Escherichia coli

J Biol Chem, 1999 Mar 26, 274(13), 8764 - 9
Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity; Matkovic-Calogovic D et al.; Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined . The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus . Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75% . The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry . On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered . The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors . Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.

J Biol Chem, 1999 Mar 26, 274(13), 8723 - 9
Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy; Agrawal RK et al.; The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy . Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration {10-15 mM}; and polyamine buffer: containing Hepes, lower Mg2+ concentration {6 mM}, and polyamines) . Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome . The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions . The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i . e . in the P/P state, under the polyamine buffer conditions.

J Biol Chem, 1999 Mar 26, 274(13), 8717 - 22
Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase; Shenoy SK et al.; The smallest membrane-anchoring subunit (QPs3) of bovine heart succinate:ubiquinone reductase was overexpressed in Escherichia coli JM109 as a glutathione S-transferase fusion protein using the expression vector pGEX2T/QPs3 . The yield of soluble active recombinant glutathione S-transferase-QPs3 fusion protein was isopropyl-1-thio-beta-D-galactopyranoside concentration-, induction growth time-, temperature-, and medium-dependent . Maximum yield of soluble recombinant fusion protein was obtained from cells harvested 3.5 h post-isopropyl-1-thio-beta-D-galactopyranoside (0.4 mM)-induction growth at 25 degrees C in 2.0% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose (SOC medium) containing 440 mM sorbitol and 2.5 mM betaine . QPs3 was released from the fusion protein by proteolytic cleavage with thrombin . Isolated recombinant QPs3 shows one protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that corresponds to subunit V of mitochondrial succinate:ubiquinone reductase . Although purified recombinant QPs3 is dispersed in 0.01% dodecylmaltoside, it is in a highly aggregated form, with an apparent molecular mass of more than 1 million . The recombinant QPs3 binds ubiquinone, causing a spectral blue shift . Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that recombinant QPs3 may be in the functionally active state . Two amino acid residues, serine 33 and tyrosine 37, in the putative ubiquinone binding domain of QPs3 are involved in ubiquinone binding because the S33A- or Y37A-substituted recombinant QPs3s do not cause the spectral blue shift of ubiquinone . Although recombinant QPs3 contains little cytochrome b560 heme, the spectral characteristics of cytochrome b560 are reconstituted upon addition of hemin chloride . Reconstituted cytochrome b560 in recombinant QPs3 shows a EPR signal at g = 2.92 . Histidine residues at positions 46 and 60 are responsible for heme ligation because the H46N- or H60N-substituted QPs3 fail to restore cytochrome b560 upon addition of hemin chloride.

J Biol Chem, 1999 Mar 26, 274(13), 8359 - 62
Caspase-9 can be activated without proteolytic processing; Stennicke HR et al.; The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP . Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms . To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen . These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors . The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells . Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity.

J Biol Chem, 1999 Mar 26, 274(13), 8351 - 4
A model of the transition state in the alkaline phosphatase reaction; Holtz KM et al.; A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 A resolution . The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile . A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site . This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction.

Infect Immun, 1999 Apr, 67(4), 2045 - 9
beta1-chain integrins are not essential for intimin-mediated host cell attachment and enteropathogenic Escherichia coli-induced actin condensation; Liu H et al.; Intimin is a bacterial outer membrane protein required for intimate attachment of enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) to mammalian cells . beta1-chain integrins have been proposed as candidate receptors for intimin . We found that binding of mammalian cells to immobilized intimin was not detectable unless mammalian cells were preinfected with EPEC or EHEC . beta1-chain integrin antagonists or inactivation of the gene encoding the beta1-chain did not affect binding of preinfected mammalian cells to intimin or the actin condensation associated with the attachment of EPEC . The results indicate that beta1-chain integrins are not essential for intimin-mediated cell attachment or EPEC-mediated actin polymerization.

Infect Immun, 1999 Apr, 67(4), 1821 - 7
Antibodies reactive with the N-terminal domain of Plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts; Pang XL et al.; The serine repeat antigen (SERA) is a vaccine candidate antigen of Plasmodium falciparum . Immunization of mice with Escherichia coli-produced recombinant protein of the SERA N-terminal domain (SE47') induced an antiserum that was inhibitory to parasite growth in vitro . Affinity-purified mouse antibodies specific to the recombinant protein inhibited parasite growth between the schizont and ring stages but not between the ring and schizont stages . When Percoll-purified schizonts were cultured with the affinity-purified SE47'-specific antibodies, schizonts and merozoites were agglutinated . Indirect-immunofluorescence assays with unfixed parasite cells showed that SE47'-specific immunoglobulin G (IgG) bound to SERA molecules on rupturing schizonts and merozoites but the IgG did not react with the schizont-infected erythrocytes (RBC) . Furthermore, double-fluorescence staining against SE47'-specific IgG and anti-human RBC membrane IgG showed that the RBC membrane disappeared from SE47'-specific-IgG-bound schizonts after cultivation . These observations suggest that the SE47'-specific antibodies inhibit parasite growth by cross-linking SERA molecules that are associated with merozoites in rupturing schizonts with partly broken RBC and parasitophorous vacuole membranes, blocking merozoite release.

Infect Immun, 1999 Apr, 67(4), 1633 - 9
Lipopolysaccharide enhances the production of vascular endothelial growth factor by human pulp cells in culture; Matsushita K et al.; We investigated whether vascular endothelial growth factor (VEGF) production by human pulp cells (HPC) is regulated by lipopolysaccharide (LPS) in relation to the pathogenesis of pulpitis . Although HPC incubated with medium alone only marginally expressed VEGF mRNA and produced a low level of VEGF as detected by enzyme-linked immunosorbent assay, the VEGF mRNA expression and VEGF production were markedly enhanced upon stimulation with LPS from Escherichia coli . Prevotella intermedia LPS, phorbol 12-myristate 13-acetate, and interleukin-6 also induced VEGF mRNA expression in HPC . A simian virus 40-infected HPC line also exhibited increased VEGF mRNA expression in response to E . coli LPS, but lung and skin fibroblasts did not . Fetal bovine serum (FBS) increased the sensitivity of HPC to LPS in a dose-dependent manner . HPC did not express membrane CD14 on their surfaces . However, the anti-CD14 monoclonal antibody MY4 inhibited VEGF induction upon stimulation with LPS in HPC cultures in the presence of 10% FBS but not in the absence of FBS . LPS augmented the VEGF production in HPC cultures in the presence of recombinant human soluble CD14 (sCD14) . To clarify the mechanisms of VEGF induction by LPS, we examined the possible activation of the transcription factor AP-1 in HPC stimulated with LPS, by a gel mobility shift assay . AP-1 activation in HPC was clearly observed, whereas that in skin fibroblasts was not . The AP-1 inhibitor curcumin strongly inhibited LPS-induced VEGF production in HPC cultures . In addition, a protein synthesis inhibitor, cycloheximide, inhibited VEGF mRNA accumulation in response to LPS . These results suggest that the enhanced production of VEGF in HPC induced by LPS takes place via an sCD14-dependent pathway which requires new protein synthesis and is mediated in part through AP-1 activation.

Genet Anal, 1999 Feb, 14(5-6), 181 - 6
Enzymatic methods for mutation scanning; Taylor GR et al.; Enzymatic methods for mutation scanning still lack the sensitivity and specificity of the chemical cleavage of mismatch method . However developments in our understanding of the mismatch recognition process should lead to improvements . Several promising candidates exist with potential for more specific and sensitive mutation detection.

Biochim Biophys Acta, 1999 Mar 19, 1430(2), 290 - 301
Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain; Kimura S et al.; The cDNA encoding solubilized porcine liver NADH-cytochrome b5 reductase catalytic domain (Pb5R) was cloned and overexpressed in Escherichia coli . A highly conserved His49 and a C-terminal Phe272 of Pb5R, which are located near the isoalloxazine moiety of the FAD, were systematically modulated by site-directed mutagenesis . Large structural change was not detected on the absorption and circular dichroism spectra of mutant proteins . Drastic changes in enzymatic properties were not observed, but the apparent Km value for soluble form of porcine liver cytochrome b5 (Pb5) was affected by the substitutions of His49 with glutamic acid and with lysine, deletion of C-terminal Phe272, and addition of Gly273 . The values of the catalytic constant (kcat) were obviously decreased by the substitution of His49 with glutamic acid or the addition of Gly273 . In these two mutants, the rate for reduction of FAD was decreased, and the rate for autoxidation of reduced FAD was increased . These results showed that His49 and C-terminal carboxyl group in Pb5R are not critical for the electron transfer to Pb5, but the electrostatic environmental changes at these positions could affect the recognition of Pb5 and modulate the catalytic function of the enzyme by changing the stability of reduced FAD.

Biochim Biophys Acta, 1999 Mar 19, 1430(2), 191 - 202
Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5; Wibbenmeyer JA et al.; Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced . The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding . Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains . The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG . Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.

Biochim Biophys Acta, 1999 Feb 10, 1430(1), 119 - 26
Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions; D'Silva I et al.; Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions . DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively . In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions . PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM) . Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends . The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively . These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP . The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.

Biochim Biophys Acta, 1999 Feb 10, 1430(1), 95 - 102
Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli; Hsu NC et al.; P450c21 catalyzes an important step in steroid synthesis . Its deficiency leads to symptoms of steroid imbalance . To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli . The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification . Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells . One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity . The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal . The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra . The D380 mutant protein had 3-fold reduction in peak heights in both spectra . This reduced heme binding resulted in 3-fold lower enzymatic activity.

Mutat Res, 1999 Mar 10, 425(1), 55 - 69
Use of log-linear analysis to construct explanatory models for TDBP- and AFB1-induced mutation spectra in lacI transgenic animals; Brackley ME et al.; Mutation spectra recovered from lacI transgenic animals exposed in separate experiments to tris-(2,3-dibromopropyl)phosphate (TDBP) or aflatoxin B1 (AFB1) were examined using log-linear analysis . Log-linear analysis is a categorical procedure that analyses contingency table data . Expected contingency table cell counts are estimated by maximum likelihood as effects of main variables and variable interactions . Evaluation of hierarchical models of decreasing complexity indicates when significant explanatory power is lost by the sequential omission of interactions between variables . Use of this technique allows construction of the most parsimonious models to account for mutation spectra obtained in the two experiments . The resulting statistical models are consistent with previous analyses of these data and with biological explanations for causes of the observed spectra .

Mutat Res, 1999 Mar 10, 425(1), 47 - 54
The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques; Hill KA et al.; There are mutational artifacts in the Big Blue(R) assay and it is important to characterize the source and nature of these mutations . Differences were reported in the mutation patterns of a small sample of 23 sectored and 91 circular mutant plaques derived from skin using the Big Blue(R) transgenic mouse mutation detection system {G . R . Stuart, N.J . Gorelick, J.L . Andrews, J.G . de Boer, B.W . Glickman, The genetic analysis of lacI mutations in sectored plaques from Big Blue transgenic mice, Environ . Mol . Mutagen 28 (1996) 385-392.} . We have extended these observations by analyzing 46 sectored and 224 circular mutant plaques derived from seven tissues . The frequency of sectored mutant plaques is estimated to be 16% with no significant variation with tissue type . However, the patterns of mutation for sectored mutants and mouse-derived mutations differed significantly (p=0.04) . Base substitutions in sectored mutant plaques do not show the asymmetries found in circular mutants consistent with integration of a GC rich transgene into the AT rich mammalian genome . Sectored mutants have mutation patterns consistent with a mixture of mouse, in vitro and Escherichia coli-derived mutations . Data on the relative frequencies of different mutant plaque morphologies suggests that overlapped plaques are substantially contaminated by sectored plaques at recommended plating densities .

Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 123 - 8
Cloning and expression of two carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase cDNAs in ovarian follicles of rainbow trout (Oncorhynchus mykiss); Guan G et al.; In salmonid fish, 20beta-hydroxysteroid dehydrogenase (20beta-HSD) is a key enzyme involved in the production of oocyte maturation-inducing hormone (MIH), 17alpha, 20beta-dihydroxy-4-pregnen-3-one . Here we report the isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like 20beta-HSD (CR/20beta-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles . Genomic DNA analysis showed that the two CR/20beta-HSD cDNAs are derived from two different genes . Northern blot and RT PCR analysis demonstrated that trout CR/20beta-HSDs are broadly expressed in various tissues . Enzymatic characterization using recombinant CR/20beta-HSD proteins produced in E . coli showed that the product of one of the two cDNAs had both 20beta-HSD and CR activity, but the other had neither activity . Although the functional significance of the two genes remains unresolved, these results clearly demonstrate the presence of two distinct CR/20beta-HSD transcripts in the trout ovary .

Biochem Biophys Res Commun, 1999 Feb 5, 255(1), 1 - 5
R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase; Ohsato T et al.; Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells . The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase . A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA . Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication . This mitochondrial R-loop is resolved by RecG in a dose-dependent manner . Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG . A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop . This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase . The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG .

Proteins, 1999 Mar 1, 34(4), 533 - 9
Characterization of internal motions of Escherichia coli ribonuclease H by Monte Carlo simulation; Haliloglu T; The backbone dynamics of Escherichia coli ribonuclease H (RNase H) is studied by a recently developed off-lattice Monte Carlo/Metropolis simulation technique . A low-resolution model (virtual-bond model) is used together with knowledge-based potentials . The calculated mean-square fluctuations in alpha carbons are in good agreement with crystallographic temperature factors . The conformations generated around the native state are analyzed by time-dependent orientational and conformational correlation functions to study the internal motions of RNase at different time windows . A correlation between the free-energy changes for native-state hydrogen exchange (HX) and the extent of the autocorrelation in the rotations of the virtual bonds at long times has been observed . Cross-correlations between the rotations of the bonds, which are near-neighbor in the sequence, are effective in all time windows and help the secondary structures to preserve their kinetic stability . On the other hand, the existence of cross-correlations at long times help the tertiary contacts be maintained . The order parameter of NH bond vector for each residue has been calculated and compared with 15N-NMR relaxation measurements.

Mol Cell Biol, 1999 Apr, 19(4), 3177 - 83
Mutator phenotypes conferred by MLH1 overexpression and by heterozygosity for mlh1 mutations; Shcherbakova PV et al.; Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer . In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1 function . First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation . Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function . Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis . Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations . Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented . Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain . This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.

Virus Res, 1999 Feb, 59(2), 203 - 10
Immunological characterization of two major secreted forms of recombinant hepatitis B virus e antigens; Hwang GY et al.; Plasmids containing PCR-amplified hepatitis B virus e antigen (HBeAg) genes (HBeAg-MV and HBeAg-SV) were constructed and expressed in E . coli strain DH5alpha . The induced intracellular glutathione S-transferase (GST) fusion proteins of HBeAg-MV and HBeAg-SV were recovered and purified from bacterial lysates by affinity chromatography with glutathione-sepharose beads . The HBeAg-MV protein contained an additional 19 amino acids at its amino terminus . These two proteins were specifically cleaved from GST by the protease factor Xa and recognized by a monoclonal antibody against HBeAg . HBeAg-MV and HBeAg-SV were found to be the two major components of the post-modified HBcAg during viral infection . The antigenic specificities of the fusion and purified HBeAgs (factor Xa-digested) were confirmed by the Abbott HBe enzyme immunoassay (EIA) detection system . Sera from patients with confirmed hepatocellular carcinoma (HCC) specifically reacted only with HBeAg moiety of fusion proteins . HCC sera bound more strongly to the HBeAg-SV protein than to the HBeAg-MV one . This indicates that HBeAg-SV is either more antigenic than -MV or is the major target protein for the elicitation of antibody production after HBV infection . Thus, the two recombinant HBeAgs expressed and obtained in this study are appropriate immunological agents for the diagnostic detection of hepatitis B virus infection in humans.

Virus Res, 1999 Feb, 59(2), 165 - 77
Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant; Osterrieder N; An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC) . The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene . On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC) . However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus . Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations . These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell . Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used . L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells . Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia . Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.

Protein Sci, 1998 Aug, 7(8), 1839 - 42
Overexpression of recombinant proteins with a C-terminal thiocarboxylate: implications for protein semisynthesis and thiamin biosynthesis; Kinsland C et al.; A facile and rapid method for the production of protein C-terminal thiocarboxylates on DNA-encoded polypeptides is described . This method, which relies on the mechanism of the cleavage reaction of intein-containing fusion proteins, can produce multi-milligram quantities of protein C-terminal thiocarboxylate quickly and inexpensively . The utility of this method for protein semisynthesis and implications for studies on the biosynthesis of thiamin are discussed.

Protein Sci, 1998 Aug, 7(8), 1836 - 8
Crystallization of recombinant human heme oxygenase-1; Schuller DJ et al.; Heme oxygenase catalyzes the NADPH, O2, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide . One of two primary isozymes, HO-1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C-terminus . While full-length human HO-1 consists of 288 residues, a truncated version with residues 1-265 has been expressed as a soluble active enzyme in Escherichia coli . The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies . SDS gel analysis indicated that the protein had undergone proteolytic degradation . An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize . N-terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1-226 and 1-237 . Expression of the 1-226 and 1-233 versions of human HO-1 provided active enzyme that crystallizes in a form suitable for diffraction studies . These crystals belong to space group P2(1), with unit cell dimensions a = 79.3 A, b = 56.3 A, c = 112.8 A, and beta = 101.5 degrees.

Protein Sci, 1998 Aug, 7(8), 1821 - 8
Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein; Kim K et al.; We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein . IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds . A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets . Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically . These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse . We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997) . Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions . For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants . In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step . Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.

Protein Sci, 1998 Aug, 7(8), 1796 - 801
Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry; Kelleher NL et al.; The 10(5) resolving power and MS/MS capabilities of Fourier-transform mass spectrometry provide electrospray ionization mass spectra containing >100 molecular and fragment ion mass values of high accuracy . Applying these spectra to the detection and localization of errors and modifications in the DNA-derived sequences of proteins is illustrated with the thiCEFSGH thiamin biosynthesis operon from Escherichia coli . Direct fragmentation of the multiply-charged intact protein ions produces large fragment ions covering the entire sequence; further dissociation of these fragment ions provides information on their sequences . For ThiE (23 kDa), the entire sequence was verified in a single spectrum with an accurate (0.3 Da) molecular weight (Mr) value, with confirmation from MS/MS fragment masses . Those for ThiH (46 kDa) showed that the Mr value (1 Da error) represented the protein without the start Met residue . For ThiF (27 kDa), MS/MS localized a sequence discrepancy to a 34 residue peptide . The first 107 residues of ThiC (74 kDa) were shown to be correct, with C-terminal heterogeneity indicated . For ThiG (predicted Mr = 34 kDa), ESI/FTMS showed two components of 7,310.74 (ThiS) and 26,896.5 Da (ThiG); MS/MS uncovered three reading frame errors and a stop codon for the first protein . MS/MS ions are consistent with 68 fragments predicted by the corrected ThiS/ThiG DNA sequences.

Protein Sci, 1998 Aug, 7(8), 1781 - 8
The role of the 6 lysines and the terminal amine of Escherichia coli single-strand binding protein in its binding of single-stranded DNA; Chen J et al.; Differential chemical modification of the lysines and amino-terminus of Escherichia coli single-strand binding (SSB) protein was used to determine their roles in the binding of SSB to single-stranded DNA (ssDNA) . A combination of isotope labeling and mass spectrometry was used to determine the rates at which SSB was acetylated by acetic anhydride . First, SSB was labeled by deuterated acetic anhydride for given lengths of time in the presence or absence of single-stranded ssDNA . Then, the protein was denatured and completely acetylated by nondeuterated acetic anhydride . Enzymatic digests of the completely acetylated, isotopically labeled SSB were analyzed by electrospray ionization mass spectrometry . The intensities of the deuterated and nondeuterated forms of acetylated peptides provided accurate quantification of the reactivity of the amines in native SSB, either free or bound to ssDNA . Acetylation rate constants were determined from time course measurements . In the absence of ssDNA, the terminal alpha-amine of SSB was 10-fold more reactive than Lys residues at positions 43, 62, 73, and 87 . The reactivities of Lys 7 and 49 were much lower yet, suggesting that they have very limited access to solution under any condition . In the presence of ssDNA, the reactivities of the amino-terminus and Lys residues 43, 62, 73, and 87 were reduced by factors of 3.7-25, indicating that the environments around all of these amines is substantially altered by binding of SSB to ssDNA . Three of these residues are located near putative ssDNA binding sites, whereas Lys 87 is located at the monomer-monomer interface.

Protein Sci, 1998 Aug, 7(8), 1757 - 67
Probing enzyme quaternary structure by combinatorial mutagenesis and selection; MacBeath G et al.; Genetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants . We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain-swapped enzyme . By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity . The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure . The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.

Protein Sci, 1998 Aug, 7(8), 1728 - 37
Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator; Vindigni A et al.; The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media . The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1) . Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin . In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2 . A specific inhibition of tPA by Cu2+ was discovered . The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM . In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases . The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI . Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme . Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E . coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr . These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.

J Vet Med Sci, 1999 Feb, 61(2), 171 - 3
Expression of bovine cytokines in Escherichia coli; Kashima T et al.; Glutathione S-transferase fusion proteins of bovine interleukin-2 (IL-2), IL-4, IL-6 and interferon-gamma (IFN-gamma) were expressed in Escherichia coli . Complementary DNA (cDNA) for open reading frame of each cytokine without signal peptide encoding region was amplified by reverse transcriptional polymerase chain reaction method and was subcloned into pGEX-5X-1 . In result, IL-6 and IFN-gamma fusion proteins in bacteria were soluble, but IL-2 and IL-4 fusion proteins were insoluble . The insoluble IL-2 fusion protein successfully refolded by urea became soluble . The recombinant IL-2, IL-6 and IFN-gamma could be obtained by the batch method using Glutathione Sepharose 4B and Factor Xa digestion, which may be useful for preparation of antisera as antigens and functional studies.

Domest Anim Endocrinol, 1999 Jan, 16(1), 69 - 80
Effect of exogenous FSH on ovulation rate in homozygous carriers or noncarriers of the Booroola FecB gene after hypothalamic-pituitary disconnection or after treatment with a GnRH agonist; Hudson NL et al.; We have tested the hypothesis "that the ovulation rate in homozygous carriers (BB) and noncarriers (+2) of the Booroola FecB gene would not be different if the plasma concentrations of follicle-stimulating hormone (FSH) in the two genotypes were similar." For this purpose we used two experimental animal models: 1) the hypothalamic-pituitary disconnected (HPD) ovary-intact ewe; and 2) and GnRH agonist (i.e., Deslorelin)-treated ewe . Following HPD or Deslorelin treatment, the animals had low plasma concentrations of gonadotropins and were anovulatory . In both animal models, BB and +2 ewes were treated with exogenous pregnant mares serum gonadotropin (PMSG) and varying doses of FSH to induce preovulatory follicular growth, and human chorionic gonadotropin (hCG) to induce ovulation . HPD or Deslorelin-treated animals administered with pregnant mares serum gonadotropin without FSH followed by human chorionic gonadotropin failed to ovulate . However for both animal models, the proportion of BB and +2 ewes ovulating to various doses of FSH differed such that significantly greater proportions of +2 animals ovulated relative to the BB genotype (P < 0.05) . When HPD or Deslorelin-treated BB and +2 ewes were administered identical doses of FSH, the mean ovulation rate and plasma concentrations of FSH in those animals which ovulated was the same in both genotypes . These findings confirm, at least in part, the aforementioned hypothesis . The results also demonstrated that higher ovulation rates were obtained in both genotypes as the FSH dose was increased . Collectively, these findings infer that the higher mean ovulation rate in normal intact BB ewes compared to the +2 genotype is attributable to effects of the FecB gene at the level of ovarian follicular development as well as at the level of pituitary FSH release.

Gesundheitswesen, 1999 Jan, 61(1), 38 - 44
{Environmental studies of asymptomatic kindergarten children as carriers of enterohemorrhagic Escherichia coli (EHEC) in the Ammerland district}; Vogelsang E et al.; The increase of serious EHEC diseases in the northwest Lower Saxony in summer 1997 was accompanied by a lively discussion on the hazard represented by this pathogen which gained a large amount of media attention . The Lower Saxony Public Health Department initiated a study of this case in the Weser-Ems government district to investigate the spread of EHEC by children and their supervisors in kindergartens receiving certified raw milk supply . This established that there were ten kindergarten children in the Ammerland rural district who were asymptomatik EHEC carriers . The Local Public Health Department immediately carried out the necessary epidemic hygiene measures . First of all, control investigations were performed on the affected children, including environmental investigations on their families and contracts . The results of these investigations revealed the need to carry out additional environmental investigations in two kindegartens (follow-up investigations) as well as amongst employees in a hospital canteen . Within the families, a mother of an affected child was also identified as another asymptomatic EHEC carrier . However, the strains that were isolated reflected different serotypes . In total, the investigation of 337 people in contact with the eleven asymptomatic EHEC carriers did not confirm any person-to-person transmission--even though three of the kindergarten children shedding the organisms for 6-10 weeks attended the kindergartens for at least 4 weeks . No EHEC disease occurred in the communal facilities, neither were any positive cases identified by additional control investigations . The results indicate a probable lower rate of infectiousness by healthy EHEC carriers than was previously thought to be the case . Further studies are needed to decide if in future one should proceed on a case by case basis when considering the reauthorization of affected children and other people to enter communal facilities.

Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 532 - 6
Recognition of sequence-directed structure of the ssDNA backbone by nucleases; Akaboshi E; Escherichia coli endonuclease I and exonuclease VII appear to recognize sequence-dependent conformations in the ssDNA backbone . ssDNAs, containing either A- and/or T-tract or a CAP binding region, were digested with these nucleases under conditions which minimize the formation of secondary structures . The digestion patterns were examined in relation to previous results of biochemical and crystallographic studies on dsDNA, and showed broad agreement . Endonuclease I cleaved ssDNA at sites corresponding to bent sites in dsDNA .

Biochem Biophys Res Commun, 1999 Mar 24, 256(3), 500 - 4
Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis; Timofeevski SL et al.; Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium . Despite structural similarity, these peroxidases oxidize different substrates . Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+ . By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli . The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3 . The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes . The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3 . Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation . The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis .

J Mol Biol, 1999 Mar 26, 287(2), 383 - 94
The crystal structure of Escherichia coli class II fructose-1, 6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity; Hall DR et al.; The structure of a class II fructose-1,6-bisphosphate aldolase in complex with the substrate analogue and inhibitor phosphoglycolohydroxamate (PGH) has been determined using X-ray diffraction terms to a resolution of 2.0 A (1 A=0.1 nm) . The crystals are trigonal, space group P3121 with a=b=78.24 A, c=289.69 A . The asymmetric unit is a homodimer of (alpha/beta)8 barrels and the model has refined to give R-work 19.2 %, R-free (based on 5 % of the data) 23.0 % . PGH resembles the ene-diolate transition state of the physiological substrate dihydroxyacetone phosphate . It is well ordered and bound in a deep polar cavity at the C-terminal end of the (alpha/beta)8 barrel, where it chelates the catalytic zinc ion using hydroxyl and enolate oxygen atoms . Trigonal bipyramidal coordination of the zinc ion is completed by three histidine residues . The complex network of hydrogen bonds at the catalytic centre is required to organise the position of key functional groups and metal ion ligands . A well-defined monovalent cation-binding site is observed following significant re-organisation of loop structures . This assists the formation of a phosphate-binding site on one side of the barrel that tethers PGH in the catalytic site . The positions of functional groups of substrate and putative interactions with key amino acid residues are identified . Knowledge of the complex structure complements the results of spectroscopic and site-directed mutagenesis studies, and contributes to our understanding of the mechanism and substrate specificity of this family of enzymes . A reaction mechanism distinct from that proposed for other class II aldolases is discussed . The results suggest that the class II aldolases should be sub-divided into two groups on the basis of both distinct folds and mechanism .

J Mol Biol, 1999 Mar 26, 287(2), 331 - 46
Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase; Putnam CD et al.; Uracil-DNA glycosylase (UDG), which is a critical enzyme in DNA base-excision repair that recognizes and removes uracil from DNA, is specifically and irreversably inhibited by the thermostable uracil-DNA glycosylase inhibitor protein (Ugi) . A paradox for the highly specific Ugi inhibition of UDG is how Ugi can successfully mimic DNA backbone interactions for UDG without resulting in significant cross-reactivity with numerous other enzymes that possess DNA backbone binding affinity . High-resolution X-ray crystal structures of Ugi both free and in complex with wild-type and the functionally defective His187Asp mutant Escherichia coli UDGs reveal the detailed molecular basis for duplex DNA backbone mimicry by Ugi . The overall shape and charge distribution of Ugi most closely resembles a midpoint in a trajectory between B-form DNA and the kinked DNA observed in UDG:DNA product complexes . Thus, Ugi targets the mechanism of uracil flipping by UDG and appears to be a transition-state mimic for UDG-flipping of uracil nucleotides from DNA . Essentially all the exquisite shape, electrostatic and hydrophobic complementarity for the high-affinity UDG-Ugi interaction is pre-existing, except for a key flip of the Ugi Gln19 carbonyl group and Glu20 side-chain, which is triggered by the formation of the complex . Conformational changes between unbound Ugi and Ugi complexed with UDG involve the beta-zipper structural motif, which we have named for the reversible pairing observed between intramolecular beta-strands . A similar beta-zipper is observed in the conversion between the open and closed forms of UDG . The combination of extremely high levels of pre-existing structural complementarity to DNA binding features specific to UDG with key local conformational changes in Ugi resolves the UDG-Ugi paradox and suggests a potentially general structural solution to the formation of very high affinity DNA enzyme-inhibitor complexes that avoid cross- reactivity .

J Mol Biol, 1999 Mar 26, 287(2), 277 - 85
A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila; Simmons AJ et al.; In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis . Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus . We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e . CPS.B2 domain) of CPSase . This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases . When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype . Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition . The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP . However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values . From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site . This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters .

J Mol Biol, 1999 Mar 26, 287(2), 265 - 76
Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling; Hansson LO et al.; A library of variant enzymes was created by combined shuffling of the DNA encoding the human Mu class glutathione transferases GST M1-1 and GST M2-2 . The parental GSTs are 84 % sequence identical at the protein level, but their specific activities with the substrates aminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold . Aminochrome is of particular interest as an oxidation product of dopamine and of possible significance in the etiology of Parkinson's disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds . GST M2-2 has at least two orders of magnitude higher catalytic activity with both of the substrates than any of the other known GSTs, including GST M1-1 . The DNA library of variant Mu class GST sequences contained "mosaic" structures composed of alternating segments of both parental sequences . All clones contained the 5'-end of a GST M1-1 clone optimized for high-level expression in Escherichia coli . The remainder of the sequences derived from segments of GST M2-2 and GST M1-1 DNA . All of the clones analyzed contained between two and seven distinct DNA segments . In addition, each clone contained an average of approximately one point mutation . None of the library clones analyzed was identical with either of the two parental structures . Variant GST sequences were expressed in E . coli, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determined in bacterial lysates . Such screening of more than 70 clones demonstrated a continuous range of activities covering at least two orders of magnitude for each of the substrates . For a given clone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB . This functional correlation suggests a common structural basis for the enzymatic mechanisms for conjugation of aminochrome and denitrosation of cyanoDMNG . From an evolutionary perspective, the results show that recombination of segments from homologous proteins gives rise to a large proportion of functionally competent proteins with a range of activities . The data support the proposal that natural evolution of protein functions may involve recombination of DNA segments followed by selection for advantageous functional properties of the resulting proteins . Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution .

Plant Mol Biol, 1999 Jan, 39(2), 381 - 6
Random mutagenesis in the large extrinsic loop E and transmembrane alpha-helix VI of the CP 47 protein of Photosystem II; Wu J et al.; The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution . Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane alpha-helix VI . Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered . The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands . Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47 . These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters . Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations . Finally, a mutant cell line exhibiting a substitution at position 342G was recovered . The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution . In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced . These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen.

Mol Biochem Parasitol, 1999 Jan 25, 98(2), 225 - 37
Molecular characterization of a calponin-like protein from Schistosoma japonicum; Yang W et al.; The gene for a Schistosoma japonicum (Philippine strain origin) (Sjp) calponin-like protein has been cloned and characterised . The clone, designated P14, was isolated from a Sjp adult worm lambda ZAP cDNA library by immunoscreening, and was shown to contain a full-length cDNA encoding a 38.3 kDa protein that shared significant sequence similarity to a number of previously reported calponins and 22 kDa smooth-muscle proteins . Northern analysis indicated the P14 transcript was approximately 2.2 kb in both Sjp and Chinese strain S . japonicum (Sjc) adult worms . Southern blot analysis of genomic DNA suggested that several copies of the P14 gene are present in the Sjc and Sjp genomes but only one copy was evident in the S . mansoni (Sm) genome . Western blot analysis indicated that the product of P14 occurs as a 38 kDa protein in adult Sjp worms and homologues are present in adult worms of Sjc and Sm . At least six isoforms, all with a similar molecular size of approximately 38 kDa and isoelectric points ranging from 8.1 to 9.5, were present in adult Sjc worms . The protein was immunolocalized to the muscle of male and female Sjc adult worms . Recombinant protein was expressed in E . coli and purified under denaturing conditions, and in yeast to produce a soluble protein in purified form . The availability of purified, correctly folded protein will allow investigations into its biological functions and potential involvement in host immunity.

Bioorg Khim, 1998 Dec, 24(12), 920 - 5
{A new inhibitor of pyrimidine phosphorylase}; Dmitrieva NA et al.; 5,5-Bis(hydroxymethyl)-2-oxo-{1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases . Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly . Its LD50 in mice was 40 mg/kg . Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.

FEMS Microbiol Lett, 1999 Mar 1, 172(1), 91 - 7
Interaction of Escherichia coli heat-stable enterotoxin B with rat intestinal epithelial cells and membrane lipids; Chao KL et al.; The binding of 125I-labeled Escherichia coli heat-stable enterotoxin B to rat intestinal epithelial cells was unsaturable and nonspecific, at concentrations well above that required to mediate biological events . Following its interaction with intestinal cells, approximately 50-80% of heat-stable enterotoxin B remained stably associated with the cells, implying that it was partitioned into the membrane and/or internalized by the cell . The toxin bound with different affinities to lipids isolated from intestinal epithelial cells, phospholipids, glycolipids, neutral lipids and to model membrane vesicles containing negatively charged lipids . These results indicate that heat-stable enterotoxin B utilizes the membrane bilayer, rather than a surface protein or glycoprotein in modulating toxin-induced enterotoxicity.

FEMS Microbiol Lett, 1999 Mar 1, 172(1), 29 - 34
An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli; Lai XH et al.; Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line . All the seven virotypes of E . coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response . With the exception of the enterotoxigenic E . coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level . These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development.

Dev Genes Evol, 1999 Mar, 209(3), 145 - 54
Segmentation gene expression in the mothmidge Clogmia albipunctata (Diptera, psychodidae) and other primitive dipterans; Rohr KB et al.; To obtain a clearer understanding of the evolutionary transition between short- and long-germ modes of embryogenesis in insects, we studied the expression of two gap genes hunchback (hb) and Kruppel (Kr) as well as the pair-rule gene even-skipped (eve) in the dipteran Clogmia albipunctata (Nematocera, Psychodidae) . This species has features of both short- and long-germ mode of embryogenesis . In Clogmia hb expression deviates from that known in Drosophila in two main respects: (1) it shows an extended dorsal domain that is linked to the large serosa anlage, and (2) it shows a terminal expression in the proctodeal region . These expression patterns are reminiscent of the hb expression pattern in the beetle Tribolium, which has a short germ mode of embryogenesis . Kruppel expression, on the other hand, was found to be rather similar to the Drosophila expression, both at early and late stages . eve expression starts with six stripes formed at blastoderm stage, while the seventh is only formed after the onset of gastrulation and germband extension . Surprisingly, no segmental secondary Eve stripes could be observed in Clogmia although such segmental stripes are known from higher dipterans, beetles and hymenopterans . We therefore also studied another nematoceran, Coboldia, to address this question and found that some segmental stripes form by intercalation as in Drosophila, although belatedly . Our results suggest that Clogmia embryogenesis, both with respect to morphological and molecular characteristics represents an intermediate between the long-germ mode known from higher dipterans such as Drosophila, and the short-germ mode found in more ancestral insects.

Nature, 1999 Mar 4, 398(6722), 84 - 90
Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase; Meng W et al.; Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface . The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity . Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential . In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70 . Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70 . The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein . The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation . In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket . Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

Nature, 1999 Mar 4, 398(6722), 39 - 46
Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport; Vetter IR et al.; The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport . The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes . The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex . The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end . This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP . Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.

J Mol Recognit, 1998 Winter, 11(1-6), 91 - 3
Phage viability in organic media: insights into phage stability; Olofsson L et al.; The stability of the filamentous phages derived from phagemid pG8H6 has been examined in a range of solvents and solvent mixtures . The results show an enhanced capacity to infect E . coli after exposure to various organic solvent-water mixtures . The dependence of stability upon solvent hydrophobicity was demonstrated . Furthermore, conditions have been identified which should allow the application of phage display libraries based upon pG8H6 in organic media.

Biochemistry, 1999 Mar 16, 38(11), 3421 - 5
Curvature of dinucleotide poised for formation of trinucleotide in transcription with Escherichia coli RNA polymerase; Garland CS et al.; A frequently used schematic model of transcriptional elongation shows an RNA polymerase molecule moving along a linear DNA . This model is of course highly idealized and not compatible with promoter sequences {Gralla, J . D . (1991) Cell 66, 415-418; Schleif, R . (1992) Annu . Rev . Biochem . 61, 199-223} and regulatory proteins {Koleske, A . J., and Young, R . A . (1995) Trends Biochem . Sci . 20, 113-116; Dunaway, M., and Droge, P . (1989) Nature 341, 657-659; Muller, H . P., Sogo, J . M., and Schaffner, W . (1989) Cell 58, 767-777} located some distance away from the point of transcription initiation {Karsten, R., von Hippel, P . H., and Langowski, J . (1995) Trends Biochem . Sci . 20, 500-506} . These circumstances lead to the expectation of curvature along the DNA strand and require looping between sometimes distant points . We have now shown curvature in a dinucleotide formed at the very onset of transcription when it is poised for reaction with a mononucleotide to form a trinucleotide . The curvature became evident from the demonstration that a metal ion bound with a mononucleotide in the i+1 (elongation) site is approximately equidistant from bases at the 5' end (i-1 site) and 3' end (i site) of the dinucleotide . Similar results were obtained with three different dinucleotides and four mononucleotides . Curvature of the RNA initiate may reflect curvature of the DNA to which it is bound . These studies show curvature to be a significant feature in the interaction between DNA template and RNA elongate even at the very beginning of transcription.

Biochemistry, 1999 Mar 16, 38(11), 3393 - 400
Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin; Blanchard CZ et al.; Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids . The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein . In this report a system for site-directed mutagenesis of the biotin carboxylase component is described . The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid . Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography . This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase . In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function . The mutations did not affect the bicarbonate-dependent ATPase reaction . In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin . The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations . The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes . The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme . Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.

Biochemistry, 1999 Mar 16, 38(11), 3335 - 44
Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: kinetic and mechanistic aspects; D'Ham C et al.; Oligonucleotides that contain a single modified pyrimidine, i.e., thymine glycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were synthesized in order to investigate the substrate specificity and the excision mechanism of two Escherichia coli repair enzymes: endonuclease III and formamidopyrimidine DNA glycosylase (Fpg) . Three techniques of analysis were employed . A gas chromatography-mass spectrometry (GC-MS) assay with HPLC prepurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage . Values of Vm/Km constants lead to the conclusion that the substrates are processed by endonuclease III with the following preference: Tg >> 5-OHC > DHT . This confirms that Tg is an excellent substrate for endonuclease III . Fpg-mediated cleavage of the 5-OHC-containing oligonucleotide is processed at the same rate than endonuclease III . Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificity of this enzyme to a new modified pyrimidine . While 5-OHC-containing oligonucleotides are cleaved by the two enzymes, no or a small amount of the modified base was found to be released, as determined by GC-MS . From these data it may be suggested that 5-OHC could be modified during its enzymatic excision . Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-OHC- and DHT-containing oligonucleotides showed that endonuclease III cleaves the DNA backbone mainly through a hydrolytic process and that no beta-elimination product was detected.

Biochemistry, 1999 Mar 16, 38(11), 3327 - 34
Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site; Lundegaard C et al.; Phosphoribosyltransferases catalyze the formation of nucleotides from a nitrogenous base and 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) . These enzymes and the PRPP synthases resemble each other in a short homologous sequence of 13 amino acid residues which has been termed the PRPP binding site and which interacts with the ribose 5-phosphate moiety in structurally characterized complexes of PRPP and nucleotides . We show that each class of phosphoribosyltransferases has subtle deviations from the general consensus PRPP binding site and that all uracil phosphoribosyltransferases (UPRTases) have a proline residue at a position where other phosphoribosyltransferases and the PRPP synthases have aspartate . To investigate the role of this unusual proline (Pro 131 in the E . coli UPRTase) for enzyme activity, we changed the residue to an aspartate and purified the mutant P131D enzyme to compare its catalytic properties with the properties of the wild-type protein . We found that UPRTase of E . coli obeyed the kinetics of a sequential mechanism with the binding of PRPP preceding the binding of uracil . The basic kinetic constants were derived from initial velocity measurements, product inhibition, and ligand binding assays . The change of Pro 131 to Asp caused a 50-60-fold reduction of the catalytic rate (kcat) in both directions of the reaction and approximately a 100-fold increase in the KM for uracil . The KM for PRPP was strongly diminished by the mutation, but kcat/KM,PRPP and the dissociation constant (KD,PRPP) were nearly unaffected . We conclude that the proline in the PRPP binding site of UPRTase is of only little importance for binding of PRPP to the free enzyme, but is critical for binding of uracil to the enzyme-PRPP complex and for the catalytic rate.

Biochemistry, 1999 Mar 16, 38(11), 3280 - 4
Familial prion disease mutation alters the secondary structure of recombinant mouse prion protein: implications for the mechanism of prion formation; Cappai R et al.; A considerable body of data supports the model that the infectious agent (called a prion) which causes the transmissible spongiform encephalopathies is a replicating polypeptide devoid of nucleic acid . Prions are believed to propagate by changing the conformation of the normal cellular prion protein (PrPc) into an infectious isoform without altering the primary sequence . Proteins equivalent to the mature form of the wild-type mouse prion protein (residues 23-231) or with a mutation equivalent to that associated with Gerstmann-Straussler-Scheinker disease (proline to leucine at codon 102 in human; 101 in mouse) were expressed in E . coli . The mutation did not alter the relative proteinase K susceptibility properties of the mouse prion proteins . The wild-type and mutant proteins were analyzed by circular dichroism under different pH and temperature conditions . The mutation was associated with a decrease in alpha-helical content, while the beta-sheet content of the two proteins was unchanged . This suggests the mutation, while altering the secondary structure of PrP, is not sufficient to induce proteinase K resistance and could therefore represent an intermediate isoform along the pathway toward prion formation.

Eur J Pharmacol, 1999 Feb 19, 367(2-3), 351 - 9
Central effects of cromoglycate sodium salt in rats treated with lipopolysaccharide; Nava F et al.; In 24-h water- and food-deprived rats, we have evaluated the effects of cromoglycate sodium salt, an inhibitor of the mast cell degranulation with anti-inflammatory and membrane-stabilizating activity, on the central effects induced by Escherichia coli lipopolysaccharide (LPS) . Intraperitoneal (i.p.) injection of LPS (0.25, 0.50 and 1 mg/kg) induced a dose-dependent inhibition of water and food intake, fever, reduction in locomotor activity as well as increased anxiety levels . All these LPS effects were antagonized by a prior intracerebroventricular (i.c.v.) injection of cromoglycate sodium salt (100, 150 and 200 microg/rat) . Our findings suggest that peripheral LPS administration may activate brain mast cells and indicate an involvement of these cells in brain pathophysiology.

Bull Soc Pathol Exot, 1998, 91(5 Pt 1-2), 450 - 1
{Traveller's diarrhea: progress and lessons drawn from recent surveys}; Steffen R; Diarrhea among travellers continues to be as widespread as ever . A multicentric investigation carried out in Jamaica, Kenya, Goa (India) and Fortaleza (Brazil) indicates high incidence rates and enterotoxigenic Escherichia coli are the most common etiology for each destination.

Mol Cell, 1999 Feb, 3(2), 255 - 61
hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA; Gradia S et al.; Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch . Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular . We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone . Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.

Mol Cell, 1999 Feb, 3(2), 239 - 45
ISWI is an ATP-dependent nucleosome remodeling factor; Corona DF et al.; The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin . We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions . The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes . Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes . The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.

FEMS Microbiol Lett, 1999 Feb 15, 171(2), 141 - 6
Increased heavy metal sensitivity of Escherichia coli producing the expression product of priA gene derived from the basidiomycete Lentinus edodes; Ishizaki T et al.; We have previously isolated a developmentally regulated novel gene, priA, from the basidiomycete Lentinus edodes . The deduced PRIA protein contains the two set of motifs similar to a 'zinc finger' typified by transcription factor TFIIIA and the motif of a 'zinc cluster' observed in metallothioneins . It also contains a hydrophobic N-terminal sequence . Here Escherichia coli cells producing PRIA were found to show a remarkable sensitivity to zinc ion and other heavy metal ions such as nickel and cadmium . Deletion analysis of PRIA revealed that the zinc-binding motifs and the hydrophobic N-terminal sequence are responsible for conferring the heavy metal sensitivity on the host cells.

Ital J Gastroenterol Hepatol, 1998 Oct, 30 Suppl 3, S261 - 3
Experimental model of Helicobacter pylori infection; Del Giudice G et al.; Critical issues in the development of a vaccine against Helicobacter pylori are represented by the definition of molecules important in the pathogenesis of the infection, by the availability of an animal model reproducing several aspects of the human infection, and lastly by the availability of powerful adjuvants allowing strong protection after mucosal delivery of the antigens . A mouse model of Helicobacter pylori infection was established in our laboratories . Vaccination of these animals with Helicobacter pylori antigens, such as VacA, CagA, etc., induced protection, both prophylactic and therapeutic, when antigens were administered orally together with fully non toxic mutants of Escherichia coli heat-labile enterotoxin, as mucosal adjuvants . This experimental mouse model allows the study of the pathogenesis of Helicobacter pylori infection and the development of vaccines.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 3211 - 6
Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus; Wilson SP et al.; To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter . This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice . After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression . Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses . Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection . Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission . This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord . The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene . Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2964 - 9
Microsatellite instability in Drosophila spellchecker1 (MutS homolog) mutants; Flores C et al.; We have cloned a mutS homolog from Drosophila melanogaster called spellchecker1 (spel1) and have constructed spel1 mutant flies . MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair . The spel1 gene belongs to a subfamily of mutS first characterized by the MSH2 gene of yeast and which also includes hMSH2, one of the two major hereditary nonpolyposis colon cancer loci of humans . Like msh2 mutants in other species, we find that flies lacking the spel1 gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10-12 fly generations . Using a new assay, we have also discovered that mutations in spel1 decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion . Contrary to the case in mammalian cells, spel1 deficiency does not affect tolerance of flies to a methylating agent nor does it affect resistance to gamma-irradiation.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2752 - 7
Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors; Sata M et al.; Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release . ARFs are approximately 20-kDa GTPases that are active with bound GTP and inactive with GDP bound . Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins . All known ARF guanine nucleotide-exchange proteins contain a Sec7 domain of approximately 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA) . To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7) . Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity . A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d) . Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect . As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2692 - 7
Identification of a transcription factor, encoded by two vaccinia virus early genes, that regulates the intermediate stage of viral gene expression; Sanz P et al.; Vaccinia virus early, intermediate, and late stage genes are sequentially transcribed by the viral RNA polymerase within the cytoplasm of infected cells . We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcription of a template with an intermediate stage promoter . Coexpression of the A8R and A23R genes in Escherichia coli was required for in vitro activity . In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor . This factor, which we named VITF-3, complemented three viral proteins-namely, the RNA polymerase, capping enzyme, and a 30-kDa protein called VITF-1 that is also a subunit of the RNA polymerase-and an unidentified cell factor called VITF-2 . Expression of the A8R and A23R genes occurred between 1 and 5 h after vaccinia virus infection and was not prevented by an inhibitor of DNA replication, consistent with a role for VITF-3 in specifically regulating intermediate transcription in vivo . The vaccinia virus A8R and A23R genes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified.

Proc Natl Acad Sci U S A, 1999 Mar 16, 96(6), 2682 - 6
GroES in the asymmetric GroEL14-GroES7 complex exchanges via an associative mechanism; Horowitz PM et al.; The interaction of the chaperonin GroEL14 with its cochaperonin GroES7 is dynamic, involving stable, asymmetric 1:1 complexes (GroES7.GroEL7-GroEL7) and transient, metastable symmetric 2:1 complexes {GroES7.GroEL7-GroEL7.GroES7} . The transient formation of a 2:1 complex permits exchange of free GroES7 for GroES7 bound in the stable 1:1 complex . Electrophoresis in the presence of ADP was used to resolve free GroEL14 from the GroES7-GroEL14 complex . Titration of GroEL14 with radiolabeled GroES7 to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex . No stable 2:1 complex was detected . Preincubation of the asymmetric GroES7.GroEL7-GroEL7 complex with excess unlabeled GroES7 in the presence of ADP demonstrated GroES7 exchange . The rates of GroES7 exchange were proportional to the concentration of unlabeled free GroES7 . This concentration dependence points to an associative mechanism in which exchange of GroES7 occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL14 . Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES7 . In agreement with recent structural studies, this indicates that conformational changes in GroEL14 following the dissociation of GroES7 must precede ADP release . These results explain how the GroEL14 cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes.

Bioconjug Chem, 1999 Mar-Apr, 10(2), 155 - 8
Paramagnetic oligonucleotides: contrast agents for magnetic resonance imaging with proton relaxation enhancement effects; Hines JV et al.; An antisense paramagnetic oligonucleotide analogue targeted to a model macromolecular receptor (5S rRNA) was prepared . The paramagnetic agent's relaxivity (dependence of the relaxation rate on paramagnetic agent concentration) in the presence and absence of the macromolecular receptor was measured at 1.5 and 6.3 T . The relaxivity of the targeted agent increased specifically in the presence of the macromolecular receptor (16% at 6.3 T and 15% at 1.5 T) . This effect was specific for a paramagnetic oligonucleotide targeted to the receptor and was larger than the relaxivity enhancement due simply to receptor-induced viscosity differences . Maximizing this relaxivity enhancement of tumor targeted paramagnetic oligonucleotides will aid in contrast agent development for magnetic resonance imaging.

Int J Biol Macromol, 1999 Jan, 24(1), 65 - 7
Size and folding in globular proteins; Yan BC et al.; We have modeled protein folding by packing a unified length of regular structural elements (alpha-helices and beta-sheets) into a 'cube' . In a globular protein with m alpha-helices and n beta-strands, this unified length is expressed in units of heptapeptides in alpha-helices, and in units of tripeptides in beta-strands . Calculations using published data show that a 4-helix bundle (m = 4, n = 0) has at least 2 x 2 x 2 helical heptapeptides; the 16-strand beta-barrel of porin (m = 0, n = 16) is at most 4 x 4 x 4 tripeptides in beta-strands . Compact, recurring protein modules with mixed helices and beta-strands are the ones that actually acquire a geometrically quasi-spherical, or cubic, shape.

Int J Biol Macromol, 1999 Jan, 24(1), 11 - 4
Structural and functional heterogeneity of the amino-terminal receptor-binding domain of human interferon-alpha 2; Kontsekova E et al.; Structural immunoanalysis of human interferon (IFN)-alpha 2c revealed antigenic and functional heterogeneity in its N-terminal receptor-binding domain (loop AB) . Monoclonal antibodies (mAbs) mapped to the region 30-53 of IFN-alpha 2 defined three partially overlapping antigenic sites designated here as 'a', 'b' and 'c' . For the high-affinity binding of IFN-alpha 2c to the cellular receptor, site b located in segment 34-41 and site c (residues 43-53) appeared to be most important . Only the part of site a (amino acids 30-33) seemed to be involved in the interaction with receptor . The segment of residues 30-46 forms a relatively straight structure on the protein surface, according to the three-dimensional model of human IFN-alpha 2.

FEMS Immunol Med Microbiol, 1999 Feb, 23(2), 125 - 33
Aspergillus fumigatus catalases: cloning of an Aspergillus nidulans catalase B homologue and evidence for at least three catalases; Takasuka T et al.; The presence of catalases in the water soluble fractions of three Aspergillus fumigatus strains was investigated using non-denaturing and denaturing polyacrylamide gel electrophoresis and Western analysis . Using non-denaturing polyacrylamide gel electrophoresis and staining for catalase activity, three separate catalases were identified . An A . fumigatus catalase gene (catB) was cloned from genomic DNA using the Aspergillus niger catR gene as a probe . Polyclonal antibodies were raised to a glutathione S-transferase-CatB fusion product expressed in Escherichia coli . Western analysis indicated that, under denaturing conditions, the polyclonal antibody recognised a 90-kDa band and under non-denaturing conditions, two separate bands were identified . These results indicate that A . fumigatus in addition to CatB, produces at least two other catalases, one of which is similar in size to CatB . The polyclonal antibody was also used to observe catalase expression in mice, experimentally infected with A . fumigatus . Staining was observed heterogeneously throughout the fungal hyphae . This result indicates that catalase is produced by A . fumigatus during invasive aspergillosis.

Surgery, 1999 Mar, 125(3), 339 - 44
Effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit; Cullen JJ et al.; BACKGROUND: The disrupted intestinal transit during endotoxemia may be mediated by nitric oxide (NO) . We hypothesized that the isoforms of nitric oxide synthase (NOS) are up-regulated in intestinal smooth muscle during endotoxemia and that the scavenging of NO will normalize transit . METHODS: Rats were given Escherichia coli lipopolysaccharide (LPS) 10 mg/kg intravenously and were killed 4 hours later . To determine the activity of NOS isoforms in the jejunum and ileum, the conversion of tritiated L-arginine to tritiated L-citrulline was measured . Western immunoblots were performed by incubating the extracted protein with specific polyclonal antibodies . To determine intestinal transit, rats were divided into 4 groups: 0.9% sodium chloride 1 mL/h intravenously for 5 hours, LPS 10 mg/kg intravenous bolus plus 1 mL/h 0.9% sodium chloride intravenously, LPS plus oxyhemoglobin 0.5 g/kg/h intravenously, and oxyhemoglobin 0.5 g/kg/h intravenously . RESULTS: LPS increased the constitutive and inducible NOS enzyme activities in the jejunum and ileum . Western blots demonstrated that LPS up-regulates both the NOS1 and NOS2 isoforms in jejunal and ileal smooth muscle . Oxyhemoglobin alone increased intestinal transit compared with controls, whereas endotoxemia increased intestinal transit, which was ameliorated with infusions of oxyhemoglobin . CONCLUSIONS: NO may play a major role in mediating the rapid intestinal transit induced by endotoxemia.

Surgery, 1999 Mar, 125(3), 280 - 7
C1-esterase inhibitor and its effects on endotoxin-induced leukocyte adherence and plasma extravasation in postcapillary venules; Schmidt W et al.; BACKGROUND: C1-esterase inhibitor (C1-INH) has been shown to have beneficial effects in patients with sepsis . However, the microcirculatory effects of C1-INH during sepsis are unknown . This study investigated the influence of C1-INH on leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics in postcapillary venules of rat mesentery during endotoxemia . METHODS: Thirty-two anesthetized Wistar rats randomly received 1 of 4 treatments: pretreatment with infusion of C1-INH in a concentration of 7.5 U.kg-1 body weight (C1-INH-7.5 group, n = 8) or in a concentration of 15 U.kg-1 body weight (C1-INH-15 group, n = 8) followed by continuous infusion of Escherichia coli lipopolysaccharide (LPS) . The LPS group (n = 8) was pretreated with saline solution 30 minutes before LPS infusion . The control group (n = 8) received equivalent amounts of saline infusion . Leukocyte adherence, red blood cell velocity, and vessel diameters in postcapillary venules of rat mesentery were determined every 60 minutes during a period of 120 minutes using in vivo videomicroscopy . Vascular permeability was determined by measuring the extravasation of fluorescence-labeled albumin . Venular wall shear rate was calculated from mean red blood cell velocity and vessel diameter . RESULTS: LPS infusion induced a decrease in venular wall shear rate and an increase in leukocyte adherence and vascular permeability in postcapillary venules of rat mesentery . All microcirculatory disturbances were attenuated by pretreatment with C1-INH, showing no significant difference between the 2 concentrations . CONCLUSIONS: Pretreatment with C1-INH attenuates endotoxin-induced leukocyte adherence and macromolecular leakage in postcapillary venules of rat mesentery, indicating that complement inhibition might be a therapeutic tool in the treatment of sepsis.

Biochim Biophys Acta, 1999 Feb 4, 1417(1), 37 - 50
A transfection compound series based on a versatile Tris linkage; Cameron FH et al.; The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants . The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group . The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties . We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents . A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Biochim Biophys Acta, 1999 Jan 27, 1410(1), 32 - 50
Sequence analysis of cytochrome bd oxidase suggests a revised topology for subunit I; Osborne JP et al.; Numerous sequences of the cytochrome bd quinol oxidase (cytochrome bd) have recently become available for analysis . The analysis has revealed a small number of conserved residues, a new topology for subunit I and a phylogenetic tree involving extensive horizontal gene transfer . There are 20 conserved residues in subunit I and two in subunit II . Algorithms utilizing multiple sequence alignments predicted a revised topology for cytochrome bd, adding two transmembrane helices to subunit I to the seven that were previously indicated by the analysis of the sequence of the oxidase from E . coli . This revised topology has the effect of relocating the N-terminus and C-terminus to the periplasmic and cytoplasmic sides of the membrane, respectively . The new topology repositions I-H19, the putative ligand for heme b595, close to the periplasmic edge of the membrane, which suggests that the heme b595/heme d active site of the oxidase is located near the outer (periplasmic) surface of the membrane . The most highly conserved region of the sequence of subunit I contains the sequence GRQPW and is located in a predicted periplasmic loop connecting the eighth and ninth transmembrane helices . The potential importance of this region of the protein was previously unsuspected, and it may participate in the binding of either quinol or heme d . There are two very highly conserved glutamates in subunit I, E99 and E107, within the third transmembrane helix (E . coli cytochrome bd-I numbering) . It is speculated that these glutamates may be part of a proton channel leading from the cytoplasmic side of the membrane to the heme d oxygen-reactive site, now placed near the periplasmic surface . The revised topology and newly revealed conserved residues provide a clear basis for further experimental tests of these hypotheses . Phylogenetic analysis of the new sequences of cytochrome bd reveals considerable deviation from the 16sRNA tree, suggesting that a large amount of horizontal gene transfer has occurred in the evolution of cytochrome bd.

Nucleic Acids Res, 1999 Apr 1, 27(7), 1683 - 9
The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria; Tomita K et al.; It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU . Nucleotide sequencing of starfish mt tRNAAsn revealed that the anticodon is GPsiU, U35 at the anticodon second position being modified to pseudouridine (Psi) . In contrast, mt tRNALys, corresponding to another lysine codon, AAG, has the anticodon CUU . mt tRNAs possessing anti-codons closely related to that of tRNAAsn, but responsible for decoding only two codons each-tRNAHis, tRNAAsp and tRNATyr-were found to possess unmodified U35 in all cases, suggesting the importance of Psi35 for decoding the three codons . Therefore, the decoding capabilities of two synthetic Escherichia coli tRNAAla variants with the anticodon GPsiU or GUU were examined using an E.coli in vitro translation system . Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was approximately 2-fold higher in the case of tRNAAlaGPsiU than tRNAAlaGUU . These findings imply that Psi35 of echinoderm mt tRNAAsn actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.

Nucleic Acids Res, 1999 Apr 1, 27(7), 1674 - 82
Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells; Wade-Martins R et al.; We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay . The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication . This element provides nuclear retention in cells expressing the EBV protein EBNA-1 . We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line . Episomal mitotic stability was quantitatively analysed using flow cytometry . The episomes were also tracked by time course photography of expanding colonies . A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth . Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days . Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.

EMBO J, 1999 Mar 15, 18(6), 1712 - 21
Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control; Lobner-Olesen A; A novel method was devised to measure the number of plasmids in individual Escherichia coli cells . With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured . Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes . When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased . At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected . This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome . However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome . At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.

EMBO J, 1999 Mar 15, 18(6), 1689 - 700
Self assembly of NuMA: multiarm oligomers as structural units of a nuclear lattice; Harborth J et al.; NuMA is a nuclear matrix protein in interphase and relocates to the spindle poles in mitotis . Different NuMA constructs, in which either N- or C-terminal domains were deleted, and the full-length construct were expressed in Escherichia coli, and the NuMA polypeptides were purified to homogeneity and allowed to assemble in vitro . Electron microscopy showed that NuMA can build multiarm oligomers by interaction of the C-terminal globular domains . Each arm of the oligomer corresponds to a NuMA dimer . Oligomers with up to 10 or 12 arms have been observed for both full-length NuMA and for constructs that still contain the proximal part of the C-terminal tail domain . Other results from this laboratory have shown that transient overexpression of NuMA in HeLa cells induces a nuclear scaffold with a quasi-hexagonal organization that can fill the nuclei . Here we show that computer modelling of the three-dimensional packing of NuMA into such scaffolds can explain the different spacing of the hexagons seen when constructs with different coiled-coil lengths are used . Thus, the 12 arm oligomer, for which we have in vitro evidence, may be the structural unit from which the nuclear scaffold in transfected cells is built.

EMBO J, 1999 Mar 15, 18(6), 1653 - 9
Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions; Brandi A et al.; The most characteristic event of cold-shock activation in Escherichia coli is believed to be the de novo synthesis of CspA . We demonstrate, however, that the cellular concentration of this protein is > or = 50 microM during early exponential growth at 37 degrees C; therefore, its designation as a major cold-shock protein is a misnomer . The cspA mRNA level decreases rapidly with increasing cell density, becoming virtually undetectable by mid-to-late exponential growth phase while the CspA level declines, although always remaining clearly detectable . A burst of cspA expression followed by a renewed decline ensues upon dilution of stationary phase cultures with fresh medium . The extent of cold-shock induction of cspA varies as a function of the growth phase, being inversely proportional to the pre-existing level of CspA which suggests feedback autorepression by this protein . Both transcriptional and post-transcriptional controls regulate cspA expression under non-stress conditions; transcription of cspA mRNA is under the antagonistic control of DNA-binding proteins Fis and H-NS both in vivo and in vitro, while its decreased half-life with increasing cell density contributes to its rapid disappearance . The cspA mRNA instability is due to its 5' untranslated leader and is counteracted in vivo by the cold-shock DeaD box RNA helicase (CsdA).

EMBO J, 1999 Mar 15, 18(6), 1447 - 58
X-ray structure of T4 endonuclease VII: a DNA junction resolvase with a novel fold and unusual domain-swapped dimer architecture; Raaijmakers H et al.; Phage T4 endonuclease VII (Endo VII), the first enzyme shown to resolve Holliday junctions, recognizes a broad spectrum of DNA substrates ranging from branched DNAs to single base mismatches . We have determined the crystal structures of the Ca2+-bound wild-type and the inactive N62D mutant enzymes at 2.4 and 2.1 A, respectively . The Endo VII monomers form an elongated, highly intertwined molecular dimer exhibiting extreme domain swapping . The major dimerization elements are two pairs of antiparallel helices forming a novel 'four-helix cross' motif . The unique monomer fold, almost completely lacking beta-sheet structure and containing a zinc ion tetrahedrally coordinated to four cysteines, does not resemble any of the known junction-resolving enzymes, including the Escherichia coli RuvC and lambda integrase-type recombinases . The S-shaped dimer has two 'binding bays' separated by approximately 25 A which are lined by positively charged residues and contain near their base residues known to be essential for activity . These include Asp40 and Asn62, which function as ligands for the bound calcium ions . A pronounced bipolar charge distribution suggests that branched DNA substrates bind to the positively charged face with the scissile phosphates located near the divalent cations . A model for the complex with a four-way DNA junction is presented.

EMBO J, 1999 Mar 15, 18(6), 1435 - 46
The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds; Hall DR et al.; The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum . We have determined the structure of ModE using multi-wavelength anomalous dispersion . Selenomethionyl and native ModE models are refined to 1 . 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator . ModE is a homodimer and each subunit comprises N- and C-terminal domains . The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization . The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified . This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies . The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions . This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription . Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation.

Biotechnol Appl Biochem, 1999 Apr, 29 ( Pt 2), 165 - 84
Mutational analysis of sickle haemoglobin (Hb) gelation; Li X et al.; The use of recombinant Hb has provided the advantage that any amino acid substitution can be made at sites not represented by natural mutants or that cannot be modified by chemical procedures . We have recently reported the expression of human sickle Hb (HbS) in the yeast Saccharomyces cerevisiae that carries a plasmid containing the human alpha- and beta-globin cDNA sequences; N-terminal nascent protein processing is correct and a soluble correctly folded Hb tetramer is produced . The yeast system produces a recombinant sickle Hb that is identical by about a dozen biochemical and physiological criteria with the natural sickle Hb purified from the red cells of sickle-cell anaemia patients . Most importantly, the gelling concentration of this recombinant sickle Hb is the same as that of the HbS purified from human sickle red cells . The misfolding of Hb reported for the Escherichia coli-expressed protein is not apparent for Hb expressed in yeast by any of the criteria that we have used for characterization . These findings indicate that this system is well suited to the production of HbS mutants to explore those areas of the HbS tetramer whose roles in the gelation process are not yet defined and to measure quantitatively the strength of such interactions at certain inter-tetrameric contact sites in the deoxy-HbS aggregate . This article reviews our studies on a number of sickle Hb mutants, including polymerization-enhancing HbS mutants and polymerization-inhibiting HbS mutants.

Anal Biochem, 1999 Mar 15, 268(2), 343 - 53
Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL); Schmitz AA et al.; The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures . We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase . We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate) . The partition coefficients describing the association of unmyristoylated and myristoylated MARCKS-related protein with membranes of different phospholipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-related protein mediate the binding . However, no significant cooperativity is observed between these two domains . Interestingly, MARCKS-related protein binds to TRANSIL membranes more strongly at temperatures below their phase-transition temperature . Taking advantage of this property, MARCKS-related protein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSIL column at 4 degrees C and eluting MRP at room temperature . In conclusion, TRANSIL is a versatile tool to determine the affinity of compounds for phospholipid membranes and to purify membrane-bound proteins . TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes .

Anal Biochem, 1999 Mar 15, 268(2), 330 - 6
Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?
Oehler S, Alex R, Barker A.
The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study . This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli . Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions . However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain . Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose . Investigation of this loss of binding suggests that LacR requires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA . In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices . Dimeric LacR variants will only bind if they have certain C-terminal extensions . These experiments sound a cautionary note for the use of filter binding as an assay of choice, particularly in applications involving screening for the DNA-binding site of putative DNA-binding proteins .

Anal Biochem, 1999 Mar 15, 268(2), 318 - 29
A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors; McDonald OB et al.; We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway . The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2 . cRaf1 was expressed as a his-tagged protein in insect cells in an active form . MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms . ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease . When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA . Phosphorylation of the peptide using {gamma-33P}ATP is detected following binding to streptavidin-coated SPA beads . The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds . The specific target of inhibition was subsequently identified with secondary assays described herein .

Anal Biochem, 1999 Mar 15, 268(2), 193 - 200
A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro; Zaika A et al.; Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper) . Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus . They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper . After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper . Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta) . Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site . In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins .

J Biol Chem, 1999 Mar 19, 274(12), 8191 - 8
Antisense oligonucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin; Compagno D et al.; Complementary oligodeoxynucleotides (ODNs) that contain 2-aminoadenine and 2-thiothymine interact weakly with each other but form stable hybrids with unmodified complements . These selectively binding complementary (SBC) agents can invade duplex DNA and hybridize to each strand (Kutyavin, I . V., Rhinehart, R . L., Lukhtanov, E . A., Gorn, V . V., Meyer, R . B., and Gamper, H . B . (1996) Biochemistry 35, 11170-11176) . Antisense ODNs with similar properties should be less encumbered by RNA secondary structure . Here we show that SBC ODNs strand invade a hairpin in the mini-exon RNA of Leishmania amazonensis and that the resulting heteroduplexes are substrates for Escherichia coli RNase H . SBC ODNs either with phosphodiester or phosphorothioate backbones form more stable hybrids with RNA than normal base (NB) ODNs . Optimal binding was observed when the entire hairpin sequence was targeted . Translation of L . amazonensis mRNA in a cell-free extract was more efficiently inhibited by SBC ODNs complementary to the mini-exon hairpin than by the corresponding NB ODNs . Nonspecific protein binding in the cell-free extract by phosphorothioate SBC ODNs rendered them ineffective as antisense agents in vitro . SBC phosphorothioate ODNs displayed a modest but significant improvement of leishmanicidal properties compared with NB phosphorothioate ODNs.

J Biol Chem, 1999 Mar 19, 274(12), 8199 - 207
Sorting of furin at the trans-Golgi network . Interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins; Teuchert M et al.; The eukaryotic subtilisin-like endoprotease furin is found predominantly in the trans-Golgi network (TGN) and cycles between this compartment, the cell surface, and the endosomes . There is experimental evidence for endocytosis from the plasma membrane and transport from endosomes to the TGN, but direct exit from the TGN to endosomes via clathrin-coated vesicles has only been discussed but not directly shown so far . Here we present data showing that expression of furin promotes the first step of clathrin-coat assembly at the TGN, the recruitment of the Golgi-specific assembly protein AP-1 on Golgi membranes . Further, we report that furin indeed is present in isolated clathrin-coated vesicles . Packaging into clathrin-coated vesicles requires signal components in the furin cytoplasmic domain which can be recognized by AP-1 assembly proteins . We found that besides depending on the phosphorylation state of a casein kinase II site, interaction of the furin tail with AP-1 and its mu1subunit is mediated by a tyrosine motif and to less extent by a leucine-isoleucine signal, whereas a monophenylalanine motif is only involved in binding to the intact AP-1 complex . This study implies that high affinity interaction of AP-1 or mu1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal.

J Biol Chem, 1999 Mar 19, 274(12), 8169 - 74
Inhibition of DNA supercoiling-dependent transcriptional activation by a distant B-DNA to Z-DNA transition; Sheridan SD et al.; Negative DNA superhelicity can destabilize the local B-form DNA structure and can drive transitions to other conformations at susceptible sites . In a molecule containing multiple susceptible sites, superhelicity can couple these alternatives together, causing them to compete . In principle, these superhelically driven local structural transitions can be either facilitated or inhibited by proteins that bind at or near potential transition sites . If a DNA region that is susceptible to forming a superhelically induced alternate structure is stabilized in the B-form by a DNA-binding protein, its propensity for transition will be transferred to other sites within the same domain . If one of these secondary sites is in a promoter region, this transfer could facilitate open complex formation and thereby activate gene expression . We previously proposed that a supercoiling-dependent, DNA structural transmission mechanism of this type is responsible for the integration host factor-mediated activation of transcription from the ilvPG promoter of Escherichia coli (Sheridan, S . D., Benham, C . J . & Hatfield, G . W . (1998) J . Biol . Chem . 273, 21298-21308) . In this report we confirm the validity of this mechanism by demonstrating the ability of a distant Z-DNA-forming site to compete with the superhelical destabilization that is required for integration host factor-mediated transcriptional activation, and thereby delay its occurrence.

J Biol Chem, 1999 Mar 19, 274(12), 8029 - 38
Heparin-induced conformational change in microtubule-associated protein Tau as detected by chemical cross-linking and phosphopeptide mapping; Paudel HK et al.; In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments . Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments . The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M . G., Hasegawa, M., Smith, M . J., and Crowther, R . A . (1996) Nature 383, 550-553) . The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear . In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin . In the absence of heparin only dimeric tau was cross-linked by DSS . Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS . These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species . Heparin-induced structural changes in tau molecule depended on time of heparin exposure . Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure . Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species . Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase . Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin . Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau . Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule.

J Biol Chem, 1999 Mar 19, 274(12), 7705 - 13
Modulation of the remote heme site geometry of recombinant mouse neuronal nitric-oxide synthase by the N-terminal hook region; Iwasaki T et al.; The role of two essential residues at the N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in nitric-oxide synthase activity was investigated . Full-length mouse nNOS proteins containing single-point mutations of Thr-315 and Asp-314 to alanine were produced in the Escherichia coli and baculovirus-insect cell expression systems . The molecular properties of the mutant proteins were analyzed in detail by biochemical, optical, and electron paramagnetic resonance spectroscopic techniques and compared with those of the wild-type enzyme . Replacement of Asp-314 by Ala altered the geometry around the heme site and the substrate-binding pocket of the heme domain and abrogated the ability of nNOS to form catalytically active dimers . Replacement of Thr-315 by Ala reduced the protein stability and altered the geometry around the heme site, especially in the absence of bound (6R)-5,6,7, 8-tetrahydro-L-biopterin cofactor . These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit interactions in mouse nNOS.

J Biol Chem, 1999 Mar 19, 274(12), 7695 - 8
New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase; Lillig CH et al.; Reduction of inorganic sulfate to sulfite in prototrophic bacteria occurs with 3'-phosphoadenylylsulfate (PAPS) as substrate for PAPS reductase and is the first step leading to reduced sulfur for cellular biosynthetic reactions . The relative efficiency as reductants of homogeneous highly active PAPS reductase of the newly identified second thioredoxin (Trx2) and glutaredoxins (Grx1, Grx2, Grx3, and a mutant Grx1C14S) was compared with the well known thioredoxin (Trx1) from Escherichia coli . Trx1, Trx2, and Grx1 supported virtually identical rates of sulfite formation with a Vmax ranging from 6.6 units mg-1 (Trx1) to 5.1 units mg-1 (Grx1), whereas Grx1C14S was only marginally active, and Grx2 and Grx3 had no activity . The structural difference between active reductants had no effect upon Km PAPS (22.5 microM) . Grx1 effectively replaced Trx1 with essentially identical Km-values: Km trx1 (13.7 microM), Km grx1 (14.9 microM), whereas the Km trx2 was considerably higher (34.2 microM) . The results agree with previous in vivo data suggesting that Trx1 or Grx1 is essential for sulfate reduction but not for ribonucleotide reduction in E . coli.

Microbiology, 1999 Feb, 145 ( Pt 2), 495 - 501
Cloning and characterization of the thiD/J gene of Escherichia coli encoding a thiamin-synthesizing bifunctional enzyme, hydroxymethylpyrimidine kinase/phosphomethylpyrimidine kinase; Mizote T et al.; A 1.7 kb DNA fragment isolated from an E . coli genomic library was able to complement the thiamin requirement of strains carrying the thiM, thiJ and thiD mutations . The three genes encode hydroxyethylthiazole kinase, hydroxymethylpyrimidine (HMP) kinase and phosphomethylpyrimidine (HMP-p) kinase, respectively . Sequence analysis revealed that the 1.7 kb fragment contained two ORFs of 708 bp and 801 bp . The former ORF complemented the thiM mutation and the latter ORF both the thiJ and thiD mutations . The latter ORF was cloned into the expression vector pET3a, and the encoded protein was purified through three successive column chromatographies . The purified protein was able to convert HMP to its monophosphate and the monophosphate to its pyrophosphate . These results suggest that the two distinct enzyme activities, HMP kinase and HMP-P kinase, are indeed a bifunctional enzyme encoded by a single gene, designated thiDIJ.

Microbiology, 1999 Feb, 145 ( Pt 2), 419 - 25
The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: and in vivo study using differential scanning calorimetry; Niven GW et al.; Differential scanning calorimetry of whole Escherichia coil cells allowed the detection in vivo of changes in ribosome conformation . This enabled for the first time an analysis of the effects of high hydrostatic pressures on ribosomes in living cells . A correlation was observed between loss of cell viability and decrease in ribosome-associated enthalpy in cells subjected to pressures of 50-250 MPa for 20 min . Cell death and ribosome damage were therefore closely related phenomena . In pressure-treated cells, the thermogram peak temperatures decreased, suggesting that the remaining ribosomes had adopted a less stable conformation . During subsequent incubation of the cultures at 37 degrees C, peak temperatures and enthalpies gradually increased over a period of 5 h . This change in ribosome conformation had no apparent effect on cell survival, as viability continued to decrease . The addition of 5 mM MgCl2 before pressure treatment of cells prevented the reduction in stability of surviving ribosomes but had no effect on the initial loss of enthalpy or on cell viability.

Microbiology, 1999 Feb, 145 ( Pt 2), 283 - 91
Evidence that halogenated furanones from Delisea pulchra inhibit acylated homoserine lactone (AHL)-mediated gene expression by displacing the AHL signal from its receptor protein; Manefield M et al.; Acylated homoserine lactone (AHL)-mediated gene expression controls phenotypes involved in colonization, often specifically of higher organisms, in both marine and terrestrial environments . The marine red alga Delisea pulchra produces halogenated furanones which resemble AHLs structurally and show inhibitory activity at ecologically realistic concentrations in AHL bioassays . Evidence is presented that halogenated furanones displace tritiated OHHL {N-3-(oxohexanoyl)-L-homoserine lactone} from Escherichia coli cells overproducing LuxR with potencies corresponding to their respective inhibitory activities in an AHL-regulated bioluminescence assay, indicating that this is the mechanism by which furanones inhibit AHL-dependent phenotypes . Alternative mechanisms for this phenomenon are also addressed . General metabolic disruption was assessed with two-dimensional PAGE, revealing limited non-AHL-related effects . A direct chemical interaction between the algal compounds and AHLs, as monitored by 1H NMR spectroscopy, was shown not to occur in vitro . These results support the contention that furanones, at the concentrations produced by the alga, can control bacterial colonization of surfaces by specifically interfering with AHL-mediated gene expression at the level of the LuxR protein.

Crit Care Med, 1999 Feb, 27(2), 385 - 93
Porcine endotoxemic shock is associated with increased expired nitric oxide; Mehta S et al.; OBJECTIVES: Nitric oxide (NO) is believed to decrease systemic vascular resistance in sepsis, but the data are mainly from studies on rats and mice . We tested this hypothesis in pigs and also whether there is induction of the inducible form of nitric oxide synthase (iNOS) . DESIGN: Animal study . SETTING: University center . SUBJECTS: Ten pigs . INTERVENTIONS: The pigs were anesthetized and mechanically ventilated . MEASUREMENTS AND MAIN RESULTS: Pulmonary and systemic hemodynamics were monitored and mixed expired NO was measured by chemiluminescence . Animals received 20 microg/kg of endotoxin over 2 hrs . We then infused 25 mg/kg of N(omega)-nitro-L-arginine methyl ester (L-NAME) over 10 mins, followed by 0.5 g/kg of L-arginine, the precursor of NO, for 30 mins more to reverse the effects of L-NAME . Five additional pigs were treated with 20 microg/kg of endotoxin for 2 hrs and followed for another hour . Plasma nitrite/nitrate was measured by Greiss reaction . The animals were then killed and tissues were sampled for iNOS by Western blot, and iNOS messenger RNA by reverse transcriptase polymerase chain reaction . After endotoxin infusion, arterial pressure (BP) initially increased, then decreased to 62+/-1 mm Hg from the baseline of 115+/-4 mm Hg (p<.001) . Cardiac output initially decreased, then increased slightly from the baseline of 3.7+/-0.2 to 4.2 +/-0.3 L/min (p<.05) . The BP pattern was mirrored by an increase in expired NO concentration from 6.4+/-0.8 to 10.4+/-1.4 parts per billion (p<.05) and increased rate of pulmonary NO excretion in expired gas (VeNO) from 71+/-10 to 146+/-24 pmol/kg/min (p<.05) . Inhibition of NOS with L-NAME decreased expired NO concentration and VeNO and increased BP; however, cardiac output decreased . The vasoconstriction produced by L-NAME was partially reversed by L-arginine, and this also increased VeNO from 80+/-18 after L-NAME to 132+/-31 pmol/kg/min (p<.05) . Plasma nitrite (n = 5) did not change and there was no iNOS by Western blot analysis in multiple tissues . However, there was a small increase in messenger RNA by reverse transcriptase polymerase chain reaction . CONCLUSIONS: The time course and pattern of changes in expired NO during endotoxemia followed the change in systemic hemodynamics, which supports a causal role for NO in sepsis . However, this is not due to a large production of NO by iNOS induction . The hemodynamic pattern, nitrite in blood, and changes in expired NO also differed markedly from those findings in rodent models and caution should be used in extrapolating from rodents to higher order animals.

Crit Care Med, 1999 Feb, 27(2), 373 - 9
Enteral infusion of sodium 2-ketoisocaproate in endotoxic rats; Hirokawa M et al.; OBJECTIVE: In view of our previous finding that the intravenous infusion of 2-ketoisocaproate (KIC) improved survival in septic rats, we endeavored to determine whether the enteral infusion of KIC improves survival in endotoxic rats, and, if so, the mechanism of this effect . SUBJECTS: Eighty-five rats were given 15 mg/kg of Escherichia coli lipopolysaccharide (026:B6) . INTERVENTIONS: KIC, sodium pyruvate (PYR), or sodium bicarbonate (HCO3) was infused continuously intragastrically at 18.75 mmol/kg/day . MEASUREMENTS AND MAIN RESULTS: KIC administration increased circulating concentrations of KIC and ketone bodies . Survival rates were: KIC 17/32; PYR 2/22; and HCO3 8/31 . The significant improvement in survival with KIC, in contrast with HCO3 (p<.04) or PYR (p<.002), points to an effect specific to KIC rather than to ketoacids generally, and argues against an antioxidant mechanism to explain improved survival with enteral administration . To determine whether altered nitric oxide production was responsible, plasma nitrite plus nitrate concentrations were measured sequentially in rats given a lower dose of lipopolysaccharide plus continuous intragastric KIC, PYR, or HCO3 . All rats exhibited pronounced increases in plasma nitrite plus nitrate concentrations, peaking at 8 hrs, but both KIC and PYR caused greater increases than HCO3 . Thus, differences in nitric oxide production cannot account for the different effects of PYR and KIC on survival . However, KIC infusion for 8 hrs substantially increased ketone bodies in blood and liver, in comparison with the infusion of HCO3 or PYR . CONCLUSION: Continuous enteral infusion of KIC improves survival in endotoxemia, probably by its conversion to ketone bodies, which serve as an alternative energy substrate.

Plant J, 1999 Jan, 17(2), 181 - 9
Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens; Saito K et al.; Anthocyanidin synthase (ANS), an enzyme of the biosynthetic pathway to anthocyanin, has been postulated to catalyze the reaction(s) from the colorless leucoanthocyanidins to the colored anthocyanidins . Although cDNAs have been isolated that encode putative ANS, which exhibits significant similarities in amino acid sequence with members of a family of 2-oxoglutarate-dependent oxygenases, no biochemical evidence has been presented which identifies the actual reaction that is catalyzed by ANS . Here we show that anthocyanidins are formed in vitro through 2-oxoglutarate-dependent oxidation of leucoanthocyanidins catalyzed by the recombinant ANS and subsequent acid treatment . A cDNA encoding ANS was isolated from red and green formas of Perilla frutescens by differential display of mRNA . Recombinant ANS tagged with maltose-binding-protein (MBP) was purified, and the formation of anthocyanidins from leucoanthocyanidins was detected by the ANS-catalyzed reaction in the presence of ferrous ion, 2-oxoglutarate and ascorbate, being followed by acidification with HCI . Equimolar stoichiometry was confirmed for anthocyanidin formation and liberation of CO2 from 2-oxoglutarate . The presumptive two-copy gene of ANS was expressed in leaves and stems of the red forma of P . frutescens but not in the green forma plant . This corresponds to the accumulation pattern of anthocyanin . The mechanism of the reaction catalyzed by ANS is discussed in relation to the molecular evolution of a family of 2-oxoglutarate-dependent oxygenases.

Perfusion, 1999 Jan, 14(1), 49 - 57
Lipid peroxidation during initiation of extracorporeal membrane oxygenation after hypoxia in endotoxemic rabbits; Trittenwein G et al.; Initiation of extracorporeal membrane oxygenation (ECMO) in septic children with severe respiratory failure often improves oxygenation but not pulmonary function . The factors affecting pulmonary function following onset of ECMO are not completely understood, but are thought to involve injury mediated, in part, by reactive oxygen species . We hypothesized that induction of ECMO using 100% oxygen as the sweep gas through the oxygenator would increase lipid peroxidation in endotoxin-primed animals after severe hypoxia . We further speculated that provision of oxygenated blood to the pulmonary circulation via venovenous ECMO would promote a greater degree of oxidative damage to the lung as compared to venoarterial ECMO . Eighteen New Zealand White rabbits were assigned to a control group (control) or two intervention groups subjected to 60 min of venoarterial or venovenous ECMO . ECMO was initiated following an intravenous challenge with 0.5 mg/kg of E . coli endotoxin and a period of global hypoxia leading to an arterial pH of 6.99 +/- 0.09, PaCO2 of 103 +/- 31 mmHg and PaO2 of 27 +/- 5 mmHg . Malondialdehyde (MDA), a marker of lipid peroxidation, was measured in lung tissue homogenates and in arterial plasma . Lung tissue MDA demonstrated a strong trend towards an increase in the venoarterial group (1884 +/- 945 nmol/g protein) and in the venovenous group (1905 +/- 758 nmol/g protein) in comparison to the control group (644 +/- 71 nmol/g protein) (p = 0.1; significance at 95% in Scheffe test) . Lung tissue MDA in the venovenous group had a significant correlation with mean PaO2 during ECMO by regression analysis (r2 = 0.678, p = 0.044) . The change in blood MDA concentration between pre-ECMO and post-ECMO values was greater in the venovenous group (pre 1.62 +/- 0.61 versus post 5.12 +/- 0.2.07 mumol/l, p = 0.043) compared with that seen in the venoarterial group (pre 1.46 +/- 0.38 versus post 3.9 +/- 0.93 mumol/l) . Our data support the hypothesis that initiation of ECMO with a circuit gas oxygen concentration of 100% after global hypoxia enhances oxidative damage to lipids in endotoxin-challenged animals . During venovenous ECMO this finding is dependent on PaO2.

Chem Biol, 1999 Mar, 6(3), 133 - 41
Molecular forceps from combinatorial libraries prevent the farnesylation of Ras by binding to its carboxyl terminus; Dong DL et al.; INTRODUCTION: Ras is one of the major oncogenes . In order to function properly it has to undergo post-translational processing at its carboxyl terminus . It has been shown that inhibitors of farnesyl transferase, the first enzyme in the processing chain, can suppress the transforming activity of oncogenic Ras . RESULTS: We have identified molecular forceps, branched peptidic molecules, from combinatorial libraries that bind to the carboxyl terminus of Ras and interfere with its farnesylation without inhibiting the farnesyl transferase . The active molecules were selected by a screening against the carboxy-terminal octapeptide of Ras . CONCLUSIONS: The implications of our findings are twofold . First, we demonstrate that it is possible to prevent enzymatic transformations by blocking the enzyme's access to its substrate using a synthetic small molecule to mask the substrate . Second, we show that it is feasible to derive molecules from combinatorial libraries that bind a specific epitope on a protein by selecting these molecules with the isolated peptide epitope.

J Mol Biol, 1999 Mar 19, 287(1), 47 - 57
Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease; Izumi T et al.; The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs . The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor . Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R . Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity . These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme . The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable . No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation . The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity . These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis . The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies . It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein . These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure .

J Mol Biol, 1999 Mar 19, 287(1), 21 - 31
The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein; Zaitsev EN et al.; We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes . Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament . We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP . In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration . The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2 . Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion . For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA . These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes .

J Theor Biol, 1999 Mar 21, 197(2), 193 - 205
Characterization and comparison of Escherichia coli transfer RNAs by graph theory based on secondary structure; Bermudez CI et al.; We have developed a model to characterize the tRNA structures of Escherichia coli using graph theory . First of all, tRNAs were represented as graphs, whose vertices correspond to nucleotides and the edges to phosphodiester and hydrogen bond linkages . Vertices and edges were weighted using the results of a preliminary quantum study of the nucleotides and the possible coupling between pair bases using the semiempirical method AM1 . For each vertex, we defined a nucleotide valence that measures the capability of forming hydrogen bonds . Edges were differentiated by using bond orders.We have proposed weighted structural descriptors-closely related to molecular Randic connectivity and Balaban distance indices-as a distinctive characteristic of each structure . Molecules were characterized by a set of weighted structural descriptors and classified by a clustering method and discriminant function analysis . Two main groups of tRNAs that correspond to the biosynthetic amino acid pathways, in agreement with Wong's coevolution theory of the genetic code, were obtained .

Biochemistry, 1999 Mar 9, 38(10), 3175 - 86
RecA protein of Mycobacterium tuberculosis possesses pH-dependent homologous DNA pairing and strand exchange activities: implications for allele exchange in mycobacteria; Vaze MB et al.; To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein . The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values . In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks . At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity . Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange . Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion . Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner . Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA . Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected . We discuss how these results relate to inefficient allele exchange in mycobacteria.

Biochemistry, 1999 Mar 9, 38(10), 3120 - 6
Helix packing in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking: helix I is close to helices V and XI; Wang Q et al.; Coexpression of lacY gene fragments encoding the first two transmembrane domains and the remaining 10 transmembrane domains complement in the membrane and catalyze active lactose transport {Wrubel, W., Stochaj, U., et al . (1990) J . Bacteriol . 172, 5374-5381} . Accordingly, a plasmid encoding contiguous, nonoverlapping permease fragments with a discontinuity in the cytoplasmic loop between helices II and III (loop II/III) was constructed (N2C10 permease) . When Phe27 (helix I) is replaced with Cys, cross-linking is observed with two native Cys residues, Cys148 (helix V) and Cys355 (helix XI) . Cross-linking of a Cys residue at position 27 to Cys148 occurs with N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), with N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A), or with 1,6-bis(maleimido)hexane (BMH; flexible 16 A) . On the other hand, with the Phe27-->Cys/Cys355 pair, cross-linking is observed with p-PDM or BMH but not o-PDM . In neither case is cross-linking observed with iodine . It is suggested that a Cys residue at position 27 is within 6-10 A from Cys148 and about 10 A from Cys355 . The results provide evidence for proximity between helix I and helices V or XI in the tertiary structure of the permease . In addition, the findings are consistent with other results {Venkatesan, P., Kaback, H . R . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 9802-9807} indicating that Glu126 (helix IV) and Arg144 (helix V) are within the membrane, rather than at the membrane-water interface on the cytoplasmic face.

Biochemistry, 1999 Mar 9, 38(10), 3100 - 5
Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling; Sun J et al.; Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII {Sun, J., and Kaback, H . R . (1997) Biochemistry 36, 11959-11965} . To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label . The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins . Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction . Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR . Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.

Biochemistry, 1999 Mar 9, 38(10), 3043 - 54
Asn249Tyr substitution at the coenzyme binding domain activates Sulfolobus solfataricus alcohol dehydrogenase and increases its thermal stability; Giordano A et al.; A mutant of the thermostable NAD+-dependent homotetrameric alcohol dehydrogenase from Sulfolobus solfataricus (SsADH), which has a single substitution, Asn249Tyr, located at the coenzyme binding domain, was obtained by error prone PCR . The mutant enzyme, which was purified from Escherichia coli to homogeneous form, exhibits a specific activity that is more than 6-fold greater than that of the wild type enzyme, as measured at 65 degrees C with benzyl alcohol as the substrate . The oxidation rate of aliphatic alcohols and the reduction rate of aromatic aldehydes were also higher . The dissociation constants for NAD+ and NADH determined at 25 degrees C and pH 8.8 were 3 orders of magnitude greater compared to those of the wild type enzyme . It is thought that the higher turnover of the mutant SsADH is due to the faster dissociation of the modified enzyme-coenzyme complex . Spectroscopic studies showed no relevant changes in either secondary or tertiary structure, while analysis with fluorescent probes revealed a significant increase in surface hydrophobicity for the mutant, with respect to that of the wild type molecule . The mutant SsADH displays improved thermal stability, as indicated by the increase in Tm from 90 to 93 degrees C, which was determined by the apparent transition curves . Kinetic thermal stability studies at pH 9.0 for mutant SsADH showed a marked increase in activation enthalpy compensated by an entropy gain, which resulted in a higher activation barrier against thermal unfolding of the enzyme . Ammonia analysis showed that the Asn249Tyr substitution produced the effect of markedly reducing the extent of deamidation during thermoinactivation, thus suggesting that Asn249 plays a significant role in the mechanism of irreversible thermal denaturation of the archaeal ADH . Furthermore, the decrease in the activating effect by moderate concentrations of denaturants and studies with proteases and chelating agents point to an increase in structural rigidity and a tightening of structural zinc as additional factors responsible for the improved thermal resistance of the mutant enzyme.

Biochemistry, 1999 Mar 9, 38(10), 2969 - 81
Intercalation of the (-)-(1R,2S,3R, 4S)-N6-{1-benz{a}anthracenyl}-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence; Li Z et al.; The solution structure of the (-)-(1R,2S,3R,4S)-N6-{1-(1,2,3, 4-tetrahydroxy-benz{a}anthracenyl)}-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined . This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz{a}anthracenyl-3,4-diol by the exocyclic N6 of adenine . Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR . The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms . The refined structure was a right-handed duplex, in which the benz{a}anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17 . In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction . The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17 . These two base pairs also buckled in opposite directions away from the intercalated BA moiety . The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-{10-(7,8,9, 10)-tetrahydrobenzo{a}pyrenyl)}-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide {Zegar, I . S., Kim, S . J., Johansen, T . N., Horton, P . J., Harris, C . M., Harris, T . M., and Stone, M . P . (1996) Biochemistry 35, 6212-6224} . Both adducts intercalated toward the 5'-direction from the site of adduction . The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.

Biochemistry, 1999 Mar 9, 38(10), 2930 - 40
Tumor suppressor INK4: determination of the solution structure of p18INK4C and demonstration of the functional significance of loops in p18INK4C and p16INK4A; Li J et al.; Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently, the detailed structures and the functional roles of the loops have drawn considerable interest . This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects . First, the solution structure of p18INK4C was determined by NMR, and the loop structures were analyzed in detail . The loops adapt nascent antiparallel beta-sheet structures, but the positions are slightly different from those in the crystal structure . A detailed comparison between the solution structures of p16 and p18 has also been presented . The determination of the p18 solution structure made such detailed comparisons possible for the first time . Second, the {1H,15N}HSQC NMR experiment was used to probe the interactions between p18INK4C and other proteins . The results suggest that p18INK4C interacts very weakly with dna K and glutathione S-transferase via the loops . The third aspect employed site-specific mutagenesis and functional assays . Three mutants of p18 and 11 mutants of p16 were constructed to test functional importance of loops and helices . The results suggest that loop 2 is likely to be part of the recognition surface of p18INK4C or p16INK4A for CDK4, and they provide quantitative functional contributions of specific residues . Overall, our results enhance understanding of the structural and functional roles of the loops in INK4 tumor suppressors in particular and in ankyrin-repeat proteins in general.

Biochemistry, 1999 Mar 9, 38(10), 2919 - 29
Structural analysis of phospholipase A2 from functional perspective . 2 . Characterization of a molten globule-like state induced by site-specific mutagenesis; Yuan C et al.; Previous NMR studies have shown that many phospholipase A2 (PLA2, from bovine pancreas, overexpressed in Escherichia coli) mutants display some properties reminiscent of a molten globule state . Further NMR analyses for some of the mutants indicated that formation of the "molten globule-like state" is a pH-dependent phenomenon . The mutants I9Y and I9F showed perturbed NMR properties throughout the pH range studied, while the mutants H48A and C44A/C105A displayed native-like spectra at neutral pH but molten globule-like ones under acidic conditions, with a "transition pH" around 4 . On the other hand, wild-type PLA2 exhibits exceptional pH stability and turns into a similar molten globule-like state only under highly acidic conditions such as 1 M HCl . The H48A mutant was used to rigorously establish the property of the molten globule-like state of PLA2 mutants . The results of far-UV CD, near-UV CD, and ANS-binding fluorescence suggest that H48A retains native-like secondary structures but loses tertiary structure during the conformational transition . However, the tertiary structure is not completely lost, as evidenced by the retention of some long-range NOEs in two-dimensional NOESY spectra . The conclusion was further substantiated by three-dimensional NOESY-HSQC experiments on a 15N-labeled H48A sample . It was revealed that the molten globule-like state at mildly acidic pH retained some rigid tertiary structure, which consisted of partial alpha-helix II (Y52-L58), alpha-helix III (D59-V63), beta-wing (S74-S85) and partial alpha-helix IV (A90-N97) . These residual tertiary structures grouped in half of the protein could be attributed to stabilization by some of the disulfide bonds . The extreme sensitivity of the PLA2 structure to site-directed mutagenesis is unprecedented . It is interesting to note that most of the functional residues (the active site, the hydrophobic channel, the interfacial binding site, and the calcium-binding loop) are located in the remainder of the protein, which is well disrupted in tertiary interactions.

J Bacteriol, 1999 Mar, 181(6), 1779 - 85
Organization of biogenesis genes for aggregative adherence fimbria II defines a virulence gene cluster in enteroaggregative Escherichia coli; Elias WP Jr et al.; Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers . Among these factors are the enterotoxins Pet and EAST and the fimbrial antigen aggregative adherence fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2 . Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map . The cluster is 23 kb in length and includes two regions required for expression of the AAF/II fimbria . These fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family fimbrial clusters . This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.

J Virol, 1999 Apr, 73(4), 3410 - 7
Disease-inducible transgene expression from a recombinant adeno-associated virus vector in a rat arthritis model; Pan RY et al.; Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 1% of the world's population, with significant morbidity and mortality . In this study, we investigated a recombinant adeno-associated virus (rAAV) vector for its potential application in RA gene therapy . rAAV encoding Escherichia coli beta-galactosidase was injected into rat joints which had already been induced into acute arthritis after local lipopolysaccharide (LPS) administration, and the efficiency of in vivo transduction was evaluated . We observed a striking correlation between vector transgene expression and disease severity in arthritic joints . The inflammatory reaction peaked at 3 to 7 days after LPS treatment, and, at the same time, 95% of the synoviocytes had high-level transgene expression . Gene expression diminished to the basal level (5%) when the inflammation subsided at 30 days after LPS treatment . More importantly, the diminished transgene expression could be efficiently reactivated by a repeated insult . The transgene expression in normal joints transduced with rAAV remained low for a long period of time (30 days) but could still be induced to high levels (95%) at 3 to 7 days after LPS treatment . This is the first demonstration of disease state-regulated transgene expression . These findings strongly support the feasibility of therapeutic as well as preventative gene transfer approaches for RA with rAAV vectors containing therapeutic genes, which are expected to respond primarily to the disease state of the target tissue.

J Virol, 1999 Apr, 73(4), 3108 - 16
The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids; Li H et al.; NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues . NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor . The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases . To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified . Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities . However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity . The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs . Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors . Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.

J Virol, 1999 Apr, 73(4), 3032 - 9
Bamboo mosaic potexvirus satellite RNA (satBaMV RNA)-encoded P20 protein preferentially binds to satBaMV RNA; Tsai MS et al.; A satellite RNA of 836 nucleotides {excluding the poly(A) tail} depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation . The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20) . The P20 protein with eight histidine residues at the C terminus was overexpressed in Escherichia coli . Experiments of gel retardation, UV cross-linking, and Northwestern hybridization demonstrated that purified P20 was a nucleic-acid-binding protein . The binding of P20 to nucleic acids was strong and highly cooperative . P20 preferred binding to satBaMV- or BaMV-related sequences rather than to nonrelated sequences . By deletion analysis, the P20 binding sites were mainly located at the 5' and 3' untranslated regions of satBaMV RNA, and the RNA-protein interactions could compete with the poly(G) and, less efficiently, with the poly(U) homopolymers . The N-terminal arginine-rich motif of P20 was the RNA binding domain, as shown by in-frame deletion analysis . This is the first report that a plant virus satellite RNA-encoded nonstructural protein preferentially binds with nucleic acids.

J Virol, 1999 Apr, 73(4), 2909 - 15
RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3; Gwack Y et al.; Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins . In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography . The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities . Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins . However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins . The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well . In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes . We also found that the conserved motif VI was important for RNA binding . Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR . Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.

J Virol, 1999 Apr, 73(4), 2658 - 66
Expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides; Teng H et al.; Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication . In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2 . Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cis and trans cleavage activities for PLP-1 . We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein . With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates . Subsequent characterization of the recombinant PLP-1 revealed that in vitro trans cleavage is more efficient at 22 degrees C than at higher temperatures . Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa . In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed . The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans . In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage in cis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.

J Virol, 1999 Apr, 73(4), 2633 - 40
Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer; Watanabe T et al.; The complete nucleotide sequence was determined for the putative RNA polymerase (183K protein) gene of tobacco mosaic virus (TMV) OM strain, which differed from the related strain, vulgare, by 51 positions in its nucleotide sequence and 6 residues in its amino acid sequence . Three segments of this 183K protein, each containing the sequence motif of methyltransferase (M), helicase (H), or RNA-dependent RNA polymerase (P), were expressed in Escherichia coli as fusion proteins with hexahistidine tags, and domain-specific antibodies were raised against purified His-tagged M and P polypeptides . By immunoaffinity purification, a template-specific RNA-dependent RNA polymerase containing a heterodimer of the full-length 183K and 126K (an amino-terminal-proximal portion of the 183K protein) viral proteins was isolated . We propose that the TMV RNA polymerase for minus-strand RNA synthesis is composed of one molecule each of the 183- and 126-kDa proteins, possibly together with two or more host proteins.

J Bacteriol, 1999 Mar, 181(6), 1958 - 62
Expression of the Methanobacterium thermoautotrophicum hpt gene, encoding hypoxanthine (Guanine) phosphoribosyltransferase, in Escherichia coli; Sauer J et al.; The hpt gene from the archaeon Methanobacterium thermoautotrophicum, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into Escherichia coli . The hpt-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine . The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the hpt gene . The recombinant enzyme was purified to apparent homogeneity.

J Bacteriol, 1999 Mar, 181(6), 1944 - 6
A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant; Heath RJ et al.; The sn-glycerol-3-phosphate acyltransferase (plsB) catalyzes the first step in membrane phospholipid formation . A conditional Escherichia coli mutant (plsB26) has a single missense mutation (G1045A) predicting the expression of an acyltransferase with an Ala349Thr substitution . The PlsB26 protein had a significantly reduced glycerol-3-phosphate acyltransferase specific activity coupled with an elevated Km for glycerol-3-phosphate.

J Bacteriol, 1999 Mar, 181(6), 1920 - 3
Site-directed mutagenesis of loop L3 of sucrose porin ScrY leads to changes in substrate selectivity; Ulmke C et al.; The difference in substrate selectivity of the maltodextrin (LamB) and sucrose (ScrY) porins is attributed mainly to differences in loop L3, which is supposed to constrict the lumen of the pores . We show that even a single mutation (D201Y) in loop L3 leads to a narrowing of the substrate range of ScrY to that resembling LamB . In addition, we removed the putative N-terminal coiled-coil structure of ScrY and studied the effect of this deletion on sucrose transport.

J Bacteriol, 1999 Mar, 181(6), 1906 - 11
Regulation of autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein; Jiang P et al.; The nitrogen regulator II (NRII or NtrB)-NRI (NtrC) two-component signal transduction system regulates the transcription of nitrogen-regulated genes in Escherichia coli . The NRII protein has both kinase and phosphatase activities and catalyzes the phosphorylation and dephosphorylation of NRI, which activates transcription when phosphorylated . The phosphatase activity of NRII is activated by the PII signal transduction protein . We showed that PII was also an inhibitor of the kinase activity of NRII . The data were consistent with the hypothesis that the kinase and phosphatase activities of two-component system kinase/phosphatase proteins are coordinately and reciprocally regulated . The ability of PII to regulate NRII is allosterically controlled by the small-molecule effector 2-ketoglutarate, which binds to PII . We studied the effect of 2-ketoglutarate on the regulation of the kinase and phosphatase activities of NRII by PII, using a coupled enzyme system to measure the rate of cleavage of ATP by NRII . The data were consistent with the following hypothesis: when not complexed with 2-ketoglutarate, PII cannot bind to NRII and has no effect on its competing NRI kinase and phosphatase activities . Under these conditions, the kinase activity of NRII is dominant . At low 2-ketoglutarate concentrations, PII trimers complexed with a single molecule of 2-ketoglutarate interact with NRII to inhibit its kinase activity and activate its phosphatase activity . However, at high 2-ketoglutarate concentrations, PII binds additional ligand molecules and is rendered incapable of binding to NRII, thereby releasing inhibition of NRII's kinase activity and effectively inhibiting its phosphatase activity (by failing to stimulate it).

J Bacteriol, 1999 Mar, 181(6), 1900 - 5
Nucleoid-independent identification of cell division sites in Escherichia coli; Cook WR et al.; The mechanism used by Escherichia coli to determine the correct site for cell division is unknown . In this report, we have attempted to distinguish between a model in which septal position is determined by the position of the nucleoids and a model in which septal position is predetermined by a mechanism that does not involve nucleoid position . To do this, filaments with extended nucleoid-free regions adjacent to the cell poles were produced by simultaneous inactivation of cell division and DNA replication . The positions of septa that formed within the nucleoid-free zones after division was allowed to resume were then analyzed . The results showed that septa were formed at a uniform distance from cell poles when division was restored, with no relation to the distance from the nearest nucleoid . In some cells, septa were formed directly over nucleoids . These results are inconsistent with models that invoke nucleoid positioning as the mechanism for determining the site of division site formation.

J Bacteriol, 1999 Mar, 181(6), 1892 - 9
Catabolic repression of secB expression is positively controlled by cyclic AMP (cAMP) receptor protein-cAMP complexes at the transcriptional level; Seoh HK et al.; SecB, a protein export-specific chaperone, enhances the export of a subset of proteins across cytoplasmic membranes of Escherichia coli . Previous studies showed that the synthesis of SecB is repressed by the presence of glucose in the medium . The derepression of SecB requires the products of both the cya and crp genes, indicating that secB expression is under the control of catabolic repression . In this study, two secB-specific promoters were identified . In addition, 5' transcription initiation sites from these two promoters were determined by means of secB-lacZ fusions and primer extension . The distal P1 promoter appeared to be independent of carbon sources, whereas the proximal P2 promoter was shown to be subject to control by the cyclic AMP (cAMP) receptor protein (CRP)-cAMP complexes . Gel-mobility shift studies showed that this regulation results from direct interaction between the secB P2 promoter region and the CRP-cAMP complex . Moreover, the CRP binding site on the secB gene was determined by DNase I footprinting and further substantiated by mutational analysis . The identified secB CRP binding region is centered at the -61.5 region of the secB gene and differed from the putative binding sites predicted by computer analysis.

J Bacteriol, 1999 Mar, 181(6), 1853 - 60
Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli; Jarvill-Taylor KJ et al.; Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained . As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis . A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed . Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli . This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease") . The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region . Antisera raised against two MnuA peptide sequences identified an M . pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size . Maxicell experiments with E . coli confirmed that mnuA coded for a nuclease of unknown specificity . Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma.

J Bacteriol, 1999 Mar, 181(6), 1838 - 46
Characterization of the ssnA gene, which is involved in the decline of cell viability at the beginning of stationary phase in Escherichia coli; Yamada M et al.; When grown in rich medium, Escherichia coli exhibits a drastic reduction of the number of viable cells at the beginning of stationary phase . The decline of cell viability was retarded by disruption of the ssnA gene, which was identified as a gene subject to RpoS-dependent negative regulation . Moreover, ssnA expression was induced at the time of decline of cell viability at early stationary phase . The viability decline was augmented in the rpoS background, and this augmentation was suppressed by ssnA mutation . Cloning of the ssnA gene in a multicopy plasmid, pBR322, caused small colony formation and slow growth in liquid medium . Cells harboring the ssnA clone showed aberrant morphology that included enlarged and filamentous shapes . The gene product was identified as a 44-kDa soluble protein, but its function could not be deduced by homology searching . From these results, we conclude that ssnA is expressed in response to a phase-specific signal(s) and that its expression level is controlled by RpoS, by a mechanism which may contribute to determination of cell number in the stationary phase.

J Bacteriol, 1999 Mar, 181(6), 1827 - 30
CspA, CspB, and CspG, major cold shock proteins of Escherichia coli, are induced at low temperature under conditions that completely block protein synthesis; Etchegaray JP et al.; CspA, CspB, and CspG, the major cold shock proteins of Escherichia coli, are dramatically induced upon temperature downshift . In this report, we examined the effects of kanamycin and chloramphenicol, inhibitors of protein synthesis, on cold shock inducibility of these proteins . Cell growth was completely blocked at 37 degrees C in the presence of kanamycin (100 microgram/ml) or chloramphenicol (200 microgram/ml) . After 10 min of incubation with the antibiotics at 37 degrees C, cells were cold shocked at 15 degrees C and labeled with {35S}methionine at 30 min after the cold shock . Surprisingly, the synthesis of all these cold shock proteins was induced at a significantly high level virtually in the absence of synthesis of any other protein, indicating that the cold shock proteins are able to bypass the inhibitory effect of the antibiotics . Possible bypass mechanisms are discussed . The levels of cspA and cspB mRNAs for the first hour at 15 degrees C were hardly affected in the absence of new protein synthesis caused either by antibiotics or by amino acid starvation.

J Bacteriol, 1999 Mar, 181(6), 1811 - 9
Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid; Athanasopoulos V et al.; Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA . Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot . Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB . The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD) . The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

Vaccine, 1999 Feb 5, 17(5), 497 - 507
Immunization with recombinant Semliki Forest virus induces protection against influenza challenge in mice; Berglund P et al.; The replicon of Semliki Forest virus (SFV) offers the possibility to direct high-level, transient expression of heterologous proteins in vivo . We initiated studies to determine the possibility of employing the SFV expression system for recombinant vaccine purposes . Mice immunized with recombinant SFV encoding Influenza A nucleoprotein (NP) or E . coli LacZ developed long-lasting antigen-specific IgG levels and induction of cytotoxic T-cell (CTL) memory that persisted for over one year . Predominantly type 1 T-helper cells were induced as shown by IgG subclass ELISA . Humoral and cell-mediated immune responses could be induced upon delivery by several administration routes and mucosal immunizations induced secretory IgA in the respiratory tract . Development of immune responses against the vector itself did not inhibit boost responses by subsequent immunizations with recombinant SFV . Immunization of mice with vectors encoding the Influenza A virus antigens nucleoprotein (NP) and hemagglutinin (HA) resulted in immune responses that were protective against challenge infection with Influenza virus.

J Gen Virol, 1999 Feb, 80 ( Pt 2), 493 - 500
Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143); Evans JT et al.; LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays . LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases . In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses . Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165 . Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

J Gen Virol, 1999 Feb, 80 ( Pt 2), 345 - 53
Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein; Matthews DA et al.; An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures . To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein . The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG . Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently . The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells . Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron . These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells.

Radiat Res, 1999 Mar, 151(3), 334 - 42
Yield of DNA strand breaks after base oxidation of plasmid DNA; Milligan JR et al.; We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide . These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants . Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species {(SCN)2*- and Br2*-, respectively} which are capable of reacting with the bases in DNA . The oxidized bases were detected after incubation of the irradiated plasmid with the two E . coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III . Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage) . By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH . The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.

Zhonghua Bing Li Xue Za Zhi, 1997 Apr, 26(2), 78 - 81
{Uptake of Escherichia coli lipopolysaccharide and expression of tumor necrosis factor-alpha-mRNA by isolated rat intrahepatic bile duct epithelial cells}; Chen X et al.; OBJECTIVE: To study the uptake of E . coli lipopolysaccharide (LPS) and expression of tumor necrosis factor-alpha-mRNA in isolated intrahepatic bile duct epithelial cells . METHODS: Fluorescent immunohistochemical and in situ hybridization techniques and observations with a confocal laser scanning microscope . RESULTS: Positive reactions to LPS were found in the cytoplasm of isolated intrahepatic bile duct epithelial cells after incubation with S type LPS (S-LPS) for 15 minutes, and the FITC fluorescent intensity against LPS was significantly higher than that of controls . After incubation with S-LPS for 3 hours, FITC fluorescent intensities to the expression of tumor necrosis factor (TNF)-alpha-mRNA by flurescent in situ hybridization in the cytoplasm and nuclei of cultured bile duct epithelial cells were significantly higher than that of controls . The increase of FITC fluorescent intensity to TNF-alpha-mRNA expression showed a peak at 6 hours after incubation and at various time points after incubation with LPS, the increase of fluorescent intensities in the cytoplasm were much higher than that in the nuclei . CONCLUSION: It is suggested that LPS could act on and enter into isolated intrahepatic bile duct epithelial cells and stimulate the expression of TNF-alpha-mRNA.

Mech Dev, 1999 Feb, 80(2), 191 - 5
Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped; Schroder R et al.; The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious . We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium . We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments . Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila . However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species . In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species . We conclude that the primary function of eve as a pair rule gene is conserved between the two species .

Gene, 1999 Mar 4, 228(1-2), 61 - 71
A novel A-isoform-like inositol 1,4,5-trisphosphate 3-kinase from chicken erythrocytes exhibits alternative splicing and conservation of intron positions between vertebrates and invertebrates; Bertsch U et al.; Based on the partial peptide sequence of inositol 1,4, 5-trisphosphate 3-kinase purified with 135 000-fold enrichment from chicken erythrocytes, cDNA-fragments were cloned by RT-PCR using degenerate oligonucleotides . Subsequent hybridization screening of an embryonic chicken cDNA library and 5'-RACE yielded a cDNA-contig of 2418 bp, encoding a 452 amino acid protein . The amino acid sequence shows the highest degree of homology with A-isoforms of inositol 1,4,5-trisphosphate 3-kinase (65% identities), whereas homology towards B and C isoforms was lower (57% and 52% amino acid identities respectively) . These findings reveal a new tissue-specific pattern of A-isoform expression, a form which so far has only been found in brain and testes.Two overlapping lambda-genomic clones for chicken inositol 1,4,5-trisphosphate 3-kinase, isolated by hybridization screening, covered 18 499 bp of genomic sequence . This contig included four exons: three of them were present in all cDNA clones, whereas one was only represented in a single cDNA clone . In addition, the sequence of the latter differed from the other cDNAs by an in-frame deletion of 72 bp within the coding region for the catalytic domain of the enzyme . This divergent cDNA suggests the existence of alternative splice products, at least in embryonic tissue.A comparison of the position of introns, with the respective introns known from the corresponding gene from Caenorhabditis elegans, revealed a high degree of conservation of intron positions between vertebrates and invertebrates . Functional data for the enzyme suggests that the conserved exons represent defined functional protein modules.

J Basic Microbiol, 1999, 39(1), 51 - 60
A polylinker-derived sequence, PL, highly increased translation efficiency in Escherichia coli; Xu J et al.; Pokeweed (Phytolacca americana) antiviral protein (PAP) is a highly specific ribosome-inactivating glycosidase . The PAP gene was isolated and cloned in an expression vector containing a polylinker-derived sequence (PL) but devoid of a Shine-Dalgarno (SD) sequence . Surprisingly, E . coli cells transformed with this vector produced over twice the amount of PAP than that with the consensus SD sequence . Computer analysis of the 5' terminal region of PAP mRNA revealed a nucleotide sequence (ACCUACUCGAGUUAG) which was complementary to two domains in 16S rRNA . The heptanucleotide ACCUACU (box I) is complementary to nucleotides 1434-1440 and the GAGUUAG (box II) to nucleotides 507-513 in 16S rRNA of E . coli . To examine the role of this sequence in the translation of PAP mRNA, single or both boxes were mutated and the protein yield was measured . Mutation of box I and of box II resulted in a 2.7 and 5.3 fold decrease in protein yield respectively, indicating that the PAP gene expression was dependent on the presence of both boxes . To investigate whether PL also increases expression of other genes, human calcitonin monomeric and tetrameric genes were used as reporters . It was found that the expression level was doubled compared to that by SD . These results demonstrate that the PL is an efficient translational initiator and may be used for high level expression of certain genes in E . coli . The possible mechanisms for the high level expression are discussed.

Plant Cell, 1999 Mar, 11(3), 335 - 48
The Arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme oxygenase; Muramoto T et al.; The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light . We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation . The product of the HY1 gene shows significant similarity to animal heme oxygenases and contains a possible transit peptide for transport to plastids . Heme oxygenase activity was detected in the HY1 protein expressed in Escherichia coli . Heme oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis . The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts . The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum . These results indicate that the Arabidopsis HY1 gene encodes a plastid heme oxygenase necessary for phytochrome chromophore biosynthesis.

Arch Pharm Res, 1999 Feb, 22(1), 13 - 7
The kinetic characteristics of K228G mutant horse liver alcohol dehydrogenase; Cho SH et al.; The kinetic constants and the reaction mechanism of the K228G mutant horse liver alcohol dehydrogenase isoenzyme E (HLADH-E) were compared to the wild-type enzyme . All the Km and Ki constants of the mutant enzyme for NAD+, ethanol, acetaldehyde and NADH were larger than those of the wild-type enzyme . The dissociation constants for the NADH and NAD+ (Kiq and Kia) were greatly increased by 130- and 460-fold, respectively . The product inhibition patterns suggested that the reaction mechanism of the mutant enzyme was changed to Random Bi Bi . These results could attribute to the increase in the dissociation rate of coenzyme with the substitution at Lys-228 residue.

J Protein Chem, 1999 Jan, 18(1), 117 - 25
Kinetics and equilibrium studies of Tet repressor-operator interaction; Kedracka-Krok S et al.; Binding of a Tet repressor mutant containing a single Trp43 residue in the tet operator recognition alpha-helix leads to the quenching of the protein fluorescence down to about 23% in the case of the tet O1 operator and to 40% in the case of the tet O2 operator . We have used fluorescence detection to describe the binding equilibrium and kinetics of the Tet repressor interaction with the 20-bp DNA operators tet O1 and tet O2 . Stopped-flow measurements in an excess of the tet operators performed in 5 mM NaCl or 150 mM NaCl indicate that the reaction can be described by at least three exponentials characterized by different relaxation times . The mechanism of interaction for both operators as well as for two salt concentrations used can be described as TetR + Operator <==> Complex 1 <==> Complex 2 <==> Complex 3 . Only the much faster process can be described as a second-order reaction characterized by a bimolecular rate constant equal to 2.8X10(6) M(-1) sec(-1) for both operators . The medium and slow processes may be described by relaxational times ranging from 50 msec to seconds . The results of the binding equilibrium measurements extrapolated to 1 M NaCl concentration, which reflects the specific nonionic interaction between TetR and tet operators, indicate Kas equal to 3.2x10(4) and 4.0x10(5) M(-1) for tet O1 and tet O2, respectively . The number of monovalent ions replaced upon binding can be calculated as about 5 and 3 for tet O1 and tet O2, respectively . The binding of Tet repressor to the operators leads to changes in the circular dichroism spectra of the DNA which could indicate transitions of B-DNA into A-like DNA structure.

J Protein Chem, 1999 Jan, 18(1), 79 - 87
Participation of chaperonin GroEL in the folding of D-glyceraldehyde-3-phosphate dehydrogenase . An approach based on the use of different oligomeric forms of the enzyme immobilized on sepharose; Bulatnikov IG et al.; The binding of denatured B . stearothermophilus D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the E . coli chaperonin GroEL was investigated in two systems: (1) GroEL immobilized on Sepharose via a single subunit was titrated with urea-denatured soluble GAPDH and (2) a Sepharose-bound denatured GAPDH monomer was titrated with soluble GroEL . Similar apparent KD values for the complex GroEL x GAPDH were obtained in both cases (0.04 and 0.03 microM, respectively), the stoichiometry being 1.0 mol chaperonin per GAPDH subunit in the system with the immobilized GroEL and 0.2 mol chaperonin per Sepharose-bound GAPDH monomer . Addition of GroEL and Mg x ATP to a reactivation mixture increased the yield of reactivation of both E . coli and B . stearothermophilus GAPDHs . Incubation of the Sepharose-bound catalytically active tetrameric and dimeric GAPDH forms with the protein fraction of a wild-type E . coli cell extract resulted in the binding of GroEL to the dimer and no interaction with the tetrameric form . These data suggest that GroEL may be capable of interacting with the interdimeric contact regions of the folded GAPDH dimers.

J Protein Chem, 1999 Jan, 18(1), 39 - 46
Protein structure prediction in a 210-type lattice model: parameter optimization in the genetic algorithm using orthogonal array; Sun Z et al.; We have applied the orthogonal array method to optimize the parameters in the genetic algorithm of the protein folding problem . Our study employed a 210-type lattice model to describe proteins, where the orientation of a residue relative to its neighboring residue is described by two angles . The statistical analysis and graphic representation show that the two angles characterize protein conformations effectively . Our energy function includes a repulsive energy, an energy for the secondary structure preference, and a pairwise contact potential . We used orthogonal array to optimize the parameters of the population, mating factor, mutation factor, and selection factor in the genetic algorithm . By designing an orthogonal set of trials with representative combinations of these parameters, we efficiently determined the optimal set of parameters through a hierarchical search . The optimal parameters were obtained from the protein crambin and applied to the structure prediction of cytochrome B562 . The results indicate that the genetic algorithm with the optimal parameters reduces the computing time to reach a converged energy compared to nonoptimal parameters . It also has less chance to be trapped in a local energy minimum, and predicts a protein structure which is closer to the experimental one . Our method may also be applicable to many other optimization problems in computational biology.

Biopolymers, 1999 Feb, 49(2), 145 - 65
Complete suboptimal folding of RNA and the stability of secondary structures; Wuchty S et al.; An algorithm is presented for generating rigorously all suboptimal secondary structures between the minimum free energy and an arbitrary upper limit . The algorithm is particularly fast in the vicinity of the minimum free energy . This enables the efficient approximation of statistical quantities, such as the partition function or measures for structural diversity . The density of states at low energies and its associated structures are crucial in assessing from a thermodynamic point of view how well-defined the ground state is . We demonstrate this by exploring the role of base modification in tRNA secondary structures, both at the level of individual sequences from Escherichia coli and by comparing artificially generated ensembles of modified and unmodified sequences with the same tRNA structure . The two major conclusions are that (1) base modification considerably sharpens the definition of the ground state structure by constraining energetically adjacent structures to be similar to the ground state, and (2) sequences whose ground state structure is thermodynamically well defined show a significant tendency to buffer single point mutations . This can have evolutionary implications, since selection pressure to improve the definition of ground states with biological function may result in increased neutrality.

Mol Gen Genet, 1999 Feb, 261(1), 201 - 7
A putative sigma factor from Streptomyces sp . strain A21 can activate the expression of the cryptic operon bgl in Escherichia coli K-12; Marri L et al.; Streptomyces sp A21 is a cellulolytic strain isolated from soil which was assigned to the genus Streptomyces on the basis of distinctive morphological features . A genomic library of A21 DNA has been constructed and transformed into Escherichia coli K-12 using a high-copy-number vector . One of the recombinant plasmids activates the cryptic bgl operon when inserted into appropriate strains . The complete sequence of the 1629-bp A21 DNA fragment has been determined . The analysis revealed the presence of an ORF whose putative product shows a high degree of similarity to RNA polymerase sigma factors; we therefore designated the gene psfS (Putative sigma factor, Streptomyces) . Mapping of the 5' terminus of transcript by primer extension indicated that PsfS induces transcription initiation within the bgl promoter-silencer region.

Mol Gen Genet, 1999 Feb, 261(1), 170 - 6
A mutational study of the ArcA-P binding sequences in the aldA promoter of Escherichia coli; Pellicer MT et al.; The aldA gene (encoding aldehyde dehydrogenase) of Escherichia coli is anaerobically repressed by ArcA-P, the phosphorylated response regulator of the ArcB/A two-component signal transduction system . The promoter region of aldA contains two 10-bp sequences (5'-TGTTAATTAA-3') that perfectly match the proposed ArcA-P binding consensus (5'-{A/T}GTTAATTA{A/T}-3') . One consensus sequence is on the coding strand (-13 to -4 from the transcriptional start point), whereas the other is on the template strand (position -2 to -11) . In this study we used the aldA promoter to test the validity of the proposed consensus sequence . DNase I protection experiments confirmed the 10-bp sequence to be a strong ArcA-P binding site . Alteration of the wild-type sequence from 5'-TGTTAATTAAC-3' to 5'-TCTTAATTAAG-3' or 5'-TATTAATTAAT-3' by site-directed mutagenesis markedly decreased the in vitro affinity of the promoter region for ArcA-P, and abolished the anaerobic repression of mutant att lambda::phi (aldA'-lacZ) transcriptional reporter constructs . Both the in vitro and in vivo results therefore support the proposed consensus sequence.

Insect Biochem Mol Biol, 1999 Jan, 29(1), 71 - 9
Mechanism of an insect glutathione S-transferase: kinetic analysis supporting a rapid equilibrium random sequential mechanism with housefly I1 isoform; Nay B et al.; The steady-state kinetics of glutathione S-transferase I1 (GST I1) from housefly Musca domestica expressed in Escherichia coli were investigated with glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) . Concentrations of the varied substrates were from 0.03 to 1 mM for GSH and 0.05 to 1 mM for CDNB . Within this range, Michaelis-Menten behaviour was observed and convergent straight lines in double reciprocal plots excluded a ping-pong kinetic mechanism . Instead, data were consistent either with rapid-equilibrium random or with steady-state ordered sequential mechanisms because of abscissa convergence . Discrimination was achieved by studying the reaction with another electrophilic partner, p-nitrophenyl-acetate (PNPA) . Concentrations of PNPA and GSH varied within the ranges 0.5 to 10 mM and 0.03 to 0.6 mM, respectively . The complete set of data supports the proposal of a rapid-equilibrium random-sequential model with strictly independent sites for GSH and CDNB or PNPA . Kinetic parameters are thus true dissociation equilibrium constants with values of 0.15 mM for GSH, 0.15 mM for CDNB, and 7 mM for PNPA . Analysis of the inhibition by the product (S-(2,4-dinitrophenyl)-glutathione, 10 to 100 microM), on the coupling reaction between GSH and CDNB with either GSH (0.05 to 0.5 mM, CDNB 0.2 mM) or CDNB (0.05 to 0.5 mM, GSH 0.2 mM) varied, consistent with the proposed mechanism . Binding of product to the free enzyme excludes GSH (competitive inhibition pattern with Kp = 12 microM) but only slightly hinders binding of CDNB . Binding free energies, together with the inhibition pattern, suggest that the non-peptidic moiety of product interacts with an alternative sub-site within the large open pocket accommodating the various electrophilic substrates . These results lead us to propose a model for intra-pocket shifting of the non-peptidic moiety upon product formation which contributes to the product release.

Chemosphere, 1999 Mar, 38(6), 1331 - 7
Characterization of genes for enzymes involved in the phenanthrene degradation in Nocardioides sp . KP7; Saito A et al.; The nucleotide sequence of the gene cluster, phdEFABGHCD, encoding enzymes responsible for the transformation of phenanthrene to 1-hydroxy-2-naphthoate in Nocardioides sp . strain KP7 was determined . This gene cluster, which may constitute a single operon, resided at 6.1-kb downstream of the phdIJK gene cluster encoding the enzymes for the transformation of 1-hydroxy-2-naphthoate to o-phthalate . In general, the phd products exhibited moderate degrees of homology with isofunctional enzymes found in pathways for the degradation of other aromatic compounds . Remarkably, the phdC gene product had features of the {3Fe-4S} type ferredoxin, which has not been found so far as a component of the ring-hydroxylating dioxygenase . Escherichia coli carrying the genes for phenanthrene dioxygenase, phdABCD, was capable to oxidize phenanthrene.

Virology, 1999 Mar 15, 255(2), 354 - 65
In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV); Marcos JF et al.; Biochemical and structural characterization studies on the p7 putative movement protein from a Spanish isolate of carnation mottle carmovirus (CarMV) have been conducted . The CarMV p7 gene was fused to a sequence coding for a six-histidine tag and expressed in bacteria, allowing the purification of CarMV p7 and the production of a specific antiserum . This antiserum led to the immunological identification of CarMV p7 in infected leaf tissue from the experimental host Chenopodium quinoa . Putative nucleic acid-binding properties of the CarMV p7 have been explored and demonstrated with both electrophoretic mobility shift and RNA-protein blot in vitro assays using digoxigenin-labeled riboprobes . CarMV p7 did not show preferential binding to any of the different regions of the CarMV genomic RNA tested, suggesting that RNA binding was sequence nonspecific . Quantitative analyses of the data allowed calculation of the apparent dissociation constant of the p7-RNA complex (Kd approximately 0.7 microM) and supported a cooperative type of binding . A small 19-amino-acid synthetic peptide whose sequence corresponds to the putative RNA-binding domain of CarMV p7, at the basic central part of the protein, was synthesized, and it was demonstrated that it binds viral RNA probes . Peptide RNA binding was as stable as p7 binding, although data indicated it was not cooperative, thus suggesting that this cooperative binding requires another motif or motifs within the p7 amino acid sequence . The peptide could be induced to fold into an alpha-helix structure in which amino acids that are conserved among carmovirus p7-like proteins are distributed on one side . This alpha-helix motif could define a new and previously uncharacterized RNA-binding domain for plant virus movement proteins .

Plant Physiol, 1999 Mar, 119(3), 925 - 34
GTPase activity and biochemical characterization of a recombinant cotton fiber annexin; Shin H et al.; A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library . The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified . We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins . An "add-back experiment" was performed to study the effect of the recombinant annexin on beta-glucan synthase activity, but no effect was found . However, it was found that the recombinant annexin could display ATPase/GTPase activities . The recombinant annexin showed much higher GTPase than ATPase activity . Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory . A photolabeling assay showed that this annexin could bind GTP more specifically than ATP . The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin . Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.

Trends Microbiol, 1999 Jan, 7(1), 29 - 36
Escherichia coli mutator genes; Horst JP et al.; The isolation and characterization of Escherichia coli mutator genes have led to a better understanding of DNA replication fidelity mechanisms and to the discovery of important DNA repair pathways and their relationship to spontaneous mutagenesis . Mutator strains in a population of cells can be beneficial in that they allow rapid selection of variants during periods of stress, such as drug exposure.

Curr Microbiol, 1999 Apr, 38(4), 210 - 6
Isolation and characterization of Synechococcus PCC7942 promoters: tRNApro gene functions as a promoter; Chungjatupornchai W et al.; Promoter-active fragments of Synechococcus PCC7942 were isolated by transcriptional gene fusion to the promoterless beta-glucuronidase (GUS) gene of E . coli, which was used as a reporter gene . Several of the isolated promoter-active fragments expressed GUS activity in Synechococcus comparable to that of the lambdaPR promoter . Only 10% of the isolated promoter-active fragments also functioned in E . coli . The transcription initiation sites of the two promoter-active fragments, D13 and E3, were identified . The major transcription initiation sites of D13 and E3 in Synechococcus were located within the nucleotides TTTG and TTG respectively, which were identical to those corresponding to E . coli . The inferred -10 and -35 regions of D13 were TAAACT and TTGTAG respectively, which conformed to the E . coli sigma70 promoter . Immediately upstream of the E3 transcription initiation sites was the tRNApro (GGG) gene, which contained two regions exhibiting strong homology to the major promoter elements in eukaryotic tRNA genes, but did not contain the E . coli promoter element . Thus, the tRNApro gene can act as a promoter.

Plant Physiol, 1999 Mar, 119(3), 961 - 78
A multisubunit acetyl coenzyme A carboxylase from soybean; Reverdatto S et al.; A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized . The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis . The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT) . At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT . Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts . Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase . Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences . Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria . The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma . Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD . A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD . By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored . Even higher ACCase activities were recovered by mixing complexes from pea and soybean . The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.

Clin Nephrol, 1999 Feb, 51(2), 73 - 6
Erythrocyte P1 group antigen expression in VTEC-associated hemolytic uremic syndrome; Ashida A et al.; BACKGROUND: The P1 blood group antigen has been postulated as a protective host factor against the development of the verotoxin-associated with hemolytic uremic syndrome (HUS) . PATIENTS AND METHODS: In this study the P1 blood group antigen in 59 Japanese children with a past history of hemorrhagic colitis (HC) was scored with a direct agglutination method using a commercial anti P1 antibody . RESULTS: The P1 antigen was positive in 4 (21.1%) of 19 patients with a history of postdiarrheal HUS and in 11 (28.0%) of 40 patients with no history . There were no significant differences in the frequency of the P1 phenotype between the 2 groups (p = 0.629) . CONCLUSION: We concluded that the P1 blood group antigen is not a protective factor against the development of HUS from verotoxin-producing Escherichia coli(VTEC)-infected hemorrhagic colitis in Japanese individuals.

Carcinogenesis, 1999 Feb, 20(2), 261 - 8
The major, N2-dG adduct of (+)-anti-B{a}PDE induces G-->A mutations in a 5'-AGA-3' sequence context; Shukla R et al.; Previously, in a random mutagenesis study, the (+)-anti diol epoxide of benzo{a}pyrene {(+)-anti-B{a}PDE} was shown to induce a complex mutational spectrum in the supF gene of an Escherichia coli plasmid, which included insertions, deletions and base substitution mutations, notably a significant fraction of GC-->TA, GC-->AT and GC-->CG mutations . At some sites, a single type of mutation dominated and to understand individual mutagenic pathways these sites were chosen for study by site-specific means to determine whether the major adduct, {+ta}-B{a}P-N2-dG, was responsible . {+ta}-B{a}P-N2-dG was shown to induce approximately 95% G-->T mutations in a 5'-TGC-3' sequence context and approximately 80% G-->A mutations in a 5'-CGT-3' sequence context . (+)-anti-B{a}PDE induced principally GC-->CG mutations in the G133 sequence context (5'-AGA-3') in studies using both SOS-uninduced or SOS-induced E . coli . Herein, {+ta}-B{a}P-N2-dG is shown to induce principally G-->A mutations (>90%) either without or with SOS induction in a closely related 5'-AGA-3' sequence context (identical over 7 bp) . This is the first time that there has been a discrepancy between the mutagenic specificity of (+)-anti-B{a}PDE versus {+ta}-B{a}P-N2-dG . Eight explanations for this discordance are considered . Four are ruled out; e.g . the second most prevalent adduct {+ca}-B{a}P-N2-dG also induces a preponderance of G-->A mutations (>90%), so it also is not responsible for (+)-anti-B{a}PDE-induced G133-->C mutations . The four explanations not ruled out are discussed and include that another minor adduct might be responsible and that the 5'-AGA-3' sequence context differed slightly in the studies with {+ta}-B{a}P-N2-dG versus (+)-anti-B{a}PDE . In spite of the discordance, {+ta}-B{a}P-N2-dG induces G-->A mutations in the context studied herein and this result has proven useful in generating a hypothesis for what conformations of {+ta}-B{a}P-N2-dG are responsible for G-->T versus G-->A mutations.

Prep Biochem Biotechnol, 1999 Feb, 29(1), 49 - 54
An improved alkaline lysis method for minipreparation of plasmid DNA; Liou JT et al.; This study is to improve the digestion pattern of miniprepped plasmid analyzed on gel . Frequently, some ambiguous DNA bands, which are suspected to be denatured DNA molecules, appear during electrophoresis of enzyme digested miniprepped plasmids . By employing Southern hybridization of two identical gels, one had been treated with denaturation-neutralization step and another without such treatment, we confirmed that many of these ambiguous DNA bands were single-stranded (SS) DNA molecules . The presence of SS DNA was due to the use of excess amount of NaOH during plasmid DNA purification with the conventional alkaline lysis method . We, therefore, modified the procedure and recommend that a half amount of NaOH (0.1N instead of 0.2N) should be used when isolating small quantity of plasmid DNA with the method.

FEBS Lett, 1999 Feb 19, 445(1), 192 - 6
Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae; Wyllie S et al.; We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel . In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C . psittaci and Chlamydia pneumoniae and analysed them at the single channel level . Both form porin-like ion channels that are functionally similar to those formed by native C . psittaci major outer membrane protein . Also, like the native channels, recombinant C . psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody . This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins . Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

FEBS Lett, 1999 Feb 19, 445(1), 111 - 4
A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon; Rashid N et al.; A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues . Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity . This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped . To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined . The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities . The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities . In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities . These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common . It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

Plant J, 1999 Jan, 17(1), 1 - 9
Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone; Gubler F et al.; GAMYB is an MYB transcription factor which is expressed in cereal aleurone cells in response to gibberellin (GA) . HvGAMYB binds to the TAACAAA box of a high-pl alpha-amylase gene promoter and transcriptionally activates its expression . In this study, we examined the role of HvGAMYB in activating expression of other GA-regulated genes encoding hydrolytic enzymes . In transient expression experiments, HvGAMYB transactivated expression of reporter genes fused to a low-pl alpha-amylase gene promoter, an EII (1-3, 1-4)-beta-glucanase gene promoter and a cathepsin B-like protease promoter . HvGAMYB DNA binding specificity was determined using a PCR-based random site selection using HvGAMYB fusion protein isolated from E . coli . The deduced consensus closely resembled gibberellin response elements in alpha-amylase promoters . Functional analysis of HvGAMYB by transient expression of C terminal HvGAMYB deletions in barley aleurone cells identified two transcriptional activation domains (TADs) which function in transcriptional regulation of both high- and low-pl alpha-amylase promoters . The same TADs were identified using a heterologous yeast expression system . Together, these results indicate that HvGAMYB has two TADs . These domains are C-terminal to its DNA-binding domain.

Folia Microbiol (Praha), 1998, 43(6), 601 - 4
Overexpression of the FNR protein of Escherichia coli with T7 expression system; Stuchlik S et al.; We have used the T7 expression system for expression of E . coli FNR protein . The fnr gene was cloned from its initiation codon ATG into the NdeI site of an expression vector and filamentous phage mGP1-2 was used as a donor of T7 RNA polymerase gene . The level of FNR expression attained by this expression arrangement was about 45% of total cell proteins.

Folia Microbiol (Praha), 1998, 43(6), 589 - 99
In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli; Burian J et al.; A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53 . The strain formed typical black colonies on solid LB medium with tellurite . The determinant was located on a large conjugative plasmid designated pTE53 . Electron-dense particles were observed in cells harboring pTE53 by electron microscopy . X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures . Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals . In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism . Tellurite accumulation rates for E . coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain . In addition, no efflux mechanism was detected . The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46 . Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful . During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite . All these clones were Mucts62 lysogens . Their tellurite resistance levels were in the same order as the wild type strains . Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain . Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype . However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype . It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Trends Microbiol, 1999 Jan, 7(1), 37 - 45
Small RNAs in Escherichia coli; Wassarman KM et al.; Bacterial cells contain several small RNAs (sRNAs) that are not translated . These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.

Mol Genet Metab, 1999 Feb, 66(2), 80 - 90
Mechanism of biotin responsiveness in biotin-responsive multiple carboxylase deficiency; Dupuis L et al.; Holocarboxylase synthetase (HCS) catalyses the biotinylation of the four biotin-dependent carboxylases found in humans . A deficiency in HCS results in biotin-responsive multiple carboxylase deficiency . We have evaluated the biotin responsiveness associated with six missense mutations previously identified in affected patients by expression of plasmids containing the mutated HCS in an Escherichia coli strain mutated in the corresponding BirA gene . We demonstrate that the mutations identified in the MCD patients are indeed responsible for their reduced HCS activity . Four of the mutations, clustering in the putative biotin binding domain as deduced from the structure of the E . coli enzyme, are consistent with an explanation for biotin responsiveness based on altered affinity for biotin . The remaining mutations, located outside the biotin binding region, were associated with a more limited biotin responsiveness that may be explained by the degree of residual enzyme activity present . The data suggest that the concentration of circulating biotin is as low as 100 times below the Km of the enzyme, so that any increase in biotin concentration through dietary supplementation would result in saturation of the available mutant enzyme . We suggest that these alternative explanations are sufficient to account for the apparent universality of biotin responsiveness in biotin responsive multiple carboxylase deficiency .

Arch Biochem Biophys, 1999 Mar 15, 363(2), 237 - 45
Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems; Federici C et al.; Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells . Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family . Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems . However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E . coli cells . These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody . An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography . Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30-31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme .

Arch Biochem Biophys, 1999 Mar 15, 363(2), 219 - 26
Vitamin D receptor interacts with DnaK/heat shock protein 70: identification of DnaK interaction site on vitamin D receptor; Swamy N et al.; Vitamin D receptor (VDR) regulates the expression of vitamin D-dependent genes upon binding to its cognate ligand, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) . This process represents a complex interaction of ligand-bound VDR with nuclear proteins like retinoid X receptor, nuclear accessory factors, and regulatory elements of the target gene . Expression of full-length VDR in Escherichia coli revealed that VDR binds DnaK, a member of heat-shock protein (Hsp) family, with high affinity . By systematic N-terminal truncation of VDR, the interaction site of DnaK on VDR was localized within a 17-amino-acid segment (105-122) representing the "hinge region" between the DNA-binding and hormone-binding domains of VDR . The putative DnaK-binding site was further localized between residues 105 to 109 of VDR by using binding-energy-minimization studies . The interaction of DnaK with VDR did not influence the binding of 1,25(OH)2D3 or nuclear accessory factor(s) to VDR . Furthermore, bovine brain Hsp 70, similar to DnaK, interacted with VDR-ligand-binding domain (105-427) . These results suggest that DnaK/Hsp 70 may interact with VDR prior to the activation of the latter by 1,25(OH)2D3-binding .

Biotechnol Bioeng, 1999 Mar 20, 62(6), 659 - 65
Biocatalytic nerve agent detoxification in fire fighting foams; LeJeune KE et al.; Current events across the globe necessitate rapid technological advances to combat the epidemic of nerve agent chemical weapons . Biocatalysis has emerged as a viable tool in the detoxification of organophosphorus neurotoxins, such as the chemical weapons VX and sarin . Efficient detoxification of contaminated equipment, machinery, and soils are of principal concern . This study describes the incorporation of a biocatalyst (organophosphorus hydrolase, E.C . 3.1.8.1) into conventional formulations of fire fighting foam . The capacity of fire fighting foams to decrease volatilization of contained contaminants, increase surface wettability, and control the rate of enzyme delivery to large areas makes them useful vehicles for enzyme application at surfaces . The performance of enzyme containing foams has been shown to be not only reproducible but also predictable . An empirical model provides reasonable estimations for the amounts of achievable surface decontamination as a function of the important parameters of the system . Theoretical modeling illustrates that the enzyme-containing foam is capable of extracting agent from the surface and is catalytically active at the foam-surface interface and throughout the foam itself . Biocatalytic foam has proven to be an effective, "environmentally friendly" means of surface and soil decontamination.

FEMS Microbiol Lett, 1999 Jan 15, 170(2), 307 - 12
Intracellular location of catalase-peroxidase hydroperoxidase I of Escherichia coli; Hillar A et al.; The catalase-peroxidase hydroperoxidase I of Escherichia coli has been confirmed to be located in the cytoplasm using two independent methods . Catalase activity was found predominantly (> 95%) in the cytoplasmic fraction following spheroplast formation . The cytoplasmic enzyme glucose-6-phosphate dehydrogenase and the periplasmic enzyme alkaline phosphatase were used as controls . The second method of immunogold staining for the enzyme in situ revealed an even distribution of the enzyme across the cell.

Mol Immunol, 1998 Nov, 35(16), 1069 - 77
Conversion of an antibody into an enzyme which cleaves the protein HPr; Liu E et al.; Jel 42 is an IgG which binds to the small bacterial protein, HPr and the structure of the complex is known at high resolution . The IgG was expressed as a single chain variable fragment (scFv) and the binding to HPr was assessed by fluorescence polarization of fluorescein-labelled HPr . The binding constant for the IgG was about 20-fold higher than the scFv . Inspection of the structure of the complex suggested that it might be possible to convert the scFv into a bond-specific protease by the introduction of three catalytic residues: a glutamate to increase the nucleophilicity of a nearby water molecule, a lysine to increase the polarizability of the carbonyl group and a histidine to provide a proton to convert the amine into a better leaving group . By trial and error it was found that a fourth residue had to be converted into glycine in order to maintain the integrity of complimentarity-determining region three of the heavy chain (CDRH3) at the binding interface . The resulting quadruple mutant still bound to HPr and unlike other mutants, showed weak protease activity as judged from the fluorescence polarization assay . The activity was maximum at pH 6 consistent with a requirement for a protonated histidine residue . With the aid of HPr fluorescein-labelled at two different positions, it was demonstrated that the size of the products was consistent with cleavage occurring in the vicinity of the target peptide bond . The activity was specific for HPr since an excess of bovine serum albumin did not interfere with the reaction.

Mol Immunol, 1998 Nov, 35(16), 1033 - 43
Baculoviral expressed HLA class I heavy chains used to screen a synthetic peptide library for allele-specific peptide binding motifs; Smith MH et al.; Recombinant baculoviruses encoding truncated HLA-A*0101 and HLA-A*0201 class I heavy chains have been isolated and used to infect lepidopteran cells . Proteins overexpressed in this system were glycosylated, and consisted of 282 amino acid residues after signal sequence cleavage . These class I heavy chains could fold into their native conformation in the presence of recombinant human beta2-microglobulin expressed in Escherichia coli and a synthetic peptide library of nonamers bound to resin-support beads . Reconstitution into native ternary complexes was detected using a conformation specific monoclonal antibody followed by isolation and sequencing of the bound peptides . The motifs obtained for HLA-A1.1 and HLA-A2.1 peptides are similar although more extensive than those derived from sequencing endogenous peptides . This approach selects peptides which form very stable complexes regardless of whether these peptides are generated under physiological conditions, thereby providing unique supplementary data for predicting and designing CTL epitopes . This method is based solely on peptide binding to the class I molecule and is therefore independent of any constraints imposed by endogenous intracellular processing or transport systems . A comparison of the two motifs provides an opportunity to distinguish between the requirements of binding from those arising as a function of intracellular processing or transport . Our findings are not consistent with a recent report suggesting that constraints on the COOH termini of these peptides can be attributed to the effects of either intracellular processing or transport . We find that the carboxy termini in the class I peptides analyzed to date mimic the endogenous data, suggesting that residues in this position contribute to binding affinity.

Mol Immunol, 1998 Nov, 35(16), 1017 - 23
Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor; Olsson S et al.; To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed . Mutants with one or more cysteine residues altered were constructed with a histidine residue tag for purification purposes and expressed as recombinant proteins in E . coli . Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72 77) and one mutant with all 6 cysteines altered (6 Cys) . The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding . Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2 . However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered . A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72 77 or the 6 Cys mutant . Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 microg/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved . The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy.

Vaccine, 1999 Feb 26, 17(7-8), 695 - 704
The adjuvants MF59 and LT-K63 enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administered intranasally in mice; Barchfeld GL et al.; Commercial influenza vaccines generate serum antibody, but not local IgA . Influenza vaccines that induce both serum and secretory antibody are more likely to protect against infection and disease progression . The adjuvants MF59 and LT-K63 were tested intramuscularly and intranasally with subunit HA . In naive mice, intranasal adjuvant effect was more apparent when included with the first than second immunization . In previously infected mice, intranasal adjuvants had little effect on serum antibodies and were most effective for nasal antibodies after the second immunization . Overall, both adjuvants enhanced anti-HA IgA and IgG by intranasal vaccination whereas, by intramuscular vaccination, they only enhanced serum IgG.

Yi Chuan Xue Bao, 1998, 25(4), 375 - 80
{Thermostable alkaline phosphatase from Thermus sp . FD3041: cloning of the gene and expression in Escherichia coli}; Yuan YZ et al.; A genomic library of Thermus sp . FD3041 which produces thermostable alkaline phosphatase (FD-TAP) was constructed with the vector pUC118 and the host E . coli TG1 . 3-10kb inserted fragments of foreign DNA were identified in 90 percent of the 12,000 clones thus obtained . Five positive clones were detected after screening the plated library by the method of colony coloration for TAP in situ . Preliminary analysis of the enzyme expressed from one recombinant plasmid pTAP362 showed that the properties of the recombinant enzyme, such as the thermal stability and optimal temperature of reaction, were identical to those of the native enzyme . The gene of FD-TAP was located on the 2.0kb BamHI-HindIII fragment of the pTAP362, determined by its physical map and the change of enzyme activity in different partially deleted plasmids . Results of thermostability experiment in PCR thermal cycle showed that the FD-TAP would be suitable for labelling of primers and detection of PCR amplified products.

Yi Chuan Xue Bao, 1998, 25(4), 301 - 7
{Expression and biochemical characterization of human G6PD gene 1376 and 1388 mutation in G6PD-deficient Escherichia coli}; Jiang WY et al.; Nine types of human G6PD gene mutated at the positions of nt 1376 and nt 1388 by site-directed mutagenesis were transformed into the strain of G6PD dificent E . coli HB 351(DE3) . The mutated gene was expressed successfully and the enzyme kinetic studies undertaken according to WHO standardization . The results showed that the arginine residues at the positions of 459 and 463 of G6PD gene play an important role in maintaining activity of the enzyme . The amino acid structure, polarity, and electronic property may be responsible for it . The arginine residues at the positions of 459 and 463 are also important for the enzyme-NADP+ binding, but it was not interfered by the lysine-arginine substitution . By inducing a non-sense mutation, it was further demonstrated that the amino acids residueds behind the position of 459 were extremely significant for G6PD activity.

J Biol Chem, 1999 Mar 12, 274(11), 7516 - 27
Evolution of plant defense mechanisms . Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases; Gang DR et al.; Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers . Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds . In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli . The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects . It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases . A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa . Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination . A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

J Biol Chem, 1999 Mar 12, 274(11), 7137 - 45
Mapping binding domains of kininogens on endothelial cell cytokeratin 1; Shariat-Madar Z et al.; Human cytokeratin 1 (CK1) in human umbilical vein endothelial cells (HUVEC) is expressed on their membranes and is able to bind high molecular weight kininogen (HK) (Hasan, A . A . K., Zisman, T., and Schmaier, A . H . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 3615-3620) . New investigations have been performed to demonstrate the HK binding domain on CK1 . Four overlapping recombinant (r) CK1 proteins were produced in Escherichia coli by a glutathione S-transferase gene fusion system . Biotin-HK specifically bound to rCK128 and rCK131 in the presence of Zn2+ but not to Deleted1-6rCK131 . Recombinant CK128 and rCK131 also inhibited biotin-HK binding to HUVEC with IC50 of 0.4 and 0.5 microM, respectively . Alternatively, rCK114 and Deleted1-6rCK131 did not inhibit binding at concentrations >/=1 microM . Seven sequential 20 amino acid peptides of CK1 were prepared to cover the protein coded by exons 1-3 . Only the first peptide (GYG20) coded by exon 1 significantly inhibited HK binding to HUVEC with an IC50 of 35 microM . Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC50 of 18 and 9 microM, respectively . A sequence scrambled peptide of PGG15 did not block binding to HUVEC and biotin-GPV20 specifically bound to HK . Peptides GPV15 and PGG15 also blocked prekallikrein activation on endothelial cells . However, inhibition of PK activation by peptide PGG15 occurred at 10-fold lower concentration (IC50 = 1 microM) than inhibition of biotin-HK binding to HUVEC (IC50 = 10 microM) . These studies indicate that HK binds to a region of 20 amino acids coded by exon 1 on CK1 which is carboxyl-terminal to its glycine-rich amino-terminal globular domain . Furthermore, HK binding to CK1 modulates PK activation on HUVEC.

J Biol Chem, 1999 Mar 12, 274(11), 7128 - 36
Purification and characterization of a mitochondrial thymine glycol endonuclease from rat liver; Stierum RH et al.; Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation . Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases . The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects . Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism . Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo) . By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts . After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of approximately 37 kDa on a silver-stained gel . MtTGendo is active within a broad KCl concentration range and is EDTA-resistant . Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity . MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA . Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein.

J Biol Chem, 1999 Mar 12, 274(11), 7052 - 8
Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase; Nadanaciva S et al.; Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone . beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites . Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three . Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding . The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state . Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se . The fluorescence technique should prove valuable for future transition state studies of F1-ATPase.

J Biol Chem, 1999 Mar 12, 274(11), 6992 - 7001
Characterization of the nucleoside triphosphatase activity of poliovirus protein 2C reveals a mechanism by which guanidine inhibits poliovirus replication; Pfister T et al.; The highly conserved non-structural protein 2C of picornaviruses is involved in viral genome replication and encapsidation and in the rearrangement of intracellular structures . 2C binds RNA, has nucleoside triphosphatase activity, and shares three motifs with superfamily III helicases . Motifs "A" and "B" are involved in nucleotide triphosphate (NTP) binding and hydrolysis, whereas a function for motif "C" has not yet been demonstrated . Poliovirus RNA replication is inhibited by millimolar concentrations of guanidine hydrochloride (GdnHCl) . Resistance and dependence to GdnHCl map to 2C . To characterize the nucleoside triphosphatase activity of 2C, we purified poliovirus recombinant 2C fused to glutathione S-transferase (GST-2C) from Escherichia coli . GST-2C hydrolyzed ATP with a Km of 0.7 mM . Other NTPs, including GTP, competed with ATP for binding to 2C but were poor substrates for hydrolysis . Mutation of conserved residues in motif A and B abolished ATPase activity, as did mutation of the conserved asparagine residue in motif C, an observation indicating the involvement of this motif in ATP hydrolysis . GdnHCl at millimolar concentrations inhibited ATP hydrolysis . Mutations in 2C that confer poliovirus resistant to or dependent on GdnHCl increased the tolerance to GdnHCl up to 100-fold.

Clin Diagn Lab Immunol, 1999 Mar, 6(2), 224 - 30
Genetic and serological analysis of lipoprotein LppA in Mycoplasma mycoides subsp . mycoides LC and Mycoplasma mycoides subsp . capri; Monnerat MP et al.; The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp . mycoides large-colony type (LC) strain Y-goat and the M . mycoides subsp . capri strain PG3 were cloned and analyzed by sequencing . These two lipoproteins have been named LppA{MmymyLC} and LppA{Mmyca}, and their corresponding genes have been named lppA{MmymyLC} and lppA{Mmyca}, respectively . The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas . Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M . mycoides subsp . mycoides small-colony type and P67 of the Mycoplasma species bovine group 7 . Based on lppA gene sequences of M . mycoides subsp . mycoides LC and M . mycoides subsp . capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M . mycoides cluster . Analysis of the PCR-amplified lppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies . This PCR therefore allows a rapid identification of M . mycoides subsp . mycoides LC and M . mycoides subsp . capri but does not distinguish between these two closely related subspecies . LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum . Antibodies against recombinant LppA{MmymyLC} reacted with a 62-kDa protein in all M . mycoides subsp . mycoides LC and M . mycoides subsp . capri type strains and field strains tested but not with the other members of the M . mycoides cluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.

Curr Opin Microbiol, 1998 Apr, 1(2), 204 - 9
Proteolysis and chaperones: the destruction/reconstruction dilemma; Herman C et al.; Cytoplasmic proteases, although necessary for proper cell functioning, must be strictly regulated . In fact, they resemble chaperones, ancient protein folding devices . These molecules recognise exposed hydrophobic regions of unfolded or denatured proteins . For most substances it is not known how the cell chooses between the refolding and proteolytic pathways . In Escherichia coli, however, a carboxy-terminal proteolysis tag and binding site for the chaperone DnaK have recently been identified.

Curr Opin Microbiol, 1998 Apr, 1(2), 152 - 9
Positive activation of gene expression; Rhodius VA et al.; Most bacterial transcription activators function by making direct contact with RNA polymerase at target promoters . Some activators contact the carboxy-terminal domain of the RNA polymerase alpha subunit, some contact region 4 of the sigma70 subunit, whilst others interact with other contact sites . A number of activators are ambidextrous and can, apparently simultaneously, contact more than one target site on RNA polymerase . Expression from many promoters is co-dependent on two or more activators . There are several different mechanisms for coupling promoter activity to more than one activator: in one such mechanism, the different activators make independent contacts with different target sites on RNA polymerase.

Curr Opin Microbiol, 1998 Apr, 1(2), 216 - 22
The Sec system; Driessen AJ et al.; Proteins designated to be secreted by Escherichia coli are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB . Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecF . Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism or preprotein translocation . It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction.

Curr Opin Microbiol, 1998 Feb, 1(1), 103 - 8
Enterohemorrhagic Escherichia coli; Kaper JB; Enterohemorrhagic Escherichia coli has been responsible for an increasing number of large food-borne outbreaks of bloody diarrhea and hemolytic uremic syndrome . Recent developments in our understanding of the pathogenesis of disease due to enterhemorrhagic E . coli include the description of a pathogenicity island, a type III secretion system and potential plasmid-encoded virulence factors . Recent developments in our understanding of the epidemiology include a recognition of a widening spectrum of vehicles.

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 84 - 8
Photosensitization of wild and mutant strains of Escherichia coli by meso-tetra (N-methyl-4-pyridyl)porphine; Valduga G et al.; Wild type Escherichia coli cells as well as some mutant strains lacking specific DNA repair systems are efficiently killed upon visible light-irradiation after 5 min-incubation with meso-tetra(4N-methyl-pyridyl)porphine (T4MPyP) . The presence of oxygen is necessary for cell photoinactivation . The porphyrin appears to exert its phototoxic activity largely by impairing some enzymic and transport functions at the level of both the outer and cytoplasmic membrane . Thus, SDS-PAGE electrophoresis shows a gradual attenuation of some transport protein bands as the irradiation proceeds, while a complete loss of lactate and NADH dehydrogenase activities is caused by 15 min-exposure to light . On the other hand, DNA does not represent a critical target of T4MPyP photosensitization as suggested by the closely similar photosensitivity of the wild E . coli and E . coli strains defective for two different DNA repair mechanisms, as well as by the lack of any detectable alteration of the pUC19 plasmids extracted from photosensitized E . coli TG1 cells .

Fertil Steril, 1999 Mar, 71(3), 564 - 6
Testicular sperm aspiration and intracytoplasmic sperm injection for persistent infection of the ejaculate; Seidman DS et al.; OBJECTIVE: To report the successful use of testicular sperm aspiration and intracytoplasmic sperm injection in the presence of an Escherichia coli-infected ejaculate that previously caused repeated embryo degeneration . DESIGN: Case report . SETTING: University medical center . PATIENT(S): A 38-year-old woman who did not conceive for 6 years with repeated IVF attempts . Escherichia coli was isolated from both the oocyte culture dish and her male partner's ejaculate . INTERVENTION(S): Testicular sperm aspiration and intracytoplasmic sperm injection followed by ET . MAIN OUTCOME MEASURE(S): Clinical outcome . RESULT(S): Establishment of a pregnancy delivered at term . CONCLUSION(S): Patients undergoing IVF treatment who have repeated embryo degeneration caused by bacterial infection originating in the ejaculate may be treated successfully with testicular sperm aspiration and intracytoplasmic sperm injection.

Cell Mol Life Sci, 1999 Jan, 55(1), 135 - 41
Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site; Burgin J et al.; Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affinity for omega-aminocarboxylic acids . Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys311 was altered by site-directed mutagenesis into Asp311 in accordance with the consensus sequence of the LBS . Cys297 involved in the interkringle disulfide bridge was mutated into Ser297 to minimize dimerization and aggregation . The mutated K3 TYQ{K3HPg/C297S/K311D}DS (r-K3mut) was expressed in Escherichia coli, isolated on an Ni2(+)-nitrilotriacetic acid-agarose column, refolded and purified on a lysine Bio-Gel column . Fluorescence titration indicates affinity of r-K3mut for omega-aminocarboxylic acids with the following association constants (Kass, mM-1): 5-aminopentanoic acid: 1.3; 6-aminohexanoic acid: 4.2; 7-aminoheptanoic acid: 0.5; trans-(aminomethyl)cyclohexanecarboxylic acid: 12.7; p-benzylaminesulfonic acid: 11.8 . r-K3mut exhibits an affinity similar to native and mutated (R220G, E221D) K2 . The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg . We were able to demonstrate for the first time that an appropriate mutation in the LBS of a kringle produced a weak but distinct affinity for omega-aminocarboxylic acids.

Cell Mol Life Sci, 1999 Jan, 55(1), 131 - 4
Production of functional rat liver PSP protein in Escherichia coli; Oka T et al.; An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed . Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1 . After the plasmid introduced into E . coli was expressed by isopropyl 1-thio-beta-D-galaetopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography . The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.

Biochim Biophys Acta, 1999 Feb 25, 1437(2), 182 - 93
Sequence, expression in Escherichia coli, and characterization of lysophospholipase II; Toyoda T et al.; Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form lysophospholipase named lysophospholipase II from mouse embryo . The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of lysophospholipase I previously cloned from rat liver (H . Sugimoto et al., J . Biol . Chem . 271 (1996) 7705-7711) . The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24794, and showed 64% identity to that of lysophospholipase I with the Gly-X-Ser-X-Gly esterase/lipase consensus . The lacZ fusion protein expressed in E . coli cells exhibited lysophospholipase activity and reacted with antibody raised against previously purified pig gastric lysophospholipase II (H . Sunaga et al., Biochem . J . 308 (1995) 551-557), but not with antibody against rat liver lysophospholipase I . The expressed enzyme was purified to a specific activity of 0.15 micromol/min per mg by DEAE-Sepharose A-500 chromatography . The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid . Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated . Northern blot analysis and reverse transcription-polymerase chain reaction revealed that lysophospholipase II transcript as well as lysophospholipase I transcript was widely distributed in mouse tissues.

Biochim Biophys Acta, 1999 Feb 25, 1437(2), 157 - 69
A specific human lysophospholipase: cDNA cloning, tissue distribution and kinetic characterization; Wang A et al.; Lysophospholipases are critical enzymes that act on biological membranes to regulate the multifunctional lysophospholipids; increased levels of lysophospholipids are associated with a host of diseases . Herein we report the cDNA cloning of a human brain 25 kDa lysophospholipid-specific lysophospholipase (hLysoPLA) . The enzyme (at both mRNA and protein levels) is widely distributed in tissues, but with quite different abundances . The hLysoPLA hydrolyzes lysophosphatidylcholine in both monomeric and micellar forms, and exhibits apparent cooperativity and surface dilution kinetics, but not interfacial activation . Detailed kinetic analysis indicates that the hLysoPLA binds first to the micellar surface and then to the substrate presented on the surface . The kinetic parameters associated with this surface dilution kinetic model are reported, and it is concluded that hLysoPLA has a single substrate binding site and a surface recognition site . The apparent cooperativity observed is likely due to the change of substrate presentation . In contrast to many non-specific lipolytic enzymes that exhibit lysophospholipase activity, hLysoPLA hydrolyzes only lysophospholipids and has no other significant enzymatic activity . Of special interest, hLysoPLA does not act on plasmenylcholine . Of the several inhibitors tested, only methyl arachidonyl fluorophosphonate (MAFP) potently and irreversibly inhibits the enzymatic activity . The inhibition by MAFP is consistent with the catalytic mechanism proposed for the enzyme - a serine hydrolase with a catalytic triad composed of Ser-119, Asp-174 and His-208.

Brain Res Mol Brain Res, 1999 Mar 5, 65(2), 143 - 50
Expression of intercellular adhesion molecule 1 (ICAM-1) is reduced in permanent focal cerebral ischemic mouse brain using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist; Yang GY et al.; Our previous studies have demonstrated that overexpression of recombinant human interleukin-1 receptor antagonist protein (IL-1ra) via gene transfer can reduce ischemic brain injury . However, the mechanism of action of IL-1ra in ischemia is unclear . Since interleukin-1 can up-regulate intercellular adhesion molecules in endothelium, the present study was designed to determine whether overexpression of the IL-1ra can reduce the expression of intercellular adhesion molecule-1 (ICAM-1) after ischemic injury . Normal saline or adenovirus vector (1x109 particles) encoding the human IL-1ra gene (Ad.RSVIL-1ra) or the Escherichia coli LacZ gene (Ad.RSVlacZ) was injected into the right lateral cerebral ventricle of adult CD-1 mice . After five days, permanent middle cerebral artery occlusion (MCAO) was achieved for 24 h using an intraluminal suture . Cerebral blood flow was monitored by transcranial laser Doppler flowmetry to verify the occlusion . ICAM-1 protein was quantified using Western blot analysis and localized using immunohistochemistry . After MCAO, surface blood flow in the ischemic hemisphere was decreased to 9-11% of the baseline . There were fewer ICAM-1 positive vessels in the ischemic cortex of the Ad.RSVIL-1ra transfected mice than in the Ad.RSVlacZ transfected and saline treated mice (138+/-19 vs . 249+/-25, 284+/-22, p<0.05) . Western blot analysis shows that ICAM-1 protein decreased 50-60% in the Ad . RSVIL-1ra group compared to the other two groups . There were no significant differences in the numbers of positive vessels in the ischemic basal ganglia and contralateral hemisphere among the three groups . Our studies suggest that IL-1ra overexpression can down-regulate the expression of ICAM-1 in the ipsilateral cortex in ischemic mice . Interleukin-1 may play an important role in the activation of inflammatory reaction during focal cerebral ischemia by promoting leukocyte adhesion on the endothelium cells .

Mutat Res, 1999 Mar 8, 424(1-2), 221 - 36
DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions; Eckert KA et al.; DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision . Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms . Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs . The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA . However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase . With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis . For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis . However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities . Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed . In vitro reaction conditions also influence DNA polymerase processing of lesions . Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates . The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM {dNTP}, relative to 50 microM {dNTP}, consistent with an increased efficiency of extension of the etO6G.T mispair . Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP . The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide . Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes . In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates . However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease . We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay . We observed no significant reduction in the magnitude of the mutant frequency vs . dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions . Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied .

J Mol Biol, 1999 Mar 12, 286(5), 1609 - 19
Increased helix and protein stability through the introduction of a new tertiary hydrogen bond; Peterson RW et al.; In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr . Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution . Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue . Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein . However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr . Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability . We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability .

J Mol Biol, 1999 Mar 12, 286(5), 1597 - 608
Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9; Ferguson N et al.; The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and Im9 have been characterised at pH 7.0 and 10 degrees C . These proteins are 60 % identical in sequence and have the same three-dimensional structure, yet appear to fold by different kinetic mechanisms . The logarithm of the folding and unfolding rates of Im9 change linearly as a function of urea concentration and fit well to an equation describing a two-state mechanism (with a folding rate of 1500 s-1, an unfolding rate of 0 . 01 s-1, and a highly compact transition state that has approximately 95 % of the native surface area buried) . By contrast, there is clear evidence for the population of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a significant burst phase amplitude in the refolding kinetics . Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 remains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 10 degrees C) the intermediate populated during Im7 refolding is significantly stabilised (KUI=125) . Equilibrium denaturation experiments, under the conditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (DeltaDeltaG 9.3 kJ/mol) and the DeltaG and m values determined accord with those obtained from the fit to the kinetic data . The results show, therefore, that the population of an intermediate in the refolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein . We discuss the possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general .

J Mol Biol, 1999 Mar 12, 286(5), 1547 - 65
Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme; Mine S et al.; A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme . Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins . Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz . Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions . On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes . A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them . In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free . Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased . From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity .

J Mol Biol, 1999 Mar 12, 286(5), 1519 - 31
Structure of D-allose binding protein from Escherichia coli bound to D-allose at 1.8 A resolution; Chaudhuri BN et al.; ABC transport systems for import or export of nutrients and other substances across the cell membrane are widely distributed in nature . In most bacterial systems, a periplasmic component is the primary determinant of specificity of the transport complex as a whole . We report here the crystal structure of the periplasmic binding protein for the allose system (ALBP) from Escherichia coli, solved at 1.8 A resolution using the molecular replacement method . As in the other members of the family (especially the ribose binding protein, RBP, with which it shares 35 % sequence homology), this structure consists of two similar domains joined by a three-stranded hinge region . The protein is believed to exist in a dynamic equilibrium of closed and open conformations in solution which is an important part of its function . In the closed ligand-bound form observed here, D-allose is buried at the domain interface . Only the beta-anomer of allopyranose is seen in the crystal structure, although the alpha-anomer can potentially bind with a similar affinity . Details of the ligand-binding cleft reveal the features that determine substrate specificity . Extensive hydrogen bonding as well as hydrophobic interactions are found to be important . Altogether ten residues from both the domains form 14 hydrogen bonds with the sugar . In addition, three aromatic rings, one from each domain with faces parallel to the plane of the sugar ring and a third perpendicular, make up a hydrophobic stacking surface for the ring hydrogen atoms . Our results indicate that the aromatic rings forming the sugar binding cleft can sterically block the binding of any hexose epimer except D-allose, 6-deoxy-allose or 3-deoxy-glucose; the latter two are expected to bind with reduced affinity, due to the loss of some hydrogen bonds . The pyranose form of the pentose, D-ribose, can also fit into the ALBP binding cleft, although with lower binding affinity . Thus, ALBP can function as a low affinity transporter for D-ribose . The significance of these results is discussed in the context of the function of allose and ribose transport systems .

J Mol Biol, 1999 Mar 12, 286(5), 1285 - 91
Structure and structural variations of the Escherichia coli 30 S ribosomal subunit as revealed by three-dimensional cryo-electron microscopy; Gabashvili IS et al.; A three-dimensional reconstruction of the 30 S subunit of the Escherichia coli ribosome was obtained at 23 A resolution . Because of the improved resolution, many more structural details are seen as compared to those obtained in earlier studies . Thus, the new structure is more suitable for comparison with the 30 S subunit part of the 70 S ribosome, whose structure is already known at a better resolution . In addition, we observe relative and, to some extent, independent movements of three main structural domains of the 30 S subunit, namely head, platform and the main body, which lead to partial blurring of the reconstructed volume . An attempt to subdivide the data set into conformationally defined subsets reveals the existence of conformers in which these domains have different orientations with respect to one another . This result suggests the existence of dynamic properties of the 30 S subunit that might be required for facilitating its interactions with mRNA, tRNA and other ligands during protein biosynthesis .

J Nutr, 1999 Feb, 129(2S Suppl), 477S - 484S
Molecular biology of biotin attachment to proteins; Chapman-Smith A et al.; Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification . Both biotin domains and biotin protein ligases are very strongly conserved throughout biology . This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins.

FASEB J, 1999 Mar, 13(3), 563 - 71
Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-alpha production and accelerated IL-10 expression; Spittler A et al.; Cytokines play a pivotal role in the pathogenesis of septic shock . Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) stimulate the progression of septic shock whereas the anti-inflammatory cytokine IL-10 has counterregulative potency . The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF-alpha production . In the current study we investigated the role of GLY on lipopolysaccharide (LPS) -induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors . GLY did not modulate the expression of HLA-DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E . coli phagocytosis (P<0.05) decreased significantly . GLY decreased LPS-induced TNF-alpha production (P<0.01) and increased IL-10 expression of purified monocytes . Similarly, in a whole blood assay, GLY reduced TNF-alpha (P<0.0001) and IL-1beta (P<0.0001) synthesis and increased IL-10 expression (P<0.05) in a dose-dependent manner . The inhibitory effects of GLY were neutralized by strychnine, and the production of IL-10 and TNF-alpha was augmented by anti-IL-10 antibodies . Furthermore, GLY decreased the amount of IL-1beta and TNF-alpha-specific mRNA . Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.

EMBO J, 1999 Mar 1, 18(5), 1415 - 24
P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities; Bouet JY et al.; The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS . parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex . ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process . In this study we demonstrate a direct interaction between ParA and the P1 partition complex . The interaction was strictly dependent on ParB and ATP . The consequence of this interaction depended on the ParB concentration . At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex . ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon . Conversely, ATP could not support a stable interaction of ParA with parOP in this assay . Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition . Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.

EMBO J, 1999 Mar 1, 18(5), 1407 - 14
Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity; Arnold PH et al.; Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites . Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination . We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining) . Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I . A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro . In these novel site Ixsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision . Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.

EMBO J, 1999 Mar 1, 18(5), 1192 - 8
Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway; Kobayashi T et al.; Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm . Functional respiratory chain is required for the cell to keep DsbA oxidized . In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain . Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions . While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44 . Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent . This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated . Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.

Biol Chem, 1999 Jan, 380(1), 89 - 94
Homo-dimeric spherulin 3a: a single-domain member of the beta gamma-crystallin superfamily; Kretschmar M et al.; The beta gamma-crystallin superfamily of eye lens proteins comprises a class of structurally related members with a wide variety of different functions . Common features of these proteins are 1 . the Greek-key motif of antiparallel beta-sheets, called the crystallin fold, and 2 . the high intrinsic long-term stability . Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within the beta gamma-crystallin family . Based on sequence homology and 'domain swapping', it has been proposed to represent an evolutionary ancestor of present-day eye lens crystallins . Since S3a is highly expressed in spherulating plasmodia of P . polycephalum under a variety of stress conditions, it can be assumed that the protein may serve as a compatible solute in the cytosol of the slime mold . In order to investigate the stability and other physicochemical properties of a single-domain all-beta protein, we isolated natural S3a . For the large-scale purification, the recombinant protein was cloned and expressed in Escherichia coli . The detailed spectral and biochemical analysis proved the recombinant protein to be authentic . In its native form, S3a is dimeric . Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the formation of higher oligomers . In order to preserve the native quaternary structure without aggregation artifacts in denaturation/renaturation experiments, the Cys4-->Ser mutant (S3a C4S) was produced . Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties . At pH 3 - 4, both proteins form a stable compact intermediate (A-state) . Concentration-dependent thermal and chemical denaturation showed that the equilibrium unfolding of S3a obeys the simple two-state model with no significant occurrence of folding intermediates.

Biol Chem, 1999 Jan, 380(1), 47 - 54
Studies with lysine N6-hydroxylase . Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity; Stehr M et al.; The proposed FAD binding site of L-lysine N6-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases . We have studied the role of this proline by mutating it to glycine in {P14G}aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity . The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine . Compared to the wild-type enzyme, the activity vs . pH profile of the mutant protein indicates a shift of the apparent pK'(a)s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme) . While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme . These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.

Biol Chem, 1999 Jan, 380(1), 19 - 29
Efficient control of raf gene expression by CAP and two Raf repressors that bend DNA in opposite directions; Muiznieks I et al.; The plasmid-borne raf operon of Escherichia coli encodes proteins involved in the uptake and utilisation of the trisaccharide raffinose . The operon is subject to dual regulation; to negative control by the binding of RafR repressor to twin operators, O1 and O2, and to positive control by the cAMP-binding protein, CAP . We have identified the CAP binding site (CBS) as a 22 bp palindromic sequence with incomplete dyad symmetry by deletion analysis, DNasel footprinting and electrophoretic mobility shift assays (EMSA) of CAP-DNA complexes . The CBS is centred 60.5 bp upstream of the transcription start point and partially overlaps O1 . In vivo, CAP increases rafA (alpha-galactosidase) gene expression up to 50-fold . The 28 bp spacing between the centres of CBS and the - 35 box is essential, since insertions of 4, 8, 12 or 16 bp completely eliminated rafA gene expression . In vitro binding studies revealed that the CBS, O1 and O2 sites, can be simultaneously occupied by their cognate proteins . However, no cooperativity between binding of CAP and RafR was detected . EMSA with circularly permuted DNA fragments demonstrated that CAP and RafR proteins bend raf promoter (rafP) DNA by 75 degrees +/- 5 degrees and 95 degrees +/- 5 degrees, respectively, in opposite directions . Among sugar catabolic operons, the compact arrangement of three protein-binding sites, a CBS and two operators bounding the - 35 promoter box, is unique and provides a sensitive and highly efficient device for transcriptional control.

J Anim Sci, 1999 Jan, 77(1), 137 - 47
Manipulation of the type of fat consumed by growing pigs affects plasma and mononuclear cell fatty acid compositions and lymphocyte and phagocyte functions; Thies F et al.; To investigate the immunological effect of feeding pigs different dietary lipids, 3-wk-old, weaned pigs were fed for 40 d on one of five diets, which differed only in the type of oil present (the oil contributed 5% by weight of the diet and the total fat content of the diets was 8% by weight) . The oils used were soybean (control diet), high-oleic sunflower oil (HOSO), sunflower oil (SO), canola oil (CO), and fish oil (FO; rich in long-chain {n-3} polyunsaturared fatty acids) . There were no significant differences in initial or final animal weights, weight gains, or health scores among the groups . There were no significant differences in the concentration of anti-Escherichia coli vaccine antibodies in the gut lumens of pigs fed the different diets . The fatty acid composition of the diet markedly affected the fatty acid composition of the plasma and of mononuclear cells (a mixture of lymphocytes, monocytes, and macrophages) prepared from the blood, lymph nodes, or thymus . The FO feeding resulted in a significant increase in the number of circulating granulocytes . The FO feeding significantly decreased the proportion of phagocytes engaged in uptake of E . coli and decreased the activity of those phagocytes that were active . The proliferation of lymphocytes in cultures of whole blood from pigs fed the HOSO, SO, or FO diets was less than in those from pigs fed the CO diet . Proliferation of lymph node lymphocytes from SO- or FO-fed pigs was less than that from control, CO-, or HOSO-fed pigs . The natural killer cell activity of blood lymphocytes from pigs fed the FO diet was significantly reduced compared with those from pigs fed the CO diet . The concentration of PGE2 in the medium of cultured blood, lymph node, or thymic mononuclear cells was lower if the cells came from pigs fed the FO diet . Thus, the type of oil included in the diet of growing pigs affects the numbers and functional activities of immune cells in different body compartments.

Photochem Photobiol, 1999 Jan, 69(1), 108 - 13
A putative blue-light receptor from Drosophila melanogaster; Okano S et al.; A gene encoding a 62.5 kDa homolog of Drosophila melanogaster photolyase was isolated . Purified recombinant protein contained a flavin adenine dinucleotide chromophore . The recombinant protein did not show photolyase activity for either cyclobutane pyrimidine dimers or 6-4 photoproducts in vitro as well as in vivo in Escherichia coli host cells, suggesting that the protein is not a DNA repair enzyme but a blue-light photoreceptor . Reverse transcription polymerase chain reaction analysis showed that the gene is more expressed in head than in body and that it is more expressed in antennae than in legs, wings and mouth appendages . In a phylogenetic tree of the photolyase family, the Drosophila photolyase homolog is located in a cluster containing 6-4 photolyases and mammalian photolyase homologs, which is only distantly related to the clade of higher plant blue-light photoreceptors . The mammalian photolyase homologs are more closely related to Drosophila 6-4 photolyase than to the Drosophila photolyase homolog, suggesting different roles of the photolyase homologs.

Photochem Photobiol, 1999 Jan, 69(1), 77 - 85
Light activates reduction of methotrexate by NADPH in the ternary complex with Escherichia coli dihydrofolate reductase; Chen YQ et al.; Methotrexate (MTX), a strong inhibitor of dihydrofolate reductase (DHFR), has been widely used for chemotherapy for many types of cancer as well as for juvenile rheumatoid arthritis . It mimics folate substrates and binds tightly to the active site of DHFR, perhaps in a conformation close to the transition state of the folate catalyzed reaction . Absorption, fluorescence and ultrasensitive Raman difference spectroscopies show that light-activated MTX reacts with NADPH in the enzyme active site, producing 5,8-dihydromethotrexate (5,8-dihydro-MTX) and NADP+ . The reaction, which proceeds with a hydride transfer between C4 (pro-R side) of the nicotinamide ring and N5 of the pteridine ring, is similar to that between folate and NADPH except that the hydride is transferred to C6 in this case . Hence, MTX is catalytically competent in its excited state . Most experiments were performed on the Escherichia coli enzyme, but preliminary studies show that the reaction also occurs with human DHFR.

J Appl Microbiol, 1999 Feb, 86(2), 237 - 44
Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor for chick embryos; Krinos C et al.; The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E . coli LP 116 clone expressed the Legionella-MOMP . Compared with the parent E . coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells . Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg . pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile . After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established . The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence . These studies suggest that the 25 kDa MOMP of Leg . pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.

J Appl Microbiol, 1999 Feb, 86(2), 231 - 6
Rapid enumeration of Escherichia coli in oysters by a quantitative PCR-ELISA; Gonzalez I et al.; Direct enumeration of Escherichia coli from oysters was achieved using a polymerase chain reaction (PCR) amplification of the lamB gene coupled with an enzyme-linked immunosorbent assay (ELISA) . Amplified PCR products generated using a digoxigenin-labelled primer were heat denatured before being quantified by an ELISA . A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase antidigoxigenin conjugate . Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying oyster samples containing E . coli in the range 10-10(5) cfu g-1.

J Magn Reson, 1999 Mar, 137(1), 285 - 8
A new multi-quantum version of the HBHA(CBCACO)NH experiment with enhanced sensitivity for partially deuterated samples; Gschwind RM et al.; A new multi-quantum version of the HBHA(CBCACO)NH experiment for partially deuterated protein samples is presented . The method is based on the significant reduction of the proton and carbon relaxation rates due to multi-quantum delays in highly deuterated proteins recently published by our group . The introduction of a multi-quantum period in the coherence transfer pathway of the HBHA(CBCACO)NH experiment yields a dramatic increase of sensitivity-on average 46% with a 75% deuterated sample of the homodimeric 31 kDa E . coli IIAMan domain . Additional resolution in the proton dimension can be achieved by a double time shared approach keeping the 1H single-quantum period at a minimum .

Gynecol Oncol, 1999 Mar, 72(3), 292 - 7
A phase II double-blind randomized study of the simultaneous administration of recombinant human interleukin-6 and recombinant human granulocyte colony-stimulating factor following paclitaxel and carboplatin chemotherapy in patients with advanced epithelial ovarian cancer; Hochster H et al.; PURPOSE: Recombinant human interleukin-6 (rhuIL-6) is a glycosylated cytokine with hematopoietic stimulatory effects . In particular, preclinical studies suggest the agent can stimulate thrombopoiesis, even in conjunction with chemotherapy . We attempted to determine whether higher dose chemotherapy for ovarian cancer was possible given the pharmacologic use of this important growth factor . METHODS: We conducted a randomized, double-blind phase II study of IL-6 plus granulocyte colony-stimulating factor (G-CSF) versus placebo plus G-CSF in combination with a standard chemotherapy regimen . Patients with epithelial ovarian cancer, stages Ic to IV, were eligible . All patients were previously untreated with chemotherapy and had Karnofsky performance status >/=60 . rhuIL-6 (Escherichia coli, SDZ ILS 969) 1.0 micrograms/kg or placebo was given subcutaneously on days 2-8 every cycle together with G-CSF 5.0 micrograms/kg subcutaneously days 2-15, following administration of paclitaxel 175 mg/m2 as a 3-h infusion and carboplatin given to a desired AUC of 7.5 on day 1 every 21 days . RESULTS: Fifty patients were entered in this study, although the study was temporarily suspended by the FDA in midstudy over manufacturing concerns . Therefore, 37 patients were evaluable for efficacy of growth factor; 19 patients received placebo plus G-CSF and 18 rhIL-6 plus G-CSF . There was no difference in prognostic variables between these two groups . Platelet nadirs were lower in the first cycle for the placebo group (P = 0.004, Wilcoxon sum-rank test) but not in other cycles . There was no statistically significant difference in cycle treatment delays, carboplatin dose delivered, number of patients with grade 4 thrombocytopenia, or platelet transfusion . Nonetheless, the trend of the data favored IL-6 in all cases . CONCLUSIONS: This study demonstrated a minimal effect (statistically significant in the first cycle only) on thrombopoiesis in women undergoing paclitaxel and carboplatin therapy of ovarian cancer . No clinically significant effect on actual chemotherapy delivery was demonstrated, however . Future studies, if warranted, to ameliorate thrombocytopenia should be carried out with regimens producing even greater thrombocytopenia than the current regimen in the control arm .

Am J Hum Genet, 1999 Mar, 64(3), 698 - 705
Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames; Stallmeyer B et al.; A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase . The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway . The involved enzymes exhibit homologies, even between bacteria and humans . We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase . This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo . The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides . In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected . In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.

Biochemistry, 1999 Mar 2, 38(9), 2842 - 8
Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center; Sun L et al.; Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate . The rate-determining step for the wild-type enzyme is pH dependent . At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate . When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release . The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme . The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme . Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme . These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E . coli alkaline phosphatase.

Biochemistry, 1999 Mar 2, 38(9), 2669 - 78
Identification of two important heme site residues (cysteine 75 and histidine 77) in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum; Shelver D et al.; The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands . Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron . In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA . However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA . Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription . On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA . These results imply a ligand switch from Cys75 to His77 upon reduction of CooA . In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism . Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

J Chromatogr B Biomed Sci Appl, 1999 Jan 22, 721(2), 187 - 95
Preparation of poly(epsilon-lysine) adsorbents and application to selective removal of lipopolysaccharides; Hirayama C et al.; To remove endotoxins (lipopolysaccharides; LPS) from cell products used as drugs, water-insoluble poly(epsilon-lysine) (PL) particles were prepared by cross-linking with PL originating from Streptomyces albulus and chloromethyloxirane (CMO) . The apparent pKa (pK(a,app)) and the anion-exchange capacity of the particles were easily adjusted by changing the PL ratio and the CMO ratio . The higher the pK(a,app), the greater the LPS-adsorption capacity of the particles . On the other hand, when the PL ratio (in the particles) increased to 75 unit-mol% or higher, the adsorption of bovine serum albumin by the particles also increased, but decreased with increasing ionic strength of the buffer to mu = 0.2 or higher . The adsorption of gamma-globulin increased with decreasing PL ratio to 65 unit-mol% or lower . As a result, when the PL ratio was 70 unit-mol% and the pK(a,app) was 6.7, the PL/CMO particles selectively removed LPS from various protein solutions that were naturally contaminated with LPS, at pH 6.0 and mu = 0.05.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 206 - 9
Cloning and sequencing of beta-mannosidase gene from Aspergillus aculeatus no . F-50; Takada G et al.; The manB gene, coding for a unique beta-mannosidase (MANB) of Aspergillus aculeatus, was cloned from genomic and cDNA libraries, and sequenced . The gene consists of 2,811 bp encoding a polypeptide of 937 amino acid residues with a molecular mass of 104,214 Da . The A . aculeatus MANB shared amino acid sequence identity with MANB of human (24%), goat (24%), bovine (24%), and Caenorhabditis elegans (22%) . When the A . aculeatus MANB was compared with other related enzymes, a Glu residue corresponding to the active site identified by the Escherichia coli beta-galactosidase and the human beta-guclonidase was conserved . This is the first fungal gene that encodes MANB.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 180 - 3
Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system; Kanemori Y et al.; Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose . The A . oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A . oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase . To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A . nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter . Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose . The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III . The sequence between -233 to -181 containing Region II had no effect on the expression . These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.

Anal Chem, 1999 Feb 15, 71(4), 763 - 8
Green fluorescent protein in the design of a living biosensing system for L-arabinose; Shetty RS et al.; Analysis of monosaccharides is typically performed using analytical systems that involve a separation step followed by a detection step . The separation step is usually necessary because of the high degree of structural similarity between different monosaccharides . A novel sensing system for monosaccharides is described here in which living bacteria were designed to detect a model monosaccharide, L-arabinose, without the need for a separation step . In such sensing systems, analytes are detected by employing the selective recognition properties found in certain bacterial proteins . These systems are designed so that a reporter protein is expressed by the bacteria in response to the analyte . The concentration of the analyte can be related to the signal generated by the reporter protein . In the sensing system described here, the green fluorescent protein (GFP) was used as the reporter protein . L-Arabinose concentrations can be determined by monitoring the fluorescence emitted by the bacteria at 509 nm after excitation of GFP at 395 nm . The system can detect L-arabinose at concentrations as low as 5 x 10(-7) M and is selective over D-arabinose, the stereoisomer of the analyte, as well as over a variety of pentose and hexose sugars.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2503 - 7
Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins; Levay K et al.; The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization . To detect the protein targets of Gbeta5 in vivo, we have isolated a native Gbeta5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gbeta5 as the regulator of G protein signaling (RGS) protein, RGS7 . Here we show that complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins . The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies . The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits . Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained . Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1 . The interaction of Gbeta5 with RGS7 blocked the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2339 - 44
A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase; Sarkissian CN et al.; Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development . Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease . However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic . Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC 4.3.1.5) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC 1.14.16.1), which is deficient in PKU . PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite . We describe (i) an efficient recombinant approach to produce PAL enzyme, (ii) testing of PAL in orthologous N-ethyl-N'-nitrosourea (ENU) mutant mouse strains with HPA, and (iii) proofs of principle (PAL reduces HPA)-both pharmacologic (with a clear dose-response effect vs . HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs . HPA) . These findings open another way to facilitate treatment of this classic genetic disease.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2141 - 6
Human deafness dystonia syndrome is a mitochondrial disease; Koehler CM et al.; The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function . We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space . Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p . DDP is most similar to Tim8p . Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p . The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial protein-import system.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2122 - 8
The small GTPase RalA targets filamin to induce filopodia; Ohta Y et al.; The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin . Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton . Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA's effects, which, unlike Cdc42, are Rac independent . RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response . RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1953 - 8
Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces; Briesewitz R et al.; A general strategy is described for improving the binding properties of small-molecule ligands to protein targets . A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein . When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event . We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide . This approach provides a way to modulate the potency and specificity of biologically active compounds.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1941 - 6
Mg-chelatase of tobacco: the role of the subunit CHL D in the chelation step of protoporphyrin IX; Grafe S et al.; The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway . The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H) . We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H . We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein-protein interaction and enzyme activity . Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity . In addition, it could be demonstrated that CHL D is capable of forming homodimers . Moreover, it interacted with both CHL I and CHL H . Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1915 - 20
Native display of complete foreign protein domains on the surface of hepatitis B virus capsids; Kratz PA et al.; The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence . Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes . The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence . Because of its central position, however, any c/e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation . According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces . We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently . Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded . We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP . Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles . Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 1858 - 62
Proteasome-dependent degradation of the human estrogen receptor; Nawaz Z et al.; In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives . The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease . In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner . The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway . In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro . Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER . The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor beta proteins.

Plant Cell Physiol, 1998 Dec, 39(12), 1375 - 9
Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression; Yamada K et al.; Partial amino acid sequences of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were used to identify a full-length cDNA from apple fruit . This clone consisted of 1,433 bp containing an open reading frame of 1,137 bp that could code for a polypeptide with 379 amino acids . To our knowledge, this is the first report about cloning of NAD-SDH cDNA from a plant source . The deduced amino acids from cDNA revealed 43.7% identity to human NAD-SDH . The activity of this enzyme to convert sorbitol to fructose with the reduction of NAD was certified by the fusion protein of this clone expressed in Escherichia coli . Northern blot analysis showed that the mRNA was expressed in matured apple fruit.

Plant Cell Physiol, 1998 Dec, 39(12), 1337 - 41
An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11; Nakai T et al.; A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E) . Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase . The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes . These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose . Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized . The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity . We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.

Plant Cell Physiol, 1998 Dec, 39(12), 1269 - 80
Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.); Kaminaka H et al.; We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.) . The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform . The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo . Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol . mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues . Southern blot analysis showed that the RGRC2 gene exists as a single copy gene . Here, we have also isolated a genomic clone completely corresponding to RGRC2 . The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon . From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity . The expression of RGRC2 was strongly induced by all these treatments . We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.

Plant Cell Physiol, 1998 Dec, 39(12), 1258 - 68
Histidine-containing phosphotransfer (HPt) signal transducers implicated in His-to-Asp phosphorelay in Arabidopsis; Suzuki T et al.; His to Asp phosphorelay signal transduction mechanisms involve three types of widespread signaling components: a sensor His-kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) domain . In Arabidopsis, several sensor His-kinases have recently been discovered (e.g., ETR1 and CKI1) through extensive genetic studies . Furthermore, a recent search for response regulators in this higher plant revealed that it possesses a group of response regulators (ARR-series), each of which exhibits the phospho-accepting receiver function . However, no signal transducer containing the HPt domain has been reported . Here we identify three distinct Arabidopsis genes (AHP1 to AHP3), each encoding a signal transducer containing a HPt domain . Both in vivo and in vitro evidence that each AHP can function as a phospho-transmitting HPt domain with an active histidine site was obtained by employing both the Escherichia coli and yeast His-Asp phosphorelay systems . It was demonstrated that AHP1 exhibits in vivo ability to complement a mutational lesion of the yeast YPD1 gene, encoding a typical HPt domain involved in an osmosensing signal transduction . It was also demonstrated that AHPs can interact in vitro with ARRs through the His-Asp phosphotransfer reaction . It was thus suggested that the uncovered sensors-AHPs-ARRs lineups may play important roles in propagating environmental stimuli through the multistep His-Asp phosphorelay in Arabidopsis.

Genetics, 1999 Mar, 151(3), 929 - 34
Identification of RNase T as a high-copy suppressor of the UV sensitivity associated with single-strand DNA exonuclease deficiency in Escherichia coli; Viswanathan M et al.; There are three known single-strand DNA-specific exonucleases in Escherichia coli: RecJ, exonuclease I (ExoI), and exonuclease VII (ExoVII) . E . coli that are deficient in all three exonucleases are abnormally sensitive to UV irradiation, most likely because of their inability to repair lesions that block replication . We have performed an iterative screen to uncover genes capable of ameliorating the UV repair defect of xonA (ExoI-) xseA (ExoVII-) recJ triple mutants . In this screen, exonuclease-deficient cells were transformed with a high-copy E . coli genomic library and then irradiated; plasmids harvested from surviving cells were used to seed subsequent rounds of transformation and selection . After several rounds of selection, multiple plasmids containing the rnt gene, which encodes RNase T, were found . An rnt plasmid increased the UV resistance of a xonA xseA recJ mutant and uvrA and uvrC mutants; however, it did not alter the survival of xseA recJ or recA mutants . RNase T also has amino acid sequence similarity to other 3' DNA exonucleases, including ExoI . These results suggest that RNase T may possess a 3' DNase activity capable of substituting for ExoI in the recombinational repair of UV-induced lesions.

Appl Environ Microbiol, 1999 Mar, 65(3), 1320 - 4
Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli; Chen ZY et al.; The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector . The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro . This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme . The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species . It also inhibited A . flavus and F . moniliforme simultaneously when they were tested together . The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops.

Appl Environ Microbiol, 1999 Mar, 65(3), 929 - 35
Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism; Carsolio C et al.; The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42 . We constructed several transgenic T . harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants . The level of extracellular endochitinase activity when T . harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity . The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type . Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R . solani or Sclerotium rolfsii were observed in greenhouse experiments.

J Struct Biol, 1998 Dec 15, 124(2-3), 257 - 75
Chemotaxis receptors: a progress report on structure and function; Mowbray SL et al.; Recent biochemical and structural studies have provided many new insights into the structure and function of bacterial chemoreceptors . Aspects of their ligand binding, conformational changes, and interactions with other members of the signaling pathway are being defined at the structural level . It is anticipated that the combined effort will soon provide a detailed, unified view of an entire response system .

J Struct Biol, 1998 Dec 15, 124(2-3), 151 - 63
Crystal structure determination of Escherichia coli ClpP starting from an EM-derived mask; Wang J et al.; Large ATP-dependent proteolytic complexes carry out the majority of intracellular proteolysis . To begin to understand the function of these proteases at a structural level, we have combined the information from a number of biophysical techniques such as electron microscopy (EM), small-angle scattering, and x-ray crystallography . In this study, we exploited the inherent symmetry of Escherichia coli ClpP, the proteolytic component of the ClpAP/XP ATP-dependent protease, to determine its x-ray crystal structure to 2.3-A resolution starting with a phase set derived from a low-resolution mask obtained from EM and small-angle x-ray scattering analysis . Sevenfold and 14-fold noncrystallographic symmetry averaging facilitated phase extension beyond 20 A and in combination with mask redetermination and matrix refinement was sufficient for completely determining the structure . The structure of ClpP is a homo-tetradecamer composed of two heptameric rings enclosing a cavity of approximately 50 A in diameter that compartmentalizes the 14 serine proteolytic active sites . Comparison of the ClpP structure with those of the 20S proteasome and HslV reveals a striking example of evolutionary convergence, despite them being unrelated in sequence and fold . Moreover, similarity in their overall architecture suggests a common model for their action .

J Struct Biol, 1998 Dec 15, 124(2-3), 142 - 50
The ribosome-structure and functional ligand-binding experiments using cryo-electron microscopy; Frank J; Cryo-electron microscopy has greatly advanced our understanding of the basic steps of protein synthesis in the bacterial ribosome . This article gives an overview of what has been achieved so far . Through three-dimensional visualization of complexes that represent the ribosome in defined binding states, locations were derived for the tRNA in A, P, and E sites, as well as the elongation factors . In addition, the pathways of messenger RNA and the exiting polypeptide chain could be inferred .

J Struct Biol, 1998 Dec 15, 124(2-3), 129 - 41
GroEL/GroES: structure and function of a two-stroke folding machine; Xu Z et al.; Recent structural and functional studies have greatly advanced our understanding of the mechanism by which chaperonins (Cpn60) mediate protein folding, the final step in the accurate expression of genetic information . Escherichia coli GroEL has a symmetric double-toroid architecture, which binds nonnative polypeptide substrates on the hydrophobic walls of its central cavity . The asymmetric binding of ATP and cochaperonin GroES to GroEL triggers a major conformational change in the cis ring, creating an enlarged chamber into which the bound nonnative polypeptide is released . The structural changes that create the cis assembly also change the lining of the cavity wall from hydrophobic to hydrophilic, conducive to folding into the native state . ATP hydrolysis in the cis ring weakens it and primes the release of products . When ATP and GroES bind to the trans ring, it forms a stronger assembly, which disassembles the cis complex through negative cooperativity between rings . The opposing function of the two rings operates as if the system had two cylinders, one expelling the products of the reaction as the other loads up the reactants . One cycle of the reaction gives the polypeptide about 15 s to fold at the cost of seven ATP molecules . For some proteins, several cycles of GroEL assistance may be needed in order to achieve their native states .

J Struct Biol, 1998 Dec 15, 124(2-3), 115 - 22
Insights into Escherichia coli RNA polymerase structure from a combination of x-ray and electron crystallography; Darst SA et al.; Our goal is to understand the mechanism of transcription and its regulation . Determining structures of RNA polymerase and transcription complexes is an essential step . Because of their large size and complexity, determination of these structures will require a combination of electron microscopy, biophysical methods, and biochemical methods to identify functionally and structurally relevant subassemblies and domains and x-ray crystallography to determine high-resolution structures of RNA polymerase components and accessory factors . We recently solved the 2.5-A crystal structure of the Escherichia coli RNA polymerase alpha subunit N-terminal domain, which is the first high-resolution structure of a core component required for RNA polymerase assembly and basal transcription . This structure, combined with a new 19-A resolution structure determined by cryo-electron microscopy of helical crystals of E . coli core RNAP embedded in vitreous ice, leads to a model for the organization of the RNAP subunits .

Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 657 - 62
A cellulose-binding domain-fused recombinant human T cell connective tissue-activating peptide-III manifests heparanase activity; Rechter M et al.; The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts . However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and degrade heparan sulfate . This suggests that CTAP-III may also function as a proinflammatory mediator . We have successfully cloned CTAP-III from a lambdagt11 cDNA library of PHA-activated human CD4(+) T cells and produced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety . This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, naturally produced extracellular matrix . We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III detected the CTAP-III molecule in human T cells, polymorphonuclear leukocytes, and placental extracts . Thus, our study provides tools to examine further immune cell behavior in inflamed sites rich with extracellular moieties and proinflammatory mediators .

Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 412 - 5
cDNA cloning and expression of acutin; Pan H et al.; Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies . Its first 15 N-terminal amino acid residues sequence <VIGGVECDINEHRFL> was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR . Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time . The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms . Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200 . The recombinant acutin has been expressed in E . coli and purified by affinity column . The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase .

Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 307 - 11
Roles of the four cysteine residues in the function of the integral inner membrane Hg2+-binding protein, MerC; Sahlman L et al.; The roles of the four cysteine residues of the integral inner membrane Hg2+-binding protein, MerC, have been examined using site-directed mutagenesis . Residues Cys-22 and Cys-25 have previously been predicted to lie within the membrane . Substitution of each of these residues in turn with alanine resulted in complete abolition of specific Hg2+ uptake by vesicles . In contrast, substitution by alanine of the other two cysteine residues, Cys-127 and Cys-132, predicted to lie with within a C-terminal cytoplasmic tail, did not significantly affect Hg2+ uptake . Since previous results indicated that native MerC tends to form intermolecular disulfide-bonded dimers, the effects of these substitutions on dimer formation were also examined . Only the Cys-127 and Cys-132 variants spontaneously formed significant amounts of disulfide-bonded dimer . Further experiments using copper-1,10-phenanthroline indicated that each variant with an unpaired cysteine residue was more susceptible to dimer formation than native MerC .

Protein Expr Purif . 1999 Mar;15(2):IV.
Papers to Appear in Forthcoming Issues; Affinity purification of recombinant interferon-alpha on a mimetic ligand adsorbent; Recombinant Gene Products Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, IndiaA method for improved refolding and purification of recombinant human interferon-alpha (rh-IFN-alpha) from inclusion bodies is described . The optimal conditions of refolding were obtained by the addition of 0.5 M l-arginine to the refolding buffer . The rh-IFN-alpha was purified to near homogeneity utilizing a single-step chromatography on a mimetic dye-ligand matrix . Improved refolding, coupled to a single-column affinity purification strategy, resulted in a 10-fold increase in the yield of rh-IFN-alpha . This single-step purification protocol yielded approximately 50 mg of purified rh-IFN-alpha from 1 liter of shake flask culture . The rh-IFN-alpha prepared by this protocol was found to be essentially monomeric based on HPLC gel filtration and nonreducing SDS-PAGE . It had a specific activity of approximately 2.8 x 10(8) IU/mg, measured as inhibition of cytopathic effect of encephalomyocarditis virus on A549 human lung carcinoma cells .

Protein Expr Purif, 1999 Mar, 15(2), 228 - 35
Overexpression, oxidative refolding, and zinc binding of recombinant forms of the murine S100 protein MRP14 (S100A9); Raftery MJ et al.; Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system . The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da) . Thrombin digestion of fusion protein was expected at a cloned thrombin consensus sequence ( . LVPRGS . ) located between glutathione S-transferase and mMRP14 . Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/Western blotting revealed two immunoreactive cleavage products with molecular weights around 13, 000 . Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da . The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da) . Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence ( . NNPRGH . ) located close to the C-terminus . The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) with a calculated mass of 11,918.6 Da . Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14 . Native mMRP14 contains one intramolecular disulfide bond between Cys79 and Cys90 . The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form . MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding .

Protein Expr Purif, 1999 Mar, 15(2), 213 - 20
Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease; D'alessio KJ et al.; We have constructed and optimized a high yielding Escherichia coli expression system to produce glycosylation-free human procathepsin K and have developed conditions for refolding this enzyme . Recombinant human procathepsin K (EC 3.4.22.38) was expressed in E . coli, refolded from inclusion bodies, and further purified by Superdex 75 size-exclusion chromatography . Purified procathepsin K had a {MH}+ of 35,063 Da which is in agreement with the predicted mass of the construct . Amino-terminal sequence analysis matched the predicted sequence with no secondary sequence detected . Purified procathepsin K activated under autocatalytic conditions to a final specific activity of 23 micromol 7-amido-4-methylcoumarin liberated/min/mg of enzyme using the fluorescent peptide substrate benzyloxycarbonyl-phenylalanine-arginine-7-amido-4-methylcoumarin . This expression and refolding procedure yielded 50 mg of purified, glycosylation-free human procathepsin K from 1 liter of E . coli cell culture and enabled the determination of the structure of human procathepsin K at 2.6 A resolution .

Protein Expr Purif, 1999 Mar, 15(2), 202 - 6
Expression and purification of full-length human Bax alpha; Montessuit S et al.; Bax is a proapoptotic ion channel forming protein of the Bcl-2 family . In cells the protein is found in the cytosol and in the mitochondria membrane where it presumably is involved during apoptosis in disruption of the mitochondrial membrane potential and release of cytochrome c . The protein has a hydrophobic domain at the C-terminus, which renders it a limited solubility . Thus, all studies on recombinant Bax has so far been performed on C-terminal truncated protein . We have expressed and purified the full-length human Bax alpha . The protein was expressed with a His tag at the N-terminus and purified by affinity chromatography on Ni-NTA-agarose followed by ion-exchange chromatography on Q-Sepharose . The protein was more than 98% pure on SDS-PAGE and in the presence of 1% (w/v) octyl glucoside it could be concentrated up to 0.5 mg/ml . Full-length Bax was 25-fold more efficient, compared to C-terminal truncated Bax, in forming ion channels and trigger carboxyfluorescein release from liposomes .

Protein Expr Purif, 1999 Mar, 15(2), 178 - 87
A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate; Skorey KI et al.; The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification . In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H . pomatia . No enzyme was retained without vanadate or with arsenate or phosphate . Arylsulfatase was eluted from the column matrix by removing the vanadate to less than 50 microM with buffer containing EDTA to chelate the vanadate . Escherichia coli alkaline phosphatase and potato acid phosphatase, two enzymes which are inhibited by vanadate but not by the vanadate-TEE complex, were not retained by the immobilized TEE under any conditions used . The sulfatase activity was completely separated from contaminating glucuronidase activity present in the crude enzyme extracts . The Ki for the immobilized vanadate-TEE system was found to be 5.0 x 10(-7) M with a capacity of 25 mg/ml swollen gel . A purification of greater than 40-fold from the lyophilized powder of H . pomatia (Sigma Type H-5) was achieved using this technique . The Ki/Keq of other phenols with vanadate were determined in a 96-well plate format as an example of a rapid screening technique that could be extended to other phosphoryl and sulfuryl-transfer enzyme classes .

Arch Biochem Biophys, 1999 Mar 1, 363(1), 182 - 90
Mutation of conserved polar residues in the transmembrane domain of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; The pyridine nucleotide transhydrogenase carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD+ and NADP+ . Previous workers (E . Holmberg et al . Biochemistry 33, 7691-7700, 1994; N . A . Glavas et al . Biochemistry 34, 7694-7702, 1995) had examined the role in proton translocation of conserved charged residues in the transmembrane domain . This study was extended to examine the role of conserved polar residues of the transmembrane domain . Site-directed mutagenesis of these residues did not produce major effects on hydride transfer or proton translocation activities except in the case of betaAsn222 . Most mutants of this residue were drastically impaired in these activities . Three phenotypes were recognized . In betaN222C both activities were impaired maximally by 70% . The retention of proton translocation indicated that betaAsn222 was not directly involved in proton translocation . In betaN222H both activities were drastically reduced . Binding of NADP+ but not of NADPH was impaired . In betaN222R, by contrast, NADP+ remained tightly bound to the mutant transhydrogenase . It is concluded that betaAsn222, located in a transmembrane alpha-helix, is part of the conformational pathway by which NADP(H) binding, which occurs outside of the transmembrane domain, is coupled to proton translocation . Some nonconserved or semiconserved polar residues of the transmembrane domain were also examined by site-directed mutagenesis . Interaction of betaGlu124 with the proton translocation pathway is proposed .

Arch Biochem Biophys, 1999 Mar 1, 363(1), 163 - 70
Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction; Lin A et al.; Glycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis . As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an alpha-1,4-glycan of around 10 residues attached to Tyr194 . Whether this self-glucosylation reaction is inter- or intramolecular has been debated . We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-1 to address this question . Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1-2% of the self-glucosylating activity of wild-type enzyme . Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation . The Y194F mutant was able to glucosylate the K85Q mutant . However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins . The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 microM . We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types . The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction .

Arch Biochem Biophys, 1999 Mar 1, 363(1), 19 - 26
Reduction of peroxides and dinitrobenzenes by Mycobacterium tuberculosis thioredoxin and thioredoxin reductase; Zhang Z et al.; The thioredoxin (Trx) and thioredoxin reductase (TR) of Mycobacterium tuberculosis have been expressed in Escherichia coli and shown to reduce peroxides and dinitrobenzenes . The reduction of H2O2 requires both Trx and TR and is more efficient under anaerobic than aerobic conditions . In contrast, cumene hydroperoxide is reduced to cumyl alcohol and acetophenone in a process that requires NADPH and TR but not Trx . Cumene hydroperoxide reduction is partially inhibited by chelation of trace metals in the medium . The reduction of cumene hydroperoxide by TR is more effective under anaerobic than aerobic conditions due to a competing oxidase reaction in which electrons are transferred from TR to O2 . Under anaerobic conditions, dinitrobenzenes also serve as electron acceptors and are reduced by TR to nitroanilines, but the enzyme does not reduce mononitrobenzenes or mononitroimidazoles such as metronidazole . The reductive activity of the Trx-TR system may modify the antioxidant defenses of M . tuberculosis .

Protein Sci, 1999 Feb, 8(2), 307 - 17
Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase; Yao X et al.; The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis . In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification . Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP . Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin . Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA . The determinants of protein-protein interaction specificity in this system are unknown . The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined . Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein . These structural changes may be important for regulating specific protein-protein interactions . Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein . The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.

J Bacteriol, 1999 Mar, 181(5), 1694 - 7
The level of expression of the minor pilin subunit, CooD, determines the number of CS1 pili assembled on the cell surface of Escherichia coli; Sakellaris H et al.; CooD, the minor subunit of CS1 pili of enterotoxigenic Escherichia coli, is essential for the assembly of stable, functional pili . We previously proposed that CooD is a rate-limiting initiator of CS1 pilus assembly and predicted that the level of CooD expression should therefore determine the number of CS1 pili assembled on the cell surface . In this study, we confirm that CooD is required for the initiation of pilus assembly rather than for the stabilization of pili after they are assembled by demonstrating that specific modulation of cooD expression also modulates the number of CS1 pili on bacterial cells.

J Bacteriol, 1999 Mar, 181(5), 1636 - 42
Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines; Park OK et al.; The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase {DAHPS(Phe)} from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism . DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate . The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state . The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent . Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation . Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation . In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+ . Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme . Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack . Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues . All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor . A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.

J Bacteriol, 1999 Mar, 181(5), 1617 - 22
Inhibition of translation and cell growth by minigene expression; Tenson T et al.; A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest . Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids . Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest . Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis . Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene . Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.

J Bacteriol, 1999 Mar, 181(5), 1603 - 9
CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock; Wang N et al.; Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible . The cspI gene is located at 35.2 min on the E . coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively . Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible . The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C . The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock . Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C . Taking these results together, E . coli possesses a total of four cold shock-inducible proteins in the CspA family . Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).

J Bacteriol, 1999 Mar, 181(5), 1576 - 84
Direct selection for mutators in Escherichia coli; Miller JH et al.; We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium . The plate selection is based on using two different markers whose reversion is enhanced by a given mutator . Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs . Because two successive mutational events are required, mutator cells are favored to generate full-size colonies . Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies . We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step . Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD) . We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population . Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations . We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.

J Bacteriol, 1999 Mar, 181(5), 1544 - 54
The Bradyrhizobium japonicum nolA gene encodes three functionally distinct proteins; Loh J et al.; Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons . Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins . Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain . Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites . Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted . Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed . Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B . japonicum . Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B . japonicum nod genes . The expression of both NolA2 and NolA3 requires the presence of NolA1 . NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578.

J Bacteriol, 1999 Mar, 181(5), 1537 - 43
Protein ProQ influences osmotic activation of compatible solute transporter ProP in Escherichia coli K-12; Kunte HJ et al.; ProP is an osmoregulatory compatible solute transporter in Escherichia coli K-12 . Mutation proQ220::Tn5 decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter proP transcription or the ProP protein level . Allele proQ220::Tn5 was isolated, and the proQ sequence was determined . Locus proQ is upstream from prc (tsp) at 41.2 centisomes on the genetic map . The proQ220::Tn5 and prc phenotypes were different, however . Gene proQ is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids) . The insertion of PCR-amplified proQ into vector pBAD24 produced a plasmid containing the wild-type proQ open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa . Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in proQ+ bacteria but not in proQ220::Tn5 bacteria . ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.

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