Escherichia coli

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(-4) microM(-1) min(-1) . The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine . Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out . Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity . A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis . Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+ . Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity . When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold . Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.

J Bacteriol, 1997 Oct, 179(19), 6048 - 52
The Vsr endonuclease of Escherichia coli: an efficient DNA repair enzyme and a potent mutagen; Macintyre G et al.; The Vsr endonuclease of Escherichia coli initiates the repair of T/G mismatches caused by deamination of 5-methylcytosine to thymine . In this paper, we examine the capacity of Vsr to prevent CG-to-TA mutations in cells with increased transcription of the cytosine methylase gene (dcm) . We find that sufficient Vsr is produced by a single chromosomal copy of vsr to prevent mutagenesis . We also investigate the cause of the transition and frameshift mutations in cells overproducing Vsr . Neither the absence of the dcm methylase nor its overproduction affects Vsr-stimulated mutagenesis . However, addition of mutS, mutL, or mutH on multicopy plasmids has a significant effect: mutL or mutH decreases the number of mutations, while mutS stimulates mutagenesis . The mut-containing plasmids have the same effect in cells treated with 2-aminopurine and in cells made defective in DNA proofreading, two experimental situations known to cause transition and frameshift mutations by saturating mismatch repair.

J Bacteriol, 1997 Oct, 179(19), 5999 - 6004
Regulation of the dnaK operon of Streptomyces coelicolor A3(2) is governed by HspR, an autoregulatory repressor protein; Bucca G et al.; The dnaK operon of Streptomyces coelicolor contains four genes (5'-dnaK-grpE-dnaJ-hspR) . The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G+C actinomycete group of bacteria . HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S . coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation . Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon . Disruption of hspR leads to high-level constitutive transcription of the dnaK operon . Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type . The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca . 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator . His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon . These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S . coelicolor--one controlling the dnaK operon and another controlling the groE genes.

Immunology, 1997 Aug, 91(4), 572 - 8
Modulation of B-cell activation by the B subunit of Escherichia coli enterotoxin: receptor interaction up-regulates MHC class II, B7, CD40, CD25 and ICAM-1; Nashar TO et al.; The B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin (EtxB) are non-toxic lectins that bind and cross-link a ubiquitous cell glycolipid receptor, ganglioside GM1, and are recognized as potent mucosal and systemic immunogens . Here we examine the role of EtxB receptor occupancy in modulating the activation of B cells, in vitro, in primary lymphocyte cultures containing B and T cells . When 48-hr spleen cell cultures containing EtxB were compared with those in the presence of a non-receptor binding mutant, EtxB(G33D), a marked shift in the ratio of CD4+ T cells: B cells was noted . Evidence suggested that this was the result of either enhanced survival or proliferation of B cells associated with receptor occupancy by EtxB . Investigation revealed that EtxB induced only a minimal increase in proliferation above that of EtxB(G33D), in mixed cell cultures, and failed to induce any cell division of purified B cells or T cells . In contrast, receptor-binding by EtxB markedly up-regulated the expression of major histocompatability complex (MHC) class II, B7, intracellular adhesion molecule-1 (ICAM-1), CD40 and CD25 on the B-cell surface . These results indicate that the polyclonal effects of EtxB on B cells are not associated with wide-scale proliferation, but more likely with maintenance of B-cell survival by activation of molecules essential for B-cell differentiation . The findings also highlight the essential role of GM1-interaction with EtxB in the regulation of lymphocyte responses.

Biochem J, 1997 Aug 15, 326 ( Pt 1), 197 - 203
Studies on recombinant Acetobacter xylinum alpha-phosphoglucomutase; Kvam C et al.; The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied . After expression, the enzyme was purified from the E . coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography . The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA . The isoelectric point for A . xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1 . The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4 . When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+ . The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A . xylinum . Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+ . The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1 . The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.

Biochem J, 1997 Aug 15, 326 ( Pt 1), 173 - 9
Interaction of nitric oxide with non-haem iron sites of Escherichia coli bacterioferritin: reduction of nitric oxide to nitrous oxide and oxidation of iron(II) to iron(III); Le Brun NE et al.; The bacterioferritin (BFR) of Escherichia coli consists of 24 identical subunits, each containing a dinuclear metal-binding site consisting of two histidines and four carboxylic acid residues . Earlier studies showed that the characterization of iron binding to BFR could be aided by EPR analysis of iron-nitrosyl species resulting from the addition of NO to the protein {Le Brun, Cheesman, Andrews, Harrison, Guest, Moore and Thomson (1993) FEBS Lett . 323, 261-266} . We now report data from gas chromatographic head space analysis combined with EPR spectroscopy to show that NO is not an inert probe: iron(II)-BFR catalyses the reduction of NO to N2O, resulting in oxidation of iron(II) at the dinuclear centre and the subsequent detection of mononuclear iron(III) . In the presence of excess reductant (sodium ascorbate), iron(II)-BFR also catalyses the reduction of NO to N2O, giving rise to three mononuclear iron-nitrosyl species which are detectable by EPR . One of these, a dinitrosyl-iron complex of S = 1/2, present at a maximum of one per subunit, is shown by EPR studies of site-directed variants of BFR not to be located at the dinuclear centre . This is consistent with a proposal that the diferric form of the centre is unstable and breaks down to form mononuclear iron species.

Biochem J, 1997 Aug 15, 326 ( Pt 1), 31 - 8
The effects of truncations of the small subunit on m-calpain activity and heterodimer formation; Elce JS et al.; In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured . It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit . cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10) . The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+ . A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability . NHis10-delta 125 formed a heterodimer which was inactive and unstable . Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation . The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding . Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.

Photochem Photobiol, 1997 Oct, 66(4), 541 - 6
An insertion or deletion in the extramembrane loop connecting helices E and F of archaerhodopsin-1 affects in vitro refolding and slows the photocycle; Sugiyama Y et al.; Upon addition of retinal, archaeopsin-1 expressed in Escherichia coli (ecaO-1002) regenerated the chromophore in dimyristoyl phosphatidylcholine (DMPC), 3-{(3-cholamidopropyl) dimethylammonio}-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) mixed micelles as efficiently as the same opsin prepared from halobacteria . Introduction of an insertion or a deletion of five amino acids into the surface loop connecting helices E and F changed the secondary and tertiary structures of ecaO-1002 in SDS, and diminished regeneration of the chromophore . The effect of the insertion and deletion on the in vitro refolding was specific to archaeopsin because the same insertion introduced at the corresponding position of bacterioopsin (bO) did not affect chromophore regeneration . The photocycle of the regenerated ecaR-1002 decreased in DMPC/CHAPS/SDS mixed micelles compared with that of aR-1 in the claret membrane, which was consistent with the reported behavior of bO . Unexpectedly, the insertion and deletion in loop EF perturbed the photocycle of the regenerated ecaR-1002 . The accumulation of long-lived N- and O-like intermediates suggested that the insertion and deletion slowed down the proton uptake steps at the cytoplasmic surface.

Rocz Akad Med Bialymst, 1997, 42 Suppl 1, 363 - 71
Comparative studies on the effect of TNF-alpha and endotoxin on the ultrastructural picture of pulmonary capillaries in pregnant rabbits; Sulkowska M et al.; The aim of the present study was the comparative analysis of morphological changes found in the pulmonary microcirculation of pregnant rabbits in the course of experimental septic shock induced by endotoxin or human recombinant TNF-alpha administration . The experiments used 30 female rabbits, white Dutch, c.3 kg mean body weight . The endotoxin Escherichia coli, serotype S.0127: 138 (Sigma) was applied in a single dose of 100 micrograms/kg b.w . intraperitoneally . The human recombinant TNF-alpha (biological activity 2-4 x 10(7) U/mg of protein) was injected intraperitoneally, also once, in a dose of 100 micrograms/kg b.w . Morphological examinations were based on the ultrastructural analysis in the transmission electron microscope . The ultrastructural analysis revealed no morphological differences between pregnant and non-pregnant rabbits which received TNF-alpha . However, significant differences were observed between animals given TNF-alpha and those given the endotoxin . These differences referred to endothelium damage degree and to the cellular composition of inflammatory infiltrations . More severe damage to endothelial cells (necrosis inclusive) was observed in the endotoxin-treated rabbits . Blood vascular lumen in these animals showed cellular aggregation, of neutrophils and platelets in particular as well as microthrombi . An increased tendency towards the development of these changes was noted in pregnant rabbits . The lumen of pulmonary capillaries in TNF-alpha treated animals showed domination of monocytic cells . Features of endothelial cell stimulation were observed, although without a tendency to form microthrombi.

Protein Sci, 1997 Oct, 6(10), 2188 - 95
Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation; Mulrooney SB et al.; Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain . The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138 . TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin . This domain rotation model was investigated by using a Cys 138 Ser active-site mutant . The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation . Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence . Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation . Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer . These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium . PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.

J Biotechnol, 1997 Sep 16, 57(1-3), 181 - 90
Definition of the substrate specificity of the 'sensing' xylanase of Streptomyces cyaneus using xylooligosaccharide and cellooligosaccharide glycosides of 3,4-dinitrophenol; Zhao Y et al.; The title compounds, (Xylp beta (1-->4))nXylp beta-3,4-DNP (n = 0-4) have been made by selective anomeric deprotection of peracetylated xylose oligosaccharides with hydrazine, followed by formation of the trichloroacetimidate, uncatalysed reaction with 3,4-dinitrophenol, and Zemplen deacetylation . The values of k(cat)/K(m) for 3,4-dinitrophenol release from these substrates by xylanase III of Streptomyces cyaneus, expressed in Escherichia coli, increase with increasing n up to n = 2 and then slightly decrease . Since it is known from previous work that in its normal host, the enzyme is produced constitutively at low levels and excreted, these results suggest that the biological function of the enzyme may be to produce small molecule inducers, predominantly xylotriose, from the non-reducing end of the xylan . Activity on cellooligosaccharide glycosides (Glcp beta (1-->4))nGlcp beta-3,4-DNP (n = 0-3) was detected, at a rate about two-and-a-half orders of magnitude less than that observed on the corresponding xylooligosaccharides, indicating that the enzyme is a true xylanase.

Nat Biotechnol, 1997 Oct, 15(10), 988 - 91
Overexpression of glutathione S-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress; Roxas VP et al.; Transgenic tobacco seedlings that overexpress a cDNA encoding an enzyme with both glutathione S-transferase (GST) and glutathione peroxidase (GPX) activity had GST- and GPX-specific activities approximately twofold higher than wild-type seedlings . These GST/GPX overexpressing seedlings grew significantly faster than control seedlings when exposed to chilling or salt stress . During chilling stress, levels of oxidized glutathione (GSSG) were significantly higher in transgenic seedlings than in wild-types . Growth of wild-type seedlings was accelerated by treatment with GSSG, while treatment with reduced glutathione or other sulfhydryl-reducing agents inhibited growth . Therefore, overexpression of GST/GPX can stimulate seedling growth under chilling and salt stress, and this effect could be caused by oxidation of the glutathione pool.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11201 - 4
Converting a transmembrane receptor to a soluble receptor: recognition domain to effector domain signaling after excision of the transmembrane domain; Ottemann KM et al.; The bacterial aspartate receptor was reconstructed to eliminate the transmembrane domain, thus connecting the recognition domain directly to the effector domain . The resulting soluble receptor folded correctly and was no longer an integral membrane protein . Upon aspartate binding, this soluble receptor was stabilized to a similar extent as that of the native receptor . Of interest, this soluble receptor retained the ability to signal from the recognition to the effector domain . This result defines more clearly the role of the membrane and transmembrane domains in signal transduction and suggests that some ligand-induced motions in receptor proteins do not require the membrane or transmembrane domain for information transmission.

Am J Hum Genet, 1997 Oct, 61(4), 852 - 61
Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency; Kikawa Y et al.; Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death . We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA . In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA . Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency . Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families . The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report {46% of the 22 mutant alleles}), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles {14%}), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele {4%}), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles {9%}) . FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E . coli . Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency . Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients . Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.

Am J Gastroenterol, 1997 Oct, 92(10), 1853 - 7
Possibility of chemoprevention by the eradication of Helicobacter pylori: oxidative DNA damage and apoptosis in H . pylori infection; Hahm KB et al.; OBJECTIVES: The purpose of this study was to study the changes of 8-hydroxydeoxyguanosine (8-OH-dG) contents of DNA from human gastric mucosa with or without Helicobacter pylori and the changes of two biomarkers, iNOS and apoptosis, in gastric biopsies obtained before and after the eradication of H . pylori . METHODS: DNA isolated from the biopsied human gastric mucosa was digested to deoxynucleotides by nuclease P1, then with Escherichia coli alkaline phosphatase, and analyzed by HPLC-ECD system . 8-OH-dG content was expressed as the number of residues per 10(5) deoxyguanosine . iNOS immunohistochemical staining was performed with antihuman iNOS antiserum generated in mice at a dilution of 1:500, and in situ apoptosis was detected by in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling . Both the density of H . pylori and the degree of inflammation were scored . RESULTS: The 8-OH-dG contents of healthy normal controls with negative H . pylori were 4.31 +/- 2.33 (8-OH-dG/10(5) dG), whereas those of patients with positive H . pylori were 10.40 +/- 7.25 . The difference between these two values was statistically significant (p < 0.01) . The 8-OH-dG contents were significantly decreased after the eradication of H . pylori (12.22 +/- 2.09 vs . 2.42 +/- 1.22, p < 0.001) . After the eradication of H . pylori, both the apoptotic index and the iNOS scores were significantly decreased, compared with those before eradication (3.72 +/- 1.74 vs . 1.17 +/- 1.06 for apoptosis and 10.34 +/- 6.79 vs . 1.43 +/- 1.14 for iNOS, p < 0.001) . Statistically significant correlations were observed among apoptotic index, iNOS score, degree of inflammation, and density of H . pylori (p < 0.05) . CONCLUSIONS: The increased levels of oxidative DNA damage, increased occurrences of apoptosis, and increased expressions of iNOS suggest mechanistic links between H . pylori infection and gastric carcinogenesis.

Virus Res, 1997 Sep, 51(1), 65 - 79
Immunological characterisation of glycoprotein E of Aujeszky's disease virus; Morenkov OS et al.; A panel of 14 monoclonal antibodies (MAbs) against glycoprotein E (gE) of Aujeszky's disease (pseudorabies) virus (ADV), which constitutes a representative sample of naturally occurring gE-specific antibodies in sera from infected animals, was produced and characterised . Eleven topologically distinct antigenic domains represented by one or more MAbs were identified on gE by using these MAbs and three additional gE-specific MAbs . Three of the MAbs available recognised conformation-independent epitopes on gE, while the other 14 MAbs bound to conformation-dependent epitopes . By using the recombinant protein encompassing the N-terminal part of gE, which was expressed in Escherichia coli, all the conformation-independent epitopes of gE were mapped within the first 125 amino-terminal amino acids of gE . The epitopes of gE were demonstrated to be conserved among gE-positive laboratory, field and vaccine ADV strains . Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gE in naturally infected swine and immunised mice . Most of the infected animals responded weakly to the identified conformation-independent epitopes of gE, while the group of immunodominant epitopes of gE was represented exclusively by conformation-dependent antigenic determinants from different antigenic domains . The results clearly demonstrated that conformation-dependent epitopes play a crucial role in inducing the humoral immune response to gE of ADV during the natural infection of swine and immunisation of mice . The application of MAbs of our panel as research and diagnostic tools is discussed.

J Immunol, 1997 Oct 15, 159(8), 4024 - 34
Attenuation of IL-5-mediated signal transduction, eosinophil survival, and inflammatory mediator release by a soluble human IL-5 receptor; Monahan J et al.; A soluble form of the human IL-5R alpha-chain (IL-5Ra) that contains the extracellular IL-5 binding domain has been evaluated for its effect on IL-5 binding to and activation of human eosinophils and basophils . The truncated receptor was expressed in Escherichia coli and recovered in biologically active form following renaturation and anion exchange chromatography . The soluble receptor formed a 1/1 complex with IL-5 in solution and bound IL-5 with affinity comparable to that of cell-associated IL-5Ra . Soluble IL-5Ra also competed with IL-5 for binding to the native alpha beta IL-5R on human cells and inhibited IL-5-mediated receptor activation and inflammatory mediator production . In this regard, the soluble receptor prevented IL-5-induced tyrosine phosphorylation of JAK2 kinase and IL-5R beta-chain and inhibited IL-5 priming of leukotriene C4 release by human basophils . However, the E . coli-derived receptor failed to inhibit IL-5 in longer term assays, including eosinophil survival and TF-1 cell proliferation, possibly due to its propensity to aggregate in a time- and temperature-dependent manner . In contrast, we observed that a soluble IL-5Ra derived from baculovirus-infected cells was less prone to aggregate and effectively antagonized IL-5-induced cell proliferation and survival . These findings indicate that the extracellular portion of the human IL-5Ra chain can prevent association of IL-5 with cell surface receptors and can attenuate signal transduction, mediator release, and survival of inflammatory cells . As such, soluble IL-5R may be useful in treating diseases such as human asthma, in which pulmonary injury is associated with the activity of IL-5R-bearing cells.

Crit Care Med, 1997 Oct, 25(10), 1727 - 32
Heat stress increases survival rates in lipopolysaccharide-stimulated rats; Chu EK et al.; OBJECTIVE: To examine the hypothesis that heat stress applied after the administration of bacterial endotoxin is protective . DESIGN: Prospective, randomized, laboratory study . SETTING: University research laboratory . SUBJECTS: One hundred eleven adult male Sprague-Dawley rats (weight range 250 to 400 g) . INTERVENTIONS: Production of endotoxemia by the administration of a bacterial endotoxin and exposure to heat stress by heating animals in a neonatal incubator until their rectal temperatures reached 105.8 degrees F (41 degrees C) . MEASUREMENTS AND MAIN RESULTS: The rats (n = 111) were anesthetized and were injected with 15 mg/kg of Escherichia coli endotoxin (lipopolysaccharide, LPS) intravenously to produce septic shock . Immediately thereafter, a set of 50 rats were randomly assigned to one of two treatment groups: a) LPS-treated (control); or b) LPS-treated and heated to 105.8 degrees F (41 degrees C) . The animals were then observed for the development of fever, and survival rates were monitored for 72 hrs . In another set of 40 animals, the same experimental protocol was used to determine plasma cytokine concentrations in heated and nonheated groups . Blood samples were obtained at 0, 2, 4, or 6 hrs after LPS injection for tumor necrosis factor-alpha and interleukin (IL)-1 beta detection . In a third set of animals, the same experimental protocol was applied to nine animals for the detection of heat-shock proteins of 72-kilodalton molecular weight . LPS injection in the control group did not produce fever . Heat stress increased the abundance of heat-shock proteins of 72-kilodalton molecular weight in the rats' lungs (analysis of variance, p = .016) . Twelve hours after the initiation of sepsis, the survival rates of the control group injected with LPS alone and the group heated to 105.8 degrees F (41 degrees C) were 48% and 80%, respectively (p = .039) . The peak plasma IL-1 beta concentrations occurring at 2 hrs after LPS injection were significantly reduced in rats heated to 105.8 degrees F (41 degrees C) when compared with nonheated rats (p = .003) . CONCLUSION: We conclude that heat stress applied after the initiation of endotoxemia can provide protection against an otherwise lethal stimulus and that the mechanism of protection may be related to the attenuation of plasma IL-1 beta concentrations.

Crit Care Med, 1997 Oct, 25(10), 1700 - 6
Cerebral blood flow during experimental endotoxemia in volunteers; Pollard V et al.; OBJECTIVE: To measure cerebral blood flow, cerebral metabolic rate for oxygen, cerebral oxygen delivery, and cerebral vascular resistance during experimental endotoxemia in volunteers . DESIGN: Experimental, prospective study . SETTING: University general clinical research center . SUBJECTS: Healthy volunteers (six male, four female, 30.1 +/- 1.9 yrs of age) . INTERVENTIONS: Volunteers had radial, pulmonary arterial, and jugular venous bulb catheters inserted . All volunteers received a bolus of Escherichia coli endotoxin (4 ng/kg) . Cerebral blood flow was measured, using the Kety-Schmidt technique . MEASUREMENTS AND MAIN RESULTS: Cerebral and systemic hemodynamics and oxygenation variables were measured at baseline and hourly for 5 hrs after endotoxin administration . A systemic hyperdynamic response characterized by an increase in body temperature (97.9 +/- 0.02, 100.2 +/- 0.02, and 99.7 +/- 0.02 degrees F {36.6 +/- 0.01, 37.9 +/- 0.1, and 37.6 +/- 0.1 degrees C} at baseline, 3, and 5 hrs, respectively), cardiac index (3.7 +/- 0.2, 6.2 +/- 0.2, and 5.7 +/- 0.2 L/min/m2 at baseline, 3, and 5 hrs), and heart rate (70 +/- 2.6, 96 +/- 2.6, and 93 +/- 2.9 beats/min at baseline, 3, and 5 hrs), and a decrease in mean arterial pressure (99.3 +/- 2.2, 84.4 +/- 2.8, and 84 +/- 3.4 mm Hg at baseline, 3, and 5 hrs) and systemic vascular resistance (1498 +/- 53, 788 +/- 37, 849 +/- 36 dyne.sec/cm5.m2 at baseline, 3, and 5 hrs) followed the endotoxin bolus . Cerebral blood flow (65.4 +/- 4.3, 57.7 +/- 3.1, and 58.6 +/- 3.0 mL/100 g/min at baseline, 3, and 5 hrs), cerebral oxygen delivery (11.6 +/- 0.7, 9.8 +/- 0.6, and 9.5 +/- 0.6 mL/100 g/min at baseline, 3, and 5 hrs), cerebral metabolic rate for oxygen (3.8 +/- 0.4, 3.3 +/- 0.3, and 3.0 +/- 0.3 mL/100 g/min at baseline, 3, and 5 hrs), and cerebral vascular resistance (1.4 +/- 0.2, 1.4 +/- 0.2, and 1.3 +/- 0.2 mm Hg/mL/100 g/min at baseline, 3, and 5 hrs) were unchanged throughout the 5-hr study period . Signs of cerebral dysfunction were not apparent, although the volunteers appeared drowsy during the latter part of the study . CONCLUSION: A dose of endotoxin sufficient to induce systemic vasodilation in healthy subjects does not influence cerebral blood flow or the cerebral metabolic rate for oxygen.

Cancer Lett, 1997 Aug 19, 117(2), 143 - 7
Comparative study on organ-specificity of tumorigenicity, mutagenicity and cell proliferative activity induced by dimethylnitrosamine in Big Blue mice; Nishikawa A et al.; Recently, we have shown that dimethylnitrosamine (DMN) treatments increase lacI mutant frequency in the liver, kidney and lung but not in other organs, and also enhance cell proliferation only in the bronchial epithelia . In the present study, organ specificity of tumorigenicity induced by DMN was compared to those of lacI mutation and cell proliferation in Big Blue mice . Male 8-week-old Big Blue mice were treated with daily i.p . injections of 1 or 10 mg/kg DMN for 5 days, or a single i.p . injection of 5 or 10 mg/kg DMN . Except for the 10 mg/kg x 5 DMN group, all animals survived until 78 weeks after the first treatment of DMN . In the present study, the induction of cell proliferation in the bronchial epithelia was confirmed in a dose-dependent manner . At the termination of 78 weeks, it was histopathologically shown that the DMN-treated mice developed liver cell tumor in three out of seven (43%) of the 5 mg/kg group, renal tubule dysplasia in three out of seven (43%) of the 1 mg/kg x 5 group, and duodenal adenocarcinoma in one of seven (14%) of the 1 mg/kg x 5 group, although no neoplastic or preneoplastic lesions were found in the control mice . Because non-transgenic C57BL/6 mice are resistant to developing spontaneous liver cell and duodenal tumors, it was speculated that even these low doses of DMN could be sufficient to initiate target cells . Our results thus suggest that organ specificity of tumorigenicity by DMN is in favorable agreement with that of lacI mutation but not with possibly temporal cell proliferation induced by DMN.

Circulation, 1997 Oct 7, 96(7), 2339 - 47
Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model; Thiagarajan P et al.; BACKGROUND: The procoagulant effect of anionic phospholipid may play a major role in the development of arterial thrombosis . METHODS AND RESULTS: Annexin V, a calcium-dependent anionic-phospholipid-binding protein, was expressed and isolated from Escherichia coli and its antithrombotic effect examined in a rabbit carotid artery thrombosis model . A partially occlusive thrombus was formed in the left carotid artery by application of electric current to produce an approximately 50% occlusion of the lumen . After the current was discontinued, flow ceased completely within 42+/-12 minutes (n=6) because of continuing platelet/fibrin thrombus formation . When annexin V was given at doses of 2.8 to 16.6 microg x kg(-1) x min(-1) for a period of 180 minutes, starting at the time the current was stopped, there was a dose-dependent inhibition of thrombus formation . At a dose of 5.6 microg x kg(-1) x min(-1), blood flow remained patent throughout the infusion and for an additional 60 minutes after the infusion was stopped . In addition, there was a decrease in thrombus weight (16+/-7.4 versus 2.0+/-1.0 g), (125)I-fibrin deposition (approximately 45% reduction, P<.001), and (111)In-labeled platelet accumulation (approximately 43% reduction, P<.001) . Prior mixing of annexin V with phosphatidylserine micelles abolished the antithrombotic effect of annexin V, whereas mixing with phosphatidylcholine micelles had no effect . The antithrombotic effect of annexin V was not associated with bleeding tendency, as judged by the amount of blood absorbed in a gauze pad placed in a surgical incision extending to the muscle tissue and by the standard template bleeding time . CONCLUSIONS: These observations support a potentially important role for anionic phospholipid exposure in platelets in arterial thrombosis, and inhibition of this activity could be a novel target for therapy in coronary thrombosis and stroke and after angioplasty.

Glycoconj J, 1997 Sep, 14(6), 715 - 22
Expression and sulfogalactolipid binding specificity of the recombinant testis-specific cognate heat shock protein 70; Mamelak D et al.; Immunofluorescent studies with anti-2A antisera, raised specifically against a synthetic C-terminal peptide of native murine P70, the testes-specific cognate heat shock protein 70, demonstrated that the rat homologue of P70 is expressed on the surface of testicular cells . The murine hsp 70.2 gene, encoding P70, was cloned and expressed in Escherichia coli . The recombinant P70 (rP70) protein with a 6Xhistidine affinity tag at its amino terminus was purified from E . coli via nickel affinity column chromatography . Monoclonal anti-hsp70 antisera and anti-2A antisera cross-reacted with purified rP70 . Binding of rP70 was specific for sulfogalactosylceramide (SGC) and sulfogalactosyglycerolipid (SGG) . Binding was not inhibited by the sugar, galactose 3'sulfate, nor was binding observed to desulfated derivatives of SGC and SGG, to other negatively charged lipids or other sulfated lipids . Furthermore, rP70 bound to an SGC-column and was eluted only at high salt in combination with high pH . These results show rP70 to possess a specific sulfatide binding site . Since the biochemical properties and immunoreactivity of rP70 are indistinguishable from native P70 and SLIP1 (testicular sulfoglycolipid immobilized protein 1) rP70 can be employed to examine the role of hsp70-mediated sulfatide binding in fertilization.

Can J Microbiol, 1997 Sep, 43(9), 884 - 6
On the relation of colony variants to the time dependency of colony formation during adaptive mutation of Escherichia coli FC40; MacLeod PR et al.; When Escherichia coli FC40 formed adaptive Lac+ revertants on a selective agar medium containing lactose as the carbon source, the colonies which accumulated over several days were of two readily distinguishable types . Colonies of both types appeared both early and late on the plates . Cells of colonies that appeared early and late on the plates, irrespective of the type, when grown in liquid medium and replated, all formed colonies on the selective medium within 48 h . Cells of each colony type gave rise to colonies of both types and attempts to isolate cells of each type in pure culture were unsuccessful . It was concluded that the presence of two colony types in the cultures plated did not contribute to the observed time dependency of colony formation during adaptive mutation . The proportions of the two colony types arising from cultures of the Lac+ revertants varied from culture to culture.

Can J Microbiol, 1997 Sep, 43(9), 819 - 26
Mapping of sequences required for the translation of the beta subunit of Escherichia coli RNA polymerase; Passador L et al.; Previous experiments using expression plasmids which overproduce the beta and beta' subunits of Escherichia coli RNA polymerase suggested that regions considerably upstream of the start of the rpoB gene, which encodes the beta subunit, are required for its efficient synthesis . To further delineate the required regions, a collection of genetic constructs that contained varying amounts of the region either upstream or downstream of the translational start of rpoB was assembled . Measurements of beta and beta' synthesis and rpoB mRNA production from a series of rpoBC expression plasmids indicated that sequences extending more than 43 bp but less than 79 bp upstream of rpoB are required for the efficient translation of rpoB mRNA . This result was confirmed by beta-galactosidase measurements from a series of rpoB-lacZ fusions that have the same set of end points upstream of rpoB as the expression plasmids . A second set of gene fusions containing differing amounts of the sequence distal to the start of rpoB fused in frame to lacZ revealed that more than 29 bp but less than 70 bp of rpoB was required for efficient translation.

Int J Syst Bacteriol, 1997 Oct, 47(4), 926 - 32
Genetic and phenotypic analysis of Borrelia valaisiana sp . nov . (Borrelia genomic groups VS116 and M19); Wang G et al.; To clarify the taxonomic status of two recently described Borrelia genomic groups, groups VS116 and M19, three group VS116 strains and eight group M19 strains isolated from Ixodes ricinus ticks in Switzerland, The Netherlands, and the United Kingdom were characterized . PCR-restriction fragment length polymorphism (RFLP) analysis of the 5S-23S intergenic spacer amplicon, rRNA gene restriction analysis, 16S rRNA gene sequence analysis, randomly amplified polymorphic DNA (RAPD) fingerprinting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with monoclonal antibodies were used for genetic and phenotypic analysis . The PCR-RFLP and RAPD patterns of three group VS116 strains and eight group M19 strains were identical but differed from those of Borrelia burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and Borrelia japonica . DNAs from all group VS116 and M19 strains yielded three fragments (6.9, 3.2, and 1.4 kb) and four fragments (2.1, 1.2, 0.8, and 0.6 kb) after digestion with EcoRV and HindIII, respectively, hybridizing with an Escherichia coli 16S + 23S cDNA probe . The SDS-PAGE protein profiles of group VS116 and M19 strains were heterogeneous . Phylogenetic analysis of the partial 16S rRNA gene sequences showed that group VS116 and M19 spirochetes were members of a Borrelia species distinct from previously characterized members of the genus Borrelia . Based on our present study and data from previous DNA-DNA hybridizations, a new Borrelia species, Borrelia valaisiana sp.nov., in the B . burgdorferi complex, is proposed . Strain VS116 is the type strain of this new species.

Protein Sci, 1997 Oct, 6(10), 2264 - 7
Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I; Wang CR et al.; A 35-37-kDa protease-resistant domain of synapsin Ia/ Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X-rays to approximately 10 A resolution . The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N- and C-terminal flanking segments . Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C-terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione-S-transferase . The fusion proteins were purified by affinity chromatography . Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography . SynA and SynB formed crystals within three days, which diffracted to better than 3 A using a conventional X-ray source and to about 2 A using a synchrotron X-ray source . SynA crystals have the symmetry of the trigonal space groups P3(1)21 or P3(2)21 and the unit cell dimensions a = b = 77.4 A, c = 188.5 A, alpha = beta = 90 degrees, gamma = 120 degrees . SynB crystals have the symmetry of the orthorhombic space group C222(1) with the unit cell dimension a = 104.6 A, b = 113.3 A, and c = 273.8 A.

Protein Sci, 1997 Oct, 6(10), 2180 - 7
Glutathione S-transferase can be used as a C-terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli; Tudyka T et al.; Glutathione S-transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time . For this purpose GST was linked via a flexible spacer to the C-terminus of the thiol-protease inhibitor cystatin, whose binding properties for papain were to be studied . The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin . The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST . Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction . Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose . Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep-tag had been appended to its C terminus . The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1-chloro-2,4-dinitrobenzene as substrates . Furthermore, strong binding activity for papain was detected in an ELISA . The signal with the cystatin-GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST . The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size . Thus, engineered GST is suitable as a moderately sized, secretion-competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.

Clin Microbiol Rev, 1997 Oct, 10(4), 569 - 84
Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli; Wolf MK; Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea worldwide . Four categories of antigens have been commonly studied: O serogroup, H serogroup, colonization factor antigens (CFA), and toxins . A database has been complied from published reports of nearly 1,000 ETEC isolates from 18 locations and analyzed to determine the occurrence, distribution, and associations of O serogroup, H serogroup, CFA, and toxin type . Tables listing the associations of antigens are presented . This analysis documents the widespread nature and variety of ETEC . Even the most common combination of antigens, O6:H16 CFA/II LTST, accounted for only 11% of the ETEC isolates in the database . It was isolated from 12 locations . Many phenotypes occurred only once . CFA detection based on enzyme-linked antibodies with polyclonal sera is suggested as the preferred assay . A combination of CFA and toxin-based antigens is suggested as the most practical vaccine.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4416 - 8
Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus; Chen M et al.; The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies . However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression . Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency . The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4379 - 84
Phase imaging of moving DNA molecules and DNA molecules replicated in the atomic force microscope; Argaman M et al.; Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution . In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM . Higher scan rates make it possible to image faster processes . At lower forces the molecules are imaged more gently . Moving DNA molecules are also resolved more clearly in phase images than in height images . Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface . Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4362 - 9
Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA; Driggers WJ et al.; Genomic DNA is constantly being damaged and repaired and our genomes exist at lesion equilibrium for damage created by endogenous mutagens . Mitochondrial DNA (mtDNA) has the highest lesion equilibrium frequency recorded; presumably due to damage by H2O2 and free radicals generated during oxidative phosphorylation processes . We measured the frequencies of single strand breaks and oxidative base damage in mtDNA by ligation-mediated PCR and a quantitative Southern blot technique coupled with digestion by the enzymes endonuclease III and formamidopyrimidine DNA glycosylase . Addition of 5 mM alloxan to cultured rat cells increased the rate of oxidative base damage and, by several fold, the lesion frequency in mtDNA . After removal of this DNA damaging agent from culture, the single strand breaks and oxidative base damage frequency decreased to levels slightly below normal at 4 h and returned to normal levels at 8 h, the overshoot at 4 h being attributed to an adaptive up-regulation of mitochondrial excision repair activity . Guanine positions showed the highest endogenous lesion frequencies and were the most responsive positions to alloxan-induced oxidative stress . Although specific bases were consistently hot spots for damage, there was no evidence that removal of these lesions occurred in a strand-specific manner . The data reveal non-random oxidative damage to several nucleotides in mtDNA and an apparent adaptive, non-strand selective response for removal of such damage . These are the first studies to characterize oxidative damage and its subsequent removal at the nucleotide level in mtDNA.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4301 - 6
The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo; Zhang S et al.; A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene . The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension . It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA . Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein . Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product . However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein . Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4209 - 18
An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene; Dykxhoorn DM et al.; A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations . The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease . The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette . Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide . We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase . The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta' . Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation . Also a site that appears to be involved in translational repression by excess beta' has been identified.

Biophys J, 1997 Oct, 73(4), 1925 - 31
Estimation of the pore size of the large-conductance mechanosensitive ion channel of Escherichia coli; Cruickshank CC et al.; The open channel diameter of Escherichia coli recombinant large-conductance mechanosensitive ion channels (MscL) was estimated using the model of Hille (Hille, B . 1968 . Pharmacological modifications of the sodium channels of frog nerve . J . Gen . Physiol . 51:199-219) that relates the pore size to conductance . Based on the MscL conductance of 3.8 nS, and assumed pore lengths, a channel diameter of 34 to 46 A was calculated . To estimate the pore size experimentally, the effect of large organic ions on the conductance of MscL was examined . Poly-L-lysines (PLLs) with a diameter of 37 A or larger significantly reduced channel conductance, whereas spermine (approximately 15 A), PLL19 (approximately 25 A) and 1,1'-bis-(3-(1'-methyl-(4,4'-bipyridinium)-1-yl)-propyl)-4,4'-b ipyridinium (approximately 30 A) had no effect . The smaller organic ions putrescine, cadaverine, spermine, and succinate all permeated the channel . We conclude that the open pore diameter of the MscL is approximately 40 A, indicating that the MscL has one of the largest channel pores yet described . This channel diameter is consistent with the proposed homohexameric model of the MscL.

Acta Gastroenterol Latinoam, 1996, 26(4), 231 - 6
{Genomic heterogeneity of hepatitis B virus, genotype A circulating in the metropolitan area of Buenos Aires, Argentina}; Fernandez C et al.; HVB DNA was extracted from highly purified Dane particles, from sera of HBV viremic patients, collected in the metropolitan area of Buenos Aires (Argentina) . HBV DNA was cloned in pUC18 vector, amplified in Escherichia coli DH5 F' . Plasmids were recovered and analyzed for HBV DNA inserts . Three recombinant plasmids, pHB4, pHB7 and pHB20 were selected, and HBVDNA inserts sequenced . The resulted sequences were incorporated at the GenBank, with the following accession numbers: PHB4P3=U33188; PHB4P5=U33189; PHB7P3=U33190; PHB7P5=U33191 and PHB20=U33190; PHB7P5=U33191 and PHB20=U33187 . All belongs to the genotype A, pHB4 and pHB20 have a very close relation in between each other and with L13994 sequence, from North America origin . pHB7 have a significant distance from pHB4 and pHB20 and have a discrete homology with m57633 detected in Philippines . pHB4 shows a mutation at the T 3182-Leu in the preS1 region that change Pro for Leu, this mutation is absent in 125 sequences selected (having a 65% or more of homology) from NCBI by Blast algortm . The sequence of the pre C regions of all three inserts do not show any evidence to belong to the e-or scape mutants . Type A genotypes shows to be common in the area, but a hight degree of divergence have been demonstrate between two circulating strains.

Genetics, 1997 Oct, 147(2), 743 - 53
Mismatch repair by efficient nick-directed, and less efficient mismatch-specific, mechanisms in homologous recombination intermediates in Chinese hamster ovary cells; Miller EM et al.; Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells . Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations . Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches . Product spectra were consistent with hDNA only occurring between DSBs . Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites . Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system . Discontinuous patterns, seen in approximately 10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A-->G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY . Mismatch repair was > 80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates . Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick . We also observed products resulting from two tracts of intermediate length initiated from two nicks.

Biochemistry, 1997 Oct 21, 36(42), 13027 - 33
Mapping of the residues involved in a proposed beta-strand located in the ferric enterobactin receptor FepA using site-directed spin-labeling; Klug CS et al.; Electron paramagnetic resonance (EPR) site-directed spin-labeling (SDSL) has been used to characterize a proposed transmembrane beta-strand of the Escherichia coli ferric enterobactin receptor, FepA . Each of nine consecutive residues was mutated to cysteine and subsequently labeled with the sulfhydryl-specific spin-label methanethiosulfonate (MTSL) and the purified protein reconstituted into liposomes . Continuous wave (CW) power saturation methods were used to determine exposure of the nitroxide side chains to a series of paramagnetic relaxation agents, including nickel acetylacetonate (NiAA), nickel ethylenediaminediacetate (NiEDDA), chromium oxalate (CROX), and molecular oxygen . The spin-label attached to Q245C, L247C, L249C, A251C, and Y253C had higher collision frequencies with molecular oxygen than with polar relaxation agents, indicating that these sites are exposed to the hydrophobic phase of the lipid bilayer . MTSL bound to residues S246C, E248C, E250C, and G252C had higher collision rates with the polar agents than with oxygen, suggesting that these sites are exposed to the aqueous channel . The alternating periodicity observed with the polar relaxation agents, NiAA and NiEDDA, and in opposite phase with oxygen, is consistent with beta-sheet structure . Depth measurements, based on the reciprocal concentration gradients of NiEDDA and O2 across the bilayer and calibrated for our system with phosphatidylcholine spin-labels, indicated that L249C was nearest the center of the bilayer and that Q245C and Y253C were located just below the bilayer surface in opposite leaflets of the membrane . Thus, we conclude that this approach, through mapping of individual residues, has the capability of defining beta-sheet secondary structure.

Biochemistry, 1997 Oct 21, 36(42), 12984 - 93
Analysis of some optical properties of a native and reconstituted photosystem II antenna complex, CP29: pigment binding sites can be occupied by chlorophyll a or chlorophyll b and determine spectral forms; Giuffra E et al.; The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments . Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight . A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro . The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region . Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly . These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms . In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm . The wavelength position of all subbands is unchanged . The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs . Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm . The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands . We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

Biochemistry, 1997 Oct 21, 36(42), 12961 - 9
The Escherichia coli FOF1 gammaM23K uncoupling mutant has a higher K0.5 for Pi . Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway; Al-Shawi MK et al.; The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a Pi release mode . In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed . Activation of ATP synthesis by ADP and Pi was determined for the gammaM23K FOF1 . The K0.5 for ADP was not affected, but K0.5 for Pi was approximately 7-fold higher even though the apparent Vmax was close to the wild-type level . Wild-type enzyme had a turnover number of 82 s-1 at pH 7.5 and 30 degrees C . During oxidative phosphorylation, the apparent dissociation constant (KI) for ATP was not affected and was 5-6 mM for both wild-type and gammaM23K enzymes . Thus, the apparent binding affinity for ATP in the presence of DeltamuH+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site . Arrhenius analysis of ATP synthesis for the gammaM23K FOF1 revealed that, like those of ATP hydrolysis, the transition state DeltaH was much more positive and TDeltaS was much less negative, adding up to little change in DeltaG . These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state . Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway . Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.

Biochemistry, 1997 Oct 21, 36(42), 12954 - 60
Mechanism of energy coupling in the FOF1-ATP synthase: the uncoupling mutation, gammaM23K, disrupts the use of binding energy to drive catalysis; Al-Shawi MK et al.; The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits which caused inefficient transmission of coupling information between transport and catalysis {Al-Shawi, M . K . , Ketchum, C . J., and Nakamoto, R . K . (1997) J . Biol . Chem . 272, 2300-2306} . We hypothesized that the gammaM23K mutation, because of its effect on coupling, should alter the fundamental reactions steps that are normally modulated by DeltamuH+ via the coupling mechanism . In this paper, we address this issue by studying the thermodynamics of individual catalytic steps through the use of energy profiles to gain information regarding enzyme mechanism and the effects of the mutation . Compared to wild-type enzyme, the gammaM23K F1 had significant differences of two partial reactions: the rate constant for Pi release was 49-fold faster and the rate constant for ATP release was 8.4-fold faster than wild-type . These rate constants were considered together with characteristics of a group of F1 ATPase mutant enzymes and were analyzed quantitatively by linear free energy relationships {Al-Shawi, M . K., Parsonage, D., and Senior, A . E., (1990) J . Biol . Chem . 265, 4402-4410} . We found that the gammaM23K mutation prevents the proper utilization of binding energy to drive catalysis and blocks the enzyme in a Pi release mode . This finding is consistent with the use of energy from DeltamuH+ for increasing the affinity for Pi so that the substrate binds in a catalytically competent manner for synthesis of ATP . These results support the notion that the communication of coupling information is transmitted through the gamma-beta interface near gammaMet23 and beta380DELSEED386 segment.

Biochemistry, 1997 Oct 21, 36(42), 12921 - 9
Cytoplasmic domains mediate the ligand-induced affinity shift of guanylyl cyclase C; Deshmane SP et al.; Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), exhibits multiple binding affinities, including high (RH) and low (RL) affinity sites and a ligand-induced conversion of low-affinity sites from a higher (RL1) to a lower (RL2) affinity state . Occupancy of the lowest affinity state of low-affinity sites is coupled to ligand-induced catalytic activation . In the present studies, ligand binding and catalytic activation properties of a series of intracellular deletion mutants of GCC were examined to identify the structural domains underlying expression of high- and low-affinity sites and the ligand-induced shift in low-affinity sites . These studies demonstrated that the cytoplasmic domains of GCC are not required, but extracellular and transmembrane domains are sufficient, for expression of high-affinity binding sites . In addition, the cytoplasmic juxtamembrane and kinase homology domains are required for expression of the ligand-induced affinity shift in low-affinity sites . Of significance, this shift in affinity was insensitive to adenine nucleotides, in contrast to other members of the receptor guanylyl cyclase family, such as guanylyl cyclase A (GCA) . Also, the juxtamembrane and kinase homology domains are critical for coupling ST-receptor binding and guanylyl cyclase catalytic activation . Indeed, deletion of those domains from GCC results in a constitutively inhibited enzyme, suggesting that they function as positive effectors of ligand activation, in contrast to GCA and GCB, in which the kinase homology domain represses basal catalytic activity . These data suggest that the mechanisms regulating different members of the receptor guanylyl cyclase family are overlapping but not identical.

Biochemistry, 1997 Oct 21, 36(42), 12836 - 44
Nonidentity of the alpha-neurotoxin binding sites on the nicotinic acetylcholine receptor revealed by modification in alpha-neurotoxin and receptor structures; Ackermann EJ et al.; alpha-Neurotoxins constitute a large family of polypeptides that bind with high affinity to the nicotinic acetylcholine receptor (nAChR) . Using a recombinant DNA-derived alpha-neurotoxin (Naja mossambica mossambica, NmmI) and mouse muscle nAChR expressed transiently on the surface of HEK 293 cells, we have delineated residues involved in the binding interaction on both the alpha-neurotoxin and the receptor interface . Several of the studied NmmI mutations, including two residues conserved throughout the alpha-neurotoxin family (K27 and R33), resulted in substantial decreases in the binding affinity . We have also examined 23 mutations located on the receptor alpha subunit and have identified 4 positions that appear to be important to NmmI recognition . These determinants represent a conserved aromatic residue (Y190), two positions where neuronal and muscle receptors differ (V188 and P197), and a negatively charged residue (D200) . Unlike many of the nAChR agonists and antagonists which bind to the alphadelta and alphagamma binding sites on the receptor with different affinities, the wild-type NmmI-wild-type nAChR interaction showed a single affinity . However, by mutating critical toxin or receptor residues, we were able to produce site-selectivity between the alphagamma and alphadelta interfaces . These results suggest a nonequivalence in the binding interaction at the two sites, sensitive to discrete structural changes at key contact points on either the toxin or the receptor protein, and underscore the importance of delta and gamma receptor subunits in governing binding affinity.

Biochemistry, 1997 Oct 21, 36(42), 12814 - 22
Failure of a two-state model to describe the influence of phospho(enol)pyruvate on phosphofructokinase from Escherichia coli; Johnson JL et al.; A linked-function analysis is presented of the influence of the inhibitor phospho(enol)pyruvate (PEP) on the binding of fructose 6-phosphate (Fru-6-P) and MgATP to phosphofructokinase (PFK) from Escherichia coli . The results of this analysis indicate that the previously described inhibition of Fru-6-P binding by MgATP {Johnson, J . L., & Reinhart, G . D . (1992) Biochemistry 31, 11510-11518} is almost completely independent of the inhibition by PEP . Moreover, with or without the presence of MgATP, the inhibition by PEP does not conform to the behavior expected if PEP and Fru-6-P bind exclusively to different enzyme forms since the formation of a ternary complex with both PEP and Fru-6-P bound is clearly evident at high concentrations of Fru-6-P and PEP . van't Hoff analyses of the coupling interactions between PEP and Fru-6-P in the presence and absence of MgATP indicate that these couplings are driven by enthalpy . However, the influence that PEP has on MgATP binding is small and changes from being activating to being inhibitory at temperatures above 40 degrees C, revealing the importance of a compensating entropy component to the coupling interactions . The four functionally defined enzyme forms that contribute to the coupling between Fru-6-P and PEP were evaluated structurally using the fluorescence properties of the single intrinsic tryptophan as a probe . The steady-state and dynamic fluorescence emission and polarization properties of the tryptophan, as well as its susceptibility to I- quenching, indicate that the flexibility of PFK in the vicinity of the tryptophan is perturbed by the binding of ligands . The properties of free PFK do not lie between those established by the binding of Fru-6-P and PEP individually, indicating that it is structurally distinct . The properties of the ternary complex lie between those of the singly-ligated forms . Though an equilibrium mixture of two conformations of ternary complex cannot therefore be formally ruled out, no evidence obtained to date requires the presumption of this mechanistic complication.

Biochemistry, 1997 Oct 21, 36(42), 12733 - 8
Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis; Davidson VL et al.; Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin . Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins . Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation . Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue . Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99 . Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin . These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins . These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.

Biochemistry, 1997 Oct 21, 36(42), 12722 - 32
RNA recognition by a bent alpha-helix regulates transcriptional antitermination in phage lambda; Su L et al.; A novel RNA recognition motif is characterized in an arginine-rich peptide . The motif, derived from lambda transcriptional antitermination protein N, regulates an RNA-directed genetic switch . Its characterization by multidimensional nuclear magnetic resonance (NMR) demonstrates specific RNA-dependent folding of N- and C-terminal recognition helices separated by a central bend . The biological importance of the bent alpha-helix is demonstrated by mutagenesis: binding is blocked by substitutions in the N peptide or its target (the boxB RNA hairpin) associated in vivo with loss of transcriptional antitermination activity . Although arginine side chains are essential, the peptide is also anchored to boxB by specific nonpolar contacts . An alanine in the N-terminal helix docks in the major groove of the RNA stem whereas a tryptophan in the C-terminal helix stacks against a purine in the RNA loop . At these positions all 19 possible amino acid substitutions have been constructed by peptide synthesis; each impairs binding to boxB . The pattern of allowed and disallowed substitutions is in accord with the results of random-cassette mutagenesis in vivo . The helix-bend-helix motif rationalizes genetic analysis of N-dependent transcriptional antitermination and extends the structural repertoire of arginine-rich domains observed among mammalian immunodeficiency viruses.

Biochemistry, 1997 Oct 21, 36(42), 12683 - 99
Structural characterization of an analog of the major rate-determining disulfide folding intermediate of bovine pancreatic ribonuclease A; Laity JH et al.; The major rate-determining step in the oxidative regeneration of bovine pancreatic ribonuclease A (RNase A) proceeds through des-{40-95} RNase A, a three-disulfide intermediate lacking the Cys40-Cys95 disulfide bond . An analog of this intermediate, {C40A, C95A} RNase A, has been characterized in terms of regular backbone structure and thermodynamic stability at pH 4.6 . Nearly complete backbone 1H, 15N, and 13C resonances, and most 13Cbeta side-chain resonances have been assigned for the mutant RNase A using triple-resonance NMR data and a computer program, AUTOASSIGN, for automated analysis of resonance assignments . Comparisons of chemical shift data, 3J(1HN-1Halpha) coupling constants, and NOE data for the mutant and wild-type proteins reveal that the overall chain folds of the two proteins are very similar, with localized structural perturbations in the regions spatially adjacent to the mutation sites in {C40A, C95A} RNase A . More significantly, 1H/2H amide exchange and thermodynamic data reveal a global destabilization of the mutant protein characterized by a significant difference in the midpoint of the thermal transition curves (DeltaTm of 21.8 degrees C) and a significant increase in the slowest exchanging backbone amide 1H/2H exchange rates (10(2)-10(6)-fold faster in the hydrophobic core of {C40A, C95 A} RNase A) . Comparisons of the entropy DeltaS degrees (T) and enthalpy DeltaH degrees (T) of unfolding between wild-type and {C40A, C95A} RNase A reveal that some of the global destabilization of the mutant protein arises from entropic and enthalpic changes in the folded state . Implications of these observations for understanding the role of des-{40-95} in the folding pathway of RNase A are discussed.

J Interferon Cytokine Res, 1997 Sep, 17(9), 551 - 8
In vivo effects of chicken interferon-gamma during infection with Eimeria; Lowenthal JW et al.; Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life . The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients . This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors . We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma) . Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coli, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively . Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages . Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period . Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model . Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina . Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds . The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.

J Leukoc Biol, 1997 Oct, 62(4), 517 - 23
Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM-CSF; Sweeney JF et al.; Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection . Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life-threatening infections, as well as local and systemic inflammatory responses . Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult . This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70-90% of PMN displaying features of apoptosis by 4 h after UV exposure . Bacterial lipopolysaccharide and granulocyte-macrophage colony-stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV-accelerated apoptosis . The UV-accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis.

J Bacteriol, 1997 Oct, 179(20), 6525 - 30
Transcriptional regulation of the cydDC operon, encoding a heterodimeric ABC transporter required for assembly of cytochromes c and bd in Escherichia coli K-12: regulation by oxygen and alternative electron acceptors; Cook GM et al.; The expression of the cydDC operon was investigated by using a chromosomal phi(cydD-lacZ) transcriptional fusion and primer extension analysis . A single transcriptional start site was found for cydD located 68 bp upstream of the translational start site, and Northern blot analysis confirmed that cydDC is transcribed as a polycistronic message independently of the upstream gene trxB . cydDC was highly expressed under aerobic growth conditions and during anaerobic growth with alternative electron acceptors . Aerobic expression was independent of ArcA and Fnr, but induction of cydDC by nitrate and nitrite was dependent on NarL and Fnr.

J Bacteriol, 1997 Oct, 179(20), 6518 - 21
A mutational study of the site-specific cleavage of EC83, a multicopy single-stranded DNA (msDNA): nucleotides at the msDNA stem are important for its cleavage; Kim K et al.; Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA . Such molecules are encoded by genetic elements called retrons . Unlike other retrons, retron EC83 isolated from Escherichia coli 161 produces RNA-free msDNA by site-specific cleavage of msDNA at 5'-TTGA/A-3', where the slash indicates the cleavage site . In order to investigate factors responsible for the msDNA cleavage, retron EC83 was treated with hydroxylamine and colonies were screened for cleavage-negative mutants . We isolated three mutants which were defective in msDNA cleavage and produced RNA-linked msDNA . They were all affected in msd, a gene for msDNA, with a base substitution at the bottom part of the msDNA stem . In contrast, base substitution at and around the cleavage site has no marked effect on msDNA synthesis or its cleavage . From these results, we concluded that the nucleotides at the bottom of the msDNA stem, but not the nucleotides at the cleavage site, play a major role in the recognition and cleavage of msDNA EC83.

J Bacteriol, 1997 Oct, 179(20), 6512 - 7
Two distinct models account for short and long deletions within sequence repeats in Escherichia coli; Schumacher S et al.; In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events . These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence . The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E . coli mismatch repair-deficient strains . In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat . These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E . coli . Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.

J Bacteriol, 1997 Oct, 179(20), 6472 - 9
Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2; Easter CL et al.; A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner . This region, designated par, consists of two divergently arranged operons: parCBA and parDE . The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division . The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown . It has been proposed that the parCBA operon encodes a plasmid partitioning system (M . Gerlitz, O . Hrabak, and H . Schwabb, J . Bacteriol . 172:6194-6203, 1990; R . C . Roberts, R . Burioni, and D . R . Helinski, J . Bacteriol . 172:6204-6216, 1990) . To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac) . The intact 3.2-kb region provided the highest degree of stability in the two strains tested . The ability of the parCBA or parDE region alone to promote stable maintenance in the E . coli strains was dependent on the particular strain and the growth temperature . Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon . To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing . In E . coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region . However, in the case of E . coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance . To examine the basis for the apparent differences in postsegregational killing between the two E . coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein . Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E . coli MV10delta lac . A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies . This plasmid was found to be lost from E . coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.

J Bacteriol, 1997 Oct, 179(20), 6464 - 71
Filamentous phage infection: required interactions with the TolA protein; Click EM et al.; Infection of Escherichia coli by the filamentous phage f1 is initiated by binding of the phage to the tip of the F conjugative pilus via the gene III protein . Subsequent translocation of phage DNA requires the chromosomally encoded TolQ, TolR, and TolA proteins, after the pilus presumably has withdrawn, bringing the phage to the bacterial surface . Of these three proteins, TolA is proposed to span the periplasm, since it contains a long helical domain (domain II), which connects a cytoplasmic membrane anchor domain (domain I) to the carboxyl-terminal domain (domain III) . By using a transducing phage, the requirement for TolA in an F+ strain was found to be absolute . The role of TolA domains II and III in the infective process was examined by analyzing the ability of various deletion mutants of tolA to facilitate infection . The C-terminal domain III was shown to be essential, whereas the polyglycine region separating domains I and II could be deleted with no effect . Deletion of helical domain II reduced the efficiency of infection, which could be restored to normal by retaining the C-terminal half of domain II . Soluble domain III, expressed in the periplasm but not in the cytoplasm or in the medium, interfered with infection of a tolA+ strain . The essential interaction of TolA domain III with phage via gene III protein appears to require interaction with a third component, either the pilus tip or a periplasmic entity.

J Bacteriol, 1997 Oct, 179(20), 6367 - 77
Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis; Pratt TS et al.; Fis is a small DNA-binding and -bending protein in Escherichia coli that is involved in several different biological processes, including stimulation of specialized DNA recombination events and regulation of gene expression . fis protein and mRNA levels rapidly increase during early logarithmic growth phase in response to a nutritional upshift but become virtually undetectable during late logarithmic and stationary phases . We present evidence that the growth phase-dependent fis expression pattern is not determined by changes in mRNA stability, arguing in favor of regulation at the level of transcription . DNA deletion analysis of the fis promoter (fis P) region indicated that DNA sequences from -166 to -81, -36 to -26, and +107 to +366 relative to the transcription start site are required for maximum expression . A DNA sequence resembling the integration host factor (IHF) binding site centered approximately at -114 showed DNase I cleavage protection by IHF . In ihf cells, maximum cellular levels of fis mRNA were decreased more than 3-fold and transcription from fis P on a plasmid was decreased about 3.8-fold compared to those in cells expressing wild-type IHF . In addition, a mutation in the ihf binding site resulted in a 76 and 61% reduction in transcription from fis P on a plasmid in the presence or absence of Fis, respectively . Insertions of 5 or 10 bp between this ihf site and fis P suggest that IHF functions in a position-dependent manner . We conclude that IHF plays a role in stimulating transcription from fis P by interacting with a site centered approximately at -114 relative to the start of transcription . We also showed that although the fis P region contains six Fis binding sites, Fis site II (centered at -42) played a predominant role in autoregulation, Fis sites I and III (centered at +26 and -83, respectively) seemingly played smaller roles, and no role in negative autoregulation could be attributed to Fis sites IV, V, and VI (located upstream of site III) . The fis P region from -36 to +7, which is not directly regulated by either IHF or Fis, retained the characteristic fis regulation pattern in response to a nutritional upshift.

J Bacteriol, 1997 Oct, 179(20), 6335 - 40
Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4; Stein MA et al.; The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon . This operon probably consists of all the proteins necessary for transport and metabolization of various polyols . The gene encoding SDH (smoS) was cloned and sequenced . Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family . For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter . Compared to values derived with the native host, R . sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield . SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure . Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.

J Bacteriol, 1997 Oct, 179(20), 6254 - 63
Use of an inducible regulatory protein to identify members of a regulon: application to the regulon controlled by the leucine-responsive regulatory protein (Lrp) in Escherichia coli; Bhagwat SP et al.; Procedures were developed to facilitate the identification of genes that belong to a given regulon and characterization of their responses to the regulator . The regulon controlled by the Escherichia coli leucine-responsive regulatory protein (Lrp) was studied by isolating random transcriptional fusions to lacZ, using lambda placMu53 and a strain in which lrp is under isopropylthio-beta-D-galactopyranoside (IPTG)-inducible control . Fusions exhibiting IPTG-responsive beta-galactosidase activity were cloned by integrating the suicide vector pIVET1 via homologous recombination at lacZ, followed by self-ligating digested chromosomal DNA . We verified the patterns of lacZ expression after using the plasmid clones to generate merodiploid strains with interrupted and uninterrupted copies of the same sequence . If the merodiploid expression pattern was unchanged from that shown by the original fusion strain, then the cloned fusion was responsible for the regulatory pattern of interest; a difference in the expression pattern could indicate that the original strain carried multiple fusions or that there were autogenous effects of having interrupted the fused gene . Using these procedures, we generated a fusion library of approximately 5 x 10(6) strains; approximately 3,000 of these strains were screened, yielding 84 Lrp-responsive fusions, and 10 of the 84 were phenotypically stable and were characterized . The responses of different fusions in a given operon to in vivo Lrp titrations revealed variations in expression with the position of insertion . Among the newly identified members of the regulon is an open reading frame (orf3) between rpiA and serA . Also, expression of a fusion just downstream of dinF was found to be Lrp dependent only in stationary phase.

J Bacteriol, 1997 Oct, 179(20), 6238 - 43
Functional domains of the InsA protein of IS2; Lei GS et al.; The InsA protein is a transcriptional regulator . It binds to the promoter region of insA and insAB' . To understand the molecular mechanism for the interaction between InsA and its binding sequence, the functional domains of InsA were identified . The glutaraldehyde cross-linking method and the two-hybrid expression system were used to study the protein-protein interaction of InsA . The results of these experiments showed that InsA forms homodimers . Deletion of the last 44 amino acid residues at its C terminus, but not the first 12 or 57 residues at the N terminus, abolished the ability of InsA to form homodimers, indicating that the protein-protein interaction domain of InsA is located at its C terminus . Gel retardation assays revealed that deletion of the last 29 amino acid residues at its C terminus had no effect on the DNA binding ability of InsA . In contrast, deletion of the first N-terminal 12 residues abolished the DNA binding capability of InsA . These results indicate that the DNA binding domain of InsA is located at its N terminus.

J Bacteriol, 1997 Oct, 179(20), 6228 - 37
Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization; Link AJ et al.; We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli . Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome . Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA . These deletions are placed in the E . coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision . Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted . Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach . When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions . When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen . Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ . The two genes proved to be nonessential in both rich and glucose-minimal media . In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ . These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E . coli . Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

J Biomol NMR, 1997 Jul, 10(1), 53 - 62
Backbone dynamics of oxidized and reduced D . vulgaris flavodoxin in solution; Hrovat A et al.; Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli . A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site . A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein . The backbone dynamics of both the oxidized and reduced forms of D . vulgaris flavodoxin were investigated using two-dimensional 15N-1H correlation NMR spectroscopy . T1, T2 and NOE data are obtained for 95% of the backbone amide groups in both redox states . These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo {(1982) J . Am . Chem . Soc., 104, 4546-4559, 4559-4570} . A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN . Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state.

J Biomol NMR, 1997 Jul, 10(1), 9 - 19
Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase; Moy FJ et al.; Fibroblast collagenase (MMP-1), a 169-residue protein with a molecular mass of 18.7 kDa, is a matrix metalloproteinase which has been associated with pathologies such as arthritis and cancer . The assignments of the 1H, 15N, 13CO and 13C resonances, determination of the secondary structure and analysis of 15N relaxation data of the inhibitor-free catalytic fragment of recombinant human fibroblast collagenase (MMP-1) are presented . It is shown that MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and antiparallel arrangement (residues 13-19, 48-53, 59-65, 82-85 and 94-99) and three alpha-helices (residues 27-43, 112-124 and 150-160) . This is nearly identical to the secondary structure determined from the refined X-ray crystal structures of inhibited MMP-1 . The major difference observed between the NMR solution structure of inhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is the dynamics of the active site . The 2D 15N-1H HSQC spectra, the lack of information in the 15N-edited NOESY spectra, and the generalized order parameters (S2) determined from 15N T1, T2 and NOE data suggest a slow conformational exchange for residues comprising the active site (helix B, zinc ligated histidines and the nearby loop region) and a high mobility for residues Pro138-Gly144 in the vicinity of the active site for inhibitor-free collagenase . In contrast to the X-ray structures, only the slow conformational exchange is lost in the presence of an inhibitor.

Nat Biotechnol, 1997 Oct, 15(10), 997 - 1001
Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist; Ciapponi L et al.; Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations . A viable alternative is to induce a neutralizing antibody response . Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response . The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.

Rev Hosp Clin Fac Med Sao Paulo, 1997 Jan-Feb, 52(1), 32 - 4
Primary community-acquired pneumonia by Escherichia coli . Case report and review of the literature; Sztajnbok J et al.; A two year old girl with chronic neurologic convulsive disease was admitted with a six day history of pneumonia and, despite treatment, died on hospital day 3 . The X-ray revealed right upper lobar pneumonia . The results of pleural effusion and blood cultures drawn on admission yielded a non-typable Escherichia coli . No other source of infection was identified . The authors discuss the clinical and pathophysiological aspects of Escherichia coli pneumonia.

Genes Dev, 1997 Oct 1, 11(19), 2580 - 92
Topoisomerase IV, not gyrase, decatenates products of site-specific recombination in Escherichia coli; Zechiedrich EL et al.; DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregation to occur . We showed recently that topoisomerase IV (topo IV) is the only important decatenase of DNA replication intermediates in bacteria . Earlier results, however, indicated that DNA gyrase has the primary role in unlinking the catenated products of site-specific recombination . To address this discordance, we constructed a set of isogenic strains that enabled us to inhibit selectively with the quinolone norfloxacin topo IV, gyrase, both enzymes, or neither enzyme in vivo . We obtained identical results for the decatenation of the products of two different site-specific recombination enzymes, phage lambda integrase and transposon Tn3 resolvase . Norfloxacin blocked decatenation in wild-type strains, but had no effect in strains with drug-resistance mutations in both gyrase and topo IV . When topo IV alone was inhibited, decatenation was almost completely blocked . If gyrase alone were inhibited, most of the catenanes were unlinked . We showed that topo IV is the primary decatenase in vivo and that this function is dependent on the level of DNA supercoiling . We conclude that the role of gyrase in decatenation is to introduce negative supercoils into DNA, which makes better substrates for topo IV . We also discovered that topo IV has an unexpectedly strong DNA relaxation activity that, together with gyrase and topo I, is able to set the supercoiling levels in Escherichia coli.

Genes Dev, 1997 Oct 1, 11(19), 2545 - 56
Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast; Kessler MM et al.; In yeast, four factors (CF I, CF II, PF I, and PAP) are required for accurate pre-mRNA cleavage and polyadenylation in vitro . CF I can be separated further into CF IA and CF IB . Here we show that CF IB is the 73-kD Hrp1 protein . Recombinant Hrp1p made in Escherichia coli provides full CF IB function in both cleavage and poly(A) addition assays . Consistent with the presence of two RRM-type motifs, Hrp1p can be UV cross-linked to RNA, and this specific interaction requires the (UA)6 polyadenylation efficiency element . Furthermore, the CF II factor enhances the binding of Hrp1p to the RNA precursor . A temperature-sensitive mutant in HRP1 yields mRNAs with shorter poly(A) tails when grown at the nonpermissive temperature . Genetic analyses indicate that Hrp1p interacts with Rna15p and Rna14p, two components of CF 1A . The HRP1 gene was originally isolated as a suppressor of a temperature-sensitive npl3 allele, a gene encoding a protein involved in mRNA export . Like Npl3p, Hrp1p shuttles between the nucleus and cytoplasm, providing a potential link between 3'-end processing and mRNA export from the nucleus.

Genes Dev, 1997 Oct 1, 11(19), 2482 - 93
A multifunctional DNA-binding protein that promotes the formation of serum response factor/homeodomain complexes: identity to TFII-I; Grueneberg DA et al.; The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay . However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro . Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1 . We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo . Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE) . SPIN is identical to the initiator-binding protein TFII-I . Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro . We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.

Science, 1997 Oct 17, 278(5337), 446 - 9
DNA solution of the maximal clique problem; Ouyang Q et al.; The maximal clique problem has been solved by means of molecular biology techniques . A pool of DNA molecules corresponding to the total ensemble of six-vertex cliques was built, followed by a series of selection processes . The algorithm is highly parallel and has satisfactory fidelity . This work represents further evidence for the ability of DNA computing to solve NP-complete search problems.

J Biol Chem, 1997 Oct 17, 272(42), 26756 - 60
Identification of an active site alanine in mevalonate kinase through characterization of a novel mutation in mevalonate kinase deficiency; Hinson DD et al.; Sequencing of polymerase chain reaction-amplified cDNAs from cultured cells of three patients with mevalonate kinase deficiency revealed a G --> A transversion at nucleotide 1000 of the coding region, converting alanine to threonine at position 334 (A334T) . To characterize this defect, we expressed wild-type and mutant cDNAs in Escherichia coli as the glutathione S-transferase fusion proteins, with purification by affinity chromatography . SDS-polyacrylamide gel electrophoresis analysis for wild-type and mutant fusion proteins indicated an expected molecular mass of 42-43 kDa . Kinetic characterization of the wild-type fusion protein yielded Km values of 150 +/- 23 and 440 +/- 190 microM (mean +/- S.E.) for substrates (RS)-mevalonate and ATP, respectively . Expressed wild-type mevalonate kinase (MKase) had a maximum velocity of 13.6 +/- 1.4 units/mg of protein (n = 22, +/-S.E.), whereas the A334T mutation yielded an enzyme with average Vmax of 0.26 +/- 0.02 unit/mg of protein (n = 6, +/-S.E.), representing a decrease to 1.4% of control Vmax . Restriction digestion with HhaI, in conjunction with direct sequencing of cDNAs, revealed that two patients were homozygous and one heterozygous for the A334T allele, establishing autosomal recessive inheritance within families . Although the A334T enzyme had a normal Km for ATP of 680 +/- 226 microM (n = 3, +/-S.E.), the Michaelis constant for (RS)-mevalonate was increased >30-fold to 4623 +/- 1167 microM (n = 4, +/-S.E.) under standard assay conditions . Comparable kinetic results were obtained using extracts of lymphoblasts, which were homozygous for the A334T allele . Alanine 334 is invariant in MKase from bacteria to man and located in a glycine-rich region postulated to have homology with ATP-binding sequences . Our results indicate that the bacterial expression system for human MKase will provide a useful model system in which to analyze inherited mutations and identify the first active site residue in MKase associated with stabilization of mevalonate binding.

J Biol Chem, 1997 Oct 17, 272(42), 26511 - 21
High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr; Seok YJ et al.; The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system . Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP) . The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis . Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase . HPr binding increases GP activity to 250% of the level in control assays . Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation . The stimulatory effect of E . coli HPr on E . coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form . Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important . This novel mechanism for the regulation of GP provides the first evidence directly linking E . coli HPr to the regulation of carbohydrate metabolism.

J Biol Chem, 1997 Oct 17, 272(42), 26497 - 504
Characterization of an epithelial approximately 460-kDa protein that facilitates endocytosis of intrinsic factor-vitamin B12 and binds receptor-associated protein; Birn H et al.; By using receptor-associated protein (RAP) as an affinity target, an intrinsic factor-vitamin B12 (IF-B12)-binding renal epithelial protein of approximately 460 kDa was copurified together with the transcobalamin-B12-binding 600-kDa receptor, megalin . IF-B12 affinity chromatography of renal cortex membrane from rabbit and man yielded the same approximately 460-kDa protein . Binding studies including surface plasmon resonance analyses of the protein demonstrated a calcium-dependent and high affinity binding of IF-B12 to a site distinct from the RAP binding site . The high affinity binding of IF-B12 was dependent on complex formation with vitamin B12 . Light and electron microscope autoradiography of rat renal cortex cryosections incubated directly with IF-57Co-B12 and rat proximal tubules microinjected in vivo with the radioligand demonstrated binding of the ligand to endocytic invaginations of proximal tubule membranes followed by endocytosis and targeting of vitamin B12 to lysosomes . Polyclonal antibodies recognizing the approximately 460-kDa receptor inhibited the uptake . Immunohistochemistry of kidney and intestine showed colocalization of the IF-B12 receptor and megalin in both tissues . In conclusion, we have identified the epithelial IF-B12-binding receptor as a approximately 460-kDa RAP-binding protein facilitating endocytosis.

J Biol Chem, 1997 Oct 17, 272(42), 26448 - 56
Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli; Coskun-Ari FF et al.; Arrest of DNA replication in Escherichia coli is mediated by specific interactions between the Tus protein and terminator (Ter) sequences . Binding of Tus to a Ter site forms a asymmetric protein-DNA complex that arrests DNA replication in an orientation-dependent fashion . In this study, mutant Ter sites carrying single base pair substitutions at 16 different positions were examined for their ability to bind purified Tus protein and arrest DNA replication . In vitro competition assays demonstrated that base pair substitutions at positions 8-19 had significant effects on the free energy of Tus binding (DeltaDeltaG0 of 1.5 to >4 . 0 kcal/mol) . Concomitant with loss of binding affinity, mutations at these positions also showed significantly lower or undetectable replication arrest activities in vivo . Substitutions at positions 6, 20, and 21 had moderate effects on Tus-Ter interactions, suggesting that these base pairs contribute to, but are not absolutely critical for, Tus binding . Even though the effects on binding were minimal, these Ter mutants were not as efficient as wild type Tus-TerB complexes at arresting replication forks . Three new potential Ter sites, referred to as TerH, TerI, and TerJ, were identified by searching the E . coli genome for sequence similarity to a consensus Ter site sequence.

J Biol Chem, 1997 Oct 17, 272(42), 26425 - 33
Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein; Salazar D et al.; Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II . In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired . Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid beta-oxidation . In this investigation, two mutations in the alpha subunit that have been identified in patients were expressed in Escherichia coli . Of the two mutant alleles, alphaT266M and alphaG116R, the former is the most frequent mutation found in patients with ETF deficiency . The crystal structure of human ETF shows that alphaG116 lies in a hydrophobic pocket, under a contact residue of the alpha/beta subunit interface, and that the hydroxyl hydrogen of alphaT266 is hydrogen-bonded to N(5) of the FAD; the amide backbone hydrogen of alphaT266 is hydrogen-bonded to C(4)-O of the flavin prosthetic group (Roberts, D . L., Frerman, F . E . and Kim, J-J . P . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 14355-14360) . Stable expression of the alphaG116R ETF required coexpression of the chaperonins, GroEL and GroES . alphaG116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts . It is unstable and could not be extensively purified . The alphaT266M ETF was purified and characterized after stabilization to proteolysis in crude extracts . Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein . The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF . The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone . However, kcat/Km of ETF-QO in a coupled acyl-CoA:ubiquinone reductase assay with oxidized alphaT266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the alphaT266M ETF semiquinone catalyzed by ETF-QO.

J Biol Chem, 1997 Oct 17, 272(42), 26110 - 6
An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner; Pazdernik NJ et al.; A conserved motif, GXXX(D/E)(R/K)XG{X}(R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters . The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N . J., Jessen-Marshall, A . E., and Brooker, R . J . (1997) J . Bacteriol . 179, 735-741) . Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport . In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function . A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V) . A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild-type strain, but its Vmax for lactose transport was over 30-fold lower . The primary effect of the second-site mutations was to increase the Vmax for lactose transport, in some cases, to levels that were near the wild-type value . When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made . Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects . These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

Antibiot Khimioter, 1997, 42(7), 8 - 11
{Effect of promethazine hydrochloride (pipolphen) on the stability of R plasmid resistance in Escherichia coli}; Evdokimova OV et al.; The influence of promethazin hydrochloride (pipolphen) on stability of R plasmid inheritance in Escherichia coli strains of various serogroups was studied . The strains were isolated from patients with acute intestinal infection and from healthy persons . It was shown that in subbacteriostatic concentrations (100 to 450 micrograms/ml) pipolphen promoted elimination of the R plasmids . Decreased stability of the R plasmid inheritance was not associated with the pipolphen concentration . No influence of the drug on the biochemical characteristics, antigenic properties and nutritional requirements of the plasmid-free derivatives was detected . The eliminating action of pipolphen and ethidium bromide in some strans of Escherichia coli was shown to be different.

Dtsch Tierarztl Wochenschr, 1996 Dec, 103(12), 511 - 2
{Investigations of raw milk for Shiga-like toxin producing Escherichia coli by the polymerase chain reaction}; Made D et al.; This is a report about the occurrence of Shiga-like toxin (SLT) producing Escherichia coli in milk in southern Sachsen-Anhalt . Samples were taken from tanks of milk at the farms . The samples were investigated by polymerase chain reaction as recommended by the "Bundesinstitut fur gesundheitlichen Verbraucherschutz und Veterinarmedizin" . Using PCR, in 11% of the tank samples sequences coding for SLT I and/or SLT II were detected . 75% of the as positive detected samples had eae gen . Some cows in these farms spread potentially enterohemorrhagic E . coli . These farms should not sell milk for raw consumption.

Antimicrob Agents Chemother, 1997 Oct, 41(10), 2127 - 31
The oxazolidinone eperezolid binds to the 50S ribosomal subunit and competes with binding of chloramphenicol and lincomycin; Lin AH et al.; The oxazolidinones are a novel class of antibiotics that act by inhibiting protein synthesis . It as been reported that the drug exerts its primary activity on the initiation phase of translation . In order to study the possibility of direct interaction between the drug and the ribosome, we have developed a binding assay using 14C-labelled eperezolid (PNU-100592; formerly U-100592) . Eperezolid binds specifically to the 50S ribosomal subunit of Escherichia coli . The specific binding of eperezolid is dose dependent and is proportional to the ribosome concentrations . Scatchard analysis of the binding data reveals that the dissociation constant (Kd) is about 20 microM . The binding of eperezolid to the ribosome is competitively inhibited by chloramphenicol and lincomycin . However, unlike chloramphenicol and lincomycin, eperezolid does not inhibit the puromycin reaction, indicating that the oxazolidinones have no effect on peptidyl transferase . In addition, whereas lincomycin and, to some extent, chloramphenicol inhibit translation termination, eperezolid has no effect . Therefore, we conclude that the oxazolidinones inhibit protein synthesis by binding to the 50S ribosomal subunit at a site close to the site(s) to which chloramphenicol and lincomycin bind but that the oxazolidinones are mechanistically distinct from these two antibiotics.

Oncogene, 1997 Sep 18, 15(12), 1385 - 94
Gamma-heregulin: a novel heregulin isoform that is an autocrine growth factor for the human breast cancer cell line, MDA-MB-175; Schaefer G et al.; A novel neuregulin isoform, termed gamma-HRG, was cloned and characterized from the human breast cancer cell line, MDA-MB-175 . As observed with other neuregulins, gamma-HRG, is a product of alternative mRNA splicing of the neuregulin gene . Gamma-HRG contains the EGF-like and immunoglobulin-like domains that are commonly found in other family members, but lacks a transmembrane and cytoplasmic region . The new isoform possesses a unique N-terminal region that includes a hydrophobic domain that may function as a secretion signal . A purified recombinant version of gamma-HRG competes for binding to soluble ErbB3- and ErbB4-IgG fusion proteins with affinities similar to those observed for rHRGbeta1(177-244) . Gamma-HRG has a wide distribution in mesenchymal or neuronal tissues but in contrast to other neuregulins, it is not present in breast, lung, liver and small intestine . Expression of gamma-HRG with its cognate receptors, ErbB3 and ErbB2 suggested that the growth of the MDA-MB-175 cell line might be a result of the autocrine stimulation of a growth factor signaling pathway . Treatment of MDA-MB-175 cells with an anti-ErbB2 monoclonal antibody that interferes with the ligand-dependent formation of ErbB2-ErbB3 heterodimer complexes shows a strong growth inhibitory effect on this cell line . Moreover, incubation with a receptor-IgG fusion protein that neutralizes secreted gamma-HRG, also inhibits cell growth . These data suggest that the secretion of gamma-HRG by MDA-MB-175 cells leads to the formation of a constitutively active receptor complex and stimulates the growth of these cells in an autocrine manner.

Brain Res Mol Brain Res, 1997 Sep, 48(2), 206 - 14
Expression and characterization of human beta-secretase candidates metalloendopeptidase MP78 and cathepsin D in beta APP-overexpressing cells; Thompson A et al.; Human beta-secretase candidates, MP78 (h-MP78, EC 3.4.24.15) and cathepsin D (Cat D, EC 3.4.23.5), were evaluated for their ability to enhance amyloid-beta-protein (A beta) secretion when overexpressed in beta APP-containing cells . HEK-293 cells stably co-expressing h-MP78 or Cat D and h-beta APP695 were metabolically labeled with {35S}methionine and A beta secretion was quantified in the conditioned media by immunoprecipitation and ELISA without showing any significant increase in A beta production . Because Cat D is known to have a higher affinity for APP-substrate containing the Swedish familial Alzheimer's disease double mutation (SFAD, K595N and M596L substitutions in beta APP695) than for the wild type substrate {Dreyer et al., Eur . J . Biochem., 224 (1994) 265-271}, the effect of Cat D overexpression was tested in a HEK293/beta APPSFAD stable cell line . ELISA analysis of the conditioned media from these cells did also not reveal any increase in A beta generation . In addition, recombinant h-MP78 purified from E . coli cleaved an APP-derived substrate spanning the beta-secretase site (ISEVKMD1AEFRHDS) at multiple sites, but the beta-site cleavage was only a minor one; cleavage occurred predominantly at K-M and E-F bonds . Human liver Cat D also cleaved the same substrate at multiple sites, yet the major cleavage at pH 4.0 occurred at the amyloidogenic D1 site . These findings indicate that h-MP78 does not have the cleavage specificity required for a beta-secretase protease and although Cat D fulfilled the amyloidogenic cleavage specificity, the results of the co-expression experiments make both enzymes less likely candidates as relevant beta-secretases.

Gene, 1997 Sep 15, 197(1-2), 289 - 93
Molecular cloning and expression of a rat cDNA encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; Akira T et al.; The cDNA of a 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase) was isolated from rat liver RNA by reverse transcription and the polymerase chain reaction (PCR) . The rat AICARFT/IMPCHase cDNA included 1928 bp containing a coding region of 1779 bp for a 592-amino acid polypeptide (Mr = 64 200) . Rat and human AICARFT/IMPCHase cDNAs show 84 and 91% homology at the nucleotide and amino acid sequence level, respectively . The protein produced by the rat cDNA using pET-expression system catalysed the penultimate and final steps of de novo purine biosynthesis . Northern analysis identified a 2.8-kb AICARFT/IMPCHase mRNA and the level of the AICARFT/IMPCHase transcripts increased markedly at 24 h after partial (70%) hepatectomy.

Gene, 1997 Sep 15, 197(1-2), 231 - 8
Molecular cloning and characterization of a cDNA encoding a bovine butanediol dehydrogenase; Smania AM et al.; Using a polyclonal antibody against a bovine brain 30-kDa protein (p30), we isolated from a lambda gt11 bovine brain expression library a cDNA that codifies a protein with an apparent molecular mass of 30 kDa . The cDNA nucleotide sequence contained a unique open reading frame encoding a 26.7 kDa polypeptide . The 257 amino acids deduced sequence showed a significant homology with several dehydrogenases, mainly with a bacterial acetoin reductase (62%) . The cloned cDNA identity was confirmed by the determination of acetoin reductase activity in lysogens of lambda phage constructions containing the full length cDNA . The results described in this report are to our knowledge the first molecular characterization of a 2,3-butanediol dehydrogenase in mammals.

Gene, 1997 Sep 15, 197(1-2), 165 - 8
Cloning and sequencing of the dnaK and grpE genes of Legionella pneumophila; Amemura-Maekawa J et al.; A 4.4-kb DNA fragment from Legionella pneumophila (Lp) was isolated, which could complement an Escherichia coli (Ec) dnaK ts mutant, HC4102 . Nucleotide sequence analysis of the region revealed two complete open reading frames (ORFs) encoding both a predicted DnaK protein of 644 aa and a predicted GrpE protein of 199 aa, and also the 5'-end of the predicted dnaJ gene organized in the order of grpE-dnaK-dnaJ . Consensus heat shock (HS) promoter sequences were identified upstream of the start of both grpE and dnaK transcripts . However, no obvious promoter sequences were detected upstream of dnaJ . The transcription start points of grpE and dnaK were determined by primer extension analysis and the amount of each of the transcripts increased four- to eightfold after HS.

Biol Pharm Bull, 1997 Sep, 20(9), 1036 - 8
Comparison of characterization among Bordetella calmodulin-like protein, bovine brain calmodulin and Escherichia coli acyl-carrier protein; Nagai M et al.; We previously reported that Bordetella calmodulin-like protein (CLP), like bovine brain calmodulin (CaM), enhances adenylate cyclase and phosphodiesterase activities . In this communication, antigenic and biochemical characters were compared between CLP and CaM . It was found that anti-CLP and anti-CaM sera obtained reacted with CaM and CLP, respectively . The amino acid composition of CLP was similar to CaM . The similarity was also found in an Escherichia coli acyl-carrier protein (ACP), a Ca(2+)-binding protein . Though the amino acid sequence of N-terminus region of CLP has no significant homology with CaM, ACP has highly homology in its N-terminus similar to CaM . These results suggest that CLP might act as a Ca(2+)-binding protein, and/or an ACP during the activation of adenylate cyclase and phosphodiesterase.

Biol Pharm Bull, 1997 Sep, 20(9), 943 - 7
Bioassay of human granulocyte colony-stimulating factor using human promyelocytic HL-60 cells; Yamaguchi T et al.; A new method for an assay of human granulocyte colony-stimulating factor (hG-CSF) has been developed using human promyelocytic HL-60 cells . The proliferation of HL-60 cells had been suppressed by the addition of dimethyl sulfoxide (DMSO) or retinoic acid (RA) . These differentiating agent-treated HL-60 cells exhibited an increase in their number in response to recombinant hG-CSF (rhG-CSF) . Neither dibutyl-cAMP nor interferon-gamma (IFN-gamma)-treated HL-60 cells, however, showed an increase in their number in response to rhG-CSF . The proliferation rate of DMSO-pretreated HL-60 cells was linearly increased from 0.3 to 10 ng/ml of rhG-CSF . L-Value of HL-60 cells assay was 0.027 +/- 0.012 . The activities of non-glycosylated rhG-CSF produced by Escherichia coli and glycosylated rhG-CSF produced by chinese hamster ovary (CHO) cells were compared using DMSO-treated HL-60 cells; no significant difference between them . DMSO-treated HL-60 cells also responded to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), but did not respond to erythropoietin or macrophage colony-stimulating factor, suggesting that the responsiveness of these cells to growth factor is restricted to myelogenic cytokines . In conclusion, DMSO- or RA-treated HL-60 cells are useful for the measurement of bioactivity of hG-CSF.

Biochemistry (Mosc), 1997 Jul, 62(7), 732 - 41
Isolation of a strain overproducing endonuclease Eco29kI: purification and characterization of the homogeneous enzyme; Pertzev AV et al.; The physical map of the plasmid pSACII1 carrying the genes of restriction-modification system Eco29kI (isoschizomer of SacII) was determined . The cloning of the Eco29kI endonuclease and methylase genes into the plasmid vector pUC129 produced recombinant strain Escherichia coli K802{pECO29A15} with Eco29kI synthesis level about 100 times higher than in the parent strain . The restriction endonuclease was purified from Escherichia coli K802 {pECO29A15} cells to near homogeneity using column chromatography sequentially on phosphocellulose, hydroxyapatite, and heparin-Sepharose and rechromatography on phosphocellulose . Biochemical characterization of the homogeneous R Eco29kI is given . The enzyme has molecular mass 24.5 kD and is present in the solution as a monomer.

Tissue Antigens, 1997 Sep, 50(3), 265 - 76
Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments; Pichuantes S et al.; Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes . It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 . To date, each family has a distinct endoglin mutation, most of which generate premature stop codons . The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein . We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain . Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants . Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin . Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5 . Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7 . Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12 . Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.

Structure, 1997 Sep 15, 5(9), 1219 - 30
Crystal structure of the epsilon subunit of the proton-translocating ATP synthase from Escherichia coli; Uhlin U et al.; BACKGROUND: Proton-translocating ATP synthases convert the energy generated from photosynthesis or respiration into ATP . These enzymes, termed F0F1-ATPases, are structurally highly conserved . In Escherichia coli, F0F1-ATPase consists of a membrane portion, F0, made up of three different polypeptides (a, b and c) and an F1 portion comprising five different polypeptides in the stoichiometry alpha 3 beta 3 gamma delta epsilon . The minor subunits gamma, delta and epsilon are required for the coupling of proton translocation with ATP synthesis; the epsilon subunit is in close contact with the alpha, beta, gamma and c subunits . The structure of the epsilon subunit provides clues to its essential role in this complex enzyme . RESULTS: The structure of the E . coli F0F1-ATPase epsilon subunit has been solved at 2.3 A resolution by multiple isomorphous replacement . The structure, comprising residues 2-136 of the polypeptide chain and 14 water molecules, refined to an R value of 0.214 (Rfree = 0.288) . The molecule has a novel fold with two domains . The N-terminal domain is a beta sandwich with two five-stranded sheets . The C-terminal domain is formed from two alpha helices arranged in an antiparallel coiled-coil . A series of alanine residues from each helix form the central contacting residues in the helical domain and can be described as an 'alanine zipper' . There is an extensive hydrophobic contact region between the two domains providing a stable interface . The individual domains of the crystal structure closely resemble the structures determined in solution by NMR spectroscopy . CONCLUSIONS: Sequence alignments of a number of epsilon subunits from diverse sources suggest that the C-terminal domain, which is absent in some species, is not essential for function . In the crystal the N-terminal domains of two epsilon subunits make a close hydrophobic interaction across a crystallographic twofold axis . This region has previously been proposed as the contact surface between the epsilon and gamma subunits in the complete F1-ATPase complex . In the crystal structure we observe what is apparently a stable interface between the two domains of the epsilon subunit, consistent with the fact that the crystal and solution structures are quite similar despite close crystal packing . This suggests that a gross conformational change in the epsilon subunit, to transmit the effect of proton translocation to the catalytic domain, is unlikely, but cannot be ruled out.

Structure, 1997 Sep 15, 5(9), 1147 - 56
Crystal structure of the A3 domain of human von Willebrand factor: implications for collagen binding; Huizinga EG et al.; BACKGROUND: Bleeding from a damaged blood vessel is stopped by the formation of a platelet plug . The multimeric plasma glycoprotein, von Willebrand factor (vWF), plays an essential role in this process by anchoring blood platelets to the damaged vessel wall under conditions of high shear stress . This factor mediates platelet adhesion by binding both to collagen of the damaged blood vessel and to glycoprotein Ib on the platelet membrane . The A3 domain of vWF allows it to bind to collagen types I and III present in the perivascular connective tissue of the damaged vessel wall . To gain insight into the mechanism of collagen binding by vWF, we have determined the crystal structure of the human vWF A3 domain . RESULTS: The crystal structure of the 20 kDa A3 domain of human vWF (residues 920-1111), determined by the method of multiwavelength anomalous dispersion at 1.8 A resolution, exhibits a common dinucleotide-binding fold . The putative collagen-binding site of the A3 domain is rather smooth and shows a markedly high concentration of negatively charged residues . This region encompasses a potential metal-binding site containing the motif DXSXS, which is required for ligand interaction in the homologous I-type domains of integrins CR3 and LFA-1 . Although vWF A3 has considerable sequence and structural similarity with CR3 and LFA-1 in this region, one loop of A3 adopts a conformation which is incompatible with ion binding . CONCLUSIONS: The structure of the A3 domain suggests that adhesion to collagen is primarily achieved through interactions between negatively charged residues on A3 and positively charged residues on collagen . The absence of a pronounced binding groove precludes a large van der Waals surface interaction between A3 and collagen and is consistent with the low affinity for collagen of a single A3 domain and the requirement for multimeric vWF for tight association with collagen . The absence of bound metal ions upon soaking the crystal in MgCl2 and vWF A3's conformational incompatibility for metal binding is consistent with the absence of a functional role for metal ion binding in A3, which contrasts the metal ion activation required for ligand binding by the homologous integrin I type domains.

Cancer Res, 1997 Oct 1, 57(19), 4325 - 32
Molecular chemotherapy combined with radiation therapy enhances killing of cholangiocarcinoma cells in vitro and in vivo; Pederson LC et al.; Cholangiocarcinoma is a virtually incurable tumor, resistant to current surgical, chemotherapy, and radiotherapy interventions . We applied the gene therapy strategy of toxin gene conversion of nontoxic prodrug to chemotherapeutic drug in combination with radiation therapy to the treatment of cholangiocarcinoma . In this regard, 5-fluorouracil (5-FU) is an accepted radiosensitizing and chemotherapeutic agent presently used in cancer therapy . The Escherichia coli enzyme cytosine deaminase (CD) converts the prodrug 5-fluorocytosine (5-FC) to 5-FU . Therefore, our goal was to express the CD gene in the human cholangiocarcinoma cell line, SK-ChA-1, assess the cytotoxicity of intracellular production of 5-FU, and determine any enhanced cell killing by the addition of external beam radiation . The susceptibility of SK-ChA-1 cells to recombinant adenoviral infection was determined by fluorescence-activated cell sorting analysis . We used the recombinant adenoviral vector AdCMVLacZ, encoding the E . coli beta-galactosidase reporter gene under control of the human cytomegalovirus (CMV) promoter, to infect SK-ChA-1 and HeLa cells at 10 and 100 plaque forming units (pfu)/cell, followed by FACS analysis . To evaluate CD-mediated conversion of 5-FC to 5-FU and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes CD . Cells were then plated in 96-well microtiter plates and exposed to varying concentrations of 5-FC . Cell proliferation assays (tetrazolium salt conversion to formazan colorimetric assay) were performed beginning 2-8 days after plating . We evaluated the effects of external beam radiation using a single 8 Gy 60Co dose to AdCMVCD infected cells, with prior exposure to 5-FC for 2-3 days . MTS assays were performed following radiation treatment . Radiation dose-response analysis, via clonogenic assay, was used as a more sensitive assay to confirm the interaction of the treatment conditions . s.c . SK-ChA-1 tumors in athymic nude mice were established, which then received three intratumoral injections of 1 x 10(9) pfu AdCMVCD . Mice received i.p . injections of 400 mg/kg of 5-FC twice daily for 7 days beginning the day of initial AdCMVCD injection (day -2) . The radiation treatment group received 10 Gy of 60Co exposure to their tumor on day 0 . SK-ChA-1 cells were efficiently transduced (48.7 and 99.2%) by 10 and 100 pfu/cell of AdCMVLacZ, respectively . From 37.9 to 84.4% of SK-ChA-1 cells were killed following infection with 10 pfu/cell AdCMVCD and 8 days of exposure to various concentrations of 5-FC (5, 10, 30, 50, and 100 microg/ml) . Higher 5-FC concentrations and longer duration of exposure resulted in greater cell killing . Radiation treatment (8 Gy) enhanced cell killing by greater than 70% when combined with 10 or 20 microg/ml of 5-FC . Radiation dose-response analysis with clonogenic assay confirmed enhanced SK-ChA-1 cell cytotoxicity as a result of radiation treatment following AdCMVCD infection and 5-FC exposure, with radiobiological parameters alpha = 0.44 and D0 = 0.96 . Combined treatment of SK-ChA-1 tumors with AdCMVCD, 5-FC, and radiation in animals resulted in significantly greater survival, time to tumor regrowth, and doubling time compared to the nonradiation treatment group (P = 0.03, 0.015, and 0.002, respectively) . Significantly greater change in tumor size, smaller ratio of final tumor size to original tumor size, and smaller final tumor size were observed in the radiation treatment group compared to the no radiation treatment group (P = 0.02, 0.03, and 0.03, respectively) . Human cholangiocarcinoma cells were transduced with a recombinant adenovirus in vitro at high efficiency and were susceptible to CD-mediated intracellular 5-FU production . Radiobiological survival curve parameters confirmed an interactive cytotoxic effect when viral infection and prodrug therapy were combined with external beam radiation exposure . (ABSTRACT TRUNCATED)

Cancer Res, 1997 Oct 1, 57(19), 4279 - 84
In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma; Lan KH et al.; Previously, we reported that adenoviral vectors carrying the carcinoembryonic antigen (CEA) promoter sequences to direct the Echerichia coli beta-galactosidase gene (AdCEA-lacZ) or cytosine deaminase (CD) gene (AdCEA-CD) confer selective gene expression on a CEA-positive gastric cancer cell line (MKN45) in vitro . Here, adenovirus-mediated tumor-specific gene therapy for CEA-positive gastric carcinoma in vivo was investigated . Using an animal model with i.p . disseminated MKN45 tumors, adenovirus-mediated tumor-specific transgene expression and therapeutic efficacy were analyzed . After an i.p . injection of AdCEA-lacZ, beta-galactosidase activity was confined to tumor xenografts . Moreover, CD mRNA was expressed exclusively in MKN45 tumor xenografts after infection with AdCEA-CD, despite the fact that an adenovirus-mediated transfer of CD DNA was detected in all tissues tested . In contrast, CD mRNA was detected not only in tumor xenografts but also in other organs of mice infected with AdCA-CD, in which CD gene expression is governed by an ubiquitous promoter . Suppression of tumor growth and prolongation of survival were noted in tumor-bearing mice treated with AdCEA-CD and 5-fluorocytosine (5FC) without observable adverse effects . In contrast, significant hepatic toxicity was noted in animals treated with AdCA-CD . These results reveal that the CEA promoter restricts CD gene expression to CEA-positive tumor cells in the adenoviral context in vivo, along with the beneficial therapeutic effects of 5FC treatment, suggesting the i.p . AdCEA-CD/5FC system may provide a novel approach to treatment of i.p . disseminated gastric cancer.

DNA Res, 1997 Jun 30, 4(3), 185 - 92
Differences in dinucleotide frequencies of human, yeast, and Escherichia coli genes; Nakashima H et al.; Nucleotide sequences coding proteins in human, yeast and Escherichia coli genes were analyzed in terms of dinucleotide occurrences . Every gene is plotted as a point in the dinucleotide space, which is spanned by 16 axes corresponding to the 16 components of the dinucleotide . The metric unit in the space is defined using the log-odds ratio of dinucleotide occurrences in a gene . The distribution of points showed that genes from the same organism are clustered in the space . The clusters of human and E . coli are completely separated, and the yeast cluster sits between, implying that individual genes are classified into the three sources from their location . In fact, they could be identified with accuracy of 90%, using the DNA data alone . Even genes encoding homologous proteins belonging to the same protein superfamily were discriminated by the DNA data, and were correctly identified into their sources with the same accuracy as above . DNA sequences of non-coding regions, including human introns, as well as human genes of GC-rich and GC-poor types, were also analyzed in the same manner . The most significant finding is that human genomic DNA sequences, including genes and introns together, exhibit the largest deviation of dinucleotide occurrence from the random expectation . Possible origins for this phenomenon are discussed.

Mutat Res, 1997 Sep 5, 379(1), 13 - 20
Spontaneous mutation frequencies and spectra in p53 (+/+) and p53 (-/-) mice: a test of the 'guardian of the genome' hypothesis in the Big Blue transgenic mouse mutation detection system; Buettner VL et al.; TSG-p53/Big Blue double transgenic mice offer a powerful tool for examining the effect of a p53 germline mutation on spontaneous somatic mutation in vivo . After sequencing the DNA-binding domain of the lacI gene, we previously reported no differences in mutant frequency between p53 nullizygous (-/-) and p53 wild-type (+/+) mice in liver, spleen and brain . However, jackpot mutations elsewhere in the gene may have obscured a real difference in mutation frequency and the small sample size of mutations not at CpG dinucleotides (n = 23) may have been insufficient to reveal differences in mutation spectra . Herein we have sequenced the entire lacI gene, including the promoter and lacZ operator regions . 123 additional independent mutations have been found including 70 mutations not at CpG sites . The mutation frequency was determined by correcting for jackpot mutations . There were no statistically significant differences in mutation frequency or spectrum between the p53 (+/+) and p53 (-/-) genotypes in any of the three tissues.

Mutat Res, 1997 Sep, 384(3), 195 - 204
Characterization of a human homolog of (6-4) photolyase; Todo T et al.; (6-4)Photolyase catalyzes light-dependent repair of UV-induced pyrimidine (6-4) pyrimidone photoproducts . A human cDNA clone which has high sequence homology to the (6-4)photolyase gene (H64PRH gene) was identified . In this paper we also isolated a genomic clone corresponding to the H64PRH cDNA and mapped it to chromosome 12q24.1 by fluorescence in situ hybridization (FISH) . Northern-blot analysis revealed transcription of this gene in all human tissues examined . The H64PRH protein was overproduced in E . coli, partially purified and characterized . Like (6-4)photolyase, the enzyme contains two chromophores, one of which is FAD . However, the enzyme does not show any detectable photolyase activity.

Mutat Res, 1997 Sep, 384(3), 181 - 94
Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity; Yakushiji H et al.; 8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is produced during cellular metabolism, and its misincorporation into DNA causes mutation . Human cells possess an enzyme that hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing misincorporation of 8-oxo-7,8-dihydroguanine into DNA . Sequence analyses of the MTH1 gene, encoding the 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphatase (8-oxo-dGTPase) protein in human cell lines revealed that a G to A base substitution frequently occurs at codon 83, which causes a change of valine to methionine in the MTH1 protein {Wu, C . et al., Biochem . Biophys . Res . Commun . 214 (1995) 1239-1245} . Here we isolated cDNAs for the two types of MTH1 protein and expressed them in Escherichia coli mutT- . cells, devoid of their own 8-oxo-dGTPase activity . The two forms of proteins were purified to physical homogeneity, and amino acid analyses confirmed that the variant protein, Met83-MTH1, indeed carries the corresponding amino acid substitution . Met83-MTH1, but not normal type Val83-MTH1, was separated into two peaks in hydrophobic interacting chromatography . 8-Oxo-dGTPase activity of Met83-MTH1 is more thermolabile than that of Val83-MTH1 . Circular dichroism (CD) and fluorescence spectroscopic analyses confirmed this conclusion . CD further indicated that Met83-MTH1 has a higher alpha-helix content.

Methods Enzymol, 1997, 287, 162 - 74
Expression of chemokine RANTES and production of monoclonal antibodies; Krensky AM et al.; RANTES was first identified as a cDNA in a search for genes expressed late (3-5 days) after T-cell activation . Definition of RANTES function depended on the generation of protein . This chapter describes the various techniques used to make recombinant RANTES protein, to test its activity, and to generate monoclonal antibodies to assess RANTES protein cell distribution.

J Protein Chem, 1997 Oct, 16(7), 721 - 32
A model for the unusual kinetics of thermal denaturation of rubredoxin; Wampler JE et al.; The thermal denaturation of the simple, redox-active iron protein rubredoxin is characterized by a slow, irreversible decay of the characteristic red color of the iron center at elevated temperatures in the presence of oxygen at pH 7.8 . The denaturation rate is essentially constant and the time period for complete bleaching is nearly independent of protein concentration . These two characteristics of the kinetics can be fit by a simple self-catalyzed kinetics model consisting of the combination of a first-order decay and catalysis by some product of that decay, i.e., dP/dt = k1{A} + (k2{P}{A})/(K(m) + {A}), where A is native rubredoxin, P, is unspecified product, k1 is a first-order rate constant, and k2 and K(m) are the catalytic constants . In order for the second term to be of this simple form over the full course of a decay, the model must include the condition that the reaction is effectively irreversible . This model has properties which suggest other biological roles in regulation (changes in k1 or k2 can dramatically modulate the kinetics), in timing (titer-independent fixed reaction time), and in self-activation reactions . At one extreme (k1 >> k2) the kinetics becomes exponential, but at the other extreme (k2 >> k1) they show a dramatic and rapid terminal increase after a lag period . Some obvious possible roles in the kinetics of programmed cell death, prion disease, and protease autoactivation are discussed.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11339 - 44
The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain; Moser C et al.; Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY) . In E . coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA . Ffh and FtsY each contain a conserved GTPase domain (G domain) with an alpha-helical domain on its N terminus (N domain) . The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques . Methods to describe the reaction kinetically are presented . The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases . The intrinsic guanine nucleotide dissociation rates of FtsY are about 10(5) times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor . Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase-nucleotide exchange factor complex not only in its structure but also kinetically . The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor . From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11274 - 8
Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors; Murakami K et al.; Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase alpha subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to alpha . Each CRP dimer was found to direct the positioning of one of the two alpha subunit CTDs . Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two alpha subunits and two different species of transcription factors.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11221 - 6
Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein; Li Z et al.; Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described . We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA . The recombinant enzyme had DNA-dependent ATPase activity with an estimated kcat of 1.5 min-1 . DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues . HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides . The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPgammaS.

Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11210 - 5
Inorganic polyphosphate and the induction of rpoS expression; Shiba T et al.; Inorganic polyphosphate {poly(P)} levels in Escherichia coli were reduced to barely detectable concentrations by expression of the plasmid-borne gene for a potent yeast exopolyphosphatase {poly(P)ase} . As a consequence, resistance to H2O2 was greatly diminished, particularly in katG (catalase HPI) mutants, implying a major role for the other catalase, the stationary-phase KatE (HPII), which is rpoS dependent . Resistance was restored to wild-type levels by complementation with plasmids expressing ppk, the gene for PPK {the polyphosphate kinase that generates poly(P)} . Induction of expression of both katE and rpoS (the stationary-phase sigma factor) was prevented in cells in which the poly(P)ase was overproduced . Inasmuch as this inhibition by poly(P)ase did not affect the levels of the stringent-response guanosine nucleotides (pppGpp and ppGpp) and in view of the capacity of additional rpoS expression to suppress the poly(P)ase inhibition of katE expression, a role is proposed for poly(P) in inducing the expression of rpoS.

Biochem Biophys Res Commun, 1997 Sep 29, 238(3), 784 - 9
The conserved serine-threonine-serine motif of the carnitine acyltransferases is involved in carnitine binding and transition-state stabilization: a site-directed mutagenesis study; Cronin CN; There has been speculation that the carnitine acyltransferase reaction mechanism may involve the formation of an acyl-serine intermediate . A serine-threonine-serine (STS) motif that is conserved throughout the carnitine acyltransferase family, and is present also in the choline acetyltransferases, contains the only two conserved serines . The functional role of this motif in carnitine octanoyltransferase was probed by using a site-directed mutagenesis strategy to generate all seven possible alanine substitutions: single, double and triple mutants . Kinetic analyses of these mutant enzymes demonstrated that the STS motif is not essential for catalysis, thereby excluding an acyl-serine intermediate from the reaction mechanism . The kinetic analyses support, however, substantial roles for the STS motif in carnitine binding and transition-state stabilization.

Biochem J, 1997 Sep 15, 326 ( Pt 3), 883 - 9
Molecular cloning of cDNA species for rat and mouse liver alpha-methylacyl-CoA racemases; Schmitz W et al.; cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized . The rat liver lambdagt11 cDNA expression library was screened with anti-racemase IgG {Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur . J . Biochem.231, 815-822} . Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da . The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence . The cDNA coding for mouse racemase was cloned from a mouse liver lambdaZAP cDNA expression library and sequenced . The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein . Expression of the rat racemase as are combinant protein in Escherichia coli with the pTrcHisB-expression vector yielded enzymically active protein . The amino acid sequences of alpha-methylacyl-CoA racemases do not resemble any known sequence of beta-oxidation or auxiliary enzymes, supporting the view of a highly diverse evolutionary origin of enzymes acting on fatty acyl-CoA S-esters.

Biochem J, 1997 Sep 15, 326 ( Pt 3), 785 - 9
Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica; Bruchhaus I et al.; The 29 kDa protein of Entamoeba histolytica (Eh29), as well as a truncated variant of this protein, which lacks a cysteine-rich N-terminal region of 40 amino acid residues (Eh29mut), were recombinantly expressed in Escherichia coli and purified to homogeneity . Both recombinant proteins (recEh29, recEh29mut) were found to have hydrogen peroxide (H2O2)-removing activity, but recEh29 was twice as active as recEh29mut . For the consumption of exogenous H2O2, activity was dependent on the presence of reducing equivalents, such as dithiothreitol (DTT), indicating that Eh29 constitutes a thiol-dependent peroxidase . DTT was not required to remove H2O2 by recEh29 or recEh29mut when H2O2 was generated enzymically by the E . histolytica NADPH:flavin oxidoreductase . This enzyme produces H2O2 under aerobic conditions and simultaneously serves as a hydrogen donor for Eh29 . Peroxidase activity of the recombinant proteins was further supported by complementation of an E . coli strain that lacks the entire alkyl hydroperoxide reductase locus . The high sensitivity of these bacteria against cumene hydroperoxide was significantly reduced by the introduction of the genes encoding recEh29 or recEh29mut . Using antisera raised against the recombinant proteins, native Eh29 was localized within the cytoplasm of the amoebae . In addition, the antisera reacted with proteins of E . histolytica lysates with apparent molecular masses of 35 kDa and 160-300 kDa . All of them exhibited thiol-peroxidase activity.

Biochem J, 1997 Sep 15, 326 ( Pt 3), 763 - 72
Functional and biophysical characterization of recombinant human hepatocyte growth factor isoforms produced in Escherichia coli; Stahl SJ et al.; Hepatocyte growth factor (HGF) is a pluripotent secreted protein that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and haematopoietic cells . Multiple mRNA species transcribed from a single HGF gene encode at least three distinct proteins: the full-length HGF protein and two truncated HGF isoforms that encompass the N-terminal (N) domain through kringle 1 (NK1) or through kringle 2 (NK2) . We report the high-level expression in Escherichia coli of NK1 and NK2, as well as the individual kringle 1 (K1) and N domains of HGF . All proteins accumulated as insoluble aggregates that were solubilized, folded and purified in high yield using a simple procedure that included two gel-filtration steps . Characterization of the purified proteins indicated chemical and physical homogeneity, and analysis by CD suggested native conformations . Although the K1 and N-terminal domains of HGF have limited biological activity, spectroscopic evidence indicated that the conformation of each matched that observed when the domains were components of biologically active NK1 . Both NK1 and NK2 produced in bacteria were functionally equivalent to proteins generated by eukaryotic systems, as indicated by mitogenicity, cell scatter, and receptor binding and activation assays . These data indicate that all four bacterially produced HGF derivatives are well suited for detailed structural analysis.

Biochem J, 1997 Sep 15, 326 ( Pt 3), 717 - 24
Biophysical and mutagenic analysis of Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase activity and specificity; Burdette DS et al.; The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein . The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation . Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding . X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere . Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit . The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding . The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn . All five mutant enzymes had </= 3% of wild-type catalytic activity, suggesting that the T . ethanolicus secondary ADH requires a properly co-ordinated catalytic Zn atom . The His-59 and Asp-150 mutations also altered secondary ADH affinity for propan-2-ol over a 140-fold range, whereas the overall change in affinity for ethanol spanned a range of only 7-fold, supporting the importance of the metal in secondary ADH substrate binding . The lack of significant changes in cofactor affinity as a result of these catalytic Zn ligand mutations suggested that secondary ADH substrate-and cofactor-binding sites are structurally distinct . Altering Gly198 to Asp reduced the enzyme specific activity 2.7-fold, increased the Km(app) for NADP+ 225-fold, and decreased the Km(app) for NAD+ 3-fold, supporting the prediction that the enzyme binds nicotinamide cofactor in a Rossmann fold . Our data indicate therefore that, unlike the liver primary ADH,the Rossmann-fold-containing T . ethanolicus secondary ADH binds its catalytic Zn atom using a sorbitol dehydrogenase-like Cys-His-Asp motif and does not bind a structural Zn atom.

J Clin Pathol, 1997 Jul, 50(7), 573 - 9
Frequency of pathogenic and enteroadherent Escherichia coli in patients with inflammatory bowel disease and controls; Schultsz C et al.; AIMS: To determine whether inflammatory bowel disease (IBD) is associated with pathogenic or enteroadherent Escherichia coli . METHODS: A least two stool specimens and one rectal biopsy were taken from 30 patients with IBD and from 20 controls . A large number of E coli-like colonies cultured from each stool sample and biopsy was tested, using DNA probes, for the presence of genes encoding shiga-like toxins, invasiveness, attachment-effacement and the ability to adhere to HEp-2 cells . Similarity among isolates from stool samples and rectal biopsies was determined by random amplified polymorphic DNA (RAPD) analysis . RESULTS: Enterohaemorrhagic and enteroinvasive E coli were not found in samples from either patients or controls . No significant difference in the detection rate of enteroadherent E coli between patients and controls was found . Rectal biopsies from 11 of 28 patients with IBD and 4 of 18 controls contained E coli, which hybridised with probes for detection of genes encoding diffuse adherence to HEp-2 cells, or encoding P-pili (p = 0.2) . Enteroadherent E coli isolated from two or three stool specimens from the same patient or control appeared to be identical by RAPD analysis, and are considered to be residents in the colon . Probe positive isolates obtained from stool specimens and corresponding rectal biopsies were always identical on RAPD analysis . CONCLUSIONS: E coli strains possessing adherence factors reside in the large intestine and adhere to the rectal mucosa, irrespective of the presence of colitis.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3944 - 9
Localization of the major ethidium bromide binding site on tRNA; Chu WC et al.; Binding of ethidium bromide to Escherichia coli tRNAVal and an RNA minihelix based on the acceptor stem and T-arm of tRNAVal was investigated by 19F and 1H NMR spectroscopy of RNAs labeled with fluorine by incorporation of 5-fluorouracil . Ethidium bromide selectively intercalates into the acceptor stem of the tRNAVal . More than one ethidium bromide binding site is found in the acceptor stem, the strongest between base pairs A6:U67 and U7:A66 . 19F and 1H spectra of the 5-fluorouracil-substituted minihelix RNA indicate that the molecule exists in solution as a 12 base-paired stem and a single-stranded loop . Ethidium bromide no longer intercalates between base pairs corresponding to the tRNAVal acceptor stem in this molecule . Instead, it intercalates between base pairs at the bottom of the long stem-loop structure . These observations suggest that ethidium bromide has a preferred intercalation site close to the base of an RNA helical stem.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3912 - 6
In vitro suppression as a tool for the investigation of translation initiation; Karginov VA et al.; An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase.Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3881 - 8
A DNA helicase purified by replication protein A (RPA) affinity chromatography from mouse FM3A cells; Hughes P et al.; In an effort to identify cellular helicases that mimic the action of SV40 large T-antigen, we performed replication protein A (RPA) affinity chromatography on cell extracts from the mouse mammary carcinoma cell line FM3A . In this way, a novel DNA helicase was isolated and purified to near homogeneity . The most purified fractions showed the presence of two proteins of 28 and 21 kDa . Both proteins interacted with 32P-labeled partially duplex DNA when bound to nitrocellulose membranes and were efficiently UV crosslinked to {alpha-32P}dATP . Helicase activity was strongly stimulated by RPA on DNA substrates containing duplex regions longer than 18 bp . Only weak stimulation was observed in the presence of Escherichia coli single strand DNA binding protein (SSB) . The enzyme unwinds DNA in the 5'-3' direction in relation to the strand to which it binds . Only ATP and dATP were efficient as nucleoside triphosphate co-factors, and showed similar Km values of approximately 0.6 mM . The properties of this enzyme suggest that it may take part in reactions mediated by RPA such as those predicted to occur at replication forks or alternatively may function during DNA repair or recombination.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3875 - 80
DNA binding and helicase domains of the Escherichia coli recombination protein RecG; Mahdi AA et al.; The Escherichia coli RecG protein is a unique junction-specific helicase involved in DNA repair and recombination . The C-terminus of RecG contains motifs conserved throughout a wide range of DNA and RNA helicases and it is thought that this C-terminal half of RecG contains the helicase active site . However, the regions of RecG which confer junction DNA specificity are unknown . To begin to assign structure-function relationships within RecG, a series of N- and C-terminal deletions have been engineered into the protein, together with an N-terminal histidine tag fusion peptide for purification purposes . Junction DNA binding, unwinding and ATP hydrolysis were disrupted by mutagenesis of the N-terminus . In contrast, C-terminal deletions moderately reduced junction DNA binding but almost abolished unwinding . These data suggest that the C-terminus does contain the helicase active site whereas the N-terminus confers junction DNA specificity.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3868 - 74
Interaction of p53 with the human Rad51 protein; Buchhop S et al.; p53 is thought to function in the maintenance of genomic stability by modulating transcription and interacting with cellular proteins to influence the cell cycle, DNA repair and apoptosis . p53 mutations occur in >50% of human cancers, and cells which lack wild type p53 accumulate karyotypic abnormalities such as amplifications, deletions, inversions and translocations . We propose that p53 hinders these promiscuous recombinational events by interacting with cellular recombination and repair machinery . We recently reported that p53 can directly bind in vivo to human Rad51 (hRad51) protein and in vitro to its bacterial homologue RecA . We used GST-fusion and his-tagged protein systems to further investigate the physical interaction between p53 and hRad51, homologue of the yeast Rad51 protein that is involved in recombination and DNA double strand repair . The hRad51 binds to wild-type p53 and to a lesser extent, point mutants 135Y, 249S and 273H . This binding is not mediated by a DNA or RNA intermediate . Mapping studies using a panel of p53 deletion mutants indicate that hRad51 could bind to two regions of p53; one between amino acids 94 and 160 and a second between 264 and 315 . Addition of anti-p53 antibody PAb421 (epitope 372-381 amino acids) inhibited the interaction with hRad51 . In contrast, p53 interacts with the region between aa 125 and 220 of hRad51, which is highly conserved among Rad51 related proteins from bacteria to human . In Escherichia coli ecA protein, this region is required for homo-oligomerization, suggesting that p53 might disrupt the interaction between RecA and Rad51 subunits, thus inhibiting biochemical functions of Rad51 like proteins . These data are consistent with the hypothesis that p53 interaction with hRAD51 may influence DNA recombination and repair and that additional modifications of p53 by mutation and protein binding may affect this interaction.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3840 - 6
Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000; Stolt P et al.; The regulatory region of the Mycobacterium fortuitum plasmid pAL5000 was studied in vivo and in vitro by mutational analysis . This region comprises the origin of replication for the plasmid and the start point of transcription for the repA/B genes, which encode the two replication proteins RepA and RepB . In this region there are two binding sites for RepB: a low-affinity site which is probably the origin of replication and a high-affinity-site which overlaps the promoter and implies an autoregulated expression of RepB . The high-affinity site contains two 8 bp palindromes, as well as an inverted repeat structure . By introducing point mutations into each of these motifs and monitoring changes to RepB binding in a gel-retardation assay, it was shown that the central, GC-rich palindrome (the GC-box) is the most important motif for protein binding . Mutations in the second, AT-rich palindrome (the AT-box) had no effect on protein binding and the inverted repeat structure per se was not needed, though some single-base changes affected binding to one or other of the DNA strands . These mutations were subsequently tested in vivo for their effects on plasmid replication in Mycobacterium smegmatis . Any change to the GC-box abolished replication, but changes to the other motifs were dependent on the position of the changed base, again indicating that the inverted repeats are not essential and that the AT-box is part of the promoter and not primarily recognised by RepB . The mutated plasmids did not show any changes in copy number to that of the wild-type . The expression of RepB was boosted by introducing a stronger promoter upstream of the repA/B genes . The resulting plasmid was capable of increasing to a degree in trans the copy number of other plasmids carrying the ori region, but was unstable when present on its own in M.smegmatis.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3832 - 9
Stimulation of DNA inversion by FIS: evidence for enhancer-independent contacts with the Gin-gix complex; Deufel A et al.; Efficient DNA inversion catalysed by the invertase Gin requires the cis-acting recombinational enhancer and the Escherichia coliFIS protein . Binding of FIS bends the enhancer DNA and, on a negatively supercoiled DNA inversion substrate, facilitates the formation of a synaptic complex with specific topology . Previous studies have indicated that FIS-independent Gin mutants can be isolated which have lost the topological constraints imposed on the inversion reaction yet remain sensitive to the stimulatory effect of FIS . Whether the effect of FIS is purely architectural, or whether in addition direct protein contacts between Gin and FIS are required for efficient catalysis has remained an unresolved question . Here we show that FIS mutants impaired in DNA binding are capable of either positively or negatively affecting the inversion reaction both in vivo and in vitro . We further demonstrate that the mutant protein FIS K25E/V66A/M67T dramatically enhances the cleavage of recombination sites by FIS-independent Gin in an enhancer-independent manner . Our observations suggest that FIS plays a dual role in the inversion reaction and stimulates both the assembly of the synaptic complex as well as DNA strand cleavage.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3777 - 82
Effects of 5-(N-aminohexyl)carbamoyl-2'-deoxyuridine on endonuclease stability and the ability of oligodeoxynucleotide to activate RNase H; Ueno Y et al.; To evaluate an endonuclease resistance property of oligodeoxynucleotides (ODNs) containing 5-(N-aminohexyl)carbamoyl-2'-deoxyuridines (Hs) and to elucidate whether a duplex consisting of the ODN analogue and its complementary RNA induces RNase H activity, the ODNs containing the deoxyuridine analogues, Hs, at intervals of one, two, three, four and five natural nucleosides were synthesized . From partial hydrolysis of these ODNs with nuclease S1 (an endonuclease), it was found that the ODNs became more stable towards nucleolytic hydrolysis by the enzyme as the number of H increased . Furthermore, to examine whether the duplexes composed of the ODNs containing Hs and their complementary RNAs are substrates for RNase H or not, the duplexes of these ODNs and their complementary RNA strands were treated with Escherichia coliRNase H . It was found that cleavage of the RNA strands by the enzyme was kinetically affected by the introduction of Hs into the duplexes.

Nucleic Acids Res, 1997 Oct 1, 25(19), 3767 - 76
Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli; Wang J et al.; I- CreI is a member of the LAGLI-DADG family of homing nucleases; however, unlike most members of this family it contains only a single copy of this signature motif . I- CreI was over-expressed in Escherichia coli, and a simple purification protocol developed that gave reasonably pure protein in high yield . Size-exclusion chromatography and chemical cross-linking indicated that the protein is a dimer in solution . DNA cleavage by I- CreI was absolutely dependent on Mg2+(or Mn2+), and was inhibited by monovalent cations . I- CreI displayed a surprisingly high temperature optimum (>50 degrees C), with full activity occurring even at 70 degrees C . Interestingly, SDS was needed for efficient release of the cleavage products from the protein, indicating formation of very stable DNA-protein complexes . In contrast to these robust characteristics, purified I- CreI was unstable; however, it could be stabilized by the addition of either target or non-target DNA . Mobility shift assays revealed that I- CreI binds to DNA in the absence of Mg2+ . Hydroxyl radical footprinting showed that I- CreI strongly protected the backbone of a continuous stretch of at least 12 nt on each strand that were shifted, relative to each other, by 2 bp in the 3'direction . Methylation protection and interference analyses were also performed, and together with the hydroxyl radical footprinting, indicate that I- CreI binds in both the major and minor grooves of its target DNA.

FEBS Lett, 1997 Sep 15, 414(3), 562 - 6
Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv); Zhou N et al.; The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B . The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced . Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded . Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side . The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative . This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

FEBS Lett, 1997 Sep 15, 414(3), 521 - 6
Selection and identification of single domain antibody fragments from camel heavy-chain antibodies; Arbabi Ghahroudi M et al.; Functional heavy-chain gamma-immunoglobulins lacking light chains occur naturally in Camelidae . We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders . The recombinant binders are expressed well in E . coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens . This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.

FEBS Lett, 1997 Sep 15, 414(3), 514 - 20
Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG-D; Dow LK et al.; HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster . During overexpression and purification of HMG-D from E . coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide . Oxidation of this critical residue decreases the affinity of HMG-D for DNA by three-fold, altering the structure of the HMG-D-DNA complex without affecting the structure of the free protein . This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.

Hypertension, 1997 Sep, 30(3 Pt 2), 708 - 13
Gene transfer to carotid sinus in vivo: a novel approach to investigation of baroreceptors; Meyrelles SS et al.; Baroreceptor nerve endings are located in the adventitia of the carotid sinuses and aortic arch . The goal of the present study was to develop a method for gene transfer to the carotid sinus adventitia . Replication-deficient adenovirus containing the gene for Escherichia coli beta-galactosidase (beta-Gal) was applied topically to the carotid sinuses of anesthetized rabbits . Transgene expression was localized by histochemical staining and quantified by chemiluminescence assay (Galacto-Light) . Possible effects of adenovirus on baroreceptor sensitivity were investigated by recording baroreceptor activity from the vascularly isolated carotid sinus over a pressure range of 0 to 160 mm Hg . Beta-Gal expression in carotid sinus was evident 1 day after virus application, was dose dependent, and was markedly enhanced after 4 days . Expression was restricted to the adventitia of the vessel wall and was not present in vehicle-treated carotid sinuses . Baroreceptor sensitivity measured from carotid sinuses exposed to adenovirus 4 to 5 days beforehand was not altered compared with that measured from control carotid sinuses . In summary, topical application of adenoviral vectors to the carotid sinus provides transgene expression restricted to the region of baroreceptor innervation . The technique provides a novel approach to delineate mechanisms involved in baroreceptor activation and to deliver neuroactive gene products to the baroreceptors.

Endocrinology, 1997 Oct, 138(10), 4273 - 81
Endotoxin inhibits the reproductive neuroendocrine axis while stimulating adrenal steroids: a simultaneous view from hypophyseal portal and peripheral blood; Battaglia DF et al.; This study was designed to test the hypothesis that systemic immune challenge with endotoxin inhibits the reproductive axis centrally by suppressing GnRH pulsatile release into hypophyseal portal blood . Using alert, normally behaving, ovariectomized ewes, we sampled hypophyseal portal blood at 10-min intervals beginning 4 h before and continuing 10 h after endotoxin (400 ng/kg, iv bolus, n = 6) or saline (vehicle, iv, n = 6) . Simultaneous jugular samples for measurement of LH, cortisol, and progesterone were taken, and core body temperature was monitored by telemetry . Saline had no effect on any of the parameters in control ewes . In contrast, endotoxin dramatically inhibited the reproductive neuroendocrine axis coincident with stimulating the adrenal steroids, cortisol and progesterone, and elevating body temperature . Mean GnRH collection rate and GnRH pulse amplitude were suppressed (pre- vs . 7 h postendotoxin: collection rate 0.93 +/- 0.31 vs . 0.34 +/- 0.13 pg/min; amplitude 4.13 +/- 1.33 vs . 1.30 +/- 0.41 pg/min per pulse; P < 0.05 and P = 0.01) . However, endotoxin did not have a significant effect on GnRH pulse frequency . Along with inhibited GnRH secretion, endotoxin significantly suppressed mean LH concentrations (P = 0.001) and LH pulse amplitude (P < 0.05) . In addition, endotoxin suppressed LH pulse frequency (P = 0.01) . Coincident with reproductive inhibition, endotoxin stimulated cortisol (P < 0.001), progesterone (P < 0.01), and core body temperature (P < 0.001) . We conclude that the suppressive effects of endotoxin on the reproductive axis can be mediated centrally through an inhibition of GnRH and thus LH pulsatile secretion . The coincident stimulation of cortisol, progesterone, and temperature raises the possibility that the central inhibition of the reproductive system may be a consequence of any or all of these activated parameters.

Endocrinology, 1997 Oct, 138(10), 4069 - 80
Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity; Helman D et al.; Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors . To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli . Circular dichroic spectrum analyses indicated proper refolding in all cases . Biological activity of these analogs was tested in vitro . In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly . In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL . In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs . In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding . Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity . In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced . Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values . The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex . In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD . These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.

Gene, 1997 Sep 1, 196(1-2), 231 - 7
Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon; Satchidanandam V et al.; Secondary structure analysis of the mRNA from a nonproductive construct carrying the nonstructural gene 3 (NS3) of Japanese Encephalitis Virus revealed the presence of a potential 28 nucleotide long stem and loop beginning with the guanine of the initiation codon AUG that had a calculated stabilization energy of -13 kcal/mol (delta Gfzero) . Provision of an additional AUG along with three codons upstream resulted in complete relief of inhibition . N-terminal amino acid sequence of the recombinant protein was consistent with initiation of protein synthesis having occurred from the upstream AUG . Similar levels of NS3 specific RNA in E . coli cells carrying the expressing and nonexpressing constructs and restoration of recombinant protein expression following deletion of segments beginning with the stem and loop confirmed the role of this structure in blocking expression at the level of translation initiation . Our approach exploits the ability of a ribosome in motion to open up downstream secondary structural elements of considerable stability and represents a novel and widely applicable strategy to overcome a block in translation initiation caused by mRNA secondary structure around the translation start site.

Gene, 1997 Sep 1, 196(1-2), 175 - 80
Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme; Kroger M et al.; The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components . Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV . As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E . coli as a minienzyme . By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells . The cDNA fragment obtained was cloned in E . coli and sequenced . With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported . The protein was expressed in E . coli using the vector pET-12b . The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea . The product was purified by affinity chromatography and gel filtration . Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase . The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity . It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase . The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Gene, 1997 Sep 1, 196(1-2), 95 - 8
Monovalent cations differ in their effects on transcription initiation from a sigma-70 promoter of Escherichia coli; Wang JY et al.; Initiation of transcription from the sigma-70 rep promoter of plasmid pBR322 was measured by abortive transcription assays at various concentrations of potassium, rubidium, and sodium acetate . When linear and negatively supercoiled templates were compared, each salt generated a characteristic response . Increasing the salt concentration decreased transcription from a linear template but produced an increase (potassium) or a bell-shaped response (rubidium) with a supercoiled template . In the case of sodium ions, increasing concentration inhibited transcription initiation from both linear and supercoiled templates . These results are discussed with respect to effects of monovalent cations on DNA twist.

Gene, 1997 Sep 1, 196(1-2), 19 - 23
Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression; Sun B et al.; A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced . The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx . 118 kDa . The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms . Northern blot analysis reveals a single transcript of approx . 3.5 kb in all the rabbit tissues examined . The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein . The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies . Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional.

Gastroenterology, 1997 Oct, 113(4), 1323 - 33
Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats; Vos TA et al.; BACKGROUND & AIMS: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased . NO has been suggested to have a hepatoprotective function . The aim of this study was to investigate the distribution of iNOS and the effect of different NO synthase inhibitors on liver damage and hemodynamics during endotoxemia . METHODS: Rats were injected with lipopolysaccharide (LPS) and received the NOS-inhibitor S-methylisothiourea (SMT) or NG-nitro-L-arginine methyl ester (L-NAME) . iNOS induction was assessed by Western blot, immunohistochemistry, and measurement of NO metabolites in plasma and bile . Liver damage was determined by aspartate aminotransferase and alanine aminotransferase and by histology . The effects of both inhibitors on systemic and portal pressure were measured in normal and LPS-treated rats . RESULTS: LPS treatment strongly induced iNOS in inflammatory cells, macrophages, bile duct epithelium, and hepatocytes, especially at the canalicular membrane . LPS-induced liver damage strongly increased after L-NAME . SMT caused a similar reduction of NO production without enhancing liver damage . In LPS-treated rats, SMT increased the systemic and portal pressure significantly more than L-NAME . CONCLUSIONS: During endotoxemia, administration of the NOS-inhibitor L-NAME aggravates liver damage . This liver damage does not seem to be caused by hemodynamic changes . In contrast, SMT caused significant hemodynamic changes but did not increase LPS-induced liver damage.

Gastroenterology, 1997 Oct, 113(4), 1110 - 7
Helicobacter pylori lipopolysaccharide stimulates histamine release and DNA synthesis in rat enterochromaffin-like cells; Kidd M et al.; BACKGROUND & AIMS: Helicobacter pylori alterations in gastric acid output and mucosal proliferation may involve the enterochromaffin-like (ECL) cell . To test whether H . pylori affects ECL cell histamine secretion and proliferation, the effect of lipopolysaccharide (LPS) on ECL cell function in vitro was investigated . METHODS: Using a rat ECL cell preparation of high purity (+/-95%), basal and stimulated histamine secretion and DNA synthesis were measured by enzyme immunoassay and bromodeoxyuridine (BrdU) uptake, respectively . RESULTS: Escherichia coli LPS (10(-12) to 10(-6) mol/L) had no effect on basal and stimulated histamine secretion at concentrations of > 10(-6) mol/L . H . pylori LPS stimulated basal and gastrin-stimulated histamine secretion . These effects were completely inhibited by somatostatin (10(-10) mol/L) but not by the gastrin receptor antagonist L365,260 at 10(-6) mol/L . E . coli LPS had a weak stimulatory effect on ECL cell BrdU uptake at 10(-6) mol/L but had no effect on gastrin-stimulated BrdU uptake . H . pylori LPS did not stimulate basal synthesis but significantly increased (1.5-fold) gastrin-stimulated BrdU uptake . CONCLUSIONS: H . pylori influences both ECL cell proliferation and secretion in vitro . An interaction between H . pylori LPS and ECL cells may contribute to the reported abnormalities in acid secretion and gastric pathobiology noted in H . pylori infections.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 167 - 78
Detection of distant structural similarities in a set of proteins using a fast graph-based method; Koch I et al.; We introduce a method for finding weak structural similarities in a set of protein structures . Proteins are considered at their secondary structure level . The method uses a rigorous graph-theoretical algorithm which finds all structural similarities . Protein structures are modelled as undirected labelled graphs, the so-called protein graphs . We suggest that for detecting the similarities between two protein structures it is sufficient to find similarities in the protein core which consists of tightly packed secondary structure elements . Therefore, we can restrict ourselves to solving the maximal common connected subgraph problem instead of the maximal common subgraph problem . We have modified the algorithm by Bron and Kerbosch for solving that problem . The speed of the algorithm increases drastically . After calculating all maximal common connected substructures for all pairwise comparisons in a set of protein graphs the common substructure in all proteins can be calculated by intersecting them . In this paper we characterize the method briefly and explain the modelling of the protein structure in detail . For the pairwise alignment the similarity of porin (1OMF) with bacteriochlorophyll a (3BCL) and BirA protein (1BIB) with DNA polymerase III (2POL) will be discussed . In the case of the multiple structure alignment the similarity in variants of four phosphatases and in subtilisin Carlsberg, carboxypeptidase, elongation factor Tu, and flavodoxin will be represented . Our first experiments show that the method works correctly and fast . The method can be used for arbitrary graphs . Thus, different graph-theoretical models of protein structures can be examined.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 147 - 52
Better prediction of protein cellular localization sites with the k nearest neighbors classifier; Horton P et al.; We have compared four classifiers on the problem of predicting the cellular localization sites of proteins in yeast and E . coli . A set of sequence derived features, such as regions of high hydrophobicity, were used for each classifier . The methods compared were a structured probabilistic model specifically designed for the localization problem, the k nearest neighbors classifier, the binary decision tree classifier, and the naive Bayes classifier . The result of tests using stratified cross validation shows the k nearest neighbors classifier to perform better than the other methods . In the case of yeast this difference was statistically significant using a cross-validated paired t test . The result is an accuracy of approximately 60% for 10 yeast classes and 86% for 8 E . coli classes . The best previously reported accuracies for these datasets were 55% and 81% respectively.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 84 - 7
RIBOWEB: linking structural computations to a knowledge base of published experimental data; Chen RO et al.; The world wide web (WWW) has become critical for storing and disseminating biological data . It offers an additional opportunity, however, to support distributed computation and sharing of results . Currently, computational analysis tools are often separated from the data in a manner that makes iterative hypothesis testing cumbersome . We hypothesize that the cycle of scientific reasoning (using data to build models, and evaluating models in light of data) can be facilitated with resources that link computations with semantic models of the data . Riboweb is an on-line knowledge-based resource that supports the creation of three-dimensional models of the 30S ribosomal subunit . It has three components: (I) a knowledge base containing representations of the essential physical components and published structural data, (II) computational modules that use the knowledge base to build or analyze structural models, and (III) a web-based user interface that supports multiple users, sessions and computations . We have built a prototype of Riboweb, and have used it to refine a rough model of the central domain of the 30S subunit from E . coli . procedure . Our results suggest that sophisticated and integrated computational capabilities can be delivered to biologists using this simple three-component architecture.

Am J Physiol, 1997 Sep, 273(3 Pt 2), R1067 - 71
Erythropoietin gene expression is suppressed after lipopolysaccharide or interleukin-1 beta injections in rats; Frede S et al.; Proinflammatory cytokines play an important role in the pathogenesis of anemia in inflammatory diseases . Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been reported to inhibit the synthesis of erythropoietin (EPO) in vitro . To evaluate the in vivo significance of this observation, we have investigated effects of the administration of bacterial lipopolysaccharide (LPS) and IL-1 beta on renal EPO production in rats . Measurements by competitive reverse-transcription polymerase chain reaction showed that EPO mRNA levels were significantly reduced in the kidneys of normoxic rats 6 h after the injection of LPS (0.1 or 1 mg/kg) . In addition, LPS and IL-1 beta (1 microgram/kg) inhibited the increase in EPO mRNA and plasma EPO levels when administered to rats before hypoxia exposure (8% O2 in the inspiratory gas) . Evidence for an inflammatory reaction in the kidneys of LPS-treated rats was provided by measurements of greatly elevated renal TNF-alpha mRNA levels . Furthermore, kidneys isolated from LPS-created rats produced less immunoreactive EPO when perfused hypoxically in vitro for 2 h . Thus mediators of the immune response inhibit renal EPO gene expression in vivo, which is relevant with respect to the impaired synthesis of EPO in inflammatory diseases in humans.

Am J Physiol, 1997 Sep, 273(3 Pt 2), R1046 - 52
Interrelated adrenocortical and neurohypophysial responses associated with fever in endotoxin-treated pigs; Parrott RF et al.; Low intravenous doses of endotoxin {lipopolysaccharide (LPS), 0.7 microgram/kg} induce monophasic fever, increase anterior and posterior pituitary hormone release, and enhance hypothalamic c-Fos expression in pigs, all of which can be prevented by indomethacin (Ind) . The present study shows that the synthetic corticosteroid dexamethasone (Dex, 5 mg/kg) has a similar action to Ind and, when given alone, lowers core temperature . In addition, the corticosteroid synthesis inhibitor metyrapone (Met, 3.3 mg/kg, every one-half hour) reduces LPS fever and amplifies the effect of LPS on vasopressin, but not on oxytocin, release . The similar actions of Dex and Ind suggest that phospholipase A2 pathways controlling prostaglandin synthesis mediate the responses of prepubertal pigs to immunological challenge with LPS . The increased vasopressin release induced when animals receiving Met are also given LPS supports findings in other nonrodent species indicating an inverse relationship between cortisol and vasopressin . The attenuation of LPS fever by Met is suggestive of an endogenous antipyretic mechanism associated with enhanced neurohypophysial vasopressin secretion.

Am J Physiol, 1997 Sep, 273(3 Pt 2), R873 - 9
Attenuation of lipopolysaccharide fever in rats by protein kinase C inhibitors; Kozak W et al.; The purpose of this study was to assess the effects of inhibitors of protein kinase C (PKC) on lipopolysaccharide (LPS)-induced fever and changes in circulating interleukin-6 (IL-6) levels in freely moving biotelemetered rats . We used PKC inhibitors with different inhibition constants (Ki): H-7 (Ki = 6 microM) and chelerythrine (Chel; Ki = 0.66 microM; a more potent PKC inhibitor) . Rats were injected subcutaneously with either 3 or 15 microM/kg of these inhibitors and then 1 h later were injected intraperitoneally with LPS (50 micrograms/kg) . Blood samples for IL-6 bioassay were collected 4 h after LPS injection . H-7 at lower doses did not significantly affect fever and LPS-induced elevation of circulating IL-6, whereas at a higher dose (15 microM/kg) H-7 reduced both fever and the increase of IL-6 (analysis of variance, Scheffe's test, P < 0.05) . Chel (3 and 15 microM/kg) significantly reduced fever and almost completely inhibited the LPS-induced elevation of plasma IL-6 . In separate experiments, we studied the effect of H-7 on antipyresis due to dexamethasone (Dex) . Dex at a dose of 0.6 microM/kg given subcutaneously 1 h before LPS partially prevented fever (approximately 55% inhibition) and attenuated the increase of IL-6 (P < 0.05) . Simultaneous pretreatment of the rats with Dex and H-7 (3 microM/kg; a dose that did not affect fever and IL-6 elevation) led to a potentiation of the antipyretic effect of Dex, resulting in no fever . H-7 did not potentiate, however, the inhibitory effect of Dex on LPS-induced elevation of circulating IL-6 . We conclude that PKC is involved in the regulation of LPS fever and constitutes a rate-limiting factor in modulation of the fever by glucocorticoids.

Am J Physiol, 1997 Sep, 273(3 Pt 2), R858 - 63
Effect of heat stress on LPS-induced fever and tumor necrosis factor; Kluger MJ et al.; Exposure to heat stress leads to both short-term and long-term effects on morbidity . Male rats were exposed to a high ambient temperature of 40 degrees C, which resulted in biotelemetered core body temperature rising to approximately 42 degrees C . This treatment led to a marked enhancement in lipopolysaccharide (LPS)-induced fever at 24 h after exposure to heat stress . The increase in fever was accompanied by a significant suppression in the circulating concentration of tumor necrosis factor . Heat-shock protein-70 measured in liver was elevated by the heat exposure (but not further elevated by the injection of LPS) . An enhanced fever to LPS and other inflammatory stimuli found in heat-stressed human subjects could explain the apparent increase in susceptibility to disease.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4106 - 10
Interaction of human recombination proteins Rad51 and Rad54; Golub EI et al.; The cDNA for human protein HsRad54, which is a structural homolog of Saccharomyces cerevisiae recombination/repair protein Rad54, was cloned and expressed in Escherichia coli . As demonstrated by analysis in vitro and in vivo, HsRad54 protein interacts with human Rad51 recombinase . The interaction is mediated by the N-terminal domain of HsRad54 protein, which interacts with both free and DNA-bound HsRad51 protein.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4093 - 7
The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity; Persson BC et al.; We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity . The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome . The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases . Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E . coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase . Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant . Insertions in the downstream recG gene did not affect RNA modification . Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification . We suggest that the spoU gene be renamed trmH (tRNA methylation).

Nucleic Acids Res, 1997 Oct 15, 25(20), 4067 - 71
Winding of the DNA helix by divalent metal ions; Xu YC et al.; When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp . An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect . When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers . The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4061 - 6
Excision of a conjugative transposon in vitro by the Int and Xis proteins of Tn916; Rudy C et al.; The roles of purified Int and Xis proteins of the conjugative transposon Tn 916 in excision of a deletion derivative of the closely related element Tn 1545 were investigated . At a low salt concentration (37.5 mM NaCl), Int alone was able to promote limited excision to produce a covalently closed circular form of the transposon, showing that Tn 916 Int can catalyze both DNA cleavage and strand exchange . This reaction was stimulated by Xis . At higher salt concentrations (150 mM NaCl), excision by Int alone was reduced to barely detectable levels and Xis was required for excision . The low salt, Xis-stimulated reaction was approximately 8-fold more efficient than the high salt, Xis-dependent reaction . These results reflect in vivo requirements for Int and Xis in excision.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4028 - 34
Transcription activation at class I FNR-dependent promoters: identification of the activating surface of FNR and the corresponding contact site in the C-terminal domain of the RNA polymerase alpha subunit; Williams SM et al.; A library of random mutations in the Escherichia coli fnr gene has been screened to identify positive control mutants of FNR that are defective in transcription activation at Class I promoters . Single amino acid substitutions at D43, R72, S73, T118, M120, F181, F186, S187 and F191 identify a surface of FNR that is essential for activation which, presumably, makes contact with the C-terminal domain of the RNA polymerase alpha subunit . This surface is larger than the corresponding activating surface of the related transcription activator, CRP . To identify the contact surface in the C-terminal domain of the RNA polymerase alpha subunit, a library of mutations in the rpoA gene was screened for alpha mutants that interfered with transcription activation at Class I FNR-dependent promoters . Activation was reduced by deletions of the alpha C-terminal domain, by substitutions known to affect DNA binding by alpha, by substitutions at E261 and by substitutions at L300, E302, D305, A308, G315 and R317 that appear to identify contact surfaces of alpha that are likely to make contact with FNR at Class I promoters . Again, this surface differs from the surface used by CRP at Class I CRP-dependent promoters.

Nucleic Acids Res, 1997 Oct 15, 25(20), 4004 - 12
p53-mediated DNA renaturation can mimic strand exchange; Jean D et al.; The process of strand exchange is considered to be the hallmark of DNA recombination . Proteins known to carry out such exchange are believed to operate via one or the other of two mechanisms . RecA-like proteins promote the formation of a three-stranded or triplex synaptic intermediate in which strand exchange occurs, whereas other proteins would allow the coordinated exonucleolytic degradation of one strand in the duplex DNA and its replacement by an invading strand of similar sequence and polarity . In view of properties ascribed to it, we have attempted to determine whether p53 belongs to one or the other of these groups of proteins . The in vitro assay used relies on a double-stranded (ds) oligonucleotide (oligo 1+2) and a single-stranded (ss) oligonucleotide (oligo 3), part of which is complementary to oligo 1 . The data collected suggest that, under the conditions of the assay, oligo 1+2 undergoes partial denaturation; p53 then catalyzes renaturation of oligo 1 with oligo 3, rather than true strand exchange . Since p53 is not known for being able to 'melt' DNA, it would seem unlikely that this protein would effect strand exchange in vivo without assistance from another, denaturing, protein.

Am J Physiol, 1997 Sep, 273(3 Pt 1), G696 - 705
Expression of oxidative stress-responsive genes and cytokine genes during caerulein-induced acute pancreatitis; Fu K et al.; Oxidative stress and the inflammatory response may play roles in the pathogenesis of acute pancreatitis . Herein, we characterized pancreatic expression of oxidative stress-responsive genes {c-fos, heme oxygenase-1 (HO-1), and metallothionein-I (MT-I)} and cytokine genes {interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha)} during caerulein-induced acute pancreatitis in the mouse . c-fos, HO-1, and MT-I mRNAs were coordinately and rapidly (3-7 h) upregulated, and HO-1 and MT-I protein levels were increased slightly in the pancreas during acute pancreatitis . In addition, IL-1 beta, IL-6, and TNF-alpha mRNAs were rapidly (7 h) upregulated in the pancreas, and intrapancreatic IL-1 beta and IL-6 protein levels rapidly increased (3-fold and 6.4-fold, respectively) during acute pancreatitis . These studies suggest that oxidative stress and inflammation each occur in the pancreas during the early stages of acute pancreatitis . However, under a limited set of experimental conditions, we found that an insult that causes pancreatic oxidative stress (diethylmaleate) or one that induces an inflammatory response (bacterial lipopolysaccharide), or a combination of these agents, did not cause the changes characteristic of acute pancreatitis . Therefore, simply inducing oxidative stress and/or inflammation may be insufficient to initiate acute pancreatitis.

Biochemistry, 1997 Sep 30, 36(39), 11564 - 73
Sulfhydryl chemistry detects three conformations of the lipid binding region of Escherichia coli pyruvate oxidase; Chang YY et al.; Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding {Chang, Y.-Y., & Cronan, J . E., Jr . (1995) J . Biol . Chem . 270, 7896-7901} . The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini . We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine . These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents . In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-{(iodoacetyl)amino}stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent, N-ethylmaleimide . Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent . In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom . In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles . For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators . An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid . Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found . The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain . These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.

Mamm Genome, 1997 Oct, 8(10), 736 - 41
Two alpha(1,2) fucosyltransferase genes on porcine chromosome 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18R) loci; Meijerink E et al.; The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6 . In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the alpha (1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library . Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11) . Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene . The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci . Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF . Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E . coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S . A high linkage disequilibrium of M307-ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E . coli F18 adhesion-resistant animals in this breed . Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

EMBO J, 1997 Oct 15, 16(20), 6314 - 22
Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites; Bjoras M et al.; The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms . We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis . Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels . The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein . Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA . The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA . Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination . However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction . Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue . It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.

EMBO J, 1997 Oct 15, 16(20), 6105 - 13
The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation; Fekkes P et al.; The chaperone SecB keeps precursor proteins in a translocation-competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli . SecA is thought to recognize SecB via its carboxy-terminus . To determine the minimal requirement for a SecB-binding site, fusion proteins were created between glutathione-S-transferase and different parts of the carboxy-terminus of SecA and analysed for SecB binding . A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB-precursor protein complex . SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB . Heterodimers of wild-type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy-termini of the SecA dimer are needed to form a genuine SecB-binding site . SecB is released from the translocase at a very early stage in protein translocation when the membrane-bound SecA binds ATP to initiate translocation . It is concluded that the SecB-binding site on SecA is confined to the extreme carboxy-terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.

J Immunol, 1997 Oct 1, 159(7), 3119 - 25
Mitogenicity of DNA from different organisms for murine B cells; Sun S et al.; Recent evidence that DNA from bacteria causes polyclonal activation of mouse B cells raises the question of whether DNA from other organisms has similar properties . Extending prior studies on bacteria and insects, we show here that the capacity of DNA to stimulate B cells correlates closely with hypomethylation of DNA CpG dinucleotide motifs . Thus strong stimulation of B cells was seen with DNA from various organisms displaying little or no methylation of CpG motifs, i.e., yeast, nematodes, and molluscs in addition to bacteria and insects . For these organisms, DNA induced nearly all B cells (including small resting B cells) to up-regulate the activation marker, CD69, and caused many B cells to enter the cell cycle, indicative of polyclonal activation; this effect was not seen after selective methylation of CpG motifs (tested on yeast DNA) . By contrast, no stimulation of B cells was seen with DNA from organisms whose CpG motifs are heavily methylated, i.e., various vertebrates (mammals, fish, and frogs) and plants (corn) . Despite this correlation, DNA prepared from two murine cell lines exhibiting hypomethylation of CpG motifs caused little or no stimulation of B cells . Thus, the idea that the stimulatory properties of DNA correlate solely with the presence of unmethylated CpG motifs may be an oversimplification.

Infect Immun, 1997 Oct, 65(10), 4309 - 18
Molecular cloning and characterization of Dr-II, a nonfimbrial adhesin-I-like adhesin isolated from gestational pyelonephritis-associated Escherichia coli that binds to decay-accelerating factor; Pham TQ et al.; Bacterial adhesins play an important role in the colonization of the human urogenital tract . Escherichia coli Dr family adhesins have been found to be frequently expressed in strains associated with pyelonephritis in pregnant females . The tissue receptor for known Dr adhesins has been localized to the short consensus repeat-3 (SCR-3) domain of decay accelerating factor (DAF), a complement regulatory protein . In this report, we identified and cloned draE2, a gene encoding a novel 17-kDa DAF-binding adhesin, Dr-II, from a strain of E . coli associated with acute gestational pyelonephritis . Despite the significant sequence diversity between Dr-II and Dr family adhesins, the receptor of Dr-II was found to be the SCR-3 domain of DAF . Sequence analysis of the 186-amino-acid Dr-II open reading frame revealed significant diversity from other members of the Dr adhesin family, including Dr, AFA-I, AFA-III, and F1845, but only an 8-amino-acid difference in sequence from that of the 17-kDa nonfimbrial adhesin NFA-I of unknown receptor specificity . N-terminal peptide sequencing of the purified adhesin confirmed the identity of the open reading frame and indicated cleavage of a 28-amino-acid signal peptide . Antibodies raised against purified Dr-II adhesin exhibited little or no cross-reactivity to Dr adhesin . Characterization of the biological properties demonstrated that like the Dr adhesins, Dr-II was associated with the ability of E . coli to bind to tubular basement membranes and Bowman's capsule and to be internalized into HeLa cells.

Infect Immun, 1997 Oct, 65(10), 4158 - 64
Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant; Karita M et al.; Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer . A previously uncharacterized region of the H . pylori genome was identified and sequenced . This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins . In E . coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate . When wild-type H . pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation . Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H . pylori despite evidence that insertional mutagenesis had occurred . The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies . The data imply that the H . pylori dapE mutant may be useful as a conditionally lethal vaccine.

Infect Immun, 1997 Oct, 65(10), 4135 - 45
Aggregative adherence fimbria II, a second fimbrial antigen mediating aggregative adherence in enteroaggregative Escherichia coli; Czeczulin JR et al.; Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world . We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced . However, not all EAEC strains express AAF/I . Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042 . AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy . TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level) . When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum . The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I . The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization . By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist . Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.

Infect Immun, 1997 Oct, 65(10), 4082 - 9
Adhesion to and invasion of HeLa cells by pathogenic Escherichia coli carrying the afa-3 gene cluster are mediated by the AfaE and AfaD proteins, respectively; Jouve M et al.; The afa-3 gene cluster, expressed by uropathogenic and diarrhea-associated Escherichia coli strains, determines the formation of an afimbrial adhesive sheath composed of the AfaD and AfaE-III adhesins . The adherence to HeLa cells by recombinant HB101 strains producing both or only one of these two adhesins was investigated . Ultrastructural analyses of the interaction and gentamicin protection assays showed adherence to HeLa cells by HB101 producing both the AfaD and AfaE-III proteins and internalization of a subpopulation of the bacteria into the cells . The interactions of HeLa cells either with HB101 mutants producing AfaD or AfaE-III or with polystyrene beads coated with purified His6-tagged AfaD or His6-tagged AfaE-III proteins were studied . These experiments demonstrated that AfaE-III allows binding to HeLa cells and that AfaD mediates the internalization of the adherent bacteria . Ultrastructural analyses of the interaction of His6-AfaD-gold complexes with HeLa cells confirmed that AfaD is able to bind to the HeLa cell surface and indicated that it penetrates the cells via clathrin vesicles . These data demonstrate that the afa gene cluster is unique among bacteria, as alone it encodes both adhesion to and invasion of epithelial cells.

Infect Immun, 1997 Oct, 65(10), 4011 - 6
Characterization of a methyl-accepting chemotaxis protein gene, dmcA, from the oral spirochete Treponema denticola; Kataoka M et al.; A gene, dmcA, expressing a methyl-accepting chemotaxis protein (MCP) from the oral spirochete Treponema denticola has been characterized . The gene was initially identified as an open reading frame immediately upstream from the previously characterized prtB protease gene of strain ATCC 35405 . Nucleotide sequencing of the dmcA gene revealed a potential 57-kDa protein product with extensive homology with the signaling regions of MCPs from a variety of bacteria . The protein expressed in Escherichia coli cross-reacted with anti-Trg (E . coli MCP) serum, confirming its homology with MCPs . Northern blot and primer extension analyses identified the transcription start site of the monocistronic dmcA mRNA . By utilizing a T . denticola gene inactivation system recently developed in this laboratory, a mutant defective in the dmcA gene, HL0501, was constructed . The mutant was demonstrated to be defective in chemotaxis toward nutrients . In addition, the methylation profiles of cellular proteins indicated altered MCPs in the mutant relative to those of the parental strain . These results indicate that we have identified an MCP gene in the oral spirochete which plays a significant role in the chemotactic response of the organism.

Anesthesiology, 1997 Sep, 87(3), 617 - 24
Influence of lidocaine on endotoxin-induced leukocyte-endothelial cell adhesion and macromolecular leakage in vivo; Schmidt W et al.; BACKGROUND: Endotoxin activates leukocyte-endothelial cell adhesion, vascular leakage, and changes in vascular micro-hemodynamics . The aim of this study was to determine whether lidocaine, which inhibits the activation of leukocytes, could attenuate microcirculatory disturbances during endotoxemia . METHODS: Thirty anesthetized male rats were randomly assigned to receive one of three treatments (n = 10 for each group): infusion of saline (control group), infusion of Escherichia coli endotoxin (LPS group: 2 mg x kg(-1) x h(-1) lipopolysaccharides) without lidocaine treatment, or infusion of endotoxin with lidocaine pretreatment 30 min before baseline measurements (lidocaine group: intravenous bolus of 2 mg/kg and continuous infusion of 2 mg x kg(-1) x h(-1)) . Leukocyte adherence, erythrocyte velocity (V(RBC), and vessel diameters (Dv) were determined at baseline and at 60 and 120 min in mesenteric postcapillary venules using in vivo videomicroscopy . Macromolecular leakage was determined by measuring the extravasation of fluorescence-labeled albumin . Venular wall shear rate (tau) was calculated according to the equation tau = 8 x V(RBC) x Dv(-1) . RESULTS: Lidocaine significantly attenuated the increase of leukocyte adherence during endotoxemia . There were no significant differences of tau within or between the groups . Macromolecular leakage exhibited the greatest increase in the LPS group . In the lidocaine group, it was significantly decreased but still increased compared with the control group . CONCLUSIONS: These results show that lidocaine attenuates endotoxin-induced alterations in leukocyte-endothelial cell adhesion and macromolecular leakage, which suggests that lidocaine may have a therapeutic role in preventing endothelial damage in sepsis.

Clin Biochem, 1997 Aug, 30(6), 455 - 63
Removal of endotoxin from recombinant protein preparations; Liu S et al.; OBJECTIVES: To develop an effective method to remove endotoxin from large scale E . coli recombinant protein purifications . DESIGN AND METHODS: Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I . Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay . The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer . In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions . RESULTS: Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography . With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90% . All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies . Troponin I also retained its ability to bind to troponin C in the presence of Ca2+ . Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E . coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies . CONCLUSION: Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.

Am J Physiol, 1997 Sep, 273(3 Pt 1), L524 - 30
Airway inflammation induced by recombinant guinea pig tumor necrosis factor-alpha; White AM et al.; We have cloned and expressed recombinant guinea pig tumor necrosis factor-alpha (gpTNF-alpha) and examined its inflammatory activities after tracheal instillation in guinea pigs . A 1,071-bp cDNA, including the region encoding the full-length 234-amino acid gpTNF-alpha protein, was cloned from concanavalin A-stimulated guinea pig splenocytes . The 154-amino acid protein corresponding to secreted gpTNF-alpha was expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and cleaved to yield a 17-kDa protein . gpTNF-alpha had a cytotoxic effect on WEHI 164 cells and was detected by goat anti-murine tumor necrosis factor-alpha (TNF-alpha) antibody in Western blots . Intratracheal instillation of gpTNF-alpha (50-150 ng) caused pronounced and dose-dependent airway eosinophilia . Incubation of gpTNF-alpha with rabbit anti-murine TNF-alpha sera or heating the gpTNF-alpha before instillation reduced bronchoalveolar lavage (BAL) eosinophils to near control levels . Maximum BAL eosinophilia was observed at 24 h, but eosinophil numbers remained significantly above vehicle-treated animals for 72 h . Hence, gpTNF-alpha elicits a pronounced and protracted eosinophil accumulation in the guinea pig lung.

Am J Physiol, 1997 Sep, 273(3 Pt 1), C1030 - 9
CD14-dependent mechanism for endotoxin-mediated nitric oxide synthesis in murine macrophages; Schroeder RA et al.; Endotoxin-mediated macrophage synthesis of nitric oxide (NO) is associated with immune effector function, intercellular communication, leukocyte adhesion, vascular integrity, and neurotransmission . However, little is known of the cellular receptor and signal transduction pathway by which endotoxin induces NO production . With the use of a model of ANA-1 murine macrophages, we stimulated NO production by incubation with increasing concentrations of endotoxin and 5% fetal calf serum . In selected instances, the anti-CD14 antibody, ED9, was added . Endotoxin-mediated NO synthesis was dependent on CD14 function and the presence of an additional serum factor . Endotoxin treatment increased plasma membrane GTPase activity and 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ({35S}GTP gamma S) binding . Conversely, coincubation of cells with endotoxin and the heterotrimeric G protein inhibitors, suramin and guanosine 5'-O-(2-thiodiphosphate) trilithium salt, was associated with decreased NO synthesis, plasma membrane GTPase activity, and {35S}GTP gamma S binding . Blockade of CD14 or G protein function was associated with ablation of endotoxin-mediated inducible NO synthase (iNOS) protein expression, iNOS mRNA levels, and iNOS gene transcription, as determined by immunoblot, reverse transcriptase-polymerase chain reaction, and nuclear run-on analyses, respectively . These results indicate that endotoxin-mediated NO synthesis is a CD14-heterotrimeric G protein-dependent process.

J Pharm Belg, 1997 Jul-Aug, 52(4), 165 - 6
Characterization of recombinant rat plasminogen activator inhibitor-1 and development of immunological tools for its quantitation; Ngo TH et al.; Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) . Elevated plasma levels of PAI-1 have been associated with several important thrombotic diseases . A large number of studies have demonstrated that rats are suitable for in vivo investigations on thrombolysis and fibronolysis . In this study, we have expressed, in Escherichia coli, purified and characterized recombinant rat PAI-1 in comparison with human PAI-1 . Subsequently this material was used to raise monoclonal antibodies using the hybridoma technology . Characterization of purified recombinant rat PAI-1 revealed that its functional and biochemical properties are similar to those of human PAI-1 . Two fusions, with spleen cells from mice immunized with recombinant rat PAI-1, yielded 118 positive hybridomas . From these, 36 monoclonal antibodies were purified and evaluated for their applicability in the construction of sandwich-type ELISAs . Out of 860 combinations tested, 2 combinations were selected for the measurement of rat PAI-1 (antigen and activity) in biological samples (e.g., plasma, platelet lysates, cell-culture media, ...).

Nucl Med Biol, 1997 Aug, 24(6), 603 - 5
The effect of antibodies against cell-surface adhesion molecules (LFA-1 alpha and ICAM-1) on the migration and localization of 99mTc-labeled leukocytes in acute infection; Amartey JK et al.; The deficiency of adhesion molecules on leukocytes could severely impair their ability to migrate and perform effective immunological functions leading to clinical situation such as LAD (leukocyte adhesion deficiency) syndrome . We investigated the effects of blocking anti-LFA-1 alpha and ICAM-1 antibody-treated 99mTc-labeled leukocytes on the migration and localization to the site of E . coli-induced acute infection in CBA/J mice . A significant inhibition of migration and localization of antibody-treated leukocytes to the site of infection was observed, reaffirming the vital role of these adhesion molecules, especially during scintigraphic examination of patients for deep infections or abscess using labeled leukocytes.

Nucl Med Biol, 1997 Aug, 24(6), 539 - 46
Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma; Carrel F et al.; Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties . Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography . Radioiodinated CE7-scFv fragments were found to bind with high affinity (Kd approximately 10(-9) M) to target cells in vitro but formed aggregates at 37 degrees C, and bound to serum proteins in vitro and in vivo . Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent "refolding" could be achieved . In contrast, chCE7- Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (Kd approximately 10(-10) M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo . Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid . Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells . Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments . Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab')2 fragment . A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab')2 fragments showed in both cases high levels of radioactivity in the kidneys . Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments . When compared with the radioiodinated forms, tumor uptake of radiocopper-labeled 67Cu-chCE7 and its F(ab')2 fragments was found to be higher . However in the case of the non internalizing 67Cu-chCE7-Fab fragment no increase in the absolute amount of radioactivity in tumor tissue compared with the radioiodinated Fab was observed, indicating an advantage of using radiocopper labeling in conjunction with internalizing antibody fragments for delivering high doses of radioactivity to neuroblastoma.

Biochemistry, 1997 Sep 30, 36(39), 11959 - 65
Proximity of periplasmic loops in the lactose permease of Escherichia coli determined by site-directed cross-linking; Sun J et al.; Out of over 60 single-Cys mutants in putative periplasmic loops in lactose permease, three mutants {Tyr101 --> Cys (loop III/IV), Leu313 --> Cys (loop IX/X), and Ser375 --> Cys (loop XI/XII)} spontaneously form disulfide-linked dimers, indicating that these loops are located on the periphery of the 12-helix bundle that comprises the permease . By using a permease construct with a factor Xa protease site in the middle cytoplasmic loop, cross-linking between paired-Cys residues in the N- and C-terminal halves of the permease was studied by spontaneous or copper-(1, 10-phenanthroline)3-catalyzed disulfide formation or by cross-linking with homo- or heterobifunctional reagents in which the distance between the reactive groups and the flexibility of the linker vary . The findings suggest that the longer loops are relatively flexible; however, cross-linking of residues between loops is specific, indicating that these domains are not simply flexible, hydrophilic connections between helices that interact randomly . More specifically, the findings indicate that the first periplasmic loop (loop I/II) is close to loops VII/VIII and XI/XII, placing helix XII in close proximity to helices II and XI . In addition, the observations are consistent with previous results {Wu, J., & Kaback, H . R . (1996) Proc . Natl . Acad . Sci . U.S.A . 93, 14498-502} demonstrating that helices I and II are close to helices VII and XI . Finally, evidence is presented indicating that conformational flexibility between loops I/II and XI/XII may be important for permease turnover.

Biochemistry, 1997 Sep 30, 36(39), 11923 - 32
Role of carbohydrate structures in the binding of beta1-latency-associated peptide to ligands; Yang Y et al.; Transforming growth factor beta1 (TGF-beta1) is a potent growth differentiation and morphogenesis factor . The amino-terminal 248 amino acid pro region of TGF-beta1, the beta1-latency-associated peptide (beta1-LAP), is noncovalently associated with TGF-beta1 in an inactive complex . Previous studies suggested that deglycosylated beta1-LAP can not form this latent complex with TGF-beta1 . To study the role of the carbohydrate structures of beta1-LAP in its biological functions, we expressed simian beta1-LAP in Escherichia coli with a 10 histidine residue tag on the N-terminus . This polypeptide was solubilized from inclusion bodies with 6 M guanidine hydrochloride and purified by metal chelate affinity chromatography . Purified beta1-LAP was refolded to its dimeric form using a chaotrope-mediated folding procedure . The dimeric beta1-LAP forms 90 kDa complexes with TGF-beta1, TGF-beta2, and TGF-beta3, and reverses the inhibitory activity of TGF-beta1 on Mv1Lu cells . Solid phase binding assays demonstrate that refolded beta1-LAP binds to heparin and thrombospondin 1 . FET cell adhesion promoted by refolded beta1-LAP was blocked by an RGD peptide . Purified beta1-LAP produced in Chinese hamster ovary cells, deglycosylated with N-glycosidase F, forms a 80-90 kDa complex with mature TGF-beta1 . The carbohydrate structures of beta1-LAP are not required for binding to ligands or for its biological activity.

Biochemistry, 1997 Sep 30, 36(39), 11851 - 7
The serine chemoreceptor from Escherichia coli is methylated through an inter-dimer process; Li J et al.; Covalent modification of receptors is a widespread phenomenon in signal transduction . In the chemosensory system of Escherichia coli, the reversible methylation of certain glutamic acid residues in the cytoplasmic domain of receptor homodimers mediates adaptation to stimuli . Here we report that the serine receptor is methylated by an inter-dimer process . Methyltransferase bound to one subunit in a serine receptor homodimer was found to catalyze the addition of methyl groups to a receptor subunit in an adjacent dimer in the membrane . These results demonstrate a role for inter-dimer interactions in transmembrane signaling.

Biochemistry, 1997 Sep 30, 36(39), 11811 - 20
{2Fe-2S} to {4Fe-4S} cluster conversion in Escherichia coli biotin synthase; Duin EC et al.; The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithionite-reduced samples using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman spectroscopies . The results confirm the presence of one S = 0 {2Fe-2S}2+ cluster in each subunit of the homodimer in aerobically purified samples, and the Fe-S stretching frequencies suggest incomplete cysteinyl-S coordination . However, absorption and resonance Raman studies show that anaerobic reduction with dithionite in the presence of 60% (v/v) ethylene glycol or glycerol results in near-stoichiometric conversion of two {2Fe-2S}2+ clusters to form one S = 0 {4Fe-4S}2+ cluster with complete cysteinyl-S coordination . The stoichiometry and ability to effect reductive cluster conversion without the addition of iron or sulfide suggest that the {4Fe-4S}2+ cluster is formed at the subunit interface via reductive dimerization of {2Fe-2S}2+ clusters . EPR and VTMCD studies indicate that more than 50% of the Fe is present as {4Fe-4S}+ clusters in samples treated with 60% (v/v) glycerol after prolonged dithionite reduction . The {4Fe-4S}+ cluster exists as a mixed spin system with S = 1/2 (g = 2 . 044, 1.944, 1.914) and S = 3/2 (g = 5.6 resonance) ground states . Subunit-bridging {4Fe-4S}2+,+ clusters, that can undergo oxidative degradation to {2Fe-2S}2+ clusters during purification, are proposed to be a common feature of Fe-S enzymes that require S-adenosylmethionine and function by radical mechanisms involving the homolytic cleavage of C-H or C-C bonds, i.e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate formate lyase, lysine 2, 3-aminomutase, and lipoic acid synthase . The most likely role for the {4Fe-4S}2+,+ cluster lies in initiating the radical mechanism by directly or indirectly facilitating reductive one-electron cleavage of S-adenosylmethionine to form methionine and the 5'-deoxyadenosyl radical . It is further suggested that oxidative cluster conversion to {2Fe-2S}2+ clusters may play a physiological role in these radical enzymes, by providing a method of regulating enzyme activity in response to oxidative stress, without irreversible cluster degradation.

Biochemistry, 1997 Sep 30, 36(39), 11757 - 61
Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase; Carreras CW et al.; The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described . Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS . Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo . ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry . A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively . In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis . Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

Biochemistry, 1997 Sep 30, 36(39), 11697 - 706
Reassessment of cytochrome P450 2B2: catalytic specificity and identification of four active site residues; Strobel SM et al.; Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity . Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 --> 2B2 substitutions . To address these discrepancies, we have characterized two 2B2 variants . A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114 . A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions . These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin . The expressed 2B2 variants metabolized most substrates at rates that were 1-9% of those of 2B1 . When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16beta- and 16alpha-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats . These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme . Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF . On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity . In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.

Biochemistry, 1997 Sep 30, 36(39), 11685 - 96
The amino-terminal portion of the Rieske iron-sulfur protein contributes to the ubihydroquinone oxidation site catalysis of the Rhodobacter capsulatus bc1 complex; Brasseur G et al.; The Rieske iron-sulfur (Fe-S) protein subunit of bc1 complexes contains in its carboxyl-terminal part two highly conserved hexapeptide motifs (box I and box II) that include the four amino acid ligands of its {2Fe-2S} cluster . In the preceding paper {Liebl, U., Sled, V., Brasseur, G., Ohnishi, T., & Daldal, F . (1997) Biochemistry 36, 11675-11684}, the effects of mutations at two of the nonliganding residues {threonine (T) 134 and leucine (L) 136 in the Rhodobactercapsulatus Rieske Fe-S protein} of box I have been described . In this work, interactions between the occupants of the Qo site of the bc1 complex (UQ/UQH2 and the inhibitors stigmatellin and myxothiazol) and the {2Fe-2S} cluster of the Rieske Fe-S protein were probed by isolating photosynthesis-proficient (Ps+) revertants of the Ps- mutants L136R, -H, -D and -G . These revertants contained either a single substitution at the original position 136 or an additional mutation located in the amino-terminal part of the Fe-S protein at either position 44 or 46 . The same-site revertants L136A and -Y grew well under photosynthetic conditions and contained highly active bc1 complexes but exhibited modified EPR spectra both in the presence and in the absence of stigmatellin . Unexpectedly, they were highly resistant to stigmatellin (StiR) and hypersensitive to myxothiazol (MyxHS) in vivo, demonstrating for the first time that mutations located in the Fe-S subunit confer resistance to stigmatellin . The {2Fe-2S} cluster of the same-site revertants responded weakly to the Qpool redox state and had redox midpoint potential (Em7) values (around 265 mV) lower than those of their wild type counterpart (about 310 mV) . On the other hand, the second-site revertants L136H/V44L, L136G/V44F, and L136G/A46T, -V, or -P supported photosynthetic growth poorly, were StiR and MyxHS, and contained barely active bc1 complexes . Like the same-site revertants, they exhibited modified EPR spectra both in the presence and in the absence of stigmatellin and had perturbed Qo site occupancy . In addition, they contained substoichiometric amounts of the Fe-S protein with respect to the other subunits of the bc1 complex . The Em7 values of the {2Fe-2S} cluster of these double mutants were lower (around 245 mV) than that of the wild type strain but appreciably higher than those of their Ps- parents (about 200 mV for L136G) . In order to define the molecular nature of the suppression mediated by the second-site mutations, the single mutants V44L and -F and A46T and -V were constructed in the absence of the original mutations at position 136 . These mutants behaved like a wild type strain with respect to their Ps+ growth ability, inhibitor sensitivity, EPR spectra of their {2Fe-2S} cluster, and response to stigmatellin or to the Qpool redox state . But surprisingly, the Em7 values of their {2Fe-2S} cluster were much higher (about 385 mV) than that of a wild type strain . These findings demonstrated for the first time that the amino-terminal part of the Rieske Fe-S protein encompassing residues 44 and 46 is important not only for the structure and function of the Qo site of the bc1 complex but also for the properties of its {2Fe-2S} cluster.

Biochemistry, 1997 Sep 30, 36(39), 11675 - 84
Conserved nonliganding residues of the Rhodobacter capsulatus Rieske iron-sulfur protein of the bc1 complex are essential for protein structure, properties of the {2Fe-2S} cluster, and communication with the quinone pool; Liebl U et al.; The iron-sulfur (Fe-S) protein subunit of the bc1 complex, known as the Rieske protein, contains a high-potential {2Fe-2S} cluster ligated by two nitrogen and two sulfur atoms to its apoprotein . Earlier work indicated that in Rhodobacter capsulatus these atoms are provided by two cysteine (C133 and C153) and two histidine (H135 and H156) residues, located at the carboxyl-terminal end of the protein {Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F . (1992) Biochemistry 31, 3342-3351} . These ligands are part of the conserved sequences C133THLGC138 (box I) and C153PCHGS158 (box II) and affect the properties of the Fe-S protein and its {2Fe-2S} cluster . In this work, the role of amino acid side chains at positions 134 and 136, adjacent to the cluster ligands in box I, was probed by using site-directed mutagenesis and biophysical analyses . These positions were substituted with R, D, H, and G to probe the effect of charged, polar, large, and small amino acid side chains on the properties of the {2Fe-2S} cluster . Of the mutants obtained T134R, -H, and -G were photosynthetically competent (Ps+) but contained Fe-S proteins with redox midpoint potentials (Em7) 50-100 mV lower than that of a wild type strain . In contrast, T134D was Ps- and contained no detectable {2Fe-2S} cluster, although it reverted frequently to Ps+ by substitution of D with N . On the other hand, all L136 mutants were Ps-, the EPR characteristics of their {2Fe-2S} cluster were perturbed, and they were unable to sense the Qpool redox state or to bind stigmatellin properly . The overall data indicated that replacement of the amino acid side chain at position 134 of the Fe-S protein affects mainly the Em7 and oxygen sensitivity of the {2Fe-2S} cluster without abolishing its function, while substitutions at position 136 perturb drastically its ability to monitor the Qpool redox state and its interaction with the Qo site inhibitor stigmatellin . These two distinct phenotypes of box I T134 and L136 mutants are discussed with regard to the recently published three-dimensional structure of the water soluble part of the bovine heart mitochondrial Rieske Fe-S protein.

Biochemistry, 1997 Sep 30, 36(39), 11665 - 74
Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-aminothiazole and covalent modification of the enzyme; Musah RA et al.; The binding and oxidation of an artificial substrate, 2-aminothiazole, by an engineered cavity of cytochrome c peroxidase is described . The W191G mutant has been shown to create a buried cavity into which a number of small heterocyclic compounds will bind {Fitzgerald, M . M., Churchill, M . J., McRee, D . E., & Goodin, D . B . (1994) Biochemistry 33, 3807-3818}, providing a specific site near the heme from which substrates might be oxidized . In this study, we show by titration calorimetry that 2-aminothiazole binds to W191G with a Kd of 0.028 mM at pH 6 . A crystal structure at 2.3 A resolution of W191G in the presence of 2-aminothiazole reveals the occupation of this compound in the cavity, and indicates that it is in van der Waals contact with the heme . The WT enzyme reacts with H2O2 to form Compound ES, in which both the iron center and the Trp-191 side chain are reversibly oxidized . For the W191F (and perhaps the W191G) mutants, the iron is still oxidized, but the second equivalent exists transiently as a radical on the porphyrin before migrating to an alternate protein radical site {Erman, J . E., Vitello, L . B., Mauro, J . M., & Kraut, J . (1989) Biochemistry 28, 7992-7995} . Two separate reactions are observed between 2-aminothiazole and the oxidized centers of W191G . In the one reaction, optical and EPR spectra of the heme are used to show that 2-aminothiazole acts as an electron donor to the ferryl (Fe4+&dbd;O) center of W191G to reduce it to the ferric oxidation state . This reaction occurs from within the cavity, as it is not observed for variants that lack this artificial binding site . A second reaction between 2-aminothiazole and peroxide-oxidized W191G, which is much less efficient, results in the specific covalent modification of Tyr-236 . Electrospray mass spectra of the W191G after incubation in 2-aminothiazole and H2O2 show a modification of the protein indicative of covalent binding of 2-aminothiazole . The site of modification was determined to be Tyr-236 by CNBr peptide mapping and automated peptide sequencing . The covalent modification is only observed for W191G and W191F which form the alternate radical center . This observation provides an unanticipated assignment of this free radical species to Tyr-236, which is consistent with previous proposals that it is a tyrosine . The oxidation of 2-aminothiazole by W191G represents an example of how the oxidative capacity inherent in the heme prosthetic group and the specific binding behavior of artificial protein cavities can be harnessed and redirected toward the oxidation of organic substrates.

Biochemistry, 1997 Sep 30, 36(39), 11655 - 64
The core domain of the tissue transglutaminase Gh hydrolyzes GTP and ATP; Iismaa SE et al.; Tissue transglutaminase (TGase II) catalyzes the posttranslational modification of proteins by transamidation of available glutamine residues and is also a guanosinetriphosphatase (GTPase) and adenosinetriphosphatase (ATPase) . Based on its homology with factor XIIIA, an extracellular transglutaminase, the structure of TGase II is likely composed of an N-terminal beta-sandwich domain, an alpha/beta catalytic core, and two C-terminally located beta-barrels . Here we used a domain-deletion approach to identify the GTP and ATP hydrolytic domains of TGase II . Full-length TGase II and two domain-deletion mutants, one retaining the N-terminal beta-sandwich and core domains (betaSCore) and the other retaining only the core domain, were expressed as glutathione S-transferase (GST) fusion proteins and purified . GST-Full and GST-betaSCore exhibited calcium-dependent TGase activity, whereas GST-Core had no detectable TGase activity, indicating the beta-sandwich domain is required for TGase activity but the C-terminal beta-barrels are not . All three GST-TGase II fusion proteins were photoaffinity-labeled with {alpha-32P}-8-azidoGTP and were able to bind GTP-agarose . The GTPase activity of GST-betaSCore was equivalent to that of GST-Full, whereas the ATPase activity was approximately 40% higher than GST-Full . GST-Core had approximately 50% higher GTPase activity and approximately 75% higher ATPase activity than GST-Full . The GTPase and ATPase activities of each of the GST-TGase II fusion proteins were inhibited in a dose-dependent manner by both GTPgammaS and ATPgammaS . These results demonstrate that the GTP and ATP hydrolysis sites are localized within the core domain of TGase II and that neither the N-terminal beta-sandwich domain nor the C-terminal beta-barrels are required for either GTP or ATP hydrolysis . Taken together with previous work {Singh, U . S., Erickson, J . W., & Cerione, R . A . (1995) Biochemistry 34, 15863-15871; Lai, T.-S., Slaughter, T . F., Koropchak, C . M., Haroon, Z . A., & Greenberg, C . S . (1996) J . Biol . Chem . 271, 31191-31195} the results of this study indicate that the GTP and ATP hydrolysis sites are localized to a 5 . 5 kDa (47 amino acid) region at the start of the core domain.

Biochemistry, 1997 Sep 30, 36(39), 11640 - 7
Role of macromolecular hydration in the binding of the Escherichia coli cyclic AMP receptor to DNA; Vossen KM et al.; The osmotic stress technique was used to measure the changes in macromolecular hydration that accompany binding of the Escherichia coli CAP protein to its transcription-regulatory site (C1) in the lactose promoter and that accompany the transfer of CAP from site C1 to nonspecific genomic DNA . Formation of the C1 complex is accompanied by the net release of 79 +/- 11 water molecules . If all water molecules were released from macromolecular surfaces, this result would be consistent with a net reduction of solvent-accessible surface area of 711 +/- 189 A2 . This area is only slightly smaller than the solvent-inaccessible macromolecular interface in crystalline CAP-DNA complexes . The transfer of CAP from site C1 to nonspecific sites is accompanied by the net uptake of 56 +/- 10 water molecules . Taken with the water stoichiometry of sequence-specific binding, this value implies that formation of a nonspecific complex is accompanied by the net release of 2-44 water molecules . The enhanced stabilities of CAP-DNA complexes with increased osmolality (decreased water activity) may contribute to the ability of E.coli cells to tolerate dehydration and/or high external salt concentrations.

Biochemistry, 1997 Oct 7, 36(40), 12252 - 8
Pre-steady-state kinetic analysis of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase: kinetic evidence for enol/keto tautomerization; Henderson IM et al.; The reaction catalyzed by 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) was analyzed by stopped-flow UV-visible kinetics at 317 nm (substrate depletion) and 270 nm (product formation) at pH 5.0 and 4.0 . Comparison of the rates and amplitudes of product formation versus substrate depletion provided evidence for the formation of a discrete keto-intermediate, as predicted from previous isotope exchange experiments {Lam, W . W . Y., & Bugg, T . D . H . (1997) Biochemistry, 36, 12242-12251} . Accurate modeling of the concentration data could only be achieved using a branched kinetic mechanism in which the intermediate is released at a rate comparable to its catalytic turnover, consistent with the earlier isotope exchange data . The apparent "leakiness" of the active site and relatively weak substrate binding (Kd = 30 microM) are consistent with a mechanism in which the enzyme binds the dienol substrate in a strained, nonplanar conformation which promotes ketonization in the C-5 position to give a keto-intermediate.

Biochemistry, 1997 Oct 7, 36(40), 12242 - 51
Purification, characterization, and stereochemical analysis of a C-C hydrolase: 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase; Lam WW et al.; 2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli has been purified to near homogeneity from an overexpressing strain of E . coli . The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity . The enzyme has a Km of 2.1 microM and a kcat of 36 s-1 for its natural substrate and shows high selectivity for the propionate side chain of the substrate . The stereochemical course of the MhpC reaction was elucidated by conversion of protiosubstrate in 2H2O and conversion of deuteriated substrate in 1H2O, revealing that the reaction proceeds with overall replacement of a succinyl moiety by a proton from water in the H-5E position, with retention of regiochemistry . Isotope exchange was also observed in the H-5Z position of the product, which was rationalized by enzyme-catalyzed exchange of 2H into C-5 of the substrate from 2H2O . These data are consistent with a reversible keto-enol tautomerization taking place as the first step of the enzyme mechanism.

Biochemistry, 1997 Oct 7, 36(40), 12235 - 41
Substrates with charged P1 residues are efficiently hydrolyzed by serine carboxypeptidases when S3-P1 interactions are facilitated; Olesen K et al.; The high activity of carboxypeptidase S1 with substrates having basic P1 residues is predicted to depend on the size of residue 312 in combination with the presence of a counter-charge in an alpha-helix above the S1 binding pocket . This hypothesis is tested by the construction of 32 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic residue at either position 241 or position 245 . Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P1 demonstrates that most of these enzymes exhibit drastically altered catalytic properties . One mutant enzyme, N241D + W312L, hydrolyzes FA-Arg-Ala-OH with a kcat/KM value of 13 000 min-1 mM-1 corresponding to a 930-fold increase relative to the wild-type enzyme . This increased activity is due to an increase in kcat and is independent of ionic strength . The pH profile of kcat/KM exhibits an optimum around pH 5.5 similar to that observed for CPD-S1 . Another mutant enzyme, L245R + W312S, hydrolyzes FA-Glu-Ala-OH and FA-Asp-Ala-OH with kcat/KM values of 5100 and 5300 min-1 mM-1, respectively, corresponding to 120 and 170-fold increases relative to wild-type values . With the latter substrate, a 280-fold reduction of KM is observed . The activity of L245R + W312S is also independent of ionic strength and displays a virtually unaltered dependence on pH . The P1 substrate preference of these two mutant enzymes for Arg versus Asp differs 2.5 x 10(6)-fold . values of single and double mutants demonstrate that the effects of reducing the size of Trp312 and introducing a charged residue at position 241 or 245 in some cases exceed 100% additivity . Thus, the double mutant enzyme gains more activation energy than can be accounted for by each individual single mutation.

Biochemistry, 1997 Oct 7, 36(40), 12147 - 54
Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching; Rolli V et al.; Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents . Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism . Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain . Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site . Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching . The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites . Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching . This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.

Biochemistry, 1997 Oct 7, 36(40), 12120 - 37
Probing the catalytic mechanism of sulfite reductase by X-ray crystallography: structures of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates, and products; Crane BR et al.; To further understand the six-electron reductions of sulfite and nitrite catalyzed by the Escherichia coli sulfite reductase hemoprotein (SiRHP), we have determined crystallographic structures of the enzyme in complex with the inhibitors phosphate, carbon monoxide, and cyanide, the substrates sulfite and nitrite, the intermediate nitric oxide, the product sulfide (or, most likely, an oxidized derivative thereof), and an oxidized nitrogen species (probably nitrate) . A hydrogen-bonded cage of ligand-binding arginine and lysine side chains, ordered water molecules, and siroheme carboxylates provides preferred locations for recognizing the common functional groups of these ligands and accommodates their varied sizes, shapes, and charged without requiring substantial structural changes . The coordination geometries presented here suggest that the successively deoxygenated sulfur and nitrogen species produced during catalysis need not alter their orientation in the active site to adopt new stable coordination states . Strong pi-acid ligands decrease the bond length between the siroheme and the proximal cysteine thiolate shared with the iron-sulfur cluster, emphasizing the ability of the coupled cofactors to promote electron tranfer into substrate . On binding the siroheme, the substrate sulfite provides an oxygen atom in a unique location of the binding site compared to all other ligands studied, induces a spin transition in the siroheme iron, flips an active-site arginine, and orders surrounding active-center loops . The loop that coalesces over the active center shields the positively charged ligand-coordinating residues from solvent, enhancing their ability to polarize the substrate . Hydrogen bonds supplied by active-site arginine and lysine residues facilitate charge transfer into the substrate from the electron-rich cofactors, activate S-O bonds for reductive cleavage, and provide potential proton sources for the formation of favorable aquo leaving groups on the substrate . Strong interactions between sulfite and ordered water molecules also implicate solvent as a source of protons for generating product water . From the structures reported here, we propose a series of key structural states of ligated SiRHP in the catalytic reduction of sulfite to sulfide.

Biochemistry, 1997 Oct 7, 36(40), 12101 - 19
Structures of the siroheme- and Fe4S4-containing active center of sulfite reductase in different states of oxidation: heme activation via reduction-gated exogenous ligand exchange; Crane BR et al.; The active center of the Escherichia coli sulfite reductase hemoprotein (SiRHP) is exquisitely designed to catalyze the six-electron reductions of sulfite to sulfide and nitrite to ammonia . Refined high-resolution crystallographic structures of oxidized, two-electron reduced, and intermediately reduced states of SiRHP, monitored by single-crystal electron paramagnetic resonance (EPR) spectroscopy, reveal that a bridging cysteine thiolate supplied by the protein always covalently links the siroheme (iron isobacteriochlorin) to the Fe4S4 cluster, facilitating their ability to transfer electrons to substrate . The reduction potential and reactivity of the cluster are tuned by association with the siroheme, accessibility to solvent, and hydrogen bonds supplied by the protein loops containing the four cluster-ligating cysteines . The distorted conformation of the siroheme recognized by the protein potentially destabilizes the electronic conjugation of the isobacteriochlorin ring and produces axial configurations for some propionate side chains that promote interactions with exogenous ligands and active-site residues . An extensive hydrogen-bond network of positively charged side chains, ordered water molecules, and siroheme carboxylates coordinates, polarizes, and influences the protonation state of anionic ligands . In the oxidized (siroheme Fe3+, Fe4S42+) SiRHP crystal structure, the high density of positive charges in the binding pocket is stabilized by the siroheme's sixth axial ligand-an exogenous phosphate anion . Binding assays with H32PO42- demonstrate that oxidized SiRHP binds phosphate in solution with a dissociation constant of 14 microM at pH 7.7, suggesting that phosphate anions play an important role in stabilizing and sequestering the active-site of the oxidized enzyme in vivo . Reduction of the cofactors couples changes in siroheme iron coordination geometry to changes in active-site protein conformation, leading to phosphate release both in the crystal and in solution . An intermediately reduced enzyme, where the siroheme is mainly ferrous (+2) and the cluster cubane is mainly oxidized (+2), appears to have the lowest affinity for phosphate in the crystal . Reduction-gated release of phosphate from the substrate-binding site may explain the 10(5)-fold increase in rates of ligand association that accompany reduction of SiRHP.

FEBS Lett, 1997 Sep 8, 414(2), 449 - 53
Cloning, sequencing and expression of ribonucleotide reductase R2 from Trypanosoma brucei; Dormeyer M et al.; Ribonucleotide reductase (RR) is an attractive drug target molecule . The gene of the R2 protein of Trypanosoma brucei RR (nrd B) has been cloned . It encodes a protein of 337 residues which shows about 60% identity with other eukaryotic R2 proteins . All residues which bind the iron center, the tyrosyl radical or are supposed to participate in the radical transfer are conserved in the trypanosomal protein sequence . Overexpression of the gene in E . coli resulted in 2-5 mg pure R2 protein from 100 ml bacterial cell culture . Northern blot analysis revealed a transcript of 1.85 kb in bloodstream and procyclic forms of the parasite.

FEBS Lett, 1997 Sep 8, 414(2), 402 - 4
Overexpression of the hslVU operon suppresses SOS-mediated inhibition of cell division in Escherichia coli; Khattar MM; A multicopy clone was isolated which conferred resistance to the SOS inducer nitrofurantoin in an Escherichia coli lon mutant . Plasmid pHL1 was found to contain a 7-8 kbp HindIII DNA insert from a region of the chromosome at 88.5 minutes . Further characterisation of pHL1 revealed that resistance to nitrofurantoin was due to the overexpression of the hslV-hslU operon which encodes an ATP-dependent protease complex in E . coli . The overexpression of hslVU also conferred resistance to ultraviolet irradiation in the lon mutant . It is proposed that when overproduced, the HslV-HslU protease complex can degrade SulA which is an endogenous inhibitor of the essential cell division protein FtsZ . The ability of HslVU to degrade SulA in vivo suggests that Lon and HslVU may share a range of substrates . Furthermore, the suppression of lon could be used as a simple genetic test of proteolytic activity of cloned HslVU.

FEBS Lett, 1997 Sep 8, 414(2), 373 - 6
Aerobic and anaerobic regulation of the ubiCA operon, encoding enzymes for the first two committed steps of ubiquinone biosynthesis in Escherichia coli; Soballe B et al.; The ubiCA operon of Escherichia coli encodes enzymes for the first two steps of ubiquinone biosynthesis . A monolysogen (ubiC-lacZ operon fusion) was constructed to study ubiCA regulation . Expression was higher during aerobic growth than anaerobically, and increased with rate of oxygen supply . Although ubiquinone is implicated in antioxidant roles, ubiC expression was not elevated in response to hydrogen peroxide or the redox cycling agent, paraquat . Glucose repressed expression and mutation of cya (encoding adenylate cyclase) increased expression . Anaerobically utilised electron acceptors (nitrite, nitrate, fumarate) did not affect expression . ubiC expression appears to be negatively regulated by Fnr and IHF.

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