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| Biochemistry, 1996 Dec 17, 35(50), 16165 - 73 Effects of alpha-deuteration and of aza and thia analogs of L-tryptophan on formation of intermediates in the reaction of Escherichia coli tryptophan indole-lyase; Sloan MJ et al.; Tryptophan indole-lyase catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate . After the enzyme is mixed with L-tryptophan in the rapid-scanning stopped-flow spectrophotometer, there is an absorbance increase at 505 nm in the pre-steady state attributed to formation of a quinonoid intermediate, which occurs in at least three consecutive first-order phases . Reaction with {alpha-2H}-L-tryptophan results in significant primary kinetic isotope effects on the first two phases, and there is a significant isotope effect on the amplitude of the absorbance increase in the second phase . This result suggests that proton transfer to carbon to form the indolenine intermediate is relatively slow and is probably at least partially rate-determining . Reaction of L-tryptophan in the presence of benzimidazole results in a rapid increase in absorbance in the first phase, followed by a decrease in absorbance in the second phase, with rate constants very similar to those observed without benzimidazole . We have also examined aza and thia analogs of L-tryptophan, with the benzene ring of the indole replaced by pyridine or thiophene . Both 4,5-thiatryptophan and 6,7-thiatryptophan form quinonoid intermediates in the reaction with tryptophan indole-lyase; however, 6,7-thiatryptophan is a better substrate (kcat/K(m) = 32% of L-trp) for tryptophan indole-lyase than is 4,5-thiatryptophan (kcat/K(m) = 4% of L-trp) . Benzimidazole affects the pre-steady-state reaction of 6,7-thiatryptophan in a way similar to L-tryptophan, while benzimidazole does not affect the pre-steady-state reaction of 4,5-thiatryptophan . 4-Aza-, 5-aza-, 6-aza-, and 7-aza-L-tryptophan are all very slow substrates (kcat < 1% of L-trp) for Escherichia coli tryptophan indole-lyase . beta-Indazolyl-L-alanine is a relatively good substrate and exhibits a quinonoid intermediate in its reaction with tryptophan indole-lyase . 6-Aza- and 7-azatryptophan accumulate quinonoid intermediates in the reaction with tryptophan indole-lyase, whereas 4-aza- and 5-azatryptophans do not significantly accumulate quinonoid intermediates, and these latter compounds exhibit very high K(m) values . Addition of benzimidazole does not change the rapid-scanning stopped-flow spectra of 6-aza- and 7-azatryptophan . This suggests that the rate-determining step in the reaction changes depending on the position and type of heteroatom substitution . For 6-aza- and 7-azatryptophan, the very slow rates of elimination may be due to slow C-protonation of the azaindole, while for 4,5-thiatryptophan, the elimination of thienopyrrole is probably slow . Of all analogs examined, 6,7-thiatryptophan is most similar to tryptophan in its reaction with E . coli tryptophan indole-lyase. Biochemistry, 1996 Dec 17, 35(50), 16153 - 64 Rapid formation of the native 14-38 disulfide bond in the early stages of BPTI folding; Dadlez M et al.; Using recombinant variants of BPTI, we have determined the rate constants corresponding to formation of each of the fifteen possible disulfide bonds in BPTI, starting from the reduced, unfolded protein . The 14-38 disulfide forms faster than any of the other 14 possible disulfides . This faster rate results from significantly higher intrinsic chemical reactivities of Cys-14 and Cys-38, in addition to local structure in the reduced protein that facilitates formation of the 14-38 disulfide bond . This disulfide bond is found in native BPTI . Our results suggest that a significant flux of folding BPTI molecules proceed through the one-disulfide intermediate with the 14-38 disulfide bond, denoted {14-38}, that has recently been detected on the BPTI folding pathway . In addition to providing a detailed picture of the early events in the folding of BPTI, our results address quantitatively the effect of local structure in the unfolded state on folding kinetics. Biochemistry, 1996 Dec 17, 35(50), 16116 - 24 Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence . 1 . Tryptophanyl residues of the carrier can be mutated without impairing protein activity; Le Saux A et al.; During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier . To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts . The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus . A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange . The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined . Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity . The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses . When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed . Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al . {(1996) Biochemistry 35, 16125-16131}. Biochemistry, 1996 Dec 17, 35(50), 16077 - 84 Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding; Kiefer H et al.; An olfactory receptor has been expressed in bacterial cells as a fusion protein with glutathione S-transferase (GST) . Overexpression of receptor protein yielding about 10% of the cell protein was achieved with mutants lacking the N-terminus and the first transmembrane region or with mutants carrying three positively charged residues in the first intracellular loop . The overexpressed fusion protein accumulated in inclusion bodies and could be solubilized in detergent . It was purified by metal chelation chromatography based on a C-terminal 6-histidine tag, and the GST portion was removed after proteolytic cleavage . The purified receptor was reconstituted into lipid vesicles and specific binding of odor ligands was shown by photoaffinity labeling and tryptophan fluorescence measurements . Thus, for the first time, an odorant receptor/ligand pair becomes available in large amounts for biophysical and screening studies. Biochemistry, 1996 Dec 17, 35(50), 16009 - 23 Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales; Mandel AM et al.; The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation . Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches . The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales . The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively . The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein . The effective correlation times for internal motions do not show significant temperature dependence . Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D . Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms . The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol . Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2 . The present results obtained at 300 K are compared to those reported previously {Mandel, A . M., Akke, M., & Palmer, A . G., III (1995) J . Mol . Biol . 246, 144-163} to establish the reproducibility of the experimental techniques. Biochemistry, 1996 Dec 17, 35(50), 15949 - 61 Interactions of the RecBCD enzyme from Escherichia coli and its subunits with DNA, elucidated from the kinetics of ATP and DNA hydrolysis with oligothymidine substrates; Chamberlin M et al.; Oligothymidines eight nucleotides or longer stimulate ATP hydrolysis by the RecBC and RecBCD enzymes, and they are substrates for the ATP-stimulated nuclease activity of RecBCD . The steady-state kinetics of ATP hydrolysis by the RecBC enzyme are consistent with a single ATPase and DNA binding site . Results with RecBCD and RecBCD-K177Q {an enzyme with a Lys-to-Gln mutation in the ATP binding motif of the RecD subunit {Korangy, F., & Julin, D . A . (1992) J . Biol . Chem . 267, 1727-1732}} indicate that ATP hydrolysis by the RecB subunit is stimulated by pd(T)12 binding to a high-affinity site, while the RecD subunit hydrolyzes ATP stimulated by pd(T)12 binding to a low-affinity site . The site which stimulates RecB has about 50-fold greater affinity for DNA in either RecBCD or RecBCD-K177Q than does the corresponding site in RecBC . The rates of ATP hydrolysis observed for the RecBCD enzyme at low concentrations of pd(T)12 are best explained by a mechanism where the enzyme binds to the DNA and catalyzes multiple rounds of ATP hydrolysis before dissociating . Larger DNA molecules {pd(T)25-30 and poly(dT)} are bound more tightly by RecBCD, are hydrolyzed more rapidly, and are much more effective in stimulating ATP hydrolysis than is pd(T)12 . The results at low ATP concentrations where the nuclease activity is minimal (5 microM) suggest that ATP hydrolysis is stimulated by the DNA ends, but there is no evidence that the RecBCD enzyme moves along these DNA molecules in an ATP-dependent manner under these conditions. EMBO J, 1996 Dec 16, 15(24), 7178 - 87 Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription; Lanchy JM et al.; We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996) . Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes . Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex . Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation . As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme . This processivity change is reminiscent of the transition from initiation to elongation of transcription . Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme. EMBO J, 1996 Dec 16, 15(24), 6899 - 909 Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli; Missiakas D et al.; Heat shock response in Escherichia coli is autoregulated . Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures . A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin . We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin . One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon . HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome . HslU/ClpY is 50% identical to the ClpX protein of E . coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP . We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides . Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP. FEBS Lett, 1996 Dec 16, 399(3), 313 - 6 Mutations in the amino-terminal domain of the human poly(ADP-ribose) polymerase that affect its catalytic activity but not its DNA binding capacity; Trucco C et al.; Poly-ADP ribosylation of nuclear proteins is activated when poly(ADP-ribose) polymerase (PARP), a nuclear zinc-finger enzyme, binds to single-strand DNA breaks . To understand how the signal emerging from its DNA-binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure-function relationship of the DBD was investigated . We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants . In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA . The results obtained demonstrate that the DNA-dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an 'activation signal' to the catalytic domain. FEBS Lett, 1996 Dec 16, 399(3), 307 - 9 Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle; de Gier JW et al.; Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep-inv) that inserts with an inverted topology compared to the wild-type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E . coli SRP . Depletion of either component strongly affected the insertion of both Lep and Lep-inv into the cytoplasmic membrane . This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E . coli. FEBS Lett, 1996 Dec 16, 399(3), 283 - 9 Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin; Prytulla S et al.; A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique . The protein has been expressed with high efficiency and a purification protocol has been developed . The holoprotein is readily reconstituted by the addition of heme . 15N- and 15N,13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy . Comparison of the chemical shifts of 13C(alpha) and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography. FEBS Lett, 1996 Dec 16, 399(3), 232 - 6 Biosynthetically lipid-modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes; de Kruif J et al.; A human anti-CD22 single chain (sc) Fv antibody fragment from a synthetic phage antibody display library was biosynthetically lipid-tagged by using Escherichia coli lipoprotein sequences . The purified anti-CD22 scFv lipoprotein was incorporated into liposomes by detergent dilution . Anti-CD22 immunoliposomes were shown to bind specifically in a dose- and time-dependent manner to CD22+ cell lines and CD22+ B-lymphocytes present in freshly isolated samples of blood mononuclear cells . The immunoliposomes were demonstrated to accumulate in intracellular compartments . Biosynthetically lipid-tagged human scFv antibody fragments isolated from phage display libraries may facilitate the construction of immunoliposomes with improved properties. FEBS Lett, 1996 Dec 16, 399(3), 223 - 6 Interaction of 10Sa RNA with ribosomes in Escherichia coli; Tadaki T et al.; 10Sa RNA is a bacterial small stable RNA, in which the 5'- and 3'-end sequences are folded into a tRNA-like structure . The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells . In this study, we examined the ribosome-binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes . 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes . Recently, E . coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C-termini of truncated peptides synthesized in vivo . The present results are consistent with the 'trans-translation' model, which has been proposed for tag-peptide synthesis. FEBS Lett, 1996 Dec 16, 399(3), 215 - 9 Expression of active, human lysyl oxidase in Escherichia coli; Ouzzine M et al.; Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross-linking in collagens and elastin . Human LO was expressed in Escherichia coli . At 37 degrees C, large amounts of protein were obtained, but in the form of insoluble aggregates . Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a degrees {3H}lysine-labeled elastin substrate . LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide . The periplasmic enzyme was soluble, active and inhibited by beta-aminopropionitrile . Production of the carbonyl co-factor is therefore not a limitation in the expression of active LO in bacteria. Eur J Biochem, 1996 Dec 15, 242(3), 712 - 9 Cloning, sequencing, mapping and hyperexpression of the ribC gene coding for riboflavin synthase of Escherichia coli; Eberhardt S et al.; The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6-kb fragment that has been sequenced . The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a mass of 23.4 kDa . It was mapped to a position at 37.5 min on the physical map of the E . coli chromosome . The 3' end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane-fatty-acid synthase . This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively . The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein . The protein specified by ydhB shows sequence similarity to a large family of DNA-binding proteins and probably represents a helix-turn-helix protein . The ydhB gene is directly adjacent to the regulatory gene purR . A 288-bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min . The ribC gene was hyperexpressed in recombinant E . coli strains to a level of about 30% of cellular protein . The protein was purified to homogeneity by chromatography . The specific activity was 26000 nmol.mg-1.h-1 . The protein sediments at a velocity of S20 = 3.8 S . Sedimentation-equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure . The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C-terminal and N-terminal parts suggesting two structurally similar folding domains. Eur J Biochem, 1996 Dec 15, 242(3), 648 - 56 Two potential indole-3-acetaldehyde dehydrogenases in the phytopathogenic fungus Ustilago maydis; Basse CW et al.; The phytopathogenic basidiomycetc Ustilago maydis produces indole-3-acetic acid (IndCH2COOH) and indole-3-pyruvic acid (Ind-Prv) from tryptophan . Indole-3-acetaldehyde (IndCH2CH2O) is the common intermediate in the conversion of Ind-Prv and tryptamine to IndCH2COOH . We purified an enzyme (Iad1) from U . maydis that catalyzes the NAD(+)-dependent conversion of IndCH2CH2O to IndCH2COOH and isolated corresponding cDNA and genomic clones . The identity of the cDNA clone was confirmed by expression in Escherichia coli and demonstration of enzymatic activity . In U . maydis, iad1-null mutants were generated by gene replacement . The ability to convert IndCH2CH2O to IndCH2COOH was at least 100-fold reduced in U . maydis iad1-null mutants grown in medium with glucose as carbon source . However, the iad1-null mutants were not diminished in their capacity to produce IndCH2COOH from tryptophan, indicating that IndCH2COOH formation from tryptophan apparently proceeds in the absence of IndCH2CH2O dehydrogenase activity under these conditions . Iad1 expression was strongly induced during growth on ethanol while under these conditions iad1-null mutants were unable to grow . This reveals that iad1 is primarily engaged in the conversion of ethanol to acetate . In iad1-null mutants we detected an additional NAD(+)-dependent IndCH2CH2O dehydrogenase activity that was induced during growth on L-arabinose but repressed in the presence of D-glucose . In arabinose-containing medium the conversion of tryptophan to IndCH2COOH was approximately 5-fold reduced in wild-type strains but 10-15-fold reduced in iad1-null mutant strains compared to IndCH2COOH formation in glucose-containing medium . In addition, the formation of Ind-Prv from tryptophan was abolished in wild-type and iad1-null mutant strains . During growth on arabinose, the conversion of tryptamine to IndCH2COOH was strongly favored suggesting that the glucose-repressible IndCH2CH2O dehydrogenase is required to convert IndCH2CH2O derived from tryptamine to IndCH2COOH. Eur J Biochem, 1996 Dec 15, 242(3), 585 - 91 Purification, characterization and subcellular localization of a type-1 ribosome-inactivating protein from the sarcocarp of Cucurbita pepo; Yoshinari S et al.; The flesh of the fruit of Cucurbita pepo contains a type-1 ribosome-inactivating protein (RIP), which we named pepocin . Pepocin was purified to apparent homogeneity by acid fractionation, ion-exchange chromatography and adsorption chromatography . The protein was found to have a molecular mass of 26 kDa and a pI of about 9.9 . It does not contain glycosidic linkages . The protein inhibits protein synthesis in a rabbit-reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 15.4 pM, and depurinates 28S rRNA in the ribosomes of the lysate in a manner identical to that of ricin A-chain and other RIP . The enzyme is also active on wheat-germ ribosomes and on Escherichia coli ribosomes . The sequence of the N-terminal 20 amino acids of the protein reveals a close relationship to other RIP . Immunoelectron-microscopic localization of pepocin in the sarcocarp shows that the protein is predominantly localized in intercellular spaces . In addition, the immunolocalized signals are observed in leaf intercellular spaces. Eur J Biochem, 1996 Dec 15, 242(3), 567 - 75 NMR studies of the Escherichia coli Trp repressor.trpRs operator complex; Evans PD et al.; To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy . We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor . Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor . Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence . The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules . We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex . The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA . This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator. Eur J Biochem, 1996 Dec 15, 242(3), 519 - 28 The recombinant alpha isoform of protein kinase CK1 from Xenopus laevis can phosphorylate tyrosine in synthetic substrates; Pulgar V et al.; The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library . The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da . The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein . Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X . laevis possesses a family of related CK1 genes . CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined . The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme . The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20) . This inhibition is competitive and shows an approximate K1 of 5 microM . CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE . This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine. Eur J Biochem, 1996 Dec 15, 242(3), 499 - 505 Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins; Yoshida N et al.; Fructosyl amino acid oxidase (FAOD), which is active toward model compounds of the glycated proteins in blood, N epsilon-fructosyl N sigma-Z-lysine and N-fructosyl valine, was purified to homogeneity from Aspergillus terreus GP1 . Though the enzyme did not use glycated proteins directly as its substrate, it used glycated human serum albumin (HSA) when HSA was treated with a protease . Linear relationships between both the concentration and the increase in absorbance and the glycation rate of glycated HSA and the increase in absorbance were observed . cDNAs coding for FAODs were cloned from cDNA libraries of A . terreus GP1 and Penicillium janthinellum AKU 3413 . The coding region for both fungal FAODs consisted of 1314 bp encoding 437 amino acids . The sequence of a dinucleotide-binding motif, GXGXXG, was in the deduced N-terminal region and a similar sequence to that the active site of bacterial sarcosine oxidases was found near the C-terminal region of FAOD . The of C-terminal tripeptides SKL and AKL of FAODs from A . terreus and P . janthinellum, respectively, represent typical peroxisomal-targeting signals . Finally, FAOD protein was produced in Escherichia coli transformants in an active form, and at the same level as in the original fungi. Nucleic Acids Res, 1996 Dec 15, 24(24), 4933 - 9 Interaction of the RecA protein of Escherichia coli with single-stranded oligodeoxyribonucleotides; Bianco PR et al.; The RecA protein of Escherichia coli performs a number of ATP-dependent, in vitro reactions and is a DNA-dependent ATPase . Small oligodeoxyribonucleotides were used as DNA cofactors in a kinetic analysis of the ATPase reaction . Polymers of deoxythymidilic acid as well as oligonucleotides of mixed base composition stimulated the RecA ATPase activity in a length-dependent fashion . Both the initial rate and the extent of the reaction were affected by chain length . Full activity was seen with chain lengths > or = 30 nt . Partial activity was seen with chain lengths of 15-30 nt . The lower activity of shorter oligonucleotides was not simply due to a reduced affinity for DNA, since effects of chain length on KmATP and the Hill coefficient for ATP hydrolysis were also observed . The results also suggested that single-stranded DNA secondary structure frequently affects the ATPase activity of RecA protein with oligodeoxyribonucleotides. Nucleic Acids Res, 1996 Dec 15, 24(24), 4845 - 52 Deoxy- and dideoxynucleotide discrimination and identification of critical 5' nuclease domain residues of the DNA polymerase I from Mycobacterium tuberculosis; Mizrahi V et al.; The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag . The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity . In accordance with recent predictions {Tabor, S . and Richardson, C.C . (1995) Proc . Natl . Acad . Sci . USA, 92, 6339-6343}, both recombinant forms of the M . tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis . This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design . A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated . Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues. J Physiol, 1996 Dec 15, 497 ( Pt 3), 773 - 9 Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms; Doring F et al.; l . Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport . PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes . In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity . In addition, both transporters show differences in pH dependence and substrate specificity . 2 . As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1 . Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique . 3 . Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters . We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region. Biochem J, 1996 Dec 15, 320 ( Pt 3), 879 - 84 Competitive inhibition of calcineurin phosphatase activity by its autoinhibitory domain; Sagoo JK et al.; Calcineurin (protein phosphatase 2B), a calmodulin- and calcium-dependent serine/threonine phosphatase, appears to be regulated by a C-terminal autoinhibitory domain . A 25 amino acid peptide derived from this domain inhibits calcineurin phosphatase activity in vitro . Here we show that a 97 amino acid fragment of the calcineurin A alpha C-terminus is approx . 8-fold more potent than the shorter peptide in calcineurin inhibition experiments . Mutation of an evolutionarily conserved Asp to Asn, previously shown to disrupt calcium-dependent signalling and calcineurin regulation in T-lymphocytes, greatly reduced inhibition by the autoinhibitory domain in vitro . Kinetic analysis of wild-type and mutated autoinhibitory domains show that both are competitive inhibitors of calcineurin phosphatase activity with Ki values of 5.0 +/- 0.2 microM and 36.0 +/- 3.7 microM respectively . This suggests intrasteric regulation of calcineurin, with the autoinhibitory domains interacting at the active site of the enzyme . The competitive behaviour of the autoinhibitory domains contrasts with the mechanism of calcineurin inhibition by immunosuppressant-immunophilin complexes, which have been shown to bind to calcineurin at a region removed from the active site. Biochem J, 1996 Dec 15, 320 ( Pt 3), 801 - 6 Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2C alpha; Marley AE et al.; The use of protein phosphatase inhibitors has been instrumental in defining the intracellular roles of protein phosphatase 1 (PP1), PP2A and PP2B . Identification of the role of PP2C in vivo has been hampered, in part, by the unavailability of specific inhibitors . In order to facilitate the identification of novel and specific inhibitors of PP2C by random screening of compounds, and to further characterize this enzyme at the molecular level by site-directed mutagenesis and X-ray crystallography, we have expressed active recombinant human PP2C alpha (rPP2C alpha) in Escherichia coli . Biochemical characterization of rPP2C alpha showed that it could hydrolyse p-nitrophenyl phosphate (pNPP) although, in contrast with native PP2C, this was not stimulated by Mg2+ . As with native PP2C, okadaic acid failed to inhibit rPP2C alpha, whereas 50 mM NaF dramatically inhibited its activity . An alignment of the amino acid sequence of AMP-activated protein kinase (AMPK) with those of other serine/threonine protein kinases around the regulatory phosphorylation site (subdomains VII-VIII) revealed a high degree of conservation . Phosphopeptides derived from this region of AMPK and containing the almost invariant threonine (Thr172 in AMPK) were found to be good substrates for rPP2C alpha . We also showed that rPP2C alpha can inactivate AMPK, but only in the presence of Mg2+ . To define the regions of PP2C alpha important for catalytic activity, we expressed a number of truncated proteins based on the sequence and proposed domain structure of the PP2C alpha homologue from Paramecium tetraurelia . Deletion of 75 residues (9 kDa) from the C-terminus appeared to have little effect on the catalytic activity using pNPP, phosphopeptides or AMPK as substrates . This suggests that the residues important in catalysis lie elsewhere in the protein . A further deletion of the C-terminus led to a completely inactive and very poorly soluble protein. Biochem J, 1996 Dec 15, 320 ( Pt 3), 713 - 6 The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration . Evidence for altered subunit interaction in all the bacteriocupreins; Battistoni A et al.; Gel-filtration chromatography experiments performed at high protein concentrations demonstrate that the Cu,Zn superoxide dismutase from Escherichia coli is monomeric irrespective of the buffer and of ionic strength . The catalytic activity of the recombinant enzyme is comparable with that of eukaryotic isoenzymes, indicating that the dimeric structure commonly found in Cu,Zn superoxide dismutases is not necessary to ensure efficient catalysis . The analysis of the amino acid sequences suggests that an altered interaction between subunits occurs in all bacterial Cu,Zn superoxide dismutases . The substitution of hydrophobic residues with charged ones at positions located at the dimer interface of all known Cu,Zn superoxide dismutases could be specifically responsible for the monomeric structure of the E . coli enzyme. Structure, 1996 Dec 15, 4(12), 1401 - 12 The UmuD' protein filament and its potential role in damage induced mutagenesis; Peat TS et al.; BACKGROUND: Damage induced 'SOS mutagenesis' may occur transiently as part of the global SOS response to DNA damage in bacteria . A key participant in this process is the UmuD protein, which is produced in an inactive from but converted to the active form, UmuD', by a RecA-mediated self-cleavage reaction . UmuD', together with UmuC and activated RecA (RecA*), enables the DNA polymerase III holoenzyme to replicate across chemical and UV induced lesions . The efficiency of this reaction depends on several intricate protein-protein interactions . RESULTS: Recent X-ray crystallographic analysis shows that in addition to forming molecular dimers, the N- and C-terminal tails of UmuD' extend from a globular beta structure to associate and produce crystallized filaments . We have investigated this phenomenon and find that these filaments appear to relate to biological activity . Higher order oligomers are found in solution with UmuD', but not with UmuD nor with a mutant of UmuD' lacking the extended N terminus . Deletion of the N terminus of UmuD' does not affect its ability to form molecular dimers but does severely compromise its ability to interact with a RecA-DNA filament and to participate in mutagenesis . Mutations in the C terminus of UmuD' result in both gain and loss of function for mutagenesis . CONCLUSIONS: The activation of UmuD to UmuD' appears to cause a large conformational change in the protein which allows it to form oligomers in solution at physiologically relevant concentrations . Properties of these oligomers are consistent with the filament structures seen in crystals of UmuD'. Experientia, 1996 Dec 15, 52(12), 1069 - 76 Regulated protein degradation in mitochondria; Langer T et al.; Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis . The matrix-localized PIM1 protease, a homologue of the Escherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity . Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria . The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, the m- and the i-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins . In addition to its proteolytic activity, the m-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes . It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes. J Clin Invest, 1996 Dec 15, 98(12), 2683 - 7 Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes; Bosch A et al.; Retroviral gene transfer to liver without prior injury has not yet been accomplished . We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors . This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers . When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced . Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 451 - 5 Allelic divergence at B alpha 1 pheromone receptor genes of Schizophyllum commune; Wendland J et al.; The multiallelic mating type locus B alpha 1 of Schizophyllum commune encodes a pheromone receptor and putative pheromone genes . A comparison of two alleles encoding receptors that share the same specificity B alpha 1 was performed using strains of different geographic origin . The amino acid sequence alignment revealed strong conservation of the largest part of the receptor . Only in the distal C-terminus major amino acid was divergence encountered . This C-terminal region of 117 of the total of 639 amino acids was shown to be unnecessary for function in vivo by transformation experiments. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 315 - 24 A new role for RNase II in mRNA decay: striking differences between RNase II mutants and similarities with a strain deficient in RNase E; Cruz AA et al.; The effect of Escherichia coli ribonuclease II and polynucleotide phosphorylase was analysed on the degradation of Desulfovibrio vulgaris cytochrome c3 (cyc) mRNA . In the absence of these exoribonucleolytic activities, cyc mRNA was stabilised but the two enzymes had a different role in its decay . Surprisingly, a temperature-sensitive mutation in ribonuclease II gave a degradation pattern similar to what had been observed in the absence of endoribonuclease E activity . In an RNase II deletion mutant this was not observed . We propose and verify a model in which the temperature-sensitive ribonuclease II interferes with the action of ribonuclease E. FEMS Microbiol Lett, 1996 Dec 15, 145(3), 309 - 14 Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo; Aron L et al.; A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera . Restriction enzyme analysis indicated that this DNA fragment was located on the B . burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A . Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichia coli chromosomal open reading frame. Genomics, 1996 Dec 15, 38(3), 264 - 72 Isolation and characterization of GT335, a novel human gene conserved in Escherichia coli and mapping to 21q22.3; Lafreniere RG et al.; As part of efforts to identify candidate genes for disorders mapped to 21q22.3, we have constructed a 405-kb cosmid contig encompassing five tightly linked markers mapping to this region . A subset of these cosmids was used to identify cDNA fragments by the method of hybrid selection . We present here the cDNA sequence of one such gene (GT335) mapping to this region . The gene is expressed as a 1.7-kb transcript predominantly in heart and skeletal muscle, potentially displays alternate splicing, and is predicted to encode a protein 268 amino acids in length . GT335 spans an estimated 13 kb of genomic DNA and is split into seven exons . Five of the six introns conform to the GT . . . AG consensus for intronic splice junctions; the sixth contains nonconventional (AT . . . AC) intronic junctions . We screened this gene for single-basepair mutations using single-strand conformation polymorphism and sequence analysis of both cDNA and genomic DNA from a number of unrelated individuals and have identified several sequence variations, two of which cause conservative amino acid substitutions . This gene is well conserved evolutionarily, with homologs identified in zebrafish and Escherichia coli, suggesting that it plays an important role in basic cellular metabolism. Cancer Res, 1996 Dec 15, 56(24), 5571 - 5 Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine; Edara S et al.; Inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O6-benzylguanine is currently undergoing clinical trials for development as an agent to enhance chemotherapy . It has been reported recently that a polymorphism in the human O6-alkylguanine-DNA alkyltransferase gene exists, with about 15% of the population studied having arginine at codon 160 instead of glycine (Y . Imai et al., Carcinogenesis (Lond.), 16: 2441-2445, 1995) . We have studied the effects of mutations of this glycine to arginine, tryptophan, or alanine on the interaction of human alkyltransferase with O6-benzylguanine using direct determination of the amount of activity remaining after incubation with various concentrations of the inhibitor and measurement of the rate of production of {8-3H}guanine from O6-benzyl{8-3H}guanine as assays . These mutations had little effect on the alkyltransferase activity in repairing O6-methylguanine in methylated DNA . Alteration of glycine 160 to tryptophan or alanine slightly increased the sensitivity to O6-benzylguanine (by up to 4-fold) . However, alteration of glycine 160 to arginine drastically reduced the inactivation by O6-benzylguanine with at least a 20-fold increase in the ED50 value and a similar reduction in the production of guanine whether inactivation was carried out in the absence or presence of DNA . These results raise the possibility that a subpopulation of patients may be resistant to O6-benzylguanine and that higher doses or additional alkyltransferase inhibitors capable of inactivating this form of the alkyltransferase will be necessary. Arch Biochem Biophys, 1996 Dec 15, 336(2), 309 - 15 The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli; Rehman A et al.; Isocitrate lyase from Escherichia coli has been expressed in transformed E . coli JE10 cells lacking the isocitrate lyase (icl) gene . After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells . Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively . Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R . Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes . The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis . The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes . Plots of pKm vs pH were different for the wt and H356K variant . Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4 . The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage. Anal Biochem, 1996 Dec 15, 243(2), 234 - 44 Real-time fluorescence assay system for gene transcription: simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production; Dunkak KS et al.; This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production . These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-aminopurine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates . Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution . The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription. Mol Gen Genet, 1996 Dec 13, 253(3), 397 - 400 A RecA/RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only; Rashid N et al.; A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp . KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli . The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA) . The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini . The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity . Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E . coli recA mutant strain. Mol Gen Genet, 1996 Dec 13, 253(3), 342 - 52 Isolation of a carbon source-regulated gene from Ustilago maydis; Bottin A et al.; We have isolated a carbon source-regulated gene from the phytopathogenic fungus Ustilago maydis by use of a promoter-probe vector . This gene, called crg1, is strongly induced by L-arabinose and efficiently repressed by D-glucose and D-xylose . The predicted 36.5-kDa mature crg1 gene product lacks similarity to known proteins but is likely to be secreted . Sequences required for regulated expression of a reporter gene are contained within a 3.6-kb fragment upstream of the crg1 gene . The promoter of crg1 fulfils requirements for an efficient controllable gene expression system in U . maydis Mol Gen Genet, 1996 Dec 13, 253(3), 303 - 14 Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei; Ilmen M et al.; Functional analysis of the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei was carried out using the Escherichia coli lacZ gene as a reporter . An assay based on cultivation on solid medium in microtiter plates was developed that allows rapid and reliable semiquantitative analysis of beta-galactosidase expression of a large number of transformants . A series of deletions and specifically designed alterations were made covering 2.2 kb of the cbh1 promoter . Removal of sequences upstream of nucleotide -500 in relation to the initiator ATG abolished glucose repression . Mutation of a single hexanucleotide sequence 5'GTGGGG at nucleotide -720 was sufficient for derepression . This site is similar to the binding sites of the glucose repressors MIG1 of Saccharomyces cerevisiae and CREA/CREI of filamentous fungi . Removal of the glucose repressor site did not affect sophorose induction . Sophorose induction of the promoter was retained even in deletion derivatives lacking sequences upstream of position -161, which retained about 70 bp upstream of the transcription start point and only 30 bp upstream of the TATA box. Mol Gen Genet, 1996 Dec 13, 253(3), 297 - 302 Increase in synthesis and stability of sigma 32 on treatment with inhibitors of DNA gyrase in Escherichia coli; Mizushima T et al.; We report here that in Escherichia coli, the anti-bacterial agent nalidixic acid induces transient stabilization and increased synthesis of sigma 32, accompanied by the induction of heat shock proteins (Dnak and GroEL proteins) . The induction of heat shock proteins, increased synthesis of sigma 32, and stabilization of sigma 32 observed on treatment of wild-type cells with nalidixic acid were not observed in a nalA26 mutant, a strain that is resistant to nalidixic acid as the result of a mutation in the gyrA gene . Not only oxolinic acid, but also novobiocin, whose targets are the A and B subunits of DNA gyrase, respectively, also induced stabilization and increased synthesis of sigma 32 . Thus, inhibition of the activity of DNA gyrase may cause stabilization and increased synthesis of sigma 32, resulting in turn in induction of heat shock proteins. Biochim Biophys Acta, 1996 Dec 13, 1304(3), 245 - 53 Association of lipid A disaccharide synthase with aerobic glycerol-3-phosphate dehydrogenase in extracts of Escherichia coli; Milla ME et al.; Variants of the Escherichia coli UDP-GlcNAc O-acyltransferase (LpxA) and of the lipid A disaccharide synthase (LpxB) containing affinity chromatography tags (C-terminal histidine 8 {H8} tails) were constructed in order to investigate whether or not these enzymes interact with other E . coli proteins . These variants (LpxA-H8 and LpxB-H8) had specific activities in vitro that were similar to wild-type enzymes . Crude extracts made from E . coli cells expressing LpxA-H8 or LpxB-H8 were chromatographed over Ni(2+)-NTA-Agarose, and proteins purifying with the tagged proteins were identified by SDS-PAGE, followed by blotting and N-terminal microsequencing . At high levels of LpxB-H8 expression, two heat-shock proteins (DnaK and GroEL) were associated with the disaccharide synthase, but not with the acyltransferase . Another major protein recovered with LpxB-H8 (both at low and high levels of expression) was the aerobic glycerol-3-phosphate dehydrogenase (GlpD) . The latter interaction was specific, since GlpD did not bind the affinity resin when the affinity tag was present on the UDP-GlcNAc O-acyltransferase (LpxA-H8) . Velocity centrifugation experiments indicated that both wild-type LpxB and GlpD sedimented together under some conditions, but these aggregates were smaller than and distinct from inner membranes . Our findings suggest a possible new mechanism by which the biosynthetic pathways for lipid A and glycerophospholipids may be coordinated. J Mol Biol, 1996 Dec 13, 264(4), 713 - 21 The RecG branch migration protein of Escherichia coli dissociates R-loops; Vincent SD et al.; The RuvAB and RecG proteins of Escherichia coli promote branch migration of Holliday junction intermediates in genetic recombination . Both are structure-specific helicases that unwind and rewind DNA at the junction point . The helicase activities of these proteins were investigated using RNA:DNA hybrid molecules . RuvAB catalyses the unwinding of RNA:DNA partial duplexes of at least 218 bp in a reaction that requires both RuvA and RuvB, ATP and Mg2+ . RecG failed to unwind these substrates even when the duplex region was reduced to 35 bp . In contrast, RecG rapidly removes a 218 nt RNA from an R-loop substrate, whereas RuvAB does not . RecG's ability to dissociate R-loops is correlated with an ability to reduce the copy number of pUC plasmids and other constructs based on the ColE1 replicon . Copy number is reduced severely when the plasmid carries recG+ . RecG is assumed to reduce copy number by interfering with RNA II's ability to form an R-loop at the plasmid origin of replication and prime DNA synthesis . The dissociation of R-loops by RecG is discussed in terms of the functions needed to promote recombination and to prime DNA replication at D-loops formed during the early stages of RecA-mediated recombination. J Mol Biol, 1996 Dec 13, 264(4), 696 - 712 Characterization of a mutant RecA protein that facilitates homologous genetic recombination but not recombinational DNA repair: RecA423; Ishimori K et al.; A recA mutant (recA423; Arg169-->His), with properties that should help clarify the relationship between the biochemical properties of RecA protein and its two major functions, homologous genetic recombination and recombinational DNA repair, has been isolated . The mutant has been characterized in vivo and the purified RecA423 protein has been studied in vitro . The recA423 cells are nearly as proficient in conjugational recombination, transductional recombination, and recombination of lambda red- gam- phage as wild-type cells . At the same time, the mutant cells are deficient for intra-chromosomal recombination and nearly as sensitive to UV irradiation as a recA deletion strain . The cells are proficient in SOS induction, and results indicate the defect involves the capacity of RecA protein to participate directly in recombinational DNA repair . In vitro, the RecA423 protein binds to single-stranded DNA slowly, with an associated decline in the ATP hydrolytic activity . The RecA423 protein promoted a limited DNA strand exchange reaction when the DNA substrates were homologous, but no bypass of a short heterologous insert in the duplex DNA substrate was observed . These results indicate that poor binding to DNA and low ATP hydrolysis activity can selectively compromise certain functions of RecA protein . The RecA423 protein can promote recombination between homologous DNAs during Hfr crosses, indicating that the biochemical requirements for such genetic exchanges are minimal . However, the deficiencies in recombinational DNA repair suggest that the biochemical requirements for this function are more exacting. Cell, 1996 Dec 13, 87(6), 1123 - 34 Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase; Niu W et al.; At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase . We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain . We further show that the two interactions affect different steps in transcription initiation . Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 612 - 7 Molecular cloning of a novel 120-kDa TBP-interacting protein; Yogosawa S et al.; TATA-binding protein (TBP) is a central component for transcriptional regulation and is a target for various transcription regulators . Using histidine-tagged TBP as a ligand for affinity-purification of proteins bound to TBP, we purified a 120-kD protein, termed TBP-interacting protein 120 (TIP120), from rat liver nuclear extracts . The entire cDNA sequence of TIP120 contained an open reading frame encoding a novel polypeptide of 1230 amino acids . The recombinant TIP120 interacted directly with TBP under a physiological condition in vitro . Immunoprecipitation analysis indicated that TIP120 was associated with TBP in nuclear extracts . Interestingly, the N-terminal region of TIP120 exhibited sequence similarity to that of Drosophila TAF80, which was shown to bind directly to TBP . This novel TBP-binding protein is considered to participate in transcription regulation through the interaction with TBP. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 531 - 5 Poly-L-lysine activates both peptide and ATP hydrolysis by the ATP-dependent HslVU protease in Escherichia coli; Yoo SJ et al.; Hs1VU in E . coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain beta-type subunits of the 20S proteasome . Here we show that the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome . However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself . Instead, it stimulated the hydrolysis of ATP by HslU several-fold . Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine . Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV . These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU. Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 412 - 8 Evidence for GroES acting as a transcriptional regulator; Legname G et al.; Cochaperonins (cpn10) assist chaperonins (cpn60) in promoting folding and assembly of other proteins . Upon expression of Mycobacterium tuberculosis cpn10 in Escherichia coli we have purified a polypeptide which, through amino acid sequencing, was identified as the endogenous E . coli 10K-S protein . Subsequent studies showed that its expression was specifically upregulated upon cloning of different members of the cpn10 family, including GroES, the E . coli cpn10 . Pulse-chase experiments demonstrated that 10K-S is but one of several proteins whose expression is modulated upon cloning of cpn10 . Up-regulation of 10K-S was also observed after exposure of normal cells, but not of groES- mutants, to elevated temperatures (42 degrees C) . This allowed us to define 10K-S as a heat-shock protein (hsp) whose expression is dependent on the production of another hsp, GroES . Northern blot experiments showed that enhanced expression of 10K-S was consequent to increased accumulation of transcripts and that groES- mutants were devoid even of baseline levels of transcripts both at 37 degrees C and after temperature upshift . These results show that GroES, in addition to its established role in assisting protein folding may act as a transcriptional regulator and is likely to play an important role in modulating gene expression particularly in those conditions, like the stress response, in which its production is greatly enhanced. J Biol Chem, 1996 Dec 13, 271(50), 32306 - 14 B cell antigen receptor signaling induces the formation of complexes containing the Crk adapter proteins; Ingham RJ et al.; Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes . We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins . We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement . BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction . We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes . Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains . In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G . Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR. J Biol Chem, 1996 Dec 13, 271(50), 32288 - 92 Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli; Rothman A et al.; Analysis of the hydropathic profile of the amino acid sequence of NhaA, a Na+/H+ antiporter from Escherichia coli has previously suggested the existence of 11 putative transmembrane segments (Taglicht, D., Padan, E., and Schuldiner, S . (1991) J . Biol . Chem . 266, 11289-11294) . In the present work to test the location of the C terminus, right-side-out and inside-out membrane vesicles were digested with carboxypeptidase B and probed with an antibody raised against a synthetic peptide whose sequence was based on the C terminus sequence . The results demonstrate that the C terminus is facing the cell interior because it is available for digestion only from the inside . Previous evidence from an NhaA-beta-galactosidase fusion to loop 5 of NhaA indicated that this loop is also facing the cytoplasm (Karpel, R., Alon, T., Glaser, G., Schuldiner, S., and Padan, E . (1991) J . Biol . Chem . 266, 21753-21759) and therefore was not consistent with the position of the C terminus in an 11-transmembrane segment model . Therefore, the model was re-evaluated . For this purpose, 10 nhaA'-'phoA gene fusions were constructed and assayed for alkaline phosphatase activity . The results support a 12-transmembrane segment model with the N and C termini located in the cytoplasm . The evidence indicates that two very short segments, 14 and 16 amino acids long, must cross the membrane in an unknown conformation. Gene, 1996 Dec 12, 183(1-2), 259 - 63 A single vector cloning, mutagenesis and expression system; Towler EM et al.; We describe a multipurpose Escherichia coli vector, pOTSf1blue, that can be utilized for high efficiency subcloning, epitope-tagged protein overexpression, authentic protein overexpression and efficient mutagenesis. Gene, 1996 Dec 12, 183(1-2), 137 - 42 Inducible retroviral vectors regulated by lac repressor in mammalian cells; Chang BD et al.; We have developed lac repressor-regulated retroviral expression vectors that are induced by beta-galactosides upon stable transduction in mammalian cells . These vectors, derived from a Moloney-virus-based vector LNCX, contain an internal Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter, coupled with 2 to 4 lac operator sequences and placed in anti orientation relative to the retroviral long terminal repeat (LTR) . Three different vectors were tested in stably infected mass populations of mouse and human cells expressing the lac repressor, in parallel with the constitutively expressed LNCX vector . The highest expression levels from these vectors ranged from 1-4% to 25-33% of the LNCX level, and the induction by beta-galactosides ranged from 6-11-fold to 29-54-fold . These vectors should be suitable for studies requiring efficient gene transfer and regulated expression in mass populations of stably transduced mammalian cells. Mutat Res, 1996 Dec 12, 361(2-3), 165 - 72 A plasmid rescue to investigate mutagenesis in transgenic D . melanogaster; Hersberger M et al.; We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo . Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis . The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors . The resulting circular plasmids are then transformed back into E . coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal . The number of inactivated versus intact lacZ genes directly indicates the mutation frequency . By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely . Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4) . Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively. Biochim Biophys Acta, 1996 Dec 11, 1309(3), 194 - 6 Molecular cloning of a cDNA encoding an antigen which is salt-stably attached to centrosomes; Angiolillo A et al.; A monoclonal antibody (MAB 2A8) was used for expression-cloning of a complete cDNA (1133/5) to a mRNA (3 kb) encoding a murine 76 kDa polypeptide . The N-terminal section of the polypeptide is composed of domains capable to form alpha-helical coiled-coils . Its C-terminus is proline-rich and has characteristics of the Src homology region 3 (SH3) . Affinity-purified antibodies to a recombinant section of the protein show that the antigen is salt-stably associated with the centrosome throughout the cell cycle. J Biotechnol, 1996 Dec 10, 52(2), 127 - 33 Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies; Arora D et al.; A strategy is described for improved refolding and purification of recombinant human gamma interferon (rh-IFN gamma), which could warrant a higher yield and specific activity than reported previously . The optimal conditions of refolding are obtained by addition of a labilizing agent, L-arginine, in the refolding buffer . A 10-fold increase in the yield was observed with 0.5 M L-arginine, compared with renaturation in its absence . By varying renaturation parameters, the conditions that allow functional refolding of approximately 25-30% of the recombinant protein have been standardized . A simple process is also described for the purification of rh-IFN gamma . The purification involves a single-column chromatography on S-Sepharose, after refolding of rh-IFN gamma in arginine containing buffer . This procedure has consistently produced rh-IFN gamma having a purity of at least 97%, the rest being the aggregated form of gamma interferon . The purified protein is a dimer under non-denaturing conditions and has a specific activity of 2 x 10(8) IU mg-1 protein, as measured by viral cytopathic assay. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14509 - 14 Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli; Jayakumar A et al.; We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously . In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site . The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion . The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography . As expected from the coding capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein . The maltose-binding protein-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells . Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (> 90%) and minimal amounts of stearic and arachidic acids . Similarly, a human FAS cDNA encoding domain I (beta-ketoacyl synthase, acetyl-CoA and malonyl-CoA transacylases, and beta-hydroxyacyl dehydratase) was cloned and expressed in E . coli using pMAL-c2 . The expressed fusion protein, MBP-hFAS domain I, was purified to apparent homogeneity (M(r) 190,000) and exhibited the activities of the acetyl/malonyl transacylases and the beta-hydroxyacyl dehydratase . In addition, a human FAS cDNA encoding domains II and III (enoyl and beta-ketoacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E . coli as a fusion protein with thioredoxin and six in-frame histidine residues . The recombinant fusion protein, thioredoxin-human FAS domains II and III, that was purified from E . coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and beta-ketoacyl reductases and the thioesterase . Both the MBP and the thioredoxin-His-tags do not appear to interfere with the catalytic activity of human FAS or its partial activities. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14498 - 502 A general method for determining helix packing in membrane proteins in situ: helices I and II are close to helix VII in the lactose permease of Escherichia coli; Wu J et al.; It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation . We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease . After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots . A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII . Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54 . Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI) . The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI . The method should be applicable to a number of different polytopic membrane proteins. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14486 - 91 Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases; Srinivasula SM et al.; The Fas/APO-1-receptor associated cysteine protease Mch5 (MACH/FLICE) is believed to be the enzyme responsible for activating a protease cascade after Fas-receptor ligation, leading to cell death . The Fas-apoptotic pathway is potently inhibited by the cowpox serpin CrmA, suggesting that Mch5 could be the target of this serpin . Bacterial expression of proMch5 generated a mature enzyme composed of two subunits, which are derived from the pre-cursor proenzyme by processing at Asp-227, Asp-233, Asp-391, and Asp-401 . We demonstrate that recombinant Mch5 is able to process/activate all known ICE/Ced-3-like cysteine proteases and is potently inhibited by CrmA . This contrasts with the observation that Mch4, the second FADD-related cysteine protease that is also able to process/activate all known ICE/Ced-3-like cysteine proteases, is poorly inhibited by CrmA . These data suggest that Mch5 is the most upstream protease that receives the activation signal from the Fas-receptor to initiate the apoptotic protease cascade that leads to activation of ICE-like proteases (TX, ICE, and ICE-relIII), Ced-3-like proteases (CPP32, Mch2, Mch3, Mch4, and Mch6), and the ICH-1 protease . On the other hand, Mch4 could be a second upstream protease that is responsible for activation of the same protease cascade in CrmA-insensitive apoptotic pathways. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14468 - 73 Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination; Hegde SP et al.; The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis . Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO . These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins . Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded . Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins . These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein . Finally, we found that interactions of RecF with RecO are lost in the presence of ATP . We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14452 - 5 Dominant negative inhibition by fragments of a monomeric enzyme; Michaels JE et al.; Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired . By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway . Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose . We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase . Inhibition is fragment-specific, as not all fragments cause inhibition . An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation . This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive . Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14391 - 5 Phosphohistidyl active sites in polyphosphate kinase of Escherichia coli; Kumble KD et al.; In the synthesis of inorganic polyphosphate (polyP) from ATP by polyphosphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked phosphoenzyme was previously identified as the intermediate . The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine . Tryptic digests of {32P}PPK contain a predominant 32P-labeled peptide that includes His-441 . Of the 16 histidine residues in PPK of E . coli, 4 are conserved among several bacterial species . Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes. Biochemistry, 1996 Dec 10, 35(49), 15896 - 9 Determination of a transmembrane segment using cysteine-scanning mutants of transposon Tn10-encoded metal-tetracycline/H+ antiporter; Kimura T et al.; The metal-tetracycline/H+ antiporter is a bacterial plasma membrane protein belonging to the 12-transmembrane transporter family . The boundaries of membrane-embedded domains can be clearly determined by means of a method involving cysteine-scanning mutants and their reactivity to N-ethylmaleimide . The results for 37 cysteine-scanning mutants around putative transmembrane helix 9 indicate that the residues from 266 to 275 and from 296 to 298 form water extruding loops, while residues from 276 to 295 and from 299 to 302 are embedded in the membrane . This method is generally useful for determination of the transmembrane regions of a polytopic membrane protein. Biochemistry, 1996 Dec 10, 35(49), 15831 - 8 Metal and pH dependence of heptapeptide catalysis by human matrilysin; Cha J et al.; Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein . Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process . The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C . It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E . coli-expressed promatrilysin . The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme . The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C . The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline . OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex . The zinc atom is then removed from the protein in a time-dependent manner . In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom . The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively . Cadmium, on the other hand, forms an inactive Cd/Zn hybrid . The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin. Biochemistry, 1996 Dec 10, 35(49), 15753 - 9 Refined crystal structure of adenylosuccinate synthetase from Escherichia coli complexed with hydantocidin 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin; Poland BW et al.; A crystal structure of adenylosuccinate synthetase from Escherichia coli, complexed with 5'-phosphate, GDP, HPO4(2-), Mg2+, and hadacidin at 100 K, has been refined to an Rfactor of 0.195 against data to 2.6 A resolution . Bond lengths and angles deviate from expected values by 0.012 A and 1.86 degrees, respectively . Lys 16 and backbone amides 15-17 and 42 interact with the phosphates of GDP, while Ser 414, Asp 333, and backbone amides 331 and 416 interact with the base . Mg2+ is octahedrally coordinated . Oxygen atoms from GDP, phosphate, and hadacidin define the equatorial plane of coordination of the Mg2+, while backbone carbonyl 40 and the side chain of Asp 13 are the apical ligands . HPO4(2-) hydrogen bonds with Lys 16, His 41, backbone amides 13, 40, and 224, and the base moiety of the hydantocidin inhibitor . The carboxylate of hadacidin interacts with Arg 303 and Thr 301; its N-formyl group coordinates to Mg2+, and its hydroxyl group hydrogen bonds with Asp 13 . The 5'-phosphate of the hydantocidin inhibitor interacts with Asn 38, Thr 129, and Thr 239 but is approximately 3.5 A from Arg 143 (related by molecular 2-fold symmetry) . The base moiety of hydantocidin 5'-phosphate hydrogen bonds to Gln 224 and participates in a hydrogen-bonded network that includes the phosphate molecule, several water molecules, and Asp 13 . Hydantocidin 5'-phosphate, GDP, HPO4(2-), and Mg2+ may represent a set of synergistic inhibitors even more effective than the combination of IMP, GDP, NO3-, and Mg2+. Biochemistry, 1996 Dec 10, 35(49), 15646 - 53 Structural and thermodynamic characterization of the interaction of the SH3 domain from Fyn with the proline-rich binding site on the p85 subunit of PI3-kinase; Renzoni DA et al.; The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data . The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described . This indicates that the peptide binds as a poly(L-proline) type II helix . Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure . Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 microM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (delta S0 = -78 J.mol-1.K-1) . Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (delta S0 = +252 J.mol-1.K-1; delta H0 = +44 kJ.mol-1) . From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important. Biochemistry, 1996 Dec 10, 35(49), 15618 - 25 The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence; van Raaij MJ et al.; The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase . In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield . The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria . Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis . By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47 . The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable . At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution . Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase . However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region . The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit . The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase. FEBS Lett, 1996 Dec 9, 399(1-2), 135 - 9 Toxicity of expanded polyglutamine-domain proteins in Escherichia coli; Onodera O et al.; Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains . Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available . In this paper, we demonstrate in E . coli that long polyglutamine domains (59-81 residues) as GST-fusion proteins inhibit growth while smaller glutamine (10-35 residues) or polyalanine (61 residues) domains have no effect . Analogously in humans, polyglutamine repeats less than 35-40 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease . Expression of polyglutamine proteins in E . coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compound. FEBS Lett, 1996 Dec 9, 399(1-2), 99 - 102 Escherichia coli inorganic pyrophosphatase: site-directed mutagenesis of the metal binding sites; Avaeva S et al.; Aspartic acids 65, 67, 70, 97 and 102 in the inorganic pyrophosphatase of Escherichia coli, identified as evolutionarily conserved residues of the active site, have been replaced by asparagine . Each mutation was found to decrease the k(app) value by approx . 2-3 orders of magnitude . At the same time, the Km values changed only slightly . Only minor changes take place in the pK values of the residues essential for both substrate binding and catalysis . All mutant variants have practically the same affinity to Mg2+ as the wild-type pyrophosphatase. FEBS Lett, 1996 Dec 9, 399(1-2), 26 - 8 Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli; Akiyama Y et al.; Escherichia coli FtsH is a membrane-bound ATPase with a proteolytic activity against the SecY subunit of protein translocase . We now report that subunit a of the membrane-embedded Fo part of H+-ATPase is another substrate of FtsH . Pulse-chase experiments showed that subunit a is unstable when it alone (without Fo subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants . Selective and ATP-dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro . These results suggest that FtsH serves as a quality-control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane. FEBS Lett, 1996 Dec 9, 399(1-2), 21 - 5 The reaction of Escherichia coli cytochrome bo with H2O2: evidence for the formation of an oxyferryl species by two distinct routes; Brittain T et al.; We have re-examined the reaction of fast oxidised cytochrome bo with H202 in a stopped-flow spectrophotometer . Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C . and Thomson, A.J . (1994) Biochem . J . 300, 469-475 {1}) . The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence . Initially these results appeared incompatible with the report that only one equivalent of H202 is required to generate the oxyferryl species (Moody, A.J . and Rich, P.R . (1994) Eur . J . Biochem . 226, 731-737 {21} . However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used. Biochim Biophys Acta, 1996 Dec 6, 1291(3), 189 - 94 Natural and synthetic betaines counter the effects of high NaCl and urea concentrations; Randall K et al.; Escherichia coli was used as a model system to evaluate a range of betaines for their ability to protect against salt and urea stresses . Betaine structure determined the salt and urea protective effects . Dimethylthetin conferred salt protection similar to glycine betaine, whereas dimethylsulfoniopropionate (DMSP) was less effective than either glycine betaine or dimethylthetin, but similar to propionobetaine (its nitrogen analogue) . Hydrophobic alpha-substituents altered salt tolerance . Valine betaine with an aliphatic side group conferred salt tolerance similar to glycine betaine . Betaines containing phenyl groups (phenylglycine, phenylalanine and N-phenylglycine betaines) did not confer salt protection, growth being similar to, or less than the control (no betaine) . Hydrophobic groups decreased the ability to protect against urea stresses; valine betaine conferred poor urea tolerance . The addition of an hydroxyl group increased the ability of a betaine to protect against urea denaturation . Proline betaine, an effective salt protector, conferred poor urea tolerance . Increasing the charge separation in the betaine molecule decreased the ability to confer urea tolerance . Thiolanium, pyridinium and triethylglycine betaines, with larger cationic functions, conferred no urea tolerance to E . coli. Neurosci Lett, 1996 Dec 6, 220(1), 66 - 8 Neural toxicity of retroviruses; Hurley MJ et al.; Recombinant retroviruses containing the cDNA for human tyrosine hydroxylase-1 and Escherichia coli lacZ gene were used to infect primary foetal ventral mesencephalon and cortical cultures from rat brain . Severe neuronal toxicity resulted 3-4 days after infection, glial cells seemed to be much more resistant . The toxicity was likely to have resulted from an agent present within the virus-containing medium itself, rather than from the retrovirus itself . The results of this study indicate that retroviruses are not suitable vectors for the introduction of tyrosine hydroxylase into primary neuronal cultures. J Mol Biol, 1996 Dec 6, 264(3), 472 - 83 The donor substrate site within the peptidyl transferase loop of 23 S rRNA and its putative interactions with the CCA-end of N-blocked aminoacyl-tRNA(Phe); Porse BT et al.; An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study . Growth phenotypes of Escherichia coli cells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the "fragment" assay . Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa . All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site . A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA(Phe) and this region of the peptidyl-transferase loop . Single nucleotide substitutions were introduced into the -CCA end of tRNA(Phe) and the ability of the 3'-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs . No evidence was found for the occurrence of Watson-Crick base-pairing interactions . However, the data are consistent with the formation of a Hoogsteen pair between the 3'-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA. J Mol Biol, 1996 Dec 6, 264(3), 412 - 25 A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes; Winzeler E et al.; Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle . The onset of replication coincides with the stimulation of transcription of several genes involved in the replication process . Analysis of the C . crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication . We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication . Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site . We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle. J Mol Biol, 1996 Dec 6, 264(3), 407 - 11 Functional effects of mutating the closing GxA base-pair of a conserved hairpin loop in 23 S ribosomal RNA; Xu W et al.; Recently, Draper and co-workers solved the structure of a hexanucleotide hairpin loop that is conserved in large subunit ribosomal RNAs . (In Escherichia coli, the hexanucleotide consists of nucleotides 1093 to 1098, in the GTPase center of 23 S rRNA.) A major feature of that structure is a G1093xA1098 base-pair that closes the loop . Our laboratory reported previously the isolation of the mutation G1093A and its characterization as a suppressor of UGA mutations and a cause of temperature-conditional lethality . For the work reported here, we asked whether G1093A causes its phenotypes precisely because it is part of the G1093/A1098 base-pair . Using oligonucleotide-directed site-specific mutagenesis, we introduced base substitutions at nucleotides 1093 and 1098 into a plasmid-borne ribosomal RNA operon (rrnB) . Each mutant plasmid was then tested for the two mutant phenotypes, nonsense suppression and temperature-dependent growth inhibition . Our results indicate that mutations at 1093 do not cause the mutant phenotypes because G1093 is part of the G1093xA1098 base-pair . We discuss alternative avenues to the observed mutant phenotypes and, in particular, present a model in which a specific interaction of the loop is involved in peptide chain termination. J Biol Chem, 1996 Dec 6, 271(49), 31749 - 55 Expression in Escherichia coli and refolding of the malonyl-/acetyltransferase domain of the multifunctional animal fatty acid synthase; Rangan VS et al.; A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase . Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein . Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity . The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA . Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate . The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 --> Ala mutant . These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity. J Biol Chem, 1996 Dec 6, 271(49), 31580 - 4 Separable ATPase and membrane insertion domains of the SecA subunit of preprotein translocase; Price A et al.; The SecA subunit of preprotein translocase drives ATP-dependent translocation of preproteins across the bacterial inner membrane concomitant with cycles of membrane insertion and de-insertion (Economou, A., and Wickner, W . (1994) Cell 78, 835-843) . We have identified the membrane-inserting region of SecA as a 30-kDa domain in the C-terminal third of the protein beginning at aminoacyl residue 610 . Limited proteolysis in the absence of translocation ligands indicates that the SecA monomer is composed of two primary structural domains, the 30-kDa membrane-inserting domain and an N-terminal 65-kDa ATPase domain . This limited protease treatment of SecA results in constitutive ATPase activity, indicating that intramolecular constraints between the two domains may play a role in the regulation of ATP hydrolysis by SecA. J Biol Chem, 1996 Dec 6, 271(49), 31549 - 55 Contrasting enzymatic activities of topoisomerase IV and DNA gyrase from Escherichia coli; Ullsperger C et al.; DNA gyrase and topoisomerase IV (Topo IV) have distinct roles as unlinking enzymes during DNA replication despite 40% sequence identity between them . DNA gyrase unlinks replicating DNA by introducing negative supercoils while Topo IV decatenates the two daughter molecules . For this study, we measured the rates of unlinking of various topoisomers of DNA by DNA gyrase and Topo IV . Each enzyme has marked preferences for certain strand-passage reactions . DNA gyrase is a relatively poor decatenase, catalyzing strand-passage events that result in supercoiling at rates several orders of magnitude faster than those causing decatenation . Topo IV, in contrast, decatenates linked circles 10-40 times more quickly than it removes the intramolecular crossings from supercoiled DNA . Supercoiled catenanes are unlinked at an even more increased rate by Topo IV . Thus, the supercoils augment decatenation rather than compete with catenane crossings for their removal . Knot crossings and the crossings of multiply interlinked catenanes are also preferentially removed by Topo IV . This ability of Topo IV to selectively unlink catenated molecules mirrors its key role in decatenation of replicating chromosomes in vivo. J Biol Chem, 1996 Dec 6, 271(49), 31227 - 33 Transit peptides play a major role in the preferential import of proteins into leucoplasts and chloroplasts; Wan J et al.; The in vitro import characteristics of six different precursors of plastid proteins were assessed to determine differences in the protein import pathways of leucoplasts and chloroplasts . Five of these precursor proteins are destined to different subchloroplast sites, and one is a leucoplast stromal precursor protein . The results indicate that some of these precursors can be imported equally into both plastid types and others preferentially into one type of plastid versus the other . The ability of plastids to import different proteins correlates with the in vivo steady state levels of these proteins . Additional differences were also observed in the intraorganellar portion of the translocation pathway for two thylakoidal proteins . The differences in import characteristics were found to be predominantly governed by information in the transit peptides, since attachment of the various transit peptides to different plastid and foreign proteins demonstrated that the import behavior of the proteins is transferable with the transit sequence . These results indicate that the import mechanisms of leucoplasts and chloroplasts are sufficiently different such that the plastids respond differently to the information present in the transit peptides. J Biol Chem, 1996 Dec 6, 271(49), 31196 - 201 FtsH (HflB) is an ATP-dependent protease selectively acting on SecY and some other membrane proteins; Akiyama Y et al.; The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins . SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein) . We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH . We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini . Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH . Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro . Mutant proteins without ATPase activity were inactive in proteolysis . A zinc binding motif mutant showed a decreased proteolytic activity . SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation . These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity . FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation. J Biol Chem, 1996 Dec 6, 271(49), 31172 - 8 Heterotetramers of human liver mitochondrial (class 2) aldehyde dehydrogenase expressed in Escherichia coli . A model to study the heterotetramers expected to be found in Oriental people; Wang X et al.; About 50% of the Oriental population have less liver mitochondrial aldehyde dehydrogenase (ALDH2) activity than do other people . It was found that they possessed an enzyme with a lysine at position 487 (E487K) instead of glutamate (Glu487) . We previously found that the Km for NAD of recombinant human and rat E487K enzymes increased more than 150-fold (Farres, J., Wang X., Takahashi, K., Cunningham, S . J . , Wang, T.T., and Weiner, H (1994) J . Biol . Chem . 269, 13854-13860) . Many aldehyde dehydrogenase-deficient people were found to be heterozygous when genotyped for ALDH2 . In this study liver tissue from heterozygous people was analyzed and found to possess mRNAs for both the glutamate and the lysine subunits . Western blot analysis showed that the glutamate subunit was present . The cDNAs for Glu487 and E487K were coexpressed on one plasmid in Escherichia coli, and the enzyme forms were separated from each other by isoelectric focusing to show that heterotetramers were formed . Only one Km value for NAD could be measured with the purified heterotetrameric enzyme that possessed just 16-18% activity of the glutamate homotetrameric enzyme . The E487K homotetramers had 8% specific activity of the Glu487 enzyme . There was no pre-steady state burst of NADH formation with the heterotetramer, a property found with the glutamate enzyme . Similar results were found for the coexpressed rat liver enzyme, except that a higher specific activity, 48%, was obtained . Thus, we conclude that presence of the lysine subunit altered the activity of the glutamate subunit in the heterotetramer to make it function more like an E487K enzyme. J Biol Chem, 1996 Dec 6, 271(49), 31160 - 5 Binding of an N-terminal rhodanese peptide to DnaJ and to ribosomes; Kudlicki W et al.; A peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese was fluorescently labeled with a coumarin derivative at its primary amino group(s) and then purified by high performance liquid chromatography . This peptide interacted with the molecular chaperone DnaJ in the absence of other chaperones and ATP . In the presence of ATP, the molecular chaperone DnaK bound to the DnaJ-peptide complex, but not to the peptide alone . The chaperone GrpE appeared to cause the release of the peptide bound to the ternary complex in the presence of ATP but not in the presence of ADP . This nucleotide apparently stabilized the complex . The peptide also bound to salt-washed Escherichia coli 70 S ribosomes, specifically to 50 S ribosomal subunits, not to 30 S subunits . DnaJ plus DnaK interacted with the peptide on the ribosome . GrpE caused dissociation of the peptide from the ribosome; ATP was required for this reaction . It was inhibited by ADP . A comparable series of chaperone-mediated reactions is assumed to occur with the N-terminal segment of the nascent polypeptide to facilitate its folding on ribosomes. J Biol Chem, 1996 Dec 6, 271(49), 31044 - 8 Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli; Yerushalmi H et al.; EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents . EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen . Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state . For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing . As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants . Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants . To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive . When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition . We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation . A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits . The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter. J Biol Chem, 1996 Dec 6, 271(49), 31033 - 6 Mutational analysis of the functional domains of the large subunit of the isozyme form of wheat initiation factor eIF4F; Metz AM et al.; The isozyme form of plant eukaryotic initiation factor 4F (eIF(iso)4F) contains two subunits: p28, a cap-binding protein, and p86 . To identify the functional domains of p86, truncations of the p86 cDNA were made, and the protein was expressed in Escherichia coli and purified . The deletion mutants were tested for the ability to bind the p28 subunit by two methods . In addition, these deletion mutants were evaluated in vitro by the ability to catalyze eIF4A and RNA-dependent ATP hydrolysis and to support polypeptide synthesis . The loss of the ability to bind p28 occurs within the first 90 amino acids of the N terminus and abrogates the ability of p86 to participate in translation initiation and bind to eIF4A, but does not affect ATP hydrolysis . Up to 299 amino acid residues from the C terminus of p86 must be deleted before an effect is observed on the ATP hydrolysis activity . Thus, the p28 binding and ATP hydrolysis activities appear to lie on two separate domains and are functionally uncoupled . In addition, at least a portion of the eIF4A binding domain appears to be in close proximity to the p28 binding domain and is also uncoupled from the ATP hydrolysis activity. Eur J Pharmacol, 1996 Dec 5, 316(2-3), 277 - 86 Antioedematogenic and antinociceptive actions of NPC 18521, a novel bradykinin B2 receptor antagonist; De Campos RO et al.; The novel pseudopeptide bradykinin B2 receptor antagonist containing the 1,3,8-triazaspiro{4,5}decan-4-one ring system, NPC 18521 (D-Arg-Arg-{1,3-phenyl,8-triazaspiro{4,5}-decane-4-one-3-acetyl}-S er-D -tetrahydroisoquinolinyl-octahydroindolinyl-Arg) (10 and 30 nmol/kg, i.p.), given 30 min prior, produced significant and long-lasting inhibition of rat paw oedema induced by bradykinin (3 nmol/paw) and carrageenan (300 micrograms/paw), without affecting the oedema induced by the selective bradykinin B1 receptor agonist, des-Arg9-bradykinin, in rats pretreated with Escherichia coli endotoxin . In contrast, when injected locally into the rat or mouse hindpaw, NPC 18521 (1-100 nmol) elicited dose-related oedema formation . This effect was almost completely blocked by cyproheptadine (20 mg/kg, i.p.) or by compound 48/80 (12 micrograms/paw), but was unaffected by Hoe 140 (D-Arg-{Hyp5,Thi5,Tic7,Oic8}bradykinin) . NPC 18521 (0.3-10 nmol/kg, i.p.) produced significant inhibition of acetic acid, acetylcholine and kaolin- but not zymosan-induced abdominal constrictions in mice . The calculated mean ID50 values for these effects were 0.84, 0.46 and 0.55 nmol/kg, respectively . The antinociceptive action of NPC 18521 (3 nmol/kg, i.p.) had a rapid onset (15 min) and lasted for up to 120 min . Given topically (0.01-0.3 nmol), NPC 18521 produced significant attenuation of both the early and the late phase of the formalin-induced licking, as well as formalin-induced oedema formation . In addition, NPC 18521 given both systemically or topically, produced significant inhibition of the neurogenic nociception caused by topical injection of capsaicin . Given topically in the rat paw, NPC 18521 (10 nmol) caused marked hyperalgesia, an effect which was completely prevented by cyproheptadine (20 mg/kg, i.p.), but was unaffected by Hoe 140 (3 nmol/kg, i.p.) . Given intraperitoneally, 30 min prior, NPC 18521 (3-30 nmol/kg) like Hoe 140 (1-10 nmol/kg) prevented, in a dose-dependent manner, bradykinin (3 nmol/paw)-induced hyperalgesia with mean ID50 values of 13.16 and 1.36 nmol/kg, respectively . Thus, the novel pseudopeptide bradykinin B2 receptor antagonist, NPC 18521, has an effect with rapid onset, and produces potent and relatively long-lasting antioedematogenic and antinociceptive properties . However, in contrast to Hoe 140, given locally into the hindpaw, NPC 18521 elicited marked oedema formation and hyperalgesia, an effect which seems to be secondary to mast cell degranulation and histamine and/or serotonin release . Finally, the anti-bradykinin actions of NPC 18521 are quite selective towards the bradykinin B2 receptor-mediated responses. Eur J Pharmacol, 1996 Dec 5, 316(2-3), 257 - 62 Involvement of K+ channel modulation in the proabsorptive effect of nitric oxide in the rat jejunum in vivo; Schirgi-Degen A et al.; The role of K+ channels in the mediation of the nitric oxide(NO)-induced proabsorptive effect in intestinal fluid transport was investigated in a functional study, using a model of ligated jejunal loops of anaesthetized rats in vivo . The K+ channel opener cromakalim and the K+ channel blocker glibenclamide were administered under basal conditions as well as under conditions, when fluid secretion was influenced by N omega-nitro-L-arginine methyl ester (L-NAME), prostaglandin E2, Escherichia coli heat stable enterotoxin a (E . coli STa) or L-arginine . Intravenous infusion of cromakalim (63.5 micrograms/kg per min) significantly enhanced net fluid absorption compared to controls, totally abolished net fluid secretion induced by L-NAME (0.55 mg/kg per min), reversed net fluid secretion induced by intraluminal instillation of E . coli STa (10 units/ml) to absorption, but did not influence fluid secretion elicited by close i.a . infusion of prostaglandin E2 (79 ng/min) . Close i.a . infusion of glibenclamide (0.16 mg/kg per min) reversed net fluid absorption to net secretion, blocked the absorptive effect of L-arginine (8.88 mg/kg per min) and reduced the proabsorptive effect of cromakalim . The secretory effect of L-NAME was not further enhanced by glibenclamide . These results suggest that modulation of basolateral K+ channels by NO is involved in the mediation of its proabsorptive effect, since opening and closure of K+ channels mimicked, respectively counteracted, the action of NO-donors and inhibitors of NO-synthesis on intestinal fluid transport . The role of prostaglandins in the proabsorptive effect of NO remains to be elucidated . These results furthermore support the role of K+ channel openers as potential new antidiarrheal drugs. Gene, 1996 Dec 5, 182(1-2), 229 - 30 A novel plasmid vector that uses the glucose kinase gene (glkA) for the positive selection of stable gene disruptants in Streptomyces; van Wezel GP et al.; We describe an Escherichia coli plasmid, pIJ2581, that can be used for the efficient construction of stable gene disruptants and of gene deletions in Streptomyces . Integration of pIJ2581 derivatives carrying chromosomal sequences is achieved by selecting for plasmid-encoded thiostrepton resistance, while plasmid excision is secured by counter-selection of the pIJ2581 glkA gene, which confers sensitivity to 2-deoxyglucose. Gene, 1996 Dec 5, 182(1-2), 203 - 11 Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A; Ide Y et al.; Hepatitis C virus (HCV) has a positive strand RNA genome that codes for a polyprotein that is processed co-translationally and post-translationally into three structural and at least seven nonstructural (NS) proteins . To investigate the function of NS5A, a recombinant vaccinia virus was constructed in which the NS5A gene was cloned under the control of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells . In addition, the NS5A gene was also cloned under the control of cytomegalovirus (CMV) early promoter . The NS5A expressed in monkey kidney (CV-1) cells was located predominantly in the cytoplasm . Using immunohistochemical analysis, the subcellular distribution of NS5A in liver biopsy samples from chronic HCV-infected patients was also found to be in the cytoplasm . However, the NS5A protein has a stretch of positively charged domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-terminal half of the protein . To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in which regions containing the NLS were fused to the N-terminus of the E . coli beta-galactosidase (E . coli beta-Gal) . The expression of the fusion proteins in CV-1 cells resulted in their nuclear localization, indicating that the putative NLS is functional in targeting the heterologous protein, E . coli beta-Gal, to the nucleus, although the native NS5A is retained in the cytoplasm. Gene, 1996 Dec 5, 182(1-2), 81 - 7 Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking; Mythili E et al.; beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli . To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity . A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution . A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis . Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues . Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA . Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl . The Kd of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M . Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain . Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end . These findings provide a basis for further understanding of the structure and function of beta protein. Gene, 1996 Dec 5, 182(1-2), 53 - 62 Cloning and expression pattern of Hor v 9, the group 9 pollen isoallergen from barley; Astwood JD et al.; In this study we report the cloning, sequence, and characterization of Hor v 9 allergen cDNAs from barley (Hordeum vulgare) pollen . Structural homologues of Kentucky bluegrass (Poa pratensis) group 9 pollen allergens were identified in a cDNA library of barley pollen expressed mRNAs . The Hor v 9 cDNA clone (hvp9742) contained an open reading frame encoding 313 amino acids which included a putative 27-residue signal peptide and one asparagine sequon for glycosylation . The mRNA corresponding to clone hvp9742 was produced abundantly in pollen during the late stages of anther development . The protein encoded by clone hvp974 was synthesized as a fusion protein in the E . coli expression vector pMAL . Immunoblots using antibodies to this recombinant allergen, rHor v 9, showed that Hor v 9 protein accumulated during pollen development and was produced maximally at pollen maturity . Using these antibodies, we also provide evidence that Hor v 9 protein localized to the extracellular matrix of mature pollen . Southern blots suggested that Hor v 9 allergens exist as multiple isoforms in barley . Sequence comparisons showed that the Hor v 9 cDNA clones were also homologous to group 5 allergens of Timothy grass (Phleum pratense) pollen and canary grass (Phalaris aquatica) pollen, and the group 9 allergen of ryegrass (Lolium perenne) pollen. Gene, 1996 Dec 5, 182(1-2), 45 - 52 Molecular cloning of a cDNA encoding 3-ketoacyl-acyl carrier protein synthase III from leek; Chen J et al.; 3-Ketoacyl-acyl carrier protein synthase III (KAS III) catalyzes the initial condensation of malonyl-acyl carrier protein (ACP) with acetyl-CoA in plant and bacterial fatty acid biosynthesis . The first cDNA clone encoding KAS III from a monocot is reported here . A cDNA clone was isolated from a leek epidermal cDNA library by screening with spinach and Arabidopsis heterologous probes from KAS III cDNA clones . When expressed in Escherichia coli, the cloned enzyme was able to catalyze the expected condensation reaction, was insensitive to cerulenin (100 microM) and cross-reacted with spinach KAS III antibody . The 1476-bp cDNA clone contained a 1206-bp open reading frame which encoded a 402-amino acid polypeptide . The deduced amino acid sequence showed significant similarities to other KAS IIIs although the leek sequence had some notable differences in regions otherwise completely conserved in dicots . Northern blot analyses indicated that KAS III transcript levels were similar in leaf epidermis and parenchyma, although developmental changes in transcript levels differed between these two tissues . In addition, leek KAS III was expressed in a manner comparable to leek oleoyl-ACP thioesterase (OTE), another enzyme of fatty acid biosynthesis, in both tissues. Biochim Biophys Acta, 1996 Dec 5, 1298(2), 294 - 304 Mutational analysis of human uroporphyrinogen decarboxylase; Wyckoff EE et al.; Uroporphyrinogen decarboxylase (URO-D), a heme biosynthetic enzyme, catalyzes the multi-step decarboxylation reaction converting uroporphyrinogen I or III to coproporphyrinogen I or III . The URO-D protein has been purified from several sources and its gene has been cloned from many organisms . In spite of this, little is known about the active site(s) of the enzyme . Inhibitor studies suggest that cysteine and histidine residues are important for enzyme activity . We employed the Kunkel method of site-directed mutagenesis to convert each of the six cysteines in human URO-D to serine and each of the three conserved histidines to asparagine . Recombinant mutant URO-D's were expressed in Escherichia coli, partially purified, and their kinetic properties compared to recombinant wild-type URO-D . All cysteine mutants retained approx . 40% wild-type enzyme activity, indicating that no single cysteine is absolutely critical for the integrity of the catalytic site . The three histidine mutants also retained significant enzyme activity and one, (H339N), displayed unique properties . The H339N mutation resulted in an enzyme with high residual activity but decarboxylation of intermediate reaction products of the I isomer series was markedly abnormal . The histidine at residue 339 is likely important in imparting isomer specificity. Biochim Biophys Acta, 1996 Dec 5, 1298(2), 191 - 8 GroEL reversibly binds to, and causes rapid inactivation of, human carbonic anhydrase II at high temperatures; Persson M et al.; The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35 degrees C . At temperatures above 35 degrees C, the enzymatic activity is not stable, but decreases over time . If the bacterial chaperonin GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50 degrees C . However, unlike the spontaneous reactivation, the enzymatic activity with time in the presence of GroEL . In the presence of GroEL, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present . It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme . Furthermore, proline isomerization is part of the rate-limiting step of refolding even in the presence of GroEL, and it is very noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL. Biochim Biophys Acta, 1996 Dec 5, 1298(2), 141 - 7 Sequence analysis of an operon of a NAD(P)-reducing nickel hydrogenase from the cyanobacterium Synechocystis sp . PCC 6803 gives additional evidence for direct coupling of the enzyme to NAD(P)H-dehydrogenase (complex I); Appel J et al.; The sequence of a NAD(P)-reducing hydrogenase operon of Synechocystis sp . PCC 6803 containing genes for a small and a large hydrogenase subunit and six additional ORFs was determined . Until now only 11 of the 14 polypeptides of the NADH-dehydrogenase of E . coli were found in Synechocystis . By sequence homologies we suggest that the missing subunits of the peripheral part of the dehydrogenase, containing most of the FeS-clusters, are encoded by three ORFs of this operon . This hypothesis is discussed in relation to the NAD(P)-reducing hydrogenase of Synechocystis. Nature, 1996 Dec 5, 384(6608), 481 - 4 A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains; Chardin P et al.; The small G protein ARF1 is involved in the coating of vesicles that bud from the Golgi compartments . Its activation is controlled by as-yet unidentified guanine-nucleotide exchange factors . Gea1, the first ARF exchange factor to be discovered in yeast, is a large protein containing a domain of homology with Sec7, another yeast protein that is also involved in secretion . Here we characterized a smaller human protein (relative molecular mass 47K) named ARNO, which contains a central Sec7 domain that promotes guanine-nucleotide exchange on ARF1 . ARNO also contains an amino-terminal coiled-coil motif and a carboxy-terminal pleckstrin-homology (PH) domain . The PH domain mediates an enhancement of ARNO exchange activity by negatively charged phospholipid vesicles supplemented with phosphatidylinositol bisphosphate . The exchange activity of ARNO is not inhibited by brefeldin A, an agent known to block vesicular transport and inhibit the exchange activity on ARF1 in cell extracts . This suggests that a regulatory component which is sensitive to brefeldin A associates with ARNO in vivo, possibly through the amino-terminal coiled-coil . We propose that other proteins with a Sec7 domain regulate different members of the ARF family. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 334 - 40 DnaK3, one of the three DnaK proteins of cyanobacterium Synechococcus sp . PCC7942, is quantitatively detected in the thylakoid membrane; Nimura K et al.; Subcellular localization of three DnaK proteins of cyanobacterium Synechococcus sp . PCC7942 was determined . DnaK1 and DnaK2 proteins were detected mainly in the cytosolic fraction . On the other hand, the DnaK3 protein occurred in large amounts in the thylakoid membrane fraction . Furthermore, DnaK3 was found to be located on the surface of the thylakoid membrane on the cytosol side . Subcellular localization of chimeric and truncated DnaK3 proteins was also determined, and it was suggested that the region a.a . 381 to a.a . 597 of DnaK3 protein, which is considered to correspond to the peptide-binding domain, was required for the association with the thylakoid membrane. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 271 - 4 The ATPase activity of chaperonin GroEL is highly stimulated at elevated temperatures; Mendoza JA et al.; The chaperonin GroEL is a heat-shock protein that stabilizes folding intermediates by forming binary complexes . The release of bound polypeptides as active proteins requires ATP hydrolysis by GroEL . The ability of GroEL to support the folding of urea-unfolded rhodanese and to hydrolyze ATP was investigated at high temperatures . We found that the chaperonin-mediated folding of rhodanese and the ATPase activity of GroEL are temperature dependent . The GroEL ATPase activity, however, increases very strongly over the range of temperatures that is physiologically relevant for Escherichia coli growth . Further, GroES partially suppresses the GroEL ATPase activity in the same temperature range. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 219 - 24 Disruption of the hslU gene, which encodes an ATPase subunit of the eukaryotic 26S proteasome homolog in Escherichia coli, suppresses the temperature-sensitive dnaA46 mutation; Katayama T et al.; The Escherichia coli hslVU genes encode subunits of an ATP-dependent protease, a homolog of the eukaryotic 26S proteasome . We found that the hslU gene is required for the temperature (40 degrees C)-sensitive phenotype of the dnaA46 mutant, while other soluble ATP-dependent proteases, La (Lon) and Ti (Clp), were unrelated to this dnaA46 phenotype . Disruption of the hslU gene inhibited cell growth at high temperatures . These observations suggest a specific in vivo role for HslVU protease in denatured proteins . As the absence of HslU produces minicells in M9 medium, the protease may be involved in the cell cycle regulation. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 189 - 92 nrdD and nrdG genes are essential for strict anaerobic growth of Escherichia coli; Garriga X et al.; In Escherichia coli ribonucleotide reduction is catalyzed by two separate enzymes during aerobic and anaerobic growth . The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG . We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis, achieved by inclusion of sodium sulfide in the medium . Interestingly, these mutants grow well under microaerophilic conditions by overproducing the aerobic enzyme . Under such conditions wild-type bacteria turn off nrdAB and switch on nrdDG. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 121 - 7 Expression of a synthetic gene encoding the sweet-tasting protein thaumatin in Escherichia coli; Faus I et al.; A synthetic gene encoding the aminoacid sequence of the sweet-tasting protein thaumatin II has been assembled and expressed in Escherichia coli . Immunoblotting analysis shows that the expressed recombinant thaumatin has the same molecular weight as the protein from its natural source, the plant Thaumatoccocus daniellii Benth. Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 90 - 5 The carboxyl-terminal region is essential for Sec-A dimerization; Hirano M et al.; SecA, comprising 901 amino acid residues, exists as a dimer . By means of size exclusion chromatography and chemical cross-linking analysis, five truncated SecA derivatives were examined to identify the region of SecA essential for dimer formation . Among them, only N95 (delta 832-901) retained SecA activity . N95 existed as a dimer, indicating that the carboxyl-terminal three cysteine residues are dispensable for physiological dimerization . Both N76 (delta 675-901) and N66 (delta 583-901) existed as monomers . Monomeric N76 was able to bind to ATP, indicating that the dimerization of SecA is not a prerequisite for ATP binding . However, the rate of ATP hydrolysis by N76 was 25% of that by SecA . C53 (delta 1-437) and C28 (delta 1-661) formed dimers irrespective of the presence or absence of 2-mercaptoethanol . C28, but not C53, also existed as an oligomer in the absence of 2-mercaptoethanol, suggesting that the 438-661 region present in C53 prevents intermolecular disulfide bond formation at the carboxyl-terminal cysteine residue . From these results, the region essential for the physiological dimer formation was concluded to be located in the 662-831 region of SecA. Biochemistry, 1996 Dec 3, 35(48), 15485 - 93 Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp . PCC 7906 Rieske protein; Holton B et al.; The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes . We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp . PCC 7906 . A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein . Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies . The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions . Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein . Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K . Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation . The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy . These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis. Biochemistry, 1996 Dec 3, 35(48), 15391 - 6 Identification of a Tus protein segment that photo-cross-links with TerB DNA and elucidation of the role of certain thymine methyl groups in the Tus-TerB complex using halogenated uracil analogues; Duggan LJ et al.; Six potential hydrophobic sites of the Tus-TerB complex were analyzed using bromodeoxy-uridine and iododeoxyuridine as isosteric analogues of thymine . Analogues were incorporated at individual sites, and dissociation rates were measured in 150 mM potassium glutamate, pH 8.0, using a nitrocellulose filter assay . These halogenated analogues serve as a probe of the environment in which they reside . Our measurement revealed at least two types of responses . Three sites showed increases in stability with the introduction of the bromo and iodo derivatives . The enhanced stability is proposed to result from polar or charged molecules in the vicinity of the halogenated analogues through dipole-dipole, dipole-ion, or dipole-induced dipole interactions . The other three sites exhibited the opposite trend, being destabilized by the introduction of these analogues . The destabilizing effects are attributed to a hydrophobic environment which cannot accommodate polar molecules . The photoreactivity of these analogues was utilized to specifically cross-link the Tus protein and TerB DNA . Using the substitution of bromodeoxyuridine at position 8 in the TerB DNA, Tus protein was covalently attached to the DNA, and by trypsin digestion a fragment of Tus was isolated . Sequencing of the peptide fragment revealed a segment that matched the amino acid composition from 122-139 of the Tus protein. Biochemistry, 1996 Dec 3, 35(48), 15356 - 63 Functional role of the active site glutamate-368 in rat short chain acyl-CoA dehydrogenase; Battaile KP et al.; The acyl-CoA dehydrogenases are a family of flavoenzymes with similar structure and function involved in the metabolism of fatty acids and branched chain amino acids . The degree of overlap in substrate specificity is narrow among these enzymes . The position of the catalytic glutamate, identified as Glu376 in porcine medium chain acyl-CoA dehydrogenase (MCAD), Glu254 in human isovaleryl-CoA dehydrogenase (IVD), and Glu261 in human long chain acyl-CoA dehydrogenase (LCAD), has been suggested to affect substrate chain length specificity . In this study, in vitro site-directed mutagenesis was used to investigate the effect of changing the position of the catalytic carboxylate on substrate specificity in short chain acyl-CoA dehydrogenase (SCAD) . Glu368, the hypothetical active site catalytic residue of rat SCAD, was replaced with Asp, Gly, Gln, Arg, and Lys and the wild type and mutant SCADs were produced in Escherichia coli and purified . The recombinant wild type SCAD kcat/K(m) values for butyryl-hexanoyl-, and octanoyl-CoA were 220, 22, and 3.2 microM-1 min-1, respectively, while the Glu368Asp mutant gave kcat/K(m) of 81, 12, and 1.4 microM-1 min-1, respectively, for the same substrates . None of the other mutants exhibited enzyme activity . A Glu368Gly/Gly247Glu double mutant enzyme, which places the catalytic residue at a position homologous to that of LCAD, was also synthesized and purified . It showed kcat/K(m) of 9.3, 2.8, and 1.5 microM-1 min-1 with butyryl-, hexanoyl-, and octanoyl-CoA used as substrates, respectively . These results confirm the identity of Glu368 as the catalytic residue of rat SCAD and suggest that alteration of the position of the catalytic carboxylate can modify substrate specificity. Biochemistry, 1996 Dec 3, 35(48), 15340 - 8 Inhibition of tRNA aminoacylation by 2'-O-methyl oligonucleotides; Hou YM et al.; A 2'-O-methyl oligonucleotide complementary to 18 nucleotides in the dihydrouridine stemloop of Escherichia coli tRNA(Cys) has been shown to stably bind to the tRNA . The binding inhibits aminoacylation of the tRNA by cysteine tRNA synthetase . The same oligonucleotide sequence but with the DNA deoxy backbone does not bind to the tRNA . This provides the basis for the design and test of a series of 2'-O-methyl oligonucleotides for their ability to bind to E . coli tRNA(Cys) and inhibit aminoacylation . We show here that different regions of the tRNA have different sensitivities to oligonucleotides . A 10-mer that targets G15 forms a stable complex with the tRNA . The Kd of the complex is several orders of magnitude lower than that of the tRNA-synthetase complex . Measurements of dissociation rate constants indicate that the stronger affinity of the 10-mer to tRNA(Cys) is due to a significantly slower rate of dissociation (by a factor of 10(6)) than that of the synthetase from the tRNA . Only a stoichiometric amount of the 10-mer is necessary to completely inhibit aminoacylation . Because tRNA aminoacylation is fundamental to cell growth, these results provide the rationale for the 10-mer and its derivatives as pharmaceutical agents that target specific cell growth. Biochemistry, 1996 Dec 3, 35(48), 15302 - 12 Influence of the location of the cAMP receptor protein binding site on the geometry of a transcriptional activation complex in Escherichia coli; Eichenberger P et al.; The interactions between the cAMP receptor protein (CRP) and RNA polymerase during transcriptional activation at the Escherichia coli malT promoter have been analyzed using a combination of footprinting methods . We show that a closed complex is formed at this promoter in the absence of activator and that CRP merely stabilizes the open complex . The alpha-subunits of the RNA polymerase are involved in this effect as shown by KMnO4 footprinting . The open complex formed in the presence of CRP is structurally identical to the one found at a CRP-independent promoter up-mutant . UV-laser footprinting yields distinct signals for the different protein-DNA interactions within the complex and for interactions between CRP and RNA polymerase . We monitor these signals in promoter variants that place the CRP binding site at different distances upstream of the start site of transcription . Signals within the core promoter region, as well as those located just upstream of the -35 hexamer, are unaffected by the position of the CRP binding site . Contacts of RNA polymerase with the upstream promoter region change in a mutant RNA polymerase containing a truncated alpha-subunit . We conclude that at least one of the alpha-subunits of RNA polymerase binds to DNA upstream of the -35 hexamer and that this interaction is unaffected by the position of the CRP binding site . We discuss models that account for the different activities of CRP in transcriptional activation as a function of promoter geometry. Biochemistry, 1996 Dec 3, 35(48), 15236 - 43 Thermodynamics of carbohydrate binding to galectin-1 from Chinese hamster ovary cells and two mutants . A comparison with four galactose-specific plant lectins; Gupta D et al.; The thermodynamics of carbohydrate binding to the 14 kDa dimeric beta-galactoside-binding lectin galectin-1 (Gal-1) from Chinese hamster ovary cells and four galactose-specific plant lectins were investigated by isothermal titration microcalorimetry . Recombinant Gal-1 from Escherichia coli, a Cys-->Ser mutant with enhanced stability (C2S-Gal-1), and a monomeric mutant of the lectin (N-Gal-1) were studied along with the soybean agglutinin and the lectins from Erythrina indica, Erythrina crystagalli, and Erythrina corollodendrum . Although the pattern of association constants of the Erythrina lectins was similar for mono- and disaccharides, variations exist in their enthalpy of binding (-delta H) values for individual carbohydrates . While the Erythrina lectins show greater affinities and -delta H values for lactose and N-acetyllactosamine, the soybean agglutinin possesses similar affinities for methyl beta-galactopyranoside, lactose, and N-acetyllactosamine and a greater -delta H value for the monosaccharide . Gal-1 and the plant lectins possess essentially the same affinities for N-acetyllactosamine; however, the animal lectin shows a lower -delta H value and more favorable binding entropy for the disaccharide . While Gal-1, C2S-Gal-1, and N-Gal-1 all possess essentially the same affinities for N-acetyllactosamine, the two mutants possess much lower -delta H values, even though the mutation site(s) are far removed from the carbohydrate binding site . These results indicate that there are different energetic mechanisms of carbohydrate binding between galectin-1, its two mutants, and the Gal-specific plant lectins. Biochemistry, 1996 Dec 3, 35(48), 15215 - 21 Analysis of the physical properties and molecular modeling of Sec13: A WD repeat protein involved in vesicular traffic; Saxena K et al.; WD repeat proteins are a family of proteins that contain a series of highly conserved internal repeat motifs, usually ending with WD (Trp-Asp) . The G beta subunit of heterotrimeric guanine nucleotide binding protein is a member of this family, and its crystal structure has been recently solved at high resolution (Wall et al . (1995) Cell 83, 1047-1058; Sondek et al . (1996) Nature 379, 369-374) . Based on the coordinates of G beta, we have constructed a model for the structure of Sec13, a 33 kDa WD repeat protein from Saccharomyces cerevesiae essential for vesicular traffic . The model has been tested using a combination of biophysical and biochemical methods . Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity . In contrast to some other WD repeat proteins that are unable to fold into monomeric structures when expressed in E . coli, H6Sec13 was soluble and monomeric in the absence of detergent . The far-UV circular dichroism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consistent with the model which predicts primarily beta-sheets . H6Sec13 shows a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure . The CD spectrum shows an unusual positive ellipticity at 229 nm that was attributed to interactions of surface tryptophans since the 229 nm maximum could be abolished by modification of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the molecule) with N-bromosuccinimide . Our model predicts that three sets of tryptophans are clustered near the surface . As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion . The concordance of the model of Sec13 presented in this paper with the biochemical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function of Sec13 in vesicular traffic. Mol Biochem Parasitol, 1996 Dec 2, 83(1), 93 - 106 Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene; Prapunwattana P et al.; Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil . The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme . The 1912-base pair P . falciparum DHFR-TS gene was designed based on E . coli codon preference with unique restriction sites evenly placed throughout the coding sequence . The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain . Each of these domains contained numerous unique restriction sites to facilitate mutagenesis . The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence . The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E . coli about 10-fold more efficiently than from the wild-type sequence . The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study . The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria. EMBO J, 1996 Dec 2, 15(23), 6766 - 74 The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome; Vorstenbosch E et al.; Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly . GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration . Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes . The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation . Codon recognition following initial binding is also not affected by the mutation . The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding . Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates . The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome . Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges. EMBO J, 1996 Dec 2, 15(23), 6408 - 15 TolA central domain interacts with Escherichia coli porins; Derouiche R et al.; TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains . The interaction of TolA with outer membrane porins of Escherichia coli was investigated . Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA . The interaction of purified TolA domains with purified porins was also studied . TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA . Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions . These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins. FEBS Lett, 1996 Dec 2, 398(2-3), 322 - 5 Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme; Sjoblom B et al.; A murine carbonic anhydrase-related protein (CARP) has been expressed in Escherichia coli and purified to near homogeneity . The polypeptide chain consists of 290 amino acid residues and has a calculated molecular mass of 32,950 Da . By introducing two mutations, Arg117 --> His and Glu115 --> Gln, we created a metal-binding center homologous to that in the carbonic anhydrases from the animal kingdom . In contrast to unmodified CARP, this double mutant was isolated as a 1:1 zinc-protein complex . While unmodified CARP is catalytically inactive, the mutant catalyzes CO2 hydration with a significantly higher efficiency than the mammalian low-activity carbonic anhydrase isozyme III . The activity is strongly inhibited by the powerful and selective carbonic anhydrase inhibitor, acetazolamide. FEBS Lett, 1996 Dec 2, 398(2-3), 206 - 10 The catalytic domain of the cGMP-dependent protein kinase Ialpha modulates the cGMP-binding characteristics of its regulatory domain; Dostmann WR et al.; The cGMP-dependent protein kinase Ialpha (PKG Ialpha) possesses two functional moieties, the regulatory and catalytic domains, which reside on a single polypeptide chain . Here we report on the influence of the catalytic domain on the binding of cGMP to the regulatory domain . A deletion mutant, delta352-670 of PKG Ialpha, lacking the catalytic domain, was constructed and expressed in E . coli . The purified 38 kDa mutant protein showed strong reactivity toward tryptic proteolysis at residue Arg77 . Thus, a double deletion fragment delta1-77/352-670 PKG Ialpha, lacking the N-terminus, was also purified . Both proteins had functional cGMP binding, but differed kinetically from the wild-type protein . First the affinity constants for cGMP were modulated, second the constructs showed no signs of cooperative cGMP binding and third dimerization of the delta352-670 mutant was abolished . Our results provide evidence that the catalytic domain forms an intimate interaction with the regulatory domain and modulates the kinetics of cGMP binding. FEBS Lett, 1996 Dec 2, 398(2-3), 151 - 4 Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli; Shin DH et al.; HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli . To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr) . Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV . The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU . However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present . Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin . These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU. Mutat Res, 1996 Dec 2, 364(3), 227 - 33 Induction of oh8Gua glycosylase in rat kidneys by potassium bromate (KBrO3), a renal oxidative carcinogen; Lee YS et al.; It has been suggested that 8-hydroxyguanine (oh8Gua), a DNA adduct formed by active oxygens, impairs the maintenance of genetic integrity, oh8Gua glycosylase removes oh8Gua residues as a free base from DNA strands . In E . coli, it has been demonstrated that oh8Gua glycosylase is induced in response to oxidative stress, but the oxidative inducibility in mammalian tissues has not yet been studied . In the present study, the inducibility of oh8Gua glycosylase was tested by comparing activity changes of this enzyme in the kidney and the liver of rats treated with potassium bromate (KBrO3) . KBrO3 is known to cause oxidative damage to the kidney but not to other organs . With a single dose of KBrO3 (80 mg/kg, i.p.), activity in the kidney was found to increase significantly at 3 h compared to that at zero time . At 6 h, activity peaked, showing a 6-fold increase over that at zero time . Thereafter, it decreased and returned to its zero time level at 12 h . With increasing doses of KBrO3 (up to 160 mg/kg, i.p.), activity increased linearly with increased dosage, and over 40 mg/kg, i.p., activity increased to a level significantly higher than that in the control . In contrast to the time- and dose-dependent changes in activity in the kidney, no significant change was observed in the liver under the same conditions as above . These results show that oh8Gua glycosylase is also induced oxidatively in mammalian tissues . The induction in this tissue as well as in E . coli indicates that the adaptive response of this enzyme to oxidative stress is a general phenomenon in aerobic organisms and implies that the repair of oh8Gua residues in DNA is a process important for the survival of organisms in an aerobic environment. Mutat Res, 1996 Dec 2, 364(3), 183 - 92 Gene-specific nuclear and mitochondrial repair of formamidopyrimidine DNA glycosylase-sensitive sites in Chinese hamster ovary cells; Taffe BG et al.; This study examines the capacity of a mammalian cell to repair, at the gene level, DNA base lesions generated by photoactivation of acridine orange . Chinese hamster ovary fibroblasts were exposed to acridine orange and visible light, and gene-specific DNA repair was measured in the dihydrofolate reductase (DHFR) gene and in the mitochondrial genome . DNA lesions were recognized by Escherichia coli formamidepyrimidine-DNA glycosylase (FPG) which removes predominantly 8-oxodG and the corresponding formamidopyrimidine ring opened bases, and subsequently cleaves the DNA at the resulting apurinic site . FPG-recognized DNA lesions increased linearly with increasing photo-activation of AO, while cell survival was not affected by light alone and was negligibly affected by preincubation with AO in the dark . The frequency of induction of FPG-sensitive DNA damage by photoactivation of AO was similar in the transcribed and non-transcribed nuclear DNA as well as in the mitochondrial DNA . FPG-sensitive sites in the DHFR gene were repaired quickly, with 84% of adducts repaired within 4 h . The lesion frequency, kinetics and percent of repair of non-transcribed genomic DNA did not differ significantly from repair in the active DHFR gene up to 1 h postexposure . At late time points, transcribed DNA was repaired faster than the non-transcribed DNA . Mitochondrial DNA was efficiently repaired, at a rate similar to that in the active nuclear DNA. Mutat Res, 1996 Dec 2, 364(3), 171 - 82 Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA; Braun JE et al.; Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation . To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (.OH, .H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine . The exposed DNA was subsequently transfected to wild-type E . coli and mutations in the mutational target were characterized . This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZ alpha gene and a 144 bp inframe insert . The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O . In the latter case, the ratio of .OH and .H available for reacting with DNA is about the same as under N2 + cysteamine . The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of .OH and .H alone . This higher survival appeared to be accompanied with a higher mutation induction . However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum . This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different . Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages . Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence. Parassitologia, 1996 Dec, 38(3), 559 - 63 Epitope mapping on the ookinete surface antigen Pbs21 of Plasmodium berghei: identification of the site of binding of transmission-blocking monoclonal antibody 13.1; Spano F et al.; The ookinete surface protein of Plasmodium berghei Pbs21 belongs to a class of sexual stage antigens able to induce in the vertebrate host a transmission-blocking immune response . The effectors of this transmission-blocking immunity are antibody molecules directed against particular protein epitopes . The anti-Pbs21 monoclonal antibody 13.1 is known to bind a linear stretch of amino acids within the primary sequence of Pbs21 and to efficiently block the development of P . berghei in the mosquito gut . To map the 13.1 epitope along the amino acid sequence of Pbs21 we assayed the ability of 13.1 antibody to recognize, in Western blot, a series of Pbs21 deletion mutants as well as the ability of synthetic peptides to inhibit 13.1 binding to full length Pbs21 . The epitope was identified within the second EGF-like domain of the Pbs21 molecule. Mol Immunol, 1996 Dec, 33(17-18), 1345 - 58 Modulating the immunological properties of a linear B-cell epitope by insertion into permissive sites of the MalE protein; Martineau P et al.; In a previous study, a set of positions in the MalE protein from Escherichia coli were identified, which tolerated short insertions or deletions without compromising the maltose binding activity of the protein . It is now shown that these sites accommodate an insert of 13 amino acids and are, therefore, permissive . Eleven sites were used, including eight permissive sites, to display a linear neutralization B-cell epitope of poliovirus (C3 epitope) at different positions on the surface of MalE . The affinity of a monoclonal neutralizing anti-poliovirus antibody (anti-C3 mAb) for the hybrid proteins varied from undetectable, to more than 1000 times higher than for the synthetic peptide . Therefore, some MalEC3 proteins mimic interactions of the viral epitope with the monoclonal antibody more efficiently than the free peptide . The results are interpreted in terms of the mobility of the insert and its flanking regions . It was further shown that some of the purified hybrid proteins are able to induce high titer anti-C3-peptide antibodies in mice . A strong correlation exists between the capacity of a MalEC3 protein to induce anti-C3-peptide antibodies and the antigenicity of the inserted peptide, measured with a polyclonal serum raised against the synthetic peptide. Arch Ital Urol Androl, 1996 Dec, 68(5 Suppl), 79 - 82 {Lobar nephritis: echographic diagnosis and follow-up}; Rosi P et al.; Acute lobular nephritis is a focal bacterial infection localizer within the parenchyma of the kidney which may develops with abscess formation; clinical features of such evolution include, fever, chills, flank pain and the hematological findings of infective disease . Echographic pattern includes a law-level echogenic mass with a central hypoechoic or echo-free with sometimes may deform renal profile . Clinical picture and echographic pattern allow the diagnosis of acute lobular nephritis . In the present work we report 3 cases of lobular nephritis on which ultrasound study has permitted the correct diagnosis equally to TC and RM which also was performed . Furthermore the ultrasound imaging is a valid method to appreciate the clinical evolution of patient during therapy. Rinsho Shinkeigaku, 1996 Dec, 36(12), 1322 - 3 {Segawa disease (hereditary progressive dystonia with marked diurnal fluctuation-HPD) and abnormalities in pteridin metabolism}; Segawa M; From its characteristic clinical features, decrease of tyrosine hydroxylase (TH) in the terminal of the nigrostriatal (NS) dopamine (DA) neuron is considered the main lesion of HPD and the decrease of neopterin as well as biopterin in the cerebrospinal fluid suggested GTP cyclohydrolase I (GCH-I) as the responsible enzyme . By detecting the gene locus of GCH-I, Ichinose and his colleagues showed the abnormalities of GCH-I gene located on 14q 22.1 q22.2 as the cause of HPD . Since the first report of Ichinose et al, 11 mutations and frame shifts of the gene have been detected, in which the locus of abnormality differed among families but is identical in a family, but more than several families have been left with undetected abnormalities including those having linkage to 14q . However, the DNA of these families as well as those with detected gene abnormalities failed to synthesize GCH-I if inoculated with E . coli and the levels of GCH-I in mononuclear blood cells were below 20% of normal values in HPD patients while they were 37 and 38% in two asymptomatic carriers . Ratio of mutant mRNA of GCH-I gene was 28% in a patient and 8.3% in an asymptomatic case . These lines of evidence on GCH-I show HPD is a dominant inherited disorder with abnormalities of GCH-I gene . GCH-I is the limiting enzyme for synthesizing tetrahydrobiopterin (BH4), coenzyme transmitters for the synthesizing hydroxylases of aminergic neurotransmitters, but the affinity is the least for TH . This might cause a rather selective involvement of TH preserving serotonin synthesis un- or less affected . Fluoro-DOPA and {11C} racropride PET studies were normal in HPD . Studies of an autopsied case with dopa responsive dystonia, which was confirmed to have GCH-I gene abnormalities, neuropathologically revealed no abnormalities except for a decrease in melanin pigmentation in the substantia nigra and histochemically a decrease in TH enzyme activities and its protein only in the striatum . There was mild decrease of DA content, the interregional caudate/putamen and subregional rostrocaudal patterns which were similar to Parkinson disease, but subdivisionally different with predominant reduction in the ventral subdivision of the caudate nucleus . In the ventral part of the basal ganglia the striatal direct projection exists predominantly . Cases with recessive abnormalities of pteridin metabolism other than HPD, 6-pyruvoyl-tetra-hydropterin synthase (PSPS) deficiency and dihydropteridine reductase deficiency also show dystonia with diurnal fluctuation responding to levodopa, though not as marked as HPD . MPTP monkey studies revealed no involvement of striatal indirect pathway for peak dose dystonia . So it is suggested that in HPD, decrease of TH at the terminal of the NS-DA neuron due to partial reduction of GCH-I develops postural dystonia through the striatal direct projection in childhood with diurnal fluctuation depending on age and circadian variation of TH activities at the terminals. Antimicrob Agents Chemother, 1996 Dec, 40(12), 2813 - 9 Antimycobacterial action of thiolactomycin: an inhibitor of fatty acid and mycolic acid synthesis; Slayden RA et al.; Thiolactomycin (TLM) possesses in vivo antimycobacterial activity against the saprophytic strain Mycobacterium smegmatis mc2155 and the virulent strain M . tuberculosis Erdman, resulting in complete inhibition of growth on solid media at 75 and 25 micrograms/ml, respectively . Use of an in vitro murine macrophage model also demonstrated the killing of viable intracellular M . tuberculosis in a dose-dependent manner . Through the use of in vivo {1,2-14C}acetate labeling of M . smegmatis, TLM was shown to inhibit the synthesis of both fatty acids and mycolic acids . However, synthesis of the shorter-chain alpha'-mycolates of M . smegmatis was not inhibited by TLM, whereas synthesis of the characteristic longer-chain alpha-mycolates and epoxymycolates was almost completely inhibited at 75 micrograms/ml . The use of M . smegmatis cell extracts demonstrated that TLM specifically inhibited the mycobacterial acyl carrier protein-dependent type II fatty acid synthase (FAS-II) but not the multifunctional type I fatty acid synthase (FAS-I) . In addition, selective inhibition of long-chain mycolate synthesis by TLM was demonstrated in a dose-response manner in purified, cell wall-containing extracts of M . smegmatis cells . The in vivo and in vitro data and knowledge of the mechanism of TLM resistance in Escherichia coli suggest that two distinct TLM targets exist in mycobacteria, the beta-ketoacyl-acyl carrier protein synthases involved in FAS-II and the elongation steps leading to the synthesis of the alpha-mycolates and oxygenated mycolates . The efficacy of TLM against M . smegmatis and M . tuberculosis provides the prospects of identifying fatty acid and mycolic acid biosynthetic genes and revealing a novel range of chemotherapeutic agents directed against M . tuberculosis. J Physiol Pharmacol, 1996 Dec, 47(4), 591 - 9 The effect of prostacyclin and nitric oxide on deformability of red blood cells in septic shock in rats; Korbut R et al.; Six hours after administration of E . Coli endotoxin (LPS) into rats (10 mg kg-1, i.p.) a significant (P < 0.001) decline in the red blood cell deformability index (RBC Dj) was observed . The control Di value of untreated animals it was 300 +/- 39 RBC x 10(6)/min (means +/- S.D.; n = 12) while in LPS treated animals was 140 +/- 50 RBC x 10(6)/min; n = 12 . Pretreatment of the animals with the stable analogue of prostacyclin, iloprost (30 micrograms/kg, i.p.) or with the inhibitor of thromboxane A2-synthase, camonagrel (10 mg/kg, i.p.), but not with nitric oxide donor, such as GEA 5285 (10 mg/kg, i.p.), significantly increased deformability of red blood cells in the group of non-septicaemic animals, and antagonized the LPS-induced decline in red blood cell deformability of septicaemic rats . Administration of NG-nitro-L-arginine (L-NNA, 30 mg/kg, i.p.), as that of aspirin (50 mg/kg, i.p.), did not affect red blood cell deformability in non-septicaemic rats, however, in contrast with aspirin, it significantly improved deformability of red blood cells in LPS-treated animals . It is concluded that prostacyclin, camonagrel and L-NNA can act as protective agents against LPS-induced loss of red blood cell deformability . The mechanisms of this protection are complex and, possibly, related to the specific effects of these agents on biochemical function of leukocytes present in RBC suspension . While the effect of exogenous prostacyclin (iloprost) may be explained on the basis of its direct cytoprotective potency on leukocytes, the effect of camonagrel is indirect and can be attributed both to the release of endogenous prostacyclin and to the inhibition of thromboxane A2-synthase . The protection induced by NO-synthase inhibitor seems to depend upon inhibition of an increase of the generation of nitric oxide which follows administration of LPS. Z Gastroenterol, 1996 Dec, 34(12), 783 - 90 {Intestinal cytokine liberation after intestinal ischemia in the rat--studies in the Ussing chamber system}; Grotz M et al.; Intestinal ischemia, frequently found in clinical states such as aortic bypass operations or hemorrhagic shock, is associated with loss of gut barrier function . Subsequent translocation of indigenous bacteria and endotoxin have been implicated as a major contributor to a systemic immuno-inflammatory response, which finally leads to multiple organ failure . There is increasing evidence that intestinal injury can result in the gut becoming a cytokine generating organ . This study was designed to show direct evidence of the gut as a major source of proinflammatory cytokines after intestinal ischemia and to further relate this cytokine response to the extent of intestinal ischemia/reperfusion . Additionally the potential role of the altered intestinal barrier function after intestinal ischemia for this cytokine response was investigated . METHODS: Rats were subjected to occlusion of the superior mesenteric artery for 45 min . (SMAO45), 75 min . (SMAO75), SMAO for 45 min . and 30 min . reperfusion (SMAO45/30), or sham SMAO, and then killed . Mucosal membranes from the terminal ileum were mounted in a Ussing chamber . E . coli C25 was added to the mucosal side of the stripped gut epithelium in half of the chambers . TNF and IL-6 levels on mucosal and serosal side of the stripped gut epithelium were assessed serially over 3 hrs . Gut barrier function was assessed by in vitro bacterial translocation (BT) and the transepithelial resistance (TER) of the mucosal membrane . RESULTS: The TNF response was greatest in the SMAO75 group, the IL-6 response in the SMAO75 and SMAO45/30 groups . In the absence of E . coli C25 . IL-6 was produced to a greater extent on the serosal side, while addition of bacteria led to a significantly increased TNF/IL-6 response at the mucosal side of the stripped gut epithelium . BT was increased in SMAO75 and SMAO45/30 rats . Baseline TER was decreased in all experimental compared to sham SMAO groups . Although gut barrier function was impaired after intestinal ischemia/reperfusion there was no correlation between intestinal cytokine response and gut permeability . CONCLUSIONS: The gut becomes a cytokine liberating organ alter intestinal ischemia/reperfusion . This cytokine response is affected by certain conditions, but is not directly related to an impaired intestinal barrier function. Genes Cells, 1996 Dec, 1(12), 1069 - 75 DNA nicking by Escherichia coli topoisomerase IV with a substitution mutation from tyrosine to histidine at the active site; Yokochi T et al.; BACKGROUND: Escherichia coli topoisomerase IV is a type II topoisomerase composed of ParC and ParE subunits and plays a major role in the decatenation of replicated molecules . The reaction with type II topoisomerases involves the cutting through transesterification between an active-site tyrosine and a DNA phosphodiester bond and a rejoining of cleaved DNA . RESULTS: To genetically analyse the mechanism of this reaction, we isolated dominant-negative topoisomerase IV mutants . In one of them, the parC10 mutant, there was a substitution by histidine of the active-site tyrosine . Purified mutant topoisomerase IV did not show normal DNA cutting activity but retained DNA nicking activity, even in the absence of ATP . The DNA ends of the product were not covalently bound to the ParC subunits, suggesting that the DNA is not cut via transesterification but by hydrolysis . CONCLUSIONS: We have shown genetically that the 120th tyrosine residue is important for the DNA cutting step in the topoisomerase reaction . The 120th amino acid residue, tyrosine or histidine, appears to be activated and the histidine residue forces the hydrogen-bonded water to attack the phosphoryl group of the DNA in hydrolysis, while the tyrosine residue directly attacks the phosphoryl group during transesterification. Genes Cells, 1996 Dec, 1(12), 1057 - 68 Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction; Maeshima K et al.; BACKGROUND: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double-strand break repair . To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies . We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1 . RESULTS: Recombinant XRad51.1 was expressed in E . coli . The purified XRad51.1 protein showed ssDNA-dependent ATPase activity and promoted the DNA strand exchange reaction between two 55-mer oligonucleotides . The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(d epsilonA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E . coli RecA protein . The kinetics of the fluorescence change of poly(d epsilonA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein . The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1-ssDNA complex was slower than the RecA-ssDNA complex . CONCLUSIONS: Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules . While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP. Br J Biomed Sci, 1996 Dec, 53(4), 270 - 7 Mutagenesis of bovine basic fibroblast growth factor through hybrid clonemid construction: a new approach to DNA manipulation; Zhao Q; Basic fibroblast growth factor (bFGF) is a heparin-binding protein which is mitogenic for a wide variety of cells derived from the mesoderm and neuroectoderm . It is also known as an angiogenic and neurotrophic molecule . The mutagenesis of bovine bFGF gene into the human bFGF gene and the expression of human bFGF have been carried out in this laboratory . In this study, efficient linkage (ligation) of single strands of DNA at their terminal complementary areas was found to be obtained easily, at low temperature . Based upon this discovery, we have advanced a general method of DNA manipulation . The major principle is to make use of complementary sequences in a DNA linker to circularise a cut vector or use the single-strand DNA fragments to rebuild the plasmid through heteroduplex plasmid (hybrid clonemid) construction . The use of single DNA strands in DNA manipulation produces high flexibility in the course of hybrid clonemid construction . It enables DNA cloning and mutagenesis to be carried out concurrently . The primer-induced and polycation-reliant unidirectional deletion (PPUD) phenomenon is also observed in this study . The factors that affect the efficiency of mutagenesis are discussed . This work offers a new understanding of DNA manipulation. Solid State Nucl Magn Reson, 1996 Dec, 7(3), 203 - 10 Structural constraints on the complex of elongation factor Tu with magnesium guanosine diphosphate from rotational-echo double-resonance NMR; McDowell LM et al.; Rotational-echo, double-resonance (REDOR) NMR measurements of 31P-15N dipolar couplings have been made on a complex of Mg guanosine diphosphate (MgGDP) with uniformly 15N-labeled elongation factor Tu . The complex was embedded in a lyophilized buffer glass . The observed 15N REDOR dephasing by 31P was accounted for quantitatively by distances from 15N of Gly23 and Lys24 to P alpha and P beta of MgGDP as determined by X-ray crystallography of MgGDP complex formed using an elongation factor Tu that is missing a 15 residue loop in the vicinity of the binding site. Solid State Nucl Magn Reson, 1996 Dec, 7(3), 193 - 201 Dynamics of trimethoprim bound to dihydrofolate reductase--a deuterium NMR study; Yang QX et al.; We have employed deuterium NMR techniques to determine the dynamics of trimethoprim (TMP) in a binary complex with dihydrofolate reductase (DHFR) or in a ternary complex with DHFR and cofactor NADP+ in the fully hydrated state . TMP was deuterated at the following positions: (2',6'-D2)TMP, (3'-Ome-D3)TMP and (3',4'-Ome-D6)TMP . Dynamics of TMP were deduced from lineshape simulation and relaxation measurements of the deuterium NMR powder spectra of the three samples obtained at various temperatures . The results showed that in the polycrystalline state the TMP molecule is very rigid . The only detectable motion is the methyl group rotation at a rate of 10(10) s-1 at 25 degrees C, as determined from simulation of the partially relaxed powder patterns . When bound to DHFR a residual deuterium quadrupole splitting of 140 kHz was observed for (2',6'-D2)TMP at temperatures up to 30 degrees C, suggesting that the benzyl ring in the bound state is also very rigid . In contrast, in the binary complex with DHFR the methoxyl groups of TMP undergo librational motion of 10(7) s-1 about the C3-O bond at an amplitude of 54 degrees for the meta methoxyl group and about the C4-O bond at an amplitude of 70 degrees and similar rate for the para methoxyl group at 30 degrees C . The presence of the cofactor, NADP+, appears to tighten up the binding pocket such that the motion freedom of TMP is more restricted . The rigidity of TMP in a protein complex as revealed by our deuterium NMR results is in accord with the tight binding of TMP to DHFR. Res Commun Mol Pathol Pharmacol, 1996 Dec, 94(3), 227 - 38 Renal tubular epithelial cell-E . coli interaction products stimulate nitric oxide production in cultured rat renal medullary interstitial and mesangial cells; Trachtman H et al.; Tubulointerstitial and periglomerular inflammation and fibrosis are important consequences of pyelonephritis . The pathogenesis of these abnormalities is not fully understood . Renal tubular epithelial cells (RTEC) elaborate biologically active materials following incubation with bacteria . Nitric oxide (NO) is an inflammatory mediator and it modulates the accumulation of extracellular matrix proteins . Therefore, we studied whether RTEC-E . coli interaction products regulate NO production by cultured rat renal medullary interstitial cells (RMIC) and mesangial cells (MC) . RMIC and MC were maintained in media containing IFN-gamma and LPS for 24-72 h . Test media contained either no further additives or 20% supernatants from RTEC incubated with E . coli or bacterial cell products . RTEC-E . coli interaction products significantly increased NO production in RMIC and MC . This stimulation in NO production was not associated with changes in inducible nitric oxide synthase (iNOS) gene or protein expression . These findings indicate that RTEC-E . coli interaction products increase NO production in RMIC and MC by directly stimulating iNOS enzymatic activity . Altered NO production by renal cells may contribute to tubulointerstitial inflammation in acute and chronic pyelonephritis. Clin Exp Allergy, 1996 Dec, 26(12), 1401 - 10 Isolation, cDNA cloning and expression of Lig v 1, the major allergen from privet pollen; Batanero E et al.; BACKGROUND: An olive allergen-like protein has been detected in privet pollen . This protein could be involved in the allergenic cross-reactivity described for privet and olive tree pollen extracts . OBJECTIVE: Isolation and characterization of natural Lig v 1 . Cloning and expression of its cDNA in order to assess its structural similarity with the olive allergen . METHODS: Current chromatographic methods were used to isolate the privet counterpart of Ole e 1 . A pool of sera from subjects allergic to olive tree pollen was used to immunodetect the protein in the elution profiles . Ole e 1-specific polyclonal antibody and allergic sera were used in immunoblotting assays of the isolated protein . Polymerase chain reaction amplification of the first strand cDNA synthesized from the privet pollen total RNA was carried out to prepare a full-length fragment encoding Lig v 1 . After nucleotide sequencing, expression of one clone was performed in Escherichia coli, under the form of a fusion protein with glutathione S-transferase . The IgE binding capability of the recombinant protein was also analysed . RESULTS: The major allergen from privet pollen . Lig v 1, was purified to homogeneity by two gel filtration chromatographies and one reverse-phase high-performance liquid chromatography . Its amino acid composition and N-terminal amino acid sequence were determined . Two different clones encoding Lig v 1 were sequenced . Strong sequence similarity between Lig v 1 and Ole e 1 was observed, the identity being 85 and 96% . One of the sequenced clones was expressed and the recombinant product exhibited IgG and IgE binding activities against both anti-Ole e 1 polyclonal antibodies and olive-allergic sera . CONCLUSION: Privet pollen contains a protein structurally and immunologically related to the major allergen of olive pollen . The similarity exhibited by these proteins could explain the cross-reactivity observed between the two pollen extracts . Since these allergens are highly polymorphic, the expression of an immunologically active recombinant Lig v 1 will permit the preparation of well defined molecules for both research and clinical purposes. Am J Trop Med Hyg, 1996 Dec, 55(6), 672 - 9 Cocirculation of multiple hantaviruses in Texas, with characterization of the small (S) genome of a previously undescribed virus of cotton rats (Sigmodon hispidus); Rawlings JA et al.; An environmental and laboratory investigation was conducted after a fatal childhood case of hantavirus pulmonary syndrome occurred in Deaf Smith County, Texas in May 1995 . A trapping campaign was conducted to identify possible rodent carriers . Six species of murid and heteromyid rodents were collected, and at least one hantavirus-seropositive specimen was found in each of the five murid species . Tissues from a selection of 11 seropositive specimens were examined by the polymerase chain reaction (PCR) and sequencing of viral genetic material . The predominant hantavirus was El Moro Canyon virus (ELMCV), which occurred in three of three harvest mice (Reithrodontomys megalotis) and in three of four deer mice (Peromyscus maniculatus) examined . Sin Nombre virus (SNV) was found in one deer mouse and one white-footed mouse (P . leucopus) . A seropositive house mouse (Mus musculus) was negative by PCR . Two cotton rats (Sigmodon hispidus) were infected by a virus of novel genotype (Muleshoe virus {MULEV}) that bears closet resemblance to Bayou hantavirus . The sequence of the complete small genomic segment was determined for one MULEV, and high-level expression of its nucleocapsid protein was induced in Escherichia coli . Serologic studies indicated that the most likely etiologic agent in the human infection was SNV. Am J Trop Med Hyg, 1996 Dec, 55(6), 635 - 41 Immunogenicity of the nonrepetitive regions of the circumsporozoite protein of Plasmodium knowlesi; Sharma S et al.; The circumsporozoite antigen (CS) of the simian malarial parasite Plasmodium knowlesi consists of tandemly repeated immunodominant peptide units that are variable and may play a role in evading the immune system . To study the immunogenicity of this antigen in the absence of the immunodominant repeats, the entire nonrepetitive region of the antigen was expressed in Escherichia coli as two fusion proteins with glutathione-S-transferase (GST) representing the amino terminal (GST-CSN) and the carboxy terminal domains (GST-CSC) of the CS antigen . The immunogenicity of these fusion proteins was studied in rabbits and different strains of mice . Antibody raised against both the CSN and CSC domains in both rabbits and every strain of mice recognized the native protein, as detected by immunofluorescence assay (IFA) using P . knowlesi sporozoites . A positive IFA reaction was also obtained with P . vivax sporozoites using antisera raised against the CSC domain . High titer antisera were raised in rabbits against both the domains, whereas mice showed comparatively low titers . On Western blots, mice showed specific response against the CSC domain . In both rabbits and mice, significant titers of antibodies were raised against region II, which has been shown to be the putative sporozoite binding site for hepatocytes in the case of P . falciparum. Mol Cell Probes, 1996 Dec, 10(6), 423 - 5 A simple method to design PCR primer to detect genomic DNA of parasites and its application to Dirofilaria immitis; Nagano I et al.; A simple method to design a polymerase chain reaction (PCR) primer for parasites of interest was developed using Dirofilaria immitis as a test sample . The method involved the cloning and sequencing of randomly amplified DNA of the parasite, and designing a primer based on the resulting DNA sequence . Using the primer, DNA fragments of the expected length were amplified by a regular PCR with genomic DNA of Dirofilaria immitis. Bioorg Med Chem, 1996 Dec, 4(12), 2179 - 85 Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale; Park J et al.; The problems inherent in the enzymatic and chemical synthesis of S-adenosyl-L-methionine (SAM) led us to develop an efficient, simple method for the synthesis of large amounts of labeled SAM . Previously, we reported that the problem of product inhibition of E . coli SAM synthetase encoded by the metK gene was successfully overcome in the presence of sodium p-toluenesulfonate (pTsONa) . This research has now been expanded to demonstrate that product inhibition of this enzyme can also be overcome by adding a high concentration of beta-mercaptoethanol (beta ME), acetonitrile, or urea . In addition a recombinant strain of E . coli has been constructed that expresses the yeast SAM synthetase encoded by the sam2 gene . The yeast enzyme does not have the problem of product inhibition seen with the E . coli enzyme . Complete conversion of 10 mM methionine to SAM was achieved in incubations with either the recombinant yeast enzyme and 1 molar potassium ion or the E . coli enzyme in the presence of additives such as beta ME, acetonitrile, urea, or pTsONa . The recombinant yeast SAM synthetase was used to generate SAM in situ for use in the multi-enzymatic synthesis of precorrin 2. Anticancer Drug Des, 1996 Dec, 11(8), 611 - 24 Molecular origins of selectivity in the interaction of amsacrine-4-carboxamide with GC-rich sequences in DNA; Bailly C et al.; To determine the molecular origins of the preferential binding of an antitumour amsacrine-4-carboxamide derivative to GC-rich sequences in DNA, we have used the polymerase chain reaction to synthesize a series of oligodeoxynucleotides in which the position of the purine 2-amino group is varied and then investigated the binding of the drug to normal and modified DNA molecules by means of DNase I footprinting . The results indicate that the 2-amino group of guanine represents an important but not unique element which directs selective binding of amsacrine-4-carboxamides to GC-rich sequences. Microbiologia, 1996 Dec, 12(4), 557 - 62 Monitoring by PCR amplification of the polyphosphate kinase gene added to natural water samples; Lopez NI et al.; A fast, simple method for the detection of the Escherichia coli polyphosphate kinase (ppk) gene by means of PCR amplification is described . The method uses filters to recover cells from the samples, which makes it suitable for environmental studies . The detection of the ppk gene was achieved from samples containing 10(2) E . coli cells, either in saline solution or in river water. J Appl Physiol, 1996 Dec, 81(6), 2415 - 20 Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle; Thompson M et al.; Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase . To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin . In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge . Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots . Increased iNOS activity was demonstrated by conversion of arginine to citrulline . Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium . These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress. Immunology, 1996 Dec, 89(4), 483 - 7 The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide; Partidos CD et al.; The intranasal route has been shown to be effective for immunization . However, immunization via this route may require the use of potent and safe adjuvant . The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines . In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope . LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline . A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses . These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans. Ann Rheum Dis, 1996 Dec, 55(12), 895 - 900 Annexin V autoantibodies in rheumatoid arthritis; Rodriguez-Garcia MI et al.; OBJECTIVE: To investigate the occurrence of anti-annexin V autoantibodies in sera of patients with rheumatoid arthritis to assess involvement with the disease and any relation to glucocorticoid treatment . METHODS: Anti-annexin V antibodies were measured by an enzyme linked immunosorbent assay (ELISA) which used the purified human recombinant protein as antigen . RESULTS: Concentrations of anti-annexin V autoantibodies, predominantly of the IgG class, were significantly raised in sera from patients with rheumatoid arthritis compared to normal controls . This was not correlated with other indices of disease activity such as erythrocyte sedimentation rate or C reactive protein and was unrelated to glucocorticoid treatment . CONCLUSIONS: Extracellular annexin V provides an antigenic stimulus for autoantibody production and its in vivo expression is independent of glucocorticoid control . Such autoantibodies may have a detrimental role in the arthritic condition by interfering with putative functions of annexin V, including collagen type II binding, inhibition of phospholipase A2 activity, and Fc receptor activity. Vet Immunol Immunopathol, 1996 Dec, 55(1-3), 107 - 14 Recognition of ovine lentivirus gag gene products by serum from infected sheep; Kwang J et al.; In order to localize the immunodominant regions, 12 ovine lentivirus (OLV) gag-coding gene fragments were cloned and expressed in Escherichia coli and then tested in a Western blot (WB) assay against a panel of sera collected from US and Italian OLV-infected sheep . The most immunoreactive regions were mapped to the amino-terminal of p25 and carboxyl-terminal of p14 . In addition, we found that the reactivity pattern between US and Italian sheep was very similar, suggesting the antigenic domain between US and Italian isolates in the gag gene structures could be conserved . Given the broad immunoreactivity of the amino-terminal of p25, this region could serve as an ideal diagnostic antigen for the serological identification of OLV-infected sheep. Brain Res Mol Brain Res, 1996 Dec, 42(2), 181 - 92 Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice; Tsuruda LM et al.; To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene . Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression . Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed . We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF) . After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia . These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons. Dig Dis Sci, 1996 Dec, 41(12), 2482 - 92 IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage; Fukumura D et al.; Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood . The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine . Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated . Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration . Intestinal IL-1alpha levels were also significantly increased at that time . Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes . E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage . E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes . In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion . IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage. Plant J, 1996 Dec, 10(6), 1119 - 25 Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant; Koumoto Y et al.; Chaperonin (Cpn) is one of the molecular chaperones . Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding . It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells . The Escherichia coli homologue of Cpn10 is called GroES . A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E . coli groES mutant . The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids . The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES . A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment . The Cpn10 homologue was constitutively expressed in transgenic tobaccos . Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn 10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos. Protein Eng, 1996 Dec, 9(12), 1241 - 6 Synthesis of a squash-type protease inhibitor by gene engineering and effects of replacements of conserved hydrophobic amino acid residues on its inhibitory activity; Kojima S et al.; Cucurbita maxima trypsin inhibitor I (CMTI-I), a member of the squash-type protease inhibitor family, is composed of 29 amino acids and shows strong inhibition of trypsin by its compact structure . To study the structure-function relationship of this inhibitor using protein engineering methods, we constructed an expression system for CMTI-I as a fused protein with porcine adenylate kinase (ADK) . A Met residue was introduced into the junction of ADK and CMTI-I to cleave the fusion protein with CNBr, whereas a Met at position 8 of authentic CMTI-I was replaced by Leu . Escherichia coli JM109 transformed with the constructed plasmid expressed the fused protein as an inclusion body . After cleavage of the expressed protein with CNBr, fully reduced species of CMTI-I were purified by reversed-phase HPLC and then oxidized with air by shaking . For efficient refolding of CMTI-I, we used 50 mM NH4HCO3 (pH 7.8) containing 0.1% PEG 6000 at higher protein concentration . Strong inhibitory activity toward trypsin was detected only in the first of three HPLC peaks . The inhibitor constant of CMTI-I thus obtained, in which Met8 was replaced by Leu, was 1.4 x 10(-10) M . The effect of replacement of Met with Leu at position 8 was shown to be small by comparison of the inhibitor constant of authentic CMTI-III bearing Lys at position 9 (8.9 x 10(-11) M) with that of its mutant bearing Leu at position 8 and Lys at position 9 (1.8 x 10(-10) M) . To investigate the role of the well conserved hydrophobic residues of CMTI-I in its interaction with trypsin, CMTI-I mutants in which one or all of the four hydrophobic residues were replaced by Ala were prepared . The inhibitor constants of these mutants indicated that those with single replacements were 5-40 times less effective as trypsin inhibitors and that the quadruple mutant was approximately 450 times less effective, suggesting that the hydrophobic residues in CMTI-I contribute to its tight binding with trypsin . However, each mutant was not converted to a temporary inhibitor. Protein Eng, 1996 Dec, 9(12), 1233 - 9 Expression, purification and characterization of recombinant crambin; Lobb L et al.; Crambin, a small hydrophobic protein (4.7 kDa and 46 residues), has been successfully expressed in Escherichia coli from an artificial, synthetic gene . Several expression systems were investigated . Ultimately, crambin was successfully expressed as a fusion protein with the maltose binding protein, which was purified by affinity chromatography . Crambin expressed as a C-terminal domain was then cleaved from the fusion protein with Factor Xa protease and purified . Circular dichroism spectroscopy and amino acid analysis suggested that the purified material was identical to crambin isolated from seed . For positive identification the protein was crystallized from an ethanol-water solution, by a novel method involving the inclusion of phospholipids in the crystallization buffer, and then subjected to crystallographic analysis . Diffraction data were collected at the Brookhaven synchrotron (beamline-X12C) to a resolution of 1.32 A at 150 K . The structure, refined to an R value of 9.6%, confirmed that the cloned protein was crambin . The availability of cloned crambin will allow site-specific mutagenesis studies to be performed on the protein known to the highest resolution. Protein Eng, 1996 Dec, 9(12), 1173 - 80 Independent self-assembly of cadmium-binding alpha-fragment of metallothionein in Escherichia coli without participation of beta-fragment; Kurasaki M et al.; We examined the independent self-assembly of the alpha- and beta-fragments of human metallothionein (MT) into cadmium-binding conformation in an Escherichia coli expression system, in addition to wild-type MT expression . The expressed alpha-fragment formed independently the structure of a metal-binding cluster without the aid of the beta-fragment . The alpha-fragment and wild-type MT expressed in E.coli were purified and analyzed for their biochemical and spectroscopic properties . The apparent cadmium binding of the alpha-fragment was approximately 12-fold greater than that for the wild-type MT, whereas in other respects the studied biochemical properties were similar . In contrast, we were unable to obtain any independently expressed beta-fragment as the cadmium-binding form in this study . Possible explanations for this phenomenon are discussed. Protein Eng, 1996 Dec, 9(12), 1083 - 92 Crystal structure of glutathione synthetase at optimal pH: domain architecture and structural similarity with other proteins; Matsuda K et al.; The crystal structure of Escherichia coli B glutathione synthetase (GSHase) has been determined at the optimal catalytic condition pH 7.5 . The most significant structural difference from the structure at pH 6.0 is the movement of the central domain towards the N-terminal domain almost as a rigid body . As a result of this movement, new interdomain and intersubunit polar interactions are formed which stabilize the dimeric structure further . The structure of GSHase at optimal pH was compared with 294 other known protein structures in terms of the spatial arrangements of secondary structural elements . Three enzymes (D-alanine: D-alanine ligase, succinyl-CoA synthetase and the biotin carboxylase subunit of acetyl-CoA carboxylase) were found to have structures similar to the ATP-binding site of GSHase, which extends across two domains . The ATP-binding sites in these four enzymes are composed of two antiparallel beta-sheets and are different from the classic mononucleotide-binding fold . Except for these proteins, no significant structural similarity was detected between GSHase and the other ATP-binding proteins . A structural motif in the N-terminal domain of GSHase has been found to be similar to the NAD-binding fold . This structural motif is shared by a number of other proteins that bind various negatively charged molecules. J Biochem (Tokyo), 1996 Dec, 120(6), 1176 - 81 Comparison of ncd and kinesin motor domains by circular dichroism spectroscopy; Shimizu T et al.; ncd is a microtubule motor protein from Drosophila, having a 40 kDa domain homologous to the kinesin motor domain . In the present study, we investigated the circular dichroism (CD) spectra of the ncd motor domain in comparison with those of the kinesin motor domain . Although the two are about 40% identical in amino acid sequence, and recent X-ray crystallographic studies {Sablin, Kull, Cooke, Vale, and Fletterick (1996) Nature 380, 555-559; Kull, Sablin, Lau, Fletterick, and Vale (1996) Nature 380, 550-555} indicate that their core structures are nearly identical, the far UV CD spectra of ncd and kinesin motor domains, both being monomeric, were considerably different from each other, suggesting a significant difference in the secondary, especially loop structures . The motor domain of ncd, like that of kinesin, contains tightly associating ADP even after purification . We removed ADP from the ncd motor domain by gel filtration in the presence of EDTA and high salt . The resultant protein, however, was likely to be in an inactive state, since it bound ATP slowly . The far UV CD spectrum of the ncd motor domain devoid of ADP was nearly identical to that of the ncd motor domain with bound ADP . This indicated that the removal of ADP did not affect the backbone structure in the presence of high salt . On the other hand, the near UV CD spectrum of the ADP-free ncd motor domain differed from that of the ncd motor domain . ADP complex, one possibility being that the local conformation was changed upon removal of bound ADP . The near UV CD spectra of kinesin motor domain also showed a difference between the ADP-bound form and the nucleotide-free form, although the difference was much smaller. Appl Microbiol Biotechnol, 1996 Dec, 46(5-6), 610 - 4 Ozone-induced damage of Escherichia coli K-12; Komanapalli IR et al.; Escherichia coli K-12 transformed with pA-CYC184 plasmid DNA was exposed to ozone (O3) in aqueous solution . The damage to the membrane, protein, plasmid DNA, and cell survival were investigated . Cell viability was unaffected by short-term O3 exposure (1-5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation . The intracellular components, protein and DNA, remained intact . With longer durations of O3 exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage . The proteins leaking out were further oxidized by O3 . The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability . The data indicate that membrane components are the primary targets of O3 damage with subsequent reactions involving the intracellular components, protein and DNA. Vet Microbiol, 1996 Dec, 53(3-4), 295 - 302 Macrophage inhibitory factor-A3 derived from Mycobacterium avium serovar 2 inhibits candidacidal activity of murine peritoneal macrophages; Hines ME 2nd et al.; Macrophage inhibitory factor-A3 (MIF-A3), a fraction derived from Mycobacterium avium serovar 2 inhibited candidacidal activity in macrophages from C57BL/6, C57BL/10, C3H/HeJ and A/J strains of mice . Inhibition of candidacidal activity was demonstrated at MIF-A3 concentrations ranging from 100-400 micrograms/ml in macrophages without additional stimulators (exception C3H/HeJ macrophages) and in macrophages additionally stimulated with 200 U/ml interferon-gamma, 100 ng/ml phorbol myristate acetate and 0.4 ng/ml E . coli lipopolysaccharide from all mouse strains tested . The decreased candidacidal effect produced by MIF-A3 was dose-dependent and appeared greatest in macrophages treated with phorbol myristate acetate and lipopolysaccharide . This effect was neutralized by the addition of goat anti-MIF-A3 antiserum . Macrophages from the Bcgs mouse strains (C57BL/6 and C57Bl/100 were more sensitive to the effect(s) of MIF-A3 than macrophages from the Bcgr mouse strains (C3H/HeJ and A/J). Aust Vet J, 1996 Dec, 74(6), 437 - 8 Meningoencephalitis and brain abscessation due to Escherichia coli in a 2 week old alpaca cria; Tsur I et al.; An alpaca cria which received insufficient colostrum, presented with lethargy, anorexia, no passage of faeces and neurological signs . Differential diagnosis included neonatal septicaemia, meningitis and gastrointestinal obstruction . The cria died despite treatment . Necropsy revealed brain abscesses with Escherichia Coli in pure culture . The importance of the amount and timing of colostrum intake is discussed. Carcinogenesis, 1996 Dec, 17(12), 2543 - 50 7-Methoxy-2-nitronaphtho{2,1-b}furan (R7000)-induced mutation spectrum in the lacI gene of Escherichia coli: influence of SOS mutagenesis; Touati E et al.; The mutagenic specificity of 7-methoxy-2-nitronaphtho{2,1-b}furan (R7000), a very potent genotoxic 2-nitrofuran, was investigated in the lacI gene of E.coli . To analyze the influence of SOS-mutagenesis on R7000-induced mutations, 86 and 84 LacI- mutants were respectively isolated from umuC+ and umuC strains . Treatment of bacteria with increasing concentrations of R7000, affected 2-4 times more the survival rate in the umuC context, as compared to umuC+ . 80% of all mutations occurred primarily at G:C base pairs and were substitution events and single-base frameshifts (-1) in the same proportions . The six possible substitution events were observed in both strains . In the umuC+ context, they were dominated by G:C-->T:A transversions . 38% of substitutions at G:C base pairs occurred in the consensus sequence 5'TGGCG3' or 5'TGGC3' where the G was mutated . When umuC was deficient G:C-->C:G transversions were mainly observed . The proportions of substitution mutations were very similar to those that have been reported for apurinic (AP) sites, suggesting strongly that one mechanism for R7000-induced mutations is the formation of intermediate abasic sites that serve as a substrate for error-prone repair . Single frameshift events consisted essentially of deletions of one (G:C) base pair in runs of contiguous G or C residues . Frameshift frequency increased with the length of the reiterated sequence, suggesting a strand-slippage process . Other mutational classes were recovered to a lower extent, including double-base frameshifts and large deletions . In addition, 10% of the mutants presented two proximate mutations . Comparison of the mutations induced by R7000 in the umuC+/umuC backgrounds suggests an influence of the umuC product on strand specificity of R7000-induced mutations, particularly in the case of frameshift events. J Cell Sci, 1996 Dec, 109 ( Pt 13), 3001 - 12 Deficiency of protein phosphatase 2A uncouples the nuclear and centrosome cycles and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos; Snaith HA et al.; A Drosophila strain, carrying a P{lacW} element in the promoter of the protein phosphatase 2A (PP2A) catalytic subunit gene at chromosomal location 28D, has been identified using plasmid rescue of the P element and adjoining genomic DNA in Escherichia coli . Reversion mutagenesis was employed to demonstrate that the observed phenotype of the Drosophila strain was due to a single P{lacW} element insertion at 28D and to create three deficiency strains at this locus . Drosophila heterozygous for P{lacW}28D have reduced levels of PP2A mRNA and reduced PP2A catalytic activity against four different substrates compared to wild type, while homozygotes are deduced to have approximately 20% of wild-type PP2A activity . P{lacW}28D homozygotes, termed microtubule star (mts), die in embryo-genesis around the time of cellularisation, exhibiting over-condensed chromatin and a block in mitosis between prophase and the initiation of anaphase . Multiple centrosomes are visible in cellularised embryos, suggesting that PP2A may play a role in coupling the nuclear and centrosome cycles . When embryos arrest just prior to cellularisation, disorganised elongated arrays of microtubules radiate from centrosomes in all directions, but they are rarely associated with any DNA, suggesting that PP2A is required for the attachment of microtubules to chromosomal DNA at the kinetochore. Plant Mol Biol, 1996 Dec, 32(6), 1117 - 24 Cloning and analysis of the gene for cystathionine gamma-synthase from Arabidopsis thaliana; Kim J et al.; A cDNA clone, CGS1, encoding cystathionine gamma-synthase (CGS) from Arabidopsis thaliana was selected by complementation of CGS mutant strain of Escherichia coli (metB) . Cells expressing CGS1 can grow on medium lacking Met and contain CGS enzyme activity . Genomic DNA blot analysis of A . thaliana revealed that there is a single gene homologous with CGS1 . A genomic fragment carrying CGS1 was cloned and sequenced . Through combined analysis of the cDNA and genomic clone it was determined that the CGS1 coding sequence is 1692 bp, encodes a 563 amino acid, 60 kDa protein, and is interrupted by ten introns . A transcriptional initiation site was detected 260 bp 5' of the initiator codon . The predicted amino acid sequence of CGS1 contains a consensus pyridoxal phosphate-binding site and is similar to MetB of E . coli, with which it is 35 percent identical . The CGS1 product has a sequence at the amino terminus that resembles a transit peptide for localization to plastids . At least 160 amino acids from the amino terminus of the CGS1 enzyme are not essential for enzymatic activity. Acta Paediatr Jpn, 1996 Dec, 38(6), 672 - 6 Enteric pathogens in severe forms of acute gastroenteritis in Ghanaian children; Hori H et al.; Diarrheal disease is the major cause of childhood morbidity in developing countries . Although malnutrition is known as a risk factor for severe gastroenteritis, the role of enteric pathogens in the clinical severity is unclear . The present study was conducted in well nourished Ghanaian preschool children during a 3 month period of the rainy season to assess the relationship between enteric pathogens and severe gastroenteritis . Two hundred and twenty-five children with acute gastroenteritis and 64 age-matched control children were prospectively examined for the severity of dehydration and enteric pathogens in their stools . Of the 225 children with gastroenteritis, 69.8% (157/225) had mild dehydration and 30.2% (68/225) had severe dehydration . Bacteria were similarly isolated in stool samples from children with mild and severe dehydration and controls . Rotavirus accounted for 20.6% of children with severe dehydration and was more often isolated in stools from patients with severe dehydration than those from controls . Furthermore, the mixed infections associated with rotavirus and bacteria were more often found in patients with severe dehydration than those with mild dehydration or controls . Parasites were similarly found at low incidences among the three groups . The present study implied that rotavirus was more responsible for severe gastroenteritis than bacteria or parasites . However, factors other than enteric pathogens must be sought in a considerable number of severe cases . A large scale study throughout a year is recommended to obtain more precise information that would reflect the seasonal variation of rotavirus infections. J Gen Virol, 1996 Dec, 77 ( Pt 12), 3107 - 11 The dUTPases from herpes simplex virus type 1 and mouse mammary tumour virus are less specific than the Escherichia coli enzyme; Bjornberg O et al.; The enzyme dUTPase catalyses the hydrolysis of dUTP to dUMP and pyrophosphate, thereby suppressing incorporation of uracil into DNA and providing a pool of dUMP, the precursor of dTTP . Hydrolysis of other nucleotides similar in structure to dUTP would conceivably be physiologically detrimental and high specificity of the reaction seems to be necessary . In this work, we characterize the substrate specificity of the dUTPases from herpes simplex virus type 1 (HSV-1) and mouse mammary tumour virus (MMTV) in comparison to the Escherichia coli enzyme . We tested dCTP, dTTP, UTP and dUDP as substrates . Significantly higher reactivity was observed for the HSV-1 enzyme with dCTP and dTTP and for the MMTV enzyme with dTTP and UTP . The lower substrate specificity of the two virus enzymes compared with the bacterial enzyme is discussed in relation to the DNA precursor metabolism during virus replication. Chem Biol, 1996 Dec, 3(12), 1033 - 8 Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids; Cload ST et al.; BACKGROUND: Chemically aminoacylated suppressor tRNAs have previously been used in vitro to generate mutant proteins in which unnatural amino acids are incorporated site-specifically . Although the existing methodology often provides adequate quantities of mutant proteins, the suppression efficiencies of some unnatural amino acids are not high enough to yield useful amounts of protein . In an effort to make this useful mutagenesis strategy more general, we report here the results of a search to find alternative tRNAs as a way of increasing suppression efficiencies . RESULTS: Three suppressor tRNAs have been generated by runoff transcription and their ability to deliver unnatural amino acids site-specifically into proteins has been assessed in an E . coli-derived in vitro transcription/translation system . Analysis of their ability to insert both polar and nonpolar residues in response to an amber codon in two proteins suggests that an E . coli tRNAAsn-derived suppressor offers a significant improvement in suppression efficiency over other previously used tRNAs . CONCLUSIONS: Use of an E . coli tRNAAsn-derived suppressor may provide substantially higher yields of proteins containing unnatural amino acids, in addition to offering a broader tolerance for polar amino acids . A comparison of suppressor tRNAs derived from tRNAAsn, tRNAGln or tRNAAsp with that derived from tRNAPhe supports emerging evidence that the identity of an amino acid may be important in message recognition. Chem Biol, 1996 Dec, 3(12), 981 - 91 The leucine zipper domain controls the orientation of AP-1 in the NFAT.AP-1.DNA complex; Erlanson DA et al.; BACKGROUND: Heterologous transcription factors bound to adjacent sites in eukaryotic promoters often exhibit cooperative behavior . In most instances, the molecular basis for this cooperativity is poorly understood . Our efforts have been directed toward elucidation of the mechanism of cooperativity between NFAT and AP-1, two proteins that coordinately direct expression of the T-cell growth factor interleukin-2 (IL-2) . RESULTS: We have previously shown that NFAT1 orients the two subunits of AP-1, c-Jun and c-Fos, on DNA through direct protein-protein interactions . In the present study, we have constructed cJun-cFos chimeric proteins and determined their orientation using a novel affinity-cleavage technology based on chemical ligation . We find that, in the presence of NFAT, the chimeric heterodimer binds in such a way as to preserve the orientation of the AP-1 leucine zipper, but not that of the basic region . CONCLUSIONS: Protein-protein interactions between NFAT and the leucine zipper of AP-1 enable the two proteins to bind DNA cooperatively and coordinately regulate the IL-2 promoter . The chemical ligation technology presented here provides a powerful strategy for affinity cleavage studies, including those using recombinant proteins. Biol Chem, 1996 Dec, 377(12), 811 - 7 Increased stability of phage T7g10 mRNA is mediated by either a 5'- or a 3'-terminal stem-loop structure; Mertens N et al.; The mRNA encoding the major capsid protein of phage T7 (T7g10) is highly expressed in Escherichia coli . In common with other highly expressed T7 genes, the 5' end of this mRNA contains a stem-loop structure, while transcription termination at the phage T7 T phi terminator generates a stable 3'-end stem-loop structure . We assessed the influence of these structures on the expression level of T7g10 and on the functional stability of the mRNA . Each one of the 5'- or 3'-hairpin structures was sufficient to increase the functional stability of the T7g10 mRNA more than twofold . A duplication of the 3' T phi-terminator slightly increased the mRNA stability further . Also, differences in the observed functional half-life could be correlated with the expression level of the T7g10 derivatives when these were partially induced . Our data suggest that mRNA stabilization by a 5' stem-loop structure can occur even in the absence of a stem-loop structure that protects RNA against 3' exonucleases. Am J Physiol, 1996 Dec, 271(6 Pt 2), H2529 - 35 HSP induction inhibits iNOS mRNA expression and attenuates hypotension in endotoxin-challenged rats; Hauser GJ et al.; Endotoxin (lipopolysaccharide, LPS)-induced hypotension is, in part, mediated via induction of nitric oxide synthase (iNOS), release of nitric oxide, and suppression of vascular reactivity (vasoplegia) . Induction of heat shock proteins (HSP) or inhibition of iNOS expression improves survival in LPS-challenged rodents . We studied the effect of induction of HSP on LPS-mediated iNOS expression and on LPS-induced vasoplegia and hypotension . Rats were treated with the HSP inducer sodium arsenite (6 mg/kg iv) or saline control . Seventeen hours later, rats were challenged intravenously with 10 mg/kg of Escherichia coli LPS O127:B8 or saline control . Arsenite pretreatment resulted in expression of HSP 70 mRNA and of HSP 70 and heme oxygenase-1 proteins, inhibition of LPS-mediated iNOS mRNA induction, reversal of the LPS-induced hyporesponsiveness to norepinephrine ex vivo in isolated mesenteric arteries, and attenuation of LPS-induced hypotension in vivo . Our data suggest that induction of HSP expression protects rats from LPS by blocking LPS-induced iNOS expression, leading to inhibition of the overproduction of nitric oxide and thereby reversing LPS-induced vasoplegia and LPS-induced hypotension. Am J Physiol, 1996 Dec, 271(6 Pt 1), G993 - 1002 HIP/PAP is an adhesive protein expressed in hepatocarcinoma, normal Paneth, and pancreatic cells; Christa L et al.; Human hepatocarcinoma-intestine-pancreas (HIP) cDNA, isolated from a hepatocellular carcinoma, encodes a C-type lectin . According to published cDNA sequences, HIP protein is identical to human pancreatitis-associated protein (PAP) . In these sequences, a putative signal peptide and the carbohydrate recognition domain (CRD) can be recognized . In the present study, we established transgenic mice to drive the production of soluble recombinant HIP/PAP protein in the milk of lactating animals; using this model, we showed that HIP/PAP protein was secreted after suitable cleavage of the potential signal peptide . Moreover, we also produced HIP/PAP protein by Escherichia coli cultures performed to generate specific antibodies . These antibodies enabled the detection of HIP/PAP protein in normal intestine and pancreas (both in endocrine and exocrine cells), e.g., intestinal neuroendocrine and Paneth cells, pancreatic islets of Langerhans, and acinar cells . HIP/PAP protein was also identified in the cytoplasm of tumoral hepatocytes but not in nontumoral hepatocytes . Finally, HIP/PAP protein activity was tested and we showed that HIP/PAP induced the adhesion of rat hepatocytes and bound strongly to extracellular matrix proteins (laminin-1, fibronectin), less strongly to type I and IV collagen, and not at all to heparan sulfate proteoglycan . In conclusion, these results showed that HIP/PAP protein was matured on secretion . We also demonstrated that HIP/PAP protein was specifically expressed in hepatocarcinoma cells and interacted with rat hepatocytes and the extracellular matrix . Taken overall, these results suggest that HIP/PAP protein may be of potential importance to liver cell differentiation/proliferation. Am J Physiol, 1996 Dec, 271(6 Pt 1), G959 - 68 Structure, glycosylation, and localization of rat intestinal guanylyl cyclase C: modulation by fasting; Scheving LA et al.; Guanylyl cyclase C (GC-C), an intestinal receptor guanylyl cyclase, binds diarrhea-producing bacterial ligands such as the Escherichia coli heat stable enterotoxin . We examined the regulatory influence of feeding and fasting on the expression, structure, and biochemical properties of GC-C . When solubilized at 4 degrees C under nonreducing conditions, GC-C from both fed and fasted rats migrated on 7% sodium dodecyl sulfate-polyacrylamide electrophoretic gels as two extremely large aggregates that barely penetrated the stacking and resolving gels . Chemical reduction of disulfide linkages disaggregated GC-C in fed but not fasted rat samples, causing it to migrate as smaller forms (approximately 220 and 240 kDa) . Although GC-C aggregates from fasted rats resisted this disaggregating effect of chemical reduction, they rapidly acquired it within 90 min of refeeding . When solubilized at denaturing temperatures (95 degrees C) under reducing conditions, GC-C aggregates largely disassembled into four smaller proteins (relative molecular weight approximately 140,000, 131,000, 85,000, and 65,000) . However, the 131-kDa glycoprotein was disproportionately increased in fasted rat membranes . This unit and the 220-kDa unit were sensitive to endoglycosidase H . Subcellular fractionation and immunohistochemical studies revealed a major redistribution of GC-C from surface to intracellular enterocyte sites during fasting. Curr Opin Struct Biol, 1996 Dec, 6(6), 848 - 58 Glutamine repeats and inherited neurodegenerative diseases: molecular aspects; Perutz MF; Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins . These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown . In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats . Protein constructs with more than 41 repeats are toxic to E . coli and to CHO cells in culture, and they elicit ataxia in transgenic mice . These observations argue in favour of a distinct change of structure associated with elongation beyond 37-41 glutamine repeats . The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part . Poly-L-glutamines form pleated sheets of beta-strands held together by hydrogen bonds between their amides . Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers . That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping . Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins . Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures. Curr Opin Struct Biol, 1996 Dec, 6(6), 744 - 56 The relationship between structure and function for the sulfite reductases; Crane BR et al.; The six-electron reductions of sulfite to sulfide and nitrite to ammonia, fundamental to early and contemporary life, are catalyzed by diverse sulfite and nitrite reductases that share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an Fe4S4 cluster . The recently determined crystallographic structure of the sulfite reductase hemoprotein from Escherichia coli complements extensive biochemical and spectroscopic studies in revealing structural features that are key for the catalytic mechanisms and in suggesting a common symmetric structural unit for this diverse family of enzymes. Curr Biol, 1996 Dec 1, 6(12), 1599 - 608 GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion; Straight AF et al.; BACKGROUND: Precise control of sister chromatid separation is essential for the accurate transmission of genetic information . Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved . Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint . Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy . RESULTS: We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells . A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter . Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B . In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules . The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate . CONCLUSIONS: We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together. Curr Biol, 1996 Dec 1, 6(12), 1573 - 6 Molecular chaperones: clasping the prize; Gething MJ; The three-dimensional structure of the substrate-binding domain of DnaK, a bacterial Hsp70, shows how such molecular chaperones can be so promiscuous in recognizing different proteins, yet so accurate in discriminating between unfolded and folded forms of their polypeptide substrates. Crit Rev Biochem Mol Biol, 1996 Dec, 31(5-6), 405 - 47 DNA repair functions in heterologous cells; Memisoglu A et al.; Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself . The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum . Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E . coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells . The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways . In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms. Crit Rev Biochem Mol Biol, 1996 Dec, 31(5-6), 361 - 80 Chemistry and biology of DNA methyltransferases; Ahmad I et al.; Recognition of a specific DNA sequence by a protein is probably the best example of macromolecular interactions leading to various events . It is a prerequisite to understanding the basis of protein-DNA interactions to obtain a better insight into fundamental processes such as transcription, replication, repair, and recombination . DNA methyltransferases with varying sequence specificities provide an excellent model system for understanding the molecular mechanism of specific DNA recognition . Sequence comparison of cloned genes, along with mutational analyses and recent crystallographic studies, have clearly defined the functions of various conserved motifs . These enzymes access their target base in an elegant manner by flipping it out of the DNA double helix . The drastic protein-induced DNA distortion, first reported for HhaI DNA methyltransferase, appears to be a common mechanism employed by various proteins that need to act on bases . A remarkable feature of the catalytic mechanism of DNA (cytosine-5) methyltransferases is the ability of these enzymes to induce deamination of the target cytosine in the absence of S-adenosyl-L-methionine or its analogs . The enzyme-catalyzed deamination reaction is postulated to be the major cause of mutational hotspots at CpG islands responsible for various human genetic disorders . Methylation of adenine residues in Escherichia coli is known to regulate various processes such as transcription, replication, repair, recombination, transposition, and phage packaging. J Cell Biol, 1996 Dec, 135(6 Pt 2), 1727 - 39 Cell fusion during yeast mating requires high levels of a-factor mating pheromone; Brizzio V et al.; During conjugation, two yeast cells fuse to form a single zygote . Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material . The two plasma membranes then fuse to produce a continuous cytoplasm . We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory . Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion . Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent . Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain . FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor . Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype . First, overexpression of a-factor in the fus mutants suppressed the Fus- defect . Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans . Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating . We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion. Proteins, 1996 Dec, 26(4), 459 - 64 Computational design of a substrate specificity mutant of a protein; Honda N et al.; The wild-type trp repressor of E . coli bound 5-methoxytryptophan, a Trp analogue, less tightly than Trp . A mutant repressor (Val58-->Ala) that should bind 5-methoxytryptophan preferentially to Trp was computationally designed by free-energy calculations accompanied by free-energy decomposition . The designed mutant was demonstrated by experiments to bind 5-methoxytryptophan more tightly than Trp, consistent with the computational prediction . This success indicates the usefulness of free energy decomposition in protein design. Plant Cell, 1996 Dec, 8(12), 2381 - 94 Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control; Qian D et al.; Farnesylation is required for membrane targeting, protein-protein interactions, and the biological activity of key regulatory proteins, such as Ras small GTPases and protein kinases in a wide range of eukaryotes . In this report, we describe the molecular identification of a plant protein farnesyltransferase (FTase) and evidence for its role in the control of the cell cycle in plants . A pea gene encoding a homolog of the FTase beta subunit was previously cloned using a polymerase chain reaction-based strategy . A similar approach was used to clone a pea gene encoding a homolog of the FTase alpha subunit . The biochemical function of the pea FTase homologs was demonstrated by the reconstitution of FTase enzyme activity using FTase fusion proteins coexpressed in Escherichia coll . RNA gel blot analyses showed that levels of FTase mRNAs are generally higher in tissues, such as those of nodules, that are active in cell division . The relationship of FTase to cell division was further analyzed during the growth of suspension-cultured tobacco BY-2 cells . A biphasic fluctuation of FTase enzyme activity preceded corresponding changes in mitotic activity at the early log phase of cell growth . Moreover, manumycin, a specific inhibitor of FTase, was effective in inhibiting mitosis and growth in these cells . Using synchronized BY-2 cells, manumycin completely blocked mitosis when added at the early S phase but not when added at the G2 phase . These data suggest that FTase is required for the plant cell cycle, perhaps by modulating the progression through the S phase and the transition from G1 to the S phase. FEMS Microbiol Rev, 1996 Dec, 19(2), 85 - 116 Flux analysis and control of the central metabolic pathways in Escherichia coli; Holms H; The growth of the bacterial cell involves the co-ordination of the fluxes of carbon into a considerable diversity of products that are the components of the cell . Fortunately the monomers from which the cell's polymers are made are themselves synthesised from a relatively small group of precursors that are the products of the central metabolic pathways . This simplification renders cell metabolism accessible to flux analysis, a method for handling experimental data to derive metabolic fluxes . Through such analysis of the growth of Escherichia coli ML308 on 11 single carbon sources in batch, turbidostat or chemostat culture general patterns are discernible . Most significant among these are that growth on different carbon sources is achieved without any obvious enzyme acting as a regulator of metabolic flux, except when acetate is the sole source of carbon . In this case a junction is created at which iso citrate dehydrogenase (ICDH) and isocitrate lyase (ICL) compete for their common substrate and this competition is resolved by partial inactivation of ICDH to match flux through ICL and this balance limits growth rate . In this sense, flux through ICDH and ICL is 'rate-limiting' . Uptake of six of the remaining carbon inputs exceeds the capacity of the central metabolic pathways (CMPs) to sustain flux to the precursors required for growth and the CMPs are balanced by excretion of acetate . Restriction of carbon uptake by chemostat progressively diminishes growth rate and acetate excretion until acetate excretion is prevented . For the four remaining carbon sources, uptake is apparently restricted and the products are biomass, carbon dioxide and water . Carbon sources feeding the phosphorylated parts of the CMPs flux relatively more carbon to precursors (Pre-C) than CO2 when compared with carbon sources which feed into the non-phosphorylated pathways . Pre-C/CO2 ratios for the former are 1.73-3.91 and for the latter are 0.46-0.78 . Flux analysis of all 11 carbon sources shows that there is an overabundant supply of 'energy' (ATP + {2H}), generated by the CMPs, in all phenotypes and conditions down to a glucose chemostat at mu of 0.72 . This excess energy is a thermodynamic inefficiency which must be dissipated as heat . E . coli ML308 probably evolved in circumstances of 'feast' and 'famine' . The two strategies selected (excretion of surplus carbon and restriction of mu) would appear to be defences against 'feast' . Presumably there are defences against 'famine' . These are not made obvious by flux analysis but allosteric control of irreversible enzymes would protect pools of essential nutrients from rapid depletion on the sudden onset of 'famine'. Biochem Mol Med, 1996 Dec, 59(2), 187 - 91 Immunodetection of mitochondrial glycerophosphate dehydrogenase (mGDH) by a polyclonal antibody raised against a recombinant mGDH fragment product; Novials A et al.; The mitochondrial enzyme glycerophosphate dehydrogenase (mGDH) plays an essential role in the B-cell glucose-sensing device and its activity in islet homogenates is impaired in several animal models of type 2 diabetes . We have now developed a polyclonal antibody, raised against a recombinant mGDH fragment product, that could be used for the immunodetection of mGDH . Total RNA was isolated from rat pancreatic islets and used in the synthesis of cDNA . Specific primers were designed that corresponded to the FAD binding domain of mGDH . The PCR product was purified and cloned into an appropriate expression vector used for transformation of Escherichia coli cells . The fusion protein was extracted from the transformed cells, further purified, and used for immunization of rabbits . The antibody recognized a single band of 72 kDa in rat islets and testis . The recombinant mGDH product was also recognized as a single band with the expected 65-kDa reference . An ELISA procedure was designed for detection of antibodies against the recombinant mGDH fragment product . The availability of the mGDH antibody opens the way to a number of further applications such as immunocytochemis- try and mGDH quantification in biological material. Hybridoma, 1996 Dec, 15(6), 429 - 33 Characterization of monoclonal antibodies to the 26-kDa glutathione S-transferase of Schistosoma japonicum; Yan BS et al.; Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum . These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E . coli by an enzyme-linked immunosorbent assay . A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum . However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding . The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum . Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation . In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs . In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes . Thus, these select MAbs readily reacted with GST although their binding characteristics were different . Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology. DNA Cell Biol, 1996 Dec, 15(12), 1113 - 20 Construction of portable intron cassettes for the delivery and expression of foreign genes; Reilly JD et al.; The use of viral vectors to deliver foreign genes offers some promise of generating new and more efficacious vaccines . However, the insertion of foreign genes into viral genomes often results in the insertional mutagenesis of one or more genes that adversely affect replication . In an attempt to overcome this problem, we constructed two portable intron cassettes . The cassettes were derived from the adenovirus late leader 1 intron and were cloned into either the chloramphenicol acetyltransferase (CAT) gene or the LacZ gene of Escherichia coli . The intron cassettes were transfected into chicken embryo fibroblasts (CEFs) and the cell lysates were later assayed for either beta-galactosidase (beta-Gal) or CAT activity . The first intron cassette (type A) contained flanking adenovirus exon sequences . Consequently, the flanking adenovirus exon sequences remained in the spliced transcript . With the type A intron inserted in the correct orientation for splicing, CAT activity was not diminished . However, in the reverse orientation, no CAT activity could be detected . The second intron cassette (type B) had the splice donor and splice acceptor sites converted to the blunt-end restriction endonuclease sites Pml I and Pvu II, respectively . The blunt-end restriction endonuclease sites enabled the portable intron to be removed from the flanking adenovirus exon sequences and inserted into any blunt-end restriction endonuclease site in the recipient gene . After splicing, no adenovirus exon sequences remained in the recipient gene's RNA transcript . To demonstrate its usefulness, an insertion cassette was made by cloning the E . coli LacZ gene into a multiple cloning site within the type B intron . The insertion cassette was then cloned into a Pvu II site in the middle of the CAT gene . Following transfection in CEFs, high levels of both CAT and beta-Gal were detected, demonstrating that both genes were properly transcribed and translated. Plant Mol Biol, 1996 Dec, 32(5), 979 - 86 Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana; Fecker LF et al.; The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli . For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs . The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants . The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells . The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants . In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E . coli or from plants . The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots. Plant Mol Biol, 1996 Dec, 32(5), 937 - 45 Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3; Lin Q et al.; A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3chlM, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3chlH, molecular mass 22 829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins . The homology domain can be expressed in reasonable levels in E . coli . However, IF-3chlM is quite toxic and can only be produced in small amounts . Both forms of the chloroplast factor are associated with E . coli ribosomes . Purification procedures have been developed for both IF-3chlM and IF-3chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography . IF-3chlM and IF-3chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E . coli ribosomes . However, IF-3chlH has at least 5-fold more activity than either native IF-3chl or IF-3chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal . This observation suggests that regions of IF-3chl lying outside of the homology domain may down-regulate the activity of this factor. Genetics, 1996 Dec, 144(4), 1337 - 41 Adaptive mutation and slow-growing revertants of an Escherichia coli lacZ amber mutant; Prival MJ et al.; We have studied revertants, selected on lactose minimal agar medium, of the Escherichia coli lacZam strain that was first used by Cairns and his colleagues to demonstrate the phenomenon of "adaptive mutation." We have found, by performing appropriate reconstruction studies, that most of the late-arising Lac+ revertants of this lac amber strain (appearing as colonies in 3-5 days) are slow-growing ochre suppressor mutants that probably existed in the culture prior to plating and cannot, therefore, be classified as "adaptive." The appearance of a small number of fast-growing, late-arising Lac+ revertants may result from residual cell growth and turnover or from phenomena related to the fact that the lacZam mutation in strain SM195 is carried on an F' plasmid . Thus, the appearance of late-arising revertants in this lacZam system does not provide convincing evidence that selective conditions specifically increase the rate of occurrence of favorable mutations. J Crit Care, 1996 Dec, 11(4), 167 - 75 Exhaled nitric oxide as a marker for serum nitric oxide concentration in acute endotoxemia; Hussain SN et al.; PURPOSE: The main aim of this study was to assess the correlation between exhaled nitric oxide (NO) and serum NO concentrations during the course of endotoxemia . We also assessed whether or not the inducible isoform of NO synthase is responsible for the increase in NO production in endotoxemia animals . MATERIALS AND METHODS: Anesthetized and mechanically ventilated dogs were injected with either saline (control) or Escherichia coli endotoxin (LPS {Lipopolysaccharides}), and the animals were sacrificed 150 minutes later . We measured hemodynamics, exhaled NO, and serum arterial and mixed venous NO concentrations . Western blotting was performed on lung, pulmonary artery, aorta, and kidney tissue samples using anti-inducible NO synthase antibody . RESULTS: Arterial pressure, cardiac output, and pulmonary arterial pressure in the control group remained unchanged, whereas a significant decline in these parameters was observed in the LPS group . Exhaled NO and serum arterial NO concentrations rose significantly within 30 minutes of endotoxin injection and remained higher than baseline values, whereas mixed venous serum NO did not change from baseline values . There was a significant linear relationship between exhaled NO and arterial serum NO concentrations . By comparison, exhaled NO, and arterial and mixed venous serum NO levels remained unchanged in the control group . Western blotting showed no expression of inducible NO synthase (iNOS) isoform in the control or LPS groups . CONCLUSIONS: These results suggest that exhaled NO accurately reflects changes in arterial serum NO concentration and that the source of enhanced NO release in acute endotoxemia is not the iNOS isoform. Eur J Immunol, 1996 Dec, 26(12), 3029 - 34 Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells; Samuelsson A et al.; Human antibodies against HIV-1 have been sought to study neutralization events on the molecular level, and for possible use in passive immune intervention . The development of phage display techniques has opened the possibility of rapidly generating human monoclonal antibodies with desired specificities . We and others have isolated human HIV-1 neutralizing antibody fragments using this technique . Bacterial expression of isolated clones does, however, differ broadly both in expression levels and functional activity . In addition, intact IgG cannot be expressed in bacteria . By transferring the genes of isolated Fab clones to a mammalian expression system we could perform a comparison of functional activity between Fab expressed in bacterial and mammalian cells, as well as Fab and whole IgG . Fab fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in Escherichia coli, underlining the inefficiency of procaryotic expression . No difference in HIV-1 neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells . Thus, bivalency does not always confer improved neutralization efficacy. Protein Sci, 1996 Dec, 5(12), 2638 - 42 Cloning, expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor; Lebedeva MI et al.; T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs) . These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex . It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography . In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown . We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli . Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis . The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A . and diffract to beyond 2.3 A resolution . The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs. Protein Sci, 1996 Dec, 5(12), 2552 - 65 NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin; De Lorimier R et al.; Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates . We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR . Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain . The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type . Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K . The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type . Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins . There is also evidence of greater conformational exchange in the mutant . Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation . We infer that mispacking of the protein core in one location affects local dynamics and stability throughout. Protein Sci, 1996 Dec, 5(12), 2532 - 44 Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy; Reynaldo LP et al.; Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS) . These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema . Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+ . The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12 . Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A . Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E . coli GS . The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12 . The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively . Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12 . Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12) . The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities. J Interferon Cytokine Res, 1996 Dec, 16(12), 1073 - 8 Regulated expression of the interferon-induced protein kinase p68 (PKR) by vaccinia virus recombinants inhibits the replication of vesicular stomatitis virus but not that of poliovirus; Lee SB et al.; A direct antiviral role of the interferon-induced human protein kinase p68 has been shown only against encephalomyocarditis virus (EMCV) and vaccinia virus (VV) . To determine if p68 kinase (PKR) has a broad antiviral effect, we have used coinfections between VV recombinants expressing p68 kinase under regulation of the lac I operator/repressor elements of Escherichia coli and two RNA viruses, vesicular stomatitis virus (VSV) and poliovirus . In cells coinfected with VV recombinants and VSV, induction with isopropyl-B-D-thiogalactoside (IPTG) of wild-type p68 kinase or a mutant lacking the dsRNA binding domain resulted in inhibition of both VV and VSV protein synthesis . This inhibition is not observed in cells infected with a catalytically inactive point mutant lys-arg296 of p68 kinase . When cells are coinfected with VV recombinants and poliovirus, induction of active p68 kinase resulted in a decrease in VV proteins but not in poliovirus proteins or poliovirus yields . Immunoblot analysis revealed that p68 kinase was expressed during mixed infections . Our results demonstrate a differential effect of p68 kinase on the replication of VV, VSV, and poliovirus . We suggest that in a particular virus-cell system, the different sensitivity of a virus to p68 kinase is probably due to levels of active enzyme. J Interferon Cytokine Res, 1996 Dec, 16(12), 995 - 1000 Injection time of interleukin-6 determines fatal outcome in experimental endotoxin shock; Yoshizawa K et al.; Circulating interleukin-6 (IL-6) levels are directly correlated to fatal outcome in both patients and animal models with endotoxin shock . However, whether IL-6 is deleterious or protective in regard to survival is obscure . We investigated the action of IL-6 in the pathogenic progress of endotoxin shock . C3H/HeN mice received 10 micrograms of natural human IL-6 (Hu-IL-6) s.c . at various times before or after challenge with Escherichia coli lipopolysaccharide (LPS) at a lethal dose . Pretreatment with Hu-IL-6 0, 1, and 4 h before LPS administration improved the survival rate of the mice . However, no protection was observed when Hu-IL-6 was administered 24 h before or 1 h after the LPS injection . The protective mechanism of Hu-IL-6 pretreatment was not explained on the changes in the circulating levels of tumor necrosis factor and endogenous murine IL-6 (Mu-IL-6) . The induction of fibrinogen and immunosuppressive acidic protein, a type of acute-phase proteins, may have contributed to the protection . The results show that the order and the time interval in which the administered Hu-IL-6 and the Mu-IL-6 induced by LPS act are the key to the determination of fatal outcome. Eur J Biochem, 1996 Dec 1, 242(2), 315 - 9 Reexamination of hormone-binding properties of protein disulfide-isomerase; Guthapfel R et al.; Protein disulfide-isomerase (PDI), an abundant multifunctional protein, has been described as a 3,3',5-triiodo-L-thyronine (T3)-binding protein . As pointed out by several authors, the physiological significance of this hormone-binding property has not been fully addressed . To clarify this point, we have analyzed the T3-binding properties of purified PDI . At equilibrium, T3 binds PDI at two binding sites: first, at a high-affinity site with a Kd of 21 nM and a Bmax of 1.8 x 10(-3) mol T3/mol PDI monomer, and second at a very low affinity site that is unsaturated up to 100 microM T3 . Thus, T3 binding is mainly non-specific and the specific part represents only about 0.2% of the protein monomer . Cross-linking experiments at a concentration where mainly specific binding occurs indicate that PDI does not bind L-T3 exclusively; a wide variety of analogs are also bound . Refolding of reduced denatured ribonuclease A by PDI is inhibited by T3 and analogs, and the inhibition profile reflects the binding properties very closely . Since purified PDI displays neither the specificity expected for a physiological receptor, nor significant T3-binding activity, results are discussed in terms of a necessary PDI association with another component to form a T3 receptor. Eur J Biochem, 1996 Dec 1, 242(2), 228 - 34 Heterologous expression and site-directed mutagenesis of the 1-aminocyclopropane-1-carboxylate oxidase from kiwi fruit; Lay VJ et al.; A system has been developed for the expression in Escherichia coli of 1-aminocyclopropane-1-carboxylate (ACC) oxidase from kiwi fruit . In this first report of site-directed mutagenesis of ACC oxidase, seven different mutants of the enzyme have been expressed, and their activities compared to that of the heterologoulsy expressed wild-type enzyme . No great loss of activity was observed when Lys172 was substituted by either Ala or Cys, or when Gly137 was substituted by Pro . However, the mutant proteins showed only 1% of the wild-type activity when substitutions were made of Asp179, His177, and Lys158 . The results are discussed in terms of possible mechanisms by which ACC oxidase is activated by carbon dioxide, and in terms of structural motifs suggested by the known structure of isopenicillin N-synthase, an enzyme related by mechanism and sequence similarity to ACC oxidase . It is concluded that Lys172, a putative carbon dioxide binding site, has no role to play in the catalytic activity of the enzyme . The results support a previous suggestion that ACC oxidase shares important structural features with isopenicillin N-synthase. Eur J Biochem, 1996 Dec 1, 242(2), 186 - 90 Biochemical studies of two rat acyl-CoA synthetases, ACS1 and ACS2; Iijima H et al.; Two types of acyl-CoA synthetase (ACS), designated ACS1 and ACS2, are structurally similar isozymes with different tissue distributions . The two enzymes are organized into the following five regions: an NH2 terminus; two luciferase-like regions; a linker connecting the luciferase-like regions; a COOH terminus . Under the control of a lac promoter, rat ACS1 and ACS2 were overproduced in Escherichia coli and purified to homogeneity . The specific activities of the purified ACS1 and ACS2 were 26.2 mumol.min-1.mg-1 and 7.4 mumol.min-1.mg-1, respectively, and the most efficiently utilized saturated fatty acids were those with 10-18 carbon atoms . Among unsaturated fatty acids with 16-22 carbon atoms, the most preferred substrates were palmitoleate, oleate and linoleate for ACS1, and, for ACS2, oleate, arachidonate, eicosapentaenoate and docosahexaenoate . To determine the functionally important regions in the ACS isozymes, we constructed five ACS1 mutants lacking each of the five regions . Introduction of these mutants into E . coli revealed that all five regions in ACS1 are required for functional expression of the enzyme in E . coli; deletion of any one of the five regions almost completely abolished the enzyme activity. Clin Exp Immunol, 1996 Dec, 106(3), 534 - 40 Different expression of IL-2 receptor alpha-chain on a lamina propria T cell population and goblet cells in rats orally tolerized or sensitized to ovalbumin (OA) after colonization with an OA-producing Escherichia coli; Dahlman-Hoglund A et al.; The aim of this study was to compare the local gut immune response in sensitized and orally tolerized experimental animals . The development of IgE/IgG antibodies and the DTH to OA was studied in rats made orally tolerant to OA and compared with sensitized control rats after colonization with an Escherichia coli genetically engineered to produce OA . At 3 weeks of age, pups were weaned onto a standard diet without OA or an OA-containing diet for 4 weeks and then switched to a standard diet without OA . Both groups of rats were parenterally immunized with a mixture of OA and human serum albumin (HSA) in Freund's complete adjuvant when they were 8 weeks old . After DTH measurement 2 weeks later, all rats were colonized with an E . coli producing OA for 5 days . The local immune response in the small intestine was assessed, using immunohistochemistry, as the expression of MHC class II molecules and IL-2 receptor (IL-2R) alpha-chain . The OA-tolerant rats showed the classical signs of oral tolerance, with a reduced IgE and IgG antibody and DTH response to OA before colonization . The difference between the two groups in the anti-OA antibody response became even more pronounced after colonization with the E . coli that produce OA . Rats orally tolerant to OA maintained a normal villus architecture after colonization, with a normal expression of MHC class II molecules similar to non-treated adult rats, but with a significantly higher (P = 0.004) expression of IL-2R alpha-chain on T cells in the lamina propria of the villus core compared with sensitized control rats . The tolerant rats showed a very weak staining with the anti-IL-2R alpha-chain-specific antibody on a few goblet cells in only one out of seven rats . In the sensitized control rats, a marked local immune response was seen with an intense staining with a monoclonal anti-IL-2R alpha-chain-specific antibody on goblet cells in five out of seven rats (P = 0.019) and also an increased expression of MHC class II molecules in the epithelial cells and cells in the lamina propria of all rats . Rats orally tolerant to OA maintained a normal villus architecture after colonization, but with a significantly higher (P = 0.004) expression of IL-2R alpha-chain on T cells in the lamina propria of the villus core compared with sensitized control rats . The novel finding that goblet cells express IL-2R alpha-chain and the striking difference in expression of the receptor and the numbers of goblet cells between tolerant and sensitized rats may suggest a direct T cell regulation of the goblet cells . A possibility that oral tolerance might be maintained by the activated T cells expressing IL-2R alpha-chain in the lamina propria of the villus core is also discussed. Biochem J, 1996 Dec 1, 320 ( Pt 2), 519 - 30 A novel trans-spliced mRNA from Onchocerca volvulus encodes a functional S-adenosylmethionine decarboxylase; Da'Dara AA et al.; Complete cDNA and genomic sequences encoding the Onchocerca volvulus S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, have been isolated and characterized . The deduced amino acid sequence encodes a 42 kDa proenzyme with a moderate level of sequence homology to eukaryotic SAMDCs . Enzymically active O . volvulus SAMDC was expressed at a high level in an Escherichia coli mutant strain lacking endogenous SAMDC . The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)-Sepharose and Superdex S-200 chromatography . It was determined that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits . The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87 . Gel-filtration experiments demonstrated that these two subunits combine to form an active heterotetramer . Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes both cis- and trans-splicing in its 5'-untranslated region (UTR) . Anchored PCR on O . volvulus mRNA confirmed the cDNA sequence and identified two distinct trans-spliced products, a 22-nucleotide spliced-leader sequence and a 138 bp sequence containing the 22 nucleotide spliced-leader sequence . Genomic Southern-blot analysis suggests that the O . volvulus SAMDC is encoded by a single-copy gene . This gene spans 5.3 kb and is comprised of nine exons and eight introns . The first intron is located in the 5'-UTR and processing of this intron has a potential regulatory function . The 5'-flanking region of the gene contains potential transcriptional regulatory elements such as a TATA box, two CAAT boxes and AP-1-, C/EBP-, ELP-, H-APF-1-, HNF-5- and PEA3-binding sites. Biochem J, 1996 Dec 1, 320 ( Pt 2), 493 - 8 Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase; Sweetlove LJ et al.; The aim of this work was to use tubers from transgenic lines of potato (Solanum tuberosum) containing increased amounts of ADPglucose pyrophosphorylase to study the role of this enzyme in the control of starch synthesis . A 4-5-fold increase in activity of the enzyme, achieved by transformation with the Escherichia coli ADPglucose pyrophosphorylase gene glgC-16, had no detectable effect on the starch content of developing or mature tubers . No significant effects were found on the contents of ADPglucose, UDPglucose, glucose 1-phosphate, glucose 6-phosphate, PP1, ATP and ADP . Flux from {U-14C}sucrose, supplied to tubers still attached to the plant, to starch increased roughly in proportion to the increase in ADPglucose pyrophosphorylase activity . These measurements of flux gave a response coefficient close to 1 for the activity of the pyrophosphorylase in respect of starch synthesis . Pulse-chase experiments with {U-14C}sucrose showed that the increased flux into starch in the transformed tubers was accompanied by an increased rate of starch turnover . Further experiments suggested that the increased turnover was associated with an increase in the capacity of the tubers to degrade starch. Biochem J, 1996 Dec 1, 320 ( Pt 2), 487 - 92 Characterization of transgenic potato (Solanum tuberosum) tubers with increased ADPglucose pyrophosphorylase; Sweetlove LJ et al.; The aim of the work described in this paper was to characterize the tubers of potato (Solanum tuberosum var . Prairie) plants that had been transformed with the Escherichia coli ADPglucose pyrophosphorylase (EC 2.7.7.27) gene, glgC-16, under the control of a patatin promoter . Over 30 lines of transformed plants with increased ADPglucose pyrophosphorylase activity were obtained . The tubers of six of these lines were compared with those of control plants expressing the gene for beta-glucuronidase . The average increase in pyrophosphorylase activity was 200%, and the highest was 400% . Western immunoblotting of tuber extracts showed that the amounts of glgC-16 protein were linearly related to the extractable activity of the ADPglucose pyrophosphorylase . Cell fractionation studies showed that the increased activity of the pyrophosphorylase in the glgC-16 tubers had a similar intracellular location, the amyloplast fraction, to that found in the control tubers . No pleiotropic changes in the maximum catalytic activities of the following enzymes could be detected in the glgC-16 tubers: sucrose synthase, fructokinase, UDPglucose pyrophosphorylase, phosphofructokinase, soluble starch synthase, starch branching enzyme, phosphoglucomutase and alkaline inorganic pyrophosphatase . The glgC-16 tubers are held to be suitable for the study of the role of ADPglucose pyrophosphorylase in the control of starch synthesis. Biochem J, 1996 Dec 1, 320 ( Pt 2), 383 - 92 Purification and properties of cystathionine beta-lyase from Arabidopsis thaliana overexpressed in Escherichia coli; Ravanel S et al.; Cystathionine beta-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5'-phosphate-dependent beta-cleavage of cystathionine to produce homocysteine . To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine beta-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain . The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure . Physicochemical properties of the Arabidopsis cystathionine beta-lyase were similar to those previously reported for the bacterial enzymes . In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5'-phosphate . Interaction between the apoenzyme and pyridoxal 5'-phosphate is extremely tight, being characterized by a Kd value of 0.5 microM . Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278 . The substrate specificity of the recombinant cystathionine beta-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates . The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine beta-lyase . The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5'-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine. Nucleic Acids Res, 1996 Dec 1, 24(23), 4838 - 40 A tri-hybrid system for the analysis and detection of RNA--protein interactions; Putz U et al.; A modification of the two-hybrid system is described for the in vivo reconstruction of specific RNA-protein interactions . In this tri-hybrid system, the DNA binding and transcription activation domains of the yeast transcriptional activator GAL4 are brought together via the interaction of recombinant fusion proteins with a recombinant RNA . The method provides a system for studying RNA-protein interactions with the genetic advantages of the two-hybrid system . It may be used to detect specific RNA-binding proteins or target RNAs from a library of cDNAs, or to analyse the structural specificity of identified RNA-protein interactions. Nucleic Acids Res, 1996 Dec 1, 24(23), 4741 - 50 XAP2, a novel hepatitis B virus X-associated protein that inhibits X transactivation; Kuzhandaivelu N et al.; The hepatitis B virus X protein is a promiscuous transcriptional transactivator . Transactivation by the X protein is most likely mediated through binding to different cellular factors . Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2 . X and XAP2 interactions also occur in vitro . Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent molecular mass of 36 kDa . The interaction between X and XAP2 requires a small region on X containing amino acids 13-26 . From Northern blot analyses, XAP2 is ubiquitously expressed in both liver-derived and non-liver-derived cell lines as well as in normal non-liver tissues . In contrast, XAP2 is expressed in very low level in the normal human liver . In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein . Based on these results, we suggest that XAP2 is an important cellular negative regulator of the X protein, and that X-XAP2 interaction may play a role in HBV pathology. Nucleic Acids Res, 1996 Dec 1, 24(23), 4733 - 40 A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides; Suda T et al.; The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA . We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing . The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations . The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins . Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay . The difference suggests that the two activities of association-promotion and DNA bending are distinct . These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin. Nucleic Acids Res, 1996 Dec 1, 24(23), 4719 - 24 Sequential binding of DNA repair proteins RPA and ERCC1 to XPA in vitro; Saijo M et al.; Recent studies have shown that many proteins are involved in the early steps of nucleotide excision repair and that there are some interactions between nucleotide excision repair proteins, suggesting that these interactions are important in the reaction mechanism . The xeroderma pigmentosum group A protein (XPA) was shown to bind to the replication protein A (RPA) or the excision repair cross complementing rodent repair deficiency group 1 protein (ERCC1), and these interactions might be involved in the damage-recognition and/or incision steps, of nucleotide excision repair . Here we show that the XPA regions required for the binding to the 70 and 34 kDa subunits of RPA are located in the middle and on N-terminal regions of XPA, respectively . These regions do not overlap with the ERCC1-binding region of XPA, and a ternary protein complex of RPA, XPA and ERCC1 was detected in vitro . In addition, using the surface plasmon resonance biosensor, the binding of RPA and ERCC1 to XPA was investigated . The dissociation constants (KD) of RPA and ERCC1 with XPA were 1.9 x 10(-8 )and 2.5 x 10(-7) M, respectively . Moreover, our results suggest the sequential binding of RPA and ERCC1 to XPA. RNA, 1996 Dec, 2(12), 1295 - 305 X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering; Golden BL et al.; For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine . However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt . We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685) . To incorporate iodouridine, a two-piece RNA was constructed . The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end . A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA . The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays . Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6 . The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives . This approach is expected to be readily adaptable to other large, structured RNA molecules. RNA, 1996 Dec, 2(12), 1286 - 94 Functional analysis of filamentous phage f1 mRNA processing sites; Stump MD et al.; The abundant mRNAs used as templates for synthesis of filamentous phage f1 proteins are a combination of primary transcripts and 3' products of processing . The processing steps are mediated by host endoribonucleases . One of the enzymes implicated in f1 mRNA processing is RNase E, the only endonuclease thus far shown to have a global role in mRNA decay . By establishing the temperature-sensitive phenotypes of RNase E mutants and then inducing a transcription unit bearing cloned f1 processing sites, we show that RNase E is required for production of at least three of the processed RNAs . Using in vivo processing assays, we also test directly the regions implicated genetically in previous work to contain the processing sites . The sites function as discrete domains in a number of transcription units, show little influence of translation, but appear to have increased activity at the 5' terminus of an mRNA . From their functional properties, we suggest that the known processing sites from phage f1 that are dependent on RNase E may be representative of relatively late steps in rne-dependent cleavage pathways. RNA, 1996 Dec, 2(12), 1270 - 85 Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations; Lee K et al.; The interaction of bacterial mRNAs with the small ribosomal subunit is strongly promoted by Watson-Crick base pairing between a purine-rich consensus ribosomal RNA-binding sequence (RBS) on mRNA and its complementary message-binding sequence (MBS) on rRNA known as the Shine-Dalgarno interaction . To identify and characterize components of the Shine-Dalgarno interaction that contribute to translation initiation, we simultaneously and randomly mutated both the MBS of the 16S rRNA gene from Escherichia coli and the RBS of the chloramphenicol acetyl transferase (CAT) gene and selected chloramphenicol-resistant mutant combinations . Nucleotide distribution in both mutated sequences of the survivors was nonrandom and the MBSs of the surviving clones showed a preference for purines . In addition, strong interactions between specific nucleotide pairs within each of the mutated sequences were indicated . Although the contribution of free energy of duplex formation between rRNA and mRNA was highly significant (P < 0.001), only 23% of the observed activity in all of the mutants could be attributed to this variable . MBSs that were lethal upon expression were also isolated . These sequences may cause overtranslation of specific messages in the cell . These data indicate that specific sequence constraints exist (primarily within the MBS) that are necessary to establish a functional threshold for translation and that only after establishment of this threshold is the level of expression significantly affected by the free energy of MBS-RBS duplex formation. RNA, 1996 Dec, 2(12), 1228 - 40 RNase III autoregulation: structure and function of rncO, the posttranscriptional "operator"; Matsunaga J et al.; Expression of the Escherichia coli rnc-era-recO operon is regulated posttranscriptionally by ribonuclease III (RNase III), encoded in the rnc gene . RNase III initiates rapid decay of the rnc operon mRNA by cleaving a double-stranded region of the rnc leader . This region, termed rncO, is portable, conferring stability and RNase III regulation to heterologous RNAs . Here, we report the detailed analysis of rncO structure and function . The first 215 nt of the rnc leader are sufficient for its function . Dimethylsulfate (DMS) modification in vivo revealed distinct structural elements in this region: a 13-nt single-stranded 5' leader, followed by a 6-bp stem-loop structure (I), a larger stem-loop structure (II) containing the RNase III site, a single-stranded region containing the rnc translation initiation site, and a small stem-loop structure (III) at the 3' terminus of rncO, wholly within the rnc coding region . Genetic analysis revealed the function of these structural elements . The single-stranded leader is not required for stability or RNase III control, stem-loop II is required only for RNase III control, and both stem-loops I and III are required for stability . Stem-loop II effectively serves only as the site at which RNase III cleaves to remove stem-loop I and thereby initiates decay, after which RNase III plays no role . Mutations at the cleavage site underscore the importance of base pairing for efficient RNase III attack . When stem-loops I and II were replaced with an artificial hairpin structure, stability was restored only partially, but was restored almost fully when a single-stranded leader was also added. RNA, 1996 Dec, 2(12), 1213 - 27 NMR studies of the most conserved RNA domain of the mammalian signal recognition particle (SRP); Schmitz U et al.; Mammalian signal recognition particle (SRP) and its homologues exhibit a phylogenetically conserved RNA domain, whose predicted secondary structure exhibits a hairpin motif with two bulged regions . Two RNA fragments comprising one (24 nt) or two (43 nt) of the conserved bulges were studied . Each fragment binds specifically to the domain of the Escherichia coli homologue of the SRP54 protein, which is involved in signal sequence recognition . The SRP RNA fragments exhibited a pronounced structural stabilization in the presence of Mg2+ . Assignments of all base, H1', H2', and most imino proton resonances in the presence of Mg2+ were obtained for the 24mer RNA via NOE spectroscopy and correlated homonuclear NMR methods . 2D NOE patterns permitted a coarse structural description, revealing a relatively compact A-type geometry for the 24mer without any indications of looped-out nucleotides, syn-oriented bases, or base triplets . The GGAA-loop is structurally very similar to that of the GCAA tetraloop {Heus HA, Pardi A, 1991, Science 253:191-194} . Mg2+ seems to stabilize the structure of the conserved bulged region, which involves G:A and C:A mismatch pairs . Deviations from ideal A-type helicity are found for a larger region than the predicted secondary structure implies . Although no explicit assignment effort has been dedicated to the 43mer yet, striking similarity in chemical shift changes upon addition of Mg2+ allowed some structural conclusions . The bulge present in both RNA fragments exhibits a similar, pronounced flexibility in the absence of Mg2+, indicating that the additional bulge in the 43mer does not stabilize the other bulge. RNA, 1996 Dec, 2(12), 1199 - 212 Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs; Kolupaeva VG et al.; Initiation of translation of a subset of eukaryotic mRNAs results from internal ribosomal entry . This process is exemplified by encephalomyocarditis virus (EMCV), which contains an internal ribosomal entry site (IRES) within its 5' nontranslated region that is approximately 450-nt long and consists of a series of stem-loops designated H-L . We have previously identified a cellular 58-kDa polypeptide that binds specifically to this IRES and that is implicated in its function as the pyrimidine tract-binding protein PTB . We have now mapped PTB binding sites directly on the IRES elements of EMCV and the related foot-and-mouth disease virus (FMDV) using structure-specific enzymatic probes and base-specific chemical probes . PTB bound to six sites on the EMCV IRES: site 1 (UCUU401) is upstream of domain H, site 2 is the basal helix of domain H (nt 407-410 and 440-443), site 3 (UCUUU423) is the apical loop of domain H, site 4 is the apical helix and adjacent internal bulged loop of domain K, site 5 (CUUUA750) is the apical loop of domain K, and site 6 (CCUUU815) is downstream of domain L . PTB bound to sites on the FMDV IRES that correspond precisely to EMCV sites 3, 5, and 6 . These sites have the consensus sequence CUUU and form two groups that are located near to the 5' and 3' borders of these IRES elements . Their position, and the effects of mutation of them on IRES function are consistent with PTB's role in IRES-mediated initiation being to bind to multiple sites in the IRES, thereby stabilizing a specific active conformation. RNA, 1996 Dec, 2(12), 1189 - 98 Rp-deoxy-phosphorothioate modification interference experiments identify 2'-OH groups in RNase P RNA that are crucial to tRNA binding; Hardt WD et al.; Ribose 2'-hydroxyls make a key contribution to the enormous structural and functional potential of RNA molecules . Here, we report the identification of 2'-deoxy modifications in the catalytic RNA subunit of RNase P from Escherichia coli that interfere with tRNA binding . This was accomplished by modification interference employing pools of RNase P RNA that carried a low level of Rp-deoxy-phosphorothioate (Rp-deoxyNMPalpha(S) ) modifications randomly distributed over its 380 nt . A gel retardation assay allowed us to separate RNase P RNA pools into tRNA-binding and nonbinding fractions . Differences in the intensity of phosphorothioate-specific iodine hydrolysis patterns of the two RNA fractions revealed positions where the Rp-deoxyNMPalpha(S) modification interferes with tRNA binding . A comparison with interference patterns obtained for the Rp-NMPalpha(S) modification alone has identified some 20 positions in the backbone of E . coli RNase P RNA where the functional defect caused by the Rp-deoxyNMPalpha(S) double modification is attributable to the 2'-deoxy modification (or possibly the C5 methyl group in the case of U residues because we used deoxyTMPalpha(S) for partial substitution of UMP) . Most of the corresponding 2'-OH functions were localized in regions that have been reported to crosslink to photoreactive tRNA derivatives, suggesting that these 2'-hydroxyls are located along the tRNA binding interface of E . coli RNase P RNA . Our results indicate that the modification interference approach applied here will be useful generally to identify structurally and functionally important 2'-hydroxyls in large RNAs and ribozymes. RNA, 1996 Dec, 2(12), 1179 - 88 In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain; Frank DN et al.; In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes . Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme . A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218) . We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis . Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence . This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity . These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195) . The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life. Yeast, 1996 Dec, 12(15), 1535 - 48 A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica; Ramon AM et al.; A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library . The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells . The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide . We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein . Northern hybridization identified YWP1 transcript only when Y . lipolytica was growing in the mycelial form . The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of beta-glucan by Zymolyase digestion . The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture. Yeast, 1996 Dec, 12(15), 1501 - 10 Removal of an intron with unique 3' branch site creates an amino-terminal protein sequence directing the scERV1 gene product to mitochondria; Lisowsky T; The yeast scERV1 gene is of special interest because it has a dual function in mitochondrial biogenesis and in the regulation of the cell cycle . The recent discovery that the yeast scERV1 gene has a structural and functional human homologue initiated a detailed comparison of the genes and their structures . In addition the homologous ALR (augmenter of liver regeneration) genes from rat and mouse have just been identified and it has been found that the mammalian proteins have a specific function in liver regeneration and in spermatogenesis . It now turns out that the organization of the 5' regions of these genes is much more complicated than expected . The latest research has discovered an additional intron in the 5' region of the mouse gene and possible amino-terminal extensions of the reading frames . In this work, reinvestigation of the 5' region of the yeast gene identifies a putative intron with an unusual 3' branch site . It is shown that a small intron of 83 nucleotides is present in this genomic region . Analysis of cDNA clones demonstrates that the intron is correctly removed from the messenger RNA and that therefore the unusual 3' branch site is probably functional . Furthermore, studies with antibodies directed against recombinant scERV1 protein demonstrate that the gene product is associated with mitochondria, in agreement with its involvement in mitochondrial biogenesis . Complementation experiments with mutants and different 5' deletions of the gene identify the corresponding promotor for transcription and the start codon for translation. Thromb Haemost, 1996 Dec, 76(6), 1096 - 101 Comparison of the recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator with alteplase, reteplase and streptokinase in a canine model of coronary artery thrombolysis; Martin U et al.; Recent in vitro studies have shown that although recombinant Escherichia coli-produced protease domain of tissue-type plasminogen activator (t-PA) has no appreciable fibrin binding and less plasminforming activity compared to the wild-type, it is nevertheless an effective fibrinolytic agent in a dynamic in vitro plasma clot lysis system . The purpose of the present study was to evaluate the pharmacological profile of the protease in a canine model of coronary artery thrombosis . The effects of a single i.v . bolus injection of 1 mg/kg protease were compared with those of alteplase, reteplase and streptokinase at clinically relevant doses and dosing regimens in eight dogs per group . The protease rapidly restored coronary blood flow at 12 +/- 1 min in all treated dogs with a significantly higher maximal coronary blood flow than in the reference groups, but was associated with short cycles of reocclusion in 4/8 animals . Overall, the coronary blood flow quality of the protease was not significantly different from that of the reference thrombolytics . Although fibrinogen was nearly completely degraded during protease treatment, the bleeding time was not significantly more prolonged than in reference groups . In conclusion, the protease domain is a rapidly acting, effective, bolus-injectable thrombolytic agent associated with a systemic lytic state and does not appear to cause significantly more bleeding than the reference thrombolytic agents. Mol Microbiol, 1996 Dec, 22(5), 1013 - 23 Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA); Kansau I et al.; Helicobacter pylori synthesizes a heat-shock protein of the GroES class . The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure . H . pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain . Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP) . Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain . HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt) . Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 microM and a stoichiometry of 1.9 ions per molecule . The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e . a major and a minor variant present in 89% and 11% of strains, respectively . The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99-100% identity) . On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain . Functional complementation experiments were performed to test the properties of the two HspA variants . When co-expressed together with the H . pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H . pylori has a specialized function with regard to the nickel metalloenzyme urease. Mol Microbiol, 1996 Dec, 22(5), 977 - 89 Expression and regulation of the rnc and pdxJ operons of Escherichia coli; Matsunaga J et al.; Escherichia coli rnc-era-recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene . RNase III, a double-stranded RNA-specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stemloop structure in the 5' untranslated leader, initiating rapid decay of the rnc operon mRNA . Here, we examine rnc operon expression and regulation in greater detail . Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP) specifies two principal mRNAs: the 1.9 kb rnc-era transcript and the less-abundant 3.7 kb RNA encoding rnc-era-recO and the downstream pdxJ and acpS genes . A 1.3 kb pdxJ-acpS RNA is transcribed from a promoter (pdxP) located within recO . About 70% of pdxJ transcription depends on transcription from rncP . Both promoters were characterized genetically . RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions . These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis-acting elements required for RNase III-initiated decay of the rnc mRNA as well as the heterologous lacZ transcript . Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site. Mol Microbiol, 1996 Dec, 22(5), 827 - 40 The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose; Marolda CL et al.; We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E . coli GalU protein . These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi . We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive . In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose . The presence of GalF also increased the thermal stability of the enzyme in vitro . The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme . The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system . Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo . We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress. J Virol, 1996 Dec, 70(12), 8821 - 32 Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase; Levy-Mintz P et al.; Integration of viral DNA into a chromosome of the infected host cell is required for efficient replication of a retroviral genome, and this reaction is mediated by the virus-encoded enzyme integrase (IN) . As IN plays a pivotal role in establishing infection during the early stages of the retroviral life cycle, it is an attractive target for therapeutic intervention . However, the lack of effective antiviral drug therapy against this enzyme has led to the testing of other novel approaches towards its inhibition . In these studies, a panel of anti-human immunodeficiency virus type 1 (anti-HIV-1) IN hybridomas has been used in the construction of single-chain variable antibody fragments (SFvs) . The monoclonal antibodies produced by these hybridomas, and derived SFvs, bind to different domains within IN . We now demonstrate that intracellular expression of SFvs which bind to IN catalytic and carboxy-terminal domains results in resistance to productive HIV-1 infection . This inhibition of HIV-1 replication is observed with SFvs localized in either the cytoplasmic or nuclear compartment of the cell . The expression of anti-IN SFvs in human T-lymphocytic cells and peripheral blood mononuclear cells appears to specifically neutralize IN activity prior to integration and, thus, has an effect on the integration process itself . These data support our previous studies with an anti-HIV-1 reverse transcriptase SFv and demonstrate further that intracellularly expressed SFvs can gain access to viral proteins of the HIV-1 preintegration complex . This panel of anti-HIV-1 IN SFvs also provides the tools with which to dissect the molecular mechanism(s) directly involved in integration within HIV-1-infected cells. J Virol, 1996 Dec, 70(12), 8630 - 8 RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity; Schultz SJ et al.; The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction . Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations . The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts . Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction . Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction . Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain . These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase . The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation. J Virol, 1996 Dec, 70(12), 8564 - 70 Mutation of lysine residues in the nucleotide binding segments of the poliovirus RNA-dependent RNA polymerase; Richards OC et al.; The poliovirus 3D RNA-dependent RNA polymerase contains two peptide segments previously shown to cross-link to nucleotide substrates via lysine residues . To determine which lysine residue(s) might be implicated in catalytic function, we engineered mutations to generate proteins with leucine residues substituted individually for each of the lysine residues in the NTP binding regions . These proteins were expressed in Escherichia coli and were examined for their abilities to bind nucleotides and to catalyze RNA chain elongation in vitro . Replacement of each lysine residue in the NTP binding segment located in the central portion of the 3D molecule (Lys-276, -278, or -283) with leucine produced no impairment of GTP binding or polymerase activity . Substitution of leucine for Lys-61 in the N-terminal portion of the protein, however, abolished the binding of protein to GTP-agarose and all detectable polymerase activity . A nearby lysine replacement with leucine at position 66 had no effect on enzyme activity . The three mutations in the central region of 3D were introduced into full-length viral cDNAs, and the infectivities of RNA transcripts were examined in transfected HeLa cells . Growth of virus containing 3D with a mutation at residue 278 (3Dmu278) or 3Dmu283 was indistinguishable from that of the wild type; however, 3Dmu276 generated extremely slow-growing, small-plaque virus . Polyprotein processing by 3CDmu276 was unaffected . Large-plaque variants, in which the Leu-276 codon had mutated again to an arginine codon, emerged at high frequency . The results suggest that a lysine residue at position 61 of 3Dpol is essential for polymerase catalytic function and that a basic (lysine or arginine) residue at position 276 is required for some other function of 3D important for virus growth but not for RNA chain elongation or polyprotein processing. J Virol, 1996 Dec, 70(12), 8477 - 84 The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3); Tai CL et al.; To assess the RNA helicase activity of hepatitis C virus (HCV) nonstructural protein 3 (NS3), a polypeptide encompassing amino acids 1175 to 1657, which cover only the putative helicase domain, was expressed in Escherichia coli by a pET expression vector . The protein was purified to near homogeneity and assayed for RNA helicase activity in vitro with double-stranded RNA substrates prepared from a multiple cloning sequence and an HCV 5' nontranslated region (5'-NTR) or 3'-NTR . The enzyme acted successfully on substrates containing both 5' and 3' single-stranded regions (standard) or on substrates containing only the 3' single-stranded regions (3'/3') but failed to act on substrates containing only the 5' single-stranded regions (5'/5') or on substrates lacking the single-stranded regions (blunt) . These results thus suggest 3' to 5' directionality for HCV RNA helicase activity . However, a 5'/5' substrate derived from the HCV 5'-NTR was also partially unwound by the enzyme, possibly because of unique properties inherent in the 5' single-stranded regions . Gel mobility shift analyses demonstrated that the HCV NS3 helicase could bind to either 5'- or 3'-tailed substrates but not to substrates lacking a single-stranded region, indicating that the polarity of the RNA strand to which the helicase bound was a more important enzymatic activity determinant . In addition to double-stranded RNA substrates, HCV NS3 helicase activity could displace both RNA and DNA oligonucleotides on a DNA template, suggesting that HCV NS3 too was disposed to DNA helicase activity . This study also demonstrated that RNA helicase activity was dramatically inhibited by the single-stranded polynucleotides . Taken altogether, our results indicate that the HCV NS3 helicase is unique among the RNA helicases characterized so far. J Virol, 1996 Dec, 70(12), 8255 - 62 The carboxyl terminus of the human foamy virus Gag protein contains separable nucleic acid binding and nuclear transport domains; Yu SF et al.; The Gag protein of human foamy virus (HFV) lacks Cys-His boxes present in the nucleocapsid (NC) domains of other retroviruses; instead it contains three glycine-arginine-rich motifs (GR boxes) . We have expressed the carboxyl end of HFV Gag containing the GR boxes (the NC domain equivalent) and analyzed its nucleic acid binding properties . Our results show that the NC domain of HFV Gag binds with high affinity to both RNA and DNA, in a sequence-independent manner, as determined by filter binding assays . Analysis of a mutant containing a heterologous sequence in place of GR box I indicates that this motif is required for nucleic acid binding and for viral replication . A mutant in GR box II still binds to RNA and DNA in vitro, but virus containing this mutation does not replicate and no nuclear staining of the Gag protein is found in transfected cells . Surprisingly, a revertant from this mutant that completely lacks GR box II and exhibits very little nuclear transport of Gag can readily replicate in tissue culture . This finding thus provides a direct evidence that although the sequences in GR box II can serve as a nuclear transport signal, they are not required for HFV replication and it is unlikely that nuclear localization of Gag protein plays any critical role during viral infection . Taken together, our results suggest that the Gag protein of HFV may be more analogous to the core protein of the hepatitis B virus family than to conventional retroviral Gag protein. Am J Respir Crit Care Med, 1996 Dec, 154(6 Pt 1), 1689 - 93 Alpha-2 adrenoceptor blockade protects rats against lipopolysaccharide; Fessler HE et al.; Alpha-2 adrenoceptors are widely distributed in vascular and nonvascular tissue where they mediate diverse physiologic effects . We noted the laboratory anesthetic urethane, which possesses alpha-2 adrenergic blocking activity, protected rats against lethal endotoxemia (1) . Therefore, we undertook the present study to examine whether specific alpha-2 adrenoceptor antagonism would protect against lethality and organ injury induced by lipopolysaccharide (LPS) . Sprague-Dawley rats were pretreated with doses of the alpha-2 antagonist rauwolscine up to 1 mg/kg, followed by 20 mg/kg LPS . The highest rauwolscine dose decreased mortality from 100% to zero . In contrast, the alpha-2 agonists xylazine or UK 14,304 increased the lethality of a lower, 10-mg/kg dose of LPS from 20% to 80 to 100% . Rauwolscine administered after LPS had no protective effect against mortality . Rauwolscine pretreatment significantly reduced bowel hemorrhage and liver dysfunction induced by 20 mg/kg LPS, but it had no effect on hematologic changes, the rise in plasma creatinine, or lung myeloperoxidase content . Peak tumor necrosis factor-alpha levels were decreased from 1,305 +/- 333 to 493 +/- 155 pg/ml (p < 0.05) in animals pretreated with rauwolscine . Arterial pressure and heart rate was higher after LPS in animals pretreated with rauwolscine . We conclude that alpha-2 adrenergic blockade protects against LPS, either by decreasing tumor necrosis factor-alpha production or through direct effects on the target tissues of endotoxemia. Biotechnol Appl Biochem, 1996 Dec, 24 ( Pt 3), 225 - 30 Quantitative conversion of glucose into glucose 6-phosphate by intact Escherichia coli cells; van der Zee JR et al.; The use of intact Escherichia coli cells for the conversion of glucose into glucose 6-phosphate using the Uhp system for transport of the phosphorylated sugar out of the cell was investigated . The strain E . coli DF214, which is not capable of glucose 6-phosphate catabolism via glycolysis or the Entner-Douderoff pathway, was used . The efflux of glucose 6-phosphate was dependent on the presence of UhpT, the hexose-phosphate transporter, plus the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or the ionophore valinomycin . At low glucose concentrations (e.g . 2.5 mM), near-quantitative conversion (> or = 80%) of glucose into extracellular glucose 6-phosphate can be achieved . When cells are incubated for a shorter period of time, complete conversion can occur. Hum Mol Genet, 1996 Dec, 5(12), 1859 - 65 Defects in human methionine synthase in cblG patients; Gulati S et al.; Inborn errors resulting in isolated functional methionine synthase deficiency fall into two complementation groups, cblG and cblE . Using biochemical approaches we demonstrate that one cblG patient has greatly reduced levels of methionine synthase while in another, the enzyme is specifically impaired in the reductive activation cycle . The biochemical data suggested that low levels of methionine synthase activity in the first patient may result from mutations in the catalytic domains of the enzyme, reduced transcription, or generation of unstable message or protein . Using Northern analysis, we demonstrate that the molecular basis for the biochemical phenotype in this patient is associated with greatly diminished steady-state levels of methionine synthase mRNA . The biochemical data on the second patient cell line implicated mutations specific to reductive activation, a function that is housed in the C-terminal AdoMet-binding domain and the intermediate B12-binding domain, in the highly homologous bacterial enzyme . We have detected two mutations in a compound heterozygous state, one that results in conversion of a conserved proline (1173) to a leucine residue and the other a deletion of an isoleucine residue (881) . The crystal structure of the C-terminal domain of the Escherichia coli MS predicts that the Pro to Leu mutation could disrupt activation since it is embedded in a sequence that makes direct contacts with the bound AdoMet . Deletion of isoleucine in the B12-binding domain would result in shortening of a beta-sheet . Our data provide the first evidence for mutations in the methionine synthase gene being culpable for the cblG phenotype . In addition, they suggest directly that mutations in methionine synthase can lead to elevated homocysteine, implicated both in neural tube defects and in cardiovascular diseases. Hum Mol Genet, 1996 Dec, 5(12), 1851 - 8 Cloning, mapping and RNA analysis of the human methionine synthase gene; Li YN et al.; Elevated levels of plasma homocysteine is a risk factor in both birth defects and vascular disease . Methionine synthase (MS) is a cobalamin dependent enzyme which catalyzes methylation of homocysteine to methionine . Impaired MS activity is expected to lead to increased levels of plasma homocysteine . In addition, defects in this gene may underlie the methionine-dependence observed in a number of human tumor cell lines . We describe here the isolation and characterization of the human MS cDNA . It contains an open reading frame of 3798 nucleotides encoding a protein of 1265 amino acids with a predicted molecular mass of 140 kDa . The amino acid sequence of the human MS is 55% identical with that of the Escherichia coli enzyme (METH) and 64% identical with the predicted Caenorhabditis elegans enzyme . Seven peptide sequences derived from purified porcine MS have substantial similarity to the human protein . Northern analysis indicates that the MS RNA is present in a wide variety of tissues . We have mapped the human gene to chromosomal location 1q43, a region found monosomic in individuals with deletion 1q syndrome . The isolation of the MS cDNA will now allow the direct determination of whether mutations in this gene contribute to folate-related neural tube defects, cardiovascular diseases, and birth defects. Crit Care Med, 1996 Dec, 24(12), 2021 - 6 Effect of hemocarboperfusion on organ blood flow and survival in porcine endotoxic shock; Odnopozov VA et al.; OBJECTIVE: To evaluate the effects of hemocarboperfusion on hemodynamics, organ blood flow, and survival in endotoxin shock . DESIGN: Prospective, placebo-controlled, animal trial . SETTING: Research laboratory in a major university teaching hospital . SUBJECTS: Pentobarbital-anesthetized pigs . INTERVENTIONS: Twenty-eight pentobarbital-anesthetized pigs (18.5 to 22.3 kg) received 100 micrograms/kg of Escherichia coli endotoxin (lipopolysaccharide 0127) over 30 mins . Group 1 animals (n = 14) were controls and had blood diverted through an extracorporeal circuit without activated charcoal for 60 mins after lipopolysaccharide infusion . Group 2 animals (n = 14) underwent nonpulsatile hemocarboperfusion (activated charcoal SCN-1K) . MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure, cardiac output, systemic vascular resistance, mean pulmonary arterial pressure, pulmonary vascular resistance, oxygen delivery, and regional blood flow (radiolabeled microsphere technique) were determined at baseline and every 30 mins for 150 mins . Results are presented as mean +/- SD . Parameters in the two groups were compared by two-way analysis of variance . A p < .05 was considered significant . The survival rate was ten (71%) of 14 animals in group 1 compared with 14 (100%) of 14 animals in group 2 (p < .05, Fisher's exact test) . The mean cardiac output at the end of hemocarboperfusion was 1.6 +/- 0.6 L/min in group 1 compared with 3.0 +/- 0.9 L/min in group 2, and remained lower in group 1 animals throughout the experiment . Pulmonary arterial pressure and pulmonary vascular resistance were lower in the hemocarboperfusion-treated animals during and after hemocarboperfusion . Systemic vascular resistance increased by 70% after lipopolysaccharide infusion and returned to baseline values in the hemocarboperfusion group but remained increased in controls . Oxygen delivery was lower in group 1 at 90 and 150 mins (287 +/- 34 vs . 478 +/- 48 mL/min and 251 +/- 24 vs . 356 +/- 21 mL/min, respectively) . Blood flow rates to the brain (38.5 +/- 7.5 vs . 27.1 +/- 5.4 mL/min/100 g), large intestine (26.6 +/- 1.1 vs . 17.7 +/- 2.5 mL/ min/100 g), and adrenal cortex (200 +/- 45 vs . 139 +/- 41 mL/min/100 g) were higher in the hemocarboperfusion group at the completion of carboperfusion but not at later time points . CONCLUSION: These data suggest that hemocarboperfusion may be of value in the treatment of septic shock. Biometrics, 1996 Dec, 52(4), 1214 - 22 A reexamination of the cell-lineage data of E . O . Powell; Staudte RG et al.; E . O . Powell carried out numerous experiments observing cell-generation times on bacteria . His statistical methodology, though quite advanced 40 years ago, can be enhanced by better models of dependence, by advanced computational methods, and by judicious use of robust methods . We reexamine Powell's data, focussing on his main interest in establishing whether there were indeed dependencies in generation times between cells with close filial connections. Shock, 1996 Dec, 6(6), 434 - 41 Modulation of kupffer cell activity by gadolinium chloride in endotoxemic rats; Vollmar B et al.; Gadolinium chloride (GdCl3) has been reported to block Kupffer cell (KC) phagocytic activity in rats . In this study, we investigated the action of GdCl3 on Kupffer cells and related effects in response to lipopolysaccharide (LPS) exposure of rats . Using intravital fluorescence microscopy (IVFM), the hepatic microcirculation (phagocytic activity and zonal distribution of KC, sinusoidal perfusion, leukocyte-endothelial cell interaction) of rats pretreated with either saline or GdCl3 (10 mg/kg i.v . for 2 days) was studied at 1 h (n = 14) and 16 h (n = 16) after exposure to Escherichia coli LPS (10 mg/kg i.v.) . LPS-exposure (1 h) resulted in KC activation with increased phagocytic activity (IVFM), intracellular enrichment of phagocytic vacuoles, and marked rise of cytokines (tumor necrosis factor-alpha, interleukin-6) in serum, whereas GdCl3-pretreatment completely inhibited the LPS-related KC response . 16 h after LPS-exposure, saline-treated animals revealed high serum levels of LPS, associated with microvascular perfusion deficits, marked KC destruction, and hepatocellular disintegration, which finally resulted in a mortality rate of 47% (7/15) . In contrast, none of the GdCl3-treated animals died (0/8) . GdCl3-pretreatment significantly attenuated LPS-induced hepatic microvascular perfusion failure and parenchymal cell injury at 16 h after LPS exposure . Intact KC morphology and low serum levels of LPS indicated adequate clearance capacity . Based on these results, we propose that modulation of LPS-induced KC phagocytic activity and KC function by GdCl3 is effective to protect from LPS-induced hepatic injury and systemic toxicity, probably by inhibition of overwhelming inflammatory response. Shock, 1996 Dec, 6(6), 426 - 33 Protein tyrosine kinase activity and the influence of gender in phagocytosis and tumor necrosis factor secretion in alveolar macrophages and lung-recruited neutrophils; Spitzer JA et al.; The role of tyrosine phosphorylation in endotoxin-induced phagocytic and tumor necrosis factor secretory responses was studied in rat alveolar macrophages and lung-recruited neutrophils . Exploration of sexual dimorphism in some aspects of these functions was also a specific aim . Male and female rats were injected intratracheally with endotoxin or saline . Two and a half hours later the animals were subjected to bronchoalveolar lavage, and alveolar macrophages and lung-recruited neutrophils were isolated . Circulating neutrophils of endotoxin-treated rats were also isolated at this time . Phagocytosis and CD11b/c and CD18 expression were measured by flow cytometry; tumor necrosis factor was measured with a cytotoxicity assay . Using the protein tyrosine kinase inhibitor AG126 and phosphotyrosine immunoblotting, we demonstrated that tyrosine phosphorylation is an important signaling pathway in the activation of these cells by endotoxin and that it is coupled to phagocytosis and tumor necrosis factor secretion, but not to beta 2 integrin expression . Conditioned medium of alveolar macrophages of endotoxin-injected rats upregulates phagocytosis by blood neutrophils of naive rats and this upregulating activity is tyrosine phosphorylation dependent . The substrates for tyrosine phosphorylation are different in alveolar macrophages and lung neutrophils, as are their sensitivities to AG126 . Significant gender differences exist in the modulation of phagocytosis by inhibition of tyrosine phosphorylation and in tumor necrosis factor secretion by endotoxin-stimulated alveolar macrophages. Shock, 1996 Dec, 6(6), 410 - 7 Laparotomy and renal function during endotoxin shock in rats; Heemskerk AE et al.; Despite the wide use of laparotomy to study kidney function, the possible influence of this procedure on systemic and renal parameters in septic rats is unknown . We studied this in anesthetized Wistar rats with and without endotoxin shock (1 h Escherichia coli O 127.B8: 8 mg.kg-1 infusion) . We also compared clearance of creatinine and inulin to measure glomerular filtration rate (GFR) . Laparotomy attenuated the endotoxin-induced decrease in cardiac output and abolished the increase in systemic and renal vascular resistance, while renal plasma flow was maintained . Better perfusion in other organs as well was indicated by a more gradual increase in arterial lactate concentration and less intestinal damage . By contrast, GFR decreased considerably during endotoxemia, irrespective of laparotomy . This change in GFR could be reliably assessed using creatinine clearance . The ratio of creatinine-to-inulin clearance averaged between .5 and .75 . Renal ATP content did not change and the endotoxin-induced increase in the number of granulocytes lodged in glomeruli was not affected by laparotomy . In conclusion, our study indicates that laparotomy significantly influences the vascular effects caused by endotoxin . Laparotomy also revealed an effect of endotoxin on GFR, independent of renal blood flow. J Clin Invest, 1996 Dec 1, 98(11), 2640 - 7 Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats; Ilan Y et al.; Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo . However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration . We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats . Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes . Controls received intrathymic injections of normal saline . After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase {LacZ} gene) . In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95% . Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk . Similar findings were observed following repeat injections given on days 45 and 112 . In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective . In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus . Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response . This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen. J Bacteriol, 1996 Dec, 178(24), 7329 - 32 Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN; Callahan C et al.; The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis . Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN . Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth . On the basis of these data, we suggest that o290 be renamed rbn . Potential homologs of rbn in other organisms also were identified. J Bacteriol, 1996 Dec, 178(24), 7304 - 7 Inhibition of RecA-mediated cleavage in covalent dimers of UmuD; Lee MH et al.; Disulfide-cross-linked UmuD2 derivatives were cleaved poorly upon incubation with activated RecA . Reducing the disulfide bonds prior to incubating the derivatives with RecA dramatically increased their extent of cleavage . These observations suggest that the UmuD monomer is a better substrate for the RecA-mediated cleavage reaction than the dimer. J Bacteriol, 1996 Dec, 178(24), 7295 - 303 Analysis of the region between amino acids 30 and 42 of intact UmuD by a monocysteine approach; Guzzo A et al.; On the basis of characterizations of a set of UmuD monocysteine derivatives, we had suggested that positions 24, 34, and 44 are closer to the intact UmuD homodimer interface than other positions tested (M . H . Lee, T . Ohta, and G . C . Walker, J . Bacteriol . 176:4825-4837, 1994) . Because this region of UmuD also appeared to be important for interactions with RecA, we followed up on our previous study by constructing a second set of monocysteine UmuD derivatives with single cysteine substitutions at positions 30 to 42 . We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 showed deficiencies in in vivo and in vitro RecA-mediated cleavage as well as in UV mutagenesis, suggesting that the position 32 to 35 region may be important for RecA-mediated cleavage of UmuD . Interestingly, UmuD with monocysteine substitutions at residues 33 and 40 showed a reduction in UV mutagenesis while retaining the ability to be cleaved by RecA in vivo, suggesting a deficiency in the subsequent role of the UmuD' derivatives in mutagenesis . All of the UmuD monocysteine derivatives in the position 30 to 42 series purified indistinguishably from the wild-type protein . The observations that purified proteins of the UmuD derivatives RC37 and IC38 could be disulfide cross-linked quantitatively upon addition of iodine and yet were poorly modified with iodoacetate led us to suggest that the pairs of residues at positions 37 and 38 are extremely close to the UmuD2 homodimer interface . These observations indicate that the structure of the UmuD2 homodimer in solution is very different from the crystal structure of the UmuD'2 homodimer reported by Peat et al . (T . S . Peat, E . G . Frank, J . P . McDonald, A . S . Levine, R . Woodgate, and W . A . Hendrickson, Nature {London} 380:727-730, 1996). J Bacteriol, 1996 Dec, 178(24), 7285 - 94 Interactions of Escherichia coli UmuD with activated RecA analyzed by cross-linking UmuD monocysteine derivatives; Lee MH et al.; SOS mutagenesis in Escherichia coli requires the participation of a specialized system involving the activated form of UmuD (UmuD'), UmuC, RecA, and DNA polymerase III proteins . We have used a set of monocysteine derivatives of UmuD (M . H . Lee, T . Ohta, and G . C . Walker, J . Bacteriol . 176:4825-4837, 1994) and the cysteine-specific photoactive cross-linker p-azidoiodoacetanilide (AIA) to study not only the interactions of intact UmuD in the homodimer but also the interactions of UmuD with activated RecA . The reactivities of the individual UmuD monocysteine derivatives with AIA were similar to their reactivities with iodoacetate . The relative efficiencies of cross-linking of the AIA-modified monocysteine UmuD derivatives in the homodimer form are also consistent with our previous conclusions concerning the relative closeness of various UmuD residues to the dimer interface . With respect to the UmuD-RecA interface, the AIA-modified VC34 and SC81 monocysteine derivatives cross-linked most efficiently with RecA, indicating that positions 34 and 81 of UmuD are closer to the RecA interface than the other positions we tested . The AIA-modified SC57, SC67, and SC112 monocysteine derivatives cross-linked moderately efficiently with RecA . Neither C24, the wild-type UmuD that has a cysteine located at the Cys-24-Gly-25 cleavage site, nor SC60, the UmuD monocysteine derivative with a cysteine substitution at the position of the putative active-site residue, was able to cross-link with RecA, suggesting that RecA need not directly interact with residues involved in the cleavage reaction . SC19, located in the N-terminal fragment of UmuD that is cleaved, and LC44 also did not cross-link efficiently with RecA. J Bacteriol, 1996 Dec, 178(24), 7260 - 4 N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression; Legoux R et al.; The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E . coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin . This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity . The eliminase-encoding gene, designated elmA, has been cloned from E . coli K5 by expression in E . coli K-12 . The K-12 genome is devoid of the elmA sequence . The elmA gene product is 820 amino acids long . Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms . Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively. J Bacteriol, 1996 Dec, 178(24), 7241 - 7 Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange; Zientz E et al.; In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known . Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth . The dcuC gene is located at min 14.1 of the E . coli map in the counterclockwise orientation . The dcuC gene combines two open reading frames found in other strains of E . coli K-12 . The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells . The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration . DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth . The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers . DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB . Mutants producing only DcuA, DcuB, or DcuC were prepared . In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode. J Bacteriol, 1996 Dec, 178(24), 7234 - 40 Lrp is a direct repressor of the dad operon in Escherichia coli; Mathew E et al.; Expression of the degradative D-amino acid dehydrogenase (dad) operon is known to be increased when Escherichia coli is grown in the presence of D- or L-alanine . Alanine is thought to act as an inducer to block the action of a postulated repressor . This operon is also believed to be regulated by catabolite repression . We have used in vivo and in vitro experiments that show that the dad repressor is the leucine-responsive regulatory protein (Lrp) . dad expression in a dad-lacZ operon fusion strain was increased four- to sevenfold when cells were grown in minimal medium containing alanine or leucine . A strain lacking Lrp had high-level constitutive dad expression . Gel retardation and footprinting studies revealed that Lrp binds in vitro to multiple sites over a large area in the dad promoter region . This binding was reduced by alanine or leucine . In vitro transcription assays, using a plasmid template and primer extension analysis, identified three major dad transcripts (Tr1, Tr2, and Tr3) . The formation of these transcripts was differentially regulated by cyclic AMP-cyclic AMP receptor protein complex, and each was strongly repressed by Lrp . Alanine or leucine completely (for Tr1 and Tr2) or partially (for Tr3) reversed Lrp inhibition . Site-directed mutagenesis of an Lrp binding site strongly reduced Lrp binding and prevented Lrp repression of dad transcription in vivo and in vitro . Taken together, these results strongly suggest that Lrp and alanine or leucine act directly to repress and induce, respectively, transcription of the dad operon. J Bacteriol, 1996 Dec, 178(24), 7167 - 72 FtsA is localized to the septum in an FtsZ-dependent manner; Addinall SG et al.; The localization of the cell division protein FtsA in E . coli was examined . FtsA was found to localize to the septum in a ring pattern as previously shown for FtsZ . The localization of FtsA was completely dependent on the localization of FtsZ . Under a variety of conditions that prevented formation of the Z ring, FtsA was unable to localize . In mutants where FtsZ forms structures in addition to Z rings, the pattern of FtsA duplicated these structures . These results suggest that the Z ring recruits FtsA to the septum. J Bacteriol, 1996 Dec, 178(24), 7090 - 8 Action at a distance for negative control of transcription of the glpD gene encoding sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12; Yang B et al.; Aerobic sn-glycerol 3-phosphate dehydrogenase is a cytoplasmic membrane-associated respiratory enzyme encoded by the glpD gene of Escherichia coli . The glpD operon is tightly controlled by cooperative binding of the glp repressor to tandem operators (O(D)1 and O(D)2) that cover the -10 promoter element and 30 bp downstream of the transcription start site . In this work, two additional operators were identified within the glpD structural gene at positions 568 to 587 (0(D)3) and 609 to 628 (0(D)4) . The two internal operators bound the glp repressor in the presence or absence of the tandem operators (O(D)1 and O(D)2) in vitro, as shown by DNase I footprinting . To assess a potential regulatory role for the two internal operators in vivo, a glpD-lacZ transcriptional fusion containing all four operators was constructed . The response of this fusion to the glp repressor was compared with those of fusion constructs in which O(D)3 and O(D)4 were inactivated by either deletion or site-directed mutagenesis . It was found that the repression conferred by binding of the glp repressor to O(D)1 and O(D)2 was increased five- to sevenfold upon introduction of the internal operators . A regulatory role for HU was suggested when it was found that repressor-mediated control of glpD transcription was increased fourfold in strains containing HU compared with that of strains deficient in HU . The effect of HU was apparent only in the presence of all four glpD operators . The results suggest that glpD is controlled by formation of a repression loop between the tandem and internal operators . HU may assist repression by bending the DNA to facilitate loop formation. J Bacteriol, 1996 Dec, 178(24), 7025 - 30 The linker region of AraC protein; Eustance RJ et al.; AraC protein, a transcriptional regulator of the L-arabinose operon in Escherichia coli, is dimeric . Each monomer consists of a domain for DNA binding plus transcription activation and a domain for dimerization plus arabinose binding . These are connected to one another by a linker region of at least 5 amino acids . Here we have addressed the question of whether any of the amino acids in the linker region play active, specific, and crucial structural roles or whether these amino acids merely serve as passive spacers between the functional domains . We found that all but one of the linker amino acids can be changed to other amino acids individually and in small groups without substantially affecting the ability of AraC protein to activate transcription when arabinose is present . When, however, the entire linker region is replaced with linker sequences from other proteins, the functioning of AraC is impaired. J Bacteriol, 1996 Dec, 178(23), 6975 - 82 Identification of sequences necessary for transcription in vitro from the Chlamydia trachomatis rRNA P1 promoter; Tan M et al.; Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C . trachomatis rRNA P1 promoter in vitro . Stepwise mutational analysis revealed that sequences in the -10, -25, and -35 regions are necessary for promoter activity, but no sequence upstream of position -40 is required . Partially purified C . trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity. J Bacteriol, 1996 Dec, 178(23), 6913 - 20 Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop; Killmann H et al.; Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein . A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused . This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system . In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop . FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity . FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system . FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane . Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane . FhuA delta457-479 supported Ton-dependent uptake of ferrichrome . None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance . Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein . FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M . Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1 . Cells expressing FhuA delta335-355 were resistant to all FhuA ligands . These results indicate different structural requirements within the gating loop for the various FhuA ligands . Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant . It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding. J Bacteriol, 1996 Dec, 178(23), 6904 - 12 Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli; Borst DW et al.; Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains . We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp . To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I . C . Blomfield, P . J . Calie, K . J . Eberhardt, M . S . McClain, and B . I . Eisenstein, J . Bacteriol . 175:27-36, 1993) or on a multicopy plasmid . Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG . Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations . beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal . The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B . R . Ernsting, J . W . Denninger, R . M . Blumenthal, and R . G . Matthews, J . Bacteriol . 175:7160-7169, 1993) . Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression . The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates . Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level. J Bacteriol, 1996 Dec, 178(23), 6888 - 94 The primase of broad-host-range plasmid R1162 is active in conjugal transfer; Henderson D et al.; The broad-host-range plasmid R1162 is conjugally mobilized at high frequency by the IncP-1 plasmid R751 but is poorly mobilized by pOX38, a derivative of the F factor . In both cases, the origin of transfer (oriT) and the Mob proteins of R1162 are required, indicating that these plasmids are mobilized by similar mechanisms . R1162 encodes a primase, essential for vegetative replication of the plasmid, that is made both as a separate protein and as the carboxy-terminal domain of MobA, one of the R1162 mobilization proteins (P . Scholz, V . Haring, B . Wittman-Liebold, K . Ashman, M . Bagdasarian, and E . Scherzinger, Gene 75:271-288, 1989) . When R751 is the mobilizing vector, the primase is not required for mobilization of plasmids containing cloned mob-oriT R1162 DNA . However, detectable mobilization of such plasmids by pOX38 requires both the primase and its cognate initiation site, oriented for synthesis of the complement to the transferred strand . The long form of the primase is required for optimal transfer: R1162 replicons lacking this form also are not transferred detectably by pOX38 and are less well mobilized by R751 . The distance between oriT and the primase initiation site affects the frequency of mobilization, and this effect is polar in the direction of transfer . Our results indicate that the R1162 primase is active in mobilization of R1162 and suggest that the MobA-linked form is an adaptation increasing its effectiveness during transfer. J Bacteriol, 1996 Dec, 178(23), 6810 - 6 The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli; Jafri S et al.; A mutation in the rpoA gene (which encodes the alpha subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ . Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression . In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a lambda metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the -35 region of the metE promoter that interacts with the alpha subunit . Our data indicate that the glutamic acid at position 261 in the alpha subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter. J Bacteriol, 1996 Dec, 178(23), 6790 - 5 Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism; Nobelmann B et al.; In enteric bacteria, the hexitol galactitol (Gat) (formerly dulcitol) is taken up through enzyme II (II(Gat)) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), and accumulated as galactitol 1-phosphate (Gat1P) . The gat genes involved in galactitol metabolism have been isolated from the wild-type isolate Escherichia coli EC3132 and cloned on a 7.8-kbp PstI DNA fragment . They comprise six complete open reading frames and one truncated open reading frame in the order gatYZABCDR' . The genes gatABC code for the proteins GatA (150 residues) and GatB (94 residues), which correspond to the hydrophilic domains IIA(Gat) and IIB(Gat), and GatC, which represents a membrane-bound transporter domain IIC(Gat) (35 kDa, 427 residues) . The three polypeptides together constitute a II(Gat) of average size (671 residues) . Gene gatD codes for a Gat1P-specific NAD-dependent dehydrogenase (38 kDa, 346 residues), gatZ codes for a protein (42 kDa, 378 residues) of unknown function, and gatY (31 kDa, 286 residues) codes for a D-tagatose-1,6-bisphosphate aldolase with similarity to other known ketose-bisphosphate aldolases . The truncated gatR' gene, whose product shows similarity to the glucitol repressor GutR, closely resembles a gatR gene fragment from E . coli K-12 . The gat genes map in both organisms at similar positions, in E . coli K-12, where they are transcribed counterclockwise at precisely 46.7 min or 2,173 to 2,180 kbp . The genes are expressed constitutively in both strains, probably due to a mutation(s) in gatR . Transcription initiation sites for the gatYp and the gatRp promoters were determined by primer extension analysis. J Bacteriol, 1996 Dec, 178(23), 6782 - 9 Modulation of recombination and DNA repair by the RecG and PriA helicases of Escherichia coli K-12; Al-Deib AA et al.; The RecG protein of Escherichia coli is a structure-specific DNA helicase that targets strand exchange intermediates in genetic recombination and drives their branch migration along the DNA . Strains carrying null mutations in recG show reduced recombination and DNA repair . Suppressors of this phenotype, called srgA, were located close to metB and shown to be alleles of priA . Suppression depends on the RecA, RecBCD, RecF, RuvAB, and RuvC recombination proteins . Nine srgA mutations were sequenced and shown to specify mutant PriA proteins with single amino acid substitutions located in or close to one of the conserved helicase motifs . The mutant proteins retain the ability to catalyze primosome assembly, as judged by the viability of recG srgA and srgA strains and their ability to support replication of plasmids based on the ColE1 replicon . Multicopy priA+ plasmids increase substantially the recombination- and repair-deficient phenotype of recG strains and confer similar phenotypes on recG srgA double mutants but not on ruvAB or wild-type strains . The multicopy effect is eliminated by K230R, C446G, and C477G substitutions in PriA . It is concluded that the 3'-5' DNA helicase/translocase activity of PriA inhibits recombination and that this effect is normally countered by RecG. J Bacteriol, 1996 Dec, 178(23), 6759 - 65 Identification and characterization of uvrA, a DNA repair gene of Deinococcus radiodurans; Agostini HJ et al.; Deinococcus radiodurans is extraordinarily resistant to DNA damage, because of its unusually efficient DNA repair processes . The mtcA+ and mtcB+ genes of D . radiodurans, both implicated in excision repair, have been cloned and sequenced, showing that they are a single gene, highly homologous to the uvrA+ genes of other bacteria . The Escherichia coli uvrA+ gene was expressed in mtcA and mtcB strains, and it produced a high degree of complementation of the repair defect in these strains, suggesting that the UvrA protein of D . radiodurans is necessary but not sufficient to produce extreme DNA damage resistance . Upstream of the uvrA+ gene are two large open reading frames, both of which are directionally divergent from the uvrA+ gene . Evidence is presented that the proximal of these open reading frames may be irrB+. J Bacteriol, 1996 Dec, 178(23), 6752 - 8 Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli; Garzon A et al.; CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli . CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB . Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation . Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells . To find out why, we examined the effects of a purified P1 fragment, CheA{1-149}, on CheA-related signaling activities in vitro and devised in vivo assays for those same activities . Although readily phosphorylated by CheA{260-537}, the CheA catalytic domain, CheA{1-149}, was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center . CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA{1-149}, suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect . In agreement with these findings, a 40-fold excess of CheA{1-149} fragments did not impair the CheA autophosphorylation reaction . CheA{1-149} did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules . An H48Q mutant of CheA{1-149} could not participate in these reactions, indicating that His-48 is probably the substrate site . The low level of efficiency of these phosphotransfer reactions and the inability of CheA{1-149} to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells . However, an excess of CheA{1-149} fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA{1-149} fragments have both transphosphorylation and phosphotransfer capability in vivo. J Bacteriol, 1996 Dec, 178(23), 6720 - 9 TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4; Haase J et al.; TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4 . The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes . TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide . The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency . An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed . TrbK localizes predominately to the cytoplasmic membrane . Its function depends on presence in the recipient cell but not in the donor cell . TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system . The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets. J Bacteriol, 1996 Dec, 178(23), 6693 - 700 Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000; Stolt P et al.; Plasmid pAL5000 from Mycobacterium fortuitum encodes two proteins necessary for replication: RepA (307 amino acid residues) and RepB (119 residues) . A single RNA species encoding these proteins was characterized, and its 5' end was defined . The proteins were expressed as maltose-binding protein fusions in Escherichia coli . The RepB protein was shown in vitro to bind specifically to a previously defined 435-bp region of pAL5000 containing the origin of replication (ori) . The precise RepB binding sites were defined by DNase I footprinting experiments . RepB binds to two motifs in the ori region: a high-affinity site within its own promoter region, implying autoregulation of its expression, and a low-affinity site further upstream, presumably the origin of replication itself . The binding to the latter motif seems to occur on one DNA strand only . The high-affinity binding site contains several palindromic sequences . Gel retardation assays were performed with the different binding sites as templates, and the binding constant to each site was estimated from protein titrations . This is the first molecular dissection of mycobacterial DNA-binding proteins and their interactions with their targets. J Bacteriol, 1996 Dec, 178(23), 6685 - 92 Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure; Blanco DR et al.; We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp . pallidum rare outer membrane porin protein, designated Tromp1 (D . R . Blanco, C . I . Champion, M . M . Exner, H . Erdjument-Bromage, R . E . W . Hancock, P . Tempst, J . N . Miller, and M . A . Lovett, J . Bacteriol . 177:3556-3562, 1995) . Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli . rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC) . Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T . pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa . rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter . Under these conditions, rTromp1 fractionated predominantly with the E . coli soluble and outer membrane fractions, but not with the inner membrane fraction . rTromp1 isolated from the E . coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl . Whole-mount immunoelectron microscopy using infection-derived immune serum against T . pallidum indicated that rTromp1 was surface exposed when expressed in E . coli . These findings demonstrate that rTromp1 can be targeted to the E . coli outer membrane, where it has both porin activity and surface antigenic exposure. J Bacteriol, 1996 Dec, 178(23), 6658 - 64 Competition between functional signal peptides demonstrates variation in affinity for the secretion pathway; Chen H et al.; We have developed a system for examining the relative affinity of two different signal peptides for the protein secretion pathway in Escherichia coli . This system involves the expression of a modified alkaline phosphatase which possesses two signal peptides arranged in tandem . When both signal peptides have the wild-type sequence, cleavage after the first and cleavage after the second occur with nearly equal frequency . In both cases the remainder of the protein is transported to the periplasm . Thus both signal peptides effectively compete with each other for entrance to the secretion pathway . When the hydrophobicity of the second signal peptide is altered by small increments, we find that the more hydrophobic signal peptide is preferentially utilized . Thus, a more hydrophobic signal peptide can outcompete even an efficient wild-type signal sequence . The crossover point, for utilization of the second to the first signal peptide, is marked and occurs over a very small change in hydrophobicity . Our results suggest that the small differences in the hydrophobicity of wild-type signal peptides may have critical consequences: preproteins with the more hydrophobic signals could dominate one pathway, leaving those with only slightly less hydrophobic signals to require additional factors such as chaperonins, SecB, and other binding proteins. J Bacteriol, 1996 Dec, 178(23), 6651 - 7 Role of mismatch repair in the Escherichia coli UVM response; Murphy HS et al.; Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2 . This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents . Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis . To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E . coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion . Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology . The results showed normal UVM induction patterns in all the repair-defective strains tested . In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS . All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells . Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E . coli and may thus represent a previously unrecognized misrepair or misreplication pathway.
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