|
|
|
|
|
|
Protein Sci, 1997 Aug, 6(8), 1777 - 82 Multiple conformations of a human interleukin-3 variant; Feng Y et al.; Interleukin-3 (IL-3) is a cytokine that stimulates the proliferation and differentiation of hematopoietic cells . The hyperactive hIL-3 variant SC-55494 was shown to have at least two major conformations by high-resolution NMR spectroscopy . Mutants of SC-55494 were constructed in which alanine was substituted for proline in order to test the hypothesis that proline cis-trans isomerization is the source of the observed conformational heterogeneity, as well as to evaluate the effect of prolyl peptide bond configuration on biological activity . NMR spectra of four single proline-to-alamine mutants (P30A, P31A, P33A, and P37A) retain doubled resonances, while spectra of the double mutant P30A/P31A and the quadruple mutant P30A/P31A/P33A/ P37A are substantially free of heterogeneity . These observations suggest that the two major conformations in SC-55494 correspond to cis and trans isomers of either or both of the R29-P30 and P30-P31 peptide bonds . All six mutants had somewhat lower cell proliferative activity than SC-55494, with relative activities ranging from 40 to 80% . The P37A mutant has a binding affinity to the low-affinity IL-3 receptor alpha-subunit statistically equivalent to SC-55494, while P30A, P31A, and P33A each had about two-fold decreases, and P30A/P31A and P30A/P31A/P33A/P37A had four-fold decreases . These findings suggest an important role for the cis configuration of either or both of the R29-P30 and P30-P31 peptide bonds in IL-3 for optimal interaction with the receptor alpha-subunit. Protein Sci, 1997 Aug, 6(8), 1746 - 55 Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein; Smith VF et al.; Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity . To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E . coli on the oligopeptide-binding protein . Similar studies performed on the maltose-binding and galactose-binding proteins revealed centrally positioned binding frames of approximately 160 aminoacyl residues . The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein . We propose modes of binding to account for the data. Protein Sci, 1997 Aug, 6(8), 1653 - 60 The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization; Wingfield PT et al.; The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41 . The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli . These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein . Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure . SIV gp41 containing a double cysteine mutation was crystallized . The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry. Mol Endocrinol, 1997 Aug, 11(9), 1256 - 65 A 6-kb promoter fragment mimics in transgenic mice the prostate-specific and androgen-regulated expression of the endogenous prostate-specific antigen gene in humans; Cleutjens KB et al.; Prostate-specific antigen (PSA) is a kallikrein-like serine protease, which is almost exclusively synthesized in the luminal epithelial cells of the human prostate . PSA expression is androgen regulated . Previously, we characterized in vitro the proximal promoter, and a strong enhancer region, approximately 4 kb upstream of the PSA gene . Both regions are needed for high, androgen-regulated activity of the PSA promoter in LNCaP cells . The goal of the present study is the in vivo characterization of the PSA promoter . Three transgenic mouse lines carrying the Escherichia coli LacZ gene, driven by the 632-bp proximal PSA promoter, and three lines with LacZ, driven by the 6-kb PSA promoter, were generated . Expression of the LacZ reporter gene was analyzed in a large series of tissues . Transgene expression could not be demonstrated in any of the transgenic animals carrying the proximal PSA promoter . All three lines carrying the 6-kb PSA promoter showed lateral prostate-specific beta-galactosidase activity . Transgene expression was undetectable until 8 weeks after birth . Upon castration, beta-galactosidase activity rapidly declined . It could be restored by subsequent androgen administration . A search for mouse PSA-related kallikrein genes expressed in the prostate led to the identification of mGK22, which was previously demonstrated to be expressed in the submandibular salivary gland . Therefore, the 6-kb PSA-LacZ transgene followed the expression pattern of the PSA gene in humans, which is almost completely prostate-specific, rather than that of mGK22 in mice . In conclusion, the 6-kb promoter fragment appears to contain most, if not all, information for androgen regulation and prostate specificity of the PSA gene. J Med Chem, 1997 Aug 1, 40(16), 2502 - 24 Protein structure-based design, synthesis, and biological evaluation of 5-thia-2,6-diamino-4(3H)-oxopyrimidines: potent inhibitors of glycinamide ribonucleotide transformylase with potent cell growth inhibition; Varney MD et al.; The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described . The compounds were designed using the X-ray crystal structure of human GART . The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20 . The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes . The compounds were found to have human GART KiS ranging from 30 microM to 2 nM . The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway . The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety . Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity. Am J Vet Res, 1997 Aug, 58(8), 910 - 4 Use of jugular venous blood, compared with mixed venous blood, for measurement of venous oxygenation indices in a porcine model of endotoxic shock; Wohl JS et al.; OBJECTIVE: To evaluate the reliability of oxygen saturation and oxygen content values measured from jugular venous blood in estimating values measured from mixed venous blood during endotoxic shock . ANIMALS: 14 random-bred 10- to 15-kg Yorkshire pigs . PROCEDURE: 60 pairs of heparinized blood samples were simultaneously collected from the pulmonary artery and right jugular vein during an independent study, using a porcine model of endotoxic shock . Endotoxic shock was induced by infusion of Escherichia coli endotoxin . Eighteen of the sample pairs were obtained from pigs prior to infusion of endotoxin or from control pigs . Oxygen saturation and venous oxygen content were measured by direct oximetry . Analysis of bias and precision was used to compare jugular venous blood values with values obtained from mixed venous blood . Samples from endotoxemic pigs were subclassified on the basis of abnormal states of global oxygen imbalance associated with septic shock . RESULTS: Indices of venous oxygenation measured from jugular venous blood were an imprecise method of estimating values measured from mixed venous blood . There was no significant difference in bias between nonendotoxemic and endotoxemic pigs, regardless of abnormal hemodynamic states . CONCLUSION: Jugular venous blood oxygen saturation and oxygen content values should not be used to assess global oxygen transport during endotoxic shock. Nat Biotechnol, 1997 Aug, 15(8), 784 - 8 Drug metabolism by Escherichia coli expressing human cytochromes P450; Parikh A et al.; The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications . Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins . We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second . Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450 . Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system . These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors. J Nucl Med, 1997 Aug, 38(8), 1316 - 22 Localization of radiolabeled chemotactic peptide at focal sites of Escherichia coli infection in rabbits: evidence for a receptor-specific mechanism; Babich JW et al.; The infection imaging properties of a high-affinity 99mTc-labeled chemotactic peptide receptor agonist (N-formyl-methionyl-leucyl-phenylalanine-lysine; N-For-MLFK) were compared with a low-affinity agonist (N-Acetyl-MLFK; N-Ac-MLFK), a moderate-affinity antagonist (N-isobutyloxycarbonyl-MLFK; N-IBoc-MLFK) and non-specific inflammation imaging agents . METHODS: All peptides were prepared by solid-phase methods and purified by high-performance liquid chromatography . The products were assayed in vitro for N-formyl-methionyl-leucyl-phenylalanine receptor binding and superoxide production . Three types of studies were performed in rabbits with Escherichia coli infection: (Study A) Four groups of six animals were coinjected with 99mTc-N-For-MLFK-hydrazinonicotinamide (N-For-MLFK-HYNIC) plus 111In-immunoglobulin G, 111In-red blood cells or 111In-diethylene triamine pentaacetic acid . (Study B) Three groups of six rabbits were coinjected with 111In-leukocytes plus 99mTc-N-For-MLFK-HYNIC, 99mTc-N-Ac-MLFK-HYNIC or 99mTc-N-IBoc-MLFK-HYNIC . (Study C) Two groups of six rabbits were injected with 99mTc-N-For-MLFK-HYNIC and 111In-leukocytes with and without an excess of antagonist . In all three studies, the radiopharmaceuticals were injected 24 hr after infection and dual photon (99mTc and 111In) gamma camera images were acquired at 2-3 and 16-18 hr later . Target-to-background (T/B) ratios were calculated for regions of interest drawn over the infected and contralateral normal tissue . RESULTS: N-For-MLFK, N-Ac-MLFK and N-IBoc-MLFK had EC50s for receptor binding of 2.0, 830 and 150 nM, respectively . The corresponding EC50s for superoxide production were 20.0, approximately 10(3) and > 10(4) . Study A demonstrated that the T/B for 99mTc-N-For-MLFK-HYNIC was higher than for any of the nonspecific imaging agents (p < 0.001), and 111In-immunoglobulin G had a higher T/B ratio than 111In-diethylenetriamine pentaacetic acid (p < 0.01) or 111In-red blood cells (p = NS) . Study B showed that 99mTc-N-For-MLFK-HYNIC had a higher T/B ratio than the other peptides (p < 0.001) . 111In-leukocytes and 99mTc-N-IBoc-MLFK-HYNIC had comparable T/B ratios, which were higher than for 99mTc-N-Ac-MLFK-HYNIC (p < 0.05) . Study C demonstrated that coinjection with an antagonist resulted in a significant reduction in the T/B ratio for 99mTc-N-For-MLFK-HYNIC (p < 0.001), but did not affect the T/B ratio for 111In-leukocytes . CONCLUSION: Nonspecific mechanisms contribute minimally to the localization of 99mTc-chemotactic peptide analogs at sites of infection and the majority of the accumulation appears to be receptor mediated . Also, chemotactic peptide receptor antagonists can be used for infection imaging . These results provide important new insights for future radiopharmaceutical development. Nat Struct Biol, 1997 Aug, 4(8), 618 - 22 Trapping and visualization of a covalent enzyme-phosphate intermediate; Murphy JE et al.; Using a mutant version of E . coli alkaline phosphatase, we succeeded in trapping and determining the structure of the phospho-enzyme intermediate . The X-ray structure also revealed the catalytic water molecule, bound to one of the active site zinc ions, positioned ideally for the apical attack necessary for the hydrolysis of the intermediate. FEMS Microbiol Lett, 1997 Aug 1, 153(1), 83 - 8 Production of bleomycin N-acetyltransferase in Escherichia coli and Streptomyces verticillus; Matsuo H et al.; Bleomycin-producing Streptomyces verticillus ATCC 15003 has two bleomycin resistance genes, designated blmA and blmB . Bleomycin N-acetyltransferase, encoded by blmB, was overproduced in Escherichia coli as a protein fused to the maltose-binding protein . The protein (fBAT), purified to homogeneity after digestion of the fusion product with blood coagulation factor Xa protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein . The K(m) and Vmax values of purified fBAT for the substrate bleomycin were 13.0 microM and 3.4 nmol {corrected} min-1 ml-1, respectively . The optimal pH for the acetylating activity was 6.0 in 10 mM phosphate buffer . The molecular mass and pI value of fBAT were estimated by polyacrylamide gel electrophoresis to be about 34500 and 6.13, respectively . An anti-fBAT monoclonal antibody was generated and used to show that bleomycin N-acetyltransferase is expressed simultaneously with bleomycin production in S . verticillus. Hepatology, 1997 Aug, 26(2), 491 - 4 Distribution of hepatitis G viremia and antibody response to recombinant proteins with special regard to risk factors in 709 patients; Feucht HH et al.; A new virus named hepatitis G virus (HGV) has been detected recently . Until now, no assays for the detection of antibodies against different HGV proteins have been commercially available . Therefore, a strip immunoblot assay has been established to investigate seroreactivity against recombinant structural (core) and nonstructural proteins (NS3 and NS4) of HGV produced in Escherichia coli . Seropositivity for HGV was evaluated and concordanced with HGV polymerase chain reaction (PCR) results in 709 subjects . These individuals were classified into a nonrisk or a risk group, on the basis of infection with human immunodeficiency virus (HIV) or hepatitis C virus (HCV) or frequent parenteral exposure, including hemophilia, intravenous drug addiction, receipt of blood transfusion, or hemodialysis . The nonrisk group consisted of 257 healthy blood donors with normal alanine transaminase (ALT) levels (ALT < 30 U/L) and 154 patients with suspected non-A-E hepatitis (ALT > 45 U/L) . In the group of healthy blood donors, 1.9% (5 of 257) had detectable HGV viremia and 15.9% (41 of 257) showed antibody response to HGV . In the collective of patients with suspected non-A-E hepatitis, results from 1.9% of patients (3 of 154) were positive by HGV PCR, and 15.6% of patients (24 of 154) showed seropositivity against the recombinant HGV proteins . In six groups of patients (n = 298) with different risk factors, the prevalence of both HGV viremia (V) and serological reactivity (SR) was higher compared with that of the nonrisk group: V, 6.80%-35.2%; serological reactivity (SR), 25.4%-52.9% . The following conclusions can be derived from our data . HGV infection is widespread in the general population . The prevalence of antibodies against HGV or detectable HGV viremia is higher in patients with risk factors for parenteral viral transmission than in those without risk factors . The majority of HGV infections (70.2%) is self-limiting and not persistent in our collective of patients . We found no correlation between HGV viremia and clinical or biochemical signs of hepatitis in individuals without risk factors for acquiring parenterally transmitted agents. Hepatology, 1997 Aug, 26(2), 336 - 42 Role of nitric oxide in oxygen transport in rat liver sinusoids during endotoxemia; Huang TP et al.; To evaluate the role of nitric oxide (NO) in hepatic microcirculation and liver injury during endotoxemia, we studied O2 transport in the hepatic microcirculation of endotoxin-infused rats . Rats were continuously infused with Escherichia coli lipopolysaccharide (LPS) (0.8 mg/kg/h) for 7 hours . LPS increased the plasma levels of NO2- + NO3- and aspartate transaminase (AST), and decreased the bile flow rate and hepatic adenosine triphosphate (ATP) level . Hepatic microcirculation was evaluated by two methods: reflectance spectrophotometry showed a decrease in the oxygenation of hemoglobin (Hb) in the liver, and dual-spot microspectroscopy indicated that LPS administration decreased blood velocity, the oxygenation of Hb, and O2 release from sinusoids to hepatocytes . The observed decreases in the O2 transport parameters were prominent in pericentral sinusoids . All of these phenomena were further aggravated by the administration of N(w)-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg/h) plus LPS, and by aminoguanidine (AMG) (5 mg/kg/h) plus LPS, and these could be reversed by the concomitant administration of L-arginine (L-Arg) (100 mg/kg/h) . These results suggest that deterioration of hepatic oxygen transport and liver function induced by endotoxin can be ameliorated by NO. Biophys J, 1997 Aug, 73(2), 929 - 37 Conformational changes in actin induced by its interaction with gelsolin; Khaitlina S et al.; Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound . We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin . ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex . Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer . By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions . On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin . Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament . These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer. Appl Environ Microbiol, 1997 Aug, 63(8), 3268 - 73 Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification; Schonhuber W et al.; The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells . Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes . The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol, 1997 Aug, 63(8), 3205 - 10 Reduction of aerobic acetate production by Escherichia coli; Farmer WR et al.; Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production . We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion . Flux analysis of E . coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production . We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain . Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected . Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion . These results confirm the prediction of our flux analysis and further suggest that E . coli is not fully optimized for efficient utilization of glucose. Appl Environ Microbiol, 1997 Aug, 63(8), 3096 - 103 Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation; Mondello FJ et al.; The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria . The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners . Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity . Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners . BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity . The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV . Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707 . Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners . However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative . These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase. Appl Environ Microbiol, 1997 Aug, 63(8), 3043 - 50 Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes; Sorensen AH et al.; Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed . The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3' . In situ hybridization with the fluorescently labelled probes required several modifications of standard procedures . Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol . Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.85% NaCl . Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound . The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash the labelled probes out of the cells . Stable labelling could be obtained with rhodamine-labelled probes when the specimen was mounted in immersion oil, and high hybridization intensities of the Methanosarcina cells were found even in the presence of biomass from an anaerobic reactor . The inherent high autofluorescence of the biomass could be lowered by use of a highly specific narrow-band filter . The probes were found to be specific for Methanosarcina and useful for detection of this genus in samples from anaerobic reactors. Mol Biochem Parasitol, 1997 Aug, 87(2), 145 - 58 Purification, cloning, and expression of two closely related Trypanosoma brucei nucleic acid binding proteins; Zhang J et al.; Nucleic acid binding proteins in the trypanosomatid family are of particular interest because of several unusual molecular phenomena discovered in these organisms . We have purified two closely related proteins, p34 and p37, from the procyclic from of T . brucei using high salt extraction and single-stranded-DNA (ssDNA) agarose chromatography . Antibodies raised against the p34 protein showed crossreactivity with p37, suggesting relatedness . High performance liquid chromatography analysis and microsequencing of tryptic peptides derived from p34 and p37 showed that the primary structures of the two proteins are nearly identical . We have cloned and sequenced the two genes encoding these two proteins . Protein sequences predicted from the cDNAs confirm the relatedness of the two proteins but also indicate the presence of an 18 amino acid insertion unique to one of the two proteins as well as several minor differences resulting from single amino acid changes . Three sequence motifs have been identified in both proteins: an N-terminal alanine, proline, and lysine rich domain, one and a half internal RNA-binding domains, and a C-terminal KKDX repeat region . Both proteins preferentially bind to heterogenous RNA and ssDNA versus double-stranded DNA and homopolymers . Both recombinant proteins have been expressed in E . coli and show properties indistinguishable from those observed with native p34/p37. Mol Biochem Parasitol, 1997 Aug, 87(2), 137 - 44 Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii; Carter D et al.; The coding region derived from a full-length CDNA spanning the uracil phosphoribosyltransferase (UPRT) gene of Toxoplasma gondii has been ligated into a bacterial expression vector and overexpressed in E . coli . Recombinant UPRT protein migrated with a molecular mass of 27 kDa on SDS polyacrylamide gels and was purified to homogeneity by conventional protein purification techniques . In solution, UPRT behaved as a monomer and exhibited K(m)app values of 3.5 microM for uracil and 243 microM for phosphoribosylpyrophosphate, respectively . Other naturally occurring pyrimidine or purine bases were not recognized as substrates . {14C}Uracil phosphoribosylation was inhibited by 5-fluorouracil with a Ki value of 25 microM and was not activated by GTP . Ample quantities of recombinant enzyme are now available for biochemical and structural studies, facilitating evaluation of UPRT as a possible therapeutic target. Development, 1997 Aug, 124(15), 2915 - 22 Control of cell fates and segmentation in the Drosophila mesoderm; Riechmann V et al.; The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm . We show that the primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants . Simultaneously, the primordium of the visceral muscles is expanded . The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants . However, we find that even-skipped mutants retain the segmental modulation of the expression of twist . Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired . sloppy-paired thus controls segmental allocation of mesodermal cells to different fates. J Struct Biol, 1997 Aug, 119(3), 336 - 46 Stabilization of compact spermidine nucleoids from Escherichia coli under crowded conditions: implications for in vivo nucleoid structure; Murphy LD et al.; Nucleoids from Escherichia coli were isolated in the presence of spermidine at low salt concentrations . The nucleoids denature at relatively low temperatures or salt concentrations, yielding broad slowly sedimenting zones and/or macroscopic aggregates upon sucrose gradient centrifugation . Denaturation is accompanied by a loss of a characteristically compact shape as visualized by light and electron microscopy . Addition of polyethylene glycol or dextran prevents these changes, extending the range of stability of the isolated nucleoids to temperatures and ionic conditions like those which commonly occur in vivo . The effects of the polymers are consistent with stabilization by macromolecular crowding . Enzymatic digestion of the nucleoid DNA primarily releases three small proteins (H-NS, FIS, and HU) and RNA polymerase, as well as residual lysozyme from the cell lysis procedure . If isolated nucleoids are extracted with elevated salt concentrations under crowded, stabilized conditions, two of the proteins (HU and lysozyme) are efficiently removed and the compact form of the nucleoids is retained . These extracted nucleoids maintain their compact form upon reisolation into the initial uncrowded low-salt medium, indicating that HU, the most common "histone-like" protein of E . coli, is not a necessary component for maintaining compaction in these preparations. J Struct Biol, 1997 Aug, 119(3), 321 - 35 Isolation and characterization of spermidine nucleoids from Escherichia coli; Murphy LD et al.; Nucleoids isolated from Escherichia coli at low salt concentrations in the presence of spermidine (Kornberg et al., Proc . Natl . Acad . Sci . USA, 71, 3189-3193 (1974)) retain large amounts of protein and RNA and are, thus, potentially useful in structural and other studies . However, these preparations have neither been visualized nor extensively characterized with regard to their protein and other components . We have investigated this type of nucleoid preparation and here supply both light and electron microscope appearances and a description of the DNA-associated proteins . Light microscopy is used to follow the stages of nucleoid release and to demonstrate characteristically rounded nucleoids after chloramphenicol treatment of the cells from which the nucleoids were isolated . The nucleoids are "envelope-associated" particles . Electron microscopy shows an irregular central core that is partially covered with small, membranous vesicles . A significant fraction of the nucleoids have a characteristic doublet/dumbbell-shaped appearance by light microscopy . The nucleoids contain large amounts of protein and RNA in addition to DNA . The DNA and RNA are rendered acid-soluble by very low levels of nucleases, indicating an open structure . A small group of proteins, including H-NS, FIS, HU, and RNA polymerase, is released from the particles upon enzymatic digestion of the DNA. Microb Pathog, 1997 Aug, 23(2), 113 - 8 Role of type 1 fimbriae in EPEC infections; Elliott SJ et al.; Several fimbriae have been implicated as potentially important in EPEC adhesion and pathogenesis . EPEC strain E2348/69 produced only bundle forming pili and type 1 fimbriae, and did not produce other accesory adhesins identified in EPEC strain B171 . Cloning and mutagenesis of these EPEC fim genes indicated that type 1 pili had no affect on levels or patterns of adhesion to cultured human cells. Methods, 1997 Aug, 12(4), 318 - 24 Genetic selection in Escherichia coli for active human immunodeficiency virus reverse transcriptase mutants; Kim B; Most catalytically active human immunodeficiency virus (HIV) reverse transcriptase (RT) mutants characterized to date have been isolated from the virus after treatment with HIV RT inhibitors such as nucleoside analogs . However, detailed understanding of structure-function relationships, and of the roles of the several catalytic activities of HIV RT in viral replication, requires characterization of a greater diversity of mutant enzymes than has been obtained from viral variants . Coupling of a bacterial genetic selection system for functional HIV RT with random mutagenesis has yielded a large number of active mutant enzymes, most of which have not been found in viral variants . The genetic selection system, combined with biochemical characterization of active mutant proteins, affords three major benefits . First, we can increase our understanding of the roles of individual amino acids in catalysis . Second, the mutational spectrum observed among active HIV RT variants can identify amino acids that are intolerant, or relatively intolerant, of substitution . Third, this system provides us with HIV RT variants with altered biochemical properties, such as replicational fidelity and processivity . Characterization of HIV harboring these mutant RTs with defined structural and functional alterations will contribute to elucidation of the roles of each catalytic activity of HIV RT in viral replication. J Mol Biol, 1997 Aug 1, 270(5), 771 - 8 Folding and stability of a fibronectin type III domain of human tenascin; Clarke J et al.; The folding of an isolated fibronectin type III domain of human tenascin, a large extra-cellular matrix protein, has been characterised . The isolated module, which has no disulphide bonds, can be reversibly unfolded by chemical denaturant and temperature . Equilibrium unfolding, measured using a number of different probes, fits to a two-state transition, with consistent measures of DeltaGH2OD-N . Folding and refolding rate constants have been determined over a range of denaturant concentrations . The refolding kinetics are bi-phasic, and in the transition region the slow phase dominates refolding kinetics . Outside the transition region the folding of the fast-folding species fits to a two-state model . There is no evidence for significant accumulation of partially folded intermediates. J Mol Biol, 1997 Aug 1, 270(5), 648 - 62 Determinants for Escherichia coli RNA polymerase assembly within the beta subunit; Wang Y et al.; We used binding assays and other approaches to identify fragments of the Escherichia coli RNAP beta subunit involved in the obligatory interaction with the alpha subunit to form the stable assembly intermediate alpha2beta as well as in the interaction to recruit the beta' subunit into the alpha2beta sub-assembly . We show that two regions of evolutionarily conserved sequence near the C terminus of beta (conserved regions H and I) are central to the assembly of RNAP and likely make subunit-subunit contacts with both alpha and beta'. Gen Comp Endocrinol, 1997 Aug, 107(2), 229 - 39 Guanylyl cyclase receptors and guanylin-like peptides in reptilian intestine; Krause WJ et al.; Receptors for guanylin and uroguanylin were identified on the mucosal surface of enterocytes lining the intestine of the bobtail skink (Tiliqua rugosa), king's skink (Egernia kingii), and knight anole (Anolis equestris) by receptor autoradiography using 125I-ST (Escherichia coli heat-stable enterotoxin) as the radioligand . Specific, high-affinity binding of 125I-ST to receptors was found on the microvillus border of enterocytes and little or no specific binding of 125I-ST was observed in other strata comprising the gut wall . The American alligator (Alligator mississippensis) also exhibited receptor binding, but unlike the other three species had relatively high levels of apparent nonspecific binding . A comparison of intestinal cGMP accumulation responses between the American alligator and the knight anole demonstrated a greater magnitude of cGMP responses to ST and guanylin in vitro in the knight anole relative to the tissue cGMP accumulation responses of alligators . Treatment with ST resulted in markedly greater tissue cGMP accumulation responses in both species compared to treatment with guanylin . To complete a paracrine signaling pathway in reptilian intestine, guanylin-like peptides that stimulated cGMP accumulation in human T84 intestinal cells were isolated from the intestinal mucosa of alligators . We conclude that functional receptor-guanylyl cyclases and one or more endogenous guanylin/uroguanylin-like peptides occur in the intestinal tract of reptiles as well as in the intestines of mammals and birds . Thus, higher vertebrates have a conserved signaling pathway that regulates intestinal function through the first-messenger peptides, guanylin and/or uroguanylin, and the intracellular second messenger, cGMP. Anal Biochem, 1997 Aug 1, 250(2), 176 - 80 Chemical synthesis of 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid glycoside and its use for the fluorometric detection of poorly expressed natural and recombinant sialidases; Engstler M et al.; When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities . The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates . Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-alpha-D-N-acetylneuraminic acid (CF3MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources . Kinetic analysis revealed CF3MU-Neu5Ac to be a very sensitive sialidase substrate . Furthermore, this substance proves to be perfectly suitable for the in vivo examination of sialidases and for the detection of recombinant sialidase by means of expression cloning. Arch Biochem Biophys, 1997 Aug 1, 344(1), 103 - 13 Disulfide structure of the heparin binding domain in vascular endothelial growth factor: characterization of posttranslational modifications in VEGF; Keck RG et al.; Preparations of recombinant human vascular endothelial growth factor (VEGF165) expressed in Chinese hamster ovary (CHO) cells and Escherichia coli were compared using a variety of analytical methods . Amino terminal sequence analyses of both the CHO- and E . coli-derived VEGF165 confirmed the predicted amino terminal sequence for VEGF165, although the CHO VEGF165 exhibited a heterogeneous amino terminus with sequences beginning at Ala-1 (76%), Pro-2 (4%), Ala-4 (13%), and Glu-5 (7%) . Tryptic digests of reduced and carboxymethylated CHO- and E . coli-derived VEGF165 were examined by LC/MS analyses, indicating equivalent primary structure, except for the glycosylation at Asn-75 in the CHO-derived VEGF165 . The N-linked carbohydrate in the CHO-derived VEGF165 was determined to be a complex fucosylated biantennary structure . The data obtained from LC/MS analysis and amino terminal sequence analysis of VEGF165 confirmed 98% of the primary structure . Disulfide linkages for the eight cysteine residues in the carboxyl terminal heparin binding domain were assigned by amino terminal sequencing of fragments produced by tryptic digests of each native molecule . The following disulfides have been identified for both CHO- and E . coli-derived VEGF165: Cys-117 and Cys-135, Cys-120 and Cys-137, Cys-139 and Cys-158, plus Cys-146 and Cys-160 . Plasmin cleavage of VEGF165 yields an N-terminal homodimeric VEGF110 and a 55-amino-acid carboxyl terminal domain . VEGF110 was resistant to further proteolytic or chemical digestion such that the disulfide linkages were not elucidated . The 55-amino-acid carboxyl terminal region of VEGF165 appears to be a unique heparin binding domain with no known protein homology. Arch Biochem Biophys, 1997 Aug 1, 344(1), 67 - 74 Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain; Oh ES et al.; Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC . Other serine and threonine residues are present but not in a consensus sequence . We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain . A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC with high affinity . Peptide mapping and substitution studies showed that both serines were phosphoacceptors, but each had slightly different affinity, with that of serine-197 being higher than serine-198 . The efficiency of phosphorylation was concentration-dependent . At low concentrations, the cytoplasmic domain peptides were monomeric, with 2 mol/mol serine phosphorylation . At higher concentrations, however, the peptides formed dimers, with only 0.5 mol/mol phosphorylation . Concentration-dependent dimerization was not altered by phosphorylation . Phosphorylation is, therefore, dependent on the conformation of syndecan-2 cytoplasmic domain, but does not affect its oligomeric status. Arch Biochem Biophys, 1997 Aug 1, 344(1), 43 - 52 Elongation factor Ts of Chlamydia trachomatis: structure of the gene and properties of the protein; Zhang Y et al.; A putative structural gene cluster containing four open reading frames (ORFs) located downstream of the omp1 gene of Chlamydia trachomatis mouse pneumonitis (MoPn) was cloned and sequenced . A GenBank survey indicated that the identified cluster is similar to the rpsB-tsf-pyrH(smbA)-frr region of Escherichia coli . The second ORF was 846 bp encoding a 282-amino-acid polypeptide with a calculated M(r) 30,824 . Alignment of this deduced protein sequence and E . coli elongation factor Ts (EF-Ts, product of tsf) demonstrated 34% identity and an additional 14% similarity . The putative chlamydial tsf gene was expressed in E . coli as a nonfusion protein and as a 6x His-tagged fusion protein . By SDS-PAGE analysis, the molecular weights of the nonfusion recombinant protein and a protein of chlamydial elementary bodies (EBs), which was recognized by monoclonal antibodies derived from the nonfusion recombinant protein, are 34 kDa . The purified recombinant 6x His-tagged fusion protein increased the rate of GDP exchange with both Chlamydia and E . coli elongation factor Tu (EF-Tu) . These data show that the second gene of the identified cluster is tsf . Unlike EF-Ts from any other species, its activity was comparable to that of E . coli EF-Ts in exchange reaction with E . coli EF-Tu. J Bacteriol, 1997 Aug, 179(15), 4868 - 73 Involvement of Fnr and ArcA in anaerobic expression of the tdc operon of Escherichia coli; Chattopadhyay S et al.; Anaerobic expression of the tdcABC operon in Escherichia coli, as measured by LacZ activity from single-copy tdc-lacZ transcriptional and translational fusions, is greatly reduced in strains lacking two global transcriptional regulators, Fnr and ArcA . The nucleotide sequence of the tdc promoter around -145 shows significant similarity with the consensus Fnr-binding site; however, extensive base substitutions within this region had no effect on Fnr regulation of the tdc genes . A genetic analysis revealed that the effect of Fnr on tdc is not mediated via ArcA . Furthermore, addition of cyclic AMP to the anaerobic incubation medium completely restored tdc expression in fnr and arcA mutants as well as in strains harboring mutations in the Fnr- and ArcA-dependent pfl gene and the Fnr-regulated glpA and frd genes . These results, taken together with the earlier finding that tdc expression is subject to catabolite repression by intermediary metabolites, strongly suggest that the negative regulatory effects of mutations in the fnr and arcA genes are mediated physiologically due to accumulation of a metabolite(s) which prevents tdc transcription in vivo. J Bacteriol, 1997 Aug, 179(15), 4795 - 801 Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system; Taniguchi H et al.; We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis . All of the M . smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs) . This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance . Mutations from G to A or G to T at position 1473 of the M . smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies . Using the M . smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain . Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M . smegmatis. J Gen Physiol, 1997 Aug, 110(2), 173 - 84 Molecular tuning of an EF-hand-like calcium binding loop . Contributions of the coordinating side chain at loop position 3; Drake SK et al.; Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca2+ binding motif . The ion binding parameters of this motif are controlled, in part, by the structure of its Ca2+ binding loop, termed the EF-loop . The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca2+ binding parameters for their unique cellular roles . The present study uses a structurally homologous Ca2+ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning . 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics . Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity . Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain . Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position. J Biol Chem, 1997 Aug 1, 272(31), 19621 - 4 Rotation of a gamma-epsilon subunit domain in the Escherichia coli F1F0-ATP synthase complex . The gamma-epsilon subunits are essentially randomly distributed relative to the alpha3beta3delta domain in the intact complex; Aggeler R et al.; A triple mutant of Escherichia coli F1F0-ATP synthase, alphaQ2C/alphaS411C/epsilonS108C, has been generated for studying movements of the gamma and epsilon subunits during functioning of the enzyme . It includes mutations that allow disulfide bond formation between the Cys at alpha411 and both Cys-87 of gamma and Cys-108 of epsilon, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R . A . (1996) J . Biol . Chem . 271, 13888-13891) . A cross-link is also generated between the Cys at alpha2 and Cys-140 of the delta subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R . A . (1997) J . Biol . Chem . 272, 16652-16656) . CuCl2 treatment of the mutant alphaQ2C/alphaS411C/epsilonS108C generated five major cross-linked products . These are alpha-gamma-delta, alpha-gamma, alpha-delta-epsilon, alpha-delta, and alpha-epsilon . The ratio of alpha-gamma-delta to the alpha-gamma product was close to 1:2, i.e . in one-third of the ECF1F0 molecules the gamma subunit was attached to the alpha subunit at which the delta subunit is bound . Also, 20% of the epsilon subunit was present as a alpha-delta-epsilon product . With regard to the delta subunit, 30% was in the alpha-gamma-delta, 20% in the alpha-delta-epsilon, and 50% in the alpha-delta products when the cross-linking was done after incubation in ATP + MgCl2 . The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu2+ was added after preincubation in ATP + Mg2+ + azide . The delta subunit is fixed to, and therefore identifies, one specific alpha subunit (alphadelta) . A distribution of the gamma and epsilon subunits, which is essentially random with respect to the alpha subunits, can only be explained by rotation of gamma-epsilon relative to the alpha3beta3 domain in ECF1F0. J Biol Chem, 1997 Aug 1, 272(31), 19594 - 600 Functional interaction of the auxilin J domain with the nucleotide- and substrate-binding modules of Hsc70; Ungewickell E et al.; The uncoating of clathrin-coated vesicles requires the DnaJ homologue auxilin for targeting Hsc70 to clathrin coats . This function involves a transient interaction of the auxilin J domain with Hsc70 . We have now identified the structural elements of Hsc70 that are responsible for the uncoating activity, and we show that the hitherto accepted view, which implicates the 10-kDa carboxyl-terminal variable domain of Hsc70, is incorrect . A 60-kDa chymotryptic or analogous recombinant fragment of Hsc70, which contains the ATPase- and substrate-binding domains, is sufficient to liberate clathrin from coated vesicles . Consistent with this was the observation that Hsp70 uncoats coated vesicles with the same efficacy as Hsc70 and that DnaK possesses vestigial uncoating activity . Direct binding studies demonstrated that the auxilin J domain undergoes an ATP-dependent reaction only with fragments of Hsc70 that contain both the ATPase- and substrate-binding domains . The individual domains by themselves did not bind to the J domain nor did a recombinant protein that contained the substrate-binding domain attached to the 10-kDa variable domain. J Biol Chem, 1997 Aug 1, 272(31), 19471 - 9 HSP70 binding sites in the tumor suppressor protein p53; Fourie AM et al.; Mutations within conserved regions of the tumor suppressor protein, p53, result in oncogenic forms of the protein with altered tertiary structures . In most cases, the mutant p53 proteins are selectively recognized and bound by members of the HSP70 family of molecular chaperones, but the binding site(s) in p53 for these chaperones have not been clearly defined . We have screened a library of overlapping biotinylated peptides, spanning the entire human p53 sequence, for binding to the HSP70 proteins, Hsc70 and DnaK . We show that most of the high affinity binding sites for these proteins map to secondary structure elements, particularly beta-strands, in the hydrophobic core of the central DNA binding domain, where the majority of oncogenic p53 mutations are found . Although peptides corresponding to the C-terminal region of p53 also contain potential binding sites, p53 proteins with C-terminal deletions are capable of binding to Hsc70, indicating that this region is not required for complex formation . We propose that mutations in the p53 protein alter the tertiary structure of the central DNA binding domain, thus exposing high affinity HSP70 binding sites that are cryptic in the wild-type molecule. J Biol Chem, 1997 Aug 1, 272(31), 19314 - 8 Chaperone SecB from Escherichia coli mediates kinetic partitioning via a dynamic equilibrium with its ligands; Topping TB et al.; We have shown that the complexes between SecB, a chaperone from Escherichia coli, and two physiological ligands, galactose-binding protein and maltose-binding protein, are in rapid, dynamic equilibrium between the bound and free states . Binding to SecB is readily reversible, and each time the ligand is released it undergoes a kinetic partitioning between folding to its native state and re-binding to SecB . Binding requires that the polypeptide be devoid of tertiary structure; once the protein has folded, it is no longer a ligand . Conditions were established in which folding of the polypeptides was sufficiently slow so that at each cycle of dissociation rebinding was favored over folding and a kinetically stable complex between SecB and each polypeptide ligand was observed . Evidence that the ligand is continually released to the bulk solution and rebound was obtained by altering the conditions to increase the rate of folding of each ligand so that folding of the ligand was faster than reassociation with SecB thereby allowing the system to partition to free SecB and folded polypeptide ligand . We conclude that complexes between the chaperone SecB and ligands are in dynamic, rapid equilibrium with the free states . This mode of binding is simpler than that documented for chaperones that function to facilitate folding such as the Hsp70s and Hsp60s, where hydrolysis of ATP is coupled to the binding and release of ligands . This difference may reflect the fact that SecB does not mediate folding but is specialized to facilitate protein export . Without a requirement for exogenous energy it efficiently performs its sole duty: to keep proteins in a nonnative conformation and thus competent for export. Infect Immun, 1997 Aug, 65(8), 3478 - 84 A novel cryohemagglutinin associated with adherence of enteroaggregative Escherichia coli; Yamamoto T et al.; Strain O42 (serotype O44:H18) of enteroaggregative Escherichia coli (EAggEC) has been shown to be pathogenic in volunteer experiments . This strain exhibited plasmid (pO42)-encoded D-mannose-resistant hemagglutinating activity (MRHA) that was detected only at low temperatures (e.g., 0 degrees C) and only with human erythrocytes . The production of this cryogenic MRHA (cryo-MRHA) was observed when the bacteria were grown in liquid media and was strictly regulated by bacterial growth temperatures . Transposon-insertion mutagenesis revealed that this MRHA is associated with (i) bacterial clump formation in liquid cultures, (ii) bacterial adherence to HEp-2 cells as well as (Formalin-fixed) human colonic mucosa, and (iii) production of a 16-kDa outer membrane protein . The PCR designed on the basis of the determined cryo-MRHA-associated DNA sequence sharply distinguished strain O42 from eight other EAggEC strains whose MRHAs were detected at both cold and room temperatures to the same (or similar) extent . Strain O42 possessed a surface layer that may enhance the pO42-mediated adherence . The data suggest that a plasmid-encoded cryo-MRHA is a candidate for a major adhesin of EAggEC strain O42. Infect Immun, 1997 Aug, 65(8), 3465 - 8 Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin; Liu DF et al.; The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells . Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S . M . Prasad, Y . Yin, E . Rodzinski, E . I . Tuomanen, and H . R . Masure, Infect . Immun . 61:2780-2785, 1993) . We have constructed an E . coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels . Fragment A could be purified with high purity and yields and was immunogenic in mice . Both fragment A and anti-fragment A sera inhibited the binding of B . pertussis to asialo-GM2 and to rabbit ciliated cells . These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies. Infect Immun, 1997 Aug, 65(8), 3352 - 60 Immunologic and genetic analyses of VmpA of a neurotropic strain of Borrelia turicatae; Cadavid D et al.; In mice infected with serotype A but not serotype B of the relapsing fever spirochete Borrelia turicatae, early invasion of the brain occurs . Serotypes A and B are further distinguished by the abundant surface protein they produce: VmpA and VmpB, respectively . Western blotting with monoclonal antibodies, one-dimensional peptide mapping, and partial amino acid sequencing demonstrated regions of the VmpA protein that differed from VmpB . Oligonucleotide primers based on the partial amino acid sequences of unique regions were used to amplify a portion of the VmpA gene (vmpA) by PCR, and the product was used as a probe in Southern blot and Northern blot analyses . These experiments showed that (i) expression of the vmpA sequence was determined at the level of transcription and (ii) the vmpA sequence was in two locations in serotype A and one location in serotype B . The vmpA gene at the expression-linked locus of serotype A was cloned and sequenced . An open reading frame would encode a polypeptide of 214 amino acids . The polypeptide expressed by Escherichia coli was bound by VmA-specific but not VmpB-specific antibody . Primer extension analysis identified a consensus sigma70-type promoter for vmpA at the expression locus . Phylogenetic analysis revealed that VmpA is homologous to small Vmp (Vsp) proteins of B . hermsii and to OspC proteins of B . burgdorferi . These findings indicate that a function of the Vsp-OspC family of proteins of Borrelia spp . may be differential localization in organs, including the brain, during infection. Infect Immun, 1997 Aug, 65(8), 3286 - 92 Complement fragment C5a and inflammatory cytokines in neutrophil recruitment during intramammary infection with Escherichia coli; Shuster DE et al.; Generation of inflammatory mediators and leukocyte recruitment to infection at an epithelial surface were studied during Escherichia coli-induced mastitis . One uninfected gland of each of eight midlactation cows was challenged with only 30 CFU of E . coli McDonald strain 487, a serum-resistant isolate from a cow with mastitis . Bacteria grew logarithmically during the first 10 to 12 h after challenge, reaching concentrations of more than 10(5) CFU/ml with no detectable host response during this time . An intense inflammatory reaction began approximately 12 h after the challenge and was characterized by a breakdown in the blood-milk permeability barrier followed by pyrexia and a pronounced leukocytic influx . Coincident with the onset of mammary inflammation was the appearance of neutrophil chemotactic activity in the milk from infected glands . Factors able to upregulate CD18 expression on peripheral blood neutrophils also appeared in milk at this time . The lack of appearance of chemotactic and CD18-upregulating activities until 12 h after challenge indicated that delays in neutrophil recruitment resulted from an initial lack of bacterial recognition and inflammatory mediator production . Production of complement fragment C5a, tumor necrosis factor, and interleukin-1 (IL-1) occurred earlier than production of IL-6 or IL-8 . The early and intense production of C5a indicates that this chemoattractant may be more important than IL-8 during the initial recruitment and activation of neutrophils to a developing E . coli infection. Infect Immun, 1997 Aug, 65(8), 3277 - 85 Activation of host cell protein kinase C by enteropathogenic Escherichia coli; Crane JK et al.; Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E . coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive . EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production . We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells . Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min . Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively . The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells . Nonadherent laboratory E . coli strains such as HB101 and H.S . failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA) . In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells . Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E . coli heat-stable toxin . We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells. Infect Immun, 1997 Aug, 65(8), 3231 - 8 Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities; Chu L et al.; A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67 . Both the native and recombinant 46-kDa proteins were purified to homogeneity . Both proteins expressed identical biological and functional characteristics . In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate . This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1) . Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein . Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine) . The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0 . The enzymatic activity was increased by beta-mercaptoethanol . It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide . We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type . Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule. Infect Immun, 1997 Aug, 65(8), 3032 - 6 Comparison of protection induced by immunization with recombinant proteins from different regions of merozoite surface protein 1 of Plasmodium yoelii; Tian JH et al.; Vaccination with native full-length merozoite surface protein 1 (MSP1) or with recombinant C-terminal peptides protects mice against lethal challenge with virulent malaria parasites . To determine whether other regions of MSP1 can also induce protection, Plasmodium yoelii MSP1 was divided into four separate regions . Each was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) . The N-terminal fragment began after the cleavage site for the signal sequence and ended in the region comparable to the cleavage site for the C terminus of the 82-kDa peptide of Plasmodium falciparum . This expressed protein was 30 kDa smaller than the predicted peptide . One peptide from the middle region was produced, and the C terminus consisted of a 42-kDa fragment corresponding to the analogous peptide of P . falciparum and a 19-kDa fragment that extended 37 amino acids in the amino-terminal direction beyond the probable cleavage site . To test protection of mice against lethal P . yoelii challenge, three mouse strains (CAF1, BALB/c, and A/J) were vaccinated with each of the four recombinant proteins of MSP1 . Mice vaccinated with the C-terminal 19-kDa protein were highly protected (described previously), as were those vaccinated with the 42-kDa protein that contained the 19-kDa fragment . The N-terminally expressed fragment of P . yoelii was not full length because of proteolytic cleavage in E . coli . The GST-82-kDa partial fragments induced some immunity, but the surviving mice still had high parasitemias . Vaccination with the peptide from the middle region of MSP1 gave minimal to no protection . Therefore, in addition to the C-terminal 19- and 42-kDa proteins, the only other fragment to give protection was the 82-kDa protein . The protection induced by the truncated 82-kDa protein was minimal compared with that of the C-terminal fragments. Infect Immun, 1997 Aug, 65(8), 3011 - 6 Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS; Jarvis BW et al.; An early event in septic shock is the activation of macrophages by a complex consisting of lipopolysaccharide (LPS), LPS-binding protein (LBP), and the cell surface antigen CD14 . The complexes that form between {3H}ReLPS (ReLPS is deep-rough-chemotype hexacyl LPS from E . coli D31m4), soluble CD14 (sCD14), and LBP were analyzed by two independent methods, native (nondenaturing) gel electrophoresis and size-exclusion high-performance liquid chromatography (HPLC) . This is the first reported use of HPLC to purify and study LPS-protein complexes . The binding of {3H}ReLPS to LBP and sCD14 was inhibited by preincubation with diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA), a potent LPS antagonist . In addition, {3H}ReLPS bound to LBP and to a truncated form of sCD14 {sCD14(1-152)} that contained the LPS binding domain . Binding to both proteins was blocked by RsDPLA . Thus, RsDPLA competes in a 1:1 ratio for the same or nearby binding sites on ReLPS complexes . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aggregated ReLPS eluting from the HPLC indicated that only LBP, not sCD14, was bound to the aggregated ReLPS . This finding supports the binary model of LPS complex formation with LBP and sCD14. J Immunol, 1997 Aug 1, 159(3), 1482 - 9 Recombinant guinea pig and human RANTES activate macrophages but not eosinophils in the guinea pig; Campbell EM et al.; To characterize the biologic activities of potential mediators of allergic inflammation, we have cloned, expressed, and purified guinea pig RANTES (gpRANTES) . cDNA for gpRANTES was cloned from Con A-stimulated guinea pig spleen cells . A high level of gpRANTES expression in Escherichia coli was achieved by mutation of a human RANTES (hRANTES) expression construct to obtain a 68-amino acid protein identical with the predicted guinea pig amino acid sequence, assuming an equivalent amino terminus as the human protein . Purified gpRANTES was an effective stimulus of human eosinophils as assessed by increases in intracellular free calcium in fura-2-loaded cells and chemotactic responses in vitro . gpRANTES exhibits similar potency and efficacy to hRANTES . In marked contrast, neither gpRANTES nor hRANTES was able to activate guinea pig peritoneal eosinophils in these assays, even in the presence of IL-5 . However, gpRANTES was found to be a potent stimulator of guinea pig peritoneal macrophages . Following tracheal instillation of gpRANTES, a dose-dependent increase in macrophages, but not eosinophils, was observed in gpBAL . Macrophage accumulation was detectable by 6 h and sustained for at least 48 h . These results indicate that RANTES in the guinea pig may have a different cellular selectivity than that described in the human, which may be important in the use of animal models in the analysis of allergic disorders . These selectivities do not appear to be accounted for by differences in guinea pig and human RANTES sequences. J Neurochem, 1997 Aug, 69(2), 851 - 8 Lipopolysaccharide and interleukin-1beta augmented histidine decarboxylase activity in cultured cells of the rat embryonic brain; Niimi M et al.; We investigated the effect of lipopolysaccharide (LPS) and various inflammatory cytokines on the histidine decarboxylase (HDC) activity in cultured cells of the rat embryonic brain . Histaminergic neuronal cell bodies were supposed to exist in cultured cells of the diencephalon but not in those of the cortex . The HDC activity was elevated by adding LPS and interleukin-1 beta (IL-1beta) but not by tumor necrosis factor-alpha (TNF-alpha) and IL-6 to the mixed primary cultures of diencephalon . In the adherent cell fraction of the cultured diencephalon cells, HDC activity was also enhanced by LPS and IL-1beta . In a similar manner, LPS augmented HDC activity in the mixed primary culture of cerebral cortical cells and in its adherent cell fraction . The effects of IL-1beta but not LPS in the mixed primary culture of diencephalon were canceled by a prior exposure to cytosine-beta-D-arabinofuranoside . The changes in HDC activity after exposure to LPS for 12 h were not accompanied by increased mRNA levels . In these cell cultures, mast cells were not detected by Alcian Blue staining . These results indicated the presence of the third type of HDC-bearing cell besides neurons and mast cells in the brain . The increase of HDC activity by IL-1beta might be due to cell proliferation. J Clin Microbiol, 1997 Aug, 35(8), 1952 - 8 HEp-2 cell adherence patterns, serotyping, and DNA analysis of Escherichia coli isolates from eight patients with AIDS and chronic diarrhea; Polotsky Y et al.; Three morphologic patterns of interaction between bacteria and enterocytes have been observed in colonic biopsy specimens from AIDS patients with chronic diarrhea in the United States . The DNA encoding virulence factors and the HEp-2 cell adherence patterns of Escherichia coli strains isolated from the stools of eight symptomatic AIDS patients were compared with those of five control strains with known adherence patterns . One clinical isolate from a patient with attaching-and-effacing enteropathy displayed the localized adherence attaching-and-effacing pattern typical of enteropathogenic E . coli on HEp-2 cells, five isolates displayed the "stacked-brick" aggregative adherence pattern typical of enteroaggregative E . coli strains, and one isolate showed the pattern characteristic of diffusely adherent E . coli . One patient's isolate displayed features of all three patterns . No clinical isolate hybridized with standard probes for enteropathogenic, enteroaggregative, diffusely adherent, enterotoxigenic, and enteroinvasive E . coli strains . Thus, isolates from symptomatic AIDS patients in the United States can display the same interactive patterns with HEp-2 cells as the agents of pediatric or traveler's diarrhea, but lack their typical virulence factors. Nucleic Acids Res, 1997 Aug 1, 25(15), 3183 - 5 Magnetic bead capture of cDNAs from double-stranded plasmid cDNA libraries; Shepard AR et al.; We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge . A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli . Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones . Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information. Nucleic Acids Res, 1997 Aug 1, 25(15), 3131 - 4 An episomal vector for stable tetracycline-regulated gene expression; Jost M et al.; The recently introduced tetracycline (Tc)-regulatable eukaryotic gene expression system based on the Escherichia coli Tn 10 tetracycline operon has proven to be a powerful tool for controlled expression of a variety of genes in vitro as well as in vivo . Control elements of this expression system are contained in two separate plasmid vectors . The tTA vector encodes a transactivator protein and the tetP vector contains a responsive operator-promoter element (tetP) that controls gene expression depending on tTA binding . Establishment of cell lines expressing a gene of interest under tetP control requires two subsequent rounds of transfection and clonal selection after each transfection . Here we describe a modification of this system in which the tetP element is placed in an episomal EBNA-based plasmid that can be stably maintained in primate but not in rodent cells . Using HeLa and human melanoma cells, we show that upon transient or stable transfection a reporter gene is expressed in a Tc-regulated manner similar to the original system . Thus, this expression system combines the advantages of episomal vectors, such as high efficiency of transfection and time-efficient selection of mass cultures, with tight control of gene expression provided by the Tc-regulatable system. Nucleic Acids Res, 1997 Aug 1, 25(15), 3009 - 16 A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats; Lin CT et al.; Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes) . This recombinational rearrangement event is efficient and independent of RecA and RecBCD . In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats . In this model, the inverted repeats promote the formation of a DNA cruciform which is processed by an endonuclease into a linear DNA with two hairpin loops at its ends . Upon DNA replication, this linear dumbbell-like DNA is then converted to the inverted dimer . In support of this model, linear dumbbell DNA molecules with unidirectional origin of DNA replication (ColE1 ori ) have been constructed and shown to transform E.coli efficiently resulting in the formation of the inverted dimer . The ability of linear dumbbell DNA to transform E.coli suggests that the terminal loops may be important in bypassing the requirement of DNA supercoiling for initiation of replication of the ColE1 ori. Nucleic Acids Res, 1997 Aug 1, 25(15), 2973 - 8 DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system; Suzuki N et al.; The gene responsible for Werner's syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family . However, no evidence has been shown before to substantiate this prediction . Here, we show data that the product of the WRN gene is indeed a DNA helicase . The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase . Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism . Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA-DNA heteroduplex, while the latter reaction seems less efficient . Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified. J Virol, 1997 Aug, 71(8), 5990 - 6 The role of the pseudoknot at the 3' end of turnip yellow mosaic virus RNA in minus-strand synthesis by the viral RNA-dependent RNA polymerase; Deiman BA et al.; The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro . Deletions in the 5'-to-3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency . However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template . tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV . Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure . The results were compared with those obtained in an earlier study on aminoacylation with the same mutants (R . M . W . Mans, M . H . van Steeg, P . W . G . Verlaan, C . W . A . Pleij, and L . Bosch, J . Mol . Biol . 223:221-232; 1992) . Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus . Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50% . No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found. J Virol, 1997 Aug, 71(8), 5723 - 32 Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease; Sheng N et al.; During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC . Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC . Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM . Binding was not affected by the presence or absence of zinc or EDTA . Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease . In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers . In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage . Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein . These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core. Biochem Biophys Res Commun, 1997 Jul 30, 236(3), 785 - 8 Isolation of a genetic locus associated with metronidazole resistance in Helicobacter pylori; Chang KC et al.; We examined the molecular mechanism of metronidazole resistance by constructing a lambda-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain of Helicobacter pylori . Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain of E . coli . Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain of E . coli . After MTZ selection, five clones could confer metronidazole resistance consistently . According to Southern hybridization and DNA sequencing, the five clones contained a same locus, recA . In addition, transforming the five clones into BL21 strain of E . coli produced a higher resistance to MTZ . Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitive H . pylori yielded MTZ resistant strains . Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus . Above results suggest that mutation in recA may be associated with metronidazole resistance of H . pylori. Biochem Biophys Res Commun, 1997 Jul 30, 236(3), 760 - 4 Effects of two hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on Ca2+ regulation of thin filament motility; Bing W et al.; The functional properties of wild type alpha-tropomyosin expressed in E . coli with an alanine-serine N-terminal leader (AS-alpha-Tm) were compared with those of AS-alpha-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC) . Wild type AS-alpha-Tm and AS-alpha-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-tropomyosin whilst the affinity of AS-alpha-Tm(Glu180Gly) was about threefold weaker . In vitro motility assays were performed with AS-alpha-tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin . Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-alpha-tropomyosin or mutant tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to <20% . Under activating conditions (pCa5), troponin had a minor effect upon actin-AS-alpha-tropomyosin filament velocity (increased by 5 +/- 1%, n=10), whereas the velocity increased by 18 +/- 3% (n=7) with actin filaments containing AS-alpha-tropomyosin(Asp175Asn) and by 21 +/- 2% (n=8) with filaments containing AS-alpha-tropomyosin(Glu180Gly) (p<0.05 compared with AS-alpha-tropomyosin) . Thus FHC mutations in alpha-tropomyosin produce detectable changes in the Ca2+-regulation of thin filaments, presumably via altered interaction with troponin. Biochemistry, 1997 Jul 29, 36(30), 9273 - 82 Reconstitution of the holoenzyme form of Escherichia coli porphobilinogen deaminase from apoenzyme with porphobilinogen and preuroporphyrinogen: a study using circular dichroism spectroscopy; Awan SJ et al.; Porphobilinogen deaminase (PBG-D), an early enzyme of the tetrapyrrole biosynthetic pathway, catalyzes the formation of a tetrapyrrole chain, preuroporphyrinogen, from four molecules of porphobilinogen (PBG) . The PBG-D apoenzyme is responsible for the autocatalytic synthesis and covalent attachment of a dipyrromethane cofactor at its active site . In this paper an efficient method for the purification of Escherichia coli PBG-D apoenzyme using an affinity chromatography resin is reported . Circular dichroism (CD) spectra of apoenzyme and holoenzyme were recorded and significant differences in both the backbone and aromatic region of the spectra were observed . The differences in the spectra allowed the reconstitution of holoenzyme from purified apoenzyme with PBG and preuroporphyrinogen in solution to be monitored separately by CD . Apoenzyme incubated with preuroporhyrinogen gave a CD spectrum that was much more like the CD spectrum of holoenzyme than apoenzyme incubated with PBG . The results showed clearly that the cofactor was generated much more rapidly from preuroporphyrinogen than from PBG . Changes in the CD spectrum associated with the aromatic side-chain region, in particular the contribution assigned to phenylalanine-62, were found to correlate well with the activity of the reconstituted enzyme . Phenylalanine-62 is located in close proximity to the cofactor and acts as a sensitive probe to active-site changes . The stability of the holoenzyme and apoenzyme were compared with respect to both heat and susceptibility to proteolysis . The results were consistent with a model for the apoenzyme in which, in the absence of the cofactor, the three domains of the protein are held less rigidly together, thereby making the protein more susceptible to heat denaturation and proteolysis . The CD spectrum of the holoenzyme was found to be similar at both pH 5.1 and 7.4, suggesting that the crystal structure, determined at pH 5.1, is likely to be similar at physiological pH values. Biochemistry, 1997 Jul 29, 36(30), 9185 - 94 Molecular basis for the coupling ion selectivity of F1F0 ATP synthases: probing the liganding groups for Na+ and Li+ in the c subunit of the ATP synthase from Propionigenium modestum; Kaim G et al.; The conserved glutamate residue at position 65 of the Propionigenium modestum c subunit is directly involved in binding and translocation of Na+ across the membrane . The site-specific introduction of the cQ32I and cS66A substitutions in the putative vicinity to cE65 inhibited growth of the single-site mutants on succinate minimal agar, indicating that both amino acid residues are important for proper function of the oxidative phosphorylation system . This growth inhibition was abolished, however, if the cF84L/cL87V double mutation was additionally present in the P . modestum c subunit . The newly constructed Escherichia coli strain MPC848732I, harboring the cQ32I/cF84L/cL87V triple mutation, revealed a change in the coupling ion specificity from Na+ to H+ . ATP hydrolysis by this enzyme was therefore not activated by NaCl, and ATP-driven H+ transport was not affected by this alkali salt . Both activities were influenced, however, by LiCl . These data demonstrate the loss of the Na+ binding site and retention of Li+ and H+ binding sites within this mutant ATPase . In the E . coli strain MPC848766A (cS66A/cF84L/cL87V), the specificity of the ATPase was further restricted to H+ as the exclusive coupling ion . Therefore, neither Na+ nor Li+ stimulated the ATPase activity, and no ATP-driven Li+ transport was observed . The ATPase of the E . coli mutant MPC32N (cQ32N) was activated by NaCl and LiCl . The mutant ATPase exhibited a 5-fold higher Km for NaCl but no change in the Km for LiCl in comparison to that of the parent strain . These results demonstrate that the binding of Na+ to the c subunit of P . modestum requires liganding groups provided by Q32, E65, and S66 . For the coordination of Li+, two liganding partners, E65 and S66, are sufficient, and H+ translocation was mediated by E65 alone. Biochemistry, 1997 Jul 29, 36(30), 9159 - 68 Inhibition of preprotein translocation and reversion of the membrane inserted state of SecA by a carboxyl terminus binding mAb; den Blaauwen T et al.; SecA is the peripheral subunit of the preprotein translocase of Escherichia coli . SecA consists of two independently folding domains, i.e., the N-domain bearing the high-affinity nucleotide binding site (NBS-I) and the C-domain that harbors the low-affinity NBS-II . ATP induces SecA insertion into the membrane during preprotein translocation . Domain-specific monoclonal antibodies (mAbs) were developed to analyze the functions of the SecA domains in preprotein translocation . The antigen binding sites of the obtained mAbs were confined to five epitopes . One of the mAbs, i.e., mAb 300-1K5, recognizes an epitope in the C-domain in a region that has been implicated in membrane insertion . This mAb, either as IgG or as Fab, completely inhibits in vitro proOmpA translocation and SecA translocation ATPase activity . It prevents SecA membrane insertion and, more strikingly, reverses membrane insertion and promotes the release of SecA from the membrane . Surface plasmon resonance measurements demonstrate that the mAb recognizes the ADP- and the AMP-PNP-bound state of SecA either free in solution or bound at the membrane at the SecYEG protein . It is concluded that the mAb actively reverses a conformation essential for membrane insertion of SecA . The other mAbs directed to various epitopes in the N-domain were found to be without effect, although all bind the native SecA . These results demonstrate that the C-domain plays an important role in the SecA membrane insertion, providing further evidence that this process is needed for preprotein translocation. Biochemistry, 1997 Jul 29, 36(30), 9145 - 50 Evaluation of functionally important amino acids in L-aspartate ammonia-lyase from Escherichia coli; Jayasekera MM et al.; The high-resolution structure of l-aspartate ammonia-lyase from Escherichia coli has recently been determined {Shi, W., Dunbar, J., Jayasekera, M . M . K., Viola, R . E., & Farber, G . K . (1997) Biochemistry 36, 9136-9144} . An examination of the putative active site has been carried out, with the active site located in a cleft that contains the functionally significant lysine 327 . A list of potential active site residues has been generated based on their proximity to this active site lysine, sequence homology comparisons with other members of the aspartase-fumarase enzyme family, and the necessity for chemically reasonable functionalities for the proposed roles . The five most likely candidates in the putative active site cleft have been examined by site-directed mutagenesis to test their feasibility for either substrate binding or acid-base catalytic roles . Arginine and lysine residues have been identified that appear to function in the orientation and binding of aspartic acid at the enzyme active site . Some tentative assignments have also been made of the acid and base catalytic groups that are proposed to be involved in the deamination reaction. Biochemistry, 1997 Jul 29, 36(30), 9136 - 44 The structure of L-aspartate ammonia-lyase from Escherichia coli; Shi W et al.; The X-ray crystal structure of l-aspartate ammonia-lyase has been determined to 2.8 A resolution . The enzyme contains three domains, and each domain is composed almost completely of alpha helices . The central domain is composed of five long helices . In the tetramer, these five helices form a 20-helix cluster . Such clusters have also been seen in delta-crystallin and in fumarase . The active site of aspartase has been located in a region that contains side chains from three different subunits . The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate . These residues have been examined by site-directed mutagenesis, and their putative roles have been assigned {Jayasekera, M . M . K., Shi, W., Farber, G . K., & Viola, R . E . (1997) Biochemistry 36, 9145-9150}. Biochemistry, 1997 Jul 29, 36(30), 9093 - 100 EPR study of the mixed-valent diiron sites in mouse and herpes simplex virus ribonucleotide reductases . Effect of the tyrosyl radical on structure and reactivity of the diferric center; Davydov RM et al.; Reduction of ribonucleotide reductase (EC 1.17.4.1) R2 proteins in a frozen glycerol-buffer solution at 77 K by mobile electrons generated by gamma-irradiation produces EPR-detectable iron sites in mixed-valent Fe(II)/Fe(III) states . The primary EPR signals give information about the ligand arrangement of the diferric form of the iron site, whereas secondary signals observed after annealing of the sample show the effects of structural relaxation . In recombinant metR2 proteins (without free radical) from mouse and herpes virus type 1, the mixed-valent sites trapped at 77 K give rise to axial S = 1/2 EPR spectra with g values in the range 1.79-1.94, observable at temperatures up to 110 K . The spectra are assigned to mu-oxo-bridged dinuclear iron sites . In mouse metR2, the primary EPR spectrum is a mixture of two components . Annealing the R2 samples to 160-170 K transforms the primary EPR signals into rhombic spectra, characterized by gav < 1.8, and observable only below 25 K . These spectra are assigned to partially relaxed forms with a mu-hydroxo bridge, formed by protonation of the oxo bridge . Further annealing at 220 K produces new rhombic EPR spectra, which are closely similar with those observed and found to be stable after chemical reduction at room temperature . The EPR signal of the primary mixed-valent iron site in active mouse R2 protein with a tyrosyl radical also has two components . Both are different from those observed in metR2 . In herpes simplex virus type 1 protein R2, one primary mixed-valent component was observed for the met protein . The dose-yield curve for the mixed-valent state in active mouse R2 is sigmoidal in shape, indicating that the tyrosyl radical is reduced by mobile electrons before the iron site . Kinetic experiments on the reduction by dithionite on mouse R2 without and with radical show a significantly enhanced rate for reduction of the iron site in the protein without radical . The results suggest that in active mouse R2 only complete diferric sites with neighboring radicals give rise to the mixed-valent spectra, and that these sites may exist in two structurally distinct forms . The results on the mouse R2 proteins confirm and extend previous results obtained on the Escherichia coli protein R2 showing that the presence of the tyrosyl radical significantly affects not only the structure but also the reactivity of the iron site. FEBS Lett, 1997 Jul 28, 412(2), 359 - 64 The complete mature bovine prion protein highly expressed in Escherichia coli: biochemical and structural studies; Negro A et al.; According to the 'protein only' hypothesis, modification of the 3-dimensional fold of the constituent cellular protein, PrP(C), into the disease-associated isoform, PrP(Sc), is the cause of neurodegenerative diseases in animals and humans . Here we describe the high-level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine-tagged full-length mature PrP (25-249) of bovine brain, termed His-PrP . Based on biochemical and spectroscopic data, His-PrP displays characteristics expected for the PrP(C) isoform . The reported expression system should allow the production of quantities of bovine PrP(C) sufficient to permit 3-dimensional structure determinations. FEBS Lett, 1997 Jul 28, 412(2), 331 - 6 Mapping the ubiquitin-binding domains in the p54 regulatory complex subunit of the Drosophila 26S protease; Haracska L et al.; Short-lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease . This large ATP-dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome . The selective recognition of ubiquitinated proteins is ensured by the regulatory complex . Using an overlay assay a single 54-kDa multiubiquitin-chain-binding subunit was detected in the regulatory complex of the Drosophila 26S protease . Overlay assay with the recombinant p54 subunit confirmed its ubiquitin-binding property . The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates . To map the ubiquitin-binding domain of the p54 subunit different segments of the recombinant protein were expressed in E . coli and tested by the overlay assay . The p54 subunit carries two independent ubiquitin-binding domains . The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast . In the C-terminal ubiquitin-binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes . This domain, however, due to the shorter size of the yeast multiubiquitin-binding subunit, is present only in higher eukaryotes. FEBS Lett, 1997 Jul 28, 412(2), 318 - 20 The 58 kDa mouse selenoprotein is a BCNU-sensitive thioredoxin reductase; Gromer S et al.; The flavoprotein thioredoxin reductase {EC 1.6.4.5} (NADPH + H+ + thioredoxin-S2 --> NADP+ + thioredoxin-(SH)2) was isolated from mouse Ehrlich ascites tumour (EAT) cells . Like the counterpart from human placenta but unlike the known thioredoxin reductases from non-vertebrate organisms, the mouse enzyme was found to contain 1 equivalent of selenium per subunit of 58 kDa . The K(M) values were 4.5 microM for NADPH, 480 microM for DTNB and 36 microM for Escherichia coli thioredoxin, the turnover number with DTNB being approximately 40 s(-1) . As mouse is a standard animal model in cancer and malaria research, thioredoxin reductase and glutathione reductase {EC 1.6.4.2} from EAT cells were compared with each other . While both enzymes in their 2-electron reduced form are targets of the cytostatic drug carmustine (BCNU), no immunologic cross-reactivity between the two mouse disulfide reductases was observed. J Cell Biol, 1997 Jul 28, 138(2), 349 - 61 Talin-null cells of Dictyostelium are strongly defective in adhesion to particle and substrate surfaces and slightly impaired in cytokinesis; Niewohner J et al.; Dictyostelium discoideum contains a full-length homologue of talin, a protein implicated in linkage of the actin system to sites of cell-to-substrate adhesion in fibroblasts and neuronal growth cones . Gene replacement eliminated the talin homologue in Dictyostelium and led to defects in phagocytosis and cell-to-substrate interaction of moving cells, two processes dependent on a continuous cross talk between the cell surface and underlying cytoskeleton . The uptake rate of yeast particles was reduced, and only bacteria devoid of the carbohydrate moiety of cell surface lipopolysaccharides were adhesive enough to be recruited by talin-null cells in suspension and phagocytosed . Cell-to-cell adhesion of undeveloped cells was strongly impaired in the absence of talin, in contrast with the cohesion of aggregating cells mediated by the phospholipid-anchored contact site A glycoprotein, which proved to be less talin dependent . The mutant cells were still capable of moving and responding to a chemoattractant, although they attached only loosely to a substrate via small areas of their surface . With their high proportion of binucleated cells, the talin-null mutants revealed interactions of the mitotic apparatus with the cell cortex that were not obvious in mononucleated cells. J Mol Biol, 1997 Jul 25, 270(4), 574 - 86 Molecular properties of complexes formed between the prion protein and synthetic peptides; Kaneko K et al.; Complexes of the Syrian hamster cellular prion protein (PrPC) and synthetic Syrian hamster PrP peptides were found to mimic many of the characteristics of the scrapie PrP isoform (PrPSc) . Either PrPC expressed in chinese hamster ovary (CHO) cells or a C-terminal fragment of 142 residues of recombinant PrP protein (rPrP) produced in Escherichia coli was mixed with an excess of a synthetic 56 amino acid peptide, denoted PrP(90-145) . Complex formation required PrPC or rPrP to be destabilized by guanidine hydrochloride (GdnHCl) or urea and PrP(90-145) to be in a coil conformation; it was enhanced by an acidic environment, salt and detergent . If PrP(90-145) was in a beta-sheet conformation, then no complexes were formed . While complex formation was rapid, acquisition of protease resistance was a slow process . Amorphous aggregates with a PrPC/PrP(90-145) ratio of 1:1 were formed in phosphate buffer, whereas fibrils with a diameter of approximately 10 nm and a PrPC/PrP(90-145) ratio of 1:5 were formed in Tris buffer . The complexes were stable only in the presence of excess peptide in either the coil or beta-sheet conformation; they dissociated rapidly after centrifugation and resuspension in buffer without peptide . Neither a peptide having a similar hydrophobicity profile/charge distribution to PrP(90-145) nor a scrambled version, denoted hPrP(90-145) and sPrP(90-145), respectively, were able to induce complex formation . Although hPrP(90-145) could stabilize the PrPC/PrP(90-145) complexes, sPrP(90-145) could not . Studies of PrPC/peptide complexes may provide insights into how PrPC interacts with PrPSc during the formation of a nascent PrPSc molecule and into the process by which PrPC is converted into PrPSc. J Mol Biol, 1997 Jul 25, 270(4), 544 - 50 Growth rate-optimised tRNA abundance and codon usage; Berg OG et al.; The abundance of different tRNAs in Escherichia coli at different growth rates correlates strongly with the usage of the corresponding cognate codons . On the assumption that the investment in the translation system is optimised to provide a maximal growth rate, the relationship between tRNA levels and codon usage can be predicted . When the complications due to different degeneracies and different association rate constants for the different tRNA-codon combinations are accounted for, recent data from the literature indicate that the predicted relations hold up very well: the tRNA levels correlate with codon frequencies in a way that would support a maximal growth rate . The relations can also be used to predict the association rate constant between an A-site codon and the cognate ternary complex . In the cases where they can be compared, the results agree reasonably well with experimental results from the literature. Cell, 1997 Jul 25, 90(2), 207 - 16 Microtubule interaction site of the kinesin motor; Woehlke G et al.; Kinesin and myosin are motor proteins that share a common structural core and bind to microtubules and actin filaments, respectively . While the actomyosin interface has been well studied, the location of the microtubule-binding site on kinesin has not been identified . Using alanine-scanning mutagenesis, we have found that microtubule-interacting kinesin residues are located in three loops that cluster in a patch on the motor surface . The critical residues are primarily positively charged, which is consistent with a primarily electrostatic interaction with the negatively charged tubulin molecule . The core of the microtubule-binding interface resides in a highly conserved loop and helix (L12/alpha5) that corresponds topologically to the major actin-binding domain of myosin . Thus, kinesin and myosin have developed distinct polymer-binding domains in a similar region with respect to their common catalytic cores. Atherosclerosis, 1997 Jul 25, 132(2), 165 - 76 Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2; Eckey R et al.; In vitro-studies have shown that phospholipid hydrolysis of low density lipoproteins (LDL) by bee venom or porcine pancreatic phospholipase A2 (PLA2) leads to an increased uptake of these lipoproteins by macrophages transforming them into foam cells . Recently, a secretory phospholipase A2, group II, was detected in human atherosclerotic plaques . In order to investigate the role of this enzyme in the pathogenesis of atherosclerosis, a structurally identical human secretory PLA2 was purified from the medium of HepG2 cells stimulated with interleukin-6 and tumor necrosis factor-alpha . The activity of the purified enzyme towards the phospholipids of native and modified low density lipoproteins was compared with the activity towards Escherichia coli-membranes and other phospholipid substrates . Compared to E . coli-membranes, native LDL proved to be a poor substrate for group II PLA2 . After mild oxidation induced by copper ions or by 2,2-azobis(2-amidinopropane) (AAPH), the susceptibility of LDL to phospholipid hydrolysis was found to be increased by 25 and 23%, respectively, whereas extensive copper-mediated oxidation caused a decreased hydrolysis . Aging of LDL at 6 degrees C for weeks or at 37 degrees C for hours resulted in an increase in PLA2-catalyzed phospholipid hydrolysis of up to 26-fold . LDL protected from oxidation by probucol during aging showed a lesser increase in susceptibility to phospholipid hydrolysis . Our results suggest that PLA2, group II, can increase the atherogenicity of LDL by its ability to hydrolyze the phospholipids of these lipoproteins, especially after modifications that are likely to occur in vivo. J Biol Chem, 1997 Jul 25, 272(30), 18939 - 44 N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat; Lutsenko S et al.; N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized . Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals . When E . coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein . Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents . This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains . Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper . Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins). J Biol Chem, 1997 Jul 25, 272(30), 18869 - 74 Identification of CDK4 sequences involved in cyclin D1 and p16 binding; Coleman KG et al.; Activation of CDK4 is regulated, in part, by its association with a D-type cyclin . Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16(INK4A) . To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16(INK4A) we performed site-directed mutagenesis of CDK4 . The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins . As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus . In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated . Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus . All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding . In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding. J Biol Chem, 1997 Jul 25, 272(30), 18686 - 93 Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core; Akasako A et al.; Escherichia coli ribonuclease HI has a cavity within the hydrophobic core . Two core residues, Ala52 and Val74, resided at both ends of this cavity . We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu . All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified . Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains . A correlation was observed between the protein stability and the hydrophobicity of the substituted residue . As a result, a number of the mutant proteins were more stable than the wild-type protein . The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins . However, examination of the far- and near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein . These results suggest that the cavity in the hydrophobic core of E . coli RNase HI is conformationally fairly stable . This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein. J Biol Chem, 1997 Jul 25, 272(30), 18614 - 20 Mutation of a highly conserved arginine in motif IV of Escherichia coli DNA helicase II results in an ATP-binding defect; Hall MC et al.; A site-directed mutation in motif IV of Escherichia coli DNA helicase II (UvrD) was generated to examine the functional significance of this region . The highly conserved arginine at position 284 was replaced with alanine to construct UvrD-R284A . The ability of the mutant allele to function in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair was examined by genetic complementation assays . The R284A substitution abolished function in both DNA repair pathways . To identify the biochemical defects responsible for the loss of biological function, UvrD-R284A was purified to apparent homogeneity, and its biochemical properties were compared with wild-type UvrD . UvrD-R284A failed to unwind a 92-base pair duplex region and was severely compromised in unwinding a 20-base pair duplex region . The Km of UvrD-R284A for ATP was significantly greater than 3 mM compared with 80 microM for UvrD . A large decrease in ATP binding was confirmed using a nitrocellulose filter binding assay . These data suggested that the R284A mutation severely reduced the affinity of helicase II for ATP . The reduced unwinding activity and loss of biological function of UvrD-R284A was probably the result of decreased affinity for ATP . These results implicate motif IV of superfamily I helicases in nucleotide binding and represent the first characterization of a helicase mutation outside motifs I and II that severely impacted the Km for ATP. J Biol Chem, 1997 Jul 25, 272(30), 18602 - 7 Activation of R-Ras by Ras-guanine nucleotide-releasing factor; Gotoh T et al.; Ras-GRF/CDC25(Mm), mSos, and C3G have been identified as guanine nucleotide-releasing factors for Ras family proteins . We investigated in this study the guanine nucleotide-releasing activities of Ras-GRF, mSos, and C3G toward R-Ras, which shows high sequence similarity to Ras . Ras-GRF markedly stimulated the dissociation of GDP from R-Ras, and C3G also promoted the release of R-Ras-bound GDP . Under the same conditions, mSos little affected the reaction . When Ras-GRF and R-Ras were coexpressed in COS7 cells, the remarkable accumulation of the active GTP-bound form of R-Ras was observed . C3G also increased active R-Ras in COS7 cells, while mSos did not give any effect . These results indicated that Ras-GRF and C3G could activate R-Ras . Furthermore, the activation of R-Ras by Ras-GRF was enhanced when cells were treated with ionomycin, which is known to increase the intracellular calcium concentration . The examination of tissue distribution of R-Ras, Ras-GRF, and mSos by the reverse transcription-polymerase chain reaction revealed that Ras-GRF was expressed only in brain and testis, whereas R-Ras, C3G, and mSos were expressed rather ubiquitously . These findings raise the possibility that R-Ras is activated by Ras-GRF in brain and testis, and by C3G in other tissues, respectively. J Biol Chem, 1997 Jul 25, 272(30), 18550 - 7 Combinatorial mutations of lac repressor . Stability of monomer-monomer interface is increased by apolar substitution at position 84; Nichols JC et al.; To examine the monomer-monomer subunit interface in the lac repressor, a mutation that generates dimeric protein (deletion of C-terminal amino acids to disrupt the dimer-dimer interface) has been combined with amino acid substitutions that alter the monomer-monomer interface (substitution at Lys84 or Tyr282) . Dimeric proteins with significantly increased stability to urea denaturation were formed by the introduction of the apolar amino acids Ala or Leu in lieu of Lys84 in concert with the deletion of 11 C-terminal amino acids . K84A/-11 deletion protein retained wild-type affinity for operator DNA, while K84L/-11 deletion protein displayed operator affinity similar to its parent tetramer . To assess further the influence of monomer-monomer interface stability on assembly and DNA binding, triple mutants were generated with Y282D, an alteration that disrupts assembly completely in the wild-type background . The triple mutants were dimeric, but they exhibited diminished dimer stability to urea denaturation and decreased operator affinity compared with the double mutations . These results demonstrate directly the stabilizing influence of apolar substitution at position 84 on the monomer-monomer interface. Science, 1997 Jul 25, 277(5325), 574 - 8 Mitosis in living budding yeast: anaphase A but no metaphase plate; Straight AF et al.; Chromosome movements and spindle dynamics were visualized in living cells of the budding yeast Saccharomyces cerevisiae . Individual chromosomal loci were detected by expression of a protein fusion between green fluorescent protein (GFP) and the Lac repressor, which bound to an array of Lac operator binding sites integrated into the chromosome . Spindle microtubules were detected by expression of a protein fusion between GFP and Tub1, the major alpha tubulin . Spindle elongation and chromosome separation exhibited biphasic kinetics, and centromeres separated before telomeres . Budding yeast did not exhibit a conventional metaphase chromosome alignment but did show anaphase A, movement of the chromosomes to the poles. Nature, 1997 Jul 24, 388(6640), 355 - 8 On/off blinking and switching behaviour of single molecules of green fluorescent protein; Dickson RM et al.; Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging . Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria . Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo . GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies . Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm . This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level. Nature, 1997 Jul 24, 388(6640), 343 - 9 Recombination of protein domains facilitated by co-translational folding in eukaryotes; Netzer WJ et al.; The evolution of complex genomes requires that new combinations of pre-existing protein domains successfully fold into modular polypeptides . During eukaryotic translation model two-domain polypeptides fold efficiently by sequential and co-translational folding of their domains . In contrast, folding of the same proteins in Escherichia coli is posttranslational, and leads to intramolecular misfolding of concurrently folding domains . Sequential domain folding in eukaryotes may have been critical in the evolution of modular polypeptides, by increasing the probability that random gene-fusion events resulted in immediately foldable protein structures. Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8016 - 20 Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase; Roldan-Arjona T et al.; The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7, 8-dihydroguanine) . In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage . We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene . The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine . Expression of the human protein in a DNA repair-deficient E . coli mutM mutY strain partly suppresses its spontaneous mutator phenotype . The gene encoding the human enzyme maps to chromosome 3p25 . These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen. Biochemistry, 1997 Jul 22, 36(29), 9013 - 21 Regulation of oxidation-reduction potentials through redox-linked ionization in the Y98H mutant of the Desulfovibrio vulgaris {Hildenborough} flavodoxin: direct proton nuclear magnetic resonance spectroscopic evidence for the redox-dependent shift in the pKa of Histidine-98; Chang FC et al.; Flavodoxin from Desulfovibrio vulgaris is a low molecular weight (15 000 Da) acidic flavoprotein that contains a single flavin mononucleotide (FMN) cofactor . A distinguishing feature of the flavodoxin family is the exceptionally low midpoint potential of the semiquinone/hydroquinone couple . Tyrosine-98, which flanks the outer or si face of the FMN, plays an important role in establishing the oxidation-reduction properties of the bound cofactor as demonstrated by the substitution of a number of amino acids at this position {Swenson, R . P., & Krey, G . D . (1994) Biochemistry 33, 8505-8514} . The midpoint potential for the semiquinone/hydroquinone couple increases substantially when basic residues are introduced at this position . The pH dependency in the Y98H mutant is consistent with a redox-linked ionization model in which the favorable electrostatic coupling between the imidazolium cation and the flavin hydroquinone anion is responsible for the higher potential . Such a model predicts an increase in the pKa of 1.5 units for His98 upon complete reduction of the FMN . In this study, proton nuclear magnetic resonance spectroscopy was used to directly determine the intrinsic pKa of His98 as a function of the redox state of the cofactor in this flavodoxin . Values for the pKa of His98 in the oxidized and fully reduced flavodoxin are 7.02 +/- 0.08 and 8.43 +/- 0.11, respectively, an increase in the pKa by 1.41 units, which conforms with the previous prediction . These results provide direct experimental proof of the redox-linked ionization of this residue and provides further evidence of the crucial role of electrostatic interactions, in this case, in the stabilization of the flavin hydroquinone anion . This phenomenon may represent a general mechanism in the modulation of the reduction potential of the flavin cofactor within flavoenzymes in which ionizable groups such as histidine in the active center change ionization states during the catalytic cycle. Biochemistry, 1997 Jul 22, 36(29), 8962 - 8 Mutational analysis of an antigenic peptide shows recognition in a loop conformation; Rondard P et al.; We have analyzed the recognition between an antigenic undecapeptide and a monoclonal antibody through a mutational approach . Antibody mAb164 is directed against the native form of the TrpB2 subunit of Escherichia coli tryptophan synthase . It recognizes a synthetic peptide, P11, constituted of residues 273-HGRVGIYFGMK-283 of TrpB with high affinity . P11 was fused with a carrier protein, MalE, to facilitate its manipulation . The affinities between mAb164 and the MalE-P11 hybrids were measured by competition enzyme-linked immunosorbent assay (ELISA) . The changes of the P11 residues into progressively shorter residues, the comparison of changes into Pro and Ala, and the study of double mutants showed the following . Four hydrophobic residues of P11, Val276, Ile278, Tyr279, and Phe280, were predominant in the interaction . For some residues, e.g., Tyr279, most groups of the side chain contributed to the interaction . For others, only some groups played a significant role, e.g., the Cdelta group of Ile278 or the Cbeta group of Phe280 . The lack of side chain in position Gly281 and a tertiary interaction between the side chains of Ile278 and Lys283 were important . P11 was recognized in a loop conformation, close to that of residues 273-283 of TrpB in the crystal structure of the complete tryptophan synthase, TrpA2TrpB2 . Comparison of our mutational data with NMR data on the conformation of the isolated peptide P11 and with kinetic data on its interaction with mAb164 indicate that mAb164 selects a conformer of P11 that represents only a small minority of the molecules . Our results provide useful information on the mechanisms by which linear epitopes and unconstrained peptides are recognized by receptors. Biochemistry, 1997 Jul 22, 36(29), 8954 - 61 Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein; Rondard P et al.; Monoclonal antibody mAb164 is directed against the TrpB2 subunit of the Escherichia coli tryptophan synthase . It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affinity . We constructed a hybrid protein in which the C-terminal end of protein MalE was linked with the N-terminal end of P11 . Hybrid MalE-P11 was produced in E . coli from a plasmidic gene and purified in one step as MalE . MalE-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria . The NMR spectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 moiety of MalE-P11 moved independently from its MalE moiety . The chemical shifts of the protons for the P11 moiety of MalE-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values . The equilibrium constant of dissociation (KD) from mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM . The change of the C-terminal residue of MalE-P11 from Lys into Ala increased 37-fold this dissociation constant . This increase showed that the P11 moiety of MalE-P11 was not degraded . The high molecular mass of MalE-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon resonance, using the BIAcore apparatus . The rates of association with mAb164 were similar for MalE-P11 and TrpB2, but the dissociation was faster for MalE-P11 than for TrpB2, as previously observed for the isolated P11 by a fluorometric method . Thus, the fusion of peptides with the C-terminal end of MalE could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro. Biochemistry, 1997 Jul 22, 36(29), 8923 - 31 Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase; Whittaker MM et al.; Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions . Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex . Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands . However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands . In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme . This pH-coupled transition shifts once more to a higher effective pKa for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme . Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions . These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands. Biochemistry, 1997 Jul 22, 36(29), 8849 - 57 Conformational changes in G-CSF/Receptor complex as investigated by isotope-edited FTIR spectroscopy; Li T et al.; Conformations of G-CSF and the extracellular domain of its receptor as well as their complex have been investigated by employing isotope-edited FTIR spectroscopy . To determine unambiguously the protein conformations of G-CSF and the receptor in the complex, we have prepared uniformly 13C/15N isotope labeled G-CSF to resolve its amide I' band from that of the receptor in the IR spectrum of the complex . By comparing the IR spectra of the isotope-labeled G-CSF and the receptor with that of the complex, we have provided spectral evidence that the AB loop region involving the unique 310 helix segment of G-CSF likely undergoes a conformational change to a regular alpha-helix upon binding to the receptor . The IR data also indicate a possible minor increase in alpha-helical conformation for the receptor in the complex . Furthermore, FTIR spectra of G-CSF, the receptor, and their complex demonstrate clearly that protein conformations of both G-CSF and the receptor have been dramatically stabilized by complex formation . Specifically, the melting transition (Tm value) of the alpha-helix in G-CSF is increased by nearly 30 degrees C and that of the beta-strand in the receptor by nearly 15 degrees C in the G-CSF/receptor complex . We estimate from the current FTIR data that the native conformations of approximately 15% of all receptor residues are stabilized by G-CSF binding . On the other hand, the entire alpha-helical content of G-CSF appears to be stabilized in the complex . Together, these results indicate that formation of the ligand/receptor complex results in not only conformational changes in the receptor but also significant structural changes in the ligand . This adds insight to the general consensus that binding of ligand to cytokine receptors induces mostly structural changes in the receptor which lead to receptor oligomerization and signal transduction . The current data also suggest a possible physiological role of the 310 helix present in G-CSF for its receptor binding activity. Biochemistry, 1997 Jul 22, 36(29), 8831 - 9 Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis; He YA et al.; Cassette mutagenesis and site-directed mutagenesis were used to investigate the importance of individual amino acid residues at positions 364-377 of cytochrome P450 3A4 in determining steroid hydroxylation or stimulation by alpha-naphthoflavone . The mutants were expressed in an Escherichia coli system, and solubilized membranes were prepared . All mutants except R365G and R365K exhibited anti-3A immunoreactivity on Western blotting, although R372S and R375K were not detected as the Fe2+-CO complex . Replacement of Arg-372 by Lys yielded a typical P450 spectrum . The results indicate that the highly conserved Arg residues at positions 365 and 375 may play a role in stabilizing the tertiary structure or in heme binding . Catalytic activities of 12 mutants were examined using progesterone and testosterone as substrates, and residues 369, 370, and 373 were found to play an important role in determining substrate specificity . Although the three mutants hydroxylated progesterone and testosterone primarily at the 6beta-position like the wild-type, replacement of Ile-369 by Val suppressed progesterone 16alpha-hydroxylase activity, whereas substitution of Ala-370 with Val enhanced progesterone 16alpha-hydroxylation . Interestingly, substitution of Leu-373 with His resulted in production of a new metabolite from both steroids . Moreover, the mutants at positions 369 and 373 were more and less responsive, respectively, than the wild-type to alpha-naphthoflavone stimulation . Alterations in activities or expression of several mutants were interpreted using a three-dimensional model of P450 3A4 . The results suggest that analogy with mammalian family 2 and bacterial cytochromes P450 can be used to predict P450 3A residues that contribute to regiospecific steroid hydroxylation. Biochemistry, 1997 Jul 22, 36(29), 8807 - 20 Productive interactions between the two domains of pig heart CoA transferase during folding and assembly; Rochet JC et al.; The enzyme CoA transferase from porcine heart (EC 2.8.3.5) is a homodimer; each subunit consists of two domains linked by a hydrophilic "hinge" region . We have prepared separate DNA segments encoding each of these domains . Incorporation of these two DNA segments within an operon or within two separate transcription units does not preclude the synthesis and assembly of CoA transferase in Escherichia coli . When the two domain fragments are produced and purified individually from separate cultures and subsequently mixed, enzyme activity accumulates to near wild-type levels only after a lengthy incubation . Each domain is more susceptible to aggregation than wild-type CoA transferase . Circular dichroism shows that, prior to mixing, the domains possess a different secondary structural profile compared to their counterparts in the native enzyme . However, mixing and incubation of the domains produces a complex with far-UV CD, fluorescence, and ultracentrifugation properties similar to those of wild-type CoA transferase . Finally, we show that the intact hydrophilic peptide which links the two domains is essential for the recovery of activity observed upon refolding of the denatured enzyme in vitro . These results indicate that the folding and assembly of pig heart CoA transferase require a productive interaction between its two domains, involving a substantial conformational rearrangement. Biochemistry, 1997 Jul 22, 36(29), 8755 - 66 Definition of the switch surface in the solution structure of Cdc42Hs; Feltham JL et al.; Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility . Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins . Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement . These structures show overall similarity to those of other GTP-binding proteins, with some exceptions . The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an alpha-helix in switch II . The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the beta4-alpha3 loop . The insert forms one edge of a "switch surface" and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP . These studies indicate the insert region forms a stable invariant "footrest" for docking of regulatory and effector proteins. Biochemistry, 1997 Jul 22, 36(29), 8699 - 709 Interaction of tRNA with tRNA (guanosine-1)methyltransferase: binding specificity determinants involve the dinucleotide G36pG37 and tertiary structure; Redlak M et al.; The sequence G37pG36 is present in all tRNA species recognized and methylated by the Escherichia coli modification enzyme tRNA (guanosine-1)methyltransferase . We have examined whether this dinucleotide sequence provides the base specific recognition signal for this enzyme and have assessed the role of the remaining tRNA in recognition . E . coli tRNAHis and yeast tRNAAsp were substituted with G at positions 36 and 37 and were found to be excellent substrates for methylation . This suggested that the general tRNA structure can be specifically bound by the enzyme . In addition, heterologous tRNA species including fully modified tRNA1Leu are excellent inhibitors of tRNA1Leu transcript methylation . Analyses of structural variants of yeast tRNAAsp and E . coli tRNA1Leu demonstrate clearly that the core tertiary structures of tRNA are required for recognition and that G37 must be in the correct position in space relative to important contacts elsewhere in the molecule . This latter conclusion was reached because the addition of one to three stacked base pairs in the anticodon stem of tRNA1Leu dramatically alters activity . In this case, the G37 base is rotated away from the correct position in space relative to other tRNA contact sites . The acceptor stem structure is required for optimal activity since deletion of three or five base pairs is detrimental to activity; however, specific base sequence may not be important because (i) the addition of three stacked base pairs of different sequence had little effect on activity and (ii) heterologous tRNAs with little or no sequence homology in the acceptor stem are excellent substrates . Both poly G and GpG are potent and specific inhibitors of enzyme activity and are minimal substrates which can be methylated, forming m1G . Taken together, these studies suggest that 1MGT can bind the general tRNA structure and that the crucial base-pair contacts are G37 and G36. FEBS Lett, 1997 Jul 21, 412(1), 169 - 72 Binding of TNP-ATP and TNP-ADP to the non-catalytic sites of Escherichia coli F1-ATPase; Weber J et al.; Using site-directed-tryptophan fluorescence, parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to non-catalytic sites of Escherichia coli F1-ATPase were determined . All three non-catalytic sites showed the same affinity for MgTNP-ATP (Kd = 0.2 microM) or MgTNP-ADP (Kd = 6.5 microM) whereas even at concentrations of 100 microM no binding of uncomplexed TNP-ATP or TNP-ADP was observed . The results demonstrate that the three non-catalytic sites bind TNP-nucleotides non-cooperatively, and emphasize the importance of Mg2+ for non-catalytic-site nucleotide binding . Parameters for binding of (Mg)TNP-ADP to the three catalytic sites were also determined, and showed marked cooperativity . This work completes the set of thermodynamic parameters for equilibrium binding of (Mg)TNP-ATP and (Mg)TNP-ADP to all six nucleotide sites of F1, providing essential information to fully exploit the potential of these nucleotide analogs in studies of F1-ATPase. FEBS Lett, 1997 Jul 21, 412(1), 138 - 40 The glyoxysomal 3-ketoacyl-CoA thiolase precursor from Brassica napus has enzymatic activity when synthesized in Escherichia coli; Olesen C et al.; Glyoxysomal 3-ketoacyl-CoA thiolase is the last enzyme in the beta-oxidation of fatty acids in plant glyoxysomes . A full-length cDNA of the glyoxysomal 3-ketoacyl-CoA thiolase from Brassica napus and a truncated version, lacking the N-terminal targeting signal were cloned in a T7 promoter-based vector . Both recombinant proteins were expressed in Escherichia coli and activity was measured . Full-length and truncated 3-ketoacyl-CoA thiolase have comparable activity in E . coli . Moreover, full-length 3-ketoacyl-CoA thiolase was purified from E . coli and N-terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active. FEBS Lett, 1997 Jul 21, 412(1), 126 - 30 Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli; Sturla L et al.; GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-D-mannose to GDP-L-fucose in bacteria, plants and mammals . Recently, the gene coding for GMD has been identified and sequenced in E . coli . Based on this sequence, we have expressed and purified GMD in E . coli as a glutathione transferase (GST) fusion protein . The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized . The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively . The GST-GMD fusion protein has a Km of 0.22 +/- 0.04 mM and a specific activity of 2.3 +/- 0.2 micromol/h/mg . Ca2+ and Mg2+ activate GMD, while GDP-L-beta-fucose, the end-product of the pathway, inhibits it specifically . The GST-GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer . Apparently, this NADP+ is involved in the catalytic mechanism of GMD. FEBS Lett, 1997 Jul 21, 412(1), 115 - 20 Transferred nuclear Overhauser effect spectroscopy study of a peptide from the PapG pilus subunit bound by the Escherichia coli PapD chaperone; Walse B et al.; Interaction of the Escherichia coli PapD chaperone with the synthetic peptide PapG308-314 (Thr-Met-Val-Leu-Ser-Phe-Pro), corresponding to the seven C-terminal residues of the PapG pilus subunit, was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy . The observation of cross-peaks corresponding to either intraresidue or sequential C(alpha)H/NH and C(beta)H/NH TRNOEs and the absence of sequential NH(i)/NH(i+1) TRNOEs indicate that the peptide binds to PapD in an extended conformation . In addition, line-broadening effects gave information of the peptide's mode of interaction with PapD . These observations were in excellent agreement with a recent crystal structure of a PapG peptide complexed with PapD. FEBS Lett, 1997 Jul 21, 412(1), 57 - 60 Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease; Yoo SJ et al.; HslVU in E . coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes . To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr . Also each of the five internal Ser residues in HslV were replaced with Ala . The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease . The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation. FEBS Lett, 1997 Jul 21, 412(1), 48 - 52 A novel domain of fibronectin revealed by epitope mapping of a monoclonal antibody which inhibits fibroblasts-mediated collagen gel contraction; Obara M et al.; The ability of cells to organize collagen fibrils is fundamental to a variety of processes found in embryogenesis, wound healing, fibrosis, and scar formation . We previously isolated a monoclonal antibody (mAb A3A5) which inhibits human fibroblast-mediated collagen gel contraction, an in vitro model producing the process of collagen morphogenesis . Human fibronectin (FN) has been shown to be the antigen of A3A5 . The present study aimed at identifying the A3A5 epitope to reveal the mode of binding between collagen, FN, and fibroblasts in the process of gel contraction . The epitope was sought in FN fragments obtained by pepsin digestion and in recombinant FN fragments expressed in Escherichia coli by determining their immunological reactivity with A3A5, and was identified as a short segment consisting of the fourth through the amino half of the fifth FN type III . We propose a new functional domain of FN which plays a crucial role in the binding of fibroblasts to collagen fibrils and is involved in collagen morphogenesis. FEBS Lett, 1997 Jul 21, 412(1), 43 - 7 EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB; Hunter DJ et al.; The spectroscopic and ligand-binding properties of a copper-deficient cytochrome bo3, a member of the haem-copper superfamily of terminal oxidases, are reported and contrasted with those of the native enzyme . The enzyme lacks the copper atom (CuB) which is normally an integral part of the catalytic site . The consequences of loss of the CuB are the loss of antiferromagnetic coupling to the high-spin haem and an inability to form any of the integer-spin derivatives of the enzyme . Low-spin compounds of the normally high-spin haem are still formed with appropriate ligands, although these are modified. Gene, 1997 Jul 18, 194(1), 137 - 41 Cloning and expression of the Borrelia burgdorferi lon gene; Cloud JL et al.; The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis . In addition, mutations in the E . coli lon gene suppress temperature-sensitive mutations in other genes . The lon gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced . The gene encodes a protein of 806 amino acids . The deduced amino acid sequence of the B . burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases . The transcription start point of the B . burgdorferi lon gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E . coli . The 5'-end of the B . burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E . coli lpxA mutant. Biochim Biophys Acta, 1997 Jul 18, 1340(2), 235 - 44 Conformation of thermally denatured RNase T1 with intact disulfide bonds: a study by small-angle X-ray scattering; Damaschun H et al.; Small-angle X-ray scattering of RNase T1 with intact disulfide bonds was measured at 20 degrees and 60 degrees C in order to get insight into the structural changes of the protein caused by thermal denaturation . The radius of gyration increases from R(G)= 1.43 nm to R(G) = 2.21 nm . The conformations of the molecules at 60 degrees C are similar to those of ring-shaped random walk chains . However, the molecules are more compact than one would expect under theta conditions due to attractive interactions between the chain segments . The volume needed for free rotation of the thermally unfolded protein molecules about any axis in solution is five times greater than in the native state whereas the hydrodynamic effective volume is increasing only two times. Biochim Biophys Acta, 1997 Jul 18, 1340(2), 227 - 34 Purification and characterization of isocitrate dehydrogenase from a hyperthermophilic archaebacterium, Caldococcus noboribetus; Aoshima M et al.; Isocitrate dehydrogenase from a hyperthermophilic archaebacterium Caldococcus noboribetus produced in Escherichia coli was purified . The purification was performed by heat treatment at 80 degrees C followed by single column chromatography . N-terminal amino acid sequencing analysis revealed that the N-terminal methionine is removed from the purified enzyme . Gel filtration analysis suggests that the enzyme has a homodimeric structure with a molecular weight of 90,000 . The isoelectric point of the enzyme was estimated to be 5.6 by isoelectric focusing electrophoresis . The circular dichroism spectrum suggests that the enzyme has a secondary structure consisting of 23% alpha-helix and 34% beta-sheet . Enzymatic activity was observed under neutral pH, and the highest specific activity was obtained using cacodylic acid-KOH (pH 7.0) buffer . MgCl2 or MnCl2 was essential for the activity, and KCl concentrations higher than 0.33 M had an inhibitory effect on it . Apparent Km values were 72 and 43 microM for D,L-isocitrate and NADP, respectively . The enzyme showed extremely high stability against heat treatment, and no activity loss was observed by the treatment at 80 degrees C . The specific activity of the enzyme increased as temperature rose . Nearly no activity was observed at 40 degrees C or lower. Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 461 - 6 Sequence and analysis of a dnaJ homologue gene in cyanobacterium Synechococcus sp . PCC7942; Oguchi K et al.; The chromosomal region containing a dnaJ gene homologue (dnaJ7942) was sequenced from unicellular cyanobacterium Synechococcus sp . PCC7942 . The dnaJ7942 gene as well as following two orfs are located in the region immediately downstream of dnaK3, and they seem to be cotranscribed . The dnaJ7942 gene product shares, like all J homologues, homology for the highly conserved "J-domain" of DnaJ . It does not have, however, a glycine and phenylalanine (G/F)-rich region nor cysteine (Cys)-rich region unlike the Escherichia coli DnaJ protein . When this gene was expressed in E . coli, cells became filamentous in contrast to those expressing the E . coli dnaJ gene . Gene disruption experiments indicated that the dnaJ7942 gene was essential for growth . Analysis of subcellular localization revealed that the DnaJ protein is mainly located on the thylakoid membrane in the cyanobacterium. Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 360 - 4 The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast; Tanimoto A et al.; To investigate the biological function of hepatitis C virus (HCV)-NS5A, the NS5A was fused at its N-terminus with the DNA binding domain (DBD) of yeast transcriptional activator GAL4 (GAL4-DBD) . The GAL4-DBD alone had no transcriptional activation function . However, a mutant of the GAL4-DBD/NS5A fusion protein, in which 129 amino acid residues were deleted from the N-terminus of NS5A, exhibited strong transcriptional activation in yeast cells, bearing the Escherichia coli lacZ reporter gene encoding the beta-galactosidase under the transcriptional control of GAL4 promoter and TATA box . Further mutational analysis of NS5A revealed that the region between the amino acid residues 130 to 352 were critical for optimal level of transactivation . This region includes two acidic domains and one proline-rich region which have been shown to be involved in the function of several transcriptional activators. Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 253 - 6 Does the epsilon sequence of phage T7 function as an initiator for the translation of CAT mRNA in Escherichia coli? Golshani A, Golomehova V, Mironova R, Ivanov IG, AbouHaidar MG. Epsilon (epsilon) sequence {UUAACUUUA, complementary to nucleotides 458-466 of the 16S ribosomal RNA (rRNA)} which is naturally occurring at the 5'-untranslated leader of phage T7 gene 10 mRNA was originally described as a powerful translational enhancer in Escherichia coli . Recent studies with this sequence led to controversial conclusions about its translational initiation and enhancing capability . In this study different sequence derivatives of epsilon were constructed to evaluate its efficiency not only to enhance translation of the chloramphenicol acetyltransferase (CAT) mRNA in E . coli, but also to function as an independent initiator of translation . It was observed that the epsilon sequence in combination with the CAT natural Shine-Dalgarno (SDn) or the SD consensus sequences enhanced, as expected, the translation of CAT mRNA . The natural epsilon sequence without an SD sequence failed to initiate or enhance the translation of CAT mRNA . However, when the complementarity of epsilon to 16S rRNA was increased from 9 to 16 nucleotides, epsilon alone (without the SD sequence) became an independent translational initiator with an efficiency of about 80% that obtained with the SD consensus sequence. J Mol Biol, 1997 Jul 18, 270(3), 496 - 510 Three-dimensional structure of the DNA-binding domain of the fructose repressor from Escherichia coli by 1H and 15N NMR; Penin F et al.; FruR is an Escherichia coli transcriptional regulator that belongs to the LacI DNA-binding protein family . By using 1H and 15N NMR spectroscopy, we have determined the three-dimensional solution structure of the FruR N-terminal DNA-binding domain consisting of 57 amino acid residues . A total of 809 NMR-derived distances and 54 dihedral angle constraints have been used for molecular modelling with the X-PLOR program . The resulting set of calculated structures presents an average root-mean-square deviation of 0.37 A at the main-chain level for the first 47 residues . This highly defined N-terminal part of the structure reveals a similar topology for the three alpha-helices when compared to the 3D structures of LacI and PurR counterparts . The most striking difference lies in the connection between helix II and helix III, in which three additional residues are present in FruR . This connecting segment is well structured and contains a type III turn . Apart from hydrophobic interactions of non-polar residues with the core of the domain, this connecting segment is stabilised by several hydrogen bonds and by the aromatic ring stacking between Tyr19 of helix II and Tyr28 of the turn . The region containing the putative "hinge helix" (helix IV), that has been described in PurR-DNA complex to make specific base contacts in the minor groove of DNA, is unfolded . Examination of hydrogen bonds highlights the importance of homologous residues that seem to be conserved for their ability to fulfill helix N and C-capping roles in the LacI repressor family. J Mol Biol, 1997 Jul 18, 270(3), 451 - 63 Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot; Gluick TC et al.; A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4 . A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature . Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations . Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions . Binding of the latter ions provides most of the favorable free energy for the reaction . However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form . A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C . Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers . K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers . The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step. J Mol Biol, 1997 Jul 18, 270(3), 346 - 59 Activation of RpoS-dependent proP P2 transcription by the Fis protein in vitro; Xu J et al.; The proP gene, encoding a transporter of the osmoprotecting compounds proline and glycine betaine, is expressed from two promoters . Transcription of the P2 promoter occurs at a transient period in late exponential phase and is dependent upon Fis and the RpoS (sigma38) sigma factor . Here we characterize Fis-mediated activation of the P2 promoter in vitro . We find that this promoter displays unusually high specificity for sigma38 . Fis strongly activates P2 when bound to site I centered at -41 within the promoter region . There is a complex relationship involving DNA supercoiling and potassium glutamate concentration on Fis activation, but most efficient transcription occurs under high salt conditions when the superhelical density is above -0.03 . The major stimulatory effect of DNA supercoiling occurs between superhelical densities of 0 to -0.02 suggesting that, while supercoiling is mechanistically important, it may not be a physiologically relevant controlling factor . However, the stimulation of transcription by high potassium glutamate concentrations may contribute to the osmotic inducibility of the P2 promoter . We show that Fis and E sigma38 bind cooperatively on supercoiled DNA to form a stable complex at P2 that involves promoter melting . Fis also binds to a second site within the proP regulatory region . While binding to this site appears to play no role in Fis activation of the P2 promoter, it functions as a repressor of transcription initiating from the P1 promoter by either sigma70 or sigma38. J Mol Biol, 1997 Jul 18, 270(3), 321 - 7 Role of mitochondrial GrpE and phosphate in the ATPase cycle of matrix Hsp70; Dekker PJ et al.; The yeast mitochondrial GrpE homologue, Mge1, assists matrix Hsp70 in both protein translocation across the mitochondrial membranes and subsequent protein folding . We expressed mtHsp70 and Mge1 in Escherichia coli and analyzed their function in the ATP hydrolysis cycle . Mge1 stimulates ATP hydrolysis by mtHsp70 about twofold . Addition of inorganic phosphate inhibits ATP hydrolysis by preventing ADP release from mtHsp70 . Mge1 has no direct effect on gamma-phosphate release from mtHsp70, yet indirectly relieves the phosphate inhibition by stimulating ADP release . We conclude that Mge1 promotes the ATPase cycle of mtHsp70 by increasing the rate of ADP release . ATP then rapidly binds to mtHsp70 such that the total amount of mtHsp70-bound nucleotide is not changed by Mge1. J Biol Chem, 1997 Jul 18, 272(29), 18434 - 9 Hin-mediated inversion on positively supercoiled DNA; Lim HM et al.; Hin recombinase requires negatively supercoiled DNA for an efficient inversion . We have generated positively supercoiled plasmid DNA using reverse gyrase from Sulfolobus shibatae and subjected it to the Hin-mediated inversion reaction . Both Hin and Fis showed the same DNA binding activity regardless of the superhelical handedness of the substrate plasmid . However, inversion activity on positively supercoiled DNA was less than 1% of negatively supercoiled DNA . Assays designed to probe steps in inversion, showed that on positively supercoiled DNA, Hin was able to cleave the recombination sites with the same efficiency shown on negatively supercoiled DNA but was not able to exchange the cleaved DNA . Based on the theoretical differences between positive and negative supercoiling, our data may suggest that unwinding of the double helix at recombination sites is needed after DNA cleavage for strand exchange to occur. J Biol Chem, 1997 Jul 18, 272(29), 18216 - 21 Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain; Sia SK et al.; The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties . Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive . The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy . The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC . This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction . Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction. J Biol Chem, 1997 Jul 18, 272(29), 18191 - 9 The influence of antisense oligonucleotide-induced RNA structure on Escherichia coli RNase H1 activity; Lima WF et al.; The ability of Escherichia coli RNase H1 to hydrolyze structured substrates containing antisense oligonucleotides preannealed to a 47-mer RNA was compared with its ability to hydrolyze unstructured substrates containing antisense oligonucleotides duplexed with 13-mer RNA . These results demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significantly slower than when the same oligonucleotides were bound to unstructured oligoribonucleotides . Structured substrates exhibited fewer cleavage sites, and each cleavage site was cleaved less rapidly than in unstructured substrates . Furthermore, the enzymatic activity of E . coli RNase H1 for the structured substrates was most affected when the cleavage sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates . Molecular modeling suggests that the observed ablation of RNase H activity was due to the steric hindrance of the enzyme by the structured RNA, i.e . steric interference of the phosphate groups on the substrate and/or the binding site of the enzyme . When chimeric oligonucleotides composed of a five-base deoxynucleotide sequence flanked by chemically modified nucleotides were bound to structured RNA, the resultant duplexes were even worse substrates for RNase H . These results offer further insights into the role of antisense-induced RNA structure on RNase H activity and may facilitate the design of effective antisense oligonucleotides. J Biol Chem, 1997 Jul 18, 272(29), 18169 - 78 Characterization of PKIgamma, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase; Collins SP et al.; Attempts to understand the physiological roles of the protein kinase inhibitor (PKI) proteins have been hampered by a lack of knowledge concerning the molecular heterogeneity of the PKI family . The PKIgamma cDNA sequence determined here predicted an open reading frame of 75 amino acids, showing 35% identity to PKIalpha and 30% identity to PKIbeta1 . Residues important for the high affinity of PKIalpha and PKIbeta1 as well as nuclear export of the catalytic (C) subunit of cAMP-dependent protein kinase were found to be conserved in PKIgamma . Northern blot analysis showed that a 1.3-kilobase PKIgamma message is widely expressed, with highest levels in heart, skeletal muscle, and testis . RNase protection analysis revealed that in most tissues examined PKIgamma is expressed at levels equal to or higher than the other known PKI isoforms and that in several mouse-derived cell lines, PKIgamma is the predominant PKI message . Partial purification of PKI activities from mouse heart by DEAE ion exchange chromatography resolved two major inhibitory peaks, and isoform-specific polyclonal antibodies raised against recombinant PKIalpha and PKIgamma identified these inhibitory activities to be PKIalpha and PKIgamma . A comparison of inhibitory potencies of PKIalpha and PKIgamma expressed in Escherichia coli revealed that PKIgamma was a potent competitive inhibitor of Calpha phosphotransferase activity in vitro (Ki = 0.44 nM) but is 6-fold less potent than PKIalpha (Ki = 0.073 nM) . Like PKIalpha, PKIgamma was capable of blocking the nuclear accumulation of Flag-tagged C subunit in transiently transfected mammalian cells . Finally, the murine PKIgamma gene was found to overlap the murine adenosine deaminase gene on mouse chromosome 2 . These results demonstrate that PKIgamma is a novel, functional PKI isoform that accounts for the previously observed discrepancy between PKI activity and PKI mRNA levels in several mammalian tissues. J Biol Chem, 1997 Jul 18, 272(29), 17903 - 6 A new metabolic link . The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli and mitochondria; Jordan SW et al.; Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity . The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and alpha-ketoglutarate dehydrogenase complexes . The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism . However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism . We now report that E . coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor . This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria. Biochim Biophys Acta, 1997 Jul 17, 1353(1), 79 - 83 In vivo effect of DNA relaxation on the transcription of gene rpoH in Escherichia coli; Lopez-Sanchez F et al.; The in vivo effect of Novobiocin, a gyrase inhibitor, on the transcription of gene rpoH which codes for sigma32, the main positive regulator of the heat-shock response, was studied . Novobiocin induced a three-fold increase and a slight decrease in the activity of the rpoH promoters P1 and P4, respectively . The Novobiocin-induced increase in the activity of promoter P1 correlates with an increase in the amount of proteins sigma32 and DnaK . These results suggest that the increase in expression of the heat-shock proteins induced by gyrase inhibitors is probably due to the increased activity of P1 on relaxed DNA. Eur J Pharmacol, 1997 Jul 16, 331(1), 31 - 8 Characterization of des-Arg9-bradykinin-induced contraction in guinea-pig gallbladder in vitro; Cabrini DA et al.; We have reported that bradykinin induces graded contraction in guinea-pig gallbladder in vitro through activation of bradykinin B2 receptors and prostanoid release, while des-Arg9-bradykinin, a selective bradykinin B1 receptor agonist, causes only a weak contraction, suggesting the presence of badykinin B1 receptors in this tissue . In the present study, we attempted to characterise the receptor subtype and the possible mechanism by which des-Arg9-bradykinin induces contraction in this preparation . Contractions induced by des-Arg9-bradykinin in guinea-pig gallbladder (1 pM to 1 microM) increased significantly as a function of time elapsed after setting up of the preparation, reaching the maximum after 6 h of equilibration (EC50 16.4 pM and Emax 0.6 +/- 0.08 g) . Des-Arg9-bradykinin-induced contraction in guinea-pig gallbladder was totally prevented by cycloheximide (70 microM, an inhibitor of protein synthesis), indomethacin (3 microM), ibuprofen (30 microM), phenidone (30 microM) or Ca2+-free medium plus EGTA, and was partially antagonised by MK 571 ((3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3-oxo-propyl) thio) methyl) propanoic acid, 0.1 microM) or by nicardipine (1 microM), but was not affected by dazoxiben (30 microM), staurosporine (100 nM) or L 655,240 (240 (3-{1-(4-clorobenzil)-5-fluoro-3-metilhyindol-2il} 2,2-dimetilpropanoic acid, 1 microM) . Unexpectedly, des-Arg9-bradykinin-induced contraction was unaffected by the selective bradykinin B1 receptor antagonists, des-Arg9-{Leu8}-bradykinin and des-Arg9-NPC 17761 (des-Arg0-D-Arg {Hip3, D-HipE (transtiofenil)7, Oic8}-des-Arg9-bradykinin) . However, the selective bradykinin B2 receptor antagonists, HOE 140 (D-Arg0-{Hyp3, Thi5, D-Tic7, Oic8}-bradykinin) and NPC 17731 (D-Arg0 {Hyp3, DHypE (transpropyl)7, Oic8}-bradykinin), completely blocked des-Arg9-bradykinin-mediated contraction . Pre-treatment of the animals with Escherichia coli endotoxin (lipopolysaccharide, 30 microg/animal, i.v., 24 h) did not significantly change the response to des-Arg9-bradykinin induction . It is concluded that des-Arg9-bradykinin-induced contractions in guinea-pig gallbladder are mediated primarily by the release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway . These effects are unrelated to thromboxane A2 and do not seem to be coupled to activation of a protein kinase C-dependent mechanism . Response to des-Arg9-bradykinin increases as a function of the equilibration period of the preparation by a mechanism dependent on protein synthesis and seems to be mediated by activation of bradykinin B2 (but not B1) receptors . Finally, in contrast to that observed for bradykinin, the contraction induced by des-Arg9-bradykinin in guinea-pig gallbladder is fully dependent on the influx of extracellular Ca2+, partially through L-type Ca2+ channels. EMBO J, 1997 Jul 16, 16(14), 4467 - 76 MutS mediates heteroduplex loop formation by a translocation mechanism; Allen DJ et al.; Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy . In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop . Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay . These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair . In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation . The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction. EMBO J, 1997 Jul 16, 16(14), 4448 - 55 A LINE-like transposable element in Drosophila, the I factor, encodes a protein with properties similar to those of retroviral nucleocapsids; Dawson A et al.; I factors are members of the LINE-like family of transposable elements and move by reverse transcription of an RNA intermediate . Complete I factors contain two open reading frames . The amino acid sequence encoded by the first of these, ORF1, includes the motif CX2CX4HX4C that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences CX2CX4HX6C and CX2CX9HX6C . The function of this protein is unknown . We have expressed this protein in Escherichia coli and Spodoptera frugiperda cells and have shown that it binds both DNA and RNA but without any evidence for sequence specificity . The properties of deletion derivatives of the protein indicate that more than one region is responsible for DNA binding and that the CCHC motif is not essential for this . The ORF1 protein expressed in either E . coli or Spodoptera cells forms high molecular weight structures that require the region of the protein including the CCHC motif for their formation . This protein can also accelerate the annealing of complementary single-stranded oligonucleotides . These results suggest that this protein may associate with the RNA transposition intermediates of the I factor to form particles that enter the nucleus during transposition and that it may stimulate both the priming of reverse transcription and integration . This may be generally true for the product of the first open reading frame of LINE-like elements. EMBO J, 1997 Jul 16, 16(14), 4261 - 6 Anionic phospholipids are determinants of membrane protein topology; van Klompenburg W et al.; The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops . The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations . Orientations of constructs derived from the inner membrane protein leader peptidase from Escherichia coli were found to depend on the anionic phospholipid content of the membrane . Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops . On the other hand, elevated contents of acidic phospholipids in the membrane rendered translocation more sensitive to positively charged residues . The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins. EMBO J, 1997 Jul 16, 16(14), 4155 - 62 Cockayne syndrome: defective repair of transcription? van Gool AJ, van der Horst GT, Citterio E, Hoeijmakers JH. In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another . One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription . This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription-coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes . The mechanism of TCR in eukaryotes is still largely unknown . It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli . In the latter case, one protein, the transcription repair-coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed . While the E . coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins. Eur J Biochem, 1997 Jul 15, 247(2), 725 - 32 Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3; McCoubrey WK Jr et al.; Two isozymes of heme oxygenase (HO), HO-1 or HSP32 and the constitutive form HO-2, have been characterized to date . We report the discovery of a third protein species and refer to it as HO-3 . HO-3 is the product of a single transcript of approximately 2.4 kb and can encode a protein of approximately 33 kDa . The HO-3 transcript is found in the spleen, liver, thymus, prostate, heart, kidney, brain and testis and is the product of a single-copy gene . The predicted amino acid structure of HO-3 differs from both HO-1 (HSP32) and HO-2 but is closely related to HO-2 (approximately 90%) . Escherichia coli expressed and purified HO-3 protein does not cross react with polyclonal antibodies to either rat HO-1 or HO-2, is a poor heme catalyst, and displays hemoprotein spectral characteristics . The predicted protein has two heme regulatory motifs that may be involved in heme binding . These motifs and the hemoprotein nature of HO-3 suggest a potential regulatory role for the protein in cellular processes which are heme-dependent. Eur J Biochem, 1997 Jul 15, 247(2), 716 - 24 Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution; Brabetz W et al.; The chromosomal genes rfaC and rfaF of Escherichia coli W3110 were inactivated by allelic-replacement mutagenesis to generate a defined strain lacking both heptosyltransferases which catalyze in lipopolysaccharide (LPS) biosynthesis the transfer of the first two L-glycero-D-manno-heptose (Hep) residues to 3-deoxy-D-manno-2-octulosonic acid (Kdo) . The LPS of the mutant was isolated and its chemical structure was investigated by compositional analysis and nuclear magnetic resonance spectroscopy of isolated, deacylated oligosaccharide phosphates . The basic structure was a tetrasaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)-alpha-D- GlcN1P which in LPS was substituted at position 07 of Kdo II by 2-aminoethanol phosphate in non-stoichiometric amounts . 2-Aminoethanol was cleaved during deacylation of the LPS by successive hydrazinolysis and KOH treatment and, in addition, phosphate migration from 07 to 08 of Kdo II occurred . Thus, the oligosaccharides alpha-Kdo7P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P and alpha-Kdo8P-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN4P-(1-->6)- alpha-D-GlcN1P could be isolated . KOH treatment of the two trisphosphates and authentic methyl 3-deoxy-D-manno-octulopyranoside 7-(2-acetamidoethyl phosphate) proved that phosphate migration only took place when the phosphate group was substituted with 2-aminoethanol . Complementation studies with plasmid-encoded rfaC and rfaF genes revealed that the mutant strain can be used in combination with LPS-specific antibodies for the cloning and characterization of heptosytransferases which glycosylate Kdo residues of the inner core region of LPS. Eur J Biochem, 1997 Jul 15, 247(2), 567 - 71 Structural studies of the O-antigen polysaccharide from Escherichia coli O138; Linnerborg M et al.; The structure of the O-antigen polysaccharide from Escherichia coli O138 has been determined . NMR spectroscopy, together with component and methylation analyses, of native and reduced polysaccharide were the principal methods used . The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple bond connectivity (HMBC) NMR experiments . It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure: {structure: see text}. Eur J Biochem, 1997 Jul 15, 247(2), 511 - 7 Immunological localization and tissue distribution of alkyldihydroxyacetonephosphate synthase and deficiency of the enzyme in peroxisomal disorders; de Vet EC et al.; Alkyldihydroxyacetonephosphate synthase (alkylglycerone-phosphate synthase) is a peroxisomal enzyme involved in ether phospholipid biosynthesis . The recent cloning of the cDNA encoding this enzyme from guinea pig liver enabled the raising of specific antisera against this enzyme . Both a synthetic peptide corresponding to a predicted epitope and a recombinant protein expressed in Escherichia coli were used for that purpose . Using western blot techniques, the solubilization of the enzyme from the peroxisomal membrane by Triton X-100 in the presence of salt was confirmed . Neutral hydroxylamine treatment of peroxisomes resulted in almost no release of the protein from the membrane . The complete polypeptide chain of the enzyme was resistant to proteolysis by trypsin when intact peroxisomes were studied . Carbonate treatment released alkyldihydroxyacetonephosphate synthase from the membrane indicating that the enzyme is not an integral membrane protein . This idea is in accord with the absence of a clear hydrophobic transmembrane domain in the deduced amino acid sequence of the enzyme . Alkyldihydroxyacetonephosphate synthase, as well as its mRNA, could be detected in all five guinea pig tissues examined . When using the antiserum against guinea pig recombinant alkyldihydroxyacetonephosphate synthase, a cross-reactive protein was detected in a human liver homogenate that runs at a slightly higher molecular mass . The absence of this band in liver of Zellweger syndrome and Rhizomelic chondrodysplasia punctata patients provides strong evidence that it represents the human homolog of this enzyme. Structure, 1997 Jul 15, 5(7), 859 - 63 Touring the landscapes: partially folded proteins examined by hydrogen exchange; Chamberlain AK et al.; Recent studies on Escherichia coli ribonuclease H and several other proteins reveal a specific region in each protein that remains structured in partially folded conformations . These regions play a dominant role in determining the fold and stability of the protein. FEMS Microbiol Lett, 1997 Jul 15, 152(2), 307 - 12 Expression of P, S, and F1C adhesins by cytotoxic necrotizing factor 1-producing Escherichia coli from septicemic and diarrheic pigs; Dozois CM et al.; Nineteen papC-positive cytotoxic necrotizing factor 1 (CNF1)-producing Escherichia coli isolates from pigs with septicemia or diarrhea were tested for the presence of pap-, sfa-, and afa-related sequences encoding P/Prs, S/F1C, and Dr/AFA adhesins respectively . Production of adhesins by isolates was tested by mannose-resistant hemagglutination (MRHA), sialidase treatment of erythrocytes and particle agglutination tests . Production of P, S, and F1C fimbriae by isolates was also examined by immunofluorescence . All isolates were pap+ by PCR . Eighteen isolates (95%) were MRHA for ovine and human A erythrocytes and exhibited GalNac-GalNac receptor specificity associated with class III P(Prs) adhesins . Fifteen (79%) of the 19 isolates reacted with antisera specific for one or more different P fimbrial serotypes on immunofluorescence . Three of these isolates also demonstrated Gal-Gal receptor specificity associated with class I or II P fimbrial adhesins . Fifteen (79%) of the isolates were sfa+ by PCR . Seven of these isolates exhibited sialidase-sensitive MRHA of bovine and human O erythrocytes and reacted with serum specific for S fimbriae on immunofluorescence . Seven of the 8 sfa+ isolates which were MRHA-negative for bovine erythrocytes reacted with serum specific for F1C fimbriae on immunofluorescence . All isolates produced type 1 fimbriae as determined by mannose-sensitive agglutination of yeast cells . None of the isolates were afa+ by PCR or colony hybridization . Results suggest that most pap+ porcine CNF1-producing E . coli isolates express P fimbriae bearing class III (Prs) type adhesins . In addition, most of these isolates also produce S or F1C fimbriae. FEMS Microbiol Lett, 1997 Jul 15, 152(2), 219 - 25 Histidine-44 of the A subunit of Escherichia coli enterotoxin is involved in its enzymatic and biological activities; Kato M et al.; We examined the role in toxicity of histidine-44 of the A subunit of Escherichia coli enterotoxin, which is located in the active site cavity close to glutamic acid-112 . Although amino acid substitution of histidine-44 usually renders a mutant toxin unstable to trypsin, one mutant, alanine-44 (His44Ala) was found to be stable . His44Ala did not show any agmatine:ADP-ribosyltransferase activity in the presence or absence of recombinant ADP-ribosylation factor . It showed no diarrheal or rabbit skin permeability activity and was a competitor in enterotoxin-ADP-ribosyltransferase assays containing recombinant ADP-ribosylation factor . These results suggest that like glutamic acid-112, histidine-44 plays an essential role in toxicity . A tentative model, which explains NAD+ catalysis and the transfer of the ADP-ribosyl moiety to a target amino acid, is proposed for histidine-44 and glutamic acid-112. Biochem J, 1997 Jul 15, 325 ( Pt 2), 519 - 25 Inhibition of glycerol metabolism in hepatocytes isolated from endotoxic rats; Leclercq P et al.; Sepsis or endotoxaemia inhibits gluconeogenesis from various substrates, the main effect being related to a change in the phosphoenolpyruvate carboxykinase transcription rate . In addition, sepsis has been reported to affect the oxidative phosphorylation pathway . We have studied glycerol metabolism in hepatocytes isolated from rats fasted and injected 16 h previously with lipopolysaccharide from Escherichia coli . Endotoxin inhibited glycerol metabolism and led to a very large accumulation of glycerol 3-phosphate; the cytosolic reducing state was increased . Furthermore glycerol kinase activity was increased by 33% (P<<0.01) . The respiratory rate of intact cells was significantly decreased by sepsis, with glycerol or octanoate as exogenous substrates, whereas oxidative phosphorylation (ATP-to-O ratio or respirations in state 4, state 3 and the oligomycin-insensitive state as well as the uncoupled state) was unchanged in permeabilized hepatocytes . Hence the effect on energy metabolism seems to be present only in intact hepatocytes . An additional important feature was the observation of a significant increase in cellular volume in cells from endotoxic animals, which might account for the alterations induced by sepsis. Biochem J, 1997 Jul 15, 325 ( Pt 2), 441 - 7 Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major; Camacho A et al.; A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli . The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa . Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts . None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide . However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichia coli . The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure . The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c . The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L . major. Biochem J, 1997 Jul 15, 325 ( Pt 2), 401 - 4 Influence of systematic error on the shape of the scatchard plot of tRNAPhe binding to eukaryotic ribosomes; Nekhai SA et al.; Scatchard plots of tRNAPhe binding to poly(U)-programmed human 80 S ribosomes can be curved, either concave upwards or concave downwards, depending on the experimental conditions . The influence of a systematic error on the shape of the Scatchard plots has been analysed in a model experiment where the binding proceeds at two independent sites . The Scatchard plot for this binding model has a concave-upwards shape . When the concentration of the ribosomes is kept constant, a small systematic error in tRNA concentration changes this Scatchard plot markedly to a concave-downwards plot as though a co-operative interaction occurred . In contrast, when the tRNA concentration exceeds the ribosomal concentration and their concentration ratio is constant, the Scatchard plot is stable with respect to the systematic error . We suggest the latter type of experiment to be more appropriate . The results also imply a non-co-operative interaction of tRNAPhe with the 80 S ribosome. Arch Biochem Biophys, 1997 Jul 15, 343(2), 249 - 53 Characterization of human liver inducible nitric oxide synthase expressed in Escherichia coli; Gerber NC et al.; We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme . The protein has been expressed with and without a polyhistidine tail . In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers . Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein . The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM . The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1) . Similar values are obtained for the enzyme with and without the polyhistidine tail . Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+ . These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration . The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages. Arch Biochem Biophys, 1997 Jul 15, 343(2), 225 - 33 A potent superoxide dismutase mimic: manganese beta-octabromo-meso-tetrakis-(N-methylpyridinium-4-yl) porphyrin; Batinic-Haberle I et al.; Variously modified metalloporphyrins offer a promising route to stable and active mimics of superoxide dismutase (SOD) . Here we explore bromination on the pyrroles as a means of increasing the redox potentials and the catalytic activities of the copper and manganese complexes of a cationic porphyrin . Mn(II) and Cu(II) octabrominated 5,10,15,20-tetrakis-(N-methylpyridinium-4-yl) porphyrin, Mn(II)OBTMPyP4+, and Cu(II)OBTMPyP4+ were prepared and characterized . The rate constants for the porphyrin-catalyzed dismutation of O2.- as determined from the inhibition of the cytochrome c reduction are k(cat) = 2.2 x 10(8) and 2.9 x 10(6) M(-1) s(-1), i.e., IC50 was calculated to be 12 nM and 0.88 microM, respectively . The metal-centered half-wave potential was E(1/2) = +0.48 V vs NHE for the manganese compound . Cu(II)OBTMPyP4+ proved to be extremely stable, while its Mn(II) analog has a moderate stability, log K = 8.08 . Nevertheless, slow manganese dissociation from Mn(II)OBTMPyP4+ enabled the complex to persist and exhibit catalytic activity even at the nanomolar concentration level and at biological pH . The corresponding Mn(III)OBTMPyP5+ complex exhibited significantly increased stability, i.e., demetallation was not detected in the presence of a 400-fold molar excess of EDTA at micromolar porphyrin concentration and at pH 7.8 . The beta-substituted manganese porphyrin facilitated the growth of a SOD-deficient strain of Escherichia coli when present at 0.05 microM but was toxic at 1.0 microM . The synthetic approach used in the case of manganese and copper compounds offers numerous possibilities whereby the interplay of the type and of the number of beta substituents on the porphyrin ring would hopefully lead to porphyrin compounds of increased stability, catalytic activity, and decreased toxicity. Biochemistry, 1997 Jul 15, 36(28), 8628 - 33 Kinetics of oxidized cytosine repair by endonuclease III of Escherichia coli; Wang D et al.; Endonuclease III of Escherichia coli excises a broad range of oxidized, hydrated and ring-fragmented pyrimidines from DNA . The kinetic parameters were compared for repair of three potentially mutagenic oxidized cytosine lesions: 5,6-dihydroxy-5, 6-dihydro-2'-deoxyuridine (uracil glycol or Ug), 5-hydroxy-2'-deoxycytidine (5-ohC), and 5-hydroxy-2'-deoxyuridine (5-ohU) . Site-specifically modified 40-mer oligonucleotides containing each of the three lesions in the same sequence context were synthesized chemically or by a combination of chemical and enzymatic methods . Appropriately protected phosphoramidites of 5-ohC and 5-ohU were synthesized and incorporated into oligonucleotides by standard solid-phase synthetic methods . The lability of Ug made it necessary to use an alternative approach to prepare the analogous 40-mers containing Ug . An uracil containing pentamer oligonucleotide was oxidized with OsO4 to generate the corresponding Ug containing product, which was then ligated into an oligonucleotide scaffold to generate 40 base pair duplexes . Using 32P-labeled substrates and a gel electrophoresis based assay, the values of Km and Vmax for excision of 5-ohC, 5-ohU, and Ug were determined . In this experimental system, the order of repair efficiency is Ug >> 5-ohC >> 5-ohU based on ratios of Vmax/Km . Modest effects were observed when the base paired opposite the lesion was changed from G to A. Biochemistry, 1997 Jul 15, 36(28), 8530 - 8 Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions; Rodriguez-Crespo I et al.; cDNAs coding for bovine endothelial nitric oxide synthase (eNOS) with N-terminal deletions of 52, 91, and 105 amino acids were constructed, and the proteins were expressed in Escherichia coli and purified by affinity chromatography . All three truncated proteins bind heme and exhibit the ferrous-CO absorption maximum at 444 nm characteristic of thiolate heme ligation . Deletion of the first 52 amino acids yields a fully active dimeric protein with the same spectroscopic properties as the wild-type . The myristoylation, palmitoylation, and polyproline domains of the enzyme located in the deleted region are therefore not required for full catalytic activity . The delta91 and delta105 proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, respectively, of the maximal activity . Resonance Raman and UV-vis spectroscopy indicate that, in the absence of tetrahydrobiopterin (H4B) and l-Arg, the wild-type and delta52 proteins are predominantly five coordinate high spin, whereas the delta91 and delta105 proteins are six coordinate low spin . The delta91 and delta105 mutants bind H4B, as indicated by a concomitant decrease in the low-spin component of the UV-vis spectrum, but the binding of l-Arg is extremely slow ( approximately 15 min) . Dithiothreitol readily coordinates as the sixth iron ligand in the delta91 and delta105 mutants but not in the delta52 or wild-type proteins . The dithiothreitol can be completely displaced by l-Arg but not by H4B . Resonance Raman comparison of wild-type eNOS and nNOS confirms that, in the absence of H4B and l-Arg, eNOS is primarily high spin whereas nNOS is predominantly six coordinate, low spin . The results indicate that Cys-101 is not critical for the binding of H4B and imply that some of the protein residues involved in dimer formation and in preservation of active site integrity are located, probably at the monomer-monomer interface, in the N-terminal end of the protein. Biochemistry, 1997 Jul 15, 36(28), 8522 - 9 Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease; Gwizdek C et al.; The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion . To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus . Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm . In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin . The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed . The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+ . These results suggest that loop 4-5 is near or part of the cation binding site . Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone . The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation. Biochemistry, 1997 Jul 15, 36(28), 8495 - 503 Enterobactin biosynthesis in Escherichia coli: isochorismate lyase (EntB) is a bifunctional enzyme that is phosphopantetheinylated by EntD and then acylated by EntE using ATP and 2,3-dihydroxybenzoate; Gehring AM et al.; In Escherichia coli, the siderophore molecule enterobactin is synthesized in response to iron deprivation by formation of an amide bond between 2,3-dihydroxybenzoate (2,3-DHB) and l-serine and formation of ester linkages between three such N-acylated serine residues . We show that EntB, previously described as the isochorismate lyase required for production of 2,3-DHB, is a bifunctional protein that also serves as an aryl carrier protein (ArCP) with a role in enterobactin assembly . EntB is phosphopantetheinylated near the C terminus in a reaction catalyzed by EntD with a kcat of 5 min-1 and a Km for apo-EntB of 6.5 microM . This holo-EntB is then acylated with 2,3-DHB in a reaction catalyzed by EntE, previously described as the 2,3-DHB-AMP ligase, with a kcat of 100 min-1 and a Km of <<1 microM for holo-EntB . The N-terminal 187 amino acids of EntB (isochorismate lyase domain) are not needed for reaction of EntB with either EntD or EntE as demonstrated by the equivalent catalytic efficiencies of the full-length EntB (residues 1-285) and the C-terminal EntB ArCP domain (residues 188-285) as substrates for both EntD and EntE. Biochemistry, 1997 Jul 15, 36(28), 8474 - 8 Isolation and chemistry of the mixed anhydride intermediate in the reaction catalyzed by dethiobiotin synthetase; Gibson KJ; Dethiobiotin synthetase (DTBS) catalyzes the formation of the cyclic urea, dethiobiotin (DTB), from (7R,8S)-diaminononanoic acid (DAPA), CO2, and ATP; the other products of the reaction are ADP and Pi . The first intermediate in the reaction sequence is the 7-carbamate of DAPA {Huang, W., et al . (1995) Biochemistry 34, 10985-10995; Gibson, K . J., et al . (1995) Biochemistry 34, 10976-10984; Alexeev, D., et al . (1995) Structure 3, 1207-1215} . The existence of the second postulated intermediate, a mixed carbamic-phosphoric anhydride formed when the carbamate is phosphorylated by ATP, is consistent with the cleavage of the gamma-phosphoryl group of ATP seen in DTBS reaction mixtures {Baxter, R . L., & Baxter, H . C . (1994) J . Chem . Soc., Chem . Commun., 759-760} . Two more direct lines of evidence for the mixed anhydride intermediate have now been obtained . First, a DTBS reaction mixture containing {18O}CO2 produced 18O-enriched DTB and Pi, as the existence of such an intermediate would require . Second, a moderately stable intermediate that could be labeled with either 14CO2, {gamma-33P}ATP, {9-3H}DAPA, or {1,7-14C}DAPA was trapped by quenching DTBS reactions at pH 4 and isolated by thin-layer chromatography . As expected for the proposed mixed anhydride, this species underwent acid hydrolysis to DAPA, CO2, and Pi; under basic conditions, the intermediate cyclized, yielding DTB and Pi . When returned to fresh enzyme at pH 7.5, the intermediate underwent cyclization at a rate comparable to that of normal turnover. Biochemistry, 1997 Jul 15, 36(28), 8455 - 64 Structure of human isovaleryl-CoA dehydrogenase at 2.6 A resolution: structural basis for substrate specificity,; Tiffany KA et al.; Isovaleryl-CoA dehydrogenase (IVD) belongs to an important flavoprotein family of acyl-CoA dehydrogenases that catalyze the alpha,beta-dehydrogenation of their various thioester substrates . Although enzymes from this family share similar sequences, catalytic mechanisms, and structural properties, the position of the catalytic base in the primary sequence is not conserved . E376 has been confirmed to be the catalytic base in medium-chain (MCAD) and short-chain acyl-CoA dehydrogenases and is conserved in all members of the acyl-CoA dehydrogenase family except for IVD and long-chain acyl-CoA dehydrogenase . To understand this dichotomy and to gain a better understanding of the factors important in determining substrate specificity in this enzyme family, the three-dimensional structure of human IVD has been determined . Human IVD expressed in Escherichia coli crystallizes in the orthorhombic space group P212121 with unit cell parameters a = 94.0 A, b = 97.7 A, and c = 181.7 A . The structure of IVD was solved at 2.6 A resolution by the molecular replacement method and was refined to an R-factor of 20.7% with an Rfree of 28.8% . The overall polypeptide fold of IVD is similar to that of other members of this family for which structural data are available . The tightly bound ligand found in the active site of the structure of IVD is consistent with that of CoA persulfide . The identity of the catalytic base was confirmed to be E254, in agreement with previous molecular modeling and mutagenesis studies . The location of the catalytic residue together with a glycine at position 374, which is a tyrosine in all other members of the acyl-CoA dehydrogenase family, is important for conferring branched-chain substrate specificity to IVD. Nucleic Acids Res, 1997 Jul 15, 25(14), 2861 - 8 Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli; Bowater RP et al.; Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences . This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences . The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence . For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower) . In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions . These observations show that deletions within long CTG.CAG repeats contained on plasmids in E.coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA. FEBS Lett, 1997 Jul 14, 411(2-3), 378 - 82 Continuous hepatocyte growth factor supply prevents lipopolysaccharide-induced liver injury in rats; Kaido T et al.; We present a rat model in which continuous supply of hepatocyte growth factor (HGF) prevents liver injury induced by carbon tetrachloride (CCl4) and E . coli 011:B4 lipopolysaccharide (LPS) . Rat fibroblasts genetically modified to secrete rat HGF were implanted in syngenic rat spleen 7 days before administration of the hepatotoxins . Rats with HGF-secreting fibroblasts in the spleen showed a dramatic resistance to CCl4- and LPS-induced liver injury . In the LPS-induced liver injury model, blood chemical analysis revealed that the increase in serum glutamic oxalacetic transaminase level and the decrease in blood sugar level were remarkably suppressed in rats with HGF-secreting cells in the spleen . Most importantly, their survival rate was greatly improved compared to other control groups of rats . Thus our results indicate a new role of HGF in liver protection during endotoxemia and convey important clinical implications for developing new therapeutic modalities in the treatment of liver failure caused by endotoxemia. FEBS Lett, 1997 Jul 14, 411(2-3), 317 - 21 High-level expression of human c-Src can cause a spherical morphology without loss of anchorage-dependent growth of NIH 3T3 cells; Kato G et al.; To investigate whether overexpression of human c-Src leads to cell rounding in anchorage-dependent NIH 3T3 fibroblasts, we have established c-Src-inducible cell lines using a lac repressor-operator system . RN1 cells, which expressed c-Src at a high level after induction, exhibited a spherical morphology and ceased to grow in monolayer culture . RN1 cells, however, exhibited neither focus-forming ability nor anchorage-independent growth potential with or without induction . Induced RN1 c-Src was phosphorylated at Ser75, a previously reported spherical cell-associated site, and at Tyr419 . These data demonstrated for the first time that highly elevated human c-Src tyrosine kinase activity can cause NIH 3T3 cells to have a spherical morphology without loss of anchorage-dependent growth . The inducible cell line should be useful to study the mechanism for cell rounding by c-Src. FEBS Lett, 1997 Jul 14, 411(2-3), 291 - 5 Birch pollen profilin: structural organization and interaction with poly-(L-proline) peptides as revealed by NMR; Domke T et al.; The secondary structure of birch pollen profilin, a potent human allergen, was elucidated by multidimensional nuclear magnetic resonance (NMR), as a prerequisite to study the interaction of this profilin with ligands for its poly-(L-proline) (PLP)-binding site . The chemical shifts of the 15N-labeled backbone amide groups were used to monitor complex formation with various PLP peptides . Titration with deca-L-proline (P10) yielded a KD of 0.2 mM . P8 was the shortest PLP to provoke a significant reaction . (GP5)3G bound significantly, confirming the interaction between profilins and the protein VASP containing this motif . Birch profilin interacted also with GP6GP5, found in the cyclase-associated protein (CAP), a suspected profilin ligand. FEBS Lett, 1997 Jul 14, 411(2-3), 265 - 8 Specificity of different isoforms of protein phosphatase-2A and protein phosphatase-2C studied using site-directed mutagenesis of HMG-CoA reductase; Ching YP et al.; We have expressed the catalytic domain of Chinese hamster HMG-CoA reductase, and 13 point mutations involving the region around the single phosphorylation site for AMP-activated protein kinase . After phosphorylation, these were used to test the specificity of isoforms of protein phosphatase-2A {bovine PP2A(C) (catalytic subunit) and PP2A1 (ABC heterotrimer)} and protein phosphatase-2C (human alpha; mouse alpha, beta1, beta2, beta3, beta4, beta5) . PP2A1 had > 50-fold higher activity for HMG-CoA reductase variants than PP2A(C), but their relative selectivity for different variants was similar . Although the specificities of PP2A and PP2C were distinct, no dramatic differences in selectivity were observed between different PP2C isoforms. FEBS Lett, 1997 Jul 14, 411(2-3), 169 - 72 High-affinity binding of the yeast cis-Golgi t-SNARE, Sed5p, to wild-type and mutant Sly1p, a modulator of transport vesicle docking; Grabowski R et al.; Docking of ER-derived vesicles to the cis-Golgi compartment in yeast requires vesicle and target membrane receptors (v-SNAREs and t-SNAREs) and the GTPase Ypt1p . The t-SNARE Sed5p is complexed with Sly1p in vivo . The mutant form Sly1-20p rescues Ypt1p-lacking cells from lethality, suggesting an inhibitory function of Sly1p in v-SNARE/t-SNARE interaction . Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1-20p with equally high affinity (K(D) = 5.13 x 10(-9) M and 4.74 x 10(-9) M, respectively) . Deletion studies show that the N-terminal half of Sly1p rather than the C-terminus (harbouring the E532K substitution in Sly1-20p) is most critical for its binding to Sed5p . These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking. Mutat Res, 1997 Jul 14, 391(3), 153 - 64 Assessment of the in vivo mutagenicity of ethylene oxide in the tissues of B6C3F1 lacI transgenic mice following inhalation exposure; Sisk SC et al.; In the present study, the lacI mutant frequency was determined in the tissues of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO) . Groups of 15 male transgenic lacI B6C3F1 mice were exposed to either 0, 50, 100, or 200 ppm EO for 4 weeks (6 h/day, 5 days/week) and were sacrificed at 0, 2, or 8 weeks after the last EO exposure . The lacI transgene was recovered from lung, bone marrow, spleen, and germ cells for determination of the lacI mutant frequency . The tissues selected for analysis were tumor target site tissues in chronic bioassays (lung tumors and lymphomas) and germ cells . The lacI mutant frequency in lung was significantly increased at 8 weeks post exposure to 200 ppm EO (6.2 +/- 2.2 vs . 9.1 +/- 1.5 . p = 0.046) . In contrast, the lacI mutant frequency in spleen and bone marrow at 2 and 8 weeks was not significantly increased in mice exposed to 200 ppm EO . The lacI mutant frequencies in male germ cells for 200 ppm EO-exposed mice were not increased compared to air controls at 2 and 8 weeks post-exposure . In a spleen cell fraction two of three EO-exposed mice at the 200 ppm exposure level demonstrated an elevated lacI mutant frequency . The increased lacI mutant frequency in these animals was likely due to mutant siblings that contained background G:C --> A:T transitions at CpG sites . These results demonstrate that a 4-week inhalation exposure to EO is mutagenic in lung . However, EO did not increase the frequency of mutations recovered at the lacI transgene in other tissues examined under the conditions used in the present studies . Since the mutational spectrum for EO in other systems consists of an increased proportion of large deletions, the lack of a mutagenic response in the tissues examined is likely due to the lack of recovery of large deletions in lambda-based shuttle vector systems . These data indicate that a primary mechanism of EO-induced mutagenicity in vivo is likely through the induction of deletions, not specific point mutations. Brain Res, 1997 Jul 11, 762(1-2), 103 - 13 Preferential expression of kin, a nuclear protein binding to curved DNA, in the neurons of the adult rat; Araneda S et al.; The KIN17 gene product has been identified by cross immunoreactivity with anti-RecA antibodies and by DNA recombination techniques, and is probably part of the DNA recombination-repair machinery . Following Western blotting and immunocytochemistry using anti-RecA antibodies, and in situ hybridization with specific KIN17 cDNA probes, we here report the detection of high levels of KIN protein and KIN17 mRNA in the CNS of adult rats . The RecA cross-reacting protein has an apparent molecular weight of 41 kDa and is located in the nucleus of brain cells . Both the KIN17 transcript and the protein were found to be widespread, but they were present in different proportions, depending on the type of brain cells . High levels of KIN protein were seen in neurons of the motor nuclei of the brainstem, the locus coeruleus, hippocampal formation, entorhinal cortex, Purkinje cells, pyramidal cells of the cortex and mitral cells . In contrast, using a combination of KIN17 mRNA in situ hybridization and GFAP immunocytochemistry (a marker of glial cells) showed that the KIN17 messenger is preferentially transcribed in neurons, the post-mitotic and long lived brain cells . We postulate that KIN17 play a role in the illegitimate recombination of DNA sequences and/or the repair of alterations of the genome in neurons. Carbohydr Res, 1997 Jul 11, 302(1-2), 35 - 42 Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli; Samain E et al.; Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides . To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E . coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) . These two products were easily purified by charcoal adsorption and ion-exchange chromatography . One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation. J Mol Biol, 1997 Jul 11, 270(2), 285 - 93 Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking; Wu J et al.; N and C-terminal halves of lactose permease, each with a single-Cys residue, were co-expressed, and crosslinking was studied . Iodine or N,N'-o-phenylenedimaleimide (o-PDM; rigid 6 A), crosslinks Asn245 Cys (helix VII) and Ile52 --> Cys or Ser53 --> Cys (helix II) . N,N'-p-phenylenedimaleimide (p-PDM; rigid 10 A) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6-bis-maleimidohexane (BMH; flexible 16 A) crosslinks both pairs less effectively than o-PDM . Thus, 245 is almost equidistant from 52 and 53 by up to about 6 A . BMH or p-PDM crosslinks Gln242 --> Cys and Ser53 --> Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 A . Ligand binding increases crosslinking of 245/53 with p-PDM or BMH, has little effect with o-PDM and decreases iodine crosslinking . Similar effects are observed with 245/52 . Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o-PDM . Therefore, ligand induces a translational or scissors-like displacement of the helices by 3-4 A . Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function. J Mol Biol, 1997 Jul 11, 270(2), 201 - 11 Quantitation of the inhibition of Hfr x F- recombination by the mutagenesis complex UmuD'C; Boudsocq F et al.; The UmuD'C complex and RecA protein are two essential components in mutagenic repair of gaps produced by the replication of damaged DNA . In this process, the UmuD'C complex might help DNA polymerase to synthesize DNA across a lesion . Besides, a RecA polymer wrapping around single-stranded DNA could function as a directional chaperone to target the UmuD'C complex at the lesion . It was shown in our laboratory that the UmuD'C complex prevents homologous recombination and recombinational repair when expressed at elevated levels . To find out whether the UmuD'C complex inhibits recombination by interfering directly with RecA, we measured the kinetics of inhibition of Hfr x F- recombination in F- recipients in which either RecA or UmuD'C were made to vary . The cell concentrations of RecA and UmuD'C proteins were adjusted by having the recA and the umuD'C genes regulated by the arabinose P(BAD) promoter . In the absence of the UmuD'C complex, recombination was a function of RecA concentration and then reached a plateau when the RecA concentration was above 9000 monomers/cell . At a fixed RecA concentration, the yield of Hfr x F- recombinants decreased as a function of the UmuD'C cell concentration . At a given UmuD'C/RecA ratio, recombination inhibition by UmuD'C was reversed by increasing the RecA cell concentration . RecA1730, a mutant protein impaired in the chaperone activity, was insensitive to UmuD'C inhibition . We propose a model accounting for the RecA chaperone function in SOS mutagenesis and for the UmuD'C inhibitory effect on homologous recombination . We suggest that the UmuD'C complex is placed at the tip of a RecA polymer as a result of a treadmilling process . This would position the UmuD'C complex right at a lesion while the capping by UmuD'C would destabilize a RecA polymer and thereby abort the recombination process. J Mol Biol, 1997 Jul 11, 270(2), 188 - 200 oriT-processing and regulatory roles of TrwA protein in plasmid R388 conjugation; Moncalian G et al.; TrwA protein was purified from an overproducing Escherichia coli strain and characterized as a 53 kDa tetrameric DNA-binding protein . Gel shift assays showed that TrwA bound specifically to the oriT sequence of plasmid R388 . DNAse I footprinting analysis defined two DNA regions within oriT (sites A and B) that were protected by TrwA . At low TrwA concentrations only region A was protected (K(D) = 4 x 10(-8) M) while region B required higher TrwA concentrations (K(D) = 4 x 10(-7) M) . As a result of its binding to oriT, TrwA was found to perform two biochemical activities related to its role in R388 conjugation . First, TrwA binding to oriT resulted in transcriptional repression of the trwABC operon as indicated by its effect on the beta-galactosidase activity of transcriptional fusions in trwB and trwC, and by direct measurement of the trwA mRNA levels by hybridization . This result was further confirmed by the fact that TrwA overexpression resulted in lowered conjugation frequencies . Second, TrwA enhanced the relaxation activity of TrwC in vitro . This effect was correlated to a 10(5)-fold increase in the frequency of conjugation in vivo and was shown to be independent of the regulation of transcription . Thus, TrwA shows functional similarities to protein TraY of F-like plasmids, that could be correlated to a structural similarity in their DNA-binding motifs. J Mol Biol, 1997 Jul 11, 270(2), 152 - 68 A nucleoprotein activation complex between the leucine-responsive regulatory protein and DNA upstream of the gltBDF operon in Escherichia coli; Wiese DE 2nd et al.; The global regulator Lrp (leucine-responsive regulatory protein), in some cases modulated by its co-regulator leucine, has been shown to regulate more than 40 genes and operons in Escherichia coli . Leucine modulates Lrp regulation of leucine-responsive operons . The level of sensitivity of these operons to leucine varies greatly, but the basis for this variation is only partially understood . One operon controlled by Lrp that is relatively insensitive to leucine is gltBDF, which includes genes specifying the large (GltB) and small (GltD) subunits of glutamate synthase . Earlier gel mobility shift assays have demonstrated that Lrp binds to a fragment of DNA containing the gltBDF promoter region . To further define the nature of this Lrp-gltBDF interaction, DNase I footprinting experiments were performed . The results indicate that Lrp binds cooperatively to three sites quite far upstream, spanning the region from -140 to -260 base-pairs relative to the start of transcription . Phased hypersensitivity is observed throughout the entire binding region, suggesting that Lrp bends the DNA . To determine the relative importance of these three sites for the transcriptional activation of gltBDF, a series of site-directed mutations was generated . The effects of these mutations on Lrp binding were determined both by DNase I footprinting and by quantitative mobility shift assays, while their effects on transcription in vivo were examined by measuring beta-galactosidase activity levels of chromosomal gltB::lacZ operon fusions . Our results indicate that all three sites are required for maximal gene expression, as is the proper phasing of the sites with one another and with the start of transcription . Our results suggest that Lrp binds a central palindromic site, interacting predominantly with the major groove of its DNA target, and that additional dimers bind to flanking sites to form a nucleoprotein activation complex. Cell, 1997 Jul 11, 90(1), 77 - 86 The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner; Anderson DG et al.; Double-stranded DNA break repair and homologous recombination in E . coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end . This process is stimulated by cis-acting DNA elements, known as chi sites . Using both in vitro pairing and nuclease protection assays, we demonstrate that the translocating RecBCD enzyme, which has been activated by chi, coordinates the preferential loading of the homologous pairing protein, RecA, onto the resultant single-stranded DNA downstream of chi . This facilitated loading of RecA protein results in a substantial increase in both the efficiency and rate of in vitro recombination reactions and offers an explanation for stimulation of recombination and repair in vivo by chi. Cell, 1997 Jul 11, 90(1), 43 - 53 A small, stable RNA induced by oxidative stress: role as a pleiotropic regulator and antimutator; Altuvia S et al.; Exposure of E . coli to hydrogen peroxide induces the transcription of a small RNA denoted oxyS . The oxyS RNA is stable, abundant, and does not encode a protein . oxyS activates and represses the expression of numerous genes in E . coli, and eight targets, including genes encoding the transcriptional regulators FhlA and sigma(S), were identified . oxyS expression also leads to a reduction in spontaneous and chemically-induced mutagenesis . Our results suggest that the oxyS RNA acts as a regulator that integrates adaptation to hydrogen peroxide with other cellular stress responses and helps to protect cells against oxidative damage. J Biol Chem, 1997 Jul 11, 272(28), 17610 - 4 Two modes of ligand binding in maltose-binding protein of Escherichia coli . Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling; Hall JA et al.; Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes . We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels . Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes . The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP . Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin . Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J . A., Gehring, K., and Nikaido, H . (1997) J . Biol . Chem . 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane . In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive . The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex. J Biol Chem, 1997 Jul 11, 272(28), 17605 - 9 Two modes of ligand binding in maltose-binding protein of Escherichia coli . Correlation with the structure of ligands and the structure of binding protein; Hall JA et al.; Ligands that are transported by the maltose transport system of Escherichia coli must first bind to the periplasmic maltose-binding protein (MBP) . However, binding of a ligand does not always lead to its transport . As reported earlier, reduced or oxidized maltodextrins bind tightly to MBP but are not transported; some mutant MBPs, such as MalE254, bind maltodextrins tightly but cannot produce their transport . In this study, UV differential spectroscopy and fluorescence emission spectroscopy were used to study the modes by which various ligands bind to MBP . Maltose binding produced a red shift in the fluorescence emission spectrum of wild type MBP and a sharp hypochromatic trend below 265 nm in its UV spectrum (R mode (for red)) . On the other hand, binding of reduced, oxidized, or cyclic maltodextrins produced a pronounced blue shift in the fluorescence emission spectrum of wild type MBP and a peak at about 250 nm in its UV difference spectrum (B mode (for blue) . Binding of reducing maltodextrins to wild type MBP produced spectral changes that seemed to be a mixture of predominantly R mode binding and some B mode binding, whereas their binding to mutant MBP MalE254 produced changes indicative of pure B mode binding . Thus, the ligands that are bound exclusively via the B mode to either the wild type or MalE254 MBP are not transported. J Biol Chem, 1997 Jul 11, 272(28), 17444 - 9 Regulation of transducin GTPase activity by human retinal RGS; Natochin M et al.; The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal . The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin . We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity . As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha-subunit, GtalphaGDPAlF4-, while its binding to GtalphaGTPgammaS or GtalphaGDP was weak . hRGSr and Pgamma did not compete for the interaction with GtalphaGDPAlF4- . Affinity of the Pgamma-GtalphaGDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtalphaGDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PgammaBC) . Binding of hRGSr to GtalphaGDPAlF4- complexed with PgammaBC resulted in a maximal approximately 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtalphaGDPAlF4- (Kd 35 nM) . In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1 . Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1) . The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations . In ROS suspensions containing 30 microM rhodopsin these rates exceeded 0.7 s-1 . Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor . The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade. J Biol Chem, 1997 Jul 11, 272(28), 17263 - 8 The localization of the phosphorylation site of BglG, the response regulator of the Escherichia coli bgl sensory system; Chen Q et al.; BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue . We report here the localization of the phosphorylation site to histidine 208 . Localization of the phosphorylated histidine was carried out in two steps . We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate . This allowed the localization of the phosphorylation site to the last 111 residues . Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor . Histidine 208 was the only histidine which failed both tests . The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins . The bgl system represents a new family of sensory systems . Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family. Exp Cell Res, 1997 Jul 10, 234(1), 132 - 8 Uncoupling of S-phase and mitosis by recombinant cytotoxic necrotizing factor 2 (CNF2); Denko N et al.; Cytotoxic necrotizing factor 2 (CNF2) is an exotoxin identified from virulent clinical isolates of Escherichia coli . It has been characterized in adherent cell lines as an inducer of cellular death, hyperploidy (multinucleation), and cytoskeletal reorganization . The molecular mechanism of these actions is unclear . Two cellular mechanisms can be hypothesized to explain the DNA content increase (hyperploidy) induced by the toxin . The first is that the toxin interferes with cytoplasmic division without interfering with normal nuclear cycling, such that DNA is replicated in the absence of cell division . The second is that the toxin drives the nuclear machinery to replicate the DNA multiple times within one cell cycle, without interfering with cytoplasmic division . In order to investigate these phenomena, we have constructed a recombinant CNF2 gene that expresses a toxin with both an epitope tag and a polyhistidine tag . Extracts made from E . coli that express this gene have a high multinucleating activity that colocalizes with the recombinant 115-kDa protein . To distinguish between these hypotheses, we used recombinant CNF2 and several growth conditions (time, partial differentiation, and stage of growth) to establish a relationship between cellular divisions and generation of hyperploidy . It was also determined that the toxin had no effect upon in vitro DNA replication using a Xenopus egg extract system . In aggregate, these data are consistent with the hypothesis that CNF2 is affecting cytoplasmic division and thereby removing the requirement for a completed mitosis before the initiation of another S-phase . These data are discussed in relation to the generation of polyploid cells during megakaryopoeisis and the generation of aneuploid cells during tumorigenesis. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7521 - 5 The solution structure of the N-terminal domain of alpha2-macroglobulin receptor-associated protein; Nielsen PR et al.; The three-dimensional structure of the N-terminal domain (residues 18-112) of alpha2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy . The structure consists of three helices composed of residues 23-34, 39-65, and 73-88 . The three helices are arranged in an up-down-up antiparallel topology . The C-terminal 20 residues were shown not to be in a well defined conformation . A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed . It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure . Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin . Here we provide the three-dimensional structure of the pathogenic epitope in RAP . The amino acid residues known to form the epitope are in a helix-loop-helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7429 - 34 Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase; Rosenquist TA et al.; Oxidative DNA damage is generated by reactive oxygen species . The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair . Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast . We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae . Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair . The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E . coli . Extracts prepared from mutY/mutM E . coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a beta-lyase activity that nicks DNA 3' to the lesion . The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7, 8-dihydroxy-8-oxo-2'-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA . Mouse and human ogg1 genes contain a helix-hairpin-helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases . Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes . Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7221 - 6 Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding; Coleman RA et al.; The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression . Slow promoter binding might be related to TBP's ability to occlude its DNA binding domain through dimerization . Using a "pull-down" based assay, we find that TBP dimers dissociate slowly (t1/2 = 6-10 min), and thus present a formidable kinetic barrier to TATA binding . At 10 nM, TBP appears to exist as a mixed population of monomers and dimers . In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers . The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7192 - 7 Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site; Muller YA et al.; Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor beta2 (TGF-beta) . We have determined its crystal structure at a resolution of 2.5 A, and identified its kinase domain receptor (KDR) binding site using mutational analysis . Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended . The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-beta . Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer . Each site contains two functional "hot spots" composed of binding determinants presented across the subunit interface . The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-beta . Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots. Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7144 - 9 The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase; Arnez JG et al.; The crystal structure of an enzyme-substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme-product complex with histidyl-adenylate . An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase . When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively . Crystals soaked with MnCl2 reveal the existence of two metal binding sites between beta- and gamma-phosphates; these sites appear to stabilize the conformation of the pyrophosphate . The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases. Biochemistry, 1997 Jul 8, 36(27), 8422 - 7 Tetrahydrobiopterin binding to macrophage inducible nitric oxide synthase: heme spin shift and dimer stabilization by the potent pterin antagonist 4-amino-tetrahydrobiopterin; Mayer B et al.; The characteristics of tetrahydrobiopterin (H4biopterin) binding to pteridine-free recombinant macrophage inducible nitric oxide synthase expressed in Escherichia coli were investigated with a special focus given to effects caused by 2,4-diamino-5,6,7, 8-tetrahydro-6-(l-erythro-1,2-dihydroxypropyl)pteridine (4-amino-H4biopterin), a novel pterin-based inhibitor of nitric oxide synthase . The 4-amino compound completely inhibited enzyme stimulation by 10 microM H4biopterin with a half-maximally active concentration of 7.2 +/- 0.39 microM, whereas H2biopterin and sepiapterin were much less potent . Binding studies using {3H}H4biopterin at 4 degrees C revealed biphasic association of the radioligand according to two first-order reactions with apparent rate constants of 2.2 and 0.05 min-1, each accounting for approximately 50% of total binding . Dissociation of {3H}H4biopterin occurred with rate constants of 0.005 and 0.0028 min-1 in the absence and presence of l-arginine, respectively . Specific binding of 10 nM {3H}H4biopterin was antagonized by unlabeled H4biopterin and its 4-amino analog with half-maximal effects at 84 +/- 6 and 34 +/- 3.2 nM, respectively . Binding of H4biopterin and 4-amino-H4biopterin was accompanied by a partial low spin to high spin conversion of the heme that was completed by l-arginine . Similarly, the active cofactor and the inhibitory 4-amino derivative both induced significant formation of stable protein dimers that survived during SDS electrophoresis, suggesting that the allosteric effects caused by H4biopterin do not explain sufficiently the essential role of the pteridine cofactor in NO biosynthesis. Biochemistry, 1997 Jul 8, 36(27), 8340 - 8 Pre-steady-state study of recombinant sesquiterpene cyclases; Mathis JR et al.; An Escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step . Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimeric 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein . From initial velocity experiments, the Michaelis constant for HVS was 3.5 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, respectively . Steady-state catalytic constants were from 0.02 to 0.04 s-1 . A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in farnesyl diphosphate (FPP) . Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step . Dissociation constants for the enzyme-substrate complex (20-70 microM) were determined from the pre-steady-state experiments and were significantly larger than the observed Michaelis constants . A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes . Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents. Biochemistry, 1997 Jul 8, 36(27), 8332 - 9 Pre-steady-state kinetic analysis of the trichodiene synthase reaction pathway; Cane DE et al.; The pre-steady-state kinetics of the trichodiene synthase reaction were investigated by rapid chemical quench methods . The single-turnover rate was found to be 3.5-3.8 s-1, a rate 40 times faster than the steady-state catalytic rate (kcat = 0.09 s-1) for trichodiene synthase-catalyzed conversion of farnesyl diphosphate (FPP) to trichodiene at 15 degrees C . In a multiturnover experiment, a burst phase (kb = 4.2 s-1) corresponding to the accumulation of trichodiene on the surface of the enzyme was followed by a slower, steady-state release of products (klin = 0.086 s-1) which corresponds to kcat . These results strongly suggest that the release of trichodiene from the enzyme active site is the rate-limiting step in the overall reaction, while the consumption of FPP is the step which limits chemical catalysis at the active site . Single-turnover experiments with trichodiene synthase mutant D101E, for which the steady-state rate constant kcat is 1/3 of that of wild type, revealed that the mutation actually depresses the rate of FPP consumption by a factor of 100 . The deuterium isotope effect on the consumption of {1-2H,1,2-14C}FPP was found to be 1.11 +/- 0.06 . Single turnover reactions of {1,2-14C}FPP catalyzed by trichodiene synthase were carried out at 4, 15, or 30 degrees C in an effort to provide direct observation of the proposed intermediate nerolidyl diphosphate (NPP) . However, no NPP was detected, indicating that the conversion of NPP must be too fast to be observed within the detection limits of the assay . Taken together, these observations suggest that the isomerization of FPP to NPP is the step which limits the rate of chemical catalysis in the trichodiene synthase reaction pathway. Biochemistry, 1997 Jul 8, 36(27), 8269 - 75 The modified wobble base inosine in yeast tRNAIle is a positive determinant for aminoacylation by isoleucyl-tRNA synthetase; Senger B et al.; Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity . Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively . We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase . We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost . As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity . The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript . The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E . coli isoleucyl-tRNA synthetase . In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine {Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S . (1988) Nature 336, 179-181}, pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS . Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity. Biochemistry, 1997 Jul 8, 36(27), 8209 - 14 Mutational analysis of substrate recognition by protein phosphatase 1; Zhang L et al.; The role of residues that are involved in substrate recognition by rabbit muscle protein phosphatase 1alpha (PP1) was investigated by site-directed mutagenesis and kinetic analyses using phosphorylase a, RII peptide, Kemptide, and p-nitrophenyl phosphate as substrates . The atomic structure of PP1 has shown the active site to be at the confluence of three shallow grooves, a C-terminal groove, an acidic groove, and a hydrophobic groove . Mutations of residues D208, D210, D212, E218, D220, E252, D253, E256, E275, and D277 in the acidic groove, of R221, W206, and Y134, which have been suggested to be involved in substrate binding, and of residues C127, I130, and D197 in the hydrophobic groove were examined . Our results show that mutations in the acidic groove lead to modest changes in substrate binding, consistent with a role of the acidic residues in forming a negatively charged surface well for binding of peptides with basic N-termini . Severe effects on Vmax were observed for mutants of R221, D208, and W206 . These results are consistent with the proposal that the R221 plays an important role as a phosphate oxygen ligand that positions the substrate for catalysis . The kinetic behavior of mutants at W206 and D208 can be explained by the observation that, together with R221, these residues form the microenvironment which dictates the orientation of the imidazole ring of H248, one of the metal binding ligands, as well as contributing to the orientation of R221 itself. FEBS Lett, 1997 Jul 7, 411(1), 128 - 32 The propeptide of subtilisin BPN' as a temporary inhibitor and effect of an amino acid replacement on its inhibitory activity; Kojima S et al.; The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone . For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli . Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates . The main cleavage site was identified as Glu53-Lys54, with minor sites at Thr17-Met18 and Met21-Ser22, which were located in turn regions of the propeptide in the complex with subtilisin BPN' . Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin . Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor. FEBS Lett, 1997 Jul 7, 411(1), 123 - 7 Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration; Kholod NS et al.; Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E . coli phenylalanyl-tRNA synthetase (FRS) . In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E . coli tRNA(Phe) gene transcript . The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E . coli counterpart . However, the maximal aminoacylation rate for T5 and E . coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively . Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E . coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS . Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E . coli FRS at low and high Mg2+ concentrations . Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS. FEBS Lett, 1997 Jul 7, 411(1), 43 - 7 GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II; Persson M et al.; The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied . The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (E(a)) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively). Neuroreport, 1997 Jul 7, 8(9-10), 2399 - 404 Differential expression of the GABAA receptor alpha 1 subunit in developing chicken brain; Fan SS et al.; A unique segment of chicken GABAA receptor alpha 1 subunit was expressed in E . coli and used to generate an antiserum 2A specific for the subunit . The DNA fragment encoding the segment of alpha 1 was obtained by selective amplification by polymerase chain reaction (PCR) from a chicken brain cDNA library . The antiserum is characterized by its capacity to immunoprecipitate a {3H}flunitrazepam binding protein of 50 kDa, the chicken GABAA receptor alpha 1 subunit . Subsequent immunoblotting and immunocytochemistry analyses reveal that alpha 1 is expressed in the optic tectum and cerebellum as early as embryonic day 15 (E15), in various areas of telencephalon as early as E20 and distributed heterogeneously among different cell types . The early expression of alpha 1 may imply its functional significance in neurotransmission. J Mol Biol, 1997 Jul 4, 270(1), 26 - 35 Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library; Atwell S et al.; Structure-guided phage display was used to select for combinations of interface residues for antibody C(H)3 domains that promote the formation of stable heterodimers . A C(H)3 "knob" mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W . A library of C(H)3 "hole" mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner C(H)3 domain . The C(H)3 knob mutant was fused to a peptide flag and the C(H)3 hole library was fused to M13 gene III . Phage displaying stable C(H)3 heterodimers were recovered by panning using an anti-flag antibody . Phage-selected C(H)3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407'A, and most clones tested were more stable to guanidine hydrochloride denaturation . The thermal stability of individual C(H)3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry . One heterodimer, T366W-T366'S:L368'A:Y407'V, had a t(m) of 69.4 degrees C, which is 4.0 deg.C higher than that for the designed heterodimer and 11.0 deg.C lower than that for the wild-type homodimer . The phage-selected C(H)3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay . Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization. J Mol Biol, 1997 Jul 4, 270(1), 14 - 25 Regulation of the "tetCD" genes of transposon Tn10; Pepe CM et al.; In addition to the genes involved in tetracycline resistance, the loop region of the composite transposon Tn10 contains two other known genes, tetC and tetD, whose functions are unclear . Using primarily a genetic approach, we examined tetCD gene expression and regulation . The tetC gene product, TetC, is a diffusible repressor of both tetC and tetD transcription . Despite an earlier claim by others, we do not detect induction of either tetC or tetD by tetracycline (Tc) or several of its analogs . Although the 5' ends of the tetC and tetD messages overlap due to transcription from convergent promoters, we find no evidence for anti-sense RNA control . The operator for the TetC repressor has been localized . We also demonstrate that transcription from the tetD promoter probably terminates within IS10-Right and does not apparently interfere with Tn10 or IS10-Right transposition or its regulation. Biochim Biophys Acta, 1997 Jul 4, 1320(3), 275 - 84 Characterization of the alpha and beta-subunits of the F0F1-ATPase from the alga Polytomella spp., a colorless relative of Chlamydomonas reinhardtii; Atteia A et al.; The isolation and partial characterization of the oligomycin-sensitive F0F1-ATP synthase/ATPase from the colorless alga Polytomella spp . is described . Purification was performed by solubilization with dodecyl-beta-D-maltoside followed by Sepharose Hexyl ammonium chromatography, a matrix that interacts with the F1 sector of mitochondrial ATPases . The alpha-subunit, which migrates on SDS-polyacrylamide gels with an apparent molecular mass of 55 kDa, was identified by the N-terminal sequencing of 47 residues . This subunit exhibited a short extension at its N-terminus highly similar to the one described for the unicellular alga Chlamydomonas reinhardtii (Nurani, G . and Franzen L.-G . (1996) Plant Mol . Biol . 31, 1105-1116) . In whole mitochondria, the alpha-subunit was susceptible to limited proteolytic digestion induced by heat . An endogenous protease removed the first 22 residues of the mature alpha-subunit . Subunit beta was also identified by N-terminal sequencing of 31 residues . This subunit of 63 kDa exhibited a higher apparent molecular mass than alpha, as judged by its mobility on denaturing polyacrylamide gel electrophoresis . This beta-subunit is 7-8 kDa larger than the beta-subunits of other mitochondrial ATPases . It is suggested that the beta-subunit from Polytomella spp . may have a C-terminal extension similar to that described for the green alga C . reinhardtii (Franzen, L.-G . and Falk, G.(1992) Plant Mol . Biol . 19, 771-780) . In addition, it was found that the C-terminal extension of the beta-subunit of C . reinhardtii showed homology with the endogenous ATPase inhibitors from various sources and with the epsilon-subunit from the F0F1-ATP synthase from Escherichia coli, which is considered to be a functional homolog of the inhibitor proteins . The data reported here provide the first biochemical evidence for a close relationship between the colorless alga Polytomella spp . and its photosynthetic counterpart C . reinhardtii . It is also suggested that the C-terminal extensions of the beta-subunits of the ATP synthases from these algae, may play a regulatory role in these enzymes. Biochim Biophys Acta, 1997 Jul 4, 1320(3), 217 - 34 Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors; Unden G et al.; The electron-transport chains of Escherichia coli are composed of many different dehydrogenases and terminal reductases (or oxidases) which are linked by quinones (ubiquinone, menaquinone and demethylmenaquinone) . Quinol:cytochrome c oxido-reductase ('bc1 complex') is not present . For various electron acceptors (O2, nitrate) and donors (formate, H2, NADH, glycerol-3-P) isoenzymes are present . The enzymes show great variability in membrane topology and energy conservation . Energy is conserved by conformational proton pumps, or by arrangement of substrate sites on opposite sides of the membrane resulting in charge separation . Depending on the enzymes and isoenzymes used, the H+/e- ratios are between 0 and 4 H+/e- for the overall chain . The expression of the terminal reductases is regulated by electron acceptors . O2 is the preferred electron acceptor and represses the terminal reductases of anaerobic respiration . In anaerobic respiration, nitrate represses other terminal reductases, such as fumarate or DMSO reductases . Energy conservation is maximal with O2 and lowest with fumarate . By this regulation pathways with high ATP or growth yields are favoured . The expression of the dehydrogenases is regulated by the electron acceptors, too . In aerobic growth, non-coupling dehydrogenases are expressed and used preferentially, whereas in fumarate or DMSO respiration coupling dehydrogenases are essential . Coupling and non-coupling isoenzymes are expressed correspondingly . Thus the rationale for expression of the dehydrogenases is not maximal energy yield, but could be maximal flux or growth rates . Nitrate regulation is effected by two-component signal transfer systems with membraneous nitrate/nitrite sensors (NarX, NarQ) and cytoplasmic response regulators (NarL, NarP) which communicate by protein phosphorylation . O2 regulates by a two-component regulatory system consisting of a membraneous sensor (ArcB) and a response regulator (ArcA) . ArcA is the major regulator of aerobic metabolism and represses the genes of aerobic metabolism under anaerobic conditions . FNR is a cytoplasmic O2 responsive regulator with a sensory and a regulatory DNA-binding domain . FNR is the regulator of genes required for anaerobic respiration and related pathways . The binding sites of NarL, NarP, ArcA and FNR are characterized for various promoters . Most of the genes are regulated by more than one of the regulators, which can act in any combination and in a positive or negative mode . By this the hierarchical expression of the genes in response to the electron acceptors is achieved . FNR is located in the cytoplasm and contains a 4Fe4S cluster in the sensory domain . The regulatory concentrations of O2 are 1-5 mbar . Under these conditions O2 diffuses to the cytoplasm and is able to react directly with FNR without involvement of other specific enzymes or protein mediators . By oxidation of the FeS cluster, FNR is converted to the inactive state in a reversible process . Reductive activation could be achieved by cellular reductants in the absence of O2 . In addition, O2 may cause destruction and loss of the FeS cluster . It is not known whether this process is required for regulation of FNR function. J Biol Chem, 1997 Jul 4, 272(27), 17091 - 6 Recombination-dependent repair of DNA double-strand breaks with purified proteins from Escherichia coli; Morel P et al.; We have developed an in vitro system in which repair of DNA double-strand breaks is performed by purified proteins of Escherichia coli . A segment was deleted from a circular duplex DNA molecule by restriction at two sites . 3' single-stranded overhangs were introduced at both ends of the remaining linear fragment . In a first step, RecA protein catalyzed the formation of a D-loop between one single-stranded tail and a homologous undeleted supercoiled DNA molecule . In a second step, E . coli DNA polymerase II or III used the 3' end in the D-loop as a primer to copy the missing sequences of the linear substrate on one strand of the supercoiled template . Under proper conditions, the integrity of the deleted substrate was restored, as shown by analysis of the products by electrophoresis, restriction, and transformation . In this reaction, DNA synthesis is strictly dependent on recombination, and repair is achieved without formation of a Holliday junction. J Biol Chem, 1997 Jul 4, 272(27), 17038 - 44 Regulation of the AKAP79-protein kinase C interaction by Ca2+/Calmodulin; Faux MC et al.; The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (PKC) at the postsynaptic densities in neurons . Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme . This report focuses on the regulation of AKAP79-PKC interaction by calmodulin . AKAP79 binds calmodulin with high affinity (KD of 28 +/- 4 nM (n = 3)) in a Ca2+-dependent manner . Immunofluorescence staining shows that both proteins exhibit overlapping staining patterns in cultured hippocampal neurons . Calmodulin reversed the inhibition of PKCbetaII by the AKAP79(31-52) peptide and reduced inhibition by the full-length AKAP79 protein . The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31-52) peptide was shown to be partially dependent on Ca2+ . Ca2+/calmodulin reduced PKC coimmunoprecipitated with AKAP79 and resulted in a 2.6 +/- 0.5-fold (n = 6) increase in PKC activity in a preparation of postsynaptic densities . Collectively, these findings suggest that Ca2+/calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein. J Biol Chem, 1997 Jul 4, 272(27), 16962 - 71 Allosteric mechanism of induction of CytR-regulated gene expression . Cytr repressor-cytidine interaction; Barbier CS et al.; Transcription from cistrons of the Escherichia coli CytR regulon is activated by E . coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR . De-repression results when CytR binds cytidine . CytR is a homodimer and a LacI family member . A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription . To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA . Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps . To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified . Each has a substitution for Asp281 or neighboring residue . CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR . Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR . The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions . The data support the conclusion that the first cytidine binding step alone is sufficient to induce. J Biol Chem, 1997 Jul 4, 272(27), 16946 - 54 Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase; Hemmer W et al.; A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases . Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala . Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized . Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis . Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat . The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold . In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude . The thermostability of each mutant was slightly increased . Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338 . All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197 . These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family. J Biol Chem, 1997 Jul 4, 272(27), 16924 - 7 Kinetics and thioredoxin specificity of thiol modulation of the chloroplast H+-ATPase; Schwarz O et al.; The kinetics of thiol modulation of the chloroplast H+-ATPase (CF0CF1) in membrana were analyzed by employing thioredoxins that were kept reduced by 0.1 mM dithiothreitol . The kinetics of thiol modulation depend on the extent of the proton gradient . The process is an exponential function of the thioredoxin concentration and reaction time and can be described by an irreversible second order reaction . The results indicate that the formation of the complex between thioredoxin and CF0CF1 is slow compared with the subsequent reduction step . Furthermore we have compared the efficiencies of the Escherichia coli thioredoxin Trx and the two chloroplast thioredoxins Tr-m and Tr-f . The second order rate constants are 0.057 (Tr-f), 0.024 (Trx), and 0.010 s-1 microM-1 (Tr-m) suggesting that Tr-f rather than Tr-m is the physiological reductant for the chloroplast ATPase . The often employed artificial reductant dithiothreitol exhibits a second order rate constant in thiol modulation of 1.02.10(-6) s-1 microM-1. J Biol Chem, 1997 Jul 4, 272(27), 16911 - 6 Relationship of conserved residues in the IMP binding site to substrate recognition and catalysis in Escherichia coli adenylosuccinate synthetase; Wang W et al.; Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase . Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity . Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates . Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis . The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme . However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme . Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme . Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities . Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures . Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP. J Biol Chem, 1997 Jul 4, 272(27), 16798 - 806 Hairpin formation during DNA synthesis primer realignment in vitro in triplet repeat sequences from human hereditary disease genes; Ohshima K et al.; Genetic expansion of DNA triplet repeat sequences (TRS) found in neurogenetic disorders may be due to abnormal DNA replication . We have previously observed strong DNA synthesis pausings at specific loci within the long tracts (> approximately 70 repeats) of CTG.CAG and CGG.CCG as well as GTC.GAC by primer extensions in vitro using DNA polymerases (the Klenow fragment of Escherichia coli DNA polymerase I, the modified T7 DNA polymerase (Sequenase), and human DNA polymerase beta) . Herein, we have isolated and analyzed the products of stalled synthesis found at approximately 30-40 triplets from the beginning of the TRS . DNA sequence analyses revealed that the stalled products contained short tracts of homogeneous TRS (6-12 repeats) in the middle of the sequence corresponding to the flanking region of the primer-template sequence . The sequence at the 3'-side terminated at the end of the primer, indicating that the primer molecule had served as a template . In addition, chemical probe and polyacrylamide gel electrophoretic analyses revealed that the stalled products existed in hairpin structures . We postulate that these products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the TRS due to primer realignment . The consequence of these steps is DNA synthesis to the end of the primer and termination . Primer realignment including hairpin formation may play an important intermediate role in the replication of TRS in vivo to elicit genetic expansions. J Biol Chem, 1997 Jul 4, 272(27), 16746 - 52 Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin . Optical spectra and reactions with gaseous ligands; Duff SM et al.; A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli . The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa) . The recombinant Hb is expressed constitutively . Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein . Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography . Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer . Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate . The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein . The implications of these kinetic data on the in vivo function of barley Hb are discussed. Mutat Res, 1997 Jul 3, 377(2), 263 - 8 Nebularine (9-2'-deoxy-beta-D-ribofuranosylpurine) has the template characteristics of adenine in vivo and in vitro; Rahman MS et al.; Nebularine (9-beta-D-ribofuranosylpurine; Nb) is a naturally occurring nucleoside with structural features suggestive of a universal base . However, previous observations based on thermal melting characteristics of oligonucleotides suggested that Nb formed stable pairs only with thymine . To determine the template characteristics of Nb, we constructed M13 viral single-stranded DNA (ssDNA) molecules bearing a single site-specific deoxynebularine (9-2'-deoxy-beta-D-ribofuranosylpurine) residue . The ssDNA constructs were transfected into Escherichia coli cells to determine the specificity of base insertion opposite Nb, as well as to determine the effect of Nb on the replicability of the transfected DNA . Base insertion opposite Nb, analyzed by a multiplex sequencing technology, suggests that Nb has the template characteristics of adenine . Analysis of DNA replicability, measured as transfection efficiency, indicates that Nb does not block DNA replication . UV irradiation of host cells before transfection did not significantly affect survival or base insertion specificity within the limits of multiplex sequencing technology employed, suggesting that inducible mutagenic phenomena appear to have only minor effects on translesion synthesis across Nb . In addition, in vitro DNA elongation experiments on oligonucleotide templates using E . coli DNA polymerase I (Klenow fragment) as the model polymerase showed that Nb templates for T, but not for other bases under the tested conditions . The data reported in this communication underscore the importance of base-pair geometry as a specificity-determinant during base insertion by replicative polymerases. Mutat Res, 1997 Jul 3, 377(2), 255 - 62 Mutational specificity of glyoxal, a product of DNA oxidation, in the lacI gene of wild-type Escherichia coli W3110; Murata-Kamiya N et al.; To determine the mutation spectrum of glyoxal, which is produced from DNA by oxygen free-radicals, we analyzed the chromosomal lacI gene of mutants induced by treatment of a wild-type Escherichia coli strain with glyoxal . The cell death and the mutation frequency increased according to the concentration of glyoxal added to the culture medium . The majority of the spontaneous mutations (82%) and that of the glyoxal-induced mutations (50%) were the addition or deletion of a 5'-TGGC-3' sequence at positions 623-634, which was reported to be a mutational hot spot in the lacI gene . In the glyoxal-induced mutants, however, the ratio of base-pair substitutions was increased (35%) . Although all types of base-pair substitutions were detected, 78% of the base-pair substitutions occurred at G:C sites . Among them, G:C-->A:T transitions were predominant, followed by G:C-->T:A transversions . These mutations appeared to be distributed randomly within the lacI gene . These results suggest that glyoxal-induced mutations may correlate to mutations induced by oxygen free-radicals. Mutat Res, 1997 Jul 3, 377(2), 225 - 9 Food-derived heterocyclic amines potentiate the mutagenicity of a drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX); Watanabe-Akanuma M et al.; We investigated the enhancing effect of heterocyclic amines on base-substitution mutations with 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) and 2-amino-3,4-dimethyl-imidazo{4,5-f}quinoline (MeIQ) . We compared the mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the presence and absence of the heterocyclic amines in E . coli WP2 (trpE) and in excision repair-deficient strains WP2s (uvrA, trpE) and ZA500 (uvrA, rfa, trpE) . Since the assay was performed without microsomal metabolic activation, Trp-P-1 and MeIQ alone were not mutagenic . In WP2, trp+ reversions induced by MX were greatly potentiated by Trp-P-1 and slightly potentiated by MeIQ . Mutation enhancement was not observed in strains WP2s and ZA500, suggesting that a functional DNA excision repair system is necessary for the combined action of MX and heterocyclic amines . Our finding implies that the combined effect of mutagens as well as the effect of individual mutagens, should be considered in risk evaluation. Nature, 1997 Jul 3, 388(6637), 87 - 93 A structural basis for mutational inactivation of the tumour suppressor Smad4; Shi Y et al.; The Smad4/DPC4 tumour suppressor is inactivated in nearly half of pancreatic carcinomas and to a lesser extent in a variety of other cancers . Smad4/DPC4, and the related tumour suppressor Smad2, belong to the SMAD family of proteins that mediate signalling by the TGF-beta/activin/BMP-2/4 cytokine superfamily from receptor Ser/Thr protein kinases at the cell surface to the nucleus . SMAD proteins, which are phosphorylated by the activated receptor, propagate the signal, in part, through homo- and hetero-oligomeric interactions . Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other SMADs . The conserved carboxy-terminal domains of SMADs are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation . We now describe the crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumour suppressor, determined at 2.5 A resolution . The structure reveals that the Smad4/DPC4 CTD forms a crystallographic trimer through a conserved protein-protein interface, to which the majority of the tumour-derived missense mutations map . These mutations disrupt homo-oligomerization in vitro and in vivo, indicating that the trimeric assembly of the Smad4/DPC4 CTD is critical for signalling and is disrupted by tumorigenic mutations. Nature, 1997 Jul 3, 388(6637), 82 - 7 Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4; Hata A et al.; Smad2 and Smad4 are related tumour-suppressor proteins, which, when stimulated by the growth factor TGF-beta, form a complex to inhibit growth . The effector function of Smad2 and Smad4 is located in the conserved carboxy-terminal domain (C domain) of these proteins and is inhibited by the presence of their amino-terminal domains (N domain) . This inhibitory function of the N domain is shown here to involve an interaction with the C domain that prevents the association of Smad2 with Smad4 . This inhibitory function is increased in tumour-derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine residue . The mutant N domains have an increased affinity for their respective C domains, inhibit the Smad2-Smad4 interaction, and prevent TGF beta-induced Smad2-Smad4 association and signalling . Whereas mutations in the C domain disrupt the effector function of the Smad proteins, N-domain arginine mutations inhibit SMAD signalling through a gain of autoinhibitory function . Gain of autoinhibitory function is a new mechanism for inactivating tumour suppressors. Int J Cancer, 1997 Jul 3, 72(1), 149 - 54 Interleukin-6 functions as an autocrine growth factor in human bladder carcinoma cell lines in vitro; Okamoto M et al.; Interleukin (IL)-6 is reported to function as a growth factor for renal and prostatic carcinomas . We conducted the present study to define the role of IL-6 in the growth of normal and neoplastic urothelial cells . Human bladder carcinoma cell lines (253J, RT4 and T24) and primary cultured human urothelial cells derived from normal ureters were used . Recombinant human IL-6 stimulated the growth of bladder carcinoma cell lines far better than that of normal urothelial cells (p < 0.001) . All carcinoma cell lines tested produced and released IL-6, whereas normal urothelial cells did so only at marginal levels . Furthermore, treatment with lipopolysaccharide derived from Escherichia coli, tumor necrosis factor-alpha or IL-1 increased IL-6 secretion by bladder carcinoma cell lines but not by normal urothelial cells . Growth of bladder carcinoma cells was significantly inhibited by anti-IL-6 neutralizing antibody or the anti-sense oligonucleotide for IL-6 cDNA . We conclude that IL-6 functions as an autocrine growth factor for bladder carcinoma cells but not for normal urothelial cells and that it may be a factor accounting for the marked enhancement of inflammation-associated bladder carcinogenesis and tumor growth. Mol Microbiol, 1997 Jul, 25(2), 303 - 9 FtsN, a late recruit to the septum in Escherichia coli; Addinall SG et al.; The localization of FtsN in Escherichia coli was inves tigated by immunofluorescence microscopy . FtsN is an essential cell division protein with a simple bitopic topology, a short N-terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain . FtsN was found to localize to the septum in a ring pattern similar to that observed for FtsZ and FtsA, although the frequency of cells with rings was less . A MalG-FtsN fusion was also localized to the septum, indicating that the information for FtsN localization is supplied by its periplasmic domain . FtsN localization was dependent upon the prior localization of FtsZ and FtsA and required the function of FtsI and FtsQ . Consistent with FtsN functioning after FtsZ, Z rings were observed in a mutant depleted of FtsN. Mol Microbiol, 1997 Jul, 25(2), 237 - 46 The ssb-113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli; Quinones A et al.; Mutations in the dnaQ gene, which encodes the proofreading epsilon-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication . In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication . We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49 . The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions . S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter . This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling . These results support the hypothesis that the expression of the epsilon-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis. Mol Microbiol, 1997 Jul, 25(2), 205 - 10 The oxygen-responsive transcriptional regulator FNR of Escherichia coli: the search for signals and reactions; Unden G et al.; The FNR (fumarate and nitrate reductase regulation) protein of Escherichia coli is an oxygen-responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism . In the absence of oxygen, FNR changes from the inactive to the active state . The sensory and the regulatory functions reside in separate domains of FNR . The sensory domain contains a Fe-S cluster, which is of the {4Fe-4S}2+ type under anaerobic conditions . It is suggested that oxygen is supplied to the cytoplasmic FNR by diffusion and inactivates FNR by direct interaction . Reactivation under anoxic conditions requires cellular reductants . In vitro, the Fe-S cluster is converted to a {3Fe-4S}+ or a {2Fe-2S}2+ cluster by oxygen, resulting in FNR inactivation . After prolonged incubation with oxygen, the Fe-S cluster is destroyed . Reassembly of the {4Fe-4S}2+ cluster might require cellular proteins, such as the NifS-like protein of E . coli . In this review, the rationale for regulation of alternative metabolic pathways by FNR and other oxygen-dependent regulators is discussed . Only the terminal reductases of respiration, and not the dehydrogenases, are regulated in such a way as to achieve maximal H+/e- ratios and ATP yields. J Ind Microbiol Biotechnol, 1997 Jul, 19(1), 66 - 70 Linear epitopes of colonization factor antigen I and peptide vaccine approach to enterotoxigenic Escherichia coli; Cassels FJ et al.; Enterotoxigenic Escherichia coli (ETEC) cause diarrhea in infants and in travelers to developing countries . The bacteria utilize colonization factors (CF) for adherence to intestinal epithelia, then release toxins causing diarrhea . CF are strong immunogens as well as protective antigens . While 20 ETEC CF have been described in the literature, 11 CF are prominent enough to be considered for vaccine targeting . Of this group, six of the members fall into the CFA/I family of CF . Geysen pin (peptide) linear epitope analysis demonstrated that three regions containing linear epitopes exist in CFA/I, and that both B- and T-cell linear epitopes of CFA/I were concentrated at the N-terminus of the protein . We have determined N-terminal sequence of the CFA/I family members not previously sequenced . Comparison of the protein sequence of the six members of the family showed a strong homology up to residue 36 . A peptide of 36 amino acids representing a consensus of the six sequences was synthesized and used to immunize animals . The antibody induced to the peptide was reactive to the peptide as well as cross-reactive to each member of the CFA/I family in Western blots . In addition, this antibody agglutinated three of the six members of the CFA/I family when added to whole cells expressing the native CF . We are currently evaluating different carriers and conjugation methods to maximize production of high titer, agglutinating antibody . It is hoped that this and related research will result in an effective and inexpensive cross-reactive and cross-protective ETEC vaccine. Int J Parasitol, 1997 Jul, 27(7), 811 - 7 A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein; Sugiyama H et al.; A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction . A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli . The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands . By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms . The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica . At the deduced polypeptide level, the Sj23A also had similarities with the F . hepatica-protein sequence, the amino acid composition {high glycine (16%), lysine (12%) and tyrosine (11%)} and the presence of tyrosine residues flanked by glycine . The clone Sj23A also shared an extensive sequence homology with 3 S . mansoni expression sequence tags (ESTs) . The present results suggest that the protein encoded by the female-specific Sj23A gene of S . japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation. Biol Chem, 1997 Jul, 378(7), 719 - 21 Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide; Valentini G et al.; The gene encoding pyruvate kinase type I (PKI) of Escherichia coli was amplified by PCR, cloned and sequenced . The gene product was overexpressed in E . coli, using an inducible T7 RNA polymerase-based expression system . The transformed cells contained sixtyfold the enzyme activity of the reference cells and enabled the purification of 30 mg of highly active PKI from 1 liter of culture . The gene sequence was determined and found to be different from the one previously reported, i.e., the T nucleotide at position 1351 was missing . This resulted in a downstream shift of the stop codon, thus the deduced polypeptide was 470 amino acids long instead of 462 . In addition the twelve C-terminal amino acids of the former sequence were changed. Biol Chem, 1997 Jul, 378(7), 687 - 95 Expression in Escherichia coli, purification, and spectroscopic characterization of two mutant Bet v 1 proteins; Boehm M et al.; Bet v 1 is the major birch pollen allergen . A highly efficient expression and purification scheme for mutant forms of this protein was developed on the basis of the pET expression system in order to provide the high quantities of protein needed for spectroscopic and structural work . Bet v 1 (M139L) protein could be purified at high yield (approx . 30 mg from 1 liter of LB medium) in a two-step procedure by the use of metal-affinity chromatography . Matrix assisted laser desorption ionisation mass spectroscopy, and size exclusion chromatography demonstrate the homogeneity and purity of the prepared protein . Spectroscopic methods were used to show that Bet v 1 (M139L) is structurally similar to wild type Bet v 1 . Furthermore, we investigated the influence of the nature of amino acid 139 on the thermodynamic behaviour of the protein by replacing the leucine residue by alanine . While there appears to be no global structural effect of this mutation, the thermostability of Bet v 1 is greatly decreased. J Biochem (Tokyo), 1997 Jul, 122(1), 243 - 50 Molecular cloning of Ca2+/calmodulin-dependent protein kinase kinase beta; Kitani T et al.; Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous and immune systems, is markedly activated upon phosphorylation through the action of CaM-kinase kinase . Our previous immunotitration analysis suggested the existence of an isoform different from CaM-kinase kinase alpha, the beta isoform, in rat brain {Okuno, S., Kitani, T., and Fujisawa, H . (1996) J . Biochem . 119, 1176-1181} . In the present study, cDNA for CaM-kinase kinase beta was cloned from a rat cerebellar cDNA library . The coded protein consisted of 587 amino acids with a molecular weight of 64,445 . Western blot analysis revealed that CaM-kinase kinase beta significantly existed only in the brain . The enzyme was not significantly detected in the retina where CaM-kinase kinase alpha exists. J Biochem (Tokyo), 1997 Jul, 122(1), 217 - 25 Effects of amino acid side-chain volume on chain packing in genetically engineered periodic polypeptides; Cantor EJ et al.; The fidelity of bacterial protein synthesis allows the production of architecturally well-defined polymeric materials through precise control of chain length, sequence, stereochemistry, and interchain interactions . In the present paper, we examine the relation between amino acid residue volume and crystalline unit cell dimensions, in a set of periodic protein polymers of repeating unit sequence -(AlaGly)3-X-Gly-, where X is Asn, Phe, Ser, Val, or Tyr . The proteins were overexpressed in Escherichia coli, purified by simple procedures based on acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate, and processed to form oriented crystalline mats by precipitation from formic acid under mechanical shear . X-ray diffraction analyses revealed that the basic structures of the -(AlaGly)3-X-Gly- polymers are identical to that previously reported for {(AlaGly)3-GluGly}36, {Krejchi, M.T., Atkins, E.D.T., Waddon, A.J., Fournier, M.J., Mason, T.L., and Tirrell, D.A . (1994) Science 265, 1427-1432}, with the oligoalanylglycine segments forming antiparallel beta-sheets and the substituted amino acids occurring within three-residue folds at the lamellar surfaces . The X-ray diffraction signals for each member of the family index on an orthorhombic unit cell; the a-axis (hydrogen bond direction) and c-axis (chain direction) spacings remain invariant but the b-axis (sheet stacking direction) spacing increases with increasing volume of the substituted amino acid . The results obtained from a variant with alternating Glu and Lys substitution at the X position, together with the results previously reported for poly(L-alanylglycine) {Panitch, A., Matsuki, K., Cantor, E.J., Cooper, S.J., Atkins, E.D.T., Fournier, M.J., Mason, T.L., and Tirrell, D.A . (1997) Macromolecules 30, 42-49} are included for comparison . The average intersheet stacking distance (b/2) increases linearly with the volume of the amino acid inserted at position X . Because the chain-folded lamellar architecture adopted by these periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity, these results illustrate a new approach to the engineering of intermolecular interactions in polymeric solids. J Biochem (Tokyo), 1997 Jul, 122(1), 122 - 8 Characterization of a human placental fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase; Sakakibara R et al.; A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli . The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the deduced molecular weight of 59,410 . The N-terminal sequence of 15 amino acids coincided with that of the deduced sequence . The active enzyme was a dimer as judged by molecular sieve filtration . The expressed enzyme was bifunctional with Vmax values of 142 and 0.2 milliunits/mg for the kinase and phosphatase activities, respectively . The phosphatase activity was extremely low, because one phosphatase active site residue was mutated, and consequently the kinase/phosphatase ratio was the highest among the known isozymes . Furthermore, the enzyme was phosphorylated by cAMP-dependent protein kinase, protein kinase C and also by {2-32P}fructose-2,6-bisphosphate . Phosphorylation by cAMP-dependent protein kinase and protein kinase C increased the maximal Fru-6-P,2-kinase activities by 1.8- and 1.1-fold, respectively . These results suggested that placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase is important in maintaining and regulating a relatively high rate of glycolysis in placenta. J Biochem (Tokyo), 1997 Jul, 122(1), 55 - 63 Enzyme flexibility: a new concept in recognition of hydrophobic substrates; Kawaguchi S et al.; The mechanism of recognition of hydrophobic substrates was investigated using Escherichia coli aspartate aminotransferase (AspAT), E . coli aromatic amino acid aminotransferase (AroAT), and their chimeric enzyme (DY18) . Surprisingly, broad substrate specificity was observed in the reaction of aminotransferases with hydrophobic substrates . The catalytic efficiency increased with an increase in the side chain length of straight or branched-terminal aliphatic substrates . The straight-chain substrates catalysed with maximal efficiency were the 7-carbon substrate in the case of AspAT and the 8-carbon substrate for AroAT and DY18 . Consecutive addition of single methylene groups to the substrate had a constant effect on the stabilization energy of the transition state relative to the unbound state . The dependency of binding energy on each methylene group is usually interpreted as indicating hydrophobicity of the active site . However, we observed that AroAT and DY18 had different dependencies although both enzymes have the same residues in the substrate-binding pocket . For substrates with more than 7 carbons, the aminotransferases did not strictly distinguish between substrates with straight and branched side chains . These results suggest that the recognition of manifold hydrophobic substrates of different shapes might require not only the hydrophobicity of the active site but also enzyme flexibility. Zentralbl Veterinarmed A, 1997 Jul, 44(5), 289 - 99 Effect of weaning in the pig on ileal ion transport measured in vitro; Miller BG et al.; Early weaning in pigs results in small intestinal malabsorption and an increased susceptibility to E . coli infections . This is closely associated with villus shortening and crypt hyperplasia of the small intestine . The present study compared piglets either weaned at 3 weeks of age onto a high soya diet (n = 12) or an egg-based diet (n = 12) with piglets that remained on the sow (n = 12) . Prior to weaning, care was taken to ensure that piglets only had access to sows milk . Serum anti-soya IgG was measured 7 days after weaning and sections of the mid-ileum excised and fixed for determination of crypt depth and villus height . Four pieces of 'stripped' mucosa were mounted in Ussing chambers in Krebs-phosphate Ringer (with indomethacin) for determination of short circuit current (SCC) and unidirectional fluxes of Na22 and Cl36, half in mucosa-serosa (MS), and half in serosa-mucosa (SM) direction . After basal measurements of absorptive capacity of Na, supramaximal doses of prostaglandin E2 (PGE2) and theophylline were added to measure the intestinal secretory capacity for Cl . Anti-soya IgG was elevated in the group weaned onto soya when compared either with the group weaned onto egg or the unweaned group . All intestinal transport and histological parameters were similar in both the weaned groups, although some were different from the unweaned . The SCC (equal to Na absorption) and the villus height were reduced two-thirds by weaning, whereas crypt depth and Cl secretion were similar in all groups . It is suggested that weaning per se is causal for the observed changes in intestinal transport and morphology, but they are not influenced by whether the weaning diet is soya or egg based. Appl Microbiol Biotechnol, 1997 Jul, 48(1), 105 - 8 Creating auxotrophic mutants in Methylophilus methylotrophus AS1 by combining electroporation and chemical mutagenesis; Kim CS et al.; Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) across the cell membrane . By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone . MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%) . This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen. Appl Microbiol Biotechnol, 1997 Jul, 48(1), 41 - 6 Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES; Teshima T et al.; Thermally inactivated bovine deoxyribonuclease I (DNase I) and yeast enolase were reactivated by GroEL/ES from Escherichia coli . In both cases, GroEL/ ES was found to have the ability to reactivate inactivated enzymes in an ATP-dependent manner . GroEL/ ES can interact with the enzymes that were denatured at high temperature and convert them to the active conformations . To test the applicability of GroEL/ES to the reactivation processes of thermally inactivated enzymes, GroEL/ES was immobilized using formyl-Cellulofine (GroEL/ES-Cellulofine) and its performance was studied . GroEL/ES-Cellulofine retained a sufficiently high ability to reactivate enzymes . Moreover, GroEL/ES-Cellulofine could be used repeatedly, indicating high durability . These results indicate that immobilized chaperonin is effective for reactivation of enzymes that are thermally inactivated in various bioprocesses. Res Virol, 1997 Jul-Aug, 148(4), 299 - 305 Simple method for efficient production of hepatitis B virus core antigen in Escherichia coli; Naito M et al.; To obtain good antigenicity and high purity of the hepatitis B virus core antigen (HBcAg) in large quantities without using the fused protein technique employed in recombinant DNA technology, a protein molecule with the same primary sequence as that of wild-type HBcAg (subtype adr) was directly expressed in Escherichia coli JM109 (DE3) using pGd1 expression vector . Purification of the expressed HBcAg yielded high-quality protein by means of simple purification steps, such as sonication, ammonium sulphate precipitation and heat treatment, before final purification by conventional ultra-centrifugation . The HBcAg preparation thus obtained contains small round particles similar in appearance to the HBcAg particles from the HBV-infected human liver tissue. Clin Exp Rheumatol, 1997 Jul-Aug, 15(4), 439 - 44 Interleukin-1 receptor antagonist in neonates, children and adults, and in patients with pauci- and polyarticular onset juvenile chronic arthritis; Muller K et al.; OBJECTIVE: The production of interleukin (IL) -1 alpha IL-1 beta and IL-1 receptor antagonist (IL-1ra) by blood mononuclear cells (MNC), as well as the corresponding serum levels of IL-1ra were examined in blood samples from umbilical cords (n = 11), children (n = 40) and adults (n = 20), and in 42 patients with juvenile chronic arthritis (JCA) of the pauci- or polyarticular onset type . RESULTS: IL-1ra serum levels were found to differ significantly between the three age groups, being higher in neonates (569 pg/ml) than in children (70 pg/ml) and adults (177 pg/ml) . IL-1ra production in E . coli lipopolysacharide (LPS) stimulated-cultures of MNC was also significantly higher in neonates (median 2451 pg/ml) than in children (1526 pg/ml), but similar to that in adults (2107 pg/ml) . IL-1ra levels in the sera of both subgroups of JCA patients were significantly elevated (median 257 pg/ml), but did not reflect paraclinical or clinical disease parameters . In samples of synovial fluid the IL-1ra levels tended to be fairly high, up to approximately 2 ng/ml, but they did not reflect the serum levels of IL-1ra . CONCLUSION: These findings suggests that the upregulation of IL-1ra production forms part of the immunoregulatory response in JCA patients, and that the insufficient production of IL-1ra is unlikely to contribute to the pathogenesis of JCA. FEMS Immunol Med Microbiol, 1997 Jul, 18(3), 153 - 61 Reduced intestinal colonisation with F18-positive enterotoxigenic Escherichia coli in weaned pigs fed chicken egg antibody against the fimbriae; Zuniga A et al.; Newly weaned pigs were fed a basal diet containing either egg antibody against fimbriae F18 at a high or low level, control egg powder or no egg, and challenged with enterotoxigenic Escherichia coli with fimbriae F18 . The challenge was repeated after termination of the antibody treatment . Antibody-containing egg powder was produced by vaccination of hens with semi-purified fimbriae of the two variants F18ab and F18ac . Pigs eating egg powder with antibody against the same fimbrial variant were fully protected, even if the vaccine for the hens was produced with a different serotype devoid of enterotoxins . The effect was dose-dependent . The high dose of antibody against the heterologous variant of fimbriae F18 reduced colonisation at a level which was not significant . Ingestion of egg antibody partially suppressed the build-up of anti-colonisation immunity . Oral application of egg antibodies offers a promising approach for the prevention of infectious diseases of the digestive tract. Mol Gen Genet, 1997 Jul, 255(3), 302 - 10 AtUBP3 and AtUBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus; Chandler JS et al.; The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway . These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an alpha-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an epsilon-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins . We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical . These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism . Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-beta-gal but cannot act upon the natural alpha-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin . Affinity-purified antibody prepared against AtUBP3 expressed in E . coli recognizes both AtUBP3 and AtUBP4 . AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages . Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts . Possible physiological roles for these UBPs are discussed. Mol Gen Genet, 1997 Jul, 255(4), 376 - 81 Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli; Nakayashiki T et al.; A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212 . This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked . It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins . Using this system, we found that the rate of production of porphyrins depended on the availability of heme . The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated . We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme . The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA) . Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified . We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene . We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins. Mol Gen Genet, 1997 Jul, 255(4), 359 - 71 Differential expression of two functional serine/threonine protein kinases from soybean that have an unusual acidic domain at the carboxy terminus; Yoon HW et al.; Two soybean cDNA clones, SPK-3 and SPK-4, encoding putative protein kinases were isolated and characterized . Both cDNAs encoded approximately 40-kDa serine/threonine kinases with unusual stretches of acidic amino acids in their carboxy-terminal regions, which are highly homologous to PKABA1 from wheat and ASKs from Arabidopsis . These kinases are encoded by one- or two-copy genes in the soybean genome . Notably, SPK-3 and -4 showed different patterns of expression in various soybean tissues . SPK-3 is highly expressed in dividing and elongating tissues of young seedlings but relatively weakly in tissues of mature plants . In contrast, SPK-4 showed relatively high and constitutive expression in all the tissues examined except for leaf tissues of mature plants . Although various stressors, such as dehydration and high salinity, increased the expression of both genes, the induction kinetics were different . The two genes also differed in their response to abscisic acid (ABA) . SPK-3 was induced but SPK-4 was not affected by exogenously supplied abscisic acid . In accordance with these expression data analysis of the activity of a chimeric SPK-3 promoter::beta-glucuronidase (GUS) reporter gene by transient expression in tobacco leaves confirmed the inducibility of SPK-3 by salt and ABA . Polyclonal antibodies raised against a recombinant SPK-4 protein produced in Escherichia coli specifically recognized both recombinant SPK-3 and -4 proteins . Kinase assays using affinity-purified SPK-4/ antibody complexes with crude soybean extracts as substrate identified specific phosphorylation of two 41 and 170 kDa soybean proteins that were phosphorylated on serine residues . Taken together, our results suggest that SPK-3, and/or SPK-4 are functional serine protein kinase(s) . Furthermore, SPK-3 and -4 may play different roles in the transduction of various environmental stresses. Hokkaido Igaku Zasshi, 1997 Jul, 72(4), 457 - 69 {Inhibition of EGF and TGF-beta dependent transformation of NRK23 cells by Crk II-23 mutant}; Ota S; Adaptor proteins participate in many signaling pathways from cell surface receptors . Crk protein was the first example of the adaptor protein . We have examined the function of Crk II mutant, Crk II-23 . The Crk II-23 mutant contains two amino-acid substitutions in the carboxyl-terminal SH3 domain and is known to inhibit the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) . There was no remarkable difference between Crk II and Crk II-23 in EGF-dependent binding to EGF receptor (EGFR) . However, in contrast to the wild-type Crk II, the Crk II-23 mutant bound to EGFR in quiescent NIH 3T3 cells . To clarify the difference, both the Crk II and Crk II-23, proteins were expressed in E . coli and examined their binding capacity in vitro . They bound to EGFR from EGF-stimulated NIH 3T3 cells in vitro to a similar extent . Expression of Crk II-23 in NIH 3T3 cells did not affect the binding of bacterially expressed Crk II and Crk II-23 to EGFR . These results suggest that post-translational modification of Crk II-23, such as physical association to cellular proteins, induces binding of Crk II-23 to EGFR in quiescent cells . We also demonstrated that mutation of either the SH2 or the SH3 domain abolished the anti-oncogenic activity of Crk II-23, although both mutants bound to EGFR in the quiescent cells . From these results, it could be concluded that persistent signaling through Crk II-23 bound to EGFR is responsible for the suppression of transformation by EGF and TGF-beta. Arch Dermatol Res, 1997 Jul, 289(8), 448 - 56 Differential expression of the HsKin17 protein during differentiation of in vitro reconstructed human skin; Biard DS et al.; In eukaryotic cells, various proteins homologous to the E . coli RecA protein are involved in the elimination of DNA damage . These proteins contribute to the repair of double-strand breaks and to genetic recombination . The mouse Kin17 protein is recognised by antibodies directed against the RecA protein . Kin17 has a zinc-finger domain allowing binding to curved DNA stretching over illegitimate recombination junctions . In the present study, we identified the human counterpart of the mouse Kin17 protein (named HsKin17) in skin cells . We employed an in vitro reconstructed skin model composed of an epidermal sheath lying on a dermal matrix with human fibroblasts embedded in rat collagen type I . The maturation programme (proliferation versus differentiation) of keratinocytes was highly dependent on stromal cells . Immunohistochemical staining of frozen sections obtained from skin specimens was monitored by an interactive laser cytometer . In this way we analysed protein levels in both dermal and epidermal compartments . After having characterised the epithelium, we focused our attention on HsKin17 expression . We detected HsKin17 in human keratinocytes . HsKin17 protein levels increased in proliferating epithelial keratinocytes after 7 days of culture . After 2 weeks of culture, epidermal sheaths acquired most of the differentiated features of mature epithelium . At this time, HsKin17 protein dropped below measurable levels in the stratum corneum, and diminished in nucleated cells . This study showed that HsKin17 is expressed in human reconstructed epithelium under conditions of hyperproliferation. Biotechnol Prog, 1997 Jul-Aug, 13(4), 361 - 7 Inverse flux analysis for reduction of acetate excretion in Escherichia coli; Delgado J et al.; The determination of intracellular fluxes from the measurement of extracellular rates, a technique known as flux analysis, has been used successfully on several systems important to biotechnology . However, calculation of intracellular fluxes does not directly suggest ways to improve the performance of the cell . Here, we introduce a variation of this analysis, termed inverse flux analysis, which allows the prediction of the flux distribution as a function of manipulable internal fluxes . This approach is applied to analyze the acetate excretion problem commonly observed in aerobic Escherichia coli cultures . The effect of manipulating each flux is quantified by a flux distribution sensitivity coefficient, which is dependent on the stoichiometry . For the acetate excretion problem, our results suggest that the anaplerotic pathways, including the reactions catalyzed by phosphoenolpyruvate carboxylase and the glyoxylate bypass are the best choices for manipulating acetate excretion in E . coli . Increasing the anaplerotic flux will decrease acetate production while increasing the growth yield. Plant J, 1997 Jul, 12(1), 215 - 21 Structure and subcellular localization of a small RNA-binding protein from tobacco; Moriguchi K et al.; In this study, a cDNA encoding a small RNA-binding protein was isolated from a Nicotiana sylvestris cDNA library . The predicted protein (RGP-3) is 144 amino acid residues long, and contains a consensus sequence-type RNA binding domain (CS-RBD) of 83 amino acids and a short glycine-rich region of 15 amino acids . RGP-3 synthesized in Escherichia coli has high affinity for poly(U) . Immunocytochemical analysis indicated that RGP-3 is localized in the nucleoplasm, and that RGP-1b, a related protein reported previously, is localized in the nucleolus . Possible roles of these proteins in pre-mRNA or pre-rRNA processing are discussed. Plant J, 1997 Jul, 12(1), 179 - 90 The plant 2-Cys peroxiredoxin BAS1 is a nuclear-encoded chloroplast protein: its expressional regulation, phylogenetic origin, and implications for its specific physiological function in plants; Baier M et al.; 2-Cys peroxiredoxins constitute a family of enzymes which catalyze the transfer of electrons from sulfhydryl residues to peroxides and are ubiquitously distributed among all organisms . This paper characterizes the higher plant 2-Cys-peroxiredoxin BAS1 . (i) Escherichia coli over-expressing BAS1 exhibit increased tolerance for alkyl hydroperoxides in vivo . This result substantiates the peroxiredoxin function of BAS1 . (ii) BAS1 protein is associated with the soluble chloroplast fraction of mesophyll protoplasts . Import and processing of in vitro-transcribed and cell-free translated BAS1 protein into isolated chloroplasts provides conclusive evidence that the plant-specific N-terminal extension of bas1 encodes the chloroplast import signal which targets the pre-form of BAS1 to the chloroplast stroma where it is cleaved to its mature size . (iii) Genomic analysis reveals that the targeting signal is encoded by a separate exon in Arabidopsis thalina . (iv) The amino acid sequence of the BAS1 core protein of higher plants has a higher degree of similarity to open reading frames in the genome of the bluegreen algae Synechochystis PCC sp . 6803 and in the plastome of the red algae Porphyra purpurea than to any other nuclear-encoded 2-Cys peroxiredoxin . Therefore, it is tempting to speculate that the chloroplast import signal was added to an ancestor gene of endosymbiotic origin in the course of plant evolution . (v) The bas1 gene expression is regulated under the control of the cellular redox state which is in accordance with the anti-oxidant function of the enzyme . While oxidative stressors increased expression only slightly, antioxidants such as reduced thiols strongly suppressed the transcript level . The implications of these findings are discussed with respect to the possible physiological functions of BAS1. Int J Dev Neurosci, 1997 Jul, 15(4-5), 585 - 94 Novel genes expressed in the chick otocyst during development: identification using differential display of RNA; Gong TW et al.; Differential display of mRNA is a technique that enables the researcher to compare genes expressed in two or more different tissues or in the same tissue or cell under different conditions . The method is based on polymerase chain reaction amplification and comparison of specific subsets of mRNA . We have used this method to clone partial complementary DNAs (cDNAs; amplicons) for genes expressed in the otocyst in order to identify genes that may be involved in development of the inner ear . A full length cDNA was isolated from an embryonic quail head library with an amplicon (KH121) obtained from the otocyst . This avian cDNA encoded a novel, 172-amino acid acidic protein and detected a major transcript of ca 0.8 kb in RNA from chick embryos and several neonatal chick tissues . The full length avian cDNA had high sequence identity to several human cDNAs (expressed sequence tags) from human fetal tissues, including cochlea, brain, liver/spleen and lung, and from placenta . The human homologue of the avian gene encoded a protein that was 183 amino acids long and had 75.6% amino acid sequence identity to the avian protein . These results identified both the avian and human homologues of an evolutionarily conserved gene encoding a small acidic protein of unknown function; however, expression of this gene was not restricted to otocysts. J Pept Sci, 1997 Jul-Aug, 3(4), 252 - 60 Application of reversible biotinylated label for directed immobilization of synthetic peptides and proteins: isolation of ligates from crude cell lysates; Ball HL et al.; Chaperonin 10 protein from Rattus norvegicus (Rat cpn 10) has been reported to bind chaperonin 60 from Escherichia coli (GroEL) in an ATP-dependent manner . Chemically synthesized Rat cpn10 was immobilized in a defined orientation to agarose-bound monomeric avidin using a reversible biotinylated affinity label (1), attached to the N alpha-terminal residue . The resulting affinity chromatographic matrix was then used to isolate binding proteins from a crude cell lysate . Following affinity separation the bound ligand and ligate was released by treatment with organic base . Rat cpn 10 was prepared using a highly effective synthetic protocol involving HBTU/HOBt activation and capping with N-(2-chlorobenzyloxycarbonyloxy) succinimide to terminate unreacted amino groups . The biotinylated Fmoc-based molecule (1) was introduced specifically onto the N alpha-terminal amino acid as the succinimidyl carbonate, before final cleavage and deprotection of side-chain protecting groups using a low-TFMSA/high-HF procedure . Crude biotinylated Rat cpn10 (Rat cpn10 + 1) was immobilized on monomeric avidin with a binding efficiency of approximately 75% and unlabelled truncated/capped impurities eluted off the column with buffer . The biotinylated Rat cpn10-avidin affinity matrix was then used to isolate GroEL from a crude cell lysate . The identity of the purified protein was confirmed by SDS-PAGE and binding to a specific anti-GroEL monoclonal antibody (MoAb) . These results extend the applicability of the biotinylated label (1), providing a reversible non-covalent anchor for immobilization of peptide and protein ligands, thus simplifying isolation of ligates and enabling recovery of synthetic material under mild conditions. J Formos Med Assoc, 1997 Jul, 96(7), 517 - 24 Molecular cloning and expression of a fungal immunomodulatory protein, FIP-fve, from Flammulina velutipes; Ko JL et al.; FIP-fve, a fungal immunomodulatory protein, was isolated from the fruiting bodies of the edible mushroom, Flammulina velutipes . FIP-fve was shown to stimulate blast-forming activity of human peripheral blood lymphocytes and gene expression of interleukin-2, interferon-gamma and tumor necrosis factor-alpha . Repeated administration of FIP-fve to mice inhibits the Arthur and systemic anaphylaxis reactions . FIP-fve cDNA was cloned and sequenced, and the amino acid sequence of FIP-fve deduced from the nucleotide sequence is identical to that previously determined by protein sequencing . FIP-fve cDNA was amplified by polymerase chain reaction, ligated into the expression vector, pGEX-2T, and expressed in Escherichia coli as a fusion protein of glutathione S-transferase (GST) and FIP-fve . The GST-FIP-fve fusion protein was soluble, and the yield of recombinant FIP-fve was about 5 mg/L of induced culture . The recombinant FIP-fve was obtained by cleaving the GST-FIP-fve fusion protein with thrombin and purifing to homogeneity . The recombinant FIP-fve had about 50% of the immunomodulatory activity of the native FIP-fve. Hepatogastroenterology, 1997 Jul-Aug, 44(16), 1051 - 6 Morphometric study of the small bowel mucosa in infants with diarrhea due to enteropathogenic Escherichia coli strains; Fagundes-Neto U et al.; BACKGROUND/AIMS: Enteropathogenic Escherichia coli (EPEC) strains are the most important cause of gastroenteritis in infants under 1 year of age and may induce several patterns of villous atrophy in the intestinal mucosa . However, the interpretation of these abnormalities has usually been based on semiquantitative criteria, giving rise to considerably subjective results . We utilized the linear morphometry to analyze the morphological lesions of the small bowel mucosa induced by EPEC strains in infants with persistent diarrhea in comparison with those seen in infants with asymptomatic environmental enteropathy (AEE) and controls . METHODOLOGY: Fifty nine specimens of small bowel mucosa were comparatively studied and divided in the following groups: 1 . Group I: Thirty infants with persistent diarrhea due to EPEC strains, mean age 6.4 months; 2 . Group II: Sixteen infants with AEE, mean age 6.5 months with no enteropathogenic bacteria in stools; 3 . Group III: Thirteen children with short stature and no gastrointestinal complaints, mean age 15 months . Morphometric analysis of the small bowel mucosa was performed by using a x10 objective to a Zeiss light microscope, to which a measuring Zeiss ocular, t8x was adapted . The following measurements were carried out: Total mucosal thickness (TMT); Villous height (VH); Crypt length (CL); Intraepithelial lymphocyte (IEL) count . RESULTS: Except for the IEL, there was a significant difference in all the parameters analyzed among the evaluated groups . Group I revealed the lowest values for total mucosal thickness, villous height, and the ratio villous height/crypt length in comparison with the two other groups . On the other hand, the crypt length measurements for Group II were larger than those for Groups I and III . The measurements of villous height and the ratio villous height/crypt length for Group III turned out to be greater than those for Group II . CONCLUSIONS: The utilization of an accurate technique in the morphological study of the small bowel mucosa allowed us to detect severe abnormalities not only in infants with EPEC infection, but also in those counterparts who live in contaminated environments, and can therefore potentially acquire this type of intestinal infection. Hepatogastroenterology, 1997 Jul-Aug, 44(16), 923 - 6 Animal models for intra-abdominal infection; Sayek I; Animal models are widely used to study intra-abdominal infections . Experimental intra-abdominal infections . Experimental intra-abdominal infection is a bi-phasic process; initially a peritonitis or septic phace caused by E . coli and later a chronic-abscess forming stage, caused by B . fragilis similar to human beings . Various methods have been described . These methods includes challenge of the peritoneum with either endogenous bacteria or inoculation of pure bacteria or fectal material . Non-bacterial models have also been described . Each of these models have their own advantages and disadvantages . The aim and the end points should govern the selection of the right model to be used for experimental purposes . The ideal model should be reliable, standard, reproducible and resembling human disease . A single model with those specifications is yet to be described. DNA Cell Biol, 1997 Jul, 16(7), 883 - 92 Production of biologically active recombinant tilapia insulin-like growth factor-II polypeptides in Escherichia coli cells and characterization of the genomic structure of the coding region; Chen JY et al.; Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now . For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli . Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively . The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds . The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates . In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds . The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2 . The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3 . The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4 . These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals . To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector) . Tilapia recombinant IGF-II protein was purified and characterized in E . coli . The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD . The recombinant tilapia IGF-II protein biological function was measured by stimulation of {3H}thymidine incorporation . The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine . The results suggest that the recombinant IGF-II protein was dose dependent. J Virol Methods, 1997 Jul, 66(2), 219 - 26 Immunolocalization of the pseudorabies virus immediate-early protein IE180 by immunoperoxidase staining; Huang C et al.; The immediate-early (1E) gene of pseudorabies virus (PRV) expresses immediately upon infection, a phosphorylated protein (immediate-early protein, IE180) that can transactivate viral other genes and plays an essential role in regulating viral gene expression . In order to detect and localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate specific antibodies against IE180 protein . Two recombinant expression plasmids pN and pNB were constructed by cloning the IE gene onto pET 30a(+) expression vector via NcoI and BamHI sites . Plasmid pN contains the 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 616 amino acid residues, while pNB contains the 2.8-kb NcoI-Bam HI fragment coding for the rest of the IE180 protein . Both pN and pNB were transformed, respectively, into E . coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG . The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respectively . These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 protein . The specificities of the mice immune sera were confirmed by their abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay . Furthermore, immunoperoxidase staining of PRV infected cells undertaken with these antisera revealed the subcellular distribution of the IE proteins in the infected cells and also demonstrated their transportation from the cytoplasm to the nucleus during infection. Comp Biochem Physiol B Biochem Mol Biol, 1997 Jul, 117(3), 379 - 85 Molecular phylogenetics of a protein repair methyltransferase; Kagan RM et al.; Protein-L-isoaspartyl (D-aspartyl) O-methyltransferase (E.C . 2.1.1.77) is a well-conserved and widely distributed protein repair enzyme that methylates isomerized or racemized aspartyl residues in age-damaged proteins . We exploited the availability of protein sequences from 10 diverse animal, plant and bacterial taxa to construct a phylogenetic tree and determine the rates of amino acid substitution for this enzyme . We used a likelihood ratio test to show that this enzyme fulfills the conditions for a molecular clock . We found that the rate of substitution is 0.39 amino acid substitutions per site per 10(9) years and remains relatively constant from bacteria to humans . We argue that this degree of sequence conservation may result from the functional constraints necessitated by the requirement to specifically recognize altered aspartyl but not normal aspartyl residues in proteins . Relative rate analysis of the Caenorhabditis elegans sequence suggests that the amino acid substitution rate in the nematode lineage may be higher than that in other lineages and that the divergence of nematodes may have been a more recent event than suggested by previous analysis. Am J Physiol, 1997 Jul, 273(1 Pt 1), L217 - 26 Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation; Bannerman DD et al.; Bacterial lipopolysaccharide (LPS) induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in vitro . We studied whether LPS-induced increments in 14C-labeled bovine serum albumin (BSA) flux across bovine pulmonary artery endothelial cell (EC) monolayers and actin depolymerization are mediated through protein tyrosine phosphorylation . Lysates from EC exposed to LPS derived from Escherichia coli 0111:B4 (100 ng/ml, 1 h) demonstrated increased tyrosine phosphorylation of the cytoskeletal protein paxillin . Protein tyrosine kinase inhibition, with either herbimycin A (1 microM) or genistein (50 micrograms/ml), protected against LPS-induced actin depolymerization, intercellular gap formation, and increments in {14C}BSA flux . In contrast, inhibition of tyrosine phosphatases with sodium orthovanadate (2.5 microM) or phenylarsine oxide (0.1 microM) enhanced the LPS-induced increments in the G-actin pool and the transendothelial flux of {14C}BSA compared with that seen after exposure to LPS alone . Our data indicate that the influence of LPS on EC actin organization and barrier function is mediated, in part, through a signaling pathway that is dependent on tyrosine phosphorylation. J Hepatol, 1997 Jul, 27(1), 193 - 200 Effects of eicosanoids on lipopolysaccharide-induced ornithine decarboxylase activity and polyamine metabolism in the mouse liver; Matsuzaki Y et al.; BACKGROUND/AIMS: During endotoxic shock, arachidonic acid is released from the inflammatory cell membranes and is metabolized to form eicosanoids, which modify the deleterious effects of lipopolysaccharide (LPS) on liver function . However, it is not known which prostaglandins (PGs) or leukotrienes (LTs) are produced or how they affect the LPS-treated liver . As LPS treatment elevates hepatic ornithine decarboxylase (ODC) activity and affects the polyamine levels of the mouse liver, this study was carried out to examine the effects of eicosanoids and their inhibitors on the induction of ODC activity and polyamine levels in the LPS-treated mouse liver . METHODS: LPS in the presence or absence of other drugs was intraperitoneally administered to 6-week-old mice and the livers were then removed . The hepatic ODC activity, polyamine levels, and level of ODC mRNA were determined . RESULTS: The levels of LPS-induced ODC activity, the putrescine (PUT) and N1-acetylspermidine (A-SPD) were reduced by the administration of PGE1 . ODC activity was enhanced by the administration of corticosterone, AA-2414 (an antagonist of thromboxane (TX) A2) and TXB2, whereas the A-SPD level was reduced by corticosterone and AA-2414 treatment . The level of ODC mRNA changed in parallel with the change in ODC activity . CONCLUSIONS: PGE1 may reduce the LPS-induced production of inflammation-accelerating cytokines and reduce the level of ODC activation . Corticosterone and AA-2414 treatment may attenuate the LPS-induced production of eicosanoids, and enhance the LPS-induced ODC activation . It is possible that the eicosanoids produced by LPS treatment inhibit ODC activation during endotoxic shock. Am J Physiol, 1997 Jul, 273(1 Pt 2), F129 - 35 Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells; Arthur JM et al.; Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR) . We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells . The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner . mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR . G protein activation was evaluated using the GTP photoaffinity label {alpha-32P}GTP azidoanalide (AA-GTP) . After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members . Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1 . Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells . We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1. Am J Physiol, 1997 Jul, 273(1 Pt 2), R407 - 13 The vagus nerve in the thermoregulatory response to systemic inflammation; Romanovsky AA et al.; Experimentally, systemic inflammation induced by a bolus intravenous injection of lipopolysaccharide (LPS) may be accompanied by three different thermoregulatory responses: monophasic fever (the typical response to low doses of LPS), biphasic fever (medium doses), and hypothermia (high doses) . In our recent study {Romanovsky, A . A., V . A . Kulchitsky, C . T . Simons, N . Sugimoto, and M . Szekely . Am . J . Physiol . (Regulatory Integrative Comp . Physiol.) . In press}, monophasic fever did not occur in subdiaphragmatically vagotomized rats . In the present work, we asked whether vagotomy affects the two other types of thermoregulatory response . Adult Wistar rats were vagotomized (or sham operated) and had an intravenous catheter implanted . On day 28 postvagotomy, the thermal responses to the intravenous injection of Escherichia coli LPS (0, 1, 10, 100, or 1,000 micrograms/kg) were tested in either a neutral (30 degrees C) or slightly cool (25 degrees C) environment . Three major results were obtained . 1) In the sham-operated rats, the 1 microgram/kg dose of LPS caused at 30 degrees C a monophasic fever with a maximal colonic temperature (Tc) rise of approximately 0.6 degree C; this response was abated (no Tc changes) in the vagotomized rats . 2) At 30 degrees C, all responses to higher doses of LPS (10-1,000 micrograms/kg) were represented by biphasic fevers (the higher the dose, the less pronounced the first and the more pronounced the second phase was); none of these biphasic fevers was altered in the vagotomized animals . 3) In response to the 1,000 micrograms/kg dose at 25 degrees C, hypothermia occurred: Tc changed by -0.5 +/- 0.1 degree C (nadir); this hypothermia was exaggerated (-1.1 +/- 0.1 degrees C) in the vagotomized rats . It is concluded that vagal afferentation may be important in the mediation of the response to minor amounts of circulating LPS, whereas the response to larger amounts is brought about mostly (if not exclusively) by nonvagal mechanisms . This difference may be explained by the dose-dependent mechanisms of the processing of exogenous pyrogens . Vagotomized animals also appear to be more sensitive to the hypothermizing action of LPS in a cool environment; the mechanisms of this phenomenon remain speculative. Am J Physiol, 1997 Jul, 273(1 Pt 2), R371 - 8 Acute alterations in growth hormone-insulin-like growth factor axis in humans injected with endotoxin; Lang CH et al.; The purpose of the present study was to characterize the acute changes in the insulin-like growth factor (IGF) system in humans after administration of endotoxin (lipopolysaccharide; LPS) . Escherichia coli LPS (4 ng/kg) was injected intravenously into healthy adults, and serial blood samples were collected for the next 5 h; subjects injected with saline served as time-matched controls . LPS administration resulted in a gradual decrease in the total extractable IGF-I concentration, which was reduced by approximately 20% over the final 2 h of the experiment; levels of free IGF-I were not significantly altered . LPS also produced a marked but transient elevation in growth hormone (GH) concentration . IGF-binding protein (BP)-1 levels were elevated more than fivefold 2 h after LPS injection, and thereafter levels gradually returned toward baseline . IGFBP-2 concentration also increased after LPS injection, but the maximal increase (approximately 50% above basal) was observed during the final 2 h of the protocol . In contrast, IGFBP-3 levels did not vary over the period examined in response to LPS, and there was no apparent increase in number of BP-3 proteolytic fragments . Cortisol levels were increased early and remained two- to threefold above baseline throughout the protocol . No significant alterations in serum concentration of glucose or insulin were noted . LPS also produced an early elevation in tumor necrosis factor and a later increase in interleukin-6 . These data indicate that the acute changes in the GH-IGF axis in humans in response to LPS are comparable with those observed in humans in other traumatic conditions and in animal models of endotoxemia and infection. Am J Physiol, 1997 Jul, 273(1 Pt 2), H387 - 404 Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice; McLean JW et al.; This study identified the organ and cellular distribution of cationic liposome-DNA complexes injected intravenously into CD-1 mice for gene delivery . DOTIM-cholesterol liposomes were labeled with the fluorescent dye CM-Dil and complexed with plasmid DNA encoding the chloramphenicol acetyltransferase reporter gene . The distribution of the complexes was examined in 29 organs and tissues by fluorescence, confocal, and electron microscopy from 5 min to 24 h after injection . The complexes formed clusters in blood, which were cleared within 20 min . Complexes visible by fluorescence microscopy were taken up by endothelial cells, leukocytes, and macrophages and did not leave the vasculature except in the spleen . At 5 min, the complexes formed a patchy coating on the endothelial surface, but by 4 h, they were internalized into endosomes and lysosomes in organ- and vessel-specific patterns . Uptake by capillary endothelial cells was greatest in the lung, ovary, and anterior pituitary, less in muscle and the heart, and nearly absent in the brain and pancreatic islets . In lymph nodes and intestinal Peyer's patches, the uptake was sparse in capillaries but abundant in high endothelial venules . In the liver and spleen, most of the uptake was in Kupffer cells and macrophages . Measurements of chloramphenicol acetyltransferase reporter gene expression were generally consistent with the pattern of uptake by endothelial cells . The uptake and gene expression were accompanied by a decrease in circulating leukocytes and platelets . Overall, our results showed that the complexes were internalized by endothelial cells in organ- and vessel-specific patterns that did not match any previously identified properties of the microvasculature . The unusual distribution of endothelial cell uptake may be explained by a heterogeneously distributed membrane receptor for which the complexes are ligands. Am J Physiol, 1997 Jul, 273(1 Pt 2), H164 - 74 Dissociation of TNF-alpha from endotoxin-induced nitric oxide and acute-phase hypotension; Xie J et al.; We tested the concept that tumor necrosis factor-alpha (TNF-alpha) or platelet-activating factor (PAF) mediated Escherichia coli endotoxin lipopolysaccharide (LPS)-induced upregulation of nitric oxide (NO) and acute-phase hypotension (APH) in the rat . LPS (0.5 mg/kg i.v.) given to rats treated with saline or nonimmune goat-derived gamma-globulin (immunoglobulin G, 22 mg/kg i.m.) produced APH and increased plasma concentrations of TNF-alpha and nitrate and nitrite anions (reactive nitrogen intermediates; RNI) and NO in ex vivo incubates of polymorphonuclear neutrophils (PMN) and inducible NO synthase (iNOS) mRNA in PMN . Pretreatment of rats with a polyclonal TNF-alpha antibody (TNF-Ab, 22 mg/kg i.m.) abolished LPS-mediated increases in plasma TNF-alpha but failed to inhibit APH or the NO system . TNF-alpha (8.2 micrograms/kg i.v.) produced transient hypertension and sustained tachycardia and increased plasma TNF-alpha and PMN iNOS mRNA but not RNI . LPS and TNF-alpha decreased spontaneous and calcimycin (Ca2+ ionophore, 1 microM)- and PAF (1 microM)-mediated increases in head-space NO production by rings of mesenteric artery incubated ex vivo . TNF-Ab abolished all effects of TNF-alpha . PAF (25, 50, and 100 ng/kg) produced APH without increasing plasma TNF-alpha, RNI, or PMN iNOS mRNA . The PAF receptor antagonist BN-50730 (80 micrograms/kg i.v.) abolished PAF-induced APH and attenuated LPS-induced increases in RNI . We conclude that 1) LPS produces parallel but unrelated changes in TNF-alpha and RNI in plasma and PMN during the APH of endotoxemia; and 2) endogenous TNF-alpha is not required for LPS-mediated induction of iNOS mRNA, and PAF mediates LPS-induced APH. Gene, 1997 Jul 1, 193(1), 119 - 25 Cloning and expression of human NF-YC; Bellorini M et al.; The CCAAT box is an important element in eukaryotic promoters and NF-Y (CBF) is a conserved heterotrimeric protein binding to it . Two subunits, NF-YB and NF-YC, contain a histone-like motif . We cloned the complete cDNA coding for the human NF-YC gene . The ORF codes for a 335 aa protein that shows virtual identity to the rat sequence, confirming the stunning invariance of NF-Y genes across species . We expressed and purified the yeast homology domain of NF-YC in bacteria and performed EMSA together with the corresponding conserved domains of NF-YA and NF-YB, obtaining a CCAAT-binding mini-NF-Y . We evaluated the expression of NF-YC and found that mRNA levels are similar in different human tissues except in testis. Gene, 1997 Jul 1, 193(1), 105 - 14 Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms; Wang J et al.; Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized . The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region . In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle . The cloned cDNAs were expressed in E . coli as standards to identify a high M(r) to low M(r), acidic to basic fTnT isoform transition in postnatal developing skeletal muscles . Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle . The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression. Gene, 1997 Jul 1, 193(1), 97 - 103 Replication of the R6K gamma origin in vitro: dependence on wt pi and hyperactive piS87N protein variant; Levchenko I et al.; The pi protein of plasmid R6K is involved in control of replication . The aim of this study was to use an in vitro replication system dependent on an R6K-derived gamma origin of replication (gamma ori) to compare replication characteristics of wt pi and a hyperactive variant of pi protein (piS87N; Filutowicz et al., 1994b . Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution . Nucleic Acids Res . 22, 4211-4215) . The characteristics of in vitro replication from gamma ori reported in this investigation are as follows: (i) piS87N is considerably more active in comparison to wt pi . (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins . (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within gamma ori . (iv) Replication dependent on wt pi, but not piS87N, is stimulated up to 1.5-fold by rifampicin. Gene, 1997 Jul 1, 193(1), 59 - 63 Use of polymerase chain reaction to identify a leucyl tRNA in Streptomyces coelicolor; Trepanier NK et al.; Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor . Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA(Leu)CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S . coelicolor, the bldA encoded tRNA(Leu)UUA . Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation {Leskiw et al., 1993 . J . Bacteriol., 175, 1995-2005}. Eur J Biochem, 1997 Jul 1, 247(1), 372 - 9 Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase; Jagath JR et al.; The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants . These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli . The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity . The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies . Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate . The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT) . However, at 55 degrees C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively . Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52 degrees C) compared to rSHMT (56 degrees C) and des-(A1-K6)-SHMT (55 degrees C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme . Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2+/-0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8+/-0.1 M) and des-(A1-K6)-SHMT (1.7+/-0.1 M) . The apoenzyme of des-(A1-W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (approximately 75% and approximately 65%, respectively) and dimers . While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only up to 22% . The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration . These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT. Eur J Biochem, 1997 Jul 1, 247(1), 356 - 63 1.8-A crystal structure of the catalytic domain of human neutrophil collagenase (matrix metalloproteinase-8) complexed with a peptidomimetic hydroxamate primed-side inhibitor with a distinct selectivity profile; Betz M et al.; Matrix metalloproteinases (MMP) are zinc endopeptidases involved in tissue remodelling . They have been implicated in a series of pathologies, including cancer, arthritis, joint destruction and Alzheimer's disease . Human neutrophil collagenase represents one of the three interstitial collagenases that cleave triple-helical collagen of type I, II and III . Its catalytic domain (residues Phe79-Gly242) has been heterologously expressed in Escherichia coli and crystallized as a non-covalent complex with the hydroxamate inhibitor BB-1909, which has distinct selectivity against different MMP, in a crystal form . The crystal structure, refined to 0.18-nm resolution, shows that BB-1909 is a right-hand-side inhibitor that binds to the S1'-S3' subsites and coordinates to the catalytic Zn2+ in a bidentate manner via the hydroxyl and carbonyl oxygen atoms of the hydroxamate group in a similar manner to batimastat . The collagenase/BB-1909 complex is described in detail and compared with the collagenase/batimastat complex . These studies provide information on MMP specificity and thus may assist the development of more-selective MMP inhibitors. Eur J Biochem, 1997 Jul 1, 247(1), 217 - 23 Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity; Gehlhar K et al.; A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe . After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing . Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame . Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b . Within these two subgroups, only minor sequence variations were observed . Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity . Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar . Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups . Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual . IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests . The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens . We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity . Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy . Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated. Eur J Biochem, 1997 Jul 1, 247(1), 74 - 81 The replacement of Lys620 by serine desensitizes Escherichia coli phosphoenolpyruvate carboxylase to the effects of the feedback inhibitors L-aspartate and L-malate; Yano M et al.; Chemical modification of Escherichia coli phosphoenolpyruvate carboxylase (P-pyruvate carboxylase) by 2,4,6-trinitrobenzene sulfonate, a specific reagent for amino groups, causes desensitization to allosteric inhibitors, L-aspartate and L-malate, as well as inactivation . When L-malate is included in the modification mixture, P-pyruvate carboxylase was markedly protected from both desensitization and inactivation {Naide, A., Izui, K., Yoshinaga, T . & Katsuki, H . (1979) J . Biochem . (Tokyo) 85, 423-432} . To determine the lysine residue(s) involved in allosteric inhibition, the lysine residues that were protected from modification by L-malate were investigated by analyzing trinitrophenylated peptides liberated by digestion with glutamyl endopeptidase (V8-protease) . The identified residues were Lys491, Lys620, Lys650, and Lys773 . Each of these residues was individually replaced with an alanine or serine residue by site-directed mutagenesis to produce mutant enzymes . The mutant enzyme whose lysine residue was replaced with serine ({Ser620}P-pyruvate carboxylase) showed a marked desensitization to L-aspartate and L-malate, while retaining almost the same maximal catalytic activity as the wild-type P-pyruvate carboxylase . Essentially no changes in enzymatic properties were observed for the {Ala491}- and {Ala650}P-pyruvate carboxylases, while for the {Ala620}- and {Ala773}P-pyruvate carboxylases the polypeptides of the expected size were not significantly accumulated in the transformed E . coli cells, presumably due to intracellular degradation. Biochem Mol Biol Int, 1997 Jul, 42(3), 621 - 9 High level expression of recombinant plasminogen activator inhibitor-1 in Escherichia coli and generation of its mutants involving Asp125, Glu128 and Glu130; Sui GC et al.; Plasminogen activator inhibitor-1 (PAI-1) cDNA was expressed in Escherichia coli (E . coli) with high efficiency using a heat-inducible vector . About 100 mg of recombinant PAI-1 (rPAI-1) could be obtained from 1 liter of bacteria culture . rPAI-1 in inclusion bodies was purified by pI precipitation and Sephadex G-75 chromatography . After treatment with 4 mol/L guanidinium chloride and dialysis, the largely inactive PAI-1 gained considerably in activity as judged by its reaction with low molecular weight u-PA (LMW-u-PA) . Degenerated oligonucleotides containing ApaI site and mutations at Asp125, Glu128, Glu130 in PAI-1 cDNA were synthesized . To facilitate the introduction of mutations, an ApaI site was first generated in PAI-1 cDNA using one of the oligonucleotides . Taking advantage of the APaI site, thirteen PAI-1 mutants involving Asp125, Glu128 and Glu130 were produced with these oligonucleotides using PCR . Most of the PAI-1 mutants had a similar activity as compared to wild type PAI-1, while some of the triple-site mutants had completely lost their activity. Plant Mol Biol, 1997 Jul, 34(4), 583 - 92 Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides; Larsson S et al.; A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed . From this two different cDNA clones, denoted CA1a and CA1b, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced . Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs . The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ . Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles . Northern blot hybridization revealed a CA transcript of ca . 1300 bases, 140 bases shorter than in pea . Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used . Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa . The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography . Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme . Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants. Plant Mol Biol, 1997 Jul, 34(4), 563 - 72 Characterization of the putative alpha subunit of a heterotrimeric G protein in rice; Iwasaki Y et al.; A recombinant protein with a cDNA that encodes the putative alpha subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified . The recombinant protein (rGrice alpha) with an apparent molecular mass of 45 kDa was bound with guanosine 5'-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36 . The protein also hydrolyzed GTP and its kcat was 0.44 . rGrice alpha was ADP-ribosylated by activated cholera toxin . Monoclonal antibodies raised against rGrice alpha reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings . The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice alpha . A 45 kDa polypeptide in the plasma membrane, as well as rGrice alpha, was ADP-ribosylated by activated cholera toxin . The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17 . These properties were similar to those of the alpha subunits of heterotrimeric G proteins in animals, suggesting that the putative alpha subunit is truly the alpha subunit itself. J Protein Chem, 1997 Jul, 16(5), 375 - 83 Contact sites of peptide-oligoribonucleotide cross-links identified by a combination of peptide and nucleotide sequencing with MALDI MS; Urlaub H et al.; We have investigated peptide-oligoribonucleotide complexes isolated from cross-linked Escherichia coli 30S ribosomal subunits in order to identify the contact sites of these complexes at the molecular level . For this purpose, reversed-phase (RP) HPLC-purified peptide-oligoribonucleotide complexes were submitted to N-terminal amino acid sequencing in order to determine the cross-linked peptide moiety and were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for calculation of the nucleotide composition of the cross-linked complex . Subsequently, for nucleotide sequence information the complexes were partially hydrolyzed or treated with exonucleases and analyzed again by MALDI-MS . Applying this technique, we were able to identify the cross-linked oligoribonucleotide parts in contact with distinct peptide regions derived from ribosomal proteins S4, S7, S8, and S17 from E . coli. Scand J Immunol, 1997 Jul, 46(1), 59 - 66 Colostral mononuclear phagocytes are able to kill enteropathogenic Escherichia coli opsonized with colostral IgA; Honorio-Franca AC et al.; Enteropathogenic Escherichia coli (EPEC) is the main aetiological agent of acute diarrhoea among low socioeconomic level infants in developing countries . Breast-feeding provides infant protection against acute gastrointestinal and respiratory infections; however, little is known about the protective role of colostral phagocytes in the gut of newborn infants . In the present investigation we studied the ability of human colostral MN phagocytes to kill EPEC as well as the interactions between these cells and colostral and serum opsonins . The authors observed that the microbicidal activity of colostrum MN phagocytes was dependent on previous EPEC opsonization with colostral supernatant or blood serum . A defatted colostrum supernatant pool presented opsonic activity for EPEC killing at levels equivalent to those of normal serum . IgA-depleted colostrum supernatant showed significantly lower opsonic activity, whereas purified IgA from the same colostrum pool was a potent opsonin which induced EPEC killing at levels equivalent to those of untreated colostrum . Colostral MN phagocytes are able to release superoxide anion when incubated with both EPEC opsonized with untreated colostrum and purified IgA . Purified IgA was also able to restore opsonic activity of IgA-depleted colostrum . A colostrum pool without C3 and IgG induced EPEC killing by colostral MN phagocytes at rates equivalent to those of untreated colostrum supernatant . Addition of an IgM MoAb (My43) anti-human Fc alpha receptor resulted in a significant inhibition of EPEC killing when bacteria were opsonized with purified IgA, suggesting an interaction between IgA and Fc alpha R . With respect to serum opsonins, we observed that IgG plus complement component C3 were necessary to induce EPEC killing by the colostrum MN phagocytes . Colostral phagocyte killing of enteropathogenic bacteria may represent an additional mechanism of breast-feeding protein against intestinal infections during the first week of life. Microbiology, 1997 Jul, 143 ( Pt 7), 2197 - 207 The cydR gene product, required for regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii, is an Fnr-like protein; Wu G et al.; The cytochrome bd complex in the obligately aerobic diazotroph Azotobacter vinelandii is an oxidase, which, in vivo, has a low affinity for oxygen and is required for respiratory protection of nitrogenase . Mutations caused by insertion of Tn5-B20 upstream of the structural genes (cydAB) for cytochrome bd result in over-expression of this oxidase and, for unexplained reasons, inability of the organism to grow microaerobically . Cloning and sequencing of this upstream region revealed a gene, cydR . The deduced amino acid sequence of CydR indicates that it is a new member of the Fnr Class of regulators and that it represses cydAB expression . Refined mapping data for three insertions in cydR are presented . The cloned cydR gene complemented anaerobic growth of Escherichia coli fnr mutants and strongly enhanced expression of a narG-lacZ fusion in an E . coli fnr mutant. Microbiology, 1997 Jul, 143 ( Pt 7), 2179 - 87 Alanyl-tRNA synthetase gene of the extreme acidophilic chemolithoautotrophic Thiobacillus ferrooxidans is highly homologous to alaS genes from all living kingdoms but cannot be transcribed from its promoter in Escherichia coli; Guiliani N et al.; The alaS gene of Thiobacillus ferrooxidans has been cloned and sequenced and its expression in Escherichia coli and T . ferrooxidans analysed . The same genomic organization to that in E . coli (recA-recX-alaS) has been found in T . ferrooxidans . The recA and alaS genes cannot be transcribed from their own promoters in E . coli . In addition to the well-known homology at the protein level between AlaS proteins from various organisms, a strong homology was found between all the known alaS genes from bacteria, archaea and eucarya . Two regions, one of which corresponds to the catalytic core, are particularly well-conserved at the nucleotide sequence level, a possible indication of strong constraints during evolution on these parts of the genes. J Struct Biol, 1997 Jul, 119(2), 149 - 57 Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review; Muller DJ et al.; Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution . A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected . OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels . Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN . Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state . Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible. Can J Vet Res, 1997 Jul, 61(3), 193 - 9 Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family; Mainil JG et al.; Necrotoxigenic Escherichia coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2) . Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E . coli . Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%) . Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%) . A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied . They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes . PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons . pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors . f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene . Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay. Am J Trop Med Hyg, 1997 Jul, 57(1), 85 - 90 Enterotoxigenic Escherichia coli diarrhea among young children in Jakarta, Indonesia; Richie E et al.; The incidence of diarrhea and enterotoxigenic Escherichia coli (ETEC) infection was evaluated in children six months to five years of age from an urban community in Jakarta, Indonesia . From January through May 1994, 408 children were monitored in their homes for diarrheal disease . Thirty-six percent (148 of 408) of the study children had at least one episode of diarrhea during the study period . Twenty-nine (19.6%) of the 148 children with diarrhea had ETEC isolated from a rectal swab sample at least once during the surveillance period; five children had ETEC isolated from two distinct episodes of diarrhea, giving a total of 34 episodes of ETEC positive diarrhea in the study group . Ten of 34 episodes were associated with heat-labile toxin, 15 of 34 with heat-stable toxin, and seven of 34 with both toxins . The mean age of children with diarrhea (1.7 years), whether ETEC positive or negative, was significantly lower than those who did not have diarrhea (2.4 years) during the study period; 82% of the children with ETEC were less than two years of age . This study demonstrates a high incidence of ETEC diarrhea among young children in Jakarta, and suggests this site would be suitable for ETEC vaccine efficacy trialsPIP: During a 4-month period in 1994, 408 children 6 months to 5 years of age (mean, 2.4 years) from a densely populated slum section (Kapuk) of West Jakarta, Indonesia, were monitored in their homes for diarrheal disease . Many homes in this community lack running water or toilet facilities . Overall, 148 (36%) of these children had at least one diarrhea episode during the study period . 29 children (19.6%) with diarrhea had enterotoxigenic Escherichia coli (ETEC) isolated from a rectal swab sample at least once during the surveillance period and five children had ETEC isolated from two distinct diarrhea episodes, for a total of 34 episodes of ETEC-positive diarrhea . 10 of the 34 episodes were associated with heat-labile toxin, 15 with heat-stable toxin, and 7 with both toxins . Annualized rates of diarrhea and ETEC infections were estimated at 2.2 and 0.3 per child, respectively . The rate of children with diarrhea declined steadily with increasing age: 52% at 6-11 months, 48% at 12-23 months, 28% at 24-35 months, 30% at 36-47 months, and 12% at 48-60 months . 82% of children with ETEC were under 2 years of age . The high incidence of ETEC diarrhea recorded in this study suggests the feasibility of ETEC vaccine efficacy trials in this population . J Interferon Cytokine Res, 1997 Jul, 17 Suppl 1, S15 - 21 Interferon immunogenicity: technical evaluation of interferon-alpha 2a; Hochuli E; Observations from some studies with interferon-alpha 2a (IFN-alpha 2a) have shown the presence of neutralizing antibodies in a proportion of patients . As a result, an investigation into the production of antibodies to IFN-alpha 2a was undertaken . A number of technical aspects of its production and storage were investigated, including the possibility of an incorrect structure, which could affect the immunogenicity of the IFN-alpha 2a molecule . These investigations demonstrated the presence, in vials of IFN-alpha 2a, of both interferon-interferon (IFN-IFN) aggregates and aggregates of interferon with human serum albumin (HSA), the excipient of the galenical form of IFN-alpha 2a (IFN-HSA) aggregates . The amount of aggregates is temperature dependent, there being very little increase in aggregate content over time when vials are stored at 4 degrees C . The relative immunogenicity of IFN-alpha 2a increased when the vials were stored at ambient temperature but not when stored at 4 degrees C . These findings demonstrate that the immunogenicity of IFN-alpha 2a is likely to be related to the storage temperature . Storage of IFN-alpha 2a vials at 2-8 degrees C is now recommended . A new formulation has been introduced that does not contain HSA as an excipient, removing the possibility of IFN-HSA aggregation. J Vasc Surg, 1997 Jul, 26(1), 119 - 27 Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers; Zelenock JA et al.; Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically . Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction . We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer . Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods . Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes . High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume . Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume . A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively . A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase . Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion . Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods . In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury . Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats . Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats . Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity . We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles. J Mol Cell Cardiol, 1997 Jul, 29(7), 1915 - 26 Effect of endotoxin and platelet-activating factor on lipid oxidation in the rat heart; Wang X et al.; The effects of endotoxin and platelet-activating factor (PAF) administered in vivo and in vitro on lipid oxidation by isolated perfused working rat heart were investigated and compared . Endotoxin administered in vivo decreased oleate oxidation in perfused hearts and caused increased accumulation of lipid in myocardial tissue; this was accompanied by a dose-dependent inhibition of cardiac mechanical function . There was no effect on triolein (chylomicron) oxidation . By contrast, PAF administered in vivo caused a small (non-significant) increase in the oxidation rate of oleate and triolein, and also increased myocardial lipoprotein lipase activity . Cardiac mechanical function was not inhibited by PAF-indeed pretreatment of animals by PAF administered in vivo protected the heart from the decline in function associated with subsequent fatty acid perfusion . Administration of endotoxin during perfusion in vitro did not affect oleate or triolein oxidation and cardiac mechanical function was unchanged; PAF administered in vitro caused an early increase in oleate oxidation and a later increase in triolein oxidation . Administration of both endotoxin and PAF in vitro increased myocardial lipoprotein lipase activity . PAF is unlikely to be responsible for all of the effects of endotoxin on cardiac lipid metabolism.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
