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| J Biol Chem, 1994 Sep 23, 269(38), 23471 - 6 Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo; Faulkner KM et al.; Several manganic porphyrins, with substituents on the methine bridge carbons, were prepared and examined for stability, redox behavior, catalysis of the dismutation of superoxide radical (O2-), and the ability to protect a superoxide dismutase (SOD)-null strain of E . coli against dissolved oxygen and a SOD-competent strain against paraquat . All of the compounds tested exhibited reversible redox behavior and were stable to EDTA in both the oxidized and reduced states, and several were able to catalyze the dismutation of O2- with the rate constants of approximately 10(7) M-1 s-1 . The marked protective effects of some of these compounds exceeded that which could be anticipated on the basis of such rate constants . The tetrakis (1-methyl-4-pyridyl) compound was reduced enzymatically at the expense of NADPH and nonenzymatically by GSH and was kept in the reduced state within E . coli . Since the rate constant for reoxidation of the reduced form by O2- is 4 x 10(9) M-1 s-1, it appears that this compound acts in vivo as an NADPH/GSH:O2- oxidoreductase rather than as an SOD mimic . Its ability to facilitate aerobic growth of the SOD-null strain can be explained on this basis. J Biol Chem, 1994 Sep 23, 269(38), 23437 - 43 The glucose transporter of Escherichia coli . Overexpression, purification, and characterization of functional domains; Buhr A et al.; The glucose transporter of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar . It consists of a transmembrane subunit (IICBGlc) and a hydrophilic subunit (IIAGlc) . The IICBGlc subunit consists of two domains . The NH2-terminal IIC domain (residues 1-386) spans the membrane eight times and contains the substrate binding site . The COOH-terminal hydrophilic IIB domain (residues 391-476) is accessible from the cytoplasmic side of the membrane . It contains the phosphorylation site (Cys421) and together with the IIC domain catalyzes the transfer of phosphoryl groups from the IIAGlc subunit to the transported solute . Starting from a plasmid vector containing ptsG under an inducible promoter, the IIB and the IIC domains have been subcloned separately, overexpressed in Escherichia coli, and purified by Ni2+ chelate affinity chromatography . Approximately 40 mg of IIBGlc-6H and 4 mg of IICGlc-6H could be purified from 1 liter of culture . Cells expressing IIBGlc-6H and IICGlc-6H separately have a three times longer generation time on glucose minimal medium than cells expressing wild-type IICBGlc . The rate of IIBGlc-6H phosphorylation determined in a nitrocellulose filter binding assay is indistinguishable from wild-type IICBGlc . The in vitro specific activity of IICGlc-6H in the presence of excess IIBGlc-6H is 2% of the control . IIBGlc-6H also complements the activity of a IICBGlc mutant with an inactive IIB domain (C421S) indicating that IIC and IIB are flexibly linked such that a free IIB domain can displace an inactive IIB domain from its contact site on the IIC domain . Based on this work, the secondary structure of the IIBGlc domain has been determined by isotope-edited NMR spectroscopy (Golic Grdadolnik, S., Eberstadt, M., Gemmecker, G., Kessler, H., Buhr, A., and Erni, B . (1994) Eur . J . Biochem . 219, 945-952). J Biol Chem, 1994 Sep 23, 269(38), 23824 - 9 Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli; Petit MA et al.; Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response . Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria . The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M . F., Echols, H . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 10777-10781) . In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro . We treated purified and inactive UmuC with Hsp70 and Hsp60 . After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not . However, lesion bypass activity was recovered upon further treatment with Hsp60 . The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis. J Biol Chem, 1994 Sep 23, 269(38), 23808 - 16 Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli . Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker; Guy HI et al.; The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells . Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers . One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species . A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli . The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography . The purified protein had kinetic parameters that were close to those obtained for native CAD . Moreover, the CPSase activity was allosterically regulated . Gel filtration showed that the recombinant protein had the same molecular mass as native CAD . Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact . A deletion mutant that lacked the DA linker was then constructed . The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains . However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex . Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed . Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD. J Biol Chem, 1994 Sep 23, 269(38), 23575 - 82 Rapid degradation of an abnormal protein in Escherichia coli involves the chaperones GroEL and GroES; Kandror O et al.; In Escherichia coli, the molecular chaperones (DnaK, DnaJ, and GrpE) are essential for the rapid degradation of certain proteins . To see if chaperones are involved more generally in proteolysis, we studied the degradation of a short-lived fusion protein, CRAG, which associates with DnaK and GroEL in vivo . Its rapid degradation requires ATP and ClpP, the proteolytic subunit of protease Ti (Clp) . However, this process is not reduced in strains lacking the complementary ATPase subunit, ClpA, or its homologs, ClpB and ClpX . At 37 degrees C, but not at 42 degrees C, protease La also contributes partially to CRAG degradation . Nevertheless, CRAG is not degraded in cell-free extracts or upon incubation with ClpP or protease La . We tested whether the chaperones associated with CRAG might be involved in its degradation . CRAG breakdown was accelerated 2-3-fold in strains with high levels of heat-shock proteins (hsps), i.e . in those that overproduce the hsp transcription factor (sigma 32) or carry a dnaK deletion . A similar stimulation of proteolysis was observed in cells overproducing GroEL or both GroEL and GroES; in these cells, more CRAG was associated with GroEL than in the wild type . In a temperature-sensitive groEL44 mutant at the nonpermissive temperature, CRAG breakdown was accelerated, and more CRAG was found complexed with GroEL . However, in a temperature-sensitive groES mutant, CRAG was completely stable at the nonpermissive temperature and accumulated bound to GroEL . These findings indicate that the association of CRAG with GroEL is a rate-limiting step in CRAG degradation, which also requires a subsequent action of GroES . We propose that if the hsp60/hsp10 chaperonins fail to catalyze the proper folding of a protein, they can facilitate its rapid degradation. J Biol Chem, 1994 Sep 23, 269(38), 23655 - 60 Epitope mapping and functional characterization of monoclonal antibodies specific for the alpha subunit of Escherichia coli RNA polymerase; Sharif KA et al.; The epitopes have been localized for a set of monoclonal antibodies specific for the alpha subunit of the Escherichia coli RNA polymerase . The antibodies are classified into three groups based on their epitopic assignments . Group 1, mAb 123C2, maps in the N terminus of alpha between amino acids 1 and 23; Group 2 antibodies (mAb 129C4, mAb 124D1 and mAb 121C5) map in the central region between amino acids 190 and 210; Group 3 antibodies (mAb 130B1 and mAb 125C6) map in the C terminus between amino acids 310 and 320 . mAb 130C2 is anomalous since it maps to the N terminus between amino acids 1 and 23 as well as to the C terminus between amino acids 320 and 329 . The antibodies were used to investigate the role of alpha in transcription activation with cAMP receptor protein-dependent promoters . Three antibodies (130C2, 121C5, and 125C6) inhibited cAMP receptor protein-dependent initiation with lac P+ but not with lac UV5 or gal P+ . Inhibition was observed with free RNA polymerase and the closed promoter complex; the preformed open promoter complex was insensitive . Only lac P+ was sensitive to these anti-alpha antibodies supporting the concept that the mode of interaction of RNA polymerase with cAMP receptor protein differs between lac P+ and gal P+. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 65 - 74 Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana; Horn MA et al.; The RLK5 gene of Arabidopsis thaliana encodes a novel receptor-like protein kinase . DNA sequence analysis suggests that the RLK5 protein contains an extracellular domain that has 21 tandemly repeated leucine-rich motifs linked, via a transmembrane hydrophobic region, to a protein kinase catalytic domain that is related to the serine/threonine family of protein kinases . To study the intrinsic biochemical properties of this protein kinase we have expressed the catalytic domain as two different recombinant fusion proteins in Escherichia coli . Both hybrid proteins have similar kinetic properties, autophosphorylate on serine and threonine residues and have significantly greater activity in the presence of Mn2+ than Mg2+ . A lysine to glutamic acid substitution in the catalytic domain of RLK5 results in the catalytically inactive protein RLK5(Cat)K711E . The active RLK5 protein can phosphorylate the inactive K711E protein and the K711E protein can partially inhibit the autophosphorylation of RLK5 . Tryptic cleavage of the autophosphorylated proteins followed by two-dimensional thin layer electrophoresis indicates that several sites in the catalytic domain are phosphorylated. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 55 - 64 Bimodal action of spermine on ribosomal peptidyltransferase at low concentration of magnesium ions; Drainas D et al.; At 6 mM Mg2+, submillimolar concentrations of spermine affect the end-point as well as the kinetic phase of puromycin reaction in a cell-free system from Escherichia coli . When the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the absence of ribosomal wash (FWR fraction), the final degree of AcPhe-puromycin synthesis is raised from 12% to 60%, as the concentration of spermine increases from zero to 200 microM . However, spermine displays partial noncompetitive inhibition at the kinetic phase of the reaction . The inhibitory effect of spermine is related with its binding to AcPhe-tRNA . When complex C is formed in the presence of FWR fraction, spermine slightly affects the final degree of puromycin synthesis is markedly stimulated by the addition of relatively low concentrations of spermine . Kinetic analysis of the activation phase revealed that spermine attached on a specific site of complex C, acts as a nonessential, partial noncompetitive activator . The stimulatory effect of spermine seems to be due to its interaction with ribosomes . Further additions of spermine cause partial noncompetitive inhibition on the puromycin reaction . This result suggests that complex C possesses a second binding site, responsible for the inhibitory effect of spermine . Both activator and inhibitor sites can be occupied by spermine at the same time. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 179 - 85 Elucidation of the thermal stability of the neutral proteinase II from Aspergillus oryzae; Tatsumi H et al.; The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc proteinase with three intramolecular disulfide bonds . NpII is most unstable after 10 min at about 75 degrees C, but regains stability beyond this temperature and is relatively stable at 100 degrees C . We analyzed the thermal stability of wild-type NpII and apo NpII . The results suggested that NpII unfolds reversibly upon incubation up to 100 degrees C, and that the irreversible inactivation observed is mainly due to autoproteolysis . To further understand the stability, a mutant NpII (Cys78-->Ala) lacking one of the disulfide bonds, was produced in a heterologous yeast expression system . The mutant NpII showed a similar stability profile, but the most unstable temperature and the most catalytically active temperature decreased to the same extent (around 10 degrees C), confirming that autoproteolysis is the main cause of the irreversible inactivation . Several lines of evidence presented in this study demonstrated that the thermal stability of o++NpII is attributed to reversible thermal unfolding and autoproteolysis. Biochim Biophys Acta, 1994 Sep 21, 1208(1), 189 - 92 Refolding and release of tubulins by a functional immobilized groEL column; Phadtare S et al.; Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer . These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL . Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP . Similar results were obtained with glutamine synthetase . These data suggest that groEL can function while it is attached to a solid support system. Biochemistry, 1994 Sep 20, 33(37), 11391 - 403 A site-specific endonuclease derived from a mutant Trp repressor with altered DNA-binding specificity; Pfau J et al.; Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif . Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors . Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot . One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor . A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct . This nuclease has an altered specificity of DNA binding in vitro . When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency. Biochemistry, 1994 Sep 20, 33(37), 11307 - 14 Structure and function of Escherichia coli DnaB protein: role of the N-terminal domain in helicase activity; Biswas SB et al.; We have analyzed the contributions of specific domains of DnaB helicase to its quaternary structure and multienzyme activities . Highly purified tryptic fragments containing various domains of DnaB helicase were prepared . Fragment I lacks 14 amino acid (aa) residues from the N-terminal of DnaB helicase . Fragments II and III are 33-kDa C-terminal and 12-kDa N-terminal polypeptides, respectively, of fragment I . The single-stranded DNA-dependent ATPase and DNA helicase activities of DnaB helicase and its fragments were examined in detail . The ATPase activities of native DnaB helicase and fragment I were comparable; however, the ATPase activity of fragment II was somewhat diminished . Unlike the ATPase activity, the DNA helicase activity was totally abolished in fragment II and was not complemented by the addition of equimolar fragment III . Consequently, the N-terminal 17-kDa domain appeared to have an indispensable role in the DNA helicase action, but not in other enzymatic activities . Fragment I had a hexameric structure similar to that observed with DnaB helicase in both size exclusion HPLC (SE-HPLC) and chemical cross-linking studies . SE-HPLC analysis indicated that fragment II had an apparent hexameric form . However, a detailed chemical cross-linking analysis showed that it formed stable dimers but the formation of a stable hexamer was severely impaired . Thus, the N-terminal domain appeared to have a strong influence on the hexamer formation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Sep 20, 33(37), 11189 - 99 The A-state of barnase; Sanz JM et al.; The acid-induced denaturation of barnase and its mutants has been analyzed to search for partly-folded intermediates . Differential scanning calorimetry of barnase deviates from two-state behavior below pH 4.0 at low ionic strength, with the maximum discrepancy at pH 2.7 . Addition of 200 mM KCl apparently restores the two-state transitions . Thermograms of barnase mutants at pH 2.7 and low ionic strength fall into three classes: alpha, symmetric transitions which fit well to a two-state equilibrium; b, asymmetric transitions indicating deviation from two-state behavior; and c, transitions with an obvious second component . The most distorted thermograms are observed for mutants that had previously been engineered to accumulate at equilibrium the major kinetic folding intermediate state of barnase at neutral pH . Further analysis of these mutants show the existence of complex equilibria on thermal denaturation . Addition of KCl leads to the slow formation of soluble aggregated forms (A-state) which share some of the properties of the "molten globule" state, i.e., significant secondary structure, lack of fixed tertiary structure, and solvent-accessible hydrophobic patches . The far-UV CD spectrum of the A-state can be explained in terms of native-like secondary structure contributions . Kinetic and chemical cross-linking experiments show that dimerization of partly-folded molecules occurs in the transition region, and such dimerization is probably the rate-limiting step in the formation of the A-state in the presence of KCl . As the A-state has been observed clearly so far for only the mutants in which the folding intermediate has been designed to accumulate, we suggest that the A-state would be related to the main folding intermediate state of barnase . The intermediate would be highly stabilized at low pH, and it is prone to self-associate in these conditions. Biochemistry, 1994 Sep 20, 33(37), 11121 - 6 The activity of carboxypeptidase Y toward substrates with basic P1 amino acid residues is drastically increased by mutational replacement of leucine 178; Olesen K et al.; A random mutagenesis study on carboxypeptidase Y has previously suggested that Leu178 is situated in the S1 binding pocket, and this has later been confirmed by the three-dimensional structure . We here report the mutational replacement of Leu178 with Trp, Phe, Ala, Ser, Cys, Asn, Asp, or Lys and the kinetic characterization of each mutant, using substrates systematically varied at the P1 position . The general effect of these substitutions is a reduced kcat/Km for substrates with uncharged amino acid residues in the P1 position, little effect on those with acidic residues, and an increased kcat/Km for those with basic amino acid residues . There is a clear correlation between the reduction in kcat/Km for substrates with uncharged P1 side chains and the nature of the residue at position 178 . A small reduction is observed when Leu178 is replaced by another hydrophobic amino acid residue, a larger reduction when it is replaced by a polar residue, and a very large reduction when it is replaced by a charged residue . When Leu178 is replaced by Asp, kcat/Km is reduced by a factor of 2200 for a substrate with Val in the P1 position . The kcat/Km values for the hydrolysis of substrates with charged P1 side chains are increased when Leu178 is replaced by an amino acid residue with the opposite charge, and they are decreased when it is replaced by a residue with the same charge . Surprisingly, all mutants (except L178K) exhibit increased activity with substrates with basic P1 side chains.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Sep 20, 33(37), 11087 - 96 Solution structure of a low molecular weight protein tyrosine phosphatase; Logan TM et al.; Protein tyrosine phosphatases (PTPs) are important enzymes involved in signal transduction, cell cycle regulation, and the control of differentiation . Despite the importance of this class of enzymes in the control of critical cell processes, very little structural information is available for this family of proteins . In this paper, we present the first solution structure of a protein tyrosine phosphatase . This protein is a low molecular weight cytosolic PTP that was initially isolated from bovine heart . The structure that was determined from 1747 NMR-derived restraints consists of a central four-stranded parallel beta-sheet surrounded by four alpha-helices and a short 3(10) helix . The phosphate binding site, identified by chemical shift changes upon the addition of the competitive inhibitors phosphate and vanadate, is in a loop region connecting the C-terminal end of the first beta-strand with the first alpha-helix . Residues in the second, fourth, and fifth alpha-helices and in some of the loop regions connecting the elements of regular secondary structure also contribute to the binding site . The structure determined here is consistent with previous mutagenesis and chemical modification studies conducted on this protein. Biochemistry, 1994 Sep 20, 33(37), 11254 - 63 Dynamic transitions of the transmembrane domain of diphtheria toxin: disulfide trapping and fluorescence proximity studies; Zhan H et al.; Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane . The T domain, consisting of nine alpha-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized . To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8-TH9) to one of the other two layers . Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers . Reduction of the bridges abolished these effects . Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers . Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions . The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-TH9 hairpin from the other layers upon the T domain's interaction with the bilayer . The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8-TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions. FEBS Lett, 1994 Sep 19, 352(1), 67 - 70 Observation of the Fe-O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli; Hirota S et al.; Reaction intermediates in dioxygen reduction by the E . coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Raman spectroscopy using the artificial cardiovascular system . At 0-20 microseconds following photolysis of the enzyme-CO adduct in the presence of O2, we observed the Fe-O2 stretching Raman band at 568 cm-1 which shifted to 535 cm-1 with the 18O2 derivative . These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa3-type cytochrome c oxidase . In the later time range (20-40 microseconds), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-1 . Since the 781 cm-1 band exhibited a downshift by 37 cm-1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode . This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 microseconds) . The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine cytochrome c oxidase reaction. J Mol Biol, 1994 Sep 16, 242(2), 139 - 49 The activation of Escherichia coli aspartate transcarbamylase by ATP . Specific involvement of helix H2' at the hydrophobic interface between the two domains of the regulatory chains; Xi XG et al.; The regulatory chain of E . coli aspartate transcarbamylase (E.C . 2.1.3.2) is folded into two domains . The allosteric domain harbours the regulatory site where the activator ATP and the inhibitors CTP and UTP bind competitively . The zinc domain ensures the contact with the catalytic chains . The interface between these two domains is hydrophobic, and involves the carboxy-terminal part of the helix H2' of the allosteric domain and several residues of the zinc domain . This structural feature mediates the transmission of the ATP regulatory signal . In the present work, site-directed mutagenesis and molecular modelling were used to investigate the role of specific amino acid residues in this process . The modifications of the hydrophobic core which are expected to alter the position of helix H2' reduce or abolish the sensitivity of the enzyme to ATP . The properties of the mutants and the results of modelling are fully consistent and suggest that a movement of helix H2' is part of the mechanism of activation by ATP . A model is proposed to account for the transmission of the ATP signal from the regulatory site to the interface between the regulatory and catalytic chains. J Mol Biol, 1994 Sep 16, 242(2), 116 - 29 DNA-binding parameters of the HU protein of Escherichia coli to cruciform DNA; Bonnefoy E et al.; We have previously studied the binding characteristics of the HU protein of Escherichia coli to different linear DNAs . In this work, using gel-retardation and footprint analysis, we studied the specific binding of HU protein to a synthetic cruciform DNA . We have quantified our results in order to precisely define the binding and cooperativity constants of HU protein towards cruciform DNA and compare them to those obtained with linear DNA . We used stringent high-salt conditions versus non-stringent low-salt conditions in order to differentiate the non-specific-protein HU-DNA complexes from the specific, high-salt-resistant complexes . We observe that HU-protein dimers bind specifically to the cruciform DNA with a binding constant K = 2.0 x 10(8) M-1 and a value for the cooperativity constant omega = 1 corresponding to a non-cooperative phenomenon . For the first time we observe a footprint pattern of HU protein bound to DNA using the hydroxyl-radical-footprinting technique on HU-protein-cruciform-DNA complexes . The residues protected by HU protein are localized at and near the junction point but interestingly they are mainly present in two of the four oligonucleotides which constitute the cruciform DNA . These two oligonucleotides are unpaired and opposite each other . These results support a model where two HU-protein dimers specifically bind to two equivalent angles present opposite each other in the four-way-junction-DNA structure with almost no dimer-dimer interactions. J Mol Biol, 1994 Sep 16, 242(2), 107 - 15 Functional map of the alpha subunit of Escherichia coli RNA polymerase . Deletion analysis of the amino-terminal assembly domain; Kimura M et al.; The alpha subunit of Escherichia coli RNA polymerase plays a major role in the assembly of the core enzyme . The amino-terminal two-thirds of the alpha subunit, as far as position 235, are involved in this assembly . To define the site(s) within this region required for core enzyme assembly, we constructed a set of amino-terminal and internal deletion mutants of the rpoA gene . The overexpressed alpha derivatives were purified to apparent homogeneity and examined for their abilities to assemble beta and beta' subunits into active core enzymes in vitro . Among a total of 22 alpha derivatives tested, only four mutants retained the activity form active core enzyme . These mutants had deletions of the extreme amino-terminal residues as far as amino acid residue 30 . The minimum fragment with full activity of the core assembly was alpha(21 to 235), with deletions of 20 amino-terminal and 94 carboxy-terminal amino acid residues . Most of the other mutants appeared to be defective in the formation of stable alpha dimers as analyzed by high-pressure liquid chromatography gel filtration, although some formed self-aggregates . These results, taken together, suggest that the amino-terminal region of the alpha subunit with the core assembly activity is highly structured, and any deletion within this domain disrupts its ordered conformation . Deletions of the extreme amino-terminal region did not affect transcription activation by CRP at the lacP1 promoter or by OmpR at the ompC promoter. Science, 1994 Sep 16, 265(5179), 1699 - 701 iaglu, a gene from Zea mays involved in conjugation of growth hormone indole-3-acetic acid; Szerszen JB et al.; Plants contain most of the growth hormone indole-3-acetic acid (IAA) in conjugated forms believed to be inactive in promoting growth . The iaglu gene, which controls the first step in the biosynthesis of the IAA conjugates of Zea mays, encodes (uridine 5'-diphosphate-glucose:indol-3-ylacetyl)-beta-D-glucosyl transferase . Protein synthesized by Escherichia coli that contained cloned 1-O-beta-D-indol-3-ylacetyl-glucose complementary DNA (cDNA) was catalytically active . The predicted amino acid sequence of the cDNA was confirmed by amino-terminal sequencing of the purified enzyme . Homologous nucleotide sequences were found in all plants tested . The blockage or enhancement of iaglu expression may permit regulation of plant growth. J Biol Chem, 1994 Sep 16, 269(37), 23171 - 6 Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit; Davis R et al.; Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2 . We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system . To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside . After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture . The concentrated R1 protein solution had a pinkish red color . Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex . However, all attempts to demonstrate a function of this complex have been inconclusive . A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation . Circular dichroism spectroscopy indicated an alpha-helical content of 50% . A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein . Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein. J Biol Chem, 1994 Sep 16, 269(37), 23150 - 5 The catalytic subunit of bovine brain platelet-activating factor acetylhydrolase is a novel type of serine esterase; Hattori M et al.; Platelet-activating factor (PAF) acetylhydrolase is the key enzyme in PAF inactivation . We recently purified one isoform of the enzyme from a bovine brain soluble fraction and revealed it as a heterotrimeric enzyme consisting of 29-, 30-, and 45-kDa subunits . Among them, the 29-kDa subunit possesses an active serine residue since diisopropyl fluorophosphate (DFP), an inhibitor of the enzyme, labeled only this subunit (Hattori, M., Arai, H., and Inoue, K . (1993) J . Biol . Chem . 268, 18748-18753) . In the current study, we cloned the cDNA for the 29-kDa catalytic subunit . The predicted sequence of 232 amino acids is unique and is not homologous with those of any other proteins reported so far . When transfected into either Escherichia coli or COS7 cells, the cDNA produced PAF acetylhydrolase activity in both types of cells, indicating that this subunit alone is enough for catalysis . The recombinant 29-kDa protein was also inhibited and labeled by DFP . Furthermore, we isolated and sequenced the {3H}DFP-labeled peptide fragment, revealing that Ser47 is the active serine residue . The sequence surrounding it is different from the consensus sequence of the serine esterase family . Interestingly, the sequence of about 30 amino acids located 6 residues downstream from the active serine site exhibits significant homology to the first transmembrane region of the PAF receptor . These data demonstrate that the catalytic subunit of brain PAF acetylhydrolase is a novel type of serine esterase. J Biol Chem, 1994 Sep 16, 269(37), 23025 - 31 Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase; Symcox MM et al.; The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli . These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites . Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, {3H}cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of {3H}cAMP from the mutant subunits at low temperature . The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation . While the presence of cAMP in site A retards dissociation of {3H}cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of {3H}cAMP from site B of the mutant subunits . The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites . A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242. J Biol Chem, 1994 Sep 16, 269(37), 23018 - 24 Cloning and characterization of novel rat and mouse low molecular weight Ca(2+)-dependent phospholipase A2s containing 16 cysteines; Chen J et al.; A novel rat 4.4-kilobase (kb) cDNA encoding a low molecular weight Ca(2+)-dependent phospholipase A2 (PLA2) and its murine homologue were cloned . The rat and mouse cDNA predict a mature protein of 130 amino acids (M(r) = 14, 763) preceded by a 28-amino acid prepro-peptide . The deduced amino acid sequences encode a protein containing 16 cysteines which distinguishes them from both mammalian group I and II PLA2s and the recently described group of mammalian PLA2s containing 12 cysteines . A rat RNA blot hybridized with the rat cDNA exhibited an abundant 2.3-kb and a less abundant 5-kb transcript in testis . When the rat cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium . Conditioned medium optimally hydrolyzed the phospholipids of {1-14C}oleate-labeled Escherichia coli at neutral to alkaline pH with 1-7 mM Ca2+ . In assays with individual substrates, L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol was hydrolyzed more efficiently than L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, or L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine. J Biol Chem, 1994 Sep 16, 269(37), 22964 - 8 Sequence requirements for the biotinylation of carboxyl-terminal fragments of human propionyl-CoA carboxylase alpha subunit expressed in Escherichia coli; Leon-Del-Rio A et al.; Biotin-dependent enzymes play an essential role in the metabolism of all organisms . Their biotinylation is catalyzed by holoenzyme synthetases, which attach a biotin molecule to a specific lysine residue on the apoenzymes . The sequence flanking the biotin binding site is highly conserved among biotin-dependent enzymes . This sequence conservation might be related to the extensive cross-species activity showed by holoenzyme synthetases . In this study, we have expressed carboxyl-terminal fragments of the alpha subunit of human propionyl-CoA carboxylase (PCC-alpha) in Escherichia coli and used site-directed mutagenesis to determine the sequence requirements for biotinylation by the bacterial holoenzyme synthetase . We show that the carboxyl-terminal 67 amino acids of PCC-alpha act as an independent domain in the biotinylation reaction . Mutations that affect several conserved Gly residues and a Pro-Met-Pro sequence near the biotin binding site are critical for biotinylation . Substitution of the amino acids that flank the biotin acceptor Lys residue or elimination of the last 3 amino acids of the PCC-alpha peptides had little or no effect on their biotinylation despite their high conservation in biotin enzymes. J Biol Chem, 1994 Sep 16, 269(37), 22958 - 63 A novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines . cDNA cloning, expression, and protein analyses; Toh Y et al.; To understand the genes involved in breast cancer invasion and metastasis, we analyzed a novel candidate metastasis-associated gene, mta1, which was isolated by differential cDNA library screening using the 13762NF rat mammary adenocarcinoma metastatic system . Northern blot analyses showed that the mRNA expression level of the mta1 gene was 4-fold higher in the highly metastatic cell line MTLn3 than in the nonmetastatic cell line MTC.4 . The mta1 gene was expressed in various normal rat organs, especially in the testis, suggesting its essential normal function . The mRNA expression levels of the human homologue of this gene also correlated with the metastatic potential in two human breast cancer metastatic systems . The full-length mta1 cDNA sequence contained an open reading frame encoding a protein of 703 amino acid residues, and sequence analysis by data base homology search indicated that mta1 is a novel gene . The Mta1 protein contained several possible phosphorylation sites, and a proline-rich amino acid stretch at the carboxyl-terminal end completely matched the consensus sequence for the src homology 3 domain-binding motif . Using antibodies raised against glutathione S-transferase-Mta1 fusion protein or a synthetic oligopeptide, Western blots showed that the molecular mass of the Mta1 protein was approximately 80 kDa, and the levels of the Mta1 protein also correlated with the metastatic potential, results similar to those obtained from the Northern analyses . Thus, the novel gene mta1 may encode a molecule that is functional in normal cells as well as in breast cancer invasion and metastasis. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 866 - 73 A modified form of the cytochrome P450scc precursor: a new approach in studies on protein import into mitochondria; Novikova LA et al.; Recombinant DNA was constructed providing hypersynthesis of a hybrid protein with a MRGSH6GIR sequence preceding the NH2-terminus of the bovine cytochrome P450scc precursor (6His-pP450scc) in Escherichia coli cells . A large-scale procedure for isolation and purification of this protein was elaborated . 6His-pP450scc was imported into isolated rat liver mitochondria and processed to the mature-sized form . As a similar procedure can be applied to other proteins the results of this work offer new opportunities in studies of protein import into mitochondria. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 768 - 72 Xenopus laevis ribosomal protein S11: cloning and sequencing of the cDNA and primary structure of the protein; Annesi F et al.; We have cloned a Xenopus laevis cDNA coding for the 40S subunit cytoplasmic ribosomal protein S11 . The nucleotide sequence was determined and the derived amino acid sequence reveals that the protein has 158 amino acid residues and a calculated molecular mass of 18,424 Da . Amino acid sequence comparison with the homologous counterparts from very diverse groups of organisms representing animals (human and rat), fungi (yeast) and plants (maize and Arabidopsis thaliana), shows that this protein is very conserved during evolution . Furthermore, ribosomal protein S11 also shares a significant sequence homology to a set of related proteins: plastid ribosomal protein CS17 from different plants, Escherichia coli ribosomal protein S17 and Halobacterium marismortui ribosomal protein S14. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1152 - 9 Requirement of the human immunodeficiency virus type 1 env gene sequence for TAR-independent trans activation by Tat from the major immediate-early promoter of murine cytomegalovirus; Kim YS; Previously it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Tat protein mediates induction of the HIV-1 env expression through a TAR-independent manner in heterologous and homologous promoter systems (Kim and Risser, 1993, J . Virol . 67, 239; Kim and Panganiban, 1993, J . Virol . 67, 3739) . To further explore the transactivation of HIV-1 env gene, I examined expression of the env, the bacterial CAT, and the firefly luciferase genes from a heterologous promoter, the major immediate-early promoter (MIEP) of murine cytomegalovirus (MCMV) . Here we show that Tat augments gene expression from the MCMV MIEP only when linked to the env gene . Surprisingly, in contrast to the expression from an HIV-1 LTR lacking the TAR element, TAR-independent transactivation of env gene expression from MCMV MIEP did not require the full length Tat protein . In addition, deletion of the previously identified cis-acting Tat-responsive element in env did not affect Tat transactivation of the env gene expression . Thus, there are multiple distinct elements that mediate Tat responsiveness in the absence TAR. Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 1140 - 5 Stoichiometry of Sulfolobus ribosomal protein L12e in 50S subunits determined by quantification of immunoblots; Casiano CA et al.; A monoclonal antibody reactive with Sulfolobus solfataricus acidic ribosomal protein SsoL12e was prepared and employed to determine the stoichiometry of this protein in 50S ribosomal subunits by quantification of chloronaphthol-stained protein bands from immunoblots . Approximately four copies of SsoL12e were detected per 50S ribosome . This finding extends previous studies demonstrating the involvement of this protein in a multimeric protein complex in the ribosomal factor binding domain of Sulfolobus and strengthens the concept that this structural motif is a highly conserved and presumably critical feature of the ribosome. Gene, 1994 Sep 15, 147(1), 153 - 4 Isolation and sequencing of a cDNA encoding the hypoxanthine-guanine phosphoribosyltransferase from Toxoplasma gondii; Vasanthakumar G et al.; A hypoxanthine-guanine phosphoribosyltransferase-encoding gene (HGPRT) from Toxoplasma gondii has been isolated and sequenced . The identity of the enzyme was determined by sequence comparison and complementation in a mutant Escherichia coli. Gene, 1994 Sep 15, 147(1), 149 - 50 Purification of the Escherichia coli integration host factor (IHF) in one chromatographic step; Filutowicz M et al.; The integration host factor (IHF) participates in a diverse array of DNA transactions such as replication, recombination and gene expression . We describe a fast and very efficient isolation procedure which yields highly purified IHF in one chromatographic step. Blood, 1994 Sep 15, 84(6), 1887 - 95 Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy; May LT et al.; In a phase 1 study of recombinant interleukin-6 (rIL-6) in patients with advanced solid tumors (n = 15), we discovered that the endogenous IL-6 levels, in pretreatment plasma or serum samples, were distributed into two groups . One set of patients (designated "type 1"; n = 9) was characterized by low plasma IL-6 levels (48 to 1,700 pg/mL) as measured using enzyme-linked immunosorbent assays (ELISA) for IL-6 . In the second set of patients (designated "type 2"; n = 6), IL-6 ELISAs showed high levels of plasma IL-6 (50 to 600 ng/mL) . Neither group had detectable B9 hybridoma cell growth factor activity associated with the IL-6 in their pretreatment plasma or serum . Plasma C-reactive protein (CRP) levels were markedly elevated in type II patients suggesting that the circulating IL-6 was biologically active in vivo . In both groups of patients there was a small but significant increase in B9 activity in the plasma within three hours after rIL-6 administration (n = 5) . Gel filtration profiles showed that circulating IL-6 in type 1 patients, 15 to 120 minutes after rIL-6 administration was of approximate mass 20 to 40 kD, whereas in type 2 patients, the IL-6 before and after exogenous rIL-6 administration was indistinguishable and was of an approximate mass of 200 kD . IL-6 immunoaffinity purification of the 200 kD complexes showed these to contain multiple isoforms of IL-6 (14 to 31 kD) and the soluble IL-6 receptor (sIL-6R; 50 to 55 kD) . A distinguishing clinical history was that all of the type 2 patients had been actively immunized with an anti-idiotypic monoclonal antibody (MoAb) (MK2-23) 3 to 12 months before initiation of this study for advanced melanoma . An analysis of the plasma IL-6 content in other melanoma patients (n = 16) during antiidiotypic MoAb immunization indicated that marked (up to 600 ng/mL) and sustained (several months) elevations of circulating "chaperoned" IL-6 were induced by active immunization regimens. Nature, 1994 Sep 15, 371(6494), 264 - 7 Context is a major determinant of beta-sheet propensity; Minor DL Jr et al.; Residues in beta-sheets occur in two distinct tertiary contexts: central strands, bordered on both sides by other beta-strands, and edge strands, bordered on only a single side by another beta-strand . The delta delta G values for beta-sheet formation measured at an edge beta-strand of the IgG-binding domain of protein G(GB1) are quite different from those obtained previously at a central position in the same protein . In particular, there is no correlation at the edge position with statistically determined beta-sheet-forming preferences . The differences between beta-sheet propensities measured at central and edge beta-strands, delta delta delta G values, correlate with the values of water/octanol transfer free energies and side-chain non-polar surface area for the amino acids . These results strongly suggest that, unlike alpha-helix formation, beta-sheet formation is determined in large part by tertiary context, even at solvent-accessible sites, and not by intrinsic secondary structure preferences. Carbohydr Res, 1994 Sep 15, 262(2), 323 - 34 Structural elucidation of the capsular polysaccharide produced by Escherichia coli O20 : K84 : H26; Whittaker DV et al.; The primary structure of the acidic capsular antigen produced by E . coli K84 was shown by glycose and methylation analysis, and by 1D and 2D 1H and 13C NMR studies of the polysaccharide and an oligosaccharide produced by lithium-ethylenediamine degradation of the polysaccharide, to be comprised of linear hexasaccharide repeating units of the following structure: -->4)alpha-D-GaI pA-(1-->3)-alpha-D-Man p-(1-->4)-beta-D-GIc p-(1-->3)-alpha- D-Glc pNAc-(1-->2)-beta-D-Man p-(1-->3)-beta-D-Man pNAc-(1-->. Biochem Pharmacol, 1994 Sep 15, 48(6), 1105 - 12 Enzymatic phosphorylation and pyrophosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key metabolite in the pathway for activation of the anti-HIV (human immunodeficiency virus) agent 2',3'-dideoxyinosine; Nave JF et al.; 2',3'-Dideoxyadenosine-5'-monophosphate (ddAMP) is a key intermediate in the metabolic pathway involved in the activation of the anti-retroviral agent 2',3'-dideoxyinosine (ddI) to 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) . The potential phosphorylation of ddAMP by adenylate kinase (myokinase) and pyrophosphorylation by the reverse reaction of 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase were investigated . Using ATP as phosphate donor, ddAMP was phosphorylated by adenylate kinase with an efficiency of 8.8% of that for AMP, as estimated from the Vmax/Km ratios . In the presence of PRPP, Escherichia coli and rat PRPP synthetases catalysed the pyrophosphorylation of ddAMP with efficiencies of 52 and 35% of that determined for AMP, respectively . Two carbocyclic phosphonate analogues of ddAMP were not substrates of adenylate kinase . Yet, they were pyrophosphorylated by both PRPP synthetases, albeit less efficiently than ddAMP . In vivo, the usual function of PRPP synthetase is to synthesize PRPP from ribose-5-phosphate and ATP . In the forward reaction ddATP proved to be a substrate as efficient as ATP for rat PRPP synthetase . ddATP was also studied as a potential phosphate donor in the reaction catalysed by adenylate kinase with AMP as phosphate acceptor and found to be as efficient as ATP . The relevance of these in vitro results to the in vivo situation is discussed. Biochem J, 1994 Sep 15, 302 ( Pt 3), 881 - 7 Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase; Chapman-Smith A et al.; A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E . coli . The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E . coli biotin ligase . The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin . We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with {3H}biotin, which greatly facilitated development of a purification procedure . The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid . The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase . The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s . The domain was readily produced and purified in sufficient quantities for n.m.r . structural analysis. Biochem J, 1994 Sep 15, 302 ( Pt 3), 837 - 44 Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein; Warren MJ et al.; The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology . Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein . However, this truncated protein was not able to complement E . coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem . Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site . When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin . The CysGA protein on the other hand showed no such coenzyme-dependent activity . Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation . The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation. Biochem J, 1994 Sep 15, 302 ( Pt 3), 813 - 20 Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli; Bauminger ER et al.; Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores . Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation . Mossbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin . Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN) . A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III) . Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex . Mossbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins . Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components . This provides further evidence that the ferritin protein shell can influence the structure of its iron core. Eur J Biochem, 1994 Sep 15, 224(3), 983 - 90 Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius; Gleissner M et al.; The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius . In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol . The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S . acidocaldarius . With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C . Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C . The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon. Eur J Biochem, 1994 Sep 15, 224(3), 893 - 9 Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties; Zimniak P et al.; Glutathione S-transferase P1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta {Ahmad, H., Wilson, D . E., Fritz, R . R., Singh, S . V., Medh, R . D., Nagle, G . T., Awasthi, Y . C . & Kurosky, A . (1990) Arch . Biochem . Biophys . 278, 398-408} . In the present report, the enzymic properties of these two proteins are compared . {I104}glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and {V104}glutathione S-transferase P1-1 was expressed and isolated as described for {I104}glutathione S-transferase P1-1 . The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for {I-104}glutathione S-transferase P1-1 and {V-104}glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione . In addition, the two enzymes were distinguishable by their heat stability, with half-lives at 45 degrees C of 19 min and 51 min, respectively . The resistance to heat denaturation was differentially modulated by the presence of substrates . These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding. Eur J Biochem, 1994 Sep 15, 224(3), 797 - 802 Elongation on the amino-terminal part of stefin B decreases inhibition of cathepsin H; Jerala R et al.; Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily . The mutant proteins were expressed in Escherichia coli and purified to homogeneity . Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H . Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H . A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed . The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases . Variable regions were selected from fragments of other proteins from the protein data base . We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes . This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H . We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H. EMBO J, 1994 Sep 15, 13(18), 4412 - 20 Active site of the replication protein of the rolling circle plasmid pC194; Noirot-Gros MF et al.; Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids . The effect of mutations was examined by an in vivo activity test . Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays . We conclude that all three amino acids have a catalytic role . Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases . We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate . The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step. Nature, 1994 Sep 15, 371(6494), 261 - 4 Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy; Chen S et al.; Protein folding mediated by the molecular chaperone GroEL occurs by its binding to non-native polypeptide substrates and is driven by ATP hydrolysis . Both of these processes are influenced by the reversible association of the co-protein, GroES (refs 2-4) . GroEL and other chaperonin 60 molecules are large, cylindrical oligomers consisting of two stacked heptameric rings of subunits; each ring forms a cage-like structure thought to bind polypeptides in a central cavity . Chaperonins play a passive role in folding by binding or sequestering folding proteins to prevent their aggregation, but they may also actively unfold substrate proteins trapped in misfolded forms, enabling them to assume productive folding conformations . Biochemical studies show that GroES improves the efficiency of GroEL function, but the structural basis for this is unknown . Here we report the first direct visualization, by cryo-electron microscopy, of a non-native protein substrate (malate dehydrogenase) bound to the mobile, outer domains at one end of GroEL . Addition of GroES to GroEL in the presence of ATP causes a dramatic hinge opening of about 60 degrees . GroES binds to the equivalent surface of the GroEL outer domains, but on the opposite end of the GroEL oligomer to the protein substrate. J Physiol, 1994 Sep 15, 479 ( Pt 3), 441 - 9 Alteration of the physiological responses to indomethacin by endotoxin tolerance in the rat: a possible role for central vasopressin; Wilkinson MF et al.; 1 . Previous studies suggest that arginine vasopressin (AVP) is released into the ventral septal area (VSA) of the rat brain during the antipyresis induced by the cyclo-oxygenase inhibitor indomethacin . In addition, there is evidence for increased AVP transmission in the VSA of animals having a reduced pyretic response following three intravenous injections of bacterial endotoxin (LPS) (endotoxin tolerant) . Since ventral septal AVP receptors can also become 'sensitized' following exposure to AVP, we questioned whether the antipyretic action of indomethacin would increase, via an action involving central AVP, if this drug were administered into LPS-tolerant rats . 2 . Intraperitoneal indomethacin (7.5 mg kg-1) was effectively antipyretic when administered 2 h after an intravenous challenge with LPS (50 micrograms kg-1) into conscious unrestrained rats . This dose of indomethacin had no effect on the core temperature of non-febrile rats given intravenous 0.9% pyrogen-free saline . 3 . Three intravenous injections of LPS over a period of 3 days resulted in rats that were tolerant to the pyrogenic effects of LPS . When indomethacin was administered 2 h following the third LPS injection, a dose-dependent hypothermia was observed . This effect was age dependent, as profound hypothermia was seen in 8 week but not 20 week old rats . 4 . A mortality rate of 41% (P = 0.02) was observed within 24 h of indomethacin treatment in 8 week old tolerant rats compared with 0% in 8 week old non-tolerant and 20 week old tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS) J Physiol, 1994 Sep 15, 479 ( Pt 3), 433 - 40 Segmental differences in the effects of guanylin and Escherichia coli heat-stable enterotoxin on Cl- secretion in human gut; Kuhn M et al.; 1 . Mucosally added synthetic guanylin and Escherichia coli heat-stable enterotoxin (STa) increased short-circuit current (ISC) across isolated muscle-stripped human intestine in vitro . 2 . Serosal bumetanide inhibited ISC responses indicating that guanylin and STa stimulate electrogenic chloride secretion . 3 . ISC responses were markedly greater in the colon than in the jejunum . 4 . Pretreatment with indomethacin did not significantly alter the effects of guanylin and STa . 5 . Both peptides induced concentration-dependent increases in the cyclic GMP content of human intestinal mucosa in vitro; cyclic AMP levels remained unchanged . 6 . In contrast to ISC responses, increases in cyclic GMP content induced by guanylin and STa were markedly greater in the jejunum than in the colon . 7 . Sodium nitroprusside (SNP) but not human alpha-atrial natriuratic peptide (CDD/ANP(99-126)) increased chloride secretion in human intestine; both agents induced small increases in intestinal cyclic GMP content . 8 . Guanylin, STa and the nitric oxide (NO) donor SNP increased electrogenic chloride secretion across human intestinal mucosa in vitro by stimulation of cyclic GMP . The discrepancy between the effects on chloride secretion and intracellular cyclic GMP content suggest different cellular action sites of guanylin and STa in human small and large intestine. Cytometry, 1994 Sep 15, 18(3), 147 - 55 Evaluation of flow cytometric methods for diagnosis of chronic granulomatous disease variants under routine laboratory conditions; Emmendorffer A et al.; Neutrophils from 50 pediatric patients with normal phagocyte functions, from 150 healthy adults, from 10 chronic granulomatous disease (CGD)-patients (4 CGD+), and from 18 X-linked carriers for CGD have been tested for their production of H2O2 using staining with dihydrorhodamine 123 and subsequent flow cytometry . Additionally, neutrophils from three patients with myeloperoxidase deficiency were assessed . Cells were activated to produce H2O2 by the phorbol ester phorbol-myristate-acetate (PMA) and by phagocytosis of Escherichia coli bacteria . To evaluate the sensitivity of the method, H2O2-production by neutrophils which was inhibited by different concentrations of diphenyljodonium (DPI) was measured . The results were compared to those from other methods (NBT-testing, cytochrome c-reduction, and especially chemiluminescence) . Normal values and ranges of scatter profile were evaluated in terms of peak channel fluorescence: 97% > 700, x = 840 +/- 59 (S.D.), 97% < 890, for pediatric patients . Normal quantitative values also resulted from small blood samples of infants (< 1 year, n = 6, x = 830 +/- 52) . For CGD+ (n = 4) the results were clearly far below the normal range . In indicating decreased production of reactive oxygen intermediates the method was at least as sensitive as lucigenin enhanced chemiluminescence . Cytochrome b558-expression of neutrophils from patients and healthy controls was established by flow cytometry following staining with the monoclonal antibody 7D5 . The normal range was 97% > 485, 97% < 680, peak channel fluorescence . We conclude that flow cytometric routine diagnostics of CGD can easily enhance the reliability of recognition and the yield of information about this disease compared to conventional methods. Structure, 1994 Sep 15, 2(9), 853 - 68 High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin; Jeng MF et al.; BACKGROUND: Thioredoxin participates in thiol-disulfide exchange reactions and both oxidized thioredoxin (disulfide form) and reduced thioredoxin (dithiol form) are found under physiological conditions . Previous structural studies suggested that the two forms were extremely similar, although significant functional and spectroscopic differences exist . We therefore undertook high-resolution solution structural studies of the two forms of Escherichia coli thioredoxin in order to detect subtle conformational differences . RESULTS: The solution structures of reduced and oxidized thioredoxin are extremely similar . Backbone structure is largely identical in the two forms, with slight differences in the region of the active site, which includes Cys32 and Cys35 . The side chain sulfur atom of Cys32 is tilted away from that of Cys35 in the reduced form of the protein to accommodate the increase in S-S distance that occurs upon reduction of the disulfide, but the chi 1 angles of the two cysteines remain the same in the two forms . CONCLUSIONS: Only subtle conformational changes occur upon changing the oxidation state of the active site cysteines, including the positions of some side chains and in hydrogen bonding patterns in the active site region . Functional differences between the two forms are probably therefore related to differences in local conformational flexibility in and near the active site loop. Structure, 1994 Sep 15, 2(9), 833 - 8 Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177-->Asp); Hardy LW et al.; BACKGROUND: Thymidylate synthase (TS) converts deoxyuridylate to thymidylate, an essential DNA precursor . Replacement of Asn177 with aspartate (Asn177-->Asp) in Escherichia coli TS creates a novel ability to methylate 2'-deoxycytidylate (dCMP) . The dCMP-methylase activity of TS(Asn177-->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl donor . We have tested the possibility that the inactivation is due to negative cooperativity, created in the TS dimer by the Asn177-->Asp mutation . RESULTS: A heterodimeric form of TS, containing one wild type and one Asn177-->Asp active site, was created to test for negative cooperativity . Substrate inactivation still occurred, even with the mutation present at only one active site . CONCLUSIONS: Inactivation of TS(Asn177-->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation . The 'artificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other hetero-oligomeric proteins. Structure, 1994 Sep 15, 2(9), 793 - 6 The ribonucleotide reductase jigsaw puzzle: a large piece falls into place; Sjoberg BM; The three-dimensional structure of ribonucleotide reductase protein R1 from Escherichia coli reveals a novel 10-stranded alpha/beta barrel fold . A long loop penetrates the center cavity to assemble the active site cysteine triad. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 9067 - 71 Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli; Hwang DJ et al.; The bidirectional self-assembly of tobacco mosaic virus (TMV, common or U1 strain) has been studied extensively in vitro . Foreign single-stranded RNA molecules containing the TMV origin-of-assembly sequence (OAS, 75-432 nt in length) are also packaged by TMV coat protein (CP) in vitro to form helical pseudovirus particles . To study virus assembly in vivo requires an easily manipulated model system, independent of replication in plants . The TMV assembly machinery also provides a convenient means to protect and recover chimeric gene transcripts of almost any length or sequence for a variety of applications . Native TMV CP expressed in and purified from Escherichia coli formed nonhelical, stacked aggregates after dialysis into pH 5 buffer and was inactive for in vitro assembly with TMV RNA . U1 CP derivatives in which the second amino acid was changed from Ser to Ala or Pro, nonacetylated N termini found in two natural strains of the virus, failed to remediate these anomalous properties . However, in vivo coexpression of CP and single-stranded RNAs (up to approximately 2 kb) containing the TMV OAS gave high yields of helical pseudovirus particles of the predicted length (up to 7.4 +/- 1.4 micrograms/mg of total bacterial protein) . If the OAS-containing RNA was first recruited into bacterial polyribosomes, elongation of pseudovirus assembly was blocked . In vivo, E . coli expression of a full-length cDNA clone of the TMV genome (6.4 kb) resulted in high, immunodetectable levels of CP and assembly of sufficient intact genomic RNA to initiate systemic infection of susceptible tobacco plants. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8994 - 8 Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit; Marcello A et al.; Mimetic peptides capable of selectively disrupting protein-protein interactions represent potential therapeutic agents for inhibition of viral and cellular enzymes . This approach was first suggested by the observation that the peptide YAGAVVNDL, corresponding to the carboxyl-terminal 9 amino acids of the small subunit of ribonucleotide reductase of herpes simplex virus, specifically inhibited the viral enzyme in vitro . Evaluation and use of this peptide as a potential antiviral agent has, however, been thwarted by its failure to inhibit virus replication in vivo, presumably because the peptide is too large to enter eukaryotic cells unaided . Here, we show that the nontoxic B subunit of Escherichia coli heat-labile enterotoxin can be used as a recombinant carrier for the receptor-mediated delivery of YAGAVVNDL into virally infected cells . The resultant fusion protein specifically inhibited herpes simplex virus type 1 replication and ribonucleotide reductase activity in quiescent Vero cells . Preincubation of the fusion protein with soluble GM1 ganglioside abolished this antiviral effect, indicating that receptor-mediated binding to the target cell is necessary for its activity . This provides direct evidence of the usefulness of carrier-mediated delivery to evaluate the intracellular efficacy of a putative antiviral peptide. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8885 - 9 Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission; Schmidt I et al.; Cauliflower mosaic virus (CaMV) aphid transmission factor (ATF or P18) is presumed to interact with both virus particles and vector mouthparts, thereby mediating virus aphid transmission . We developed a protein-protein binding assay and our results clearly show that virus particles bind strongly and specifically to P18 whether P18 was obtained from plants, a baculovirus expression system, or the pGEX-3X Escherichia coli expression system . We overproduced, using the pGEX-3X expression system, various fragments of P18 and thereby demonstrated that the C-terminal 31 amino acid residues are responsible for the interaction . Using PCR-based mutagenesis, 2 amino acid residues essential for interaction were identified . Point substitutions (amino acids 157 from Ile to Asn or 159 from Gly to Ser) were sufficient to abolish the interaction, whereas another mutation (amino acid 158 from Ile to Ser) had no effect on P18 virus binding . We evaluated whether there was a correlation between the ability of P18 to interact with CaMV particles and its biological activity . Aphid transmission assays were carried out and we demonstrated that the loss of the virus binding capacity had a dramatic effect on the ability of P18 to mediate aphid transmission . Thus, our results suggest that binding between P18 and virus particles is likely to be one of the molecular mechanisms involved in CaMV aphid transmission. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 8747 - 51 Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein; Quinn TP et al.; How an amino acid sequence encodes the information necessary for a protein to adopt a unique tertiary structure remains unresolved . We are addressing this problem by designing "from scratch" protein molecules that will adopt predetermined three-dimensional structures . Based on this strategy, two identical four-stranded beta-sheets were designed to dimerize and form a beta-sandwich protein, called betadoublet . A synthetic gene encoding half the beta-sandwich protein was expressed in Escherichia coli, and the protein was purified to homogeneity . Biophysical characterization of betadoublet in aqueous solution demonstrated that the disulfide formed between the two sheets and that the dimer was a compact unaggregated globular protein, consisting predominantly of beta-sheet and stable to thermal denaturation . It has some backbone amide protons whose exchange is slow enough to be measured by NMR but binds more of the dye 1-anilinonaphthalene-8-sulfonate than a well-folded protein. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 73 - 80 Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates; Wenz C et al.; According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein . However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis . Guided by molecular modelling we have replaced . Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil . We have purified and characterized the resulting N188Q mutant . The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 175 - 8 Cloning and sequencing of the groESL homologue from Porphyromonas gingivalis; Hotokezaka H et al.; The homologue of groESL from Porphyromonas gingivalis was cloned and sequenced . Nucleotide sequencing suggested an operon containing two open reading frames (ORFs) homologous to groESL operon of Escherichia coli . The upstream ORF consisted of 267 bp corresponding to 89 amino acid residues . The downstream ORF consisted of 1635 bp corresponding to 545 amino acid residues. Biochemistry, 1994 Sep 13, 33(36), 10977 - 84 Independent folding of the domains in the hydrophilic subunit IIABman of the mannose transporter of Escherichia coli; Markovic-Housley Z et al.; The active form of the hydrophilic subunit (IIABman) of the mannose transporter of Escherichia coli is a homodimer of two 35-kDa subunits . Each subunit consists of two distinct domains, IIA and IIB, which can be separated by limited trypsin digestion . Separation of tryptic fragments yields monomers of IIB and dimers of IIA, which are active and stable . To test whether the domains fold as independent units, the effects of guanidine hydrochloride (GuHCl) and temperature on the structural stability of the intact IIABman were compared with those of the isolated fragments . Equilibrium GuHCl-induced reversible unfolding, measured by circular dichroism and tryptophan fluorescence, showed a biphasic transition for intact IIABman and monophasic transitions for each isolated fragment . The midpoint transitions of the isolated IIB and IIA fragments (at 1.0 and 2.3 M GuHCl) coincide with the first and second transitions of intact IIABman . Analytical ultracentrifugation studies suggested that dissociation precedes the unfolding of IIA . Thermal unfolding of IIABman, monitored by differential scanning calorimetry, showed two well-separated transitions near 52 and 95 degrees C which corresponded to the midpoint transitions of the isolated IIB and IIA fragments . The combined results demonstrate an independent stepwise unfolding of the domains in IIABman as well as the absence of stabilizing interdomain interactions . The lack of interdomain interactions suggests an unrestricted domain motion . This may play an important role in the phosphoryl transfer reaction which is catalyzed by the binding of IIABman to a phosphoryl carrier protein HPr (via the IIA domain) and to the transmembrane subunits of the mannose transporter (via the IIB domain). Biochemistry, 1994 Sep 13, 33(36), 10944 - 50 Annexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties: 31P- and 1H-NMR studies; Swairjo MA et al.; Calcium-dependent binding to phospholipid membranes is closely associated with annexin functional properties . In these studies, 31P- and 1H-nuclear magnetic resonance (NMR) experiments have been performed to study the effects of binding of recombinant rat annexin V to sonicated small unilamellar vesicles (SUVs) . High-resolution 31P-NMR spectra of SUVs containing mixtures of synthetic phosphatidic acid (PA) and phosphatidylcholine (PC) show resolvable resonances corresponding to the inner-leaflet PA, outer-leaflet PA, and PC phosphoryl groups . When annexin binding occurs, the outer-leaflet PA 31P resonance shifts while that of PC is unaffected, consistent with selective binding of the protein to the phosphoryl moiety of the PA component . Further, annexin V binding to membrane outer-leaflet phospholipids has a measurable effect on inner-leaflet phospholipids of intact vesicles . 1H-NMR T1 relaxation measurements of SUVs containing acyl-chain-perdeuterated PC show no effects on the PA hydrocarbon-chain segmental motions upon annexin binding . Circular dichroism measurements indicate that the protein does not undergo a significant conformational change upon binding to the vesicles . The observed NMR changes do not correspond to proton or calcium gradients, nor to lateral segregation of extended patches of homogeneous phospholipids . The combined evidence suggests that selective, peripheral annexin-membrane interactions influence the environment of the inner vesicular surface . The mechanism proposed is a protein-induced change in vesicle morphology that corresponds to reduced curvature. Proc Natl Acad Sci U S A, 1994 Sep 13, 91(19), 9022 - 6 An in vitro polysome display system for identifying ligands from very large peptide libraries; Mattheakis LC et al.; We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes . A pool of DNA sequences encoding 10(12) random decapeptides was incubated in an Escherichia coli S30 coupled transcription/translation system . Polysomes were isolated and screened by affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B . The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection . A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA . After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody . Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM . The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems. Biochemistry, 1994 Sep 13, 33(36), 11040 - 5 An RNA binding site in a tRNA synthetase with a reduced set of amino acids; Kim S et al.; A 30 amino acid helix-loop of known structure on the surface of the C-terminal domain of the class I Escherichia coli methionine tRNA synthetase is essential for methionine tRNA anticodon discrimination . Replacing this 30 amino acid peptide with a previously described sequence containing residues from the wild-type protein imbedded in a sequence matrix of mostly alanines and serines, we used a combinatorial mutagenesis and selection strategy to define residual wild-type residues that are not replaceable with alanine or serine, because they are needed for specific recognition of methionine tRNA . Four were identified, of which three have functional side chains (Asn, Arg, Lys) . These four and a fifth (Trp) that was previously identified are located at the end of the helix and within the loop, lie on the same side of the structure, and span a distance of about 20 A . We conclude that, within the alanine, serine sequence matrix, only a few non-alanine, non-serine residues in the specificity-determining part of the structure are essential. FEBS Lett, 1994 Sep 12, 351(3), 423 - 6 Functional and structural similarity between the X protein of hepatitis B virus and nucleoside diphosphate kinases; De-Medina T et al.; One of the four genes encoded by hepatitis B virus (HBV) is the regulatory 17 kDa protein called HBx (or pX) . HBx is a transcription transactivator of many cellular and viral regulatory elements . We report here that recombinant HBx supports transcription in vitro and has phosphotransfer enzymatic activity . In the presence of EDTA, a phosphoryl-HBx is formed that releases the phosphate residue upon the addition of Mg2+ . This two-step NTP hydrolysis reaction is characteristic of a group of enzymes termed nucleoside diphosphate kinases (NDPKs) . Remarkably, structural similarity between HBx and NDPKs is also evident . Our findings suggest that HBx has evolved from this group of enzymes but acquired additional activities that satisfy the viral needs. FEBS Lett, 1994 Sep 12, 351(3), 303 - 7 Mechanism of gene activation by the Escherichia coli positive regulator, OmpR: a mutant defective in transcriptional activation; Aiba H et al.; The OmpR protein is an activator specific for the E . coli ompC and ompF genes . This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase . In this study we isolated OmpR mutants which were suggested to be defective for transcriptional activation, but not for DNA binding . Two such mutants, that we isolated, have a single amino acid alteration at positions 131 {P131S}, and 179 {P179L}, respectively, of OmpR, comprising 239 amino acids . These altered amino acids in OmpR may be implicated, directly or indirectly, in the presumed RNA polymerase/OmpR interaction that is important for transcriptional activation. Nucleic Acids Res, 1994 Sep 11, 22(18), 3765 - 72 Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis; Devine SE et al.; We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing . A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed . Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles . A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids . Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process . Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure . We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors . Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts . Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis. Nucleic Acids Res, 1994 Sep 11, 22(18), 3793 - 800 Quantitative analysis of RNA cleavage during RNA-directed DNA synthesis by human immunodeficiency and avian myeloblastosis virus reverse transcriptases; DeStefano JJ et al.; We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs) . Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer . The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H . RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated . Results showed that HIV- and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products . Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity . Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity . The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater . They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand . In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed. Nucleic Acids Res, 1994 Sep 11, 22(18), 3681 - 4 The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516; Bakin A et al.; The number and location of pseudouridine residues in Escherichia coli 16S ribosomal RNA has been determined by a combination of direct and indirect methods . Only one residue was found, at position 516 . This site is at the 5'-end of one of the three most highly conserved long sequences of this RNA molecule . A number of experimental findings have strongly implicated this loop in the fidelity of codon recognition by A-site bound tRNA . By virtue of its location, we suggest that psi 516 may also play a role in maintaining the fidelity of protein synthesis. Cell, 1994 Sep 9, 78(5), 889 - 96 Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding; Blatter EE et al.; Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker . We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2) . Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet) . Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity . Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins. Cell, 1994 Sep 9, 78(5), 877 - 87 An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps; Stukenberg PT et al.; The replicase of E . coli, DNA polymerase III holoenzyme, is tightly fastened to DNA by its ring-shaped beta sliding clamp . However, despite being clamped to DNA, the polymerase must rapidly cycle on and off DNA to synthesize thousands of Okazaki fragments on the lagging strand . This study shows that DNA polymerase III holoenzyme cycles from one DNA to another by a novel mechanism of partial disassembly of its multisubunit structure and then reassembly . Upon completing a template, the polymerase disengages from its beta clamp, hops off DNA, and reassociates with another beta clamp at a new primed site . The original beta clamp is left on DNA and may be harnessed by other machineries to coordinate their action with chromosome replication. Cell, 1994 Sep 9, 78(5), 835 - 43 SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion; Economou A et al.; SecA, the peripheral subunit of E . coli preprotein translocase, alternates between a membrane inserted and a deinserted state as part of the catalytic cycle of preprotein translocation . When SecA is complexed with SecY/E and preprotein, ATP drives a profound conformational change, leading to membrane insertion of a 30 kDa domain of SecA . The inserted domain is protease-inaccessible from the cytosolic side of the membrane, but becomes accessible upon membrane disruption . Concomitant with 30 kDa domain insertion, approximately 20 aminoacyl residues of the preprotein are translocated . Additional ATP, which may be hydrolyzed at the second ATP site of SecA, releases the translocated preprotein and allows the 30 kDa domain to deinsert, whence it can exchange with cytosolic SecA . Thus, SecA is the mobile subunit of an integral membrane transporter, consuming ATP during both the insertion and deinsertion phases of its catalytic cycle while guiding preprotein segments across the membrane. J Mol Biol, 1994 Sep 9, 242(1), 99 - 102 Crystallization of the malonyl coenzyme A-acyl carrier protein transacylase from Escherichia coli; Serre L et al.; The malonyl coenzyme A-acyl carrier protein transacylase, a single polypeptide chain of 358 amino acid residues and a molecular mass of 32 kDa, is a key component of the fatty acid synthase multienzyme complex . The elucidation of its three-dimensional structure will help in the understanding of the molecular basis of the biosynthesis of fatty acids, as well as of polyketides and related biologically active molecules . Three X-ray-quality crystal forms of the Escherichia coli fabD gene product encoding for malonyl coenzyme A-acyl carrier protein transacylase have been obtained using the hanging-drop method and ammonium sulfate as precipitant . Two are tetragonal and each contains two molecules in the asymmetric unit (form I: space group P4(3(1))2(1)2 with a = b = 83.9 A, c = 166.5 A and form II: space group P4 with a = b = 132.64 A, c = 38.85 A), whereas the third form belongs to the hexagonal system and contains one molecule in the asymmetric unit (space group P6(1(5)) with a = b = 68.52 A, c = 117.71 A) . In each case, the diffraction pattern extends to approximately 2.0 A resolution using CuK alpha radiation from a rotating anode source. J Biol Chem, 1994 Sep 9, 269(36), 22614 - 22 Holoenzyme interaction sites in the cAMP-dependent protein kinase . Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit; Cox S et al.; Two mutations of the catalytic (C) subunit of the cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli and purified . These mutants were phosphorylated at Thr197 and were catalytically active, although some changes in their kinetic parameters were observed . The most striking differences were in their interaction with the physiological inhibitors . Both mutants were inhibited by protein kinase inhibitor with Ki values below 50 nM . Both mutants were defective in their interaction with the type I regulatory (RI) subunit as measured by (i) the rate of holoenzyme formation with cAMP bound RI-subunit and (ii) the apparent Kd with the cAMP-free RI-subunit . The rate of holoenzyme formation was impaired both in the presence and absence of ATP with the His to Asp mutant showing the greatest effect . The mutant C-subunits were also combined with RI-subunits that contained mutations in the autoinhibitor sequence at Arg94 (P-3) and Ser99 (P + 2) . Complementarity between His87 and Ser99 was established, but not between His87 and Arg94 . Holoenzyme formation with a Ser99-->Lys mutant RI-subunit was less dependent on ATP when combined with either of the C-subunit mutants than when it was combined with the wild-type C-subunit . The apparent Kd values in the presence of ATP for the mutant combinations were also measured . The Ser99-->Lys mutant was compensated for by both His87 mutants . The His87-->Ala C-subunit mutant was unable to form an inhibited holoenzyme complex with a mutant RI-subunit which was defective in cAMP binding to the A-site . This indicated that this R-subunit was defective in C-subunit recognition as well as in cAMP binding . The roles of His87 on the C-subunit and Ser99 and Arg209 on the RI-subunit in R-C interactions are discussed. J Biol Chem, 1994 Sep 9, 269(36), 22586 - 92 Molecular cloning of a protein serine/threonine phosphatase containing a putative regulatory tetratricopeptide repeat domain; Becker W et al.; Two novel protein serine/threonine phosphatases were cloned from a rat fat cell library with probes generated by a polymerase chain reaction-based cloning approach . One of these cDNAs encoded a protein presumably representing the rat homologue of PPV from Drosophila (75% identity of amino acids) . The other novel cDNA encoded a protein phosphatase of 499 amino acids and was designated PPT . Its catalytic domain contains motifs typical for protein phosphatases but is only distantly related with PP1, PP2A, and PP2B (38-42% identical amino acids) . When expressed in Escherichia coli, the catalytic domain of PPT exhibited protein phosphatase activity (dephosphorylation of phosphorylase a) that was inhibitable by okadaic acid . As a unique feature among other members of this gene family, PPT has an amino-terminal extension of 200 amino acids harboring three tandemly arranged tetratricopeptide repeat (TPR) motifs . This domain has previously been found in other proteins involved in the regulation of RNA synthesis or mitosis . mRNA of PPT was predominantly found in brain and, in lower levels, in testis, but was nearly undetectable in spleen, lung, skeletal muscle, kidney, and liver . It is suggested that the TPR domain of PPT may be involved in the regulation of the function of this novel protein phosphatase. J Biol Chem, 1994 Sep 9, 269(36), 22581 - 5 Properties and structure of spermidine acetyltransferase in Escherichia coli; Fukuchi J et al.; Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold . The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits . The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine . The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively . The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH . By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained . Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E . coli chromosome . E . coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold . The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein . Thus, the predicted molecular mass was 21,756 Da . Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141 . The results suggest that the active center of SAT may be located in the COOH-terminal portion. J Biol Chem, 1994 Sep 9, 269(36), 22557 - 64 C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450; Uhlmann H et al.; The role of the C-terminal region of adrenodoxin was studied by analyzing deletion mutants 4-114 and 4-108 lacking amino acids 1-3 and 115-128 or 109-128, respectively . Absorption spectra of these mutants were found to be identical to that of wild type adrenodoxin . However, EPR and CD studies indicated that the structure of deletion mutants 4-114 and 4-108 differs from that of wild type adrenodoxin . Mutant 4-107, which in addition to residues 109-128 lacks the unique proline 108, showed no EPR spectrum . This indicates that proline 108 plays an essential role for the formation of the iron-sulfur cluster . Deletion of residues 115-128 or 109-128 did not essentially affect adrenodoxin reductase binding as shown by nearly unchanged cytochrome c reduction activity . In a CYP11A1 assay, mutants 4-108 and 4-114 exhibited 3.2- and 5-fold decreased Km values, respectively, whilst the Kd values for CYP11A1 decreased 3- and 1.9-fold, respectively . Additionally, in a CYP11B1 assay, mutants 4-108 and 4-114 showed decreased Km values . Furthermore, the first step of electron transfer to CYP11B1, but not to CYP11A1, was accelerated up to 4.5-fold by the adrenodoxin mutants . The results suggest that the C-terminal peptide of adrenodoxin, especially proline 108, affects the structural integrity of the iron-sulfur cluster and that electron donation from adrenodoxin to CYP11A1 and CYP11B1 is determined at least in part by different features of the cytochromes. J Chromatogr A, 1994 Sep 9, 679(1), 67 - 83 Purification of recombinant human granulocyte-macrophage colony-stimulating factor from the inclusion bodies produced by transformed Escherichia coli cells; Belew M et al.; Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), produced as inclusion bodies in genetically transformed Escherichia coli cells was purified to homogeneity by a three-step chromatographic procedure involving hydrophobic interaction, ion exchange and gel filtration . Each purification step is reproducible and well suited for process-scale operations . The purification process also leads to a significant decrease in DNA and endotoxin levels in the final product . Of the three gel media used, Phenyl Sepharose 6 FF (high sub) was most effective in reducing the DNA content (by a factor of ca . 2000) while Superdex 75 prep grade was more effective for removing endotoxins (reduction factor ca . 15) . The recovery of purified rhGM-CSF was 35% by enzyme-linked immunosorbent assay and 70% by a biological assay method . The overall purification factor obtained was about 4.6, which is in the range of those reported for recombinant proteins produced in E . coli as inclusion bodies . The purified rhGM-CSF is an acidic protein (pI = 5.4) and has a specific activity of ca . 3.3 x 10(7) units/mg, which is in excellent agreement with that reported for its natural counterpart . Its monomer molecular mass of 14,605, as determined by electrospray mass spectrometry, corresponds exactly to the mass calculated from its cDNA sequence . Its amino acid composition and partial NH2-terminal sequence (up to seventeen residues) are also identical with those reported for this protein . These and other results confirm the identity of the purified rhGM-CSF with its natural counterpart . However, the results also showed that it is apparently heterogeneous from its NH2-terminal side as it is composed of three polypeptides having Met, Ala and Pro as the NH2-terminal residues in which the intact Met analogue accounts for 60% for the mixture . This heterogeneity does not seem to have any biological significance since the specific activity of the purified rhGM-CSF is identical with that of its natural counterpart. J Biol Chem, 1994 Sep 9, 269(36), 22683 - 90 Opposing adenine nucleotide-dependent pathways regulate guanylyl cyclase C in rat intestine; Parkinson SJ et al.; Opposing adenine nucleotide-dependent pathways regulating guanylyl cyclase C (GC-C) in rat intestinal membranes have been identified and characterized . ATP analogues substituted in the 2-position were potent inhibitors of basal and Escherichia coli heat-stable enterotoxin (ST)-stimulated GC-C, independent of the metal cation cofactor present . Inhibition of GC-C was associated with large changes in Vmax but only small changes in the S0.5, suggesting a noncompetitive mechanism . Also, inhibition of GC-C was associated with a concentration-dependent shift from positive to negative cooperativity when manganese served as the cation cofactor . These data support the existence of a noncompetitive allo steric regulatory mechanism mediating adenine nucleotide-dependent inhibition of GC-C . Adenine nucleotides not substituted in the 2-position potentiated the activation of GC-C by ST in intestinal membranes . The potentiating and inhibitory pathways regulating GC-C enzyme activity were separate and distinct . A specific inhibitor (2-chloroadenosine 5'-triphosphate (2ClATP)), was without effect on the potency of a selective activator (adenosine 5'-O-thiomonophosphate (AMPS)) of GC-C . Similarly, AMPS was without effect on the potency of 2ClATP to inhibit GC-C . These data suggest that adenine nucleotide-dependent activation and inhibition are mediated by independent sites which may modulate the second messenger response of GC-C to ST. J Biol Chem, 1994 Sep 9, 269(36), 22524 - 32 Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain; Silverman JA et al.; Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH . To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain . Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K . Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin . Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction . All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane . E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5 . Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain . A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action . The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model . The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain. Biochemistry, 1994 Sep 6, 33(35), 10815 - 24 Vaccinia virus expresses a novel profilin with a higher affinity for polyphosphoinositides than actin; Machesky LM et al.; We expressed in Escherichia coli the vaccinia virus gene for a protein similar to vertebrate profilins, purified the recombinant viral profilin, and characterized its interactions with actin and polyphosphoinositides . Compared with cellular profilins, this viral profilin has a low affinity (Kd > or = 35 microM) for human platelet actin monomers, a weak effect on the exchange of the nucleotide bound to the actin, and no detectable affinity for poly(L-proline) . Vaccinia profilin binds to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate in micelles and large unilamellar vesicles, but not to phosphatidylserine or phosphatidylcholine . Kinetic analysis by surface plasmon resonance showed that both vaccinia and amoeba profilins bind slowly to polyphosphoinositides, with association rate constants in the range of (1-4) x 10(4) M-1 s-1 . The higher affinity of vaccinia profilin for polyphosphoinositides (Kd = 0.2-8.5 microM) than for actin or poly(L-proline) and the concentration of vaccinia profilin expressed in infected HeLa cells (approximately 20 microM) suggest that vaccinia profilin binds preferentially to PIP and PIP2 in vivo . Consequently, vaccinia profilin is more likely to influence phosphoinositide metabolism than actin assembly . Expression of 7-105 microM vaccinia profilin in a Saccharomyces cerevisiae profilin null mutant did not rescue the null phenotype, so that the affinity of vaccinia profilin for phosphoinositides alone is insufficient for normal profilin function in yeast. Biochemistry, 1994 Sep 6, 33(35), 10809 - 14 Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition; Dunne PW et al.; The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified . Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1 . These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively . DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles . Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1 . Although PKA and PKC are the known types of protein kinase with closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhibit PKA and PKC with Ki's of 0.4-15 microM . Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH . DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively . These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone. Biochemistry, 1994 Sep 6, 33(35), 10731 - 42 Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy; Moy FJ et al.; NMR spectroscopy has been used to study recombinant Escherichia coli CheY, a 128-residue protein involved in regulating bacterial chemotaxis . Heteronuclear three- and four-dimensional (3D and 4D) experiments have provided sequence-specific resonance assignments and quantitation of short-, medium-, and long-range distance restraints from nuclear Overhauser enhancement (NOE) intensities . These distance restraints were further supplemented with measurements of three-bond scalar coupling constants to define the local dihedral angles, and with the identification of amide protons undergoing slow solvent exchange from which hydrogen-bonding patterns were identified . The current model structure shows the same global fold of CheY as existing X-ray structures (Volz & Matsumura, 1991; Stock et al . 1993) with a (beta/alpha)5 motif of five parallel beta-strands at the central core surrounded by three alpha-helices on one face and with two on the opposite side . Heteronuclear 15N-1H relaxation experiments are interpreted to show portions of the protein structure in the Mg2+ binding loop are ill-defined because of slow motion (chemical exchange) on the NMR time scale . Moreover, the presence of Mg2+ disrupts the salt bridge between the highly conserved Lys-109 and Asp-57, the site of phosphorylation. Biochemistry, 1994 Sep 6, 33(35), 10711 - 7 Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis; Weijland A et al.; Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP {Hwang & Miller (1987) J . Biol . Chem . 262, 13081-13085} . This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle {Weijland & Parmeggiani (1993) Science 259, 1311-1313} . Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process . In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP . However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems . The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt . The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system . The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C) . With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Sep 6, 33(35), 10652 - 7 Mutagenesis of rat liver arginase expressed in Escherichia coli: role of conserved histidines; Cavalli RC et al.; Rat liver arginase has been overexpressed in Escherichia coli using a T7-based expression system . The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme . The recombinant wild-type protein contains six Mn(II) ions per trimer, in good agreement with results obtained with the fully active native enzyme . However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer even after extensive dialysis . Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly conserved in arginases from evolutionarily divergent species . The replacement of His101 and His126 with Asn by site-directed mutagenesis produced only modest effects on enzymatic activity when measured in the presence of Mn(II) ions . However, EDTA treatment of these mutant enzymes reduced activity to < 0.2% of that for the wild-type enzyme . The activity of wild-type enzyme and the His141 Asn mutant was unaffected by treatment with EDTA . Thus, His101 and His126 are proposed to be ligands to the binuclear Mn(II) center of the enzyme . The His141 Asn mutation produced an enzyme which, in contrast to the native, wild-type, His101 Asn, and His126 Asn arginases, was not inactivated by diethyl pyrocarbonate . These results suggest a catalytic role for His141. Biochemistry, 1994 Sep 6, 33(35), 10638 - 45 Detection and characterization of a phospholactoyl-enzyme adduct in the reaction catalyzed by UDP-N-acetylglucosamine enolpyruvoyl transferase, MurZ; Brown ED et al.; The MurZ enzyme catalyzes enolpyruvoyl transfer from phosphoenolpyruvate to the 3-OH of UDP-N-acetylglucosamine (UDP-GlcNAc) in Escherichia coli peptidoglycan biosynthesis . The kinetic mechanism of MurZ has been shown to involve the generation of a non-covalently bound tetrahedral phospholactoyl-UDP-GlcNAc intermediate {Marquardt, J.L., et al . (1993) J . Am . Chem . Soc . 115, 10398-10399} . In the work described here, MurZ overproduced in E . coli copurified with 1 equiv of bound PEP . Enzyme free of bound PEP was prepared by incubation of purified MurZ with UDP-GlcNAc followed by removal of small molecules . Addition of either {14C}PEP or {32P}PEP to PEP-free enzyme led to stoichiometric labeling . The MurZ-PEP complex was stable to 6.7 M urea, and digestion of the {32P}PEP-labeled protein followed by SDS-PAGE generated a labeled peptide of molecular weight 5000 . Solution NMR of MurZ incubated with {2-13C}PEP suggested a tetrahedral phospholactoyl enzyme adduct attached via C-2 to an enzyme nucleophile . The kon for generation of the phospholactoyl enzyme in the absence of UDP-GlcNAc was 0.24 microM-1 s-1, too slow to represent the binding order of the kinetically preferred pathway since the lower limit of the second-order binding constant for PEP (kcat/Km) is 15 microM-1 s-1 . Rapid chemical quench analysis under single-turnover conditions using {32P}PEP in the presence of UDP-GlcNAc demonstrated that the covalent enzyme-phospholactoyl adduct appeared and decayed on a time scale consistent with catalysis.(ABSTRACT TRUNCATED AT 250 WORDS) Chem Phys Lipids, 1994 Sep 6, 73(1-2), 223 - 30 Interactions between DNA replication-related proteins and phospholipid vesicles in vitro; Sekimizu K; DNA and phospholipids share a common motif recognizable by proteins . It has been my hypothesis that there are DNA binding proteins which interact with phospholipid membranes and that their activities in DNA replication, transcription, and recombination, are likely to be regulated by phospholipids . I describe here examples of replication-related proteins, the activities of which are modified by acidic phospholipids in vitro. Biochemistry, 1994 Sep 6, 33(35), 10666 - 71 Potentiation of progesterone receptor-mediated transcription by the immunosuppressant FK506; Tai PK et al.; The nontransformed steroid receptors contain several non-steroid binding proteins, such as hsp90, hsp70, and p59 . Recently, we and others have shown that p59 (FKBP59) is an immunophilin which binds two potent immunosuppressants, FK506 and rapamycin . This raises the possibility that FK506 or rapamycin may modify the function of steroid receptors . To develop this line of inquiry, we chose a yeast model system in which the human progesterone receptor form B (hPR-B) was cotransformed with a reporter gene . The reporter contains two copies of a progesterone response element/glucocorticoid response element (PRE/GRE) upstream of the CYC1 promoter which are linked to the lacZ gene of Escherichia coli . We found that FK506 potentiated the ability of progesterone in activating transcription . To gain insight into the mechanism of FK506's regulation of PR action, we questioned whether calcineurin is involved, because it has been shown that FK506 is a specific inhibitor of calcineurin, a Ca(2+)- and calmodulin-regulated phosphatase, through the formation of an FKBP12-FK506-calcineurin-calmodulin complex . We found that 15-O-desmethyl-FK520, an FK506 analogue which is an excellent ligand of FKBP12, but a poor inhibitor of calcineurin, failed to induce the same effect as FK506 . We also found that calmidazolium, a calmodulin antagonist, mimicked FK506's action . Furthermore, immunoblot analysis showed that both FK506 and calmidazolium potentiated the effect of progesterone in decreasing the mobility of hPR-B upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . This suggests that FK506 and calmidazolium may cooperate with progesterone in increasing the level of hPR-B phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1994 Sep 5, 351(2), 257 - 62 Isolation and characterization of two cDNAs encoding for compartment specific isoforms of O-acetylserine (thiol) lyase from Arabidopsis thaliana; Hell R et al.; cDNAs encoding for two isoforms of O-acetylserine (thiol) lyase (OAS-TL), which catalyzes the synthesis of cysteine, have been isolated from Arabidopsis thaliana . Secondary structure together with expression patterns derived during photomorphogenesis indicate cellular localizations in the cytosol and plastids, thus allowing a direct comparison of compartment-specific forms within one species . The cytosolic OAS-TL complemented an E . coli auxotrophic mutant lacking cysteine synthesis . Both isoforms are represented by small gene families . They are expressed under all conditions investigated and were observed to increase in expression in plants grown with limited sulfate supply. Gene, 1994 Sep 2, 146(2), 199 - 207 The addition of 5'-coding information to a 3'-directed cDNA library improves analysis of gene expression; Matoba R et al.; Large-scale sequencing of a 3'-cDNA library permits one to analyse gene expression profiles in various tissues . However, many such sequences lack enough information about the encoded proteins . To overcome this problem, we tested a new library, consisting of a 3'-directed cDNA sequence fused to a to a 5' sequence of about 300 bp . Such 'joint molecules' of about 600 bp were amplified by PCR and directly sequenced . About 40% of these joint molecules included the 5' and 3' terminal portions of the mRNA, and most of the remaining clones contained the middle portion and 3' end of the mRNA . The upstream sequences contained sufficient information with which to search for similarity, ORFs, motifs and hydropathy, thus allowing the mRNAs to be categorized and their functions predicted . The rapid categorization of the cDNAs will help to sort those clones that merit further analysis. Gene, 1994 Sep 2, 146(2), 159 - 65 Characterization of an efficient gene cloning strategy for Aspergillus niger based on an autonomously replicating plasmid: cloning of the nicB gene of A . niger; Verdoes JC et al.; The development of an improved gene cloning strategy by complementation of mutant alleles in Aspergillus niger is described . The strategy is based on the use of a fungal autonomously replicating vector, pAB4-ARp1 . This vector was constructed by the introduction of a previously described sequence involved in autonomous replication (AMA1), into a pyrG integrative vector, pAB4-1 . With vector pAB4-ARp1, a 10-100-fold increase in transformation frequency was obtained, as compared to pAB4-1 . Furthermore, the transformation frequency of a co-transformed plasmid is also increased using pAB4-ARp1 . A . niger transformants containing pAB4-ARp1 are mitotically unstable . Co-transformed plasmids strictly co-segregated with the autonomously replicating vector, as a result of recombination between both vectors . The use of pAB4-ARp1 in gene cloning was demonstrated by the complementation of two linkage group-VII-specific A . niger mutants . Complementation of a lysF mutant was achieved by co-transformation of pAB4-ARp1 with total genomic A . niger DNA ('instant bank') . A nicB-deficient A . niger was complemented by co-transformation with pAB4-ARp1 and an A . niger cosmid library . The complementing DNA was re-isolated from a Nic+ transformant by transforming Escherichia coli with total genomic DNA of this transformant . Gene disruption and genetic analysis was carried out to prove that the previously unknown A . niger nicB gene had been cloned. J Mol Biol, 1994 Sep 2, 241(5), 739 - 43 Crystallization and preliminary X-ray diffraction study of an idiotope-anti-idiotope Fv-Fv complex; Goldbaum FA et al.; A complex between the Fv fragment of an anti-hen eggwhite lysozyme antibody (D1.3) and the Fv fragment of an antibody specific for an idiotypic determinant of D1.3 has been crystallized in a form suitable for X-ray diffraction analysis . Both Fv fragments were expressed in soluble form in Escherichia coli and purified by affinity chromatography; diffraction-quality crystals were only obtained following separation of each Fv into distinct isoelectric forms . The crystals belong to space group C2, have unit cell dimensions a = 152.8 A, b = 79.4 A, c = 51.5 A, beta = 100.2 degrees, and diffract to better than 2.2 A resolution . The solvent content of the crystals is approximately 60% (v/v) with one Fv-Fv complex in the asymmetric unit . The ability to readily express both components of an antigen-antibody system in bacteria will allow us to rigorously assess the energetic contribution of individual amino acids to complex formation through pairwise mutagenesis of interacting residues. J Mol Biol, 1994 Sep 2, 241(5), 645 - 50 Flow linear dichroism and electron microscopic analysis of protein-DNA complexes of a mutant UvrB protein that binds to but cannot kink DNA; Hsu DS et al.; (A)BC excinuclease of Escherichia coli is the enzymatic activity resulting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein . UvrA is a molecular matchmaker which promotes the formation of a stable UvrB-damaged DNA complex in which the DNA is kinked by about 130 degrees . The UvrB-DNA complex is then recognized by UvrC and two incisions are made in the DNA by the joint actions of UvrC and UvrB . A mutant of UvrB (D478A) can be loaded onto the DNA but it does not interact with UvrC to cause a nick 3' to the lesion . Based on the lack of a DNase-I-hypersensitive site in the footprint of the mutant, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA . In the current study we have investigated the interaction of the mutant UvrB with DNA using two biophysical methods, flow linear dichroism and electron microscopy . Both methods reveal that the mutant UvrB is unable to bend DNA. J Biol Chem, 1994 Sep 2, 269(35), 22358 - 65 Human apolipoprotein E . Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms; Dong LM et al.; Human apolipoprotein (apo) E4 (arginine at residue 112) preferentially associates with very low density lipoproteins (VLDL), and apoE3 (cysteine at 112) associates with high density lipoproteins . It has been postulated that the amino-terminal domain, which contains residue 112, influences the lipoprotein preference by interacting with the carboxyl-terminal domain, which contains the lipid-binding region . To delineate the region in the carboxyl-terminal domain mediating lipoprotein binding and involved in isoform preference, we produced truncated apoE3 and apoE4 variants (terminating at residues 251, 260, 266, or 272) in Escherichia coli and assessed them for lipoprotein association . This analysis suggested that residues 260-272 contain important determinants for complete lipoprotein association and isoform preferences . To determine whether positive charge at residue 112 was an absolute requirement for the apoE4 VLDL preference, we compared the distributions of rabbit apoE (equivalent to apoE3, with cysteine at a position corresponding to 112), canine apoE (arginine at the corresponding site), and cysteamine-treated rabbit apoE (cysteine converted to a positively charged residue) . Surprisingly, all distributed like human apoE3, suggesting that positive charge at a position corresponding to 112 was not directly responsible for the isoform preference and that other residues in the amino-terminal domain were involved . To determine which residues were involved, the structure of the apoE4 22-kDa fragment (the amino-terminal two-thirds of the molecule) was determined to 2.5 A by x-ray crystallography . Compared with the known four-helix bundle structure of apoE3, the only significant differences in the apoE4 structure were that glutamic acid 109 formed a salt bridge with arginine 112 and that the arginine 61 side chain was displaced to a new position . Site-directed mutagenesis of glutamic acid 109 in apoE3 and arginine 61 in apoE4 demonstrated that the position of the arginine 61 side chain in apoE4 was critical in determining apoE4 lipoprotein distribution, suggesting that arginine 61 interacted with the carboxyl-terminal domain to direct binding to VLDL. J Biol Chem, 1994 Sep 2, 269(35), 22340 - 6 Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP; Scheffler JE et al.; Four overlapping peptide fragments of human c-Raf-1 (residues 55-132, 55-117, 77-132, and 77-117) were expressed in Escherichia coli as carboxyl-terminal extensions of maltose binding protein (MBP) . The MBP-Raf fusions were purified by affinity chromatography on amylose resin and tested for binding to Ras.GTP indirectly by measuring their ability to inhibit the stimulation of Ras GTPase activity by GTPase activating protein (GAP120) in vitro . MBP-Raf(55-132) was a potent inhibitor in this assay (50% inhibition at 100 nM concentration), but the other fusion proteins had no measurable effect . The fusion partners were cleaved with Factor Xa protease and separated by gel filtration . The 8960-dalton Raf(55-132) fragment retained full activity as a competitive inhibitor of GAP120 . It also blocked Ras-stimulated germinal vesicle breakdown in frog oocytes . Raf(55-132) was further characterized by circular dichroism and nuclear magnetic resonance spectroscopy . The results indicate that this fragment of c-Raf-1 adopts a highly structured, monomeric conformation in solution. J Biol Chem, 1994 Sep 2, 269(35), 22320 - 7 A novel protein-tyrosine phosphatase with homology to both the cytoskeletal proteins of the band 4.1 family and junction-associated guanylate kinases; Banville D et al.; Reversible phosphorylation of proteins on tyrosine residues is critical in several cellular activities . The removal of the phosphate groups from tyrosine-phosphorylated proteins is accomplished by a class of enzymes which constitute a large family of related proteins, the protein tyrosine phosphatases . We report here the isolation of a complementary DNA encoding a novel protein tyrosine phosphatase, which we termed hPTP1E . Several overlapping cDNA clones were isolated to reconstruct a sequence of 8301 nucleotides . Northern blot analysis of poly(A)+ RNA from different human tissues revealed the presence of a mRNA of 8.2-8.5 kilobases suggesting the near full-length sequence had been cloned . The composite hPTP1E cDNA contains an open reading frame encoding 2490 amino acid residues . The predicted protein (M(r) = 277,567) does not contain a signal peptide or a membrane-spanning region and possesses a single catalytic domain (amino acids 2241-2470) . The recombinant phosphatase domain has been expressed in Escherichia coli, purified to near homogeneity and showed to possess specific protein tyrosine phosphatase activity . The primary structure of hPTP1E also displays homology to cytoskeleton- and membrane junction-associated proteins . Thus, the region encompassing amino acids 568-1053 is related to the cytoskeletal proteins of the band 4.1 family . In addition, hPTP1E contains five imperfect repeats possessing significant homology to the GLGF repeats of the junction-associated guanylate kinases such as the Drosophila discs-large tumor suppressor gene (dlg-1) . The structural features of hPTP1E suggest that it localizes at the junction between the plasma membrane and the cytoskeleton where it may regulate signal transduction and cytoskeletal integrity. J Biol Chem, 1994 Sep 2, 269(35), 22295 - 303 GreA-induced transcript cleavage is accompanied by reverse translocation to a different transcription complex conformation; Lee DN et al.; GreA- and GreB-induced transcript cleavage drives reverse translocation of Escherichia coli RNA polymerase on a DNA template in the absence of NTPs (Feng, G.-H., Lee, D . N., Wang, D., Chan, C . L., and Landick, R . (1994) J . Biol . Chem . 269, 22282-22294, accompanying report) . During transcript elongation, the sizes of the DNA footprint and the single-stranded transcription bubble vary markedly among transcription complexes halted at different template positions . To test whether transcription complex intermediates formed during transcript cleavage-induced reverse translocation also display heterogeneous conformations at different template positions, we examined the structures of two different transcription complexes before and after GreA treatment . Transcription complexes halted at position +16 after initiation at the T7 A1 promoter or paused at the trpL pause site exhibited strong blocks to transcript cleavage after removal of 6 to 10 nucleotides . In both cases, the down-stream contact between RNA polymerase and DNA moved little during transcript cleavage, thereby increasing its distance from the active site, whereas the upstream DNA contact and the borders of the transcription bubble moved in approximate register with the transcript 3'-end . The backward movements of halted E . coli RNA polymerase are similar to a recently postulated model for discontinuous translocation during transcription, but differ from those reported for arrested RNA polymerase II transcription complexes. J Biol Chem, 1994 Sep 2, 269(35), 22282 - 94 GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure; Feng GH et al.; The Escherichia coli GreA and GreB proteins induce cleavage of 3' fragments from nascent transcripts in halted transcription complexes . We have overproduced and purified the GreA protein and tested how it affects initiation, pausing, and termination by E . coli RNA polymerase . Recombinant GreA induced cleavage of two to three nucleotide fragments in two promoter-proximal complexes, whereas an apparently endogenous cleavage removed a single larger fragment . Both types of cleavage stopped once the transcript was shortened to approximately 10 nucleotides . However, during initiation, GreA induced cleavage of transcripts as short as four nucleotides, inhibiting their release as abortive products and stimulating both productive initiation and "primer-shifting" at a weak promoter . GreA induced repetitive cleavage over a long distance in complexes containing a long G-less nascent transcript . However, reverse translocation was inhibited in transcription complexes that contained a G-rich, C-less nascent transcript . Substituting IMP for GMP in the transcript relieved inhibition . Finally, GreA had little effect on transcription through the his and trp leader pause sites or on termination at nine different p-independent terminators . We propose that transcript cleavage and reverse translocation are controlled in part by backsliding of the nascent transcript through an RNA-binding site. J Biol Chem, 1994 Sep 2, 269(35), 22173 - 7 Role of the CCA terminal sequence of tRNA(Val) in aminoacylation with valyl-tRNA synthetase; Tamura K et al.; All known tRNAs have a universal CCA sequence at the 3'-terminal . To study the role of this terminal sequence in the aminoacylation process, base substitutions were introduced into a transcript of Escherichia coli valine tRNA and the effects on the aminoacylation activity with valyl-tRNA synthetase were evaluated . Substitution of the terminal adenosine residue at position 76 by C or U caused a 5-7-fold decrease of valine charging activity in Vmax/Km, while substitution by G resulted in about a 300-fold decrease . In addition, these mutations gave rise to an appreciable level of misaminoacylation with threonine . ATP hydrolysis activity during threonylation was lower in the terminal adenosine mutants than in the wild-type . Mutations introduced at positions 75 and 74 also caused threonylation instead of reducing valylation, albeit to a much smaller extent . These results indicate that the CCA sequence, especially the base portion of the terminal adenosine residue, plays an important role not only in amino-acylation efficiency with valine but also in preventing misaminoacylation by hydrolyzing misactivated threonyl-tRNA(Val). J Biol Chem, 1994 Sep 2, 269(35), 22080 - 6 Expression and structural analysis of a novel highly inducible gene encoding alpha 1-antitrypsin in rabbit; Ray BK et al.; alpha 1-Antitrypsin is a major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of neutrophil elastase that hydrolyzes structural proteins . This protein, in rabbit is expressed as three isoforms designated as F, S-1, and S-2 . An inducible form of this protein has been cloned from an acute-phase cDNA library of rabbit liver . Structural study has revealed that the cloned cDNA is the S-2 isoform previously identified by partial peptide sequence analysis . The amino acid sequence of the reactive center of the S-2 form is not conserved and thus can be categorized as of unorthodox type . mRNA analysis has indicated that the transcription of the S-2 isoform which is normally present at a very low concentration increases more than 100-fold under inflammatory conditions while the expression of the F and S-1 isoforms changes about 1.5-fold under similar conditions . The S-2 isoform is biologically active and capable of inhibiting both elastase and chymotrypsin . The high level of induction of this active isoform of alpha 1-antitrypsin under inflammatory conditions and its apparent resistance to oxidation-mediated inactivation, common to the orthodox forms of alpha 1-antitrypsin, make the S-2 isoform suitable for gene therapy in diseases associated with alpha 1-antitrypsin deficiency. J Biol Chem, 1994 Sep 2, 269(35), 22075 - 9 The mutant DnaAcos protein which overinitiates replication of the Escherichia coli chromosome is inert to negative regulation for initiation; Katayama T; Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30 degrees C . DnaAcos protein was purified from an overproducing strain and found to be as active as wild-type DnaA protein in initial synthesis rates of minichromosome replication in vitro at 30 degrees C . However, whereas efficient initiation occurred for only 20 min with wild-type DnaA protein, it continued for 45 min with DnaAcos protein, an indication that DnaAcos protein retained initiation activity for a longer time than wild-type DnaA protein . Also, whereas wild-type DnaA protein is inactivated by ADP binding, DnaAcos protein failed to be inactivated by ADP due to its inability to bind nucleotide . Thus, DnaAcos protein appears to lack negative regulation for its initiation activity . At 42 degrees C, a temperature at which initiation of chromosome replication is normal in the dnaAcos mutant, in vitro DnaAcos protein activity decreased to 25% of that observed at 30 degrees C . This coincident occurrence of normal initiation in vivo and reduced activity is consistent with the idea that negative control of DnaA protein activity is necessary for normal replication. J Biol Chem, 1994 Sep 2, 269(35), 22046 - 53 Uracil DNA N-glycosylase distributively interacts with duplex polynucleotides containing repeating units of either TGGCCAAGCU or TGGCCAAGCTTGGCCAAGCU; Purmal AA et al.; Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism) . Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides . Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer . Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique . To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning . The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms . The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model. J Biol Chem, 1994 Sep 2, 269(35), 22412 - 9 Ligand binding domain of granulocyte colony-stimulating factor receptor; Hiraoka O et al.; The amino-terminal domain of the cytokine receptor homologous region (BN domain; roughly 100 amino acid residues) in the receptor for murine granulocyte colony-stimulating factor (G-CSF) was secreted as a maltose-binding protein fusion into the Escherichia coli periplasm . The murine BN domain (mBN) was prepared from the fusion protein by restriction protease Factor Xa digestion and purified to homogeneity . The purified BN domain specifically and stoichiometrically bound G-CSF, with an apparent dissociation constant (Kd) of 3-8 x 10(-8) M . The CD spectrum of the mBN domain was similar to that of the extracellular region of the human growth hormone (GH) receptor, which is composed of turns and beta-sheets held together by disulfide bonds . Tertiary folding and the beta-sheet of this small domain was confirmed by NMR spectroscopy . Disulfide bonds determined by peptide mapping were in the following locations: Cys107-Cys118, Cys153-Cys162, and Cys143-Cys194 . Among them, the first and the second produce small loops (roughly 10 amino acid residues) as found in the human GH receptor . These results suggested that the mBN domain of the G-CSF receptor expressed by E . coli has a GH receptor-like structure . However, the third disulfide bond varied considerably between the G-CSF and GH receptors . Disruption of these disulfide bonds in the BN domain of the G-CSF receptor suggested that all of them are critical for maintaining a stably folded protein . Our results will facilitate understanding of the biophysical and structural properties of this receptor. Biochem J, 1994 Sep 1, 302 ( Pt 2), 527 - 34 Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta; Hildebrand A et al.; We have analysed the interactions of three proteoglycans of the decorin family, decorin, biglycan and fibromodulin, with transforming growth factor beta (TGF-beta) . The proteoglycan core proteins, expressed from human cDNAs as fusion proteins with Escherichia coli maltose-binding protein, each bound TGF-beta 1 . They showed only negligible binding to several other growth factors . Intact decorin, biglycan and fibromodulin isolated from bovine tissues competed with the fusion proteins for the TGF-beta binding . Affinity measurements suggest a two-site binding model with Kd values ranging from 1 to 20 nM for a high-affinity binding site and 50 to 200 nM for the lower-affinity binding site . The stoichiometry indicated that the high-affinity binding site was present in one of ten proteoglycan core molecules and that each molecule contained a low-affinity binding site . Tissue-derived biglycan and decorin were less effective competitors for TGF-beta binding than fibromodulin or the non-glycosylated fusion proteins; removal of the chondroitin/dermatan sulphate chains of decorin and biglycan (fibromodulin is a keratan sulphate proteoglycan) increased the activities of decorin and biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-beta . The fusion proteins competed for the binding of radiolabelled TGF-beta to Mv 1 Lu cells and endothelial cells . Affinity labelling showed that the binding of TGF-beta to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type-II receptors was unaffected . TGF-beta 2 and 3 also bound to all three fusion proteins . Latent recombinant TGF-beta 1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan . The results show that the three decorin-type proteoglycans each bind TGF-beta isoforms and that slight differences exist in their binding properties . They may regulate TGF-beta activities by sequestering TGF-beta into extracellular matrix. Biochem J, 1994 Sep 1, 302 ( Pt 2), 517 - 25 PCR-based cloning of the full-length Neurospora eukaryotic initiation factor 5A cDNA: polyhistidine-tagging and overexpression for protein affinity binding; Tao Y et al.; Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain a hypusine residue that is formed by transferring the aminobutyl moiety from spermidine to a specific lysine residue, followed by hydroxylation at the aminobutyl group . A simple PCR-based strategy was developed to obtain a full-length cDNA of Neurospora crassa eIF-5A . The strategy consists of (i) the design of a pair of key primers (21-mer) based on the highly conserved eIF-5A cDNA domains known in other species, (ii) PCR amplification of Neurospora cDNA using the two key primers to obtain the core sequence for the design of core primers, and (iii) combined use of the key primers, core primers and the universal primers, T3 and T7, to amplify the target sequence in a Neurospora cDNA library . The longest cDNA obtained was cloned into pBlueScript phagemid, and sequence analysis indicated that it encodes a polypeptide of 163 amino acid residues with a codon usage preference characteristic of abundant Neurospora genes . The Neurospora polypeptide showed 59% and 67% identity with human and yeast eIF-5A precursor protein respectively . We subcloned the Neurospora eIF-5A cDNA into pQE-30, which introduces six adjacent histidine residues to the N-terminus of the recombinant protein . The resulting plasmid, pQTy21, was overexpressed in Escherichia coli, and the soluble polyhistidine-tagged protein was purified by metal chelation chromatography . We obtained about 60 mg of purified eIF-5A precursor from 1 litre of culture in a single step using a Ni(II)-nitrilotriacetic acid (NTA)-agarose column . The histidine-tagged eIF-5A precursor protein could be recognized by anti-Neurospora crassa 21 kDa protein serum raised against wild-type eIF-5A precursor and could serve as the substrate protein for deoxyhypusine synthase . Using the histidine-tagged recombinant protein and the Ni(II)-NTA-agarose column, we constructed a protein affinity column and demonstrated an affinity binding between eIF-5A precursor and deoxyhypusine synthase in the presence of NAD+ . One-step eIF-5A precursor affinity-column chromatography could lead to a 30-fold purification of deoxyhypusine synthase. Anesthesiology, 1994 Sep, 81(3), 689 - 99 Effect of lidocaine pretreatment on endotoxin-induced lung injury in rabbits; Mikawa K et al.; BACKGROUND: It is well known that endotoxin causes acute lung injury resulting in adult respiratory distress syndrome . Numerous cellular and humoral factors such as macrophages, neutrophils, platelets, and inflammatory mediators (e.g., activated complements, cytokines, and arachidonic acid metabolites) are thought to play a pivotal role in the pathogenesis of endotoxin-induced lung injury . Furthermore, pulmonary edema in acute lung injury is associated with an increase in vascular permeability that may arise from a perturbation of the endothelial cell surface membrane . Lidocaine has been shown to inhibit function of these cells and stabilize cell membranes . The aim of the current study was to determine whether pretreatment with intravenous lidocaine could attenuate acute lung injury induced by endotoxin in rabbits . METHODS: Twenty-seven anesthetized male rabbits were randomly assigned to receive one of three treatments (n = 9 for each group); infusion of saline (as a control), infusion of Escherichia coli endotoxin (30 micrograms kg-1 over a 60-min period) without treatment with lidocaine, and infusion of endotoxin with treatment with lidocaine . A single dose of intravenous lidocaine 2 mg.kg-1 was administered 10 min before infusion of endotoxin and thereafter infused at a rate of 2 mg.kg-1.h-1 until 6 h after the start of endotoxin administration, when the animals were killed . The lungs of the rabbits were ventilated with 40% oxygen . Hemodynamics, peripheral leukocytes counts, and arterial oxygen tension were recorded during the ventilation period . After the observation, lung mechanics, cell fraction of bronchoalveolar lavage fluid (BALF), activated complements, cytokines, and arachidonic acid metabolites concentrations in BALF were measured and analyzed . The lung wet-to-dry-weight ratio and albumin concentrations in BALF were analyzed as an indices of pulmonary edema . The cypridina luciferin analog-dependent chemiluminescence (representing superoxide production) by neutrophils isolated from the pulmonary artery and light microscopic findings were compared among the three groups . RESULTS: Endotoxin caused decreases in peripheral leukocyte counts, lung compliance, and arterial oxygen tension, and increases in the lung wet-to dry-weight ratio, polymorphonuclear cell counts in BALF, and albumin, C3a, C5a, tumor necrosis factor alpha, interleukin-1 beta, and thromboxane B2 concentrations in BALF . Lidocaine pretreatment attenuated these changes . The cypridina luciferin analog--dependent chemiluminescence was greater in rabbits receiving endotoxin than in the control . Lidocaine pretreatment attenuated the increase in chemiluminescence . Endotoxin caused extensive morphologic lung damage, which was lessened by lidocaine . CONCLUSIONS: These results suggest that intravenous lidocaine pretreatment has a prophylactic effect on endotoxin-induced lung injury in rabbits . However, further studies are required to investigate the therapeutic (as an early posttreatment) effect of the drug given after lung injury because rabbits in the current study received lidocaine before endotoxemia. J Nutr, 1994 Sep, 124(9), 1588 - 96 The acute phase response of adult rats is altered by in utero exposure to maternal low protein diets; Langley SC et al.; The immune system has been previously demonstrated to be under the influence of maternal nutrition in pregnancy . Assessment was made of the effects of low protein diets (12, 9 and 6 g casein/100 g diet) fed before conception and during pregnancy on the immune system of the resulting offspring in early adulthood . Control animals were fed a diet containing 18 g casein/100 g . At the end of pregnancy all dams were fed a nonpurified diet containing 18.3 g protein/100 g . Male pups were weaned onto this diet, which they consumed until the age of 7 wk . Rats exposed to 18 g casein/100 g diet in utero mounted a typical acute phase response following E . coli endotoxin challenge at age 7 wk . Food intake was 75% lower, hepatic zinc concentrations 25% greater, and serum albumin 15% lower than in saline-injected controls . Pulmonary glutathione levels were 35% greater in endotoxin-treated rats than in saline-treated controls . In rats exposed to low protein diets in utero the trend was for the acute phase response to be blunted . This was most noticeable with respect to the anorectic response, hepatic zinc uptake and pulmonary glutathione uptake . In rats not challenged with endotoxin, maternal diet had pronounced effects on tissue zinc status at the age of 7 wk . Liver zinc concentrations were 21% and 16% lower in the groups exposed to 9 and 6 g casein/100 g diets relative to the control group exposed to 18 g casein/100 g diet . Glutathione status was altered in all groups exposed to low dietary protein in utero.(ABSTRACT TRUNCATED AT 250 WORDS) J Cell Biol, 1994 Sep, 126(6), 1445 - 53 Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family; Turunen O et al.; Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures . Ezrin is postulated to have a membrane-cytoskeleton linker role . Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O . Turunen, A . Vaheri, D . Louvard, and M . Arpin . 1993 . J . Cell Biol . 120: 129-139) . Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability . We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin . We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin . No binding of monomeric actin was detected in the assay . The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E . multilocularis, but not in merlin/schwannomin . Consequently, this site is a potential actin-binding site also in the other members of the protein family . Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain . The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker. J Cell Biol, 1994 Sep, 126(6), 1407 - 20 Microsomal aldehyde dehydrogenase is localized to the endoplasmic reticulum via its carboxyl-terminal 35 amino acids; Masaki R et al.; Rat microsomal aldehyde dehydrogenase (msALDH) has no amino-terminal signal sequence, but instead it has a characteristic hydrophobic domain at the carboxyl terminus (Miyauchi, K., R . Masaki, S . Taketani, A . Yamamoto, A . Akayama, and Y . Tashiro . 1991 . J . Biol . Chem . 266:19536-19542) . This membrane-bound enzyme is a useful model protein for studying posttranslational localization to its final destination . When expressed from cDNA in COS-1 cells, wild-type msALDH is localized exclusively in the well-developed ER . The removal of the hydrophobic domain results in the cytosolic localization of truncated proteins, thus suggesting that the portion is responsible for membrane anchoring . The last 35 amino acids of msALDH, including the hydrophobic domain, are sufficient for targeting of E . coli beta-galactosidase to the ER membrane . Further studies using chloramphenicol acetyltransferase fusion proteins suggest that two hydrophilic sequences on either side of the hydrophobic domain play an important role in ER targeting. Int Arch Allergy Immunol, 1994 Sep, 105(1), 62 - 9 Effects of amino acid variations in recombinant Der fII on its human IgE and mouse IgG recognition; Nishiyama C et al.; Amino acid sequencing of the major mite allergen Der fII purified from mite body extract revealed that it is a mixture of at least two variants . Substitutions were found only at positions in which amino acid variations were predicted from the nucleotide sequences of three cloned cDNAs . When cDNAs corresponding to the three variants were expressed in Escherichia coli, Der fII proteins were produced as inclusion bodies . Denaturation and renaturation with urea converted recombinant Der fII into a protein with three intramolecular disulfide bonds (Cys8-Cys119, Cys21-Cys27, and Cys73-Cys78), which were identical to those previously identified in native Der fII . All the variants, including native Der fII, were equally recognized by human IgE antibodies from 14 different sera of mite-allergic patients . These results suggested that recombinant Der fII proteins assumed a conformation identical to that of the native Der fII and that all the Der fII variants acted as allergens . This result also suggested that IgE antibody binds to the region where there were no amino acid variations . The binding ability of the monoclonal antibody (mAb) 18G8 for the variant clones 1, 2, and 11 Der fII was almost the same . However, mAb 15E11 bound to clone 1 Der fII more efficiently than to clones 2 and 11, whereas mAb 13A4 recognized clone 2 Der fII as the most preferable antigen . This suggested that these antibodies recognized the region of amino acid variations . The stability of Der fII variants was analyzed by measuring their IgE antibody binding activity after heating, freezing, or acid treatment, and was analyzed by resistance to trypsin digestion.(ABSTRACT TRUNCATED AT 250 WORDS) J Neurosci, 1994 Sep, 14(9), 5461 - 70 Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension; Jin LW et al.; Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor . To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP . B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium . Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75) . A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells . The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75 . Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites . The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells . The binding could be displaced with KB75 . A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding . A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension . A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations . APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems . These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function. J Bacteriol, 1994 Sep, 176(18), 5864 - 7 Is the IS1 transposase, InsAB', the only IS1-encoded protein required for efficient transposition? Escoubas JM, Lane D, Chandler M. The transposase of the bacterial insertion sequence IS1 is normally expressed by inefficient translational frameshifting between an upstream reading frame which itself specifies a transposition inhibitor, InsA, and a second consecutive reading frame located immediately downstream . A fused-frame mutant which carries an additional base pair inserted at the point of frameshifting was constructed . This mutant exhibits high transposition activity and should express the transposase, InsAB', constitutively without frameshifting . Unexpectedly, a second protein species was observed to be expressed from this mutant . We demonstrate here that this protein, InsA*, results from continued frameshifting on the modified frameshift motif . The protein retains the activities of the repressor InsA . Its elimination, by further modification of the frameshift motif, results in a further increase in various transposition activities of IS1 . These results support the hypothesis that a single IS1-encoded protein, InsAB', is necessary for transposition. J Bacteriol, 1994 Sep, 176(18), 5861 - 3 Mapping and disruption of the chpB locus in Escherichia coli; Masuda Y et al.; The chpB locus is a chromosomal homolog of the pem locus, which is responsible for stable maintenance of plasmid R100 within the host cells . Like pem, chpB codes for two genes, chpBK and chpBI, encoding a growth inhibitor and a suppressor for the killing action of the ChpBK protein, respectively . Here, we determined the precise location of the chpB locus, which is linked to ileR and ppa in the order ileR-chpB-ppa, at 95.7 min on the map of Escherichia coli . We then constructed mutants with an insertion of a (cat) fragment within chpBK or chpBI on the E . coli chromosome . These mutants grew normally, indicating that chpB is dispensable for cell growth. J Bacteriol, 1994 Sep, 176(18), 5847 - 51 Molecular cloning and characterization of the pgm gene encoding phosphoglucomutase of Escherichia coli; Lu M et al.; We report here the identification and characterization of pgm, a gene in Escherichia coli that encodes the enzyme phosphoglucomutase, specifically required for the catalysis of the interconversion of glucose 1-phosphate and glucose 6-phosphate . The predicted amino acid sequence of the pgm gene is highly conserved in E . coli, Acetobacter xylinum, Saccharomyces cerevisiae, rabbits, and humans . pgm deletion mutant strains are deficient in phosphoglucomutase activity. J Bacteriol, 1994 Sep, 176(18), 5788 - 95 Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis; Mengin-Lecreulx D et al.; The glmU gene product of Escherichia coli was recently identified as the N-acetylglucosamine-1-phosphate uridyltransferase activity which catalyzes the formation of UDP-N-acetylglucosamine, an essential precursor for cell wall peptidoglycan and lipopolysaccharide biosyntheses (D . Mengin-Lecreulx and J . van Heijenoort, J . Bacteriol . 175:6150-6157, 1993) . Evidence that the purified GlmU protein is in fact a bifunctional enzyme which also catalyzes acetylation of glucosamine-1-phosphate, the preceding step in the same pathway, is now provided . Kinetic parameters of both reactions were investigated, indicating in particular that the acetyltransferase activity of the enzyme is fivefold higher than its uridyltransferase activity . In contrast to the uridyltransferase activity, which is quite stable and insensitive to thiol reagents, the acetyltransferase activity was rapidly lost when the enzyme was stored in the absence of reducing thiols or acetyl coenzyme A or was treated with thiol-alkylating agents, suggesting the presence of at least one essential cysteine residue in or near the active site . The acetyltransferase activity is greatly inhibited by its reaction product N-acetylglucosamine-1-phosphate and, interestingly, also by UDP-N-acetylmuramic acid, which is one of the first precursors specific for the peptidoglycan pathway . The detection in crude cell extracts of a phosphoglucosamine mutase activity finally confirms that the route from glucosamine-6-phosphate to UDP-N-acetylglucosamine occurs via glucosamine-1-phosphate in bacteria. J Bacteriol, 1994 Sep, 176(18), 5711 - 7 Expression of proteins encoded by the Escherichia coli cyn operon: carbon dioxide-enhanced degradation of carbonic anhydrase; Kozliak EI et al.; Cyanase catalyzes the reaction of cyanate with bicarbonate to give 2CO2 . The cynS gene encoding cyanase, together with the cynT gene for carbonic anhydrase, is part of the cyn operon, the expression of which is induced in Escherichia coli by cyanate . The physiological role of carbonic anhydrase is to prevent depletion of cellular bicarbonate during cyanate decomposition due to loss of CO2 (M.B . Guilloton, A.F . Lamblin, E . I . Kozliak, M . Gerami-Nejad, C . Tu, D . Silverman, P.M . Anderson, and J.A . Fuchs, J . Bacteriol . 175:1443-1451, 1993) . A delta cynT mutant strain was extremely sensitive to inhibition of growth by cyanate and did not catalyze decomposition of cyanate (even though an active cyanase was expressed) when grown at a low pCO2 (in air) but had a Cyn+ phenotype at a high pCO2 . Here the expression of these two enzymes in this unusual system for cyanate degradation was characterized in more detail . Both enzymes were found to be located in the cytosol and to be present at approximately equal levels in the presence of cyanate . A delta cynT mutant strain could be complemented with high levels of expressed human carbonic anhydrase II; however, the mutant defect was not completely abolished, perhaps because the E . coli carbonic anhydrase is significantly less susceptible to inhibition by cyanate than mammalian carbonic anhydrases . The induced E . coli carbonic anhydrase appears to be particularly adapted to its function in cyanate degradation . Active cyanase remained in cells grown in the presence of either low or high pCO2 after the inducer cyanate was depleted; in contrast, carbonic anhydrase protein was degraded very rapidly (minutes) at a high pCO2 but much more slowly (hours) at a low pCO2 . A physiological significance of these observations is suggested by the observation that expression of carbonic anhydrase at a high pCO2 decreased the growth rate. J Bacteriol, 1994 Sep, 176(18), 5704 - 10 A new suppressor of a lamB signal sequence mutation, prlZ1, maps to 69 minutes on the Escherichia coli chromosome; Wei SQ et al.; Reversion analysis has been employed to isolate suppressors that restore export of a unique LamB signal sequence mutant . The mutation results in a substitution of Arg for Met at position 19, which prevents LamB export to the outer membrane and leads to a Dex- phenotype . Unlike other LamB signal sequence mutants utilized for reversion analysis, LamB19R becomes stably associated with the inner membrane in an export-specific manner . In this study, Dex+ revertants were selected and various suppressors were isolated . One of the extragenic suppressors, designated prlZ1, was chosen for further study . prlZ1 maps to 69 min on the Escherichia coli chromosome . The suppressor is dominant and SecB dependent . In addition to its effect on lamB19R, prlZ1 suppresses the export defect of signal sequence point mutations at positions 12, 15, and 16, as well as several point mutations in the maltose-binding protein signal sequence . prlZ1 does not suppress deletion mutations in either signal sequence . This pattern of suppression can be explained by interaction of a helical LamB signal sequence with the suppressor. J Bacteriol, 1994 Sep, 176(18), 5607 - 14 PrlA and PrlG suppressors reduce the requirement for signal sequence recognition; Flower AM et al.; Selection for suppressors of defects in the signal sequence of secretory proteins has led most commonly to identification of prlA alleles and less often to identification of prlG alleles . These genes, secY/prlA and secE/prlG, encode integral membrane components of the protein translocation system of Escherichia coli . We demonstrate that an outer membrane protein, LamB, that lacks a signal sequence can be exported with reasonable efficiency in both prlA and prlG suppressor strains . Although the signal sequence is not absolutely required for export of LamB, the level of export in the absence of prl suppressor alleles is exceedingly low . Such strains are phenotypically LamB-, and functional LamB can be detected only by using sensitive infectious-center assays . Suppression of the LamB signal sequence deletion is dependent on normal components of the export pathway, indicating that suppression is not occurring through a bypass mechanism . Our results indicate that the majority of the known prlA suppressors function by an identical mechanism and, further, that the prlG suppressors work in a similar fashion . We propose that both PrlA and PrlG suppressors lack a proofreading activity that normally rejects defective precursors from the export pathway. J Bacteriol, 1994 Sep, 176(18), 5601 - 6 The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes; Simon G et al.; Expression of the Escherichia coli torCAD operon encoding the trimethylamine N-oxide (TMAO) reductase system is induced by both TMAO and anaerobiosis . A torR insertion mutant unable to express the torA gene had previously been isolated . The torR gene was cloned and sequenced . It encodes a 25,000-Da protein which shares homology with response regulators of two-component systems and belongs to the OmpR-PhoB subclass . Overproduction of TorR mimics the presence of the inducer TMAO while the anaerobic control is unchanged, suggesting that TorR mediates only the TMAO induction . The overproduced TorR protein was purified to more than 90% . The torR gene is located just upstream of the torCAD operon, with an opposite transcription direction . The torR-torCAD intergenic region is unusual in that it contains four direct repeats of a 10-nucleotide motif . Part or all of these motifs could be involved in the binding of TorR . The gene encoding the sensor partner does not seem to be adjacent to torR, since the divergent open reading frame found immediately downstream of torR exhibits none of the features of a protein histidine kinase. Arch Biochem Biophys, 1994 Sep, 313(2), 346 - 50 Expression and partial characterization of Dolichos biflorus seed lectin in Escherichia coli; Chao Q et al.; The seed lectin from the legume, Dolichos biflorus, was expressed in Escherichia coli using the pET expression vector . Replacement of the 22-amino acid signal sequence of this lectin with a methionine increased the level of lectin expression greater than 100-fold . Approximately 20% of the expressed seed lectin was soluble; the remainder was solubilized in 8 M urea and renatured by rapid dilution . No difference in physicochemical properties or activity was detected between the soluble and renatured forms . NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontruncated subunit of the native heteroligomer, established that the expressed lectin has a primary structure equivalent to subunit I of the native seed lectin . The expressed seed lectin is active as evidenced by its ability to bind to blood group A + H substance-Sepharose and to be specifically eluted from this column with N-acetylgalactosamine . However, a comparison of the activity of the expressed lectin with the native seed lectin using a sensitive ELISA showed that the expressed lectin has a slightly lower affinity for blood group A + H substance than the native seed lectin . The expressed lectin also had a lower M(r) than the seed lectin as determined by molecular exclusion chromatography. Arch Biochem Biophys, 1994 Sep, 313(2), 287 - 95 Enzymatically active truncated cat brain glutamate decarboxylase: expression, purification, and absorption spectrum; Chu WC et al.; The DNA encoding the sequence for glutamate decarboxylase from cat brain was recloned into the Escherichia coli expression vector pET11a . The N-terminal 77- to 84-amino acid residues encoded by the cloned gene had been deleted from the protein which was purified to near homogeneity in 20-mg batches . The truncated protein is a dimer with a subunit molecular mass of about 59 kDa . This protein is enzymatically active and has a Km for L-glutamate of 1.37 mM and a turnover number of 7 s-1 at its optimal pH of 6.6 . The absorption spectrum, resulting from the bound coenzyme, pyridoxal phosphate, showed pH-dependent bands at 338 and 420 nm with an isosbestic point at 356 nm . A spectrophotometric pKa value of 6.92 was evaluated for the bound coenzyme . The pH-dependent kinetic data suggest the presence of two dissociable groups in the free enzyme with pKa values of about 6.45 and 7.05 and pKa value of the enzyme-substrate complex of about 6.82 in phosphate buffer . Structures for the coenzyme in the active site of brain glutamate decarboxylase are proposed. Mol Gen Genet, 1994 Sep 1, 244(5), 557 - 62 RecA, Tus protein and constitutive stable DNA replication in Escherichia coli rnhA mutants; Kogoma T et al.; Constitutive stable DNA replication (cSDR), which uniquely occurs in Escherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function . The recA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR in rnhA mutants . The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR . Despite the requirement for RecA recombinase activity, mutations in recB, recD, recJ, ruvA and ruvC neither inhibit nor stimulate cSDR . It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR in rnhA mutants without involving the homologous recombination process . The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in the ter region of the chromosome, was ruled out because introduction of the tus::kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR. Mol Gen Genet, 1994 Sep 1, 244(5), 530 - 8 The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey; Salmon MA et al.; The ccd operon of the F plasmid contributes to the high stability of the episome by postsegregational killing of plasmid-free bacteria . It contains two genes, ccdA and ccdB, which are negatively autoregulated at the level of transcription, probably by a complex comprising the two gene products . Using the bacterial gyrA462 CcdB resistance mutation and a Pccd-lacZ transcriptional fusion, we have obtained evidence that the CcdB protein by itself has no regulatory activity or operator DNA-binding affinity and needs CcdA in order to effect transcriptional control . The ccd killing mechanism is based on the poison-antidote principle . The CcdB protein is cytotoxic, poisoning DNA-gyrase complexes, while CcdA antagonizes this activity . In order to define functional domains of the CcdA antidote involved in the anti-killer effect, autoregulation or both, we introduced several missense or amber mutations into the CcdA protein by directed mutagenesis . We report on missense CcdA proteins that have lost their autoregulatory properties but are still able to antagonize the lethal activity of CcdB . We show that the five carboxy-terminal amino acid residues of the antidote protein are not required for the antidote effect or for autoregulation . Several missense CcdA polypeptides were generated by suppression of nonsense codons . Two substitutions lead to CcdB-promoted killing: glutamine 33-->cysteine and glutamine 33-->phenylalanine. Mol Gen Genet, 1994 Sep 1, 244(5), 451 - 5 Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock in Escherichia coli; Ogata Y et al.; The linking number of plasmid DNA in exponentially growing Escherichia coli increases immediately and transiently after heat shock . The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA . Neither introduction of a topA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock . Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process . However, the reaction was inhibited by nalidixic acid or by oxolinic acid in the topA mutant and the reaction was resistant to nalidixic acid in a topA mutant carrying, in addition, the nalA26 mutation . These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2421 - 5 Major core protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes; Wang LF et al.; Full-length cDNA of the RNA genome segment coding for the major core protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning . Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup . At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date . This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7 . Recombinant BTV-15 VP7 protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity . Polyclonal mouse sera raised against the recombinant VP7 protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2409 - 13 Molecular cloning, sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagic disease virus (Spanish isolate AST/89); Boga JA et al.; We describe the cloning, nucleotide sequencing and expression in Escherichia coli of the major capsid component (VP60) from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV) . The sequence of the 3'-terminal 2483 nucleotides of the genome was found to be 95.4% identical to the German RHDV strain, showing ten changes in the deduced VP60 amino acid sequence . The gene coding for this structural polypeptide has been expressed in bacteria as a beta-galactosidase fusion protein or using a T7 RNA polymerase-based system . The VP60 fusion protein showed only partial antigenic similarity with native VP60 and did not confer protective immunity . The recombinant VP60 produced in the T7 RNA polymerase-based system was antigenically similar to the viral polypeptide as determined using polyclonal and monoclonal antibodies . When used to immunize rabbits the recombinant VP60 was able to protect the animals against a lethal challenge using purified RHDV. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2349 - 54 Identification of human herpesvirus 6 uracil-DNA glycosylase gene; Sato S et al.; Uracil-DNA glycosylase encoded in many species functions as a DNA repair enzyme that removes uracil residues from DNA . To investigate the potential function of uracil-DNA glycosylase encoded by human herpes-virus 6 (HHV-6), we sequenced a DNA clone (pSTY09), identified an open reading frame of 765 bp and compared the putative amino acid sequence with other uracil-DNA glycosylases, by computer analysis . The amino acid sequence of HHV-6 had similarities to other uracil-DNA glycosylases, with the highest degree of similarity to those of human cytomegalovirus and Epstein-Barr virus . Two strongly conserved regions in uracil-DNA glycosylase of other species also existed in HHV-6 . The gene product which was expressed in Escherichia coli demonstrated uracil-DNA glycosylase activity . This is the first report to identify and characterize the uracil-DNA glycosylase gene in HHV-6. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2233 - 9 Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preferences and specificity of binding; Daenke S et al.; Human T cell leukaemia virus type 1 (HTLV-1) protease (PR14) was expressed in bacteria and purified by gel filtration . A continuous spectrophotometric assay was used to measure the kinetic parameters of substrate hydrolysis by PR14 . Several peptide substrates containing HTLV-1 sequences known to be cleaved by PR14 were used . Cleavage analysis showed that the affinity with which PR14 binds these substrates is higher than that previously reported for HTLV-1 Gag peptides . Also, the affinities of peptides containing the sites involved in autocleavage of protease from its precursor are higher than for the peptides containing sites required for structural protein maturation . This suggests that the autocatalysis of protease from its own precursor has priority over other cleavage reactions and supports similar observations of an ordered hierarchy of processing events by retroviral proteases . As the N- and C-terminal regions of retroviral aspartic proteases are known to contribute to stability of the dimer by forming antiparallel beta-strands, short peptides corresponding to these terminal sequences of HTLV-1 protease were tested for their ability to inhibit cleavage of substrates by PR14 . Inhibition was seen with a C-terminal peptide corresponding exactly to the C-terminal 11 amino acids of the processed PR14, whereas a peptide containing a sequence situated further from the C terminus was less effective . An inhibitor of the protease of human immunodeficiency virus type 1, Ro 31-8959, was found to be a poor inhibitor of PR14. J Gen Virol, 1994 Sep, 75 ( Pt 9), 2157 - 61 Detection of human antibodies to Crimean-Congo haemorrhagic fever virus using expressed viral nucleocapsid protein; Marriott AC et al.; Diagnosis of Crimean-Congo haemorrhagic fever (CCHF) virus infections is hampered by the problems of handling this human pathogen, which requires the highest levels of biological containment . Recombinant antigens were examined for their potential as non-hazardous diagnostic reagents . The nucleocapsid (N) gene of the Greek AP92 isolate of CCHF virus was sequenced from cloned PCR products and the open reading frame was identified by homology to the N protein of a Chinese isolate of CCHF virus . The N protein was expressed to high levels in a baculovirus expression system . Three N protein-derived peptides were expressed in Escherichia coli as fusions with glutathione S-transferase and the antigenicities of these proteins and the baculovirus-expressed protein were tested by ELISA . When tested with laboratory animal sera representing all seven serogroups of nairoviruses, the only reactive sera were those raised to CCHF virus (Greek, Nigerian and Chinese isolates) and, more weakly, Hazara virus . When tested with a panel of known positive and negative human sera, the baculovirus-expressed N protein, and the peptide derived from the central region of the N protein, proved to be the best for identifying CCHF virus-specific IgG. J Surg Res, 1994 Sep, 57(3), 416 - 9 Endotoxin causes early changes in glutathione concentrations in rabbit plasma and liver; Okabe H et al.; The effects of endotoxin on glutathione concentrations in rabbit plasma and liver were investigated . Lipopolysaccharide (2 mg/kg) from Escherichia coli was administered intravenously to seven male Japanese rabbits . In the liver, the concentrations of reduced glutathione (GSH) started to decrease, and those of oxidized glutathione (GSSG) started to increase 1 hr after the endotoxin administration, resulting in a progressive decline in the hepatic GSH/GSSG ratio . In the arterial plasma, the concentrations of both GSH and GSSG started to increase 1 hr after the endotoxin administration . Because the increase in the concentrations of GSSG was greater than that in the concentrations of GSH, the GSH/GSSG ratio in the plasma decreased as did that in the liver . These changes in glutathione concentrations occurred simultaneously with the increase in serum osmolality, but earlier than the decrease in the arterial ketone body ratio, both of which are thought to be useful markers for liver damage . It was concluded that endotoxin induced an increase in the plasma concentrations of GSH as well as GSSG, and that the changes in plasma glutathione status might be useful markers of endotoxin-induced damage in organs, including the liver. J Surg Res, 1994 Sep, 57(3), 337 - 43 LPS pretreatment protects from hepatic ischemia/reperfusion; Colletti LM et al.; In vivo administration of nonlethal doses of lipopolysaccharide (LPS) to rodents can result in protection from subsequent lethal doses of endotoxin or LPS . We have previously demonstrated that hepatic ischemia/reperfusion (I/R) results in a TNF-dependent lung and liver injury and we postulated that pretreatment with sublethal concentrations of LPS prior to hepatic I/R could be protective from this injury . To test this hypothesis, five groups of rats were studied . LPS-I/R received 25 micrograms of LPS i.v . 24 hr prior to I/R, VEH-I/R received an equivalent volume of vehicle iv 24 hr prior to I/R, LPS-LPS received 25 micrograms of LPS i.v . 24 hr prior to sham laparotomy at which time an additional 25 micrograms of LPS was given i.v., VEH-LPS received an equivalent volume of vehicle 24 hr prior to sham laparotomy and 25 micrograms of LPS i.v . immediately prior to sham laparotomy, and SHAM consisted of sham-operated control animals . Peak plasma tumor necrosis factor-alpha (TNF) levels occurred between 30 and 150 min of reperfusion: LPS-I/R = 778 +/- 150 pg/ml (n = 5), VEH-I/R = 145 +/- 46 pg/ml (n = 5), LPS-LPS = 970 +/- 716 pg/ml (n = 4), VEH-LPS = 15,949 +/- 10,937 (n = 5), and SHAM = 3 +/- 1 (n = 5) . As previously demonstrated by other investigators, pretreatment with LPS decreases TNF release in response to a second dose of LPS; however, TNF release was increased following hepatic I/R in those animals pretreated with LPS (LPS-I/R vs VEH-I/R, P = 0.014).(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1994 Sep, 176(17), 5560 - 4 Growth rate-dependent control of the rrnB P1 core promoter in Escherichia coli; Bartlett MS et al.; We have extended our previous studies of the DNA sequences required for growth rate-dependent control of rRNA transcription in Escherichia coli . Utilizing a reporter system suitable for evaluation of promoters with low activities, we have found that the core promoter region of rrnB P1 (-41 to +1 with respect to the transcription initiation site) is sufficient for growth rate-dependent control of transcription, both in the presence and in the absence of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) . The core promoter contains the -10 and -35 hexamers for recognition by the sigma 70 subunit of RNA polymerase but lacks the upstream (UP) element, which increases transcription by interacting with the alpha subunit of RNA polymerase . It also lacks the binding sites for the positive transcription factor FIS . Thus, the UP element, FIS, and ppGpp are not needed for growth rate-dependent regulation of rRNA transcription . In addition, we find that several core promoter mutations, including -10 and -35 hexamer substitutions, severely reduce rrnB P1 activity without affecting growth rate-dependent control . Thus, a high activity is not a determinant of growth rate regulation of rRNA transcription. J Bacteriol, 1994 Sep, 176(17), 5544 - 6 Loss of flagellation in dnaA mutants of Escherichia coli; Mizushima T et al.; A dnaA46 mutant of Escherichia coli showed loss of motility at 37 degrees C, a permissive temperature for cell growth of this mutant . Other dnaA mutations near the middle of the gene also caused an immotile phenotype . The amount of flagellin was much less in the dnaA46 mutant than in the wild-type control, as was the promoter activity . DnaA protein may play an important role in expression of the fliC gene. J Bacteriol, 1994 Sep, 176(17), 5537 - 40 The H-NS protein is involved in the biogenesis of flagella in Escherichia coli; Bertin P et al.; The function of the flagellum-chemotaxis regulon requires the expression of many genes and is positively regulated by the cyclic AMP-catabolite activator protein (cAMP-CAP) complex . In this paper, we show that motile behavior was affected in Escherichia coli hns mutants . The loss of motility resulted from a complete lack of flagella . A decrease in the level of transcription of the flhD and fliA genes, which are both required for the synthesis of flagella, was observed in the presence of an hns mutation . Furthermore, the Fla- phenotype was not reversed to the wild type in the presence of a cfs mutation which renders the flagellum synthesis independent of the cAMP-CAP complex . These results suggest that the H-NS protein acts as a positive regulator of genes involved in the biogenesis of flagella by a mechanism independent of the cAMP-CAP pathway. J Bacteriol, 1994 Sep, 176(17), 5414 - 22 Role of the transcriptional activator AppY in regulation of the cyx appA operon of Escherichia coli by anaerobiosis, phosphate starvation, and growth phase; Atlung T et al.; Transcriptional lacZ fusions have been used to analyze the regulation of the appA operon of Escherichia coli . The appA operon contains the genes cyxA and cyxB, coding for the putative third cytochrome oxidase, and appA, encoding acid phosphatase . The analysis showed that the cyxAB and the appA genes are cotranscribed from a potentially strong promoter, Pcyx, located immediately upstream of cyxA and that the operon in addition contains an internal promoter, PappA, contributing significantly to the transcription of the appA gene . The two promoters were both induced by starvation for Pi and by entry into stationary phase . The cyx promoter was in addition found to be activated by anaerobic growth conditions . The product of the previously identified appY gene, which when present on a high-copy-number plasmid stimulates synthesis of acid phosphatase, was shown to activate the cyx promoter . An insertion mutation in the appY gene was constructed in vitro and recombined into the chromosome . The appY mutation eliminated induction of the cyx promoter by anaerobiosis and severely reduced induction of this promoter by phosphate starvation and upon entry into stationary phase but had no effect on induction of the appA promoter . The appY mutation had no effect on survival in stationary phase, nor did it have any effect on growth rate or yield under aerobic or anaerobic conditions . The possibility that AppY is a third global regulator of energy metabolism genes is discussed. J Bacteriol, 1994 Sep, 176(17), 5393 - 400 Dominant negative mutator mutations in the mutS gene of Escherichia coli; Wu TH et al.; The MutS protein of Escherichia coli is part of the dam-directed MutHLS mismatch repair pathway which rectifies replication errors and which prevents recombination between related sequences . In order to more fully understand the role of MutS in these processes, dominant negative mutS mutations on a multicopy plasmid were isolated by screening transformed wild-type cells for a mutator phenotype, using a Lac+ papillation assay . Thirty-eight hydroxylamine- and 22 N-methyl-N'-nitro-N-nitrosoguanidine-induced dominant mutations were isolated . Nine of these mutations altered the P-loop motif of the ATP-binding site, resulting in four amino acid substitutions . With one exception, the remaining sequenced mutations all caused substitution of amino acids conserved during evolution . The dominant mutations in the P-loop consensus caused severely reduced repair of heteroduplex DNA in vivo in a mutS mutant host strain . In a wild-type strain, the level of repair was decreased by the dominant mutations to between 12 to 90% of the control value, which is consistent with interference of wild-type MutS function by the mutant proteins . Increasing the wild-type mutS gene dosage resulted in a reversal of the mutator phenotype in about 60% of the mutant strains, indicating that the mutant and wild-type proteins compete . In addition, 20 mutant isolates showed phenotypic reversal by increasing the gene copies of either mutL or mutH . There was a direct correlation between the levels of recombination and mutagenesis in the mutant strains, suggesting that these phenotypes are due to the same function of MutS. J Bacteriol, 1994 Sep, 176(17), 5378 - 84 Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli; Manna D et al.; Transcription of the proU operon of Escherichia coli is induced several hundred-fold upon growth at elevated osmolarity, but the underlying mechanisms are incompletely understood . Three cis elements appear to act additively to mediate proU osmoresponsivity: (i) sequences around a promoter, P1, which is situated 250 bp upstream of the first structural gene proV; (ii) sequences around another (sigma 70-dependent) promoter, P2, which is situated 60 bp upstream of proV; and (iii) a negative regulatory element present within the proV coding region . These three cis elements are designated, respectively, P1R, P2R, and NRE . trans-acting mutants with partially derepressed proU expression have been obtained earlier, and a vast majority of the mutations affect the gene encoding the nucleoid protein HNS . In this study we employed a selection for trans-acting mutants with reduced proU+ expression, and we obtained a derivative that had suffered mutations in two separate loci designated dpeA and dpeB . The dpeB mutation caused a marked reduction in promoter P1 expression and was allelic to rpoS, the structural gene for the stationary-phase-specific sigma factor of RNA polymerase . Expression from P1 was markedly induced, in an RpoS-dependent manner, in stationary-phase cultures . In contrast to the behavior of the isolated P1 promoter, transcription from a construct carrying the entire proU cis-regulatory region (P1R plus P2R plus NRE) was not significantly affected by either growth phase or RpoS . The dpeA locus was allelic to hupB, which along with hupA encodes the nucleoid protein HU . hupA hupB double mutants exhibited a pronounced reduction in proU osmotic inducibility . HU appears to affect proU regulation through the P2R mechanism, whereas the effect of HNS is mediated through the NRE. J Bacteriol, 1994 Sep, 176(17), 5290 - 6 Heterologous expression of the bchM gene product from Rhodobacter capsulatus and demonstration that it encodes S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase; Bollivar DW et al.; The bacteriochlorophyll biosynthesis gene, bchM, from Rhodobacter capsulatus was previously believed to code for a polypeptide involved in formation of the cyclopentone ring of protochlorophyllide from Mg-protoporphyrin IX monomethyl ester . In this study, R . capsulatus bchM was expressed in Escherichia coli and the gene product was subsequently demonstrated by enzymatic analysis to catalyze methylation of Mg-protoporphyrin IX to form Mg-protoporphyrin IX monomethyl ester . Activity required the substrates Mg-protoporphyrin IX and S-adenosyl-L-methionine . 14C-labeled product was formed in incubations containing 14C-methyl-labeled S-adenosyl-L-methionine . On the basis of these and previous results, we also conclude that the bchH gene, which was previously reported to code for Mg-protoporphyrin IX methyltransferase, is most likely involved in the Mg chelation step. J Bacteriol, 1994 Sep, 176(17), 5270 - 6 Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products; Darie S et al.; While many organisms synthesize delta-aminolevulinate, the precursor of heme, by condensing succinyl-coenzyme A and glycine, others use a glutamate-dependent pathway in which glutamyl-tRNA dehydrogenase catalyzes the rate-determining step . The hemeA gene that encodes this latter enzyme in Escherichia coli has been cloned and sequenced . To examine how its expression is regulated, we constructed hemA-lacZ operon and gene fusions and inserted them into the chromosome in single copy . The effect of aerobic and anaerobic growth conditions and the availability of electron acceptors and various carbon substrates were documented . Use of different types of cell culture medium resulted in a fivefold variation in hemA-lacZ expression during aerobic cell growth . Anaerobic growth resulted in 2.5-fold-higher hemA-lacZ expression than aerobic growth . This control is mediated by the fnr and arcA gene products . Fnr functions as a repressor of hemA transcription during anaerobic cell growth only, whereas the arcA gene product activates hemA gene expression under both aerobic and anaerobic conditions . Integration host factor protein was also shown to be required for control of hemA gene regulation . To determine whether an intermediate or a product of the heme biosynthetic pathway is involved in hemA regulation, hemA-lacZ expression was analyzed in a hemA mutant . Expression was elevated by 20-fold compared with that in a wild-type strain, while the addition of the heme pathway intermediate delta-aminolevulinate to the culture medium restored expression to wild-type levels . These results suggest that the heme pathway is feedback regulated at the level of hemA gene expression, to supply heme as it is required during different modes of cell growth. J Bacteriol, 1994 Sep, 176(17), 5197 - 201 In vivo footprinting analysis of Lrp binding to the ilvIH promoter region of Escherichia coli; Marasco R et al.; An in vivo footprinting analysis of the ilvIH regulatory region of Escherichia coli showed that the transcription activator Lrp binds to six sites, scattered over 250 bp upstream of the transcriptional start point . When Lrp-mediated activation was impaired by the presence of exogenous leucine, only one promoter-distal site (site 2) was partially protected by Lrp binding . Equilibrium dialysis experiments showed the formation of an Lrp-leucine complex in vitro . These results suggest that leucine negatively affects ilvIH transcription because its interaction with Lrp reduces the efficiency of binding of the regulatory protein to the promoter region. Mol Cell Biol, 1994 Sep, 14(9), 6180 - 6 petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing; Sakamoto W et al.; Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes . In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons . By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter . The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants . The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase) . When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end . When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25 . Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation . One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages. J Cell Biol, 1994 Sep, 126(5), 1231 - 40 Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin; Menkel AR et al.; Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components . In this report, we have identified and characterized a binding site for filamentous actin . These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli . In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments . The binding was saturable, with a dissociation constant value in the micromolar range . Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction . In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli . These results corroborated the sedimentation experiments . Our data support and extend previous work showing that vinculin binds directly to actin filaments . They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures. J Cell Biol, 1994 Sep, 126(5), 1127 - 32 The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase; Nilsson I et al.; Signal peptides (SPs) target proteins to the secretory pathway and are cleaved from the nascent chain once the translocase in the ER has been engaged . Signal-anchor (SA) sequences also interact transiently with the ER translocase, but are not cleaved and move laterally out of the translocase to become permanent membrane anchors . One obvious difference between SP and SA sequences is the considerably longer hydrophobic regions (h regions) of the latter . To study the interaction between SP/SA sequences and the ER translocase, we have constructed signal sequences with poly-Leu h regions ranging in length from 8 to 29 residues and have characterized their locations within the translocase using both a new assay that measures the minimum number of amino acids needed to span the distance between the COOH-terminal end of the h region and the active site of the oligosaccharyl transferase enzyme and an assay where the efficiency of signal peptidase catalyzed cleavage is measured . Our results suggest that SP and SA sequences are positioned differently in the ER translocase. Infect Immun, 1994 Sep, 62(9), 3808 - 16 The immunoprotective Anaplasma marginale major surface protein 2 is encoded by a polymorphic multigene family; Palmer GH et al.; An Anaplasma marginale Florida msp-2 gene was cloned and expressed in Escherichia coli . Pulsed-field gel electrophoresis and Southern blot analysis revealed the presence of multiple msp-2 gene copies that were widely distributed throughout the chromosomes of all three strains examined . Genomic polymorphism among copies was greatest in the 5' end of msp-2 but also occurred in 3' regions . The presence of gene-copy-specific epitopes was indicated by the reactivity of the cloned msp-2 copy with some, but not all, monoclonal antibodies that bound native MSP-2 . Multiple antigenically distinct MSP-2 molecules were expressed within strains and were coexpressed by individual A . marginale organisms . These results suggest that expression of polymorphic msp-2 gene copies is responsible for the significant percentages of A . marginale organisms within strains that do not react with individual anti-MSP-2 monoclonal antibodies . Sequence analysis revealed highly significant MSP-2 homology with two rickettsial surface proteins, A . marginale MSP-4 and Cowdria ruminantium MAP-1 . Immunization with MSP-4 has been shown to induce protective immunity in a manner similar to that of immunization with MSP-2 . These findings support the hypothesis that A . marginale surface proteins are targets of protective immune responses but are antigenically polymorphic. J Virol, 1994 Sep, 68(9), 6029 - 37 Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein; Ward P et al.; The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication . An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication . In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay . Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA . When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated . When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue . There was no evidence that replication could initiate on the products of rescue . Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site . Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus . When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA . Replication was linear for 4 h, during which time 70% of the maximal synthesis took place . An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis. J Virol, 1994 Sep, 68(9), 5863 - 70 DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein; Tsuchihashi Z et al.; Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified . The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations . We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules . Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing) . NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation . This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications. J Virol, 1994 Sep, 68(9), 5811 - 8 Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol; Neufeld KL et al.; A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol) . Highly purified 3Dpol is capable of adding {32P}AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs . The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity . Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity . Likewise, extracts from the parental E . coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products . With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer . GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction . Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence . The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication . The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. J Virol, 1994 Sep, 68(9), 5804 - 10 ATPase and GTPase activities associated with Semliki Forest virus nonstructural protein nsP2; Rikkonen M et al.; The replication of Semliki Forest virus requires four nonstructural proteins (nsP1 to nsP4), all derived from the same polyprotein . One of these, nsP2, is a multifunctional protein needed in RNA replication and in the processing of the nonstructural polyprotein . On the basis of amino acid sequence homologies, nsP2 was predicted to possess nucleoside triphosphatase and RNA helicase activities . Here, we report the engineered expression in Escherichia coli of nsP2 and of an amino-terminal fragment of it by use of the highly efficient T7 expression system . Both polypeptides were produced as fusion proteins with a histidine tag at the amino terminus and purified by immobilized-metal affinity chromatography . The two recombinant proteins exhibited ATPase and GTPase activities, which were further stimulated by the presence of single-stranded RNA . The activities were not found in similarly prepared fractions from uninduced control cells or cells expressing an unrelated polypeptide . Radiolabeled ribonucleoside triphosphates could be cross-linked to both the full-length and the carboxy-terminally truncated nsP2 protein, and both polypeptides had RNA-binding capacity . We also expressed and purified an nsP2 variant which had a single amino acid substitution in the nucleotide-binding motif (Lys-192-->Asn) . No nucleoside triphosphatase activity was associated with this mutant protein. J Virol, 1994 Sep, 68(9), 5677 - 84 Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma; Kirchweger R et al.; Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA . Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs . Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction . We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli . The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo . However, additional proteolysis products of eIF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro . The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation . The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487. Virology, 1994 Sep, 203(2), 336 - 43 Identification and characterization of the infectious laryngotracheitis virus glycoprotein C gene; Kingsley DH et al.; The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the glycoprotein C gene of herpes simplex virus has been sequenced and identified based on its genomic location, comparative analysis to other gC proteins, and the identification of a glycosylated protein product . Located near the small subunit ribonucleotide reductase gene, the ILTV gC gene is 1242 bp in length and is predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, five potential N-linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains . Antibodies raised in rabbits against a Cro-ILTV-beta-galactosidase fusion protein expressed in Escherichia coli recognize a 60-kDa ILTV-specific glycoprotein from infected cell extracts . Transcriptional analysis, using a portion of the open reading frame as a probe, identified a 1.55-kb transcript expressed with late gene kinetics . Comparison to other herpesvirus gC proteins revealed limited amino acid sequence homology and the absence of a charged extracellular region, which would normally interact with cell surface proteoglycans. Genetika, 1994 Sep, 30(9), 1175 - 83 {Decrease in the level of DeoR-dependent repression of the deo operon as a result of integration of foreign DNA fragments into the interoperator deoO1-deoO2 region of the Escherichia coli chromosome}; Mochul'skaia NA et al.; Two heterologous DNA fragments encoding the genes Kanr and cat were integrated into the regulatory region of the deo operon of Escherichia coli, located in a recombinant plasmid . As a result, plasmids pD5K and pD5C were obtained, in which the distance between the deoO1 and deoO2 operators increased from 599 bp in the deo operon of the wild type to 2019 and 1398 bp in case of integration of Kanr and cat, respectively . Then, linearized DNA of plasmids pD5K and pD5C was incorporated into the deo locus of the chromosome of E . coli through homologous recombination by the use of a recipient strain recBC sbcB . The constructed strains contained insertions in the interoperator region of the deo operon . Expression of the deo operon was studied in these strains, on a different genetic background with respect to the genes deoR, cytR, and cyaA . An increase in the interoperator distance was shown to increase the basal level of gene expression of the deo operon and to disturb cooperative interaction of the DeoR repressor and operator sites. Vaccine, 1994 Sep, 12(12), 1083 - 9 Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine; Tamura S et al.; Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice . Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n . inoculation of the vaccine alone . The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response . The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT . Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype . These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza. Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 978 - 90 {Structure of aminoacyl-tRNA-synthetase of higher eukaryotes from molecular cloning data}; Grigor'eva AIu; This review summarizes the data on the primary structure of eukaryotic aminoacyl-tRNA synthetases (EC 6.1.1) obtained during last decade by means of molecular cloning of respective cDNAs . This structural information is compared with that available for bacterial species . The alignment of E . coli, yeast and mammalian sequences are presented . The N- and C-terminal extensions of higher-eukaryotic aminoacyl-tRNA synthetases are discussed in view of their chimeric origin and potential functions . For some enzymes, the exon/intron organization of their genes are compared . A possible relationship between the domain structure of the protein and the exon/intron organization of the gene is considered. Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1098 - 105 {Synthesis and secretion of human atrial natriuretic factor in Escherichia coli cells}; Nikolaidis MN et al.; A recombinant plasmid providing for the synthesis and secretion of the human atrial natriuretic peptide (hANP) as a C-terminal hybrid with the St . aureus protein A was constructed . The level of secretion of the chimeric proteins and their proteolytic stability were shown to depend upon the genotype of the recipient strains and the cultivation conditions . The hybrid proteins were purified by chromatography on IgG Sepharose . The presence of peptides corresponding to the hANP in the acid hydrolysates of the secreted and affinity-purified proteins was confirmed by the enzyme-linked immunoassay and analytical HPLC. Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1078 - 86 {Expression of the Epstein-Barr virus terminal repeat protein 1 in Escherichia coli cells}; Afanas'eva TA et al.; A pMAL-vector-based plasmid clone with synthetic tac-promotor effectively expressing full-length terminal repeats protein 1 (TP1) of Epstein-Barr virus (EBV) in E . coli was constructed . It is important that the N-terminal region of recombinant TP1 was represented by a maltose-binding protein . The latter can be used to separate TP1 from bacterial lysate by affinity chromatography . Moreover, after treatment with the proteolytic factor Xa, full-length TP1 can be recovered in a discrete form . On the basis of the pATH tryptophan-regulated vector, several plasmid clones expressing different fragments of N- and C-terminal regions of TP1 were also constructed . This collection of recombinant proteins could be used as an important tool for obtaining corresponding antisera and for immunological mapping of the TP1 molecule. Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1061 - 8 {Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation}; Butanaev AM; The hygromycin phosphotransferase gene (hpt) from E . coli under the control of the SV40 early promoter was used as a dominant selectable marker for transformation of Chlamydomonas reinhardtii . Cells were transformed by electroporation (pulse length, 2 ms, field strength, 1 kV/cm) . The culture growth phase was a crucial parameter for transformation (optimal density approximately 10(6) cells/ml) . It was possible to obtain approximately 10(3) Hyg-resistant colonies under these conditions . Foreign DNA integrated into the Chlamydomonas genome was maintained for at least 8 months but the Hyg-resistant phenotype of the transformed clones was unstable . The frequency of codon usage in the hpt gene was compared with the one in Chlamydomonas nuclear genes . It is supposed that highly biased codon usage in Chlamydomonas does not preclude expression . Advantages of this selection system for studying Chlamydomonas transformation by heterologous genes are discussed. Int J Parasitol, 1994 Sep, 24(6), 863 - 70 Expression and analysis of the diagnostic value of an Echinococcus granulosus antigen gene clone; Ferreira HB et al.; A pool of 9 sera from Echinococcus granulosus infected patients (PSP) was used to screen an E . granulosus cDNA library constructed in the expression vector lambda gt11 . Ten reactive phage clones were isolated and 8 were confirmed in spot-lysis arrays probed with PSP . The insert of 1 of these clones (lambda AgEg4) previously characterized as an E . granulosus cytosolic malate dehydrogenase encoding gene was subcloned into the plasmid vector pGEX-1 and expressed as a fusion with glutathione S-transferase . The fusion peptide (Ag4-GST) was produced in Escherichia coli and its antigenicity was confirmed in colony immunoassay and in immunoblot using nondenaturing conditions . The lack of antigenicity of Ag4-GST in immunoblot using denaturing conditions suggests that the recognized epitopes are conformational . Ag4-GST was purified by affinity chromatography and tested in ELISA and immunodots to access its sensitivity and specificity in the diagnosis of human cystic hydatid disease . An overall sensitivity of 53.6% was obtained . Cross-reactions were observed with some sera from patients infected with Schistosoma mansoni and Wuchereria bancrofti . Ag4-GST was not recognized by any of the sera from Taenia solium infected patients tested . These preliminary results suggest that Ag4-GST could be useful as an accessory antigen to discriminate some cross-reactions with sera from cysticercosis patients, especially in regions like southern Brazil, where schistosomiasis and filariasis are not prevalent. Free Radic Biol Med, 1994 Sep, 17(3), 209 - 13 Stability of Escherichia coli sodA mRNA and identification of the transcriptional start site(s) under different environmental and oxidative stresses; Schrum LW et al.; Manganese-containing superoxide dismutase (MnSOD-sodA) in Escherichia coli (E . coli) is regulated at the transcriptional level as observed in studies using both operon and gene fusions . In this paper we examine the regulation of sodA gene at the level of mRNA . We examine the effects of several aerobic inducing conditions (i.e., nalidixic acid, paraquat, or 2,2'-dipyridyl) on mRNA stability, transcription initiation, and translation . The half-life of sodA mRNA was found to be approximately 3-4 min, showing no differences in mRNA stability between induced and uninduced cells . We also found, by reverse transcriptase, that the second putative promoter is not functional under normal or stress conditions, and the amount of mRNA was found to be proportional to active MnSOD . Thus, these results indicate that under oxidative stress/inducing conditions, the increase in aerobic transcription of sodA occurs from only one transcription start site without affecting the stability of sodA mRNA . In addition, the 1:1 ratio found between increases in sodA mRNA and active MnSOD suggests that no translational regulation occurs aerobically. J Crit Care, 1994 Sep, 9(3), 159 - 68 Regional blood flow distribution in endotoxin-treated dogs: modification by ibuprofen; Winslow C et al.; PURPOSE: The aim of this study was to determine whether the improved hemodynamic profiles reported with cyclooxygenase inhibition during sepsis include improvements in tissue perfusion is unknown . Our hypothesis was that cyclooxygenase inhibition with ibuprofen will prevent the endotoxin-induced alterations in regional blood flow distribution from developing and/or restore the endotoxin-induced loss of responsiveness to intravascular volume expansion . METHODS: We measured cardiac output, regional blood flow, and total systemic shunt flow in dogs using radiolabeled 15-microns microspheres . These parameters were assessed in control (N = 10) and endotoxin-treated animals (N = 7) . Additional endotoxin-treated animals (N = 7) were also pretreated with ibuprofen . RESULTS: Compared with controls, endotoxin-treated animals exhibited marked reductions in blood flow to most systemic organs that were not reversed with large, intravenous saline infusions (ie, isotonic saline; 40 mL/kg; N = 4) . By contrast, although ibuprofen pretreatment (12.5 mg/kg; N = 7) completely prevented the reductions in mean arterial pressure caused by endotoxin from occurring, it did little to prevent the development of endotoxin-induced alterations in regional blood flow distribution . Nonetheless, in contrast to the endotoxin-treated animals, there were significant increases in cardiac output and blood flow to several organs after saline infusion in the ibuprofen-pretreated animals . CONCLUSIONS: Cyclooxygenase inhibition with ibuprofen has few direct effects on regional blood flow distribution after endotoxin . However, cyclooxygenase inhibition with ibuprofen does attenuate the endotoxin-induced decrease in vascular responsiveness to intravenous saline infusion. Biometrics, 1994 Sep, 50(3), 853 - 8 On tests against one-sided hypotheses in some generalized linear models; Silvapulle MJ; One-sided hypotheses arise naturally in many situations . When testing against such hypotheses, it is desirable to take the available one-sided information into account, rather than simply applying a two-sided test . What we expect to gain by applying a one-sided test instead of a two-sided test is an increase in the power of the test . We consider various tests of one-sided hypotheses in a class of models that includes generalized linear and Cox regression models . The tests are likelihood ratio, Wald, score, generalized distance, and a Pearson chi-square . It is shown that these test statistics are asymptomatically equivalent in terms of local power; this is a generalization of the well-known corresponding result for two-sided alternatives . Two examples are also discussed . They are on (1) testing for interaction in binomial response models, and (2) comparison of treatments with ordinal categorical responses. Bone, 1994 Sep-Oct, 15(5), 533 - 8 Cytokine suppressive anti-inflammatory compounds inhibit bone resorption in vitro; Votta BJ et al.; Data from several laboratories suggest a role for a variety of cytokines in the process of bone resorption . SK&F 86002 {5-(4-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b) thiazole}, a potent cytokine-suppressive anti-inflammatory agent, has been shown to inhibit cyclooxygenase (CO) and 5-lipoxygenase (LO) activity and to inhibit the production of cytokines both in vitro and in vivo . In the present study, SK&F 86002 inhibited fetal rat long bone (FRLB) resorption induced by parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3, tumor necrosis factor alpha, and Escherichia coli lipopolysaccharide in a dose-dependent (IC50 of 0.5-1 microM) and reversible manner . Under identical conditions, selective CO inhibitors (indomethacin, ibuprofen, naproxen) and 5-LO inhibitors (phenidone, SK&F 107649) were inactive . Analogs of SK&F 86002, which are dual CO/LO inhibitors devoid of cytokine inhibitory activity (SK&F 81114 and SK&F 86055), also failed to significantly inhibit PTH-induced FRLB resorption . Analogs of SK&F 86002, which retain cytokine inhibitory activity (SK&F 104493 and SK&F 105561), inhibit bone resorption . These data indicate that the observed inhibition of bone resorption by compounds of this class correlates with their cytokine suppressive activity. Plant Physiol, 1994 Sep, 106(1), 353 - 8 Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli; Bernasconi P et al.; Anthranilate synthase is involved in tryptophan (Trp) biosynthesis . Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST) . The active product was purified in a single step on a glutathione-Sepharose column . The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants . Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings . GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein. Microbiol Rev, 1994 Sep, 58(3), 466 - 90 The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli; Calvo JM et al.; The leucine-responsive regulatory protein (Lrp) regulates the expression of more than 40 genes and proteins in Escherichia coli . Among the operons that are positively regulated by Lrp are operons involved in amino acid biosynthesis (ilvIH, serA)), in the biosynthesis of pili (pap, fan, fim), and in the assimilation of ammonia (glnA, gltBD) . Negatively regulated operons include operons involved in amino acid catabolism (sdaA, tdh) and peptide transport (opp) and the operon coding for Lrp itself (lrp) . Detailed studies of a few members of the regulon have shown that Lrp can act directly to activate or repress transcription of target operons . A substantial fraction of operons regulated by Lrp are also regulated by leucine, and the effect of leucine on expression of these operons requires a functional Lrp protein . The patterns of regulation are surprising and interesting: in some cases activation or repression mediated by Lrp is antagonized by leucine, in other cases Lrp-mediated activation or repression is potentiated by leucine, and in still other cases leucine has no effect on Lrp-mediated regulation . Current research is just beginning to elucidate the detailed mechanisms by which Lrp can mediate such a broad spectrum of regulatory effects . Our view of the role of Lrp in metabolism may change as more members of the regulon are identified and their regulation characterized, but at this point Lrp seems to be important in regulating nitrogen metabolism and one-carbon metabolism, permitting adaptations to feast and to famine. Microbiol Rev, 1994 Sep, 58(3), 401 - 65 Biochemistry of homologous recombination in Escherichia coli; Kowalczykowski SC et al.; Homologous recombination is a fundamental biological process . Biochemical understanding of this process is most advanced for Escherichia coli . At least 25 gene products are involved in promoting genetic exchange . At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases . The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination . In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function . The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure . This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Microbiol Rev, 1994 Sep, 58(3), 317 - 29 Mechanisms of transcription-repair coupling and mutation frequency decline; Selby CP et al.; Mutation frequency decline is the rapid and irreversible decline in the suppressor mutation frequency of Escherichia coli cells if the cells are kept in nongrowth media immediately following the mutagenic treatment . The gene mfd, which is necessary for mutation frequency decline, encodes a protein of 130 kDa which couples transcription to excision repair by binding to RNA polymerase and to UvrA, which is the damage recognition subunit of the excision repair enzyme . Although current evidence suggests that transcription-repair coupling is the cause of the preferential repair of the transcribed strand of mRNA encoding genes as well as of suppressor tRNA genes, the decline occurs under stringent response conditions in which the tRNA genes are not efficiently transcribed . Thus, the mechanism of strand-specific repair is well understood, but some questions remain regarding the precise mechanism of mutation frequency decline. Rinsho Byori, 1994 Sep, 42(9), 986 - 91 {Studies of comparison with fluorescent bioparticles and microspheres for phagocytosis by flow cytometric assay in polymorphonuclear leukocytes}; Sakai H et al.; We have determined the conditions for quantitative phagocytosis assay in human polymorphonuclear leukocytes by flow cytometry using fluorescent bioparticles (Zymosan A, S . aureus, E . coli) . The changes in the phagocytic index after an exogenous stimulation and after addition of monoclonal antibodies against receptor antigens on polymorphonuclear leukocytes were examined by using fluorescent microspheres (FM particles) and bioparticles . When bioparticles were used, the phagocytic index decreased after an exogenous stimulation with phorbol myristate acetate (PMA) . The phagocytic index obtained with the FM particles increased by the addition of PMA and the supernatant of E . coli, but decreased by the addition of the supernatant of S . aureus . After the addition of the monoclonal antibodies the phagocytosis for the S . aureus and E . coli particles was blocked by anti-CR3, that for the Zymosan A particles was blocked by anti-CR3 and IgGFcR2, and that for the PM particles was blocked by anti-CR1, CR3, and LFA-1 beta, partially . These findings implied the involvement of a complement receptor . However, the degree of its involvement varied with the target particle. J Natl Med Assoc, 1994 Sep, 86(9), 686 - 8 Hypertension-related coronary thrombosis: prothrombic role of angiotensin II; Spillert CR et al.; Although hypertension is a major risk factor in acute myocardial infarction, concomitant hypercoagulability causing thrombosis leading to myocardial infarction remains unproven for lack of an appropriate coagulation test . This study was devised to determine whether a modified recalcification time (MRT) test can demonstrate that angiotensin II, a potent vasoconstrictor, also accelerates coagulation to promote thrombosis . The MRT incorporates blood cells and chemical coagulants for maximizing sensitivity . Four groups (A, B, C, and D) of aliquots of citrated human blood were incubated for 2 hours at 37 degrees C after adding to A--20 microL saline, to B--10 micrograms Escherichia coli endotoxin, to C--20 micrograms angiotensin II, and to D--a combination of E coli endotoxin and angiotensin II . The experiment was repeated with nonincubated aliquots . Modified recalcification time values +/- standard deviation in minutes were: A--5.5 +/- 1.5, B--4.6 +/- 1.1, C--4.9 +/- 1.0, and D--3.9 +/- 1.0 . Significance (Student's t test) was as follows: B versus A P < .001; C versus A, P < .05; C versus D, P < .001; B versus C, P < .05; and B versus D, P < .001 . No significant changes occurred in nonincubated blood . We conclude that angiotensin II has a hypercoagulable effect, as does endotoxin . The hypercoagulability in concert with vasospasm can explain the role of hypertension in acute myocardial infarction . This in vitro study excludes the role of other in vivo mechanisms in the development of angiotensin II-induced hypercoagulability. J Invertebr Pathol, 1994 Sep, 64(2), 100 - 6 Phylogenetic relationships among Vairimorpha and Nosema species (Microspora) based on ribosomal RNA sequence data; Baker MD et al.; A portion (approximately 350 nucleotides) of the large subunit ribosomal RNA (rRNA) 5' to the 580 region (Escherichia coli numbering) was sequenced using the reverse transcriptase dideoxy method and compared for several species of Nosema and Vairimorpha . Comparison among Nosema species suggests that this genus is composed of several unrelated groups . The group which includes the type species, Nosema bombycis, consists of closely related species found primarily in Lepidoptera . Other Nosema species sequenced (Nosema kingi, Nosema algerae, and Nosema locustae) do not appear to be closely related to each other or to the lepidopteran Nosema group . Comparison among the Vairimorpha species indicates that two distinct but very closely related groups exist . The Lymantria group consists of species isolated from the gypsy moth, Lymantria dispar, while the Vairimorpha necatrix group consists of species isolated from other Lepidoptera . Intergeneric comparison of the sequence data suggests that the lepidopteran Nosema species are closely related to the Vairimorpha species. Gut, 1994 Sep, 35(9), 1306 - 10 Routes of spread of pathogens into the pancreas in a feline model of acute pancreatitis; Widdison AL et al.; The routes of spread of pathogens into the pancreas in acute pancreatitis were investigated . Four experiments were performed: (1) cats with and without acute pancreatitis were given 10(7) Escherichia coli (E coli) intravenously, (2) in cats with acute pancreatitis 10(8) E coli was placed in the colon . In half of them the colon was then enclosed in an impermeable bag to prevent transmural spread . (3) E coli (10(4)) was placed in the pancreatic duct in cats with and without acute pancreatitis . (4) In cats with acute pancreatitis 10(5) E coli was placed in the gall bladder . In half of them the common bile duct was ligated to prevent biliary-pancreatic reflux . After 24 hours, intravenous E coli infected the pancreas in six of nine cats with acute pancreatitis and three of 10 controls . After 72 hours E coli spread to the pancreas from the colon in six of nine cats with acute pancreatitis . This was prevented by enclosing the colon in an impermeable bag (p = 0.02) . In five of six cats with acute pancreatitis and five of six controls E coli placed in the pancreatic duct colonised the pancreas within 24 hours . Pancreatic colonisation from the gall bladder occurred in five of six cats with a patent common bile duct and in three of six with an obstructed common bile duct . In conclusion, in cats E coli can spread to the pancreas by the blood stream, transmurally from the colon, and by reflux into the pancreatic duct. Gut, 1994 Sep, 35(9), 1237 - 43 Role of the enteric nervous system in the maintained hypersecretion induced by enterotoxin STa in the nutritionally deprived intestine; Nzegwu HC et al.; Electrogenic (Cl-) secretion was measured as the short circuit current (Isc, microA/cm2) across muscle-stripped sheets of jejunum and ileum incubated in vitro after removal from fed rats, rats starved for three days, and chronically undernourished rats (50% of fed control intake for 21 days) . Concentration and Isc response curves for serially-added mucosal Escherichia coli STa enterotoxin showed that the rats which had undergone dietary deprivation had a larger secretory Isc maximum but the ED50 values were unchanged compared with fed animals . In fed intestine the action of STa was transient, with an Isc peak and subsequent decay to the baseline over 60 minutes but in the undernourished intestine the response consisted of a significantly greater peak than that of the fed state (jejunum = 94%; ileum = 168%) and the Isc was maintained at or near the peak for at least 60 minutes . The starved intestine had a less well developed maintenance of its enhanced peak Isc . Serosal tetrodotoxin (1 microM) had no effect on the initial peak Isc values but caused a decay of the maintained Isc down to the basal or fed levels in the starved and, especially, in the undernourished intestines . Thus, dietary deprivation, especially chronic undernutrition, enhances the maximum electrogenic secretion due to STa and creates a new neural path in the submucosal plexus that, when activated by STa, maintains its enhanced secretory action . Its putative role in exacerbating secretory diarrhoea in malnourished human subjects could be an important component underlying the known relation between malnourishment and the increased severity of diarrhoea. Genes Dev, 1994 Sep 1, 8(17), 2035 - 45 Interaction of the yeast RAD7 and SIR3 proteins: implications for DNA repair and chromatin structure; Paetkau DW et al.; We have used the two-hybrid system to identify proteins that interact with the product of RAD7, a gene involved in DNA repair . A screen of a yeast genomic DNA-GAL4 activation domain (GAD) fusion gene library allowed the isolation of plasmids containing sequences corresponding to the 3' end of the SIR3 gene . This gene is known to be involved in the production of transcriptionally silent DNA at the cryptic mating-type cassettes and at telomeres . The cloned sequences coded for amino acids 307-979 of the Sir3 protein . A sir3 deletion allele, constructed in an isogenic rad7-deletion strain, rescued approximately one-quarter of the UV sensitivity associated with the rad7 deletion, indicating that the two genes interact genetically . Radiolabeled fusion proteins, made with the glutathione S-transferase (GST) gene in the vector pGEX-2T, were purified from Escherichia coli and shown to interact in vitro . This evidence suggests that the Sir3 protein interacts with the Rad7 protein to allow the nucleotide excision repair complex access to transcriptionally inactive chromatin . The proportions of 5-FOA-resistant cells in cultures from isogenic RAD+ and rad7-delta strains containing a telomeric URA3 gene were similar, suggesting that the RAD7 gene is not involved in the production or structure of transcriptionally silent chromatin at the telomeres . RAD7-dependent DNA repair of transcriptionally silent chromatin was shown not to induce expression of a telomeric copy of the URA3 gene, suggesting that repair of transcriptionally silent chromatin differs from transcriptionally active chromatin . Expression of a telomeric copy of the URA3 gene was stimulated in a rad7-delta mutant, suggesting that repair of lesions in the absence of Rad7 can result in the activation of transcriptionally silenced genes. Can J Microbiol, 1994 Sep, 40(9), 777 - 82 Alkaline phosphatase and a cellulase reporter protein are not exported from the cytoplasm when fused to large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein; Bingle WH et al.; Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (delta CenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids) . Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250-350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus . Expression of all fusions except rsaA(315):delta cenA and rsaA(315):phoA was toxic to C . crescentus . None of the gene fusions were toxic when expressed by Escherichia coli DH5 alpha, where all the hybrid proteins accumulated as inclusion bodies . The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic . Further study of RsaA(delta C21) showed that this and presumably other truncated RsaA derivatives were neither secreted nor prone to intracellular accumulation . Although C . crescentus tolerated the expression of rsaA(315):delta cenA and rsaA(315):phoA, the encoded hybrid proteins were not exported in significant quantities from the cytoplasm . These results extend and confirm earlier work that large portions of the S-layer protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be required to properly interact with the RsaA secretion machinery. Can J Microbiol, 1994 Sep, 40(9), 699 - 704 Hypersensitivity of Rhodobacter sphaeroides ribosomes to protein synthesis inhibitors: structural and functional implications; Sanchez E et al.; The elongation cycle of protein synthesis systems of purple nonsulfur photosynthetic bacteria Rhodobacter sphaeroides, grown both phototrophically and chemotrophically, was studied using 33 inhibitors with different chemical structures and functional and domain specificities . No functional differences between phototrophic and chemotrophic ribosomal systems were detected . Rhodobacter sphaeroides ribosomes exhibited strong hypersensitivity to nine functional inhibitors when compared with Escherichia coli ribosomes . Most of the R . sphaeroides ribosomal hypersensitivities corresponded to peptidyltransferase inhibitors, implying that this important functional neighborhood must be somehow different in the two organisms. Comput Chem, 1994 Sep, 18(3), 259 - 67 Deriving non-homogeneous DNA Markov chain models by cluster analysis algorithm minimizing multiple alignment entropy; Borodovsky M et al.; Non-homogeneous Markov chain models can represent biologically important regions of DNA sequences . The statistical pattern that is described by these models is usually weak and was found primarily because of strong biological indications . The general method for extracting similar patterns is presented in the current paper . The algorithm incorporates cluster analysis, multiple alignment and entropy minimization . The method was first tested using the set of DNA sequences produced by Markov chain generators . It was shown that artificial gene sequences, which initially have been randomly set up along the multiple alignment panels, are aligned according to the hidden triplet phase . Then the method was applied to real protein-coding sequences and the resulting alignment clearly indicated the triplet phase and produced the parameters of the optimal 3-periodic non-homogeneous Markov chain model . These Markov models were already employed in the GeneMark gene prediction algorithm, which is used in genome sequencing projects . The algorithm can also handle the case in which the sequences to be aligned reveal different statistical patterns, such as Escherichia coli protein-coding sequences belonging to Class II and Class III . The algorithm accepts a random mix of sequences from different classes, and is able to separate them into two groups (clusters), align each cluster separately, and define a non-homogeneous Markov chain model for each sequence cluster. Microbiology, 1994 Sep, 140 ( Pt 9), 2467 - 73 Characterization of O-glycan moieties of the 210 and 240 kDa pig intestinal receptors for Escherichia coli K88ac fimbriae; Seignole D et al.; The porcine brush border glycoproteins of 210 and 240 kDa, recognized by Escherichia coli K88ac fimbriae, contained O-linked oligosaccharides . The carbohydrate moieties were analysed by deglycosylation, lectin-binding and agglutination assays . Neuraminidase susceptibility of the 210 kDa receptor suggested that a sialoglycoprotein may act as receptor for the K88ac fimbriae . In contrast, K88ac-binding to the 210 and 240 kDa glycoproteins totally disappeared after fucosidase treatment, indicating the critical role of fucosyl residues at the receptor sites . Among the oligosaccharides extracted from these O-glycoproteins, K88ac fimbriae showed affinity for neutral sugar chains while sialylated species were not recognized . Our data suggest a possible role of the polypeptide backbone in the definition of receptor sites . Specific agglutination by K88ac-fimbriated E . coli of the erythrocytes of the hamster Mesocricetus auratus was inhibited by the anti-T peanut lectin and the lectins of Datura stramonium, Aleuria aurantia and Maackia amurensis . Hence, we propose that Gal beta 1-3GalNAc- and Fuc alpha 1-2Gal beta 1-3/4GlcNAc- are the main sequences mediating K88ac fimbrial binding . These structures were not detected in the non-adhesive piglet brush borders characterized by a high carbohydrate content . Additional oligosaccharides probably masked the underlying receptor structures. Microbiology, 1994 Sep, 140 ( Pt 9), 2415 - 22 Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and ProP; Gouesbet G et al.; Exogenously supplied L-pipecolic acid was accumulated by Escherichia coli cells and protected them while growing at inhibitory osmolarity . Using specific uptake mutants and competitive assays, we established that the imino acid enters the cells through the ProP and ProU systems with Km values of 225 and 53 microM, respectively . Surprisingly, in spite of the requirement for the wild-type proX gene for osmoprotective ability, no binding activity of labelled pipecolate with the periplasmic protein encoded by proX could be detected . In an attempt to demonstrate whether the two porters (ProP and ProU) are the only carriers involved in osmoregulation, a variety of molecules known for their intracellular osmolarity-dependent accumulation in various organisms were investigated . N-Dimethylproline (proline betaine), N-dimethylglycine, homobetaine (beta-alanine betaine), gamma-butyrobetaine and dimethylsulfoniopropionate were found to be capable of promoting the growth of osmotically stressed E . coli . All of these molecules enter bacterial cells via ProP and ProU porters . None of the osmoprotectants except N-dimethylproline was able to bind the periplasmic protein encoded by proX, while this protein was necessary for their uptake . Apparently, ProP and ProU are the sole osmoporters involved in osmolyte influx into E . coli cells. Microbiology, 1994 Sep, 140 ( Pt 9), 2371 - 82 Immunogold localization of GyrA and GyrB proteins in Escherichia coli; Thornton M et al.; Immunogold preparations of Escherichia coli, using anti-GyrA and anti-GyrB antibodies to the subunits of DNA gyrase, showed clear labelling with both secondary antibody and protein A-gold conjugates . Both proteins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid . This partitioning of gyrase proteins between nucleoid and cytoplasm was nonrandom and was consistently observed for a range of different cell preparations . Total gold particle counts were highly variable but suggested levels of at least 1000-3000 molecules per cell for both GyrA and GyrB . Sequential treatment with both anti-GyrA and anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two groups of molecules . Treatment of cells with chloramphenicol caused marked changes in nucleoid conformation, but no reduction in cytoplasmic labelling of gyrase proteins . On the assumption that gyrase complexes within the nucleoid are not differentially masked from the monoclonal antibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DNA, but are distributed randomly throughout the cytoplasm. Microbiology, 1994 Sep, 140 ( Pt 9), 2363 - 70 Isolation of an Ustilago maydis gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase and expression of a C-terminal-truncated form in Escherichia coli; Croxen R et al.; A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was isolated from the maize fungal pathogen Ustilago maydis . This was accomplished by identifying cDNA and genomic clones that hybridized to an internal fragment of the gene, amplified from U . maydis genomic DNA by PCR . The nature of the gene was determined by nucleotide sequence analysis, and by comparing the derived amino acid sequence of the gene with HMG-CoA reductases from yeast, and from other organisms . The hydrophobic nature of the N-terminal region of the deduced protein sequence also supported the view that this gene encoded HMG-CoA reductase . A C-terminal-truncated fragment of the U . maydis HMG-CoA reductase gene was shown to be expressed in Escherichia coli in a catalytically active form . The expressed protein was also shown to be sensitive to an inhibitor of mammalian HMG-CoA reductase activity. Appl Environ Microbiol, 1994 Sep, 60(9), 3145 - 9 Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters; Lang AL et al.; A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli . This method was used to screen 377 environmental E . coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E . coli strains . Of the 377 E . coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence . None was found to contain SLT II homologous sequences . The pathogenicity of the positive environmental E . coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production . Our results suggest that toxigenic E . coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E . coli in coastal waters. Nucleic Acids Res, 1994 Sep, 22(17), 3563 - 5 The 16S ribosomal RNA mutation database (16SMDB) Triman KL. The 16S ribosomal RNA mutation database (16SMDB), provides a list of mutated positions in 16S ribosomal RNA from Escherichia coli and the identity of each alteration . Information provided for each mutation includes: (1) a brief description of the phenotype(s) associated with each mutation, (2) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (3) relevant literature citations . The database is available via ftp. Nucleic Acids Res, 1994 Sep, 22(17), 3450 - 5 ECD--a totally integrated database of Escherichia coli K12; Wahl R et al.; We have compiled the DNA sequence data for E . coli available from the GENBANK and EMBL data libraries and independently from the literature . Starting with this update of our Escherichia coli database (ECD release 20) we provide major changes compared to previous issues . This update not only represents another substantial increase in sequence information, it also allows now to find the exact physical location of each individual gene or regulatory region, even regarding discrepancies in nomenclature . In order to save space this printed version does not contain the database itself anymore, but we provide several examples . The complete database is publically available in electronic form together with a self explaining application program or as a flat file . The complete compilation including a full set of genetic map data and the E . coli protein index can be obtained in machine readable form from the EMBL data library as a part of the CD-ROM issue of the EMBL sequence database, released and updated every three months . After deletion of all detected overlaps a total of 2,878,364 individual bp is found to be determined till the end of June 1994 . This corresponds to a total of 60.98% of the entire E . coli chromosome consisting of about 4,720 kbp . This number may actually be higher by 9161 bp derived from other strains of E . coli. Mol Pharmacol, 1994 Sep, 46(3), 568 - 77 Bufuralol hydroxylation by cytochrome P450 2D6 and 1A2 enzymes in human liver microsomes; Yamazaki H et al.; Bufuralol 1'-hydroxylation is a prototypical reaction catalyzed by cytochrome P450 (P450) 2D6, an enzyme known to show debrisoquine/sparteine-type genetic polymorphism in humans . In the present study we further examined the roles of several human P450 enzymes, as well as P450 2D6, in the hydroxylation of (+/-)-bufuralol, using liver microsomes from several human samples and human P450 enzymes expressed in human lymphoblastoid cell lines or Escherichia coli . Kinetic analysis of bufuralol 1'-hydroxylation by liver microsomes showed that there were different Km and Vmax values in seven human samples examined; low Km values (approximately 0.05 mM) were observed in four samples (including sample HL-18), high Km values (approximately 0.25 mM) in two samples (including sample HL-67), and an intermediate Km value (approximately 0.1 mM) in one sample . Quinidine and anti-rat P450 2D1 antibody almost completely inhibited bufuralol 1'-hydroxylation in human sample HL-18 at a substrate concentration of 0.4 mM, whereas these effects were not so drastic when liver microsomes from human sample HL-67 were used . In contrast, a very low concentration (< 10 microM) of alpha-naphthoflavone or anti-human P450 1A2 antibody significantly inhibited bufuralol 1'-hydroxylation catalyzed by human sample HL-67, but not HL-18, with 0.4 mM bufuralol . When the relative contents of P450 2D6 and P450 1A2 in 20 human samples were determined, bufuralol 1'-hydroxylation in samples containing large amounts of P450 2D6 tended to be more sensitive to quinidine, whereas the P450 1A2-rich samples were highly susceptible to alpha-naphthoflavone . However, at low substrate concentrations bufuralol 1'-hydroxylation was shown to be catalyzed principally by P450 2D6, based on the inhibitory effects of anti-rat P450 2D1 antibody and quinidine, in both human samples HL-18 and HL-67 . At least five other, minor, bufuralol products were formed by human liver microsomes, in addition to 1'-hydroxybufuralol . Two of them were identified as 4- and 6-hydroxybufuralol by 1H NMR spectroscopy and mass spectrometry . The formation of the 4- and 6-hydroxylated products was suggested to be catalyzed by P450 1A2, based on the results of correlation with P450 1A2 contents in 60 human samples and inhibition by anti-P450 1A2 and alpha-naphthoflavone . Purified recombinant P450 1A2 (expressed in E . coli) produced 1'-, 4-, and 6-hydroxybufuralol in a reconstituted system, although P450 2D6 (expressed in human lymphoblast cell lines) was found to catalyze only bufuralol 1'-hydroxylation.(ABSTRACT TRUNCATED AT 400 WORDS) FEMS Microbiol Lett, 1994 Sep 1, 121(3), 333 - 6 Temperature shift-up leads to simultaneous and continuous plasmid DNA relaxation and induction of DnaK and GroEL proteins in anaerobically growing Escherichia coli cells; Mizushima T et al.; We previously reported that plasmid DNA in Escherichia coli cells growing under aerobic conditions relaxed immediately and transiently after heat shock (Mizushima, T., Natori, S . and Sekimizu, K., Mol . Gen . Genet . (1993) 238, 1-5) . We next examined DNA relaxation and induction of heat shock proteins after heat shock in cells growing under anaerobic conditions . DNA in these cells relaxed rapidly (2 min) after heat shock (42 degrees C), as was the case with aerobically growing cells, but full superhelicity was not recovered . The relaxed state of DNA topology was maintained for 60 min after heat shock . Induction of DnaK and GroEL proteins, which was transient in aerobically growing cells, was continuous in anaerobically growing cells . Therefore, induction of heat shock proteins correlated with DNA relaxation in both aerobic and anaerobic conditions. FEMS Microbiol Lett, 1994 Sep 1, 121(3), 293 - 6 Sequence and characterization of the Escherichia coli genome between the ndk and gcpE genes; Baker J et al.; The region of the chromosome immediately upstream of the Escherichia coli gene gcpE has been cloned and sequenced . This region contains two functional open reading frames, orf 384 and orf 337, encoding proteins of 43,082 and 36,189 Da, respectively . Sequencing analysis (this paper) and the isolation of a DNA fragment containing a functional promoter (Talukder, A.A., Yanai, S., and Yamada, M . (1994) Biosci . Biotech . Biochem . 58, 117-120) indicate that orf 337 is in an operon with gcpE . The gene orf 384 is immediately downstream of the gene ndk, which encodes nucleoside diphosphate kinase. FEMS Microbiol Lett, 1994 Sep 1, 121(3), 281 - 6 Immediate and transient inhibition of the respiration of Escherichia coli under hyperosmotic shock; Meury J; The respiration of Escherichia coli is severely inhibited, during hyperosmotic stress period, as a consequence of plasmolysis; deplasmolysis allows the cell to recover respiration . A mutant lacking all K+ transport systems can neither deplasmolyze nor recover respiration unless betaine is present in the medium . Betaine, in these conditions, increases both cytoplasmic volume and respiration; this suggests a control of respiration by cytoplasmic volume. Eur J Biochem, 1994 Sep 1, 224(2), 751 - 60 Chemical structure of the core region of Escherichia coli J-5 lipopolysaccharide; Muller-Loennies S et al.; The lipopolysaccharide of Escherichia coli J-5 was sequentially de-O-acylated, dephosphorylated, reduced, de-N-acylated, and N-acetylated . The products were separated by high-performance anion-exchange chromatography into a nonasaccharide (1), two octasaccharides (2, 3), and a heptasaccharide (4) . Compositional analysis, methylation analysis, and NMR spectroscopy revealed the structures of the products as: alpha-D-GlcpNAc-(1-7)-L-alpha-D-Hepp-(1-7)-{alpha-D-Glcp-(1-3)-}-L -alpha-D- Hepp-(1-3)-R1, (1) L-alpha-D-Hepp-(1-7)-{alpha-D-Glcp-(1-3)-}-L-alpha-D-Hepp-(1 -3)-R1, (2) alpha-D-GlcpNAc-(1-7)-L-alpha-D-Hepp-(1-7)-{alpha-D-Glcp-(1-3)-}-L -alpha-D- Hepp-(1-3)-R2, (3) alpha-D-Glcp-(1-3)-L-alpha-D-Hepp-(1-3)-R1, (4) in which 1R is L-alpha-D-Hepp-(1-5)-{alpha-Kdop-(2-4)-}-alpha-Kdop-(2 -6)- beta-D-GlcpNAc-(1-6)-D-GlcN-Acol, and 2R is L-alpha-D-Hepp-(1-5)-alpha-Kdop-(2-6)-beta-D-GlcpNAc-(1-6 )-D- GlcNAcol (LD-Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-octulopyranosonic acid; GlcNAcol, 2-acetamido-2-deoxy-glucitol) . Fast-atom-bombardment mass spectrometry of de-O-acylated and dephosphorylated lipopolysaccharide showed that the isolated oligosaccharides represented the complete carbohydrate moiety of the lipopolysaccharide, and indicated that the non-reducing terminal D-GlcN residue in lipopolysaccharide was present as the free base. Eur J Biochem, 1994 Sep 1, 224(2), 743 - 50 Acyl carrier protein (ACP) import into chloroplasts . Covalent modification by a stromal holoACP synthase is stimulated by exogenously added CoA and inhibited by adenosine 3',5'-bisphosphate; Yang LM et al.; During the import of the precursor for the acyl carrier protein (ACP) into chloroplasts, apoACP is converted to holoACP by the attachment of a phosphopantetheine group transferred from coenzyme A (CoA) by a chloroplast holoACP synthase {Fernandez, M . and Lamppa, G . (1990) Acyl carrier protein import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holoACP synthase, Plant Cell 2, 195-206} . Here it is shown that exogenous addition of CoA to intact chloroplasts in the import assay stimulates the conversion of apoACP to holoACP . If adenosine 3',5'-bisphosphate {Ado(3',5')P2}, the byproduct of the transfer reaction, was also included the extent of conversion was greatly reduced . CoA has its effect after ACP precursor (pre-ACP) import and proteolytic removal of the transit peptide, thus indicating that the chloroplast holoACP synthase resides in the stroma where fatty acid synthase is found . When Ado(3',5')P2 was added alone to the import assay, it inhibited the synthesis of holoACP . Inhibition of the conversion of apo- to holoACP with Ado(3',5')P2 made it possible to examine whether the holoform of preACP could be imported into chloroplasts . Pre-apoACP was synthesized in Escherichia coli and shown to be competent for import in an ATP- and temperature-dependent manner . A partially purified chloroplast holoACP synthase converted 60-90% of the pre-apoACP to pre-holoACP . Pre-holoACP incubated with chloroplasts in the presence of Ado(3',5')P2 yielded > 60% holoACP, whereas the control reaction with pre-apoACP gave primarily apoACP . Hence the phosphopantetheine prosthetic group of ACP does not block precursor movement through the translocation apparatus of the chloroplast envelope. Eur J Biochem, 1994 Sep 1, 224(2), 723 - 8 In vitro approaches to investigation of the early steps of colicin-OmpF interaction; el Kouhen R et al.; The OmpF porin plays a central role during the colicin uptake by sensitive Escherichia coli cells . Lipopolysaccharide-OmpF complexes (-1bLPS-OmpF), which contain one tightly bound and no loosely bound LPS molecules for each porin trimer, is able to recognize and bind to immobilized colicins . This association is specific to colicins A and N, which both use the OmpF porin as receptor, and depends on the presence of the porin-receptor domain on the bacteriocin molecule . The -1bLPS-OmpF complex protects sensitive cells against colicin A and N activity . The protection level depends on the native conformation, as demonstrated by heat denaturation of the trimeric porin which abolishes the protection . This indicates that the purified OmpF trimer presents an affinity site for the colicin which efficiently mimics the native cellular receptor site . These results are discussed with regard to the conformation of the receptor site and to the early steps of colicin uptake. Eur J Biochem, 1994 Sep 1, 224(2), 541 - 8 Molecular characterization of Escherichia coli malate synthase G . Differentiation with the malate synthase A isoenzyme; Molina I et al.; Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli . The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262-nucleotide open-reading frame, which can encode a 723-amino-acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids . Northern-blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate . Malate synthase G was purified to near homogeneity . The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure . Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms. Eur J Biochem, 1994 Sep 1, 224(2), 533 - 40 States and functions of tyrosine residues in Escherichia coli asparaginase II; Derst C et al.; The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation . While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity . However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer . pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic . 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25 . These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme. Eur J Biochem, 1994 Sep 1, 224(2), 431 - 7 Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA; Bechthold A et al.; The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector . The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity . Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E . coli (Km = 0.001 mM) were determined . S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM) . Binding of either thiostrepton or protein L11 inhibited methylation . RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase . The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand. Eur J Biochem, 1994 Sep 1, 224(2), 407 - 15 Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin; Auerswald EA et al.; Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-{Met1, Ile29, Leu89}-chicken egg white cystatin gene in Escherichia coli . The variant des-Ser1-Pro11-{Ala12, Glu13, Phe14, Met15, Ile29, Leu89}-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-{Met1, Ile29}-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied . The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge . Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar . The folding of the del-helix II variant corresponds best to a single step process . In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD . With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states . The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin . However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin. Eur J Biochem, 1994 Sep 1, 224(2), 345 - 52 Functional reconstitution of ATP-dependent transporters from the solubilized hepatocyte canalicular membrane; Buchler M et al.; The hepatocyte canalicular membrane contains several primary-active ATP-dependent export carriers including one for bile salts and one for leukotriene C4 and related conjugates . The molecular identity of both transporters has not been fully elucidated . To establish a transport assay that allows the purification and identification of the proteins involved in ATP-dependent bile salt transport and in leukotriene C4 transport, we reconstituted solubilized hepatocyte canalicular membranes into phospholipid bilayers using a rapid dilution method . The proteoliposomes formed exhibited both {3H}taurocholate and {3H}leukotriene C4 uptake, which was much higher in the presence of ATP than in the presence of the non-hydrolyzable ATP-analog AdoPP{CH2}P or in the absence of nucleotides . Nucleotide requirement and osmotic sensitivity of {3H}taurocholate transport indicates true transport into the vesicle lumen . Optimized conditions for reconstitution included the addition of a high concentration of an osmolyte (glycerol) and the presence of exogenous phospholipids (0.3%) during solubilization . Highest transport rates were obtained by reconstitution into acetone/ether-precipitated Escherichia coli phospholipid supplemented with 20% cholesterol and by use of octylglucoside concentrations between 30 mM and 50 mM . Taurocholate transport was non-competitively inhibited by vanadate (Ki = 39 microM) . The kinetic parameters of cyclosporin A inhibition (Ki = 2.6 microM for taurocholate and 4.3 microM for leukotriene C4 transport) as well as the affinities of taurocholate (Km = 12 microM) and leukotriene C4 (Km = 0.5 microM) in the proteoliposome system indicate that the reconstitution resulted in functionally active transport systems, which are representative of ATP-dependent transport in the intact plasma membrane. Eur J Biochem, 1994 Sep 1, 224(2), 335 - 43 Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase . A study with the mature enzyme expressed in Escherichia coli; Akiyoshi-Shibata M et al.; The coding region of the cDNA for rat kidney calcidiol 24-hydroxylase (P450cc24), which is involved in calcium homeostasis in animals, was inserted into an expression vector pKK223-3 . The recombinant plasmid was formed in a specific manner without deletion or substitution of any parts of the coding region of the cDNA . When the resulting plasmid was introduced into Escherichia coli JM109, the recombinant cells produced a protein which was immunoreactive to an antibody against P450cc24 . When the cell-free extract of the transformed cells was incubated with calcidiol together with bovine adrenodoxin and NADPH-adrenodoxin reductase, not only hydroxycalcidiol but also other metabolites such as oxocalcidiol and oxohydroxycalcidiol were produced . Similarly, calcitriol was converted not only to calcitetrol but also to oxocalcitriol and oxohydroxycalcitriol . These results indicate that a single enzyme expressed in the bacteria is responsible for all these successive reactions. Plant J, 1994 Sep, 6(3), 433 - 40 Synthesis of a bifunctional metallothionein/beta-glucuronidase fusion protein in transgenic tobacco plants as a means of reducing leaf cadmium levels; Elmayan T et al.; Chimeric genes under the control of a CaMV 35S promoter with a doubled enhancer (35S2) that encode a mammalian metallothionein (hMTII), or an Escherichia coli beta-glucuronidase (GUS), or a hMTII/GUS fusion protein were introduced into the genome of tobacco (Nicotiana tabacum cv . PBD6) . Transcripts and Cd-binding proteins of expected size were observed in plants expressing either the 35S2-hMTII or the 35S2-hMTII/GUS gene, and in the latter plants a protein with GUS activity that was larger than the native GUS enzyme was observed . Thus, plants expressing the hMTII-GUS gene synthesize a bifunctional protein, with both GUS and Cd-binding activity . In an in vitro assay, seedlings expressing either one of these genes had 60-70% lower Cd concentration in their shoots than controls, and Cd translocation to the shoot system was reduced (approximately 20% of Cd absorbed was translocated), compared with that in controls expressing a 35S2-GUS gene, where approximately 50% of the Cd absorbed was translocated. Plant J, 1994 Sep, 6(3), 351 - 8 The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility; Grasser KD et al.; The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail . The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments . It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding . They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles . The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites. J Steroid Biochem Mol Biol, 1994 Sep, 50(5-6), 225 - 33 Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor; Nemoto T et al.; The DNA-binding domain (DBD) of the androgen, mineralocorticoid, and glucocorticoid receptors and the steroid-binding domain (SBD) of the androgen receptor (AR) were expressed separately as fusion proteins with glutathione-S-transferase (GST) in Escherichia coli . Native polyacrylamide gel electrophoresis and gel exclusion HPLC demonstrated that the GST-ARDBD fusion protein was present as a dimer . On the other hand, the GST-ARSBD fusion protein formed a high-molecular weight oligomer, which seemed to be formed by two separate interactions, i.e . GST-GST and ARSBD-ARSBD between the fusion molecules . These findings strongly suggest that ARSBD has a potent ability to form a homodimer and that ARDBD does not . GST-ARDBD specifically interacted with the glucocorticoid response elements of the mouse mammary tumor virus long terminal repeat (GREMMTV) . Cleavage of the fusion protein by thrombin abolished the binding, while the nonspecific DNA-cellulose binding ability was retained . Therefore, the dimeric configuration of GST-ARDBD, even if accomplished through the interaction with the GST moiety, is needed for high-affinity binding to the response element . The binding of GST-ARDBD to GREMMTV was strongly competed by the glucocorticoid response element of rat tyrosine aminotransferase gene, followed by the androgen response element of the rat probasin gene . A palindromic thyroid response element showed no competition . Unexpectedly, no apparent different in the binding affinity to these response elements was observed among the DBDs of androgen, mineralocorticoid and glucocorticoid receptors. Biochem J, 1994 Sep 1, 302 ( Pt 2), 567 - 71 2-Chloro-2'-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3'-->5' exonucleases; Hentosh P et al.; 2-Chloro-2'-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3'-->5' exonuclease activity . ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase . After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates . Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3'-->5' exonuclease, but only in the vicinity of the ClAde sites . Approx . 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA . Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3'-->5' exonucleases, incorporated ClAde enhances strand degradation of duplex DNA. Biochem J, 1994 Sep 1, 302 ( Pt 2), 405 - 10 Refolding and recognition of mitochondrial malate dehydrogenase by Escherichia coli chaperonins cpn 60 (groEL) and cpn10 (groES); Hutchinson JP et al.; In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES) . When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient . C.d . spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding . Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance . The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding . Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase . A novel use is made of 100 K Centricon concentrators to study the binding of {14C}acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay . Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s) . At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx . 10 nM. J Bacteriol, 1994 Sep, 176(18), 5665 - 72 The leucine-responsive regulatory protein binds to the fim switch to control phase variation of type 1 fimbrial expression in Escherichia coli K-12; Gally DL et al.; Phase variation of type 1 fimbriation in Escherichia coli is associated with the site-specific recombination of a 314-bp DNA invertible element . The fim switch directs transcription of fimA, the major fimbrial subunit gene, in one orientation (on) but not the other (off) . Switching requires either fimB (on-to-off or off-to-on inversion) or fimE (on-to-off inversion only) and is reduced sharply in strains containing lrp::Tn10 mutations . Both fimE-promoted switching and fimB-promoted switching are stimulated by the amino acids alanine, isoleucine, leucine, and valine, and this regulation requires lrp . Here it is shown that the leucine-responsive regulatory protein (Lrp) binds in and adjacent to the fim switch . Mutations in fim that lower Lrp binding in vitro have corresponding effects on both fimB-promoted switching and fimE-promoted switching in vivo . Lrp initiates binding at one of two sites within the fim switch . Additional cooperative binding results in an extensive region of protection from both DNase I and 1,10-phenanthroline-copper complex-activated DNA cleavage . The region of protection can extend to within 12 bp of the right inverted repeat (switch off) and occupies over one-third of the switch . It is proposed that wrapping of fim DNA around an Lrp complex is required to form a recombination-proficient structure. J Bacteriol, 1994 Sep, 176(18), 5648 - 53 Induction of heat shock proteins by abnormal proteins results from stabilization and not increased synthesis of sigma 32 in Escherichia coli; Kanemori M et al.; Accumulation of abnormal proteins in cells of bacteria or eukaryotes can induce synthesis of a set of heat shock proteins . We examined such induction following addition of azetidine (a proline analog) or synthesis of a heterologous protein (human prourokinase) in Escherichia coli . Synthesis of heat shock proteins under these conditions increased almost immediately and continued with increasing rates until it reached a maximum after 30 to 60 min at 30 degrees C . The induction was closely accompanied by an increase in the cellular level of sigma 32 specifically required for transcription of heat shock genes . The increase in sigma 32 initially coincided with increased synthesis of heat shock proteins but then exceeded the latter, particularly following accumulation of prourokinase . The sigma 32 level increase upon either treatment was found to result solely from stabilization of sigma 32, which is ordinarily very unstable, and not from increased synthesis of sigma 32 . This is in contrast to what had been found when cells were exposed to a higher temperature, at which both increased synthesis and stabilization of sigma 32 contributed to the increased sigma 32 level . On the basis of these and other findings, we propose that abnormal proteins stabilize sigma 32 by a pathway or a mechanism distinct from that used for the induction of sigma 32 synthesis known to occur at the level of translation . Evidence further suggests that the DnaK chaperone plays a crucial regulatory role in induction of the heat shock response by abnormal proteins. J Bacteriol, 1994 Sep, 176(17), 5385 - 92 Regulation of rns, a positive regulatory factor for pili of enterotoxigenic Escherichia coli; Froehlich B et al.; Attachment of enterotoxigenic Escherichia coli to the human gut is considered an important early step in infection that leads to diarrhea . This attachment is mediated by pili, which belong to a limited number of serologically distinguishable types . Many of these pili require the product of rns, or a closely related gene, for their expression . We have located the major promoter for rns and found that although its sequence diverges significantly from a sigma-70 promoter consensus sequence, it is very strong . Transcription of rns is negatively regulated both at a region upstream of this promoter and at a region internal to the rns open reading frame . In addition, rns positively regulates its own transcription, probably by counteracting these two negative effects. Mol Cell Biochem, 1994 Sep, 138(1-2), 119 - 22 ADP-ribosylarginine hydrolases; Takada T et al.; ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid . NAD:arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins . The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine . An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg2+ and dithiothreitol . The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl)arginine but not phosphoribosylarginine or ribosylarginine . The hydrolase cDNA was cloned from rat and subsequently from mouse and human brain . The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides . Deduced amino acid sequences from the mouse and human hydrolase cDNAs were 94% and 83% identical, respectively to the rat . Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase . The rat hydrolase, expressed in E . coli, demonstrated enhanced activity in the presence of Mg2+ and thiol, whereas the recombinant human hydrolase was stimulated by Mg2+ but was thiol-independent . In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Mikrobiol Virusol, 1994 Sep-Oct, (5), 9 - 13 {The effect of metabolic inhibitors on resistance of mannose-specific contacts of Escherichia coli K12 and human neutrophils}; Timoshenko AV et al.; Human neutrophil aggregation induced by Escherichia coli K12 cells has been studied . D-mannose has been found to inhibit the process in the dose-dependent way causing the full blockage at 10 mM concentration . The process of disaggregation induced by the concentration is significantly higher at 10 degrees C than at 37 degrees C . The inhibitors of cellular oxygenation such as N-ethylmaleimide, nordihydroguaiaretic acid, trifluoperazine, and colchicine did not affect the aggregation process . Cellular aggregation was concomitant with production of H2O2, and this reaction was also blocked by D-mannose and depended on the number of bacteria present . The nordihydroguaiaretic acid and N-ethylmaleimide inhibited production of H2O2, while sodium azide enhanced the process . The results show the mannose-specific contact of bacteria and neutrophils to depend on the functional activity of phagocytes including their ability to produce H2O2. Virus Genes, 1994 Sep, 9(1), 77 - 83 Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression in Escherichia coli; Lopez L et al.; Total RNA from infected Physalis floridana was isolated to generate complementary DNA corresponding to the coat protein (GP) gene of a Cuban isolate of potato leaf roll virus (PLRV) . This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1-3) for fusion protein expression in E . coli . The product was detected by antibodies specific for the PLRV CP . The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD) . The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99.5% identical at both the nucleotide and amino acid sequence level of other isolates . Comparison of the deduced amino acid sequences of the PLRVcub CP revealed considerable homology to other luteoviruses . We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences. Virus Genes, 1994 Sep, 9(1), 5 - 13 Characterization of a Marek's disease virus mutant containing a lacZ insertion in the US6 (gD) homologue gene; Parcells MS et al.; We report the construction of a Marek's disease virus (MDV) mutant containing the lacZ gene of Escherichia coli inserted into a homologue of the US6 (glycoprotein D, gD) gene of herpes simplex virus . The mutant was constructed using the high-passage GAatt85 MDV strain as the parent virus, since that strain grows readily in chicken embryo fibroblasts using culture conditions conducive to mutant virus construction . The lacZ insertion site was positioned one third of the way into the US6 (gD) open reading frame . Insertion of the lacZ gene disrupted a major 6.2 kb transcript that initiated approximately 2.5 kb upstream of the gD homologue gene in the vicinity of the US3 homologue and sorf4 genes, and extended into the US7 (gI) homologue gene . The mutant virus (US6lac) and the parent virus had similar growth kinetics in cell culture at 37 degrees C and 41 degrees C . Furthermore, the US6lac mutant could be reisolated from the spleens and peripheral blood of infected chickens with a frequency comparable to that of the parent virus . Our results indicate that the gene encoding the gD homologue is nonessential for growth in cell culture or for infection of chickens following intra-abdominal inoculation with an attenuated serotype-1 MDV. Hokkaido Igaku Zasshi, 1994 Sep, 69(5), 1261 - 74 {Establishment of Chinese hamster ovary cell lines with reduced expression of glutathione reductase after antisense-oriented gene transfection and assessment of the sensitivity to oxidant injury}; Tonoki H; Glutathione reductase (GR) protects tissues from oxidant injury by catalysing the reduction of glutathione disulfide (GSSG) to glutathione (GSH) . In order to study the effect of GR in protecting cells from oxidant injury, we generated Chinese hamster ovary (CHO) cell lines stably transformed after antisense-oriented gene transfection . The coding region of the human GR was cloned using revere transcription PCR method and selected by transient expression study in mammalian cells . A clone HGR135 showed overexpression of GR in CHO cells and was proved to have no base substitution . This clone, then, was ligated into MEP4 expression vector in an antisense orientation to the human metallothionein promoter and transfected to CHO cells with polybrene . Among 12 cell lines isolated, G17 showed to have the least GR activity (48% of the control), while another four were mildly GR deficient . Southern hybridization of genomic DNA digests and transformation experiment on E . coli revealed that the promoter-antisense coding region component was integrated . Northern hybridization detected reduced amount of GR transcript but no antisense message . Baseline cellular GSH concentrations were lower in G17 than in control (25.7 +/- 2.5 vs . 36.1 +/- 1.9 nmole/mg protein, P < 0.05), while cellular GSSG concentrations were higher (0.61 +/- 0.19 vs . 0.39 +/- 0.09 nmole/mg protein, P < 0.05) . After four hours of treatment of G17 and control cells with increasing doses (1 to 10 mM) of t-butylhydroperoxide (t-BuOOH), cellular GSH concentrations in G17 decreased with an elevation of GSSG concentration at 1 mM followed by no further increase at higher t-BuOOH concentration, while GSSG concentrations increased in the control cells without reduction of GSH concentrations at 1-5 mM t-BuOOH treatment . The concentrations of GSH were lower in G17 than in controls at all doses of t-BuOOH . Four hours of exposure to 10 mM t-BuOOH resulted in greater LDH release in G17 than in control (57.3 +/- 4.7 vs . 32.1 +/- 6.5%, P < 0.05) . Similarly, G17 cells released more of their LDH to the media than did CHO cells in response to exposure to 95% O2 for 72 hours (19.3 +/- 5.9 vs . 11.9 +/- 5.4%, P < 0.05) . The partial GR deficiency in G17 cells impairs their ability to recycle GSSG and this deficiency offers the best explanation for the increased sensitivity of these cells to injury by t-BuOOH or hyperoxia. Curr Genet, 1994 Sep, 26(3), 251 - 5 Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph; Hamada W et al.; A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea . Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance, downstream from an Aspergillus nidulans promoter . Molecular analysis, showed that transformation resulted in an integration of the plasmid into different regions of the B . cinerea genome and occurred through non-homologous recombination . The frequency was 2-10 transformants per micrograms of DNA . Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype . All transformants analysed so far proved to be stable after several subcultures without any selective pressure. Curr Genet, 1994 Sep, 26(3), 217 - 24 Heterologous transformation of Zalerion arboricola; Kelly R et al.; A heterologous DNA-mediated transformation system was developed for the pneumocandin-producing fungus Z . arboricola that was based on either conferral of hygromycin B resistance or complementation of a nitrate reductase mutant . Hygromycin-resistant transformants were selected with plasmid pCSN43 which contains the E . coli hygromycin B phosphotransferase gene under the control of Aspergillus nidulans trpC transcription signals . Transformation frequencies were about four transformants per microgram of circular DNA and could be improved four- to six-fold by linearizing the transforming DNA . The transformants differed from one another with respect to the copy number of the integrated plasmid and the site of integration . Adding an autonomously-replicating sequence (AMA1) from A . nidulans to pCSN43 enhanced transformation three-fold and produced, in addition, numerous abortive transformants . However, it is unlikely that the AMA1 sequence promoted plasmid replication in Z . arboricola . Nitrate reductase mutants of Z . arboricola were isolated by positive selection on chlorate-containing medium, and one mutant was subsequently transformed with pSTA700 which contains the nitrate reductase gene (niaD) from Cephalosporium acremonium . Introduction of the niaD gene restored sensitivity to chlorate in the mutant; therefore, using the niaD gene as a selectable marker provides a system for both positive and negative selection . To our knowledge, this is the first report describing transformation of a member of the genus Zalerion. Mol Microbiol, 1994 Sep, 13(6), 987 - 1000 Regulation of R100 conjugation requires traM in cis to traJ; Dempsey WB; Deletion mutants of R100-1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM-binding sites and the traM promoters . Complementation tests showed that traM was efficiently complemented only when the trans-acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences . The conclusion is that traM and traJ constitute a complex operon . A deletion mutant lacking all of the traJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer. Mol Microbiol, 1994 Sep, 13(6), 955 - 64 Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer; Pettis GS et al.; The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating . We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt . Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA . Our results indicate that plasmid transfer in S . lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S . lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms. Mol Microbiol, 1994 Sep, 13(6), 1121 - 31 The colicin A pore-forming domain fused to mitochondrial intermembrane space sorting signals can be functionally inserted into the Escherichia coli plasma membrane by a mechanism that bypasses the Tol proteins; Espesset D et al.; Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane . The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore-forming domain of colicin A (pfColA) . Both hybrid proteins (pc1-pfCoIA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect . The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities . The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins . In addition, we showed that both the cytochrome c1 presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c1 presequence moiety of pc1-pfCoIA were translocated across inverted membrane vesicles . Our results indicated that: (i) pc1-pfCoIA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (iii) this channel activity was inhibited within the membrane by the immunity protein. Mol Microbiol, 1994 Sep, 13(6), 1111 - 20 Immunity proteins to pore-forming colicins: structure-function relationships; Espesset D et al.; Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel . By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi . The results revealed unexpectedly high structural constraints for the function of these proteins . The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be involved in topology and function of Cai . The immunity function did not seem to be confined to a particular region of the immunity proteins. Mol Microbiol, 1994 Sep, 13(6), 1021 - 32 Sigma S-dependent growth-phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by sigma 70 in the absence of the nucleoid-associated protein H-NS; Arnqvist A et al.; The stationary-phase-specific sigma factor sigma S (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin-binding curli organelles upon reaching stationary phase . We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA . The transcriptional start site of csgBA was determined and an AT-rich up-stream activating sequence (UAS) required for transcriptional activation was identified . The pcsgBA promoter is not specific for sigma S since the same promoter sequence can be used by E sigma 70 in vivo in a strain lacking nucleoid-associated protein H-NS and sigma S . Transcription remained growth-phase induced and dependent upon the UAS in such a double mutant . Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon sigma S for transcription, can be transcribed by E sigma 70 in vivo in the absence of H-NS by utilizing the phdeAB promoter . Two other genes known to be under the control of sigma S for expression, bolA and katE, remained transcriptionally silent in the absence of H-NS . It is suggested that a subset of E . coli promoters can be recognized by both E sigma S and E sigma 70 in vivo but H-NS interacting with these sequences prevents formation of successful transcription-initiation complexes with E sigma 70. Mol Microbiol, 1994 Sep, 13(6), 1001 - 12 Mutants of Escherichia coli Trp repressor with changes of conserved, helix-turn-helix residue threonine 81 have altered DNA-binding specificities; Pfau J et al.; Threonine is found at the third position of the second alpha-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins . To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressors with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis . All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function . Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities . The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressor-operator complex. J Biochem (Tokyo), 1994 Sep, 116(3), 687 - 93 Direct expression of a synthetic gene in Escherichia coli: purification and physicochemical properties of human initiation factor 4E; Morino S et al.; An artificial synthetic gene coding for human eIF-4E was cloned into an expression vector and direct expression was attempted in Escherichia coli {BL21(DE3) strain} under the control of T7 promoter . The active gene product which was induced in high yield (ca . 4 mg/100 ml) by isopropyl-beta-D-thiogalactopyranoside was purified to homogeneity by a two-step chromatographic procedure with a good yield (ca . 74%), and was confirmed to be recombinant human eIF-4E by amino acid composition and sequence analyses, isoelectric focusing, and absorption spectral measurements . The identity of three-dimensional structures between the recombinant and native human eIF-4Es was confirmed by CD and fluorescence measurements. J Biochem (Tokyo), 1994 Sep, 116(3), 502 - 7 Affinity labeling of Escherichia coli lysyl-tRNA synthetase with pyridoxal mono- and diphosphate; Hountondji C et al.; Pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphosphate (PLDP) were used to identify lysyl residues at the phosphate-binding locus in the lysS-encoded and the lysU-encoded lysyl-tRNA synthetases (LysRSs and LysRSu, respectively) from Escherichia coli . Incubation of LysRSs with either reagent, followed by borohydride reduction, resulted in a time-dependent covalent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation and lysine-dependent ATP-PPi exchange activities . By contrast, LysRSu activity was insensitive to prolonged incubation with either reagent, possibly reflecting a difference at the phosphate-binding locus in the two enzyme species . MgATP protected LysRSs against inactivation by PLP or PLDP . Complete inactivation of LysRSs corresponded to the incorporation of 2.6 +/- 0.1 mol of PLP or PLDP per mol of dimeric enzyme . Either reagent was found to label the same set of eight lysyl residues (Lys-25, Lys-82, Lys-114, Lys-132, Lys-156, Lys-185, Lys-364, and Lys-505) as adenosine di- or triphosphopyridoxal (see the preceding paper in this issue) . These lysyl residues might represent the subsite for the phosphate moiety of ATP in LysRSs . None of the identified lysyl residues is located within the three sequence motifs considered as characteristic of the class 2 aminoacyl-tRNA synthetases . The present results are discussed on the basis of the crystalline structure of the closely related aspartyl-tRNA synthetase from Saccharomyces cerevisiae. J Biochem (Tokyo), 1994 Sep, 116(3), 493 - 501 Affinity labeling of the two species of Escherichia coli lysyl-tRNA synthetase with adenosine di- and triphosphopyridoxals; Hountondji C et al.; Lysyl-tRNA synthetase (LysRS), a representative of the class 2 aminoacyl-tRNA synthetases, occurs as two species in Escherichia coli: LysRSs and LysRSu . To identify the ATP-binding site in this enzyme, we have applied affinity labeling with reactive adenine nucleotide analogs . Incubation of either enzyme species with adenosine di- or triphosphopyridoxal, followed by borohydride reduction, resulted in a time-dependent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation, and lysine-dependent isotopic ATP-PPi exchange activities . LysRSu appeared less sensitive to adenosine triphosphopyridoxal than LysRSs . Complete inactivation with either reagent corresponded to the incorporation of about 2 mol of reagent per mol of dimeric enzyme . MgATP and ATP protected both enzyme species against the inactivation, suggesting that the modification occurs at the ATP-binding site . Sequence analysis of the labeled peptide isolated from the inactivated LysRSs and LysRSu revealed that bulk of the label was distributed among six lysyl residues at positions 25, 82, 114, 156, 364, and 505, with preference for Lys-114 and Lys-156 . In LysRSs, Lys-132 and Lys-185 were also modified by both reagents, although these residues are not conserved in LysRSu . It is concluded that the folding of the LysRSs and LysRSu polypeptides and the relative locations of the identified lysyl residues with respect to the binding site for the two labels are very similar. J Biochem (Tokyo), 1994 Sep, 116(3), 488 - 92 Corn cystatin I expressed in Escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels; Abe M et al.; Corn cystatin I was expressed in Escherichia coli as a mature protein . It was purified by gel filtration on Sephadex G-50, ion-exchange HPLC, and reversed-phase HPLC . The purified protein showed strong inhibitory activities against papain (Ki: 3.7 x 10(-8) M), and cathepsins H (Ki: 5.7 x 10(-9) M) and L (Ki: 1.7 x 10(-8) M), whereas it inhibited cathepsin B to a lesser extent (Ki: 2.9 x 10(-7) M) . Western blot analysis using antibody raised against corn cystatin I revealed that in the corn kernel, the protein occurs with a molecular mass of approximately 13 kDa . Localization in the aleurone layer and embryo of the corn kernel was shown by immunostaining microscopy. J Biochem (Tokyo), 1994 Sep, 116(3), 471 - 7 Molecular structure of a heme-copper redox center of the Escherichia coli ubiquinol oxidase: evidence and model; Mogi T et al.; Biochemical, spectroscopic, and molecular biological studies on bacterial respiratory oxidases in the last decade have greatly increased our understanding of a molecular structure of the metal centers which catalyze the dioxygen reduction chemistry . Based upon the latest physicochemical and molecular biological evidence and theoretical consideration of a folding mechanics of membrane proteins, we propose here the tertiary structure of the heme-copper metal center of the Escherichia coli bo-type ubiquinol oxidase, the cytochrome bo complex . The molecular mechanism of electron transfer-coupled proton pumping in the heme-copper respiratory oxidases is reviewed on the basis of this predicted model. J Cell Sci, 1994 Sep, 107 ( Pt 9), 2509 - 21 Ezrin has properties to self-associate at the plasma membrane; Andreoli C et al.; Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J . G . and Mangeat, P . (1991) . EMBO J . 10, 2363-2373) . Human ezrin was expressed in and purified from Escherichia coli . It possesses all the major biophysical, immunological and physiological properties of natural ezrin . Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized . By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins . In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay . The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin . In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane . Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family . In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D . and Arpin, M . (1993) . J . Cell Biol . 120, 129-139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin . Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites. Environ Health Perspect, 1994 Sep, 102 Suppl 3, 41 - 4 Effects of chromium on DNA replication in vitro; Snow ET; Chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis . Chromium(VI) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable Cr(III) species . Chromium(III) forms tight complexes with biological ligands, such as DNA and proteins, which are slow to exchange . In vitro, CrCl3.6H2O primarily interacts with DNA to form outer shell charge complexes with the DNA phosphates . However, at micromolar concentrations, the Cr(III) binds to a low number of saturable tight binding sites on single-stranded M13 DNA . Additional chromium interacts in a nonspecific manner with the DNA and can form intermolecular DNA cross-links . Although high concentrations of Cr(III) inhibit DNA replication, micromolar concentrations of Cr(III) can substitute for Mg2+, weakly activate the Klenow fragment of E . coli DNA polymerase I (Pol I-KF), and act as an enhancer of nucleotide incorporation . Alterations in enzyme kinetics induced by Cr(III) increase DNA polymerase processivity and the rate of polymerase bypass of DNA lesions . This results in an increased rate of spontaneous mutagenesis during DNA replication both in vitro and in vivo . Our results indicate that chromium(III) may contribute to chromate-induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis. Chem Res Toxicol, 1994 Sep-Oct, 7(5), 659 - 65 Thioredoxin alkylation by a dihaloethane-glutathione conjugate; Meyer M et al.; Glutathione is a thiol-containing tripeptide which functions to protect cellular constituents from endogenous and xenobiotic electrophiles via conjugation and eventual excretion . In the case of compounds such as 1,2-dihaloethanes, however, conjugate formation results in bioactivation of the species rather than detoxification . The conjugate can then act as an alkylating agent toward cellular constituents including DNA, proteins, or lipids . Alkylation of protein thiols in cells exposed to dihaloethane may contribute substantially to the toxicity produced by these compounds . We examined the reactivity of the conjugate S-(2-chloroethyl)-glutathione (CEG) toward the model protein Escherichia coli thioredoxin . At physiological pH, treatment of thioredoxin by CEG resulted in the production of several bands visible on isoelectric focusing, which were determined by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to be mono-, di-, tri-, and tetra-alkylated forms of thioredoxin . A concomitant loss of in vitro enzymatic activity was observed . These products were also observed when reaction was allowed to take place at pH 11.4 . Treatment at pH 4.4 resulted in lesser alkylation of thioredoxin, with only the mono- and di-alkylated forms detected . Iodoacetic acid treatment of CEG-alkylated thioredoxin revealed that the iodoacetic acid-susceptible Cys32 was not carboxymethylated, suggesting that this is one of the sites alkylated by CEG. Chem Res Toxicol, 1994 Sep-Oct, 7(5), 628 - 32 DNA sequence changes induced by two nitric oxide donor drugs in the supF assay; Routledge MN et al.; To refine our understanding of the mutational spectra one might expect on exposure of human cells to nitric oxide (NO), we have treated the plasmid pSP189 at pH 7.4 with two compounds that generate NO spontaneously in solution, and then sequenced the mutations found when the treated plasmid was transfected into human Ad293 cells and allowed to replicate . G.C-->A.T transitions were the most abundant mutation observed with these NO donor drugs, whereas in previous work, A.T-->G.C transitions predominated when nitric oxide gas was bubbled through the plasmid solution under otherwise identical conditions . A difference in reactive intermediates formed in solution- versus gas-phase NO exposure was demonstrated by treating buffered 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) or ferrocyanide, in the presence or absence of azide, aerobically with preformed solutions of NO, with solutions of the two NO-releasing compounds, or with gaseous mixtures of equimolar NO/O2 in air; oxidation of these substrates was extensive with the gas-phase NO source whether azide was present or not, while azide almost completely quenched the oxidation pathway in the solution-phase reactions. Res Virol, 1994 Sep-Oct, 145(5), 277 - 85 Characterization and structural localization of the reovirus lambda 3 protein; Cashdollar LW; The putative reovirus RNA polymerase, protein lambda 3, was characterized using antiserum prepared against a TrpE-lambda 3 fusion protein synthesized in Escherichia coli . Immunofluorescence microscopy showed that lambda 3 accumulated in perinuclear inclusion bodies in reovirus-infected cells . Analysis of lambda 3 accumulation in infected cells indicates that, once synthesized, lambda 3 is quite stable throughout the course of infection . Anti-lambda 3 serum did not immunoprecipitate virions, core particles or iodinated surface proteins of either virions or cores . These results indicate that lambda 3 is located in the inner part of the core . Experiments involving urea denaturation of purified reovirus cores indicate that lambda 3 cannot be selectively removed from the core without total denaturation of the core structure . When the dsRNA genome was eliminated from the core, lambda 3 remained associated with the other viral proteins in the core . Thus, lambda 3 appears to be a stable, structural component of the reovirus core, not bound to genomic dsRNA or free in soluble form inside the core. Mutagenesis, 1994 Sep, 9(5), 445 - 9 On the mechanisms of genotoxicity and metabolism of quercetin; Gaspar J et al.; Quercetin has been the subject of numerous studies on its genetic toxicity and carcinogenicity . Despite its well-proven genetic damaging activity for various genetic end-points (reverse mutations, induction of SOS functions, induction of sister chromatid exchanges, chromosomal aberrations and micronuclei), the mechanisms of genetic damage by quercetin remain, by and large, unknown . The present study aims to further extend the observations on the possible active oxygen species mediated DNA-damaging activity of quercetin and the role of cytochrome P450-dependent metabolism on the genotoxicity of quercetin . The results reported in this work show that quercetin can produce the OH . radical, as assessed by deoxyribose degradation in the presence of Fe3+/EDTA (ethylenediaminetetraacetic acid), and that it induces strand breakage in isolated plasmidic DNA (pUC18) . The data support the hypothesis that the production of OH . is mediated by H2O2 . The results with genetically engineered V79 cells expressing rat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolism of quercetin, as indicated by the fact that neither an enhancement nor a decrease in the genotoxicity of quercetin was observed . Results obtained on the pH dependence of the induction of chromosomal aberrations by quercetin in V79 cells show that, as the pH value of the medium is increased to 8.0, there is a significant increase in the number of aberrant cells, as expected if oxygen radicals are responsible for the formation of chromosomal aberrations. Mutagenesis, 1994 Sep, 9(5), 407 - 10 The induction of adaptive response to alkylating agents in Escherichia coli reduces the frequency of specific C-->T mutations in chloroacetaldehyde-treated M13 glyU phage; Borys E et al.; The mutagenicity and repair of cytosine adducts formed in reactions of chloroacetaldehyde (CAA), a metabolite of the human carcinogen vinyl chloride, have been studied . The treatment of single-stranded DNA M13 JCM15472 (glyU313) phage with CAA and subsequent transfection of Escherichia coli K-12 JC15419 (trpA461) tester strain resulted in a dose-dependent increase of phage C-->T transitions and a decrease of phage survival . The induction of the adaptive response to alkylating agents in bacterial cells significantly decreased the frequency of examined C-->T transitions and increased phage survival . The results indicate that both CAA adducts to cytosine, the initially formed 3,N4-(N4-alpha-hydroxyethano)cytosine and the product of its dehydration, 3,N4-ethenocytosine, provoke C-->T transitions and are repaired in adapted bacteria . The role of 3-methyladenine-DNA glycosylase II, which is a part of the adaptive response system in E . coli, in excision of CAA adducts to cytosine, is discussed. J Biochem Biophys Methods, 1994 Sep, 29(2), 113 - 21 PCR directed preparation and single step purification of highly active histidine-tagged restriction endonuclease HgiBI (GGWCC); Blum E et al.; The polymerase-chain-reaction technique is used to produce fusion proteins via deletion of any intervening piece of DNA . Here a stretch of six histidine codons is fused to the 3'-terminus of any defined gene using a standard plasmid vector or a derivative thereof . The advantage over existing methods is that no other amino acids besides the six histidines are added to the protein terminus and only one oligonucleotide needs to be synthesized as special primer . Genes of interest must only be cloned in the correct orientation into a universal multilinker . Using just one specific primer derived from the 3'-terminus of the gene and one standard primer derived from the six histidine codons the fusion is performed by amplifying the entire vector system as described for inverse PCR . As an example, we report on the modification and purification of the restriction endonuclease HgiBI (GGWCC) . Enzymatically active protein was obtained in a single step purification under nondenaturating conditions with a purity greater than 95% according to polyacrylamide gel electrophoresis. J Appl Physiol, 1994 Sep, 77(3), 1333 - 40 Effects of ONO-5046, a specific neutrophil elastase inhibitor, on endotoxin-induced lung injury in sheep; Kubo K et al.; The purpose of the present study was to assess the role of polymorphonuclear leukocyte (neutrophil) elastase in endotoxin-induced acute lung injury in sheep with lung lymph fistula . We studied the effects of ONO-5046, a specific inhibitor of neutrophil elastase, on the lung dysfunction induced by the intravenous infusion of 1 microgram/kg of Escherichia coli endotoxin . Endotoxin alone produced a biphasic response as previously reported . Early (0.5-1 h) after endotoxin, pulmonary arterial pressure increased from 19.5 +/- 0.9 cmH2O at baseline to a peak of 46.8 +/- 2.4 cmH2O (P < 0.05) . Pulmonary vascular resistance increased from 3.03 +/- 0.17 cmH2O.l-1.min at baseline to a peak of 9.77 +/- 0.70 cmH2O.l-1.min (P < 0.05) . Circulating neutrophils decreased from 7,355 +/- 434/mm3 at baseline to a nadir of 1,762 +/- 32/mm3 (P < 0.05) . Thromboxane B2 and 6-ketoprostaglandin F1 alpha concentrations in plasma and lung lymph were significantly increased . Late (3-5 h) after endotoxin, pulmonary arterial pressure and pulmonary vascular resistance returned to baseline levels, but lung lymph flow remained increased from 4.2 +/- 0.3 ml/0.5 h at baseline to 7.3 +/- 0.7 ml/0.5 h (P < 0.05), with a slight increase in lung lymph-to-plasma protein concentration ratio, suggesting increased pulmonary vascular permeability . The histopathological features of the lungs during the early period in sheep treated with endotoxin alone revealed a large increase in neutrophils per 100 alveoli and changes of pulmonary edema such as thickening of the interstitium of the lung and alveolar flooding.(ABSTRACT TRUNCATED AT 250 WORDS) J Appl Physiol, 1994 Sep, 77(3), 1093 - 100 Pathological O2 supply dependence of diaphragmatic and systemic O2 uptake during endotoxemia; Kim WS et al.; Our aim was to assess whether endotoxemia impairs the ability of the diaphragm to extract O2 and whether this defect leads to a greater dependence of O2 uptake on O2 delivery . In two groups of anesthetized mechanically ventilated dogs, the left hemidiaphragm was vascularly isolated . Diaphragmatic blood flow and cardiac output (CO) were measured simultaneously in all animals . Saline (S group) or Escherichia coli endotoxin (100 mg; E group) was infused intravenously over 60 min . In both groups, CO was reduced in stages by controlled hemorrhage, and systemic and diaphragmatic O2 deliveries and consumptions were measured at each stage to construct the O2 delivery-O2 consumption relationships . In the S group, the average systemic O2 delivery below which O2 uptake became supply dependent was 7.2 ml.kg-1.min-1 . At this O2 delivery, systemic O2 extraction ratio (ER) averaged 67.9%, whereas the maximum O2 ER was 91.3% . Critical diaphragmatic O2 delivery and critical and maximum diaphragmatic O2 ER, by comparison, averaged 9.0 ml.kg-1.min-1, 65%, and 81.9%, respectively . Endotoxin infusion raised critical systemic O2 delivery to 16.7 ml.kg-1.min-1 (P < 0.05) and reduced critical and maximum systemic O2 ER to 55.5 and 77% (P < 0.05), respectively . Similarly, critical diaphragmatic O2 delivery in the E group increased to 14.8 ml.kg-1.min-1 (P < 0.05), whereas critical and maximum O2 ER declined to 51.8 and 72.8%, respectively (P < 0.05) . Thus, endotoxemia impairs diaphragmatic O2 extraction . This, in turn, leads to a greater dependence of diaphragmatic O2 uptake on O2 delivery. Protein Sci, 1994 Sep, 3(9), 1504 - 14 Anatomy of an engineered NAD-binding site; Mittl PR et al.; The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43) . In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%) . A comparison of these structures reveals subtle differences that explain details of the specificity change . In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker . The mutations cause a contraction of the local chain fold . Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme . A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant . Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. Protein Sci, 1994 Sep, 3(9), 1401 - 8 Equilibrium unfolding of Escherichia coli ribonuclease H: characterization of a partially folded state; Dabora JM et al.; We have examined the equilibrium unfolding of Escherichia coli ribonuclease HI (RNase H), a member of a family of enzymes that cleaves RNA from RNA:DNA hybrids . A completely synthetic gene was constructed that expresses a variant of the wild-type sequence with all 3 cysteines replaced with alanine . The resulting recombinant protein is active and folds reversibly . Denaturation studies monitored by circular dichroism and tryptophan fluorescence yield coincident curves that suggest the equilibrium unfolding reaction is a 2-state process . Acid denaturation, however, reveals a cooperative transition at approximately pH 1.8 to a partially folded state . This acid state can be further denatured in a reversible manner by the addition of heat or urea as monitored by either CD or tryptophan fluorescence . Analytical ultracentrifugation studies indicate that the acid state of RNase H is both compact and monomeric . Although compact, the acid state does not resemble the native protein: the acid state displays a near-UV CD spectrum similar to the unfolded state and binds to and enhances the fluorescence of the dye 1-anilinonaphthalene, 8-sulfonate much more than either the native or unfolded states . Therefore, the acid state of E . coli RNase H has the characteristics of a molten globule: it retains a high degree of secondary structure, remains compact, yet does not appear to contain a tightly packed core. Protein Sci, 1994 Sep, 3(9), 1392 - 400 Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions; Trinkl S et al.; beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide . Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers . It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association . In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants . In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L) . In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC) . The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation . The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly . We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers. Protein Sci, 1994 Sep, 3(9), 1383 - 91 Subunit interface mutants of rabbit muscle aldolase form active dimers; Beernink PT et al.; We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure . A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627) . The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A . To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val . Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays . All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties . In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants . In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C . Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active . Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme . The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. Cytokine, 1994 Sep, 6(5), 500 - 3 Cyclooxygenase inhibitors enhance tumour necrosis factor production and mortality in murine endotoxic shock; Pettipher ER et al.; Intraperitoneal administration of E . coli lipopolysaccharide (5-15 mg/kg) produced a dose-related increase in mortality which was maximal 72 h after induction of shock . Using a suboptimal dose of LPS (10 mg/kg i.p.), pretreatment with indomethacin (0.1-10 mg/kg p.o) or ibuprofen (1-100 mg/kg p.o) 30 min prior to induction of shock led to a significant enhancement of mortality . This enhancement was associated with a 2-3 fold increase in the peak circulating levels of tumour necrosis factor (TNF-alpha) in the serum of animals treated with indomethacin or ibuprofen compared to vehicle-treated control animals . These results indicate that TNF-alpha production is modulated by endogenous prostaglandins in vivo and that enhanced production of TNF-alpha by cyclooxygenase inhibitors may lead to exacerbation of some inflammatory processes. J Virol Methods, 1994 Sep, 49(2), 195 - 208 Quantitation of human cytomegalovirus DNA in leukocytes by end-point titration and duplex polymerase chain reaction; Kulski JK; The presence of human cytomegalovirus (CMV) DNA and cellular DNA in leukocytes was detected by duplex polymerase chain reaction (PCR) and quantitated by end-point titration . Two different duplex PCR methods were used to co-amplify CMV DNA and a 536 bp fragment of globin DNA . MIE-globin PCR amplified a 435 bp fragment of the major immediate early (MIE) gene of CMV DNA whereas the LA-globin PCR amplified a 200 bp fragment of the late antigen (LA) gene of CMV DNA . PCR products were separated by electrophoresis in 3% agarose gels and detected by ethidium bromide staining . Amplification of globin DNA was included in the PCR as a positive control to monitor the accuracy and reproducibility of the PCR assay and to provide a reference point for CMV DNA levels . End-point titration PCR using known amounts of recombinant CMV DNA and human placental DNA showed that the end-point titres of the amplified CMV DNA correlated directly with the amount of CMV DNA in the sample . The limit of detection of MIE-globin and LA-globin PCR was 1 ng for placental DNA, and 10 fg (1000 copies) for CMV-MIE DNA and 1 fg (100 copies) for CMV-LA DNA, respectively . The amount of CMV DNA was quantitated in leukocytic lysates of 16 immunocompromised patients, who were tested for the presence of CMV in blood by cell culture, and of four normal controls . The blood concentration of CMV DNA, calculated as the number of copies of CMV DNA per microgram of leukocyte DNA, varied between 10(4) and 10(7) in the seven bloods that were CMV-cell-culture-positive, and between 10(2) and 10(4) in the blood of five patients that were CMV-cell-culture-negative . CMV DNA was undetected by PCR in the blood of another eight CMV-negative cases . This study shows that end-point titration and duplex PCR can be used as a simple and rapid method to quantitate CMV DNA in blood of patients that are either CMV-positive or CMV-negative by cell culture . Quantitation of CMV DNA in blood by end-point titration PCR has potential to differentiate between asymptomatic CMV infection and symptomatic CMV disease, and to monitor viral load during viral therapy. Int J Pept Protein Res, 1994 Sep, 44(3), 278 - 87 Structural and mechanistic implications of incorporating naturally occurring aberrant mutations of human dihydropteridine reductase into a rat model; Varughese KI et al.; Phenylketonuria (PKU) is a debilitating hereditary disorder related to an individual's inability to convert phenylalanine to its usual tyrosine product . The genetic errors occur in three regions: in the cooperative enzymes phenylalanine hydroxylase (PAH) and dihydropteridine reductase (DHPR), and in the biosynthetic pathway from GTP to the hydroxylation cofactor, tetrahydrobiopterin (BH4) . Many instances of naturally occurring defects in DHPR metabolism have been identified, and in most cases the error has been equated with an altered enzyme gene sequence . Using computer graphics, this report analyses the altered structural characteristics of eight of the enzymes encoded by mutant gene sequence and provides logical explanations for their diminished enzyme activities . In one instance, that of a threonine insertion, a mutant construct of the rat analog has been expressed in Escherichia coli and the DHPR isolated and characterised, confirming the marked changes this insert can create. Res Vet Sci, 1994 Sep, 57(2), 225 - 32 Application of monoclonal antibody-based sandwich ELISAs to detect verotoxins in cattle faeces; Ball HJ et al.; b1p4wich ELISAs, which use monoclonal antibodies for both the capture and the completion stages, were developed for the detection of verotoxins I and II (non-variant) . The ELISAs were used to investigate the incidence of verotoxins in 304 samples of bovine faeces collected from cases of enteritis, and 113 samples collected from normal animals . For most samples, the tests were carried out directly on faeces, and on colony sweeps which were taken from cultures grown from the faeces . These colonies were either grown directly from faeces (mixed colony sweep) or from a coliform culture purified from this (purified colony sweep) . All the positive ELISA reactions were investigated by Vero cell cytotoxicity assays for the confirmation of verotoxins . The largest numbers of ELISA-positive reactions which were confirmed by cytotoxicity assays were obtained with mixed colony sweeps . Verotoxin activity was confirmed in 74 (24 per cent) of the samples from cases of enteritis and from 35 (31 per cent) of the normal faeces samples . The occurrence of ELISA reactions unconfirmed by cytotoxicity assays and high cytotoxicity levels with low ELISA readings indicated the presence of non-toxic verotoxin epitopes and the presence of toxic verotoxin variants in the samples. Mol Microbiol, 1994 Sep, 13(5), 887 - 96 Specific initiation of transcription at a cyanobacterial promoter with RNA polymerase purified from Calothrix sp . PCC 7601; Schyns G et al.; Although in cyanobacteria many genes have been shown to be transcriptionally controlled by specific stimuli, little is known about promoter structure and the form of RNA polymerase that recognizes individual promoters . RNA polymerase holoenzyme has been purified from Calothrix sp . PCC 7601 . Its polypeptide composition resembles that of the plant chloroplast enzymes . To study transcription in cyanobacteria further, we have analysed the promoter-recognition properties of the purified enzyme . In vitro transcription was assayed with the promoter of the phycocyanin gene (cpc1) that is expressed whatever the incident light conditions . Transcription initiation at the same start point as in vivo was obtained with the Calothrix sp . PCC 7601 purified enzyme and the Escherichia coli core enzyme supplemented with a Calothrix sp . PCC 7601 sigma factor, but not with the E . coli holoenzyme. Indian J Exp Biol, 1994 Sep, 32(9), 672 - 3 Production of ampicillin through Escherichia coli NCIM 2563 immobilized and cross-linked in alginate beads; Deshpande JV et al.; 6-Amino penicillanic acid (6-APA) was condensed with D-alpha-phenyl glycine chloride in presence of E . coli NCIM 2563 to form ampicillin . At pH 5, 30% of 6-APA was converted to ampicillin during 1 hr incubation . When E . coli cells were immobilized in calcium alginate beads, the activity remained unaffected even after six batches of bioconversion . Inclusion of glutaraldehyde as multifunctional cross-linking agent, improved the stability of the biocatalyst beads. Biophys J, 1994 Sep, 67(3), 1192 - 202 Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans; Schlyer BD et al.; The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH) . Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision . Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets . The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane) . Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein . Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions . However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety . Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers . The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation . This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments . With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s) . Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states . The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed. Biologicals, 1994 Sep, 22(3), 243 - 8 Increased levels of active pertussis toxin may aid a pertussis vaccine to pass the mouse body weight gain test; Horiuchi Y et al.; The mouse weight gain test was evaluated for its value in toxicity testing for pertussis vaccines . When the reference whole cell pertussis vaccine was tested at dilutions of 1 in 1 and 1 in 16, the mice which received the 1 in 1 dilution achieved the greatest weight gain by the seventh day of injection, although they experienced a more significant weight loss during the first 24 hours than both the normal control mice or those that received the 1 in 16 dilution . A commercial diphtheria-tetanus-acellular pertussis vaccine with an increased level of pertussis toxin activity significantly accelerated the weight gain of mice . The effect was lost by heating the vaccine at 80 degrees C for 2 hours . A 1 micrograms dose of endotoxin induced a significant weight loss in mice during the first 24 hours of injection followed by weight gain at the same rate as that of the normal control . Pertussis toxin accelerated the weight gain of mice at a dose of 2 micrograms to a level exceeding that of the normal control mice throughout the observation period of 11 days . Pertussis toxin, when inoculated with endotoxin, showed a marked effect of helping mice to recover quickly from the endotoxin-induced initial weight loss to the level of those receiving only pertussis toxin . The effect of pertussis toxin on the acceleration of the weight gain of mice showed the possibility of inappropriate interpretation of the test results . It suggests, therefore, the necessity for separate quantitative tests for controlling the vaccine's toxicities. Antimicrob Agents Chemother, 1994 Sep, 38(9), 1966 - 73 Mechanism of inhibition of DNA gyrase by cyclothialidine, a novel DNA gyrase inhibitor; Nakada N et al.; We investigated how cyclothialidine (Ro 09-1437), a novel DNA gyrase inhibitor belonging to a new chemical class of compounds, acts to inhibit Escherichia coli DNA gyrase . Cyclothialidine up to 100 micrograms/ml showed no effect on DNA gyrase when linear DNA was used as a substrate . Under the same conditions, quinolones, which inhibit the resealing reaction of DNA gyrase, caused a decrease in the amount of linear DNA used . No effect of cyclothialidine was observed on the accumulation of the covalent complex of DNA and the A subunit of DNA gyrase induced by ofloxacin in the absence of ATP . The effect of cyclothialidine on the DNA supercoiling reaction was antagonized by ATP, reducing the inhibitory activity 11-fold as the ATP concentration was increased from 0.5 to 5 mM . Cyclothialidine competitively inhibited the ATPase activity of DNA gyrase (Ki = 6 nM) . The binding of {14C}benzoyl-cyclothialidine to E . coli gyrase was inhibited by ATP and novobiocin, but not by ofloxacin . These results suggest that cyclothialidine acts by interfering with the ATPase activity of the B subunit of DNA gyrase . Cyclothialidine was active against a DNA gyrase resistant to novobiocin, suggesting that its precise site of action might be different from that of novobiocin. Anal Biochem, 1994 Sep, 221(2), 387 - 91 Nonradioactive receptor binding assay for ciliary neurotrophic factor; Saggio I et al.; A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described . The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag . Bound cytokine is revealed by alkaline phosphatase-conjugated avidin . Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively . Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution . Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis . The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF . The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems . The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants. Brain Res Mol Brain Res, 1994 Sep, 25(3-4), 359 - 63 Gene transfer and the expression of a foreign gene in vivo in post-mitotic neurons of the adult rat brain using the hemagglutinating virus of the Japan-liposome method; Kato K et al.; Neurons in the adult rat brain were transfected in vivo with a simple plasmid that harbored the gene for beta-galactosidase from Escherichia coli under control of a chicken beta-actin promoter by use of the hemagglutinating virus of Japan (HVJ) and liposomes . Cells that expressed beta-galactosidase were detected only in the target area of the central nervous system for 10 days by light microscopic analysis . Since electron microscopic analysis revealed that the products of the histochemical reaction were predominantly associated with the nuclear membrane and the endoplasmic reticulum of positive cells, it appeared that the products were translated endogenously and had not been entrapped by endocytosis . Furthermore, the products were observed in typical neuronal cells with a large, round, and pale nuclei, and with direct axo-somatic and axo-dendritic synaptic contacts . This report suggests the possibility of introducing functionally significant genes into neurons in targeted areas of the adult central nervous system. Am J Vet Res, 1994 Sep, 55(9), 1213 - 9 Evaluation of an enzyme-linked immunosorbent assay that uses the 41-kd flagellin as the antigen for detection of antibodies to Borrelia burgdorferi in cattle; Ji B et al.; An ELISA was developed to detect antibodies to the 41-kd flagellin (P41) of Borrelia burgdorferi in serum obtained from cattle . Absorption studies, immunoblot analysis, immunoelectron microscopy, and correlation of results of the P41-ELISA and the P39-ELISA as well as measurement of the antibody to P41 in calves challenge-exposed with Borrelia theileri were used to assess the specificity of the P41-ELISA . Antigens derived from Escherichia coli, Leptospira interrogans serovar hardjo, and B burgdorferi were used for absorption studies and immunoblot analysis . Antibodies to P41 of B burgdorferi cross-reacted with antigens of E coli, but were not cross-reactive with L hardjo . A value 3 SD higher than the mean of the negative-control population of cattle was defined as the minimum value (cutoff value) for a positive result by the P41-ELISA . Use of this value for classification of test results reduced the predicted rate of false-positive results attributable to E coli cross-reactivity to 1% . Immunoblot analysis revealed that test-positive serum from cattle reacted mainly with 41-, 39-, 34-, and 31-kd proteins of B burgdorferi, as well as several smaller proteins . Immunoelectron microscopy revealed that serum from cattle that was test-positive by the P41-ELISA bound to the flagellin and outer membrane of B burgdorferi . Results of absorption studies, immunoblot analysis, and immunoelectron microscopy were correlated and indicated that serum from cattle that was test-positive by P41-ELISA had stronger reactivity to B burgdorferi antigens than to antigens of E coli or L hardjo.(ABSTRACT TRUNCATED AT 250 WORDS) Poult Sci, 1994 Sep, 73(9), 1381 - 9 Blood clearance of Escherichia coli and evaluation of mononuclear-phagocytic system as influenced by supplemental dietary zinc methionine in young turkeys; Kidd MT et al.; The influence of diets containing Zn-Met on in vitro and in vivo uptake of Escherichia coli by the mononuclear-phagocytic system was evaluated . Female Nicholas turkeys reared in battery brooders were supplemented with 40 micrograms Zn/g as Zn-Met in a corn soybean meal diet from 1 to 3 wk of age . Chemical analysis of the basal diets indicated that the basal diets contained 130 micrograms Zn/g and the Zn-Met diets contained 165 micrograms Zn/g . Each diet was fed to three replicate pens of 8 birds in Experiment 1 and three pens of 16 birds in Experiment 2 . Body weight gain, feed conversion (FC), and clearance of injected E . coli from blood were determined in Experiments 1 and 2 . Abdominal exudate cells (AEC) were recruited by intra-abdominal Sephadex injection . Substrate adherence potential and incidence of macrophages in AEC, phagocytosis of E . coli in vitro in terms of percentage phagocytic macrophages, and number of internalized E . coli per phagocytic macrophage, were quantified in Experiment 1 . Plasma Zn concentrations and plasma alkaline phosphatase activity (ALKP) were determined in Experiment 2 . Supplemental Zn-Met improved 3-wk BW gain (P < or = .003) only in Experiment 2 . Dietary Zn-Met increased mean adherence of cells by 69% (P < or = .001) . The number of phagocytized E . coli per macrophage did not differ significantly between treatments; however, E . coli clearance from blood was significantly improved in poults receiving Zn-Met in Experiment 2 . Plasma Zn was higher in poults supplemented with Zn-Met prior to and after E . coli administration (P < or = .02).(ABSTRACT TRUNCATED AT 250 WORDS) J Rheumatol, 1994 Sep, 21(9), 1731 - 3 Repair characteristics of the articular cartilage surface following acute inflammatory arthritis; Noyori K et al.; OBJECTIVE . To investigate the repair characteristics of the surface protein in an acute arthritis of short duration . METHODS . Knee arthritis in rabbits was induced by intraarticular (ia) injection of 20 micrograms E . coli endotoxin into one knee of 12 rabbits . The contralateral knee served as control . Four animals were killed 3, 6, and 10 days after ia injection . The articular cartilage surface was probed by quantitation of anticollagen type II (anti-CII) antibody binding . The suprapatellar bursae were washed, and the recovered cells counted . RESULTS . A significant increase in anti-CII antibody binding compared to control was measured 3 days after ia injection, coinciding with evidence of acute arthritis (injected joint: 1.1 x 10(7), control: 4.2 x 10(4) cells/joint; percent increase in anti-CII binding: 36.7 +/- 10.7; p < 0.02) . Six days after ia injection, the acute arthritis showed a major decrease in intensity whereas anti-CII binding was still abnormal (injected joint: 1.7 x 10(6) cells/joint; percent increase in anti-CII binding: 24.7 +/- 19.6; p < 0.05) . On Day 10, there was minimal evidence of arthritis in 3 of 4 rabbits, and anti-CII antibody binding returned to normal (injected joint: 1.9 x 10(5) cells/joint; percent anti-CII binding: -5.8 +/- 3.9) . There was a strong positive correlation between individual synovial fluid cell counts and the percent increase in anti-CII binding (R = 0.72, p < 0.02) . CONCLUSION . The magnitude of binding of anti-CII antibodies to the articular cartilage surface constitutes a sensitive probe for the detection of early damage following a transient inflammatory insult . Our studies indicate that after acute injury, the cartilage surface may show evidence of damage for at least 6 days when probed with anti-CII antibodies. J Med Virol, 1994 Sep, 44(1), 49 - 53 Hepatitis C virus antibody prevalence among human immunodeficiency virus-1-infected individuals: analysis with different test systems; Nubling CM et al.; Sera of 383 human immunodeficiency virus (HIV)-1-infected individuals from Frankfurt (Main)/Germany were assayed by two hepatitis C virus (HCV) screening tests (Abbott second generation, Ortho second generation) . This population showed a prevalence for reactivity with both tests of 20.8% (80/383) . Examination of all reactive sera (91/383) by a supplemental assay (Chiron RIBA 2) gave for 46 sera a positive, for 33 sera an indeterminate, and for 12 sera a negative result . Further analysis focussed on these RIBA 2-indeterminate and -negative samples . Analysis of the sera using an in-house Western blot with three different Escherichia coli-expressed HCV proteins revealed that none of the RIBA 2-negative, but 24 of the 33 RIBA 2-indeterminate sera, including 3 of 4 c33c (NS3)-reactive samples, were reactive with a recombinant core protein . Twenty-one of 22 c22-3 (core) indeterminates stained the core antigen in the in-house Western blot and 3 of them in addition a NS5 moiety . HCV-polymerase chain reaction (PCR) was positive for 14 of the 24 RIBA 2-indeterminate sera, but for none of the RIBA 2-negative or Western blot nonreactive samples . Discrepant results between the two screening tests could not be explained by differences in the antigen compositions (i.e., a NS3-NS4 moiety of 111 amino acids present in the Ortho enzyme-linked immunosorbent assay (ELISA), not present in the Abbott or RIBA 2 assays). Southeast Asian J Trop Med Public Health, 1994 Sep, 25(3), 490 - 3 Blastocystis hominis infection, a cause of human diarrhea; Sinniah B et al.; Blastocystis hominis has long been described as a non pathogenic protozoan parasite until recently when claims have been made that it can result in pathogenic conditions . Of the 729 stool samples (614 from survey and 115 from pediatric wards) examined, 18.1% of them were found to be positive for one or more intestinal protozoan cyst . The commonest was Giardia intestinalis (8.4%) Followed by Entamoeba coli (7.1%) and Entamoeba histolytica (5.1%) in the normal children without symptoms of diarrhea . When diarrheic stools were examined, the commonest parasite encountered was Giardia (20.4%), followed by E . coli (15.9%) and E . histolytica (9.7%) . Blastocystis was observed in 4.4% of the children who had diarrhea and 1.1% among the children taken from the normal population in the rural areas. Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1694 - 9 Metal affinity engineering of proinsulin carrying genetically attached (His)10-X-Met affinity tail and removal of the tag by cyanogen bromide; Ko JH et al.; An E . coli expression clone coding for human proinsulin, which was fused to NH2-terminal beta-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH2 terminal residue (methionine) of the human proinsulin gene . Short synthetic genes encoding oligopeptide residues including (Glu)n, (His)n, (Trp)n, and (Ser)n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion . Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation . The chelating peptide covering the NH2-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide. Biotechnol Prog, 1994 Sep-Oct, 10(5), 472 - 9 Production and characterization of a novel tissue-type plasminogen activator derivative in Escherichia coli; Saito Y et al.; We have created a novel thrombolytic agent by the combination of mutation with partial deletion of tissue-type plasminogen activator (t-PA) . We constructed Escherichia coli expression vectors for (i) native t-PA (nt-PA) and its derivatives; (ii) K1K2P, consisting of kringle 1 (K1), kringle 2 (K2), and protease (P) domains; (iii) K2P, consisting of K2 and P domains; (iv) D-nt-PA; (v) D-K1K2P; and (vi) D-K2P . The latter three are point mutants of nt-PA, K1K2P, and K2P, respectively, in which Arg275 (number corresponds to that of nt-PA) has been mutated to Asp . The production of nt-PA and its derivatives was remarkably improved by (i) removal of the 3' noncoding region of nt-PA cDNA from expression vectors and (ii) expression in mutant E . coli derived from E . coli HB101, which is insensitive to heat-shock inductions . The proteins produced were precipitated as insoluble aggregates in the cells and were renatured to active forms by extraction with 8 M urea followed by dialysis against a redox buffer containing GSH and GSSG . The renaturation yield depended on the pH of the buffer and the number of disulfide bonds of the proteins (nt-PA << K1K2P < K2P) . The mutation of Arg275 (the plasmin cleavage site) caused an increase in the catalytic enhancement by fibrin and a decrease of the interaction with plasminogen activator inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Prog, 1994 Sep-Oct, 10(5), 467 - 71 Effect of preinduction specific growth rate on secretion of granulocyte macrophage colony stimulating factor by Escherichia coli; Curless CE et al.; The effect of preinduction specific growth rate on the rate of synthesis and processing of granulocyte macrophage colony stimulating factor (GMCSF) secreted by Escherichia coli was investigated . A chemostat was used to explore preinduction growth rates ranging from 0.038 to 0.2/h . The maximum yields of both total GMCSF and processed GMCSF were found to occur at a preinduction growth rate of 0.13/h . It was also discovered that if the postinduction feed rate is reduced at a preinduction growth rate near 0.13/h, then the same amount of processed GMCSF is formed, but no unprocessed GMCSF is produced . It was hypothesized that the rate of synthesis of total GMCSF increases with an increased preinduction specific growth rate, but translocation across the cytoplasmic membrane and processing is rate-limiting . Increased degradation of GMCSF during induction at higher preinduction specific growth rates decreased the amount of GMCSF produced.
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