . Mol Cell Biol, 1997 Nov, 17(11), 6367 - 78 Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon; Eissenberg JC et al.; The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis . Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents . These two mutants also had elevated rates of spontaneous mutations . The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant . In vitro, the mutant PCNAs showed defects in DNA synthesis . Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon . Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively . In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product) . A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase . Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex . Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure . A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks. Arch Biochem Biophys, 1997 Oct 15, 346(2), 303 - 11 Kinase activity of EnvZ, an osmoregulatory signal transducing protein of Escherichia coli; Kenney LJ; EnvZ is an inner membrane protein present in Escherichia coli that is important for osmosensing and required for porin gene regulation . EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression . An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reaction in vitro . Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 microM . The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature . The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive . The in vivo kinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction . Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities . These data provide support for models of EnvZ consisting of separate sensing and kinase domains. Arch Biochem Biophys, 1997 Oct 15, 346(2), 263 - 8 Superoxide-dependent peroxidase activity of H48Q: a superoxide dismutase variant associated with familial amyotrophic lateral sclerosis; Liochev SI et al.; Approximately 20% of cases of familial amyotrophic lateral sclerosis are caused by dominant mutations in the Cu,Zn superoxide dismutase . One such mutant, in which histidine #48 has been replaced by glutamine (H48Q), exhibits a novel activity . It can react sequentially with O2- and H2O2 to generate a potent oxidant at its active site, possibly Cu(II)-OH, which then can oxidize urate to the corresponding radical . This O2- -dependent peroxidase activity exerted on a substrate peculiar to motor neurons may be the toxic gain of function which leads to the deleterious consequences of this mutation . G93A, G93R, and E100G were also examined and found not to exert this O2- -dependent peroxidase activity. J Mol Endocrinol, 1997 Oct, 19(2), 183 - 90 Recognition of follicle stimulating hormone (alpha-subunit) by a recombinant receptor protein domain coded by an alternately spliced mRNA and expressed in Escherichia coli; Khan H et al.; To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli . The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor . The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes . Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol . As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones . Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit . A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action. Bull World Health Organ, 1997, 75(4), 295 - 305 The WHO Global Programme for Vaccines and Immunization Vaccine Trial Registry; Robertson SE et al.; In 1995, the WHO Global Programme for Vaccines and Immunization established a Vaccine Trial Registry . As of September 1996, this registry included 50 WHO-supported vaccine trials, of which 25 (50%) were completed studies . The vaccines most frequently tested have been against measles (9 trials), poliovirus (8 trials), cholera (8 trials), enterotoxigenic Escherichia coli (4 trials), and pneumococcus (4 trials) . Nearly 80% of these trials have been conducted in developing countries, with the largest number being in Africa . Among the 25 completed trials, outcomes measured were immune response (24 trials), adverse reactions (13 trials), morbidity (4 trials), and mortality (1 trial) . WHO's contributions to these studies include direct funding, assistance with study design, site visits, data analysis, vaccine procurement, and vaccine potency testingPIP: In 1995, the World Health Organization (WHO) Global Program for Vaccines and Immunization established a Vaccine Trial Registry . By September 1996, 50 WHO-supported human subject vaccine trials (25 completed and 16 in progress) had been entered in this registry . Altogether, 30 candidate vaccines have been tested, including ones against measles (9 trials), poliovirus (8 trials), cholera (8 trials), enterotoxigenic Escherichia coli (4 trials), and pneumococcus (4 trials) . 79% of these trials have been conducted in developing countries, primarily African . The median number of study subjects in these trials is 1022 . Most frequently assessed have been immune response, adverse reactions, and morbidity . It is projected that up to 10 new vaccines will be ready for inclusion in routine immunization schedules over the next decade . WHO's contributions to this research have included direct funding, assistance with study design, site visits, data analysis, vaccine procurement, and vaccine potency testing . Plant Physiol, 1997 Oct, 115(2), 833 - 9 DNA mismatch repair in plants . An Arabidopsis thaliana gene that predicts a protein belonging to the MSH2 subfamily of eukaryotic MutS homologs; Culligan KM et al.; Sets of degenerate oligomers corresponding to highly conserved domains of MutS-homolog (MSH) mismatch-repair proteins primed polymerase chain reaction amplification of two Arabidopsis thaliana DNA fragments that are homologous to eukaryotic MSH-like genes . Phylogenetic analysis places one complete gene, designated atMSH2, in the evolutionarily distinct MSH2 subfamily. EXS, 1997, 83, 271 - 90 Genetic variability and adaptation to stress; Taddei F et al.; Besides an immediate cellular adaptation to stress, organisms can resist such challenges through changes in their genetic material . These changes can be due to mutation or acquisition of pre-evolved functions via horizontal transfer . In this chapter we will review evidence from bacterial genetics that suggests that the frequency of such events can increase in response to stress by activating mutagenic response (e.g . the SOS response) and by inhibiting antimutagenic activities (e.g . mismatch repair system, MRS) . Natural selection, by favoring adaptations, can also select for the mechanism(s) that has/have generated the adaptive changes by hitchhiking . These mutator mechanisms can sometimes respond very specifically, though blindly, to the challenge of the environment . Such stress-induced increases in mutation rates enhance genetic polymorphism, which is the structural component of the barrier to genetic exchange . Since SOS and MRS are the enzymatic controls of this barrier, the modulation of these systems can lead to a burst of speciation. Parasitol Res, 1997, 83(8), 801 - 5 Cell-surface carbohydrates of Entamoeba invadens; Ribeiro S et al.; Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy . The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba . Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites . The agglutinating activity of E . coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment . These findings were essentially confirmed by scanning electron microscopy . After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased . These results suggest that in E . invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc. J Vet Med Sci, 1997 Sep, 59(9), 815 - 8 Apoptosis of enterocytes induced by inoculation of a strain of attaching and effacing Escherichia coli and verotoxin; Wada Y et al.; When verotoxin (VT)-producing attaching and effacing Escherichia coli (AEEC, serotype O5: H-) were inoculated perorally into 10-day-old rabbits, attaching of the E . coli to enterocytes and effecting of their microvillous portion were observed extensively from the ileum to the colon . Subsequent apoptotic changes of the infected enterocytes were demonstrated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopy . Apoptosis was also induced in cultured Vero cells by inoculation of VT extracted from the AEEC . This study clarified that VT-producing AEEC induce apoptosis of enterocytes, causing mucosal damage. Breast Cancer Res Treat, 1997 Sep, 45(2), 169 - 79 Similarity between natural and recombinant human alpha-fetoprotein as inhibitors of estrogen-dependent breast cancer growth; Bennett JA et al.; Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth . The purpose of this study was to determine whether recombinant human AFP produced in an E . coli expression system retained this function . The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated . It was detected by monoclonal and polyclonal antibodies to the natural protein . Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein . It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein . Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth. Curr Genet, 1997 Oct, 32(4), 300 - 7 Phylogeny and expression of the secA gene from a chromophytic alga--implications for the evolution of plastids and sec-dependent protein translocation; Valentin K; In bacteria many periplasmatic proteins are exported via the sec-dependent pathway . A homologous apparatus was found to be involved in the transport of proteins across the thylakoid membrane in plastids . In the present study additional data on the phylogeny and expression of one of the genes essential in this process, secA, is presented . For the first time, transcriptional activity of secA in the plastid was detected . When secA is used as a phylogenetic marker for plastid evolution it demonstrates a large phylogenetic distance between chlorophytic and non-chlorophytic (i.e . rhodophytic) primary plastids . This distance could be explained by assuming polyphyly for major plastid lineages . Moreover, it was found that two types of secA genes may exist in plastids . Whether or not these are involved in different protein translocation processes is presently unknown . In an attempt to identify further candidates, i.e . non-photosynthesis-related proteins, for sec-dependent protein transport, an SbpA protein was detected in chromophytic plastids by the use of a peptide antibody. Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11863 - 8 RecA tests homology at both pairing and strand exchange; Bazemore LR et al.; RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro . To observe the effects of mismatches on the kinetics of the RecA-catalyzed recombination reaction, we used assays based upon fluorescence energy transfer that can differentiate between the pairing and strand displacement phases . Oligonucleotide sequences that produced 2-14% mismatches in the heteroduplex product of strand exchange were tested, as well as completely homologous and heterologous sequences . The equilibrium constant for pairing decreased as the number of mismatches increased, which appeared to result from both a decrease in the rate of formation and an increase in the rate of dissociation of the intermediates . In addition, the rate of strand displacement decreased with increasing numbers of mismatches, roughly in proportion to the number of mismatches . The equilibrium constant for pairing and the rate constant for strand displacement both decreased 6-fold as the heterology increased to 14% . These results suggest that discrimination of homology from heterology occurs during both pairing and strand exchange. Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11792 - 7 Characterization of recombinant phytochrome from the cyanobacterium Synechocystis; Lamparter T et al.; The complete sequence of the Synechocystis chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in Escherichia coli . In this paper we describe this recombinant Synechocystis phytochrome in more detail . Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related . An approximately 300 residue portion that is important for plant phytochrome function is missing from the Synechocystis sequence, immediately in front of the putative kinase region . The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography . Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation . Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the Synechocystis phytochrome . According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively . Both tend to form dimers in vitro and aggregate under low salt conditions . Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography. Biochemistry, 1997 Oct 28, 36(43), 13441 - 8 Methylation of the Escherichia coli chemotaxis receptors: intra- and interdimer mechanisms; Le Moual H et al.; The mechanism(s) of methylation of the Escherichia coli chemotaxis receptors was analyzed by experiments involving the construction of a series of aspartate receptor variants . Truncation of five or more residues from the C-terminal end of the aspartate receptor, which prevents the methyltransferase from binding to the receptor, resulted in very low rates of methylation, indicating that the methyltransferase is activated by binding to the receptor . Coexpression of a receptor variant that is unmethylatable but able to C-terminally bind the methyltransferase resulted in much higher methylation rates for all of the truncated receptors . By preventing the possibility of subunit exchange between receptor variants, we showed that the truncated receptors were methylated via an interdimer mechanism . The interdimer methylation rates of the truncated receptors were found to be 3-fold lower than the methylation rate of the unaltered receptor, suggesting that intradimer methylation as well as interdimer methylation accounts for the methylation of the unaltered receptor . In addition, the presence of the cytoplasmic signaling proteins, which have been shown to cause receptor clustering, did not influence the rates of methylation. Eur J Biochem, 1997 Sep 15, 248(3), 897 - 902 Pro108 is important for folding and stabilization of adrenal ferredoxin, but does not influence the functional properties of the protein; Uhlmann H et al.; The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods . The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain . Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions . Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state . The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108) . The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction. Eur J Biochem, 1997 Sep 15, 248(3), 848 - 55 The 70-kDa heat-shock protein/DnaK chaperone system is required for the productive folding of ribulose-biphosphate carboxylase subunits in Escherichia coli; Checa SK et al.; We have studied the in vivo requirements of the DnaK chaperone system for the folding of recombinant ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli . Expression of functional dimeric or hexadecameric ribulose-bisphosphate carboxylase from different bacterial sources (including purple bacteria and cyanobacteria) was severely impaired in E . coli dnaK, dnaJ, or grpE mutants . These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild-type E . coli cells cultured in the temperature range 20-42 degrees C, but aggregated extensively in dnaK null mutants . Co-expression of dnaK, but not groESL, markedly reduced the aggregation of ribulose-bisphosphate carboxylase subunits in dnaK null mutants and restored the enzyme activity to levels found in isogenic wild-type strains . Ribulose-bisphosphate carboxylase expression in wild-type E . coli cells growing at 30 degrees C promoted an enhanced synthesis of stress proteins, apparently by sequestering DnaK from its negative regulatory role in this response . The overall results indicate that the DnaK chaperone system assists in vivo the folding pathway of ribulose-bisphosphate carboxylase large subunits, most probably at its very early stages. Eur J Biochem, 1997 Sep 15, 248(3), 834 - 9 Characterization of gelsolin truncates that inhibit actin depolymerization by severing activity of gelsolin and cofilin; Fujita H et al.; Gelsolin is a calcium-activated actin-binding protein with six subdomains . The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever . In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin . A Ca2+-sensitive truncate containing G2-6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2-3 truncate was less effective . Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics . Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-actin more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of actin filament binding domains. Eur J Biochem, 1997 Sep 15, 248(3), 820 - 6 The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability; Etchebehere LC et al.; The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core . The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved . We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI . Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI . In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins . Inhibition by the R subunit or by PKI were however unaffected . Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family . We propose that the presence of this motif may serve to identify isoforms of protein kinases. Eur J Biochem, 1997 Sep 15, 248(3), 814 - 9 Purification of the cardiac sarcoplasmic reticulum membrane protein phospholamban from recombinant Escherichia coli; Kromer WJ et al.; Phospholamban (PLN) was expressed in Escherichia coli as a protein fusion with glutathione S-transferase (GST) . GST-PLN was mostly present in the insoluble protein fraction and accounted for approximately 50% of total insoluble protein . Attempts to suppress inclusion body formation or to use GST as an affinity-purification tag failed . A successful purification method is based on preparative SDS/PAGE and electrodialysis . From 1 g cells we typically purified 13.5 mg fusion protein with a PLN content of 2.8 mg . We genetically inserted an enterokinase (EK) protease site just in front of the PLN sequence and demonstrated the proteolytical liberation of PLN from the carrier protein . The approach described represents a substantial advancement in PLN expression and purification. Eur J Biochem, 1997 Sep 15, 248(3), 731 - 40 Reduced turnover of the D1 polypeptide and photoactivation of electron transfer in novel herbicide resistant mutants of Synechocystis sp . PCC 6803; Dalla Chiesa M et al.; Two missense mutants, A263P and S264P, and a deletion mutant des-Ala263, Ser264, have been constructed in the D1 protein of the cyanobacterium Synechocystis sp PCC 6803 . All were expected to induce a significant conformational change in the QB-binding region of photosystem II (PSII) . Although the des-Ala263, Ser264-D1 mutant accumulated some D1 protein in the thylakoid membrane it was unable to grow photoautotrophically or evolve oxygen . Thermoluminescence and chlorophyll fluorescence studies confirmed that this deletion mutant did not show any functional PSII activity . In contrast, {S264P}D1 was able to grow photoautotrophically and give light-saturated rates of oxygen evolution at 60% of the rate of the wild-type control strain, TC31 . The A263P missense mutant was also able to evolve oxygen at 50% of TC31 rates although it did not readily grow photoautotrophically . Thermoluminescence, flash oxygen yield and chlorophyll fluorescence measurements indicated that in both missense mutants electron transfer from QA to QB was significantly impaired in dark adapted cells . However, QA to QB electron transfer could be photoactivated in the mutants by background illumination . Both the A263P and S264P mutants also showed an increase in resistance to the s-triazine family of herbicides although this feature did not hold for the phenolic herbicide, ioxynil . Of particular interest was that the two missense mutants, especially S264P, possessed a slower rate of turnover of the D1 protein compared with TC31 and in vivo contained detectable levels of a 41-kDa adduct consisting of D1 and the alpha subunit of cytochrome b559 . When protein synthesis was blocked by the addition of lincomycin, D1 degradation was again slower in S264P than TC31 . The results are discussed in terms of structural changes in the QB-binding region which affect herbicide and plastoquinone binding and perturb the normal regulatory factors that control the degradation of the D1 protein and its synchronisation with the synthesis of a replacement D1 protein. Eur J Biochem, 1997 Sep 15, 248(3), 692 - 9 Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria; Corti A et al.; Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells . In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation . The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis . However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size . SDS/PAGE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers . VS-2 was almost entirely dimeric at > 4 microM, but rapidly converted to monomer after dilution to 70 nM . The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action . Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides . The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments. Eur J Biochem, 1997 Sep 15, 248(3), 684 - 91 Pathway of detergent-mediated and peptide ligand-mediated refolding of heterodimeric class II major histocompatibility complex (MHC) molecules; Stockel J et al.; We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101 . Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent . Refolding was mediated by mild detergents and by peptide ligands . Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies . We found that formation of secondary structure was detectable after replacement of SDS by mild detergents . At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands . Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents . We conclude that at that stage a tertiary structure close to the native structure is formed at least locally . The nature and concentration of detergent critically determined the refolding efficiency . We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length . For each of the detergents we observed a narrow concentration range for mediating refolding . Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction . We discuss structure formation and interactions of detergents with stable folding intermediates . Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins. Protein Eng, 1997 Jul, 10(7), 757 - 61 Relations of the numbers of protein sequences, families and folds; Zhang CT; The relations among the numbers of protein sequences, families and folds have been studied theoretically . It is found that the number of families is related to the natural logarithm of the number of sequences . The logarithmic relation should not be changed regardless of what value of the homology threshold is applied in the protein sequence comparison routines . To study the relation between the numbers of families and folds, the degenerate degree of a fold has been introduced . The degenerate degree of a fold is the number of protein families which adopt the same fold . The distribution of the degenerate degrees of folds has been found to be very likely exponential . Based on the distribution, the average degenerate degree d is calculated . The number of folds is simply equal to that of families divided by the average degenerate degree of folds . It is shown that d is an increasing function of time . The current value of d is about 2 . It will continue to increase and reach the value of at least 3.3 in some years . By using the above result, the numbers of protein folds for four species have been estimated . In particular, the number of folds for human proteins is estimated to be < or =5200. Protein Eng, 1997 Jul, 10(7), 751 - 5 N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina; Anderluh G et al.; The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis . A part of this segment was classified as being amphiphilic and membrane seeking . Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector . Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity . The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes . Their specific hemolytic activity was decreased, however, with increasing truncation . The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes . It is very likely that the N-terminus play a role in the formation of functional pores. J Med Chem, 1997 Oct 10, 40(21), 3336 - 45 Effect of a chemical modification on the hydrated adenosine intermediate produced by adenosine deaminase and a model reaction for a potential mechanism of action of 5-aminoimidazole ribonucleotide carboxylase; Groziak MP et al.; Using the hydrated adenosine intermediate (6R)-6-amino-1, 6-dihydro-6-hydroxy-9-(beta-D-ribofuranosyl)purine (2) produced by adenosine deaminase (ADA, EC 3.5.4.4) as a starting point, the active site probe and inhibitor platform 5-(formylamino)imidazole riboside (FAIRs, 4) was designed by removal of the-C6(OH)(NH2)-molecular fragment of 2 generated by the early events of the enzyme-catalyzed hydrolysis . FAIRs was synthesized directly from the sodium salt of 5-amino-1-(beta-D-ribofuranosyl)imidazole-4-carboxylic acid (CAIR) along a reaction sequence involving a tandem N-formylation/decarboxylation that may have a mechanistic connection to the Escherichia coli purE-catalyzed constitutional isomerization of N5-CAIR to CAIR . The physical and spectral properties of FAIRs were elucidated, its X-ray crystal and NMR solution structures were determined, and its interaction with ADA was investigated . Crystalline FAIRs exists solely as the Z-formamide rotamer and exhibits many of the same intramolecular hydrogen bonding events known to contribute to the association of Ado to ADA . In water and various organic solvents, however, FAIRs exists as NMR-distinct, slowly interconverting Z and E rotamers . This truncated enzymatic tetrahedral intermediate analog was determined to be a competitive inhibitor of ADA with an apparent Ki binding constant of 40 microM, a value quite close to that (33 microM) of the natural substrate's K(m) . The actual species selected for binding by ADA, though, is likely the minor hydroxyimino prototropic form of Z-FAIRs possessing a far lower true Ki value . As the structural features of FAIRs appear well-suited to support its use as a template for constructing active site probes of both ADA and AIR carboxylases, a variety of carbohydrate-protected versions of FAIRs suitable for facile aglycon elaborations were synthesized . The N3-alkylation, N3-borane complexation, and C4-iodination of some of these were investigated in order to assess physicochemical properties that may assist in the elucidation of mechanisms for the AIR carboxylases . The survey of these properties taken together with a reasonable mechanism for the model CAIRs-->FAIRs synthetic transformation is interpreted to support a mechanism for the purE-catalyzed N5-CAIR-->CAIR biosynthetic one that involves a carboxylative sp3-rehybridization of the imidazole C4 atom rather than one possessing a dipole-stabilized C4 sp2 carbanionic intermediate. Eur J Immunol, 1997 Sep, 27(9), 2253 - 60 A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production; Hartmann S et al.; Filarial nematodes are a cause of chronic debilitating diseases in the tropics . A hallmark of filariasis is the marked down-regulation and polarization of host immune responses, yet molecular constituents of parasites causing this state have remained undefined . We describe a 17-kDa antigen (Av17) of the rodent filarial parasite Acanthocheilonema viteae, which shows amino acid homologies to cystatin C, a major cysteine protease inhibitor belonging to family 2 of the cystatin superfamily . Av17 is released by filariae in vitro . Exported molecules of A . viteae worms are shown to markedly suppress mitogen-induced T cell proliferation of mice and jirds . Av17 accounts for 45.5% of this suppressive activity in the murine system . Recombinant Av17 (rAv17), expressed in Escherichia coli, exhibits biological activity as a cysteine protease inhibitor and was used to examine the immunomodulatory effects, rAv17 induces down-regulation of murine T cell responses to mitogens, to T cell receptor cross-linking by anti-CD3 antibodies and to specific antigens, and at the same time up-regulation of interleukin-10 . Hence, this filarial cystatin is a likely effector molecule of immunomodulation and a potential target for antifilarial intervention. Eur J Immunol, 1997 Sep, 27(9), 2160 - 4 Elevated mutant frequencies in gene lacI in splenic lipopolysaccharide blasts after exposure to activated phagocytes in vitro; Felix K et al.; The interaction of B lymphocytes with phagocytes is critical for shaping the humoral immune response, as well as various aspects of normal and malignant B cell development, and has therefore been studied by immunologists in great detail . However, one potential outcome of this confrontation is often neglected, namely the mutagenicity of phagocytes to B lymphocytes . We are interested in phagocyte-induced B cell mutagenesis and have conducted a feasibility study on the utility of a transgenic reporter assay to evaluate mutant frequencies in B cells that have encountered phagocytes . An in vitro co-incubation system was designed in which splenic lipopolysaccharide (LPS) blasts carrying a phage lambda-derived lacI transgene were exposed to pristane-elicited peritoneal exudate cells (PEC) . Mutant frequencies in LPS blasts were significantly increased (up to 6-fold) when the cells were co-incubated with PEC that had been stimulated by phorbol myristate acetate to undergo an oxidative burst . The lacI-based transgenic mutation assay proved also useful for assessing mutagenicity in vivo, as demonstrated by the detection of elevated mutant frequencies in the spleen (3-fold) and the inflammatory granuloma (4.7-fold) obtained from pristane-treated mice . We propose to utilize the lacI-based transgenic mutagenesis assay as a tool to evaluate mutational levels during normal and aberrant B cell differentiation. Biochemistry, 1997 Oct 28, 36(43), 13389 - 95 Raman spectroscopic and light-induced kinetic characterization of a recombinant phytochrome of the cyanobacterium Synechocystis; Remberg A et al.; A phytochrome-encoding cDNA from the cyanobacterium Synechocystis has been heterologously expressed in Escherichia coli and reconstituted into functional chromoproteins by incubation with either phycocyanobilin (PCB) or phytochromobilin (PPhiB) . These materials were studied by Raman spectroscopy and nanosecond flash photolysis . The Raman spectra suggest far-reaching similarities in chromophore configuration and conformation between the Pfr forms of Synechocystis phytochrome and the plant phytochromes (e.g . phyA from oat), but some differences, such as torsions around methine bridges and in hydrogen bonding interactions, in the Pr state . Synechocystis phytochrome (PCB) undergoes a multistep photoconversion reminiscent of the phyA Pr --> Pfr transformation but with different kinetics . The first process resolved is the decay of an intermediate with red-shifted absorption (relative to parent state) and a 25-micros lifetime . The next observable intermediate grows in with 300 (+/-25) micros and decays with 6-8 ms . The final state (Pfr) is formed biexponentially (450 ms, 1 s) . When reconstituted with PPhiB, the first decay of this Synechocystis phytochrome is biexponential (5 and 25 micros) . The growth of the second intermediate is slower (750 micros) than that in the PCB adduct whereas the decays of both species are similar . The formation of the Pfr form required fitting with three components (350 ms, 2.5 s, and 11 s) . H/D Exchange in Synechocystis phytochrome (PCB) delays, by an isotope effect of 2.7, both growth (300 micros) and decay rates (6-8 ms) of the second intermediate . This effect is larger than values determined for phyA (ca . 1.2) and is characteristic of a rate-limiting proton transfer . The formation of the Pfr state of the PCB adduct of Synechocystis phytochrome shows a deuterium effect similar as phyA (ca . 1.2) . Activation energies of the second intermediate in the range 0-18 degrees C are 44 (in H2O/buffer) and 48 kJ mol-1 (D2O), with essentially identical pre-exponential factors. Biochemistry, 1997 Oct 28, 36(43), 13365 - 73 The roles of conserved carboxylate residues in IMP dehydrogenase and identification of a transition state analog; Kerr KM et al.; IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+; the enzyme is activated by K+ . This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis . In order to identify functionally important residues in IMPDH, including those involved in substrate and K+ binding, we have mutated 11 conserved Asp and Glu residues to Ala in Escherichia coli IMPDH . The values of kcat, Km, and Ki for GMP, XMP, mizoribine 5'-monophosphate (MMP), and beta-methylene-tiazofurin adenine dinucleotide (TAD) were determined . Five of these mutations caused a significant change (>/=10-fold) in one of these parameters . The Asp248 --> Ala mutation caused 100-fold decrease in the value of kcat and a 25-fold increase in the value of Kii for TAD; these observations suggest that Asp248 is in the NAD+ binding site . The Asp338 --> Ala mutation caused a 600-fold decrease in the value of kcat, but only a 5-10-fold increase in the values of Km for IMP and Kis for IMP analogs, suggesting that Asp338 may be involved in acid-base catalysis as well as IMP binding . The remaining three residues, Asp13, Asp50, and Glu469, appear to be involved in K+ activation; these residues may be ligands at one or more K+ binding sites . Interestingly, changes in the values of Ki for MMP correlate with changes in kcat/KmKm of IMPDH, while no such correlation is observed for GMP, XMP, and TAD . This observation indicates that MMP is a transition state analog for the IMPDH reaction. Biochemistry, 1997 Oct 28, 36(43), 13357 - 64 Requirement for the two AhpF cystine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase; Li Calzi M et al.; AhpF, the alkyl hydroperoxide reductase component which transfers electrons from pyridine nucleotides to the peroxidase protein, AhpC, possesses two redox-active disulfide centers in addition to one FAD per subunit; the primary goal of these studies has been to test for the requirement of one or both of these disulfide centers in catalysis . Two half-cystine residues of one center (Cys345Cys348) align with those of the homologous Escherichia coli thioredoxin reductase (TrR) sequence (Cys135Cys138), while the other two (Cys129Cys132) reside in the additional N-terminal region of AhpF which has no counterpart in TrR . We have employed site-directed mutagenesis techniques to generate four mutants of AhpF, including one which removes the N-terminal disulfide (Ser129Ser132) and three which perturb the TrR-like disulfide center (Ser345Ser348, Ser345Cys348, and Cys345Ser348) . Fluorescence, absorbance, and circular dichroism spectra show relatively small perturbations for mutations at the disulfide center proximal to the flavin (Cys345Cys348) and no changes for the Ser129Ser132 mutant; identical circular dichroism spectra in the ultraviolet region indicate unchanged secondary structures in all mutants studied . Oxidase and transhydrogenase activities are preserved in all mutants, indicating no role for cystine redox centers in these activities . Both DTNB and AhpC reduction by AhpF are dramatically affected by each of these mutations, dropping to less than 5% for DTNB reductase activity and to less than 2% for peroxidase activity in the presence of AhpC . Reductive titrations confirm the absence of one redox center in each mutant; even in the absence of Cys345Cys348, the N-terminal redox center can be reduced, although only slowly . These results emphasize the necessity for both redox-active disulfide centers in AhpF for catalysis of disulfide reductase activity and support a direct role for Cys129Cys132 in mediating electron transfer between Cys345Cys348 and the AhpC active-site disulfide. Biochemistry, 1997 Oct 28, 36(43), 13277 - 84 Yeast DNA helicase A: cloning, expression, purification, and enzymatic characterization; Biswas EE et al.; We have cloned and expressed the yeast DNA helicase A in Escherichia coli at a high level (approximately 30 mg/L of culture) in soluble form . We describe here a simple two-step purification protocol that produces reasonable quantities of homogeneous enzyme . In denaturing gel electrophoresis the enzyme behaved as a approximately 90 kDa protein . The native structure, determined by gel-filtration studies, appeared to be hexameric and its quaternary structure was salt (NaCl) dependent . In low-salt buffers (containing 50 mM NaCl), the enzyme eluted in a single activity peak at an elution volume that appeared to correlate with a possible hexameric structure . In higher salt buffer (containing greater than 150 mM NaCl), the enzyme eluted as smaller assemblies (monomer/dimer) . The recombinant helicase A was able to hydrolyze ATP or dATP with equal efficiency . The ATPase activity of the enzyme was absolutely DNA-dependent . The nucleotidase activities were comparable to those of the native enzyme . Kinetic analysis of the ATPase activity demonstrated that the Km of the enzyme was approximately 90 microM and the rate of ATP hydrolysis was approximately 20 ATP s-1 molecule-1 . DNA sequences containing pyrimidine stretches were more effective activators than those containing purine stretches . However, poly(dC) appeared to be the most effective activator of the ATPase activity . The ATPase activity was inhibited by salt (NaCl) above 50 mM with a half-maximal inhibition at approximately 110 mM . It is likely that the active state of helicase A is hexameric . The helicase activity of the recombinant enzyme was stimulated significantly by the yeast replication protein A (RPA) and to a lower extent by the single-stranded DNA binding protein of E . coli (SSB) . The DNA helicase migrated on a DNA template in a 5' --> 3' direction . Helicase A appeared to share a number of enzymatic characteristics including directionality, stimulation by RPA/SSB, and quaternary structure (monomer-hexamer) dynamics that are common to known replicative helicases such as DnaB helicase and the SV40 T-antigen. Biochemistry, 1997 Oct 28, 36(43), 13270 - 6 Purification and characterization of DNA polymerase alpha-associated replication protein A-dependent yeast DNA helicase A; Biswas SB et al.; A novel, eukaryotic, hexameric DNA helicase that was earlier identified as a component of the multiprotein polymerase alpha complex {Biswas et al . (1993) Biochemistry 32, 13393-13398} has been purified to homogeneity and characterized . Thus far, our studies demonstrated that helicase A shares certain unique features of two other hexameric DNA helicases: the DnaB helicase of Escherichia coli and the T-antigen helicase of the SV40 virus . The helicase activity was stimulated by yeast replication protein A (RPA) and to a lower extent by E . coli single-stranded DNA binding protein (SSB) . The helicase had an apparent molecular mass of 90 kDa, as determined by its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A tryptic peptide fragment of the polypeptide was sequenced followed by a BLAST search of GenBank with the tryptic peptide sequence . The search identified a 1.8 kb open reading frame previously designated as ykl017c on chromosome XI, that codes for a 78.3 kDa (683 amino acid) polypeptide . The important features of the polypeptide sequence of helicase A included a type I ATP/GTP binding motif, and a K E E R R L N V A M T R P R R sequence at the C-terminus that may be indicative of a nuclear localization signal which is required of a nuclear DNA helicase . The polypeptide sequence of helicase A appears to have homology to the DnaB helicase of E . coli (approximately 25%) . The facts that these two helicases are vastly separated by evolution and retained similar structural and functional features, as demonstrated here, point to a possible significance of this limited homology . Although the amount of purified helicase A was limited, we have carried out necessary enzymatic characterization so that these data could be correlated with that of immunoaffinity-purified helicase A and recombinant helicase A expressed in heterologous systems. Biochemistry, 1997 Oct 28, 36(43), 13195 - 200 Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli; Puustinen A et al.; Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site . At low temperatures, the C triple bond O remains bound to CuB for extended times . Here, we show that the binding of C triple bond O to CuB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys . Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm-1 for Glu286 and 1759 cm-1 for Glu286Asp . These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286 . In Glu286Cys, this IR feature is lost altogether . We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C triple bond O to CuB, which are relay ed to the 286 locus . Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations . These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O . C triple bond O binding to CuB also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm-1 . The results suggest an easily polarizable, through-bond connectivity between one of the histidine CuB ligands and the carboxylic group of Glu286 . A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases. J Biol Chem, 1997 Oct 24, 272(43), 27378 - 81 Cloning and expression of a gene encoding a protein obtained from earthworm secretion that is a chemoattractant for garter snakes; Liu W et al.; The protein ES20, derived from earthworm shock secretion, is a vomeronasally mediated chemoattractant for garter snakes (Jiang, X . C., Inouchi, J., Wang, D., and Halpern, M . (1990) J . Biol . Chem . 265, 8736-8744) . Based on its 15-residue N-terminal amino acid sequence, degenerative oligodeoxynucleotide probes were synthesized and used to screen a cDNA library that was constructed in sense orientation using a Uni-ZAPTM XR vector and XL1-Blue MRF' host . A gene was cloned from a polymerase chain reaction as well as from the cDNA library . A combination of the forward degenerative primer and T7 primer was used to obtain gene-specific DNA fragments, from which probes were synthesized and successfully used in screening the cDNA library . The ES20 gene is about 700 base pairs long and encodes 208 amino residues . The ES20 gene was excised from a recombinant plasmid pSK-ES20, ligated to pQE30 expression vector, and transformed into Escherichia coli strain JM109 . The selected recombinant plasmids were transformed into expression host cell, E . coli M15{pREP4} . Three transformants were selected, induced with isopropyl-1-thio-beta-D-galactopyranoside for fusion gene expression and an expressed 20-kDa fusion protein purified under denaturing conditions . This protein was refolded and gave a positive reaction against ES20-specific polyclonal antibodies . The fusion protein that had not been denatured remained as an aggregate and was an active chemoattractant for garter snakes. J Biol Chem, 1997 Oct 24, 272(43), 27210 - 7 The role of base flipping in damage recognition and catalysis by T4 endonuclease V; McCullough AK et al.; The process of moving a DNA base extrahelical (base flipping) has been shown in the co-crystal structure of a UV-induced pyrimidine dimer-specific glycosylase, T4 endonuclease V, with its substrate DNA . Compared with other enzymes known to use base flipping, endonuclease V is unique in that it moves the base opposite the target site extrahelical, rather than moving the target base itself . Utilizing substrate analogs and catalytically inactive mutants of T4 endonuclease V, this study investigates the discrete steps involved in damage recognition by this DNA repair enzyme . Specifically, fluorescence spectroscopy analysis shows that fluorescence changes attributable to base flipping are specific for only the base directly opposite either abasic site analogs or the 5'-thymine of a pyrimidine dimer, and no changes are detected if the 2-aminopurine is moved opposite the 3'-thymine of the pyrimidine dimer . Interestingly, base flipping is not detectable with every specific binding event suggesting that damage recognition can be achieved without base flipping . Thus, base flipping does not add to the stability of the specific enzyme-DNA complex but rather induces a conformational change to facilitate catalysis at the appropriate target site . When used in conjunction with structural information, these types of analyses can yield detailed mechanistic models and critical amino acid residues for extrahelical base movement as a mode of damage recognition. J Biol Chem, 1997 Oct 24, 272(43), 26999 - 7004 Conditions for nucleotide-dependent GroES-GroEL interactions . GroEL14(groES7)2 is favored by an asymmetric distribution of nucleotides; Gorovits BM et al.; A still unresolved question regarding the mechanism of chaperonin-assisted protein folding involves the stoichiometry of the GroEL-GroES complex . This is important, because the activities of the Escherichia coli chaperonin GroEL are modulated by the cochaperonin GroES . In this report, the binding of GroES to highly purified GroEL in the presence of ATP, ADP, and the nonhydrolyzable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (AMP-PNP), was investigated by using the fluorescence anisotropy of succinimidyl-1-pyrenebutyrate-labeled GroES . In the presence of Mg2+-ATP and high {KCl} (10 mM), two GroES7 rings bind per one GroEL14 . In contrast, in the presence of ADP or AMP-PNP only one molecule of oligomeric GroES can be tightly bound by GroEL . With AMP-PNP, binding of a small amount (<20%) of a second GroES can be detected . In the presence of ADP alone, a second GroES ring can bind to GroEL weakly and with negative cooperativity . Strikingly, addition of AMP-PNP to the solution containing preformed GroEL14(GroES7) complexes formed in the presence of ADP results in an increase in the fluorescence anisotropy . Analysis of this effect indicates that 2 mol of GroES oligomer can be bound in the presence of mixed nucleotides . A similar conclusion follows from studies in which ADP is added to an GroEL14 (GroES7) complex formed in the presence of AMP-PNP . This is the first demonstration of an asymmetric distribution of nucleotides bound on the 1:2 GroEL14 (GroES7)2 complex . The relation of the observed phenomena to the proposed mechanism of the GroEL function is discussed. J Biol Chem, 1997 Oct 24, 272(43), 26985 - 90 Identification of the cysteine residues involved in redox modification of plant plastidic glucose-6-phosphate dehydrogenase; Wenderoth I et al.; The cDNA sequences encoding cytosolic and light-modulated plastidic glucose-6-phosphate dehydrogenase (G6PDH) from potato were modified by polymerase chain reaction and subsequently overexpressed in Escherichia coli . Characterization of the recombinant enzymes showed that they closely resembled their native counterparts . Treatment with reduced dithiothreitol or glutathione led to inactivation of plastidic G6PDH, whereas the activity of the cytosolic isoenzyme was not influenced by reduction . As for the native enzyme, inactivation of recombinant plastidic G6PDH was accelerated by thioredoxin m and could be fully reversed by subsequent addition of oxidant . To identify the residues which are involved in redox regulation of plastidic G6PDH, each of the six cysteines in the mature protein sequence was exchanged separately for serine by site-directed mutagenesis . Two mutant proteins exhibited characteristics of the reduced wild-type enzyme . Exchange of either Cys149 or Cys157 to serine abolished the regulatory properties, suggesting that these cysteine residues are the sites responsible for redox-mediated inactivation of plastidic G6PDH. J Biol Chem, 1997 Oct 24, 272(43), 26947 - 52 An antagonist for the leukemia inhibitory factor receptor inhibits leukemia inhibitory factor, cardiotrophin-1, ciliary neurotrophic factor, and oncostatin M; Vernallis AB et al.; The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM) . Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signal-transducing subunit shared with interleukin-6 and interleukin-11 . The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF . In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands . On cells that express LIF-R and gp130, all LIF-R ligands were antagonized . On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R . Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist . The antagonist is therefore likely to work by preventing receptor oligomerization. J Biol Chem, 1997 Oct 24, 272(43), 26934 - 9 Mass spectrometric determination of the cleavage sites in Escherichia coli dihydroorotase induced by a cysteine-specific reagent; Daniel R et al.; Escherichia coli dihydroorotase contains six cysteines/subunit, which are potential ligands of structural and catalytic zinc metals at protein sites of the enzyme . Specific thiol reagents modify, in nondenaturing conditions only, two of these cysteines; these two residues are thought to be ligands of structural zinc . We report here on the localization of these two cysteines on the polypeptide chain through their cyanylation by 2-nitro-5-thiocyanobenzoic acid (NTCB) and the analysis by mass spectrometry of the protein adducts . This is the first study of E . coli dihydroorotase by mass spectrometry, allowing the accurate determination of the subunit molecular weight (38,695) . Treatment of dihydroorotase by NTCB induced a cleavage N-terminal to the cyanylated cysteines . The resulting fragments visualized on electrophoresis gel have been N-terminal sequenced, and their masses were determined by electrospray-ionizing mass spectrometry . This allowed the identification of cysteines 221 and 265 as the two residues cyanylated by the reagent NTCB . Results from gel filtration of dihydroorotase cyanylated on the two cysteines indicate that these residues are involved in subunit interactions leading to the active dimer . Consistent with literature data, we assume that cysteine 221 and cysteine 265, along with the neighboring cysteines 263 and 268 arranged in cluster, are potential ligands of structural zinc of E . coli dihydroorotase. J Biol Chem, 1997 Oct 24, 272(43), 26887 - 92 The C terminus of cardiac troponin I is essential for full inhibitory activity and Ca2+ sensitivity of rat myofibrils; Rarick HM et al.; Although the C terminus of troponin I is known to be important in myofilament Ca2+ regulation in skeletal muscle, the regulatory function of this region of cardiac troponin I (cTnI) has not been defined . To address this question, the following recombinant proteins were expressed in Escherichia coli and purified: mouse wild-type cTnI (WT cTnI; 211 residues), cTnI-(1-199) (missing 12 residues), cTnI-(1-188) (missing 23 residues), and cTnI-(1-151) (missing 60 residues) . The inhibitory activity of cTnI and the mutants was tested in myofibrils, from which cTnI.cTnC was extracted by exchanging endogenous cardiac troponin with exogenous cTnT causing the Ca2+ sensitivity of the myofibrils to be lost . Addition of increasing amounts of exogenous WT cTnI or cTnI-(1-199) to cTnT-treated myofibrils at pCa 8 caused a concentration-dependent inhibition of the maximum ATPase activity . However, cTnI-(1-188) and cTnI-(1-151) inhibited this activity to about 75% and 50% of that of the WT cTnI, respectively . We also formed a complex of either WT cTnI or each of the mutants with cTnC, reconstituted the complex into the cTnT-treated myofibrils, and measured the Mg2+-ATPase activity as a function of pCa . We found that the cTnI-(1-188).cTnC complex only partially restored Ca2+ sensitivity, whereas the cTnI-(1-151).cTnC complex did not restore any Ca2+ sensitivity . Each cTnI C-terminal deletion mutant was able to bind to cTnC, as shown by urea-polyacrylamide gel-shift analysis and size exclusion chromatography . Each mutant also co-sedimented with actin . Our results indicate that residues 152-199 (C-terminal to the inhibitory region) of cTnI are essential for full inhibitory activity and Ca2+ sensitivity of myofibrillar ATPase activity in the heart. J Biol Chem, 1997 Oct 24, 272(43), 26818 - 21 Designed disulfide between N-terminal domains of lactose repressor disrupts allosteric linkage; Falcon CM et al.; Substitution of Cys for Val at position 52 of the lac repressor was designed to permit disulfide bond formation between the two N-terminal DNA binding domains that comprise an operator DNA binding site . This position marks the closest approach of these domains based on the x-ray crystallographic structures of the homologous purine holorepressor-operator complex and lac repressor-operator complex (Schumacher, M . A., Choi, K . Y., Zalkin, H., and Brennan, R . G . (1994) Science 266, 763-770; Lewis, M., Chang, G., Horton, N.C., Kercher, M . A., Pace, H . C., Schumacher, M . A., Brennan, R . G., and Lu, P . (1996) Science 271, 1247-1254) . The V52C mutation was generated by site-specific methods, and the mutant protein was purified and characterized . In the reduced form, V52C bound operator DNA with slightly increased affinity . Exposure to oxidizing conditions resulted in disulfide bond formation, and the oxidized protein bound operator DNA with approximately 6-fold higher affinity than wild-type protein . Inducer binding for both oxidized and reduced forms of V52C was comparable to wild-type lac repressor . In the presence of inducer, the reduced protein exhibited wild-type, diminished DNA binding . In contrast, DNA binding for the oxidized form was unaffected by inducer, even at 1 mM . Thus, the formation of the designed disulfide between Cys52 side chains within each dimer renders the protein-operator complex unresponsive to sugar binding, presumably by disrupting the allosteric linkage between operator and inducer binding. J Biol Chem, 1997 Oct 24, 272(43), 26811 - 4 Molybdenum cofactor biosynthesis . The plant protein Cnx1 binds molybdopterin with high affinity; Schwarz G et al.; The molybdenum cofactor is an essential part of all eukaryotic molybdoenzymes . It is a molybdopterin (MPT) revealing the same core structure in all organisms . The plant protein Cnx1 from Arabidopsis thaliana is involved in the multi-step biosynthesis of molybdenum cofactor . Previous studies (Stallmeyer, B., Nerlich, A., Schiemann, J., Brinkmann, H., and Mendel, R . R . (1995) Plant J . 8, 751-762) suggested a function of Cnx1 in a late step of cofactor biosynthesis distal to the formation of MPT, i.e . conversion of MPT into molybdenum cofactor . Here we present the first biochemical evidences confirming this assumption . The protein Cnx1 consists of two domains (E and G) homologous to two distinct Escherichia coli proteins involved in cofactor synthesis . Binding studies with recombinantly expressed and purified Cnx1 and with its single domains revealed a high affinity of the G domain to MPT (kD = 0.1 microM) with equimolar binding . In contrast, the E domain of Cnx1 binds MPT with lower affinity (kD = 1.6 microM) and in a cooperative manner (nH = 1 . 5) . The entire Cnx1 showed a tight and cooperative MPT binding . Based on these data providing a common link between both domains that matches the previous characterization of plant and bacterial Cnx1 homologous mutants, we present a model for the function of Cnx1. Zh Mikrobiol Epidemiol Immunobiol, 1997 Jul-Aug, (4), 67 - 71 {An analysis of the interrelation of antilysozyme activity and reproductive function in escherichiae}; Gritsenko VA; In vitro experiments on 72 E . coli strains showed the presence of relationship between the level of their antilysozyme activity and a number of parameters of the reproduction of these bacteria . The factor analysis revealed the integration of the antilysozyme sign with the factors which determined the inhibition of the development of bacterial populations at early stages and controlled the accumulation of bacterial biomass . The study established that the beginning of the expression of the antilysozyme sign of E . coli was associated with the transition of the culture to the phase of slow growth, and the subsequent dynamics of the level of antilysozyme activity was only partially linked with the increase of biomass . The suggestion was made that the antilysozyme factor was one of the elements of the intracellular system regulating the synthesis (and/or stabilization) of peptidoglycan of bacterial cell walls. Zh Mikrobiol Epidemiol Immunobiol, 1997 Jul-Aug, (4), 123 - 6 {The interaction of Escherichia coli, possessing the antilysozyme trait, with infusoria}; Nemtseva NV; In this work isogenic E.coli strains K-12J53 and J53pAlz60, differing in the presence of the antilysozyme characteristic, were used . In the process of joint cultivation the degradation of lysozyme in protozoa by antilysozyme-active E.coli was established . The initial culture of antilysozyme-active E.coli K-12pAlz60 was found to be slightly heterogeneous with respect to the antilysozyme characteristic; this heterogeneity increased after the interaction of E.coli with protozoa . As the result of their joint cultivation with Tetrahymena, clones with low (2 micrograms/ml) antilysozyme activity (ALA) disappeared from the population and clones with medium and high values of ALA (3, 4, 5, 6 micrograms/ml) accumulated . The dynamics of the interaction of infusoria with Escherichia cells (ALA+) on the ultrastructural level in 1-6 and 24 hours revealed the gradual increase of processes leading to the destruction of most of the bacteria (up to their complete digestion) and, at the same time, the presence, observed also at an early period, of intact bacteria, resistant to lysis, whose survival was ensured by their antilysozyme characteristic. Mol Cells, 1997 Aug 31, 7(4), 468 - 72 Mutagenesis of the positively charged conserved residues in the 5' exonuclease domain of Taq DNA polymerase; Kim Y et al.; Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method . Taq DNA polymerase has a domain at the amino terminus (residue 1 to 290) that has a 5' exonuclease activity and a domain at the C-terminus that catalyzes polymerase reaction . Taq DNA polymerase is classified into the pol I family which is represented by E . coli DNA polymerase I . The alignment of amino acid sequences for the 5' exonuclease domains of the pol I family DNA polymerases shows six highly conserved sequences called motifs A to F . Motif C contains three positively charged residues such as 74Arg, 82Lys and 85Arg which might be involved in catalysis . In order to understand the function of those residues, they are mutagenized to alanine . The 5' exonucleolytic activities of those mutated 5' exonucleases decreased by 80 to 90%, thereby implying that three positively charged residues play certain roles in the 5' exonuclease catalysis. Biosci Biotechnol Biochem, 1997 Sep, 61(9), 1560 - 3 Beta ray-induced scission of DNA in tritiated water and protection by a green tea percolate and (-)-epigallocatechin gallate; Yoshioka H et al.; The beta-ray induced scission of puC18 plasmid DNA from E . coli in tritiated water was examined in the presence or absence of a green tea percolate (TP) and the main constituent, (-)-epigallocatechin gallate (EGCg) . An analysis of the ratio of the original closed-circular to the open-circular form of DNA, which was formed by the strand scission of DNA, revealed that TP and EGCg showed a protective effect on DNA scission depending on their concentrations . A new technique, named solid state spin trapping, was applied to examine this scavenging ability toward the hydroxyl (OH) radical generated in tritiated water . The result was kinetically analyzed to reveal that TP and EGCg showed the scavenging effect, suggesting that the protective effect on DNA scission was attributable to the scavenging effect on the OH radical. Biosci Biotechnol Biochem, 1997 Sep, 61(9), 1500 - 3 Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli; Nakai T et al.; The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01 . After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained . The enzyme had a tetrameric form that conserved the activity of sucrose synthase . Although the Km and Vmax of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the Km for sucrose was higher by a factor of 10 for the recombinant enzyme . This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction. Acta Physiol Pharmacol Ther Latinoam, 1997, 47(3), 147 - 56 Intracytoplasmatic and extracellular interleukin-1 production by monocytes of lung and colorectal cancer patients; Chasseing NA et al.; We studied the production of interleukin-1 (IL-1) by peripheral blood monocytes (Mo) from twelve normal subjects (NS) and eight and nine untreated lung and colorectal cancer patients (CP), respectively . No significant changes of extracellular IL-1 biological activity was observed between CP and NS by thymocyte proliferation assay . This result was independent that the cells were treated or not with lipopolisaccharide from E . coli (LPS, 10 micrograms/ml) . Moreover, CP present normal amount of antigenic IL-1 beta in LPS treated Mo culture supernatants by enzyme-linked immunosorbent assay (ELISA) . The biological activity of IL-1 released was not significant modified after indomethacin (Indo, 10(-6)M) and LPS + Indo treatments . Furthermore, patients showed a low percentage of LPS activated Mo with intracytoplasmatic IL-1 (alpha + beta) compared to normal values . These results were obtained by immuno-alkaline phosphatase staining using monoclonal antibody anti IL-1 (alpha + beta) . In conclusion, CP had a reduced number of Mo with intracytoplasmatic IL-1 (alpha + beta) and the difference observed may depend on degradation or in the rate of synthesis of this cytokine. Plant Cell, 1997 Sep, 9(9), 1673 - 82 A novel protein with DNA binding activity from tobacco chloroplast nucleoids; Nakano T et al.; A 41-kD DNA binding protein with a basic pl was purified from chloroplast nucleoids in photomixotrophically cultured tobacco cells, and its amino acid sequence was determined . Using this sequence information, its cDNA (CND41) was isolated, and its nucleotide sequence was determined . The predicted amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids . A distinctive helix-turn-helix motif in the lysine-rich N-terminal region of the mature protein and an aspartyl protease active site motif were predicted . Expression of a series of truncated CND41 proteins in Escherichia coli indicated that the lysine-rich region is essential for DNA binding and that CND41 nonspecifically binds chloroplast DNA . Protein gel blot analyses showed CND41 mainly in cells and/or tissues containing nonphotosynthesizing, actively growing plastids . In addition, the accumulation of chloroplast transcripts in these cells and/or tissues (e.g., transcripts for QB binding protein of photosystem II {psbA} and large subunit of ribulose bisphosphate carboxylase {rbcL}) was negatively correlated with the accumulation of CND41 . Analyses of cultured cells of transgenic tobacco with reduced CND41 levels showed a higher level of expression of chloroplast genes compared with that of the wild type . We discuss the possible function of CND41 as a negative regulator of chloroplast gene expression. Hepatology, 1997 Oct, 26(4), 949 - 56 Transient immunosuppression with FK506 permits long-term expression of therapeutic genes introduced into the liver using recombinant adenoviruses in the rat; Ilan Y et al.; The host immune response limits the duration of expression of adenovirally transduced genes and precludes long-term gene expression upon re-administration of the virus . In this study we wished to evaluate whether short-term immunosuppression of the host, at the time of recombinant virus administration, would allow expression of the therapeutic gene product upon virus reinjection . Gunn rats were used as recipients of recombinant adenoviruses expressing human BUGT (Ad-hBUGT) or E . coli beta-galactosidase (Ad-LacZ) . Rats were treated with FK506 (1-1.5 mg/kg, per OS daily) for three days beginning 24 hours before each virus injection . Control groups did not receive any immunosuppressant . The serum bilirubin level was reduced from 7.1 +/- 0.75 mg/dL to 2.0 +/- 0.7 mg/dL within two days of viral injection in both FK506 treated and control groups, and then gradually increased in 6 weeks . FK506-treated rats had low or undetectable antibody titers against the recombinant adenovirus and minimal or no cytotoxic T lymphocyte (CTL) response against adenovirus-infected cells . The tolerized rats received two subsequent injections 42 and 98 days after the first injection, which reduced the bilirubin levels again to 2.0 +/- 0.56 and 2.2 +/- 0.61 mg/dL, respectively . In contrast, control rats developed high titer neutralizing antibodies and a CTL response, and their serum bilirubin levels were not reduced following subsequent injections . We conclude that short-term FK506 treatment around the time of virus administration prevents the host immune response, permitting long-term gene therapy by repeated administration of the recombinant virus. Clin Exp Immunol, 1997 Sep, 109(3), 439 - 45 Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60; Deane KH et al.; An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized . Stimulatory peptides contained a nine amino acid sequence (residues 38-46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position . The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine . Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer . The sequence of the defined epitope is identical in hsp60 from both C . trachomatis and C . pneumoniae . Since the latter is a common respiratory pathogen, patients infected with C . trachomatis may already be primed for responses to hsp60 by prior infection with C . pneumoniae . Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma . Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60 . However, although an E . coli-related peptide was recognized, intact E . coli hsp60 was not, suggesting that the epitope is cryptic in E . coli hsp60 . Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized . Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient. J Bacteriol, 1997 Oct, 179(19), 6187 - 91 Amino acid residues in the alpha-subunit C-terminal domain of Escherichia coli RNA polymerase involved in activation of transcription from the mtr promoter; Yang J et al.; To examine the role of the amino acid residues (between positions 258 and 275 and positions 297 and 298) of the alpha-subunit of RNA polymerase in TyrR-mediated activation of the mtr promoter, we have carried out in vitro transcription experiments using a set of mutant RNA polymerases with a supercoiled mtr template . Decreases in factor-independent transcription in vitro by mutant RNA polymerases L262A, R265A, and K297A suggested the presence of a possible UP element associated with the mtr promoter . Mutational studies have revealed that an AT-rich sequence centered at -41 of the mtr promoter (SeqA) functions like an UP element . In vivo and in vitro analyses using a mutant mtr promoter carrying a disrupted putative UP element showed that this AT-rich sequence is responsible for interactions with the alpha-subunit which influence transcription in the absence of TyrR protein . However, the putative UP element is not needed for activator-dependent activation of the mtr promoter by TyrR and phenylalanine . The results from in vitro studies indicated that the alpha-subunit residues leucine-262, arginine-265, and lysine-297 are critical for interaction with the putative UP element of the mtr promoter and play major roles in TyrR-dependent transcription activation . The residues at positions 258, 260, 261, 268, and 270 also play important roles in TyrR-dependent activation . Other residues, at positions 259, 263, 264, 266, 269, 271, 273, 275, and 298, appear to play less significant roles or no role in activation of mtr transcription. J Bacteriol, 1997 Oct, 179(19), 6181 - 6 Transcriptional regulation of the Escherichia coli oxyR gene as a function of cell growth; Gonzalez-Flecha B et al.; The oxyR regulon plays a central role in the defense of Escherichia coli against the endogenous oxidative damage associated with active aerobic growth . Here we have studied the transcriptional regulation of oxyR in E . coli growing aerobically in rich medium . Expression of a single-copy oxyR'::lacZ reporter construct varied sixfold along the growth curve, with the highest value at 4 to 6 h of growth (approximately 14 x 10(8) cells x ml(-1)) . Direct measurements of oxyR mRNA by primer extension showed the same biphasic expression but with a peak somewhat earlier in cell growth (2 to 3 h; approximately 3.5 x 10(8) cells x ml(-1)) . The results of immunoblotting experiments demonstrated that the level of OxyR protein exhibits the same biphasic expression . Mutant strains lacking adenylate cyclase (cya) or Crp protein (crp) failed to increase oxyR expression during exponential growth . On the other hand, an rpoS mutation allowed oxyR expression to continue increasing as the cells entered stationary phase . Consistent with a biological role for increased levels of OxyR during exponential growth, the crp cya strain had lower activities of catalase hydroperoxidase I and glutathione reductase and an increased sensitivity to exogenously added hydrogen peroxide . These results suggest that the Crp-dependent upregulation of oxyR in exponential phase is a component of a multistep strategy to counteract endogenous oxidative stress in actively growing E . coli cells. J Bacteriol, 1997 Oct, 179(19), 6133 - 7 The cytoplasmic DNA-binding protein TraM binds to the inner membrane protein TraD in vitro; Disque-Kochem C et al.; The cytoplasmic protein TraM is one of four essential gene products of the F factor which are involved in DNA transfer after mating pair formation . TraM binds to three specific sites within the oriT region . Besides regulation of its own synthesis, the precise function of TraM during conjugation is not yet known . In the present work, the affinity of TraM to TraD was studied in vitro by an overlay assay and by affinity chromatography . Whether the interaction between TraM and TraD causes a transient or permanent anchoring of the F factor to the site of transfer is discussed . A 35-kDa host membrane protein of yet unknown function also shows affinity to TraM and may be involved in this anchoring process as well. J Bacteriol, 1997 Oct, 179(19), 6127 - 32 Silver-resistant mutants of Escherichia coli display active efflux of Ag+ and are deficient in porins; Li XZ et al.; Silver-resistant mutants were selected by stepwise exposure of silver-susceptible clinical strains of Escherichia coli, two of which did not contain any plasmids, to either silver nitrate or silver sulfadiazine . These mutants showed complete cross-resistance to both compounds . They showed low-level cross-resistance to cephalosporins and HgCl2 but not to other heavy metals . The Ag-resistant mutants had decreased outer membrane (OM) permeability to cephalosporins, and all five resistant mutants tested were deficient in major porins, either OmpF or OmpF plus OmpC . However, the well-studied OmpF- and/or OmpC-deficient mutants of laboratory strains K-12 and B/r were not resistant to either silver compound . Resistant strains accumulated up to fourfold less (110m)AgNO3 than the parental strains . The treatment of cells with carbonyl cyanide m-chlorophenylhydrazone increased Ag accumulation in Ag-susceptible and -resistant strains, suggesting that even the wild-type Ag-susceptible strains had an endogenous Ag efflux activity, which occurred at higher levels in Ag-resistant mutants . The addition of glucose as an energy source to starved cells activated the efflux of Ag . The results suggest that active efflux, presumably coded by a chromosomal gene(s), may play a major role in silver resistance, which is likely to be enhanced synergistically by decreases in OM permeability. J Bacteriol, 1997 Oct, 179(19), 6061 - 5 Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase; Bhattacharyya DK et al.; o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity . The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-