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