Escherichia coli

J Biol Chem, 1987 Apr 15, 262(11), 5404 - 7
Studies of the role of the Escherichia coli heat shock regulatory protein sigma 32 by the use of monoclonal antibodies; Lesley SA et al.; Purified Escherichia coli RNA polymerase containing the heat shock regulatory protein sigma 32 (gprpoH) was used to inject BALB/c mice, and the spleen cells of an immunized mouse were fused to NS1 cells . Three stable cell lines were isolated which produced monoclonal antibodies to sigma 32 . The antibodies varied in their ability to bind sigma 32 which was bound to core polymerase . Each of the antibodies was found to inhibit transcription from the rpoD heat shock promoter to a different extent . Extensive homology at the protein level has been observed between various sigma factors . The monoclonal antibodies to sigma 32 were tested for the ability to cross-react with sigma 70 on Western blots . None of the antibodies reacted with electroblotted sigma 70 . The sigma 32 content was examined in total cell extracts during heat shock Levels of sigma 32 were found to increase approximately 4-fold over pre-heat shock levels at five min after temperature upshift . These levels remained slightly elevated above pre-heat shock levels at 10 and 15 min after temperature upshift.

Eur J Biochem, 1987 Apr 15, 164(2), 389 - 96
Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance; Lane AN et al.; The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy . The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical . The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two-state transition . The circular dichroism at 222 nm decreases in two phases as urea is added . Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate . Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content . The second transition is the dissociation and unfolding of the partially unfolded dimer . At high concentrations of protein (500 microM) about 73% of the repressor exists as the intermediate in 4 M urea . The apparent dissociation constant is about 10(-4) M; the subunits are probably strongly stabilised by the subunit interaction . The native repressor is stable up to at least 70 degrees C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature approximately 60 degrees C, delta H approximately 230 kJ/mol).

J Biol Chem, 1987 Apr 15, 262(11), 5428 - 30
Crystallization of rat intestinal fatty acid binding protein . Preliminary X-ray data obtained from protein expressed in Escherichia coli; Sacchettini JC et al.; Rat intestinal fatty acid binding protein has been expressed in Escherichia coli, purified with bound long chain fatty acids and crystals grown from solutions of polyethylene glycol 4000 . The crystals are monoclinic, space group P2(1), a = 3638 A, b = 57.2 A, c = 31.9 A, and beta = 113.9 degrees . Each unit cell contains two monomers of this 132-residue, 15.1-kDa polypeptide . The crystals are remarkably resistant to x-ray damage . X-ray diffraction data have been observed to 2.0 A resolution . Platinum chloride was used to generate a potential isomorphous heavy atom derivative.

J Biol Chem, 1987 Apr 15, 262(11), 5170 - 9
Biosynthesis of lipid A precursors in Escherichia coli . A membrane-bound enzyme that transfers a palmitoyl residue from a glycerophospholipid to lipid X; Brozek KA et al.; Certain phosphatidylglycerol-deficient mutants of Escherichia coli accumulate two fatty acylated monosaccharides related to lipid A biosynthesis that have been identified as 2,3-diacylglucosamine 1-phosphate (lipid X) and triacylglucosamine 1-phosphate (lipid Y) (Raetz, C . R . H . (1984) Rev . Infect . Dis . 6, 463-472) . Lipid Y has the same structure as lipid X, except that it bears an additional palmitoyl moiety, esterified to the 3-OH of the N-linked R-3-hydroxymyristoyl residue . We now describe a membrane-associated system for the enzymatic conversion of lipid X to lipid Y . Removal of glycerophospholipids form such membranes by washing with cold ethanol abolishes the activity . The system can be reactivated by the addition of exogenous phospholipids dispersed as mixed micelles with Triton X-100 . When reconstituted in this manner, the formation of lipid Y is strictly dependent upon a glycerophospholipid donor bearing a palmitoyl residue in the sn-1 position . The enzyme system does not utilize palmitoyl coenzyme A or palmitoyl acyl carrier protein . It does not catalyze efficient transfer of fatty acids differing from palmitate by only one carbon atom . In contrast, the enzyme has relatively little specificity for the polar headgroup of the phospholipid donor, and it also appears to utilize a disaccharide precursor of lipid A as an alternative palmitoyl acceptor . Since the in vitro synthesis of lipid Y proceeds with a high yield, we have isolated the product and verified its structure by 1H NMR spectroscopy and mass spectrometry . The transesterification reaction that converts lipid X to lipid Y may be a model for the enzymatic synthesis of other acyloxyacyl structures, known to occur in mature lipid A.

J Biol Chem, 1987 Apr 15, 262(11), 5159 - 69
Biosynthesis of lipid A precursors in Escherichia coli . A cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine; Anderson MS et al.; Preliminary studies from our laboratory have suggested the existence of a novel set of fatty acyltransferases in extracts of Escherichia coli that attach two R-3-hydroxymyristoyl moieties to UDP-GlcNAc (Anderson, M.S., Bulawa, C.E., and Raetz, C.R.H . (1985) J . Biol . Chem . 260, 15536-15541) . The resulting "glucosamine-derived" phospholipids appear to be crucial precursors for the biosynthesis of the lipid A component of lipopolysaccharide . We now describe an assay and a 1000-fold purification of the first enzyme in this pathway, which catalyzes the reaction: UDP-GlcNAc + R-3-hydroxymyristoyl-acyl carrier protein----UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc + acyl carrier protein . The covalent structure of the monoacylated UDP-GlcNAc product was established by fast atom bombardment mass spectrometry and 1H-NMR spectroscopy . The UDP-GlcNAc acyltransferase has a strict requirement for R-3-hydroxymyristoyl-acyl carrier protein, since R-3-hydroxymyristoyl coenzyme A and myristoyl-acyl carrier protein are not substrates . Of various NDP-GlcNAc preparations examined, only the uridine and thymidine derivatives were utilized to a significant extent . When the product of the reaction (UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc) was isolated and reincubated with crude E . coli extracts, it was rapidly converted to more hydrophobic products in the presence of R-3-hydroxymyristoyl-acyl carrier protein . We propose that the addition of an R-3-hydroxymyristoyl residue to the 3 position of the GlcNAc moiety of UDP-GlcNAc is the first committed step in lipid A biosynthesis and that UDP-GlcNAc is situated at a biosynthetic branchpoint in E . coli leading either to lipid A or to peptidoglycan.

J Biol Chem, 1987 Apr 15, 262(11), 4943 - 6
Supercoiling facilitates lac operator-repressor-pseudooperator interactions; Whitson PA et al.; The binding affinity of the Escherichia coli lactose repressor to operator-containing plasmids was increased by negative supercoiling of the DNA . The increased affinities observed were dependent on the sequence context of the DNA as well as the degree of supercoiling . Dissociation rate constants for plasmids containing a single operator site decreased as a function of the negative supercoil density . However, the presence of pseudooperators in the plasmid DNA in addition to the primary operator sequence resulted in a significant decrease in the operator-plasmid dissociation rate at higher negative supercoil densities . Approximately eight ionic interactions were determined for both the supercoiled plasmids and the linear DNAs examined . These results suggest that the stabilization provided by the topology of supercoiled DNA affects the nonionic component of the protein-DNA interaction . The ability to form a ternary complex of protein with two DNA segments is increased by the presence of multiple operator-like sites on the DNA . Furthermore, supercoiling DNA with multiple operator-like sequences profoundly diminishes the dissociation rate and results in a remarkably stable ternary, presumably looped complex (t1/2 approximately 28 h) . These data suggest a critical role in vivo for DNA topology and pseudooperator(s) in transcriptional regulation of the lac operon.

Biochem J, 1987 Apr 15, 243(2), 345 - 50
The stimulation by salts of hexose phosphate uptake by Escherichia coli; Essenberg RC; Hexose phosphate uptake in Escherichia coli is stimulated by salts . KCl and MgCl2 stimulate to about the same extent, but Mg2+ is effective at a tenth the concentration of K+ . At higher concentrations, both salts inhibit . The stimulation by a series of salts correlates strongly with the hydrated radius of the cation, with small ions more effective than large . There are effects by the anion, but they do not correlate with any simple property . Cells accumulate glucose 6-phosphate to a higher concentration in the presence of KCl than in its absence . The maximum velocity of glucose 6-phosphate uptake is stimulated by KCl, as is the ratio V/Km.

J Biol Chem, 1987 Apr 15, 262(11), 5339 - 44
Mapping the active site tyrosine of Escherichia coli DNA gyrase; Horowitz DS et al.; We have identified tyrosine 122 of the A subunit of Escherichia coli DNA gyrase as the tyrosine that becomes covalently bound to DNA when the enzyme breaks the phosphodiester bonds of DNA . The covalent gyrase X DNA complex was isolated following cleavage of the DNA by gyrase in the presence of the gyrase inhibitor oxolinic acid . The active site tyrosine was first mapped to two overlapping peptides . Its precise position in the sequence of the A subunit of gyrase was then determined by sequencing of a peptide bound to DNA . We also present a method for mapping sites of DNA attachment in a protein of known amino acid sequence . The covalent complex of DNA and protein is treated with proteases that cut specifically . The electrophoretic mobilities of the resulting peptide-bound DNA molecules are correlated with the sizes of the bound peptides, allowing determination of the site of attachment of the DNA.

Biochem J, 1987 Apr 15, 243(2), 431 - 6
Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli; Roberts AN et al.; We report the development of a plasmid-mediated transformation system for Arthrobacter sp . NRRLB3381, using the Streptomyces cloning vector pIJ702 . Our procedure gives a transformation frequency of 10(3)/micrograms of plasmid DNA . In addition we have explored the expression of the Arthrobacter ermA gene in Streptomyces lividans and Escherichia coli, and shown that the ermA promoter is recognized in S . lividans not E . coli . The relationship between Arthrobacter, Streptomyces and E . coli promoters is discussed.

Biochem Biophys Res Commun, 1987 Apr 14, 144(1), 432 - 7
Dithiothreitol-induced oxidative damage to thymine and DNA in solution; Claycamp HG; Studies on dithiothreitol-induced oxidative damage to thymine and DNA in solution are reported . The major thymine products, cis- and trans-5,6-dihydroxy-5,6-dihydrothymine (thymine glycols), are produced rapidly in 37 degrees C neutral solutions of 10mM thymine and 10mM dithiothreitol . Iron-EDTA enhances while the iron chelator, diethylenetriaminepentaacetic acid, inhibits the reaction . In experiments using 3H-TdR-labeled Escherichia coli DNA, DNA damage was measured as increased ethanol-soluble radioactivity after treatment of the DNA with 5mM dithiothreitol at 45 degrees C . The findings are important with respect to current research interest in thiol radioprotection and thiol-plus-heat toxicity.

Biochem Biophys Res Commun, 1987 Apr 14, 144(1), 19 - 25
Insulin stimulates the phosphorylation level of v-Ha-ras protein in membrane fraction; Kamata T et al.; Insulin was found to stimulate the phosphorylation of the 21,000-dalton protein encoded by the ras oncogene of Harvey murine sarcoma virus in membrane fraction both in vivo and in vitro . When the human ras proteins expressed in E . coli were reconstituted with purified human insulin receptor, GTPase activity of normal or its mutated oncogenic ras protein was not stimulated by the addition of insulin . Likewise, tyrosine kinase activity or insulin binding capacity of the receptor was not influenced when assayed in the presence of the ras proteins . These results suggest that ras proteins may be coupled with the insulin receptor system through some unidentified membrane factors.

Nucleic Acids Res, 1987 Apr 10, 15(7), 3085 - 96
Translational regulation of the L11 ribosomal protein operon of Escherichia coli: mutations that define the target site for repression by L1; Thomas MS et al.; The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1 . The mRNA target site for this repression is located close to the Shine-Dalgarno sequence for the first cistron, rp1K (L11) . By use of a random mutagenesis procedure we have isolated and characterized a series of point mutations in the L11 leader mRNA which eliminate or greatly diminish the regulation by L1 . The mutations define a region essential for translational regulation upstream of the L11 Shine-Dalgarno sequence and identify a region of structural homology with the L1 binding site on 23S rRNA . These results are also consistent with the previously proposed model for the secondary structure of the L11 leader mRNA.

Nucleic Acids Res, 1987 Apr 10, 15(7), 3073 - 84
Nucleotide sequence of the Escherichia coli mutH gene; Grafstrom RH et al.; The complete nucleotide sequence of mutH gene from E . coli has been determined . Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein . Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene . Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced . One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).

Cell, 1987 Apr 10, 49(1), 111 - 9
The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins; Adams SE et al.; We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1 . This precursor protein is therefore functionally similar to the gag precursor of retroviruses . Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology . p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase . The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.

Nucleic Acids Res, 1987 Apr 10, 15(7), 2911 - 26
Rapid hybridization kinetics of DNA attached to submicron latex particles; Wolf SF et al.; We describe a novel method for attaching any DNA molecule to submicron latex beads and characterize the hybridization kinetic properties of these bead-DNA conjugates . The conjugates hybridize to DNA in solution with rates comparable to homogeneous hybridization reactions, are compatible with common hybridization conditions and are conveniently manipulated . They should thus serve as useful reagents for the fractionation and characterization of DNA and RNA.

Biochemistry, 1987 Apr 7, 26(7), 2047 - 54
Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu; Swart GW et al.; We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp . For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu . The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50 . For the first time we have succeeded in obtaining a tufB product in homogeneous form . Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster . Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA . However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome . Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs . 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+ . The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Apr 7, 26(7), 2039 - 46
Binding of IKe gene 5 protein to polynucleotides . Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins; de Jong EA et al.; Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other . The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides . At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer . The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT) . For most polynucleotides studied, the affinity depends linearly on the salt concentration: {d log (Kint omega)}/(d log {M+}) = -3 . The binding is highly cooperative . The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied . Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values . A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins . From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Apr 7, 26(7), 2001 - 9
Measurement of interstrand cross-link frequency and distance between interruptions in DNA exposed to 4,5',8-trimethylpsoralen and near-ultraviolet light; Matsuo N et al.; Bifunctional psoralens react photochemically with DNA to form single-strand adducts and interstrand, chemical cross-links . Cross-link formation is first order with {P}, the concentration of added psoralen, when {P} much less than Kd, the psoralen-DNA dissociation constant . DNA molecules containing interstrand cross-links are reversibly bihelical and so are readily detected . It was not heretofore possible to determine cross-link frequency in polydisperse DNA from the mass F of DNA spared cross-linkage . We have derived a statistical relation to calculate cross-link frequency at fixed light exposure and variable {P} . We show here that S, the initial slope of the curve described by -ln F as a function of {P}, is proportional to Mw, the weight-average molecular weight of nick-free DNA . The cross-link frequency at any {P} can be determined from k, a constant measured for DNA of known Mw at low cross-linkage . This relation is valid for DNA of any molecular weight distribution . In experiments with uniform length DNA, -ln F (cross-link frequency) increased in simple proportion to {P} . Intact and restriction endonuclease HindIII digested phage lambda DNA molecules have discrete lengths . S for each was proportional to Mw of the twin helix even though the molecular weight distribution of the restriction fragments was skewed . S was proportional to Mw and to the median molecular weight of sheared cellular DNA over a wide range . Also, we found that 1/S was linear with exposure of cellular DNA to gamma radiation . S can therefore be used to calculate L, the average distance between interruptions in the double helix.

Biochemistry, 1987 Apr 7, 26(7), 1987 - 95
Effect of drug-DNA interactions upon transcription initiation at the lac promoter; Straney DC et al.; We have examined the effects of six DNA binding drugs upon initiation at the lac UV5 promoter by Escherichia coli RNA polymerase . Experiments were directed at determining the influence of added drug on open complex formation, open complex stability, initiation from the open complex, and stability of the resulting initiated complex . The narrow groove binding drugs distamycin and 4',6-diamidino-2-phenylindole dihydrochloride were more effective in inhibiting initiation through their effect on the first three of these factors than were the intercalators ethidium bromide, daunomycin, and actinomycin . The bisintercalator bis(daunomycin) inhibited open complex formation better than its parent daunomycin . With the possible exception of actinomycin, the drugs tested were not able to disrupt preformed initiated complex, in contrast to their destabilizing effect upon the open complex . Combined with other results, the data suggest that the antitumor activity of daunomycin is unlikely to result from its effect on transcription . We compare the relative effectiveness of the drugs with the known physical properties of the corresponding drug-DNA interactions . The rate of open complex formation seems to be influenced by both the on and off rates of the drug, probably due to the relative slowness of open complex formation . This is in contrast to elongation, a much quicker process, which seems to be limited by the drug off rate alone; these considerations may possibly rationalize the difference in relative effect of particular drugs upon initiation and elongation . All drugs were able actively to disrupt open complex, although to substantially different extents; some possible mechanisms for this disruption, and the insensitivity of the initiated complex, are discussed.

Biochemistry, 1987 Apr 7, 26(7), 1940 - 8
Glucosamine synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location; Badet B et al.; L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli . A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate . Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations {Gosh, S., Blumenthal, H . J., Davidson, E., & Roseman, S . (1960) J . Biol . Chem . 235, 1265} . The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1 . The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E . coli glmS sequence {Walker, J . E., Gay, J., Saraste, M., & Eberle, N . (1984) Biochem . J . 224, 799} . Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue . The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide . Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate . In its presence, inactivation with {6-14C}DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit . From this behavior, a dimeric structure was tentatively assigned to the native enzyme . The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.

Biochemistry, 1987 Apr 7, 26(7), 1933 - 40
Covalent coupling of the variable loop of the elongator methionine tRNA to a specific lysine residue in Escherichia coli methionyl-tRNA synthetase; Leon O et al.; A lysine-reactive cross-linker has been coupled to the minor base 3-(3-amino-3-carboxypropyl)uridine in the variable loop of the Escherichia coli elongator methionine tRNA (tRNA(mMet} . Incubation of the derivatized tRNA with E . coli methionyl-tRNA synthetase (MetRS) resulted in covalent coupling of the protein and nucleic acid and loss of amino acid acceptor activity of the enzyme . One mole of tRNA was cross-linked per mole of enzyme inactivated . Enzyme activity was largely restored by release of the bound tRNA following cleavage of the disulfide bond in the cross-linker with a sulfhydryl reagent . The cross-linking reaction was effectively inhibited by unmodified tRNA(mMet) but not by noncognate tRNA(Phe) . The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were isolated by anion-exchange chromatography . The cross-linked peptides were released from the tRNA by cleavage in the disulfide bond of the cross-linker and purified by reverse-phase high-pressure liquid chromatography, yielding one major peptide plus several minor peptides . Amino acid analysis indicated that the major product was an octadecapeptide cross-linked to tRNA(mMet) through lysine residue 596 in the primary sequence of MetRS . The N-terminal sequence of the peptide was determined to be Val-Ala-Leu-Ile-Glu-Asn-Ala-Glu-Phe-Val, corresponding to residues 582-591 in MetRS . The procedures described here should be applicable to the determination of peptide sequences near the variable loop of other tRNAs containing the 3-(3-amino-3-carboxypropyl)uracil base when such tRNAs are bound to specific proteins.

Nature, 1987 Apr 30-May 6, 326(6116), 886 - 8
Effect of non-contacted bases on the affinity of 434 operator for 434 repressor and Cro; Koudelka GB et al.; The repressor of phage 434 binds to six operator sites on the phage chromosome . A comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (Fig . 1b) . In contrast, the sequences of the inner four base pairs are highly variable . Crystallographic analysis of the repressor-operator complex shows that at each half-site, the 'recognition alpha-helix' of the repressor is positioned in the major groove such that it could contact the outermost five base pairs, but not the innermost two (Fig . 1a) . We show in this paper that the sequence of the central base pairs of the operator (two in each half-site) have a significant role in determining operator affinity for repressor, despite the evidence presented here and in the accompanying paper that these base pairs are not contacted by repressor . We also show that these central base pairs influence operator affinity for Cro, a second gene regulatory protein encoded by phage 434 . We discuss the likely structural basis for this evidently indirect, but sequence-dependent, effect of the central base pairs of the operator on its affinity for the two regulatory proteins.

J Biol Chem, 1987 Apr 5, 262(10), 4922 - 7
The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor; Marmorstein RQ et al.; We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor . By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor . We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor . The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.

J Biol Chem, 1987 Apr 5, 262(10), 4917 - 21
Crystals of the trp repressor-operator complex suitable for X-ray diffraction analysis; Joachimiak A et al.; Crystals of a simulated trp repressor-operator complex have been grown that are large enough and are sufficiently well ordered and durable to provide a high quality molecular image of this regulatory protein X DNA complex to better than 3-A resolution . The "operator" consists of a 2-fold rotationally symmetric 18-base pair duplex that is extended by a dT residue at both 5'-termini . This system exhibits extensive crystal polymorphism . The crystal form and diffraction properties are very sensitive to the length and terminal structure of the operator fragment, as well as the type and concentration of multivalent ions . When combined with the experience reported by others, our results do not support a consistent strategy for crystallization of protein X DNA complexes.

J Biol Chem, 1987 Apr 5, 262(10), 4508 - 15
An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli; Goff SA et al.; The lon gene product in Escherichia coli is an ATP-dependent protease (La) that plays an important role in the breakdown of abnormal proteins and certain normal polypeptides . Since transcription of the lon gene rises as part of the heat-shock response, we studied the physiological effects of increased levels of protease La . In cells carrying additional copies of the lon gene under the control of the lac or tac promoter, induction of the protease resulted in a rapid cessation of cell growth and in a loss of viability at stationary phase . Similarly, cells carrying a multicopy plasmid encoding the lon gene contained 2-5-fold more protease La and grew much more slowly than did control cells . In such cells, insertion sequences appeared spontaneously in the lon gene on the plasmid and prevented the excess protease production and allowed more rapid growth . The cells with increased content of protease La (due to the lon plasmid or induction of the lon gene) exhibited severalfold higher rates of degradation of abnormal proteins containing amino acid analogs and of incomplete polypeptides containing puromycin . Also, a beta-galactosidase fusion protein with enzymatic activity was relatively stable in control cells but unstable in the cells with high protease La content . In these cells, the overall degradation of normal proteins increased 2-fold, and certain cellular polypeptides appeared particularly sensitive to proteolysis . Thus, rates of protein degradation in vivo are limited in part by the cellular content of the ATP-dependent protease, and increases in transcription of the lon gene enhance proteolysis and can be deleterious to the cell.

J Biol Chem, 1987 Apr 5, 262(10), 4477 - 85
A multiple-component, ATP-dependent protease from Escherichia coli; Katayama-Fujimura Y et al.; A new ATP-dependent, casein-degrading proteolytic complex has been identified and partially purified from Escherichia coli . The proteolytic complex can be isolated from wild-type cells as well as from mutants in which the gene for the ATP-dependent Lon protease is deleted . The complex consists of at least two components (components I and II) that can be separated from each other (and from wild-type Lon protease) by phosphocellulose chromatography . Neither component has casein-degrading activity when added separately to assay solutions with or without ATP . Both components must be present simultaneously for casein degradation to occur . Of the nucleotides tested, only ATP activates the proteolytic complex, and the ATP must be present continuously for degradation to occur . Component II copurifies with an ATPase activity and binds to a Type 4 ATP affinity column . ATP protects component II from heat inactivation, suggesting that component II interacts with ATP . Proteolysis was not inhibited by any serine protease inhibitors but was inhibited by reagents such as the organomercurial Neohydrin and N-ethylmaleimide, which react with sulfhydryl groups . Our data provide convincing evidence that E . coli possesses a previously undescribed proteolytic system composed of at least two complementary components and absolutely dependent on ATP.

J Mol Biol, 1987 Apr 5, 194(3), 391 - 6
Statistical test for the comparison of samples from mutational spectra; Adams WT et al.; The Monte Carlo estimate of the p value of the hypergeometric test is described and advocated for the testing of the hypothesis that different treatments induce the same mutational spectrum . The hypergeometric test is a generalization of Fisher's "exact" test for tables with more than two rows and two columns . Use of the test is demonstrated by the analysis of data from the characterization of nonsense mutations in the lacI gene of Escherichia coli . Unlike the chi-square test, the hypergeometric test remains valid when applied to sparse cross-classification tables . The hypergeometric test has the most discrimination power of any statistical test that could be employed routinely to compare samples from mutational spectra . Direct application of the hypergeometric test to large cross-classification tables is excessively computation intensive, but estimation of its p value via Monte Carlo techniques is practical.

J Mol Biol, 1987 Apr 5, 194(3), 385 - 90
Influence of neighbouring base sequence on N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the lacI gene of Escherichia coli; Burns PA et al.; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations within the first 540 base-pairs of the lacI gene of Escherichia coli were cloned and sequenced . In total, 167 MNNG-induced independent mutations were characterized, with G.C to A.T transitions accounting for all but three of the mutations . This mutagenic specificity is consistent with the mispairing predicted by the methylation of the O6 position of guanine . The characterization of such large numbers of mutations permitted an analysis of the influence of local DNA sequence on mutagenesis . This analysis revealed a strong influence by the 5' flanking base . On average, guanine residues preceded (5') by a guanine or an adenine residue were, respectively, nine times and five times more likely to mutate after treatment with MNNG than those preceded by a pyrimidine residue.

J Mol Biol, 1987 Apr 5, 194(3), 359 - 83
The organization and sequence of the genes for ATP synthase subunits in the cyanobacterium Synechococcus 6301 . Support for an endosymbiotic origin of chloroplasts; Cozens AL et al.; The nucleotide sequence has been determined of two regions of DNA cloned from the cyanobacterium Synechococcus 6301 . The larger, 8890 base-pairs in length, contains a cluster of seven genes for subunits of ATP synthase . The order of the genes is a:c:b':b:delta:alpha:gamma, b' being a duplicated and diverged form of b . As in the Escherichia coli unc operon, the a gene is preceded by a gene for a small hydrophobic and basic protein . The hydrophobic profile of the potential gene product suggests that its secondary structure is similar to the uncI protein . The smaller DNA fragment, 4737 base-pairs in length, is separated from the larger by at least 15 X 10(3) base-pairs of DNA . It contains a cluster of two genes encoding ATP synthase subunits beta and epsilon . Both clusters of ATP synthase genes are preceded by sequences resembling the -10 (Pribnow) box of E . coli promoters and are followed by sequences able to form stable stem-loop structures that might serve to terminate transcription . These features and the small intergenic non-coding sequences suggest that the clusters are operons, for which the names atp1 and atp2 are proposed . The order of genes within the two clusters is very similar to the gene order in the E . coli unc operon . However, it is most closely related to the arrangement of genes for ATP synthetase subunits a:c:b:alpha and beta:epsilon in two clusters in pea chloroplast DNA . This close relationship between chloroplasts and the cyanobacterium is also evident from comparisons of the sequences of ATP synthase subunits; the Synechococcus proteins are much more closely related to chloroplast homologues than to those in other bacteria or in mitochondria . It is further supported by the cyanobacterial b and b' proteins which, in common with their chloroplast counterpart, subunit I, have extra amino-terminal extensions relative to the E . coli b protein . This extension is known to be removed by post-translational processing in the chloroplast, but its function is obscure . It also seems likely that the cyanobacterial and chloroplast ATP synthases have important similarities in subunit composition . For example, the presence of two related genes, b and b', in the cyanobacterium suggests that its ATP synthase is a complex of nine polypeptides, and that it may have single copies of related b and b' proteins rather than two copies of identical b subunits as found in the E . coli enzyme.4+off

J Biol Chem, 1987 Apr 5, 262(10), 4724 - 7
alpha, beta-Dihydroxyisovalerate dehydratase . A superoxide-sensitive enzyme; Kuo CF et al.; Increasing the intracellular flux of O-2 by incubating aerobic Escherichia coli with paraquat or plumbagin markedly lowered the alpha, beta-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells . This effect was not seen in the absence of dioxygen and was exacerbated by inhibiting protein biosynthesis with chloramphenicol . These effects of paraquat and of plumbagin were both time- and concentration-dependent . Transfer of E . coli from aerobic to anaerobic conditions caused a rebound of the dehydratase activity, in the continued presence of paraquat and of chloramphenicol, indicating the presence of a mechanism for reactivating this enzyme . The instability of the dehydratase activity in cell extracts was exacerbated by selective removal of superoxide dismutase, but not of catalase, by immunoprecipitation . Addition of exogenous superoxide dismutase reversed the effect of immunoprecipitation; whereas catalase or inactive superoxide dismutase were ineffective . We conclude that the dehydratase is inactivated by O-2 . This could account for the bacteriostatic effects of dioxygen and of paraquat.

J Biol Chem, 1987 Apr 5, 262(10), 4534 - 7
31P and 13C NMR studies of oxygen transfer during catalysis by 3-deoxy-D-manno-octulosonate cytidylyltransferase from Escherichia coli; Kohlbrenner WE et al.; {18O}3-Deoxy-D-manno-octulosonate (KDO), labeled at the anomeric oxygen, was prepared by exchange with {18O}H2O and used to follow the route of oxygen transfer during cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate (CMP-KDO) formation catalyzed by 3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase) . The 31P-NMR signal of the phosphoryl group of CMP-KDO (-5.85 ppm), which appeared as a single resonance when CMP-KDO formation took place with unenriched KDO, appeared as two peaks when CMP-KDO formation took place in the presence of a mixture of {16O}-and {18O}KDO . These results demonstrate the retention of 18O during CMP-KDO formation . Confirmation that the labeled oxygen in CMP-KDO was retained in the "bridge" position between CMP and KDO came from 13C-NMR studies of CMP-KDO formed in the presence of 90% {2-13C, 18O} KDO . The prominent C-2 KDO resonance in CMP-KDO, which is normally a doublet at 101.4 ppm (Kohlbrenner, W.E., and Fesik, S.W . (1985) J . Biol . Chem . 260, 14695-14700), appeared as four peaks when a mixture of {2-13C,16O}- and {2-13C, 18O}KDO was used, confirming the direct bonding of 18O to the C-2 of KDO in CMP-KDO . These results are consistent with a nucleophilic displacement mechanism for CMP-KDO formation.

J Immunol Methods, 1987 Apr 2, 98(1), 83 - 9
Antibody chimera technique applied to the detection of Escherichia coli heat-labile enterotoxin; Germani Y et al.; Covalently prepared chimera antibodies were tested in a ganglioside GM1 erythro-immunoassay (CERIA) for E . coli heat-labile enterotoxin (LT) detection . The antibody specific for LT was conjugated with a polyclonal antibody specific for sheep erythrocytes . The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 and subsequent erythro-adsorption via chimera antibody by which the bound toxin is visualized . Enterotoxin titers determined with this CERIA method were similar to those obtained with the Vero cell assay and with ELISA . 5 ng of cholera toxin/ml may be detected with the assay . The CERIA, as described, may be used either qualitatively or quantitatively and is well suited for routine laboratory diagnosis of LT in a culture supernatant of E . coli.

Biochim Biophys Acta, 1987 Apr 2, 928(1), 83 - 91
Regulation of intestinal mucosa guanylate cyclase by hemin, heme and protoporphyrin IX; elDeib MM et al.; Mg2+-dependent activity of intestinal brush border guanylate cyclase was stimulated 4-5-fold by 50-100 microM hemin . Higher concentrations were inhibitory . In the presence of 25% dimethyl sulfoxide, which stimulated activity 9-times, 50 microM hemin further increased activity 1.7-fold . However, when activity was stimulated 32-fold by the Escherichia coli heat-stable enterotoxin, or 26-fold by Lubrol PX, hemin produced only concentration-dependent inhibition . The first type of activation was more sensitive to hemin than the second . Reduction of hemin by dithiothreitol eliminated stimulation of basal activity, while inhibition of Lubrol PX-stimulated activity remained . Protoporphyrin IX also had no effect on basal activity, however, it inhibited enterotoxin- and Lubrol PX-stimulated activities similarly, but only to half the extent of hemin . Substitution of Mn2+ for Mg2+ elevated basal activity 15-fold, and this Mn2+-dependent activity was inhibited by hemin . Mn2+-dependent activity was stimulated (43%) by enterotoxin, however, the stimulated activity was more sensitive to hemin inhibition than the basal Mn2+-dependent activity and both inhibition curves were congruent above 50 microM hemin . Hemin inhibition of Lubrol PX-stimulated activity was much less with Mn2+ than with Mg2+ . These results were interpreted as suggesting two sites of hemin inhibition; on an inhibitory regulator and on the enzyme . We also found that the secretory effect of enterotoxin in the suckling mouse bioassay was reduced 56% by the oral administration of hemin.

Carcinogenesis, 1987 Apr, 8(4), 607 - 9
Base excision repair of DNA in gamma-irradiated human cells; Moran MF et al.; Escherichia coli endonuclease IV was used to incise cellular DNA specifically at apurinic/apyrimidinic (AP) sites prior to alkaline elution to measure the resulting DNA strand breaks . gamma-Irradiated HeLa cells initially contained DNA strand breaks and no AP sites . Upon incubation at 37 degrees C the strand breaks were rapidly repaired and AP sites were generated and subsequently repaired . The transient nature of the AP sites indicates the in vivo operation of a base excision repair pathway whereby damaged bases are removed from DNA by DNA glycosylases to produce AP intermediates that are then substrates for AP endonucleases.

Br J Haematol, 1987 Apr, 65(4), 489 - 93
Characteristics of a ferritin-binding protein present in human serum; Bellotti V et al.; The ferritin present in human serum differs from the ferritins found in tissues and other body fluids in having negligible proportions of H subunits . This has been related to the possible presence of binding factors which would form complexes with H-subunit containing ferritins and thereby determine their rapid clearance and/or interference with immunoassays ('serum inhibition') . In this work we have tried to identify and characterize these binding factors . Dotting and blotting experiments demonstrated an interaction between tissue ferritins and human serum . This was stronger with human heart and recombinant H-type ferritin obtained from E . coli than with human liver ferritin . The serum binder appeared to be a glycoprotein migrating in the beta-2 region and with a molecular weight of about 200,000 and pI between 4 and 5 . Two different approaches to purification of the ferritin-binding protein yielded enriched fractions containing also the complement proteins C3 and C4, the plasma protease inhibitor alpha-2-macroglobulin, and immunoglobulins . These in vitro findings may have physiological relevance.

DNA, 1987 Apr, 6(2), 139 - 47
Comparison of the DNA sequences involved in replication and packaging of the filamentous phages IKe and Ff (M13, fd, and f1); Peeters BP et al.; The product of gene II of the distantly related, filamentous, single-stranded DNA phages IKe and Ff (M13, fd, and f1) is the only phage-encoded protein that is required for the replication of their double-stranded replicative form DNA . With the aid of recombinant plasmids containing the origins of viral strand replication {(+)-origins} of both IKe and Ff, we demonstrated that initiation but not termination of viral strand replication by gene II protein is restricted to its cognate (+)-origin . If the (+)-origins of IKe and Ff are present in the same orientation, fusion origins are generated upon gene II protein-instructed replication as a result of initiation at one origin and termination at the other . These fusion origins are only functional in the presence of the gene II protein encoded by the phage from which the sequence lying at the 3' side of the gene II protein cleavage site is derived . The nucleotides that determine the specificity of the replication initiation process are located between positions +17 and +49, or +17 and +40, with respect to the gene II protein cleavage site of IKe and Ff, respectively . The DNA sequence that forms the recognition signal for cleavage by gene II protein is probably located within the sequence that starts 3 nucleotides before and terminates 17 nucleotides after the cleavage site . Efficient packaging by phage IKe of plasmid DNA strands that contain the morphogenetic signal of Ff, or vice versa, indicates that, despite their only partial homology, the morphogenetic signals of IKe and Ff are interchangeable.

Carbohydr Res, 1987 Apr 1, 161(2), 273 - 9
Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide; Jansson PE et al.; The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r . techniques being the main methods used . It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose . ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.

Arch Biochem Biophys, 1987 Apr, 254(1), 156 - 69
Isolation and properties of a capillary injury-related protease from lung lymph; Orlowski M et al.; Lung microvascular injury induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli caused the appearance in lung lymph of high levels of a protease with trypsin-like activity . The enzyme was isolated as an apparently homogeneous protein from pooled samples of active lung lymph, after an almost 9000-fold purification by affinity chromatography on columns of Reactive Blue 2-agarose, aprotinin-agarose, and p-aminobenzamidine-agarose, and chromatography on a column of Sephadex G-100 . A molecular weight of about 70,000 to 75,000 was determined from mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The pH optimum was between 7.3 and 7.6 . The isolated enzyme was quite labile, rapidly losing activity at both 37 and 25 degrees C . Addition of albumin to enzyme solutions protected against inactivation . Inhibition by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride indicated that the enzyme belongs to the class of serine proteases . The enzyme cleaved peptide bonds on the carboxyl side of arginine residues and showed a relatively high affinity toward peptides containing several basic amino acid residues . Bonds involving the carboxyl group of lysine were cleaved at a much slower rate . The enzyme showed no plasminogen activator activity and its substrate specificity was quite different from that of several proteases of the clotting cascade . Its appearance in lymph was not influenced by lymph clotting and the isolated enzyme was not capable of correcting the clotting defect of plasmas deficient in factors XII, XI, IX, VII, and X.

Exp Parasitol, 1987 Apr, 63(2), 166 - 72
Plasmodium falciparum: immunogenicity of circumsporozoite protein constructs produced in Escherichia coli; Wirtz RA et al.; The immunogenicity of Plasmodium falciparum recombinant circumsporozoite protein constructs R16tet32, R32tet32, and R48tet32 in mice was examined by measuring antibody responses by enzyme linked immunosorbent assay, immunofluorescence, circumsporozoite precipitation, and inhibition of sporozoite invasion . All three constructs were found to be immunogenic when administered alone, but antibody responses were greater for the larger constructs, R32tet32 and R48tet32 . Increased dose, boosting, and the use of adjuvants further augmented antibody responses . R32tet32 was found to be the most immunogenic of the three constructs, and high levels of protective antibodies were found to persist for at least 44 weeks when the construct was given with alum . Clinical trials with alum adjuvanted R32tet32 have now begun.

Cornell Vet, 1987 Apr, 77(2), 107 - 18
An induced synovitis disease model in ponies; Firth EC et al.; The effects of intra-articular injection of small amounts of E . coli lipopolysaccharide (LPS) into the intercarpal joint of 5 ponies were studied . The LPS induced predictable changes all of which were analogous to acute bacterial infection, except that the development of signs occurred sooner after the LPS injection, and subsided within 36 hours . Fever was monophasic and peaked at 5-7 hours . The ponies exhibited depression, reduced or absent appetite, increased pulse and respiration rates, and lameness . The lameness became evident between 1 and 2 hours after injection, at which time warmth, articular effusion, and resentment to palpation of joint flexion were evident . Hematological changes included neutrophilic leucocytosis, and changes in copper, iron and zinc serum concentrations . The synovial fluid total protein, leucocyte, and alkaline phosphatase levels increased within 2 hours . The mucin precipitation, total protein and leucocyte counts in synovial fluid remained elevated long after clinical and hematological changes had subsided . The model is useful for the study of some aspects of infectious joint disease.

Radiat Res, 1987 Apr, 110(1), 129 - 41
Mathematical description of the interactions between cellular inactivating agents; Ager DD et al.; A mathematical technique for characterizing the interactive effects that may occur when cells are treated with two or more toxic agents is developed . This technique is used to account for the previously unexplained properties of the dose-response relations for the uv-X-ray interaction in Escherichia coli B/r.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2484 - 8
In vivo stimulation of granulopoiesis by recombinant human granulocyte colony-stimulating factor; Cohen AM et al.; Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters . Within 3 days, peripheral granulocyte counts had increased greater than 10-fold with a concomitant 4-fold increase in total leukocytes . Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased . No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment . After subcutaneous injection at rhG-CSF doses of up to 10 micrograms X kg-1 X day-1 only granulocyte counts were affected . However, at higher dose levels, a transient thrombocytopenia was noted . Erythrocyte had lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 micrograms X kg-1 X day-1) studied . Total leukocyte counts increased 3-fold within 12 hr after a single s.c . injection of rhG-CSF . This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow . A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c . injections of rhG-CSF . The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.

Proc Natl Acad Sci U S A, 1987 Apr, 84(7), 1871 - 5
Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect; Zimmerman SB et al.; Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates . Reactions tested were nick-translation and gap-filling by DNA polymerase I of Escherichia coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase . For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength . The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA . We suggest that this effect on protein-DNA complexes is only one example of a general "metabolic buffering" action of crowded solutions on a variety of macromolecular interactions.

Proc Natl Acad Sci U S A, 1987 Apr, 84(7), 1759 - 63
Inhibition of replication forks exiting the terminus region of the Escherichia coli chromosome occurs at two loci separated by 5 min; de Massy B et al.; The replication cycle of Escherichia coli strains duplicating their chromosome from the same plasmid origin placed at various locations or of strains having undergone a major inversion event along the origin-to-terminus axis was studied by marker-frequency analysis . It was observed that replication forks are unidirectionally inhibited at two loci of the termination region: counterclockwise-moving forks are inhibited at terminator T1 (28.5 min), and forks moving in the opposite direction are inhibited at terminator T2 (33.5 min) . By determining the strand preference of Okazaki fragments that are specific for markers from the T1-T2 interval, it was shown that this interval is replicated in either direction, depending upon the strain analyzed . In addition, we also observed that forks moving in the "unnatural" direction along each oriC-T1 or -T2 arm are very slow, especially in the one-third portion of the chromosome around the terminators . We propose that this phenomenon is a consequence of nucleoid organization, which is proposed to be symmetrical on the two oriC-T1 or -T2 arms and polarized with respect to the direction of replication . We also propose that T1 and T2 are the terminal limits of these two polarized half-nucleoid bodies.

Proc Natl Acad Sci U S A, 1987 Apr, 84(7), 1754 - 8
The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication; Hill TM et al.; The terminus region of the chromosome of Escherichia coli contains two separate sites, called T1 and T2, that inhibit replication forks . T1 is located near 28.5 min, which is adjacent to trp, and T2 is located at 34.5-35.7 min on the opposite side of the terminus region, near manA . The sites act in a polar fashion, and replication forks traveling in a clockwise direction with respect to the genetic map are not inhibited as they pass through T1 but are inhibited at T2 . Similarly, counterclockwise forks are not inhibited at T2 but are inhibited at T1 . Consequently, forks are not inhibited until they have passed through the terminus region and are about to leave it . Studies with deletion strains have located T2 within a 58-kilobase interval, which corresponds to kilobase coordinates 387-445 on the physical map of the terminus region.

Mutat Res, 1987 Apr, 190(4), 237 - 40
The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide; Brandi G et al.; The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure . Two modes of killing were detected following a 15-min challenge with H2O2 under either aerated or anoxic conditions . Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM . Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic cells . Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H2O2 . Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations . We suggest that hydroxyl radicals mediate mode two but not mode one killing by H2O2.

Mutat Res, 1987 Apr, 182(2), 65 - 74
Development of a yeast system to assay mutational specificity; Pierce MK et al.; We have developed a system wherein DNA alterations occurring in a target gene in the yeast Saccharomyces cerevisiae can be determined by DNA sequencing . The target gene, SUP4-o, an ochre suppressor allele of a yeast tyrosine tRNA gene, has been inserted into a shuttle vector (YCpMP2) which is maintained in yeast at a copy number of one per cell Mutations in SUP4-o are selected by virtue of their inactivation of suppressor activity . Rapid DNA preparations from these mutants are used to transform an appropriate bacterial strain . Since YCpMP2 also carries the M13 phage replication origin, superinfection of bacterial cells containing the plasmid with wild-type M13 phage yields single stranded YCpMP2 DNA suitable for dideoxynucleotide chain termination sequencing . We have used this system to examine mutations arising spontaneously in the SUP4-o gene . The spontaneous mutants occurred at a frequency of 3.2 X 10(-6)/viable cell, corresponding to a rate of 2.7 X 10(-7) events/cell division . Following bacterial transformation, 16% of the recovered plasmids tested displayed altered gel mobility consistent with loss of significant portions of the plasmid . Hybridization analysis of total yeast DNA and use of purified YCpMP2 revealed that these very large deletions were not generated in yeast but were associated with bacterial transformation . Among the SUP4-o mutants analyzed by DNA sequencing, we identified each type of single base pair substitution (transitions and transversions), small deletions of varying length (1-32 base pairs) and more extensive deletions of undetermined size . These results demonstrate that the SUP4-o system can be used to detect various types of mutation at numerous sites in a single eukaryotic gene and to characterize the DNA sequence changes responsible for the mutations selected.

Mutat Res, 1987 Apr, 177(2), 189 - 99
Transversion-specific purine analogue mutagens and the mechanism of hydroxylaminopurine mutagenesis; Murray V; The tryptophan synthetase gene A series of mutants in E . coli has been used to examine the mutational specificity of over 80 purine base analogues . 4 purine analogues have been discovered that solely cause transversions . Evidence is presented that hydroxylaminopurine mutagenesis is caused by a covalent reaction of these compounds with DNA . The transversion-causing purine analogues are derivatives of 2-aminopurine (2AP) and 2,6-diaminopurine (2,6DAP) . They stimulate the full reversion frequency of those trp A which can revert through an AT----CG transversion . 8 purine base analogues have been found that induce the AT----CG transversion at the trp A88 site; and 2-amino-6-methylaminopurine (2A6MAP) stimulates by 124-fold, 2-amino-6-ethylaminopurine by 20-fold, 2-methylaminopurine (2MAP) by 9.4-fold, 2,6-bismethylaminopurine by 25-fold, 2AP by 230-fold, 2,6DAP by 15-fold, 2.6-diaminopurine riboside by 5-fold, and 2-hydroxylaminopurine by 11-fold . The last 4 analogues also cause transitions . 2A6MAP, 2-amino-6-ethylaminopurine and 2,6-bismethylaminopurine stimulate only the AT----CG transversion while 2MAP additionally gives rise to AT----TA transversions . By testing other negative 2AP derivatives, the structural requirements necessary for AT----CG transversion mutagenesis have been determined . All 12 hydroxylaminopurine base analogues tested, 2,6-dimethoxyaminopurine and 2-hydrazinopurine were found to cause transition mutations . All of the compounds stimulated the AT----GC transition (by up to 1000-fold) and 11 of the 14 base analogues raised the GT----AT transition (by up to 450-fold) . On increasing the hydroxylaminopurine concentration, the mutation frequency also increased concomitantly . Since 6-hydroxylamino-9-methylpurine and 6-methylhydroxylaminopurine cause transitions, the mechanism of hydroxylaminopurine mutagenesis cannot be entirely due to an alteration in tautomeric equilibria or "wobble" type base mispairing . It is proposed that a major mechanism for hydroxylaminopurine mutagenesis is due to the reaction of these compounds with the O6-position of guanine and the O4-position of thymine.

J Bacteriol, 1987 Apr, 169(4), 1760 - 2
Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens; Barros ME et al.; An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200 . The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper . Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction . Enzyme production was not subject to catabolite repression by glucose.

J Bacteriol, 1987 Apr, 169(4), 1753 - 6
Involvement of the phosphate regulon and the psiD locus in carbon-phosphorus lyase activity of Escherichia coli K-12; Wackett LP et al.; Escherichia coli K-12 can readily mutate to use methylphosphonic acid as the sole phosphorus source by a direct carbon-to-phosphorus (C-P) bond cleavage activity that releases methane and Pi . The in vivo C-P lyase activity is both physiologically and genetically regulated as a member of the phosphate regulon . Since psiD::lacZ(Mu d1) mutants cannot metabolize methylphosphonic acid, psiD may be the structural gene(s) for C-P lyase.

J Bacteriol, 1987 Apr, 169(4), 1740 - 4
Determination of the promoter strength of the gene encoding Escherichia coli heat-stable enterotoxin II; Spandau DF et al.; We studied the promoter strength of the gene encoding the Escherichia coli heat-stable enterotoxin II (STII) . The promoter region and a portion of the 5' coding sequence of the STII gene were fused to the lacZ gene so that the production of beta-galactosidase was under the control of the STII gene promoter . The strength of the STII gene promoter was compared with that of the ompF and lac operons, which were similarly fused to the lacZ gene . The beta-galactosidase produced by the hybrid genes was assayed in vitro by using cell extracts . The mRNA transcribed by each promoter was assayed by Northern blot analysis and by in vitro transcription . The results suggest that the STII gene is regulated by a relatively weak promoter.

J Bacteriol, 1987 Apr, 169(4), 1554 - 63
Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B; Bishai WR et al.; We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin . These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells . Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone . We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins . The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor . The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity . Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain . High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells . It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.

J Bacteriol, 1987 Apr, 169(4), 1474 - 9
Characterization of a membrane-associated serine protease in Escherichia coli; Palmer SM et al.; Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction . On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively (M . Pacaud, J . Bacteriol . 149:6-14, 1982) . The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B . We termed this enzyme protease VI . Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V . However, all three enzymes generated acid-soluble fragments from a mixture of E . coli membrane proteins which were biosynthetically labeled with radioactive amino acids . The activity of protease VI was sensitive to serine protease inhibitors . Using {3H}diisopropylfluorophosphate as an active-site labeling reagent, we determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels . All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, and endogenous, periplasmic inhibitor of trypsin.

J Bacteriol, 1987 Apr, 169(4), 1469 - 73
Altered molecular form of acyl carrier protein associated with beta-ketoacyl-acyl carrier protein synthase II (fabF) mutants; Jackowski S et al.; Acyl carrier protein (ACP) is a required cofactor for fatty acid synthesis in Escherichia coli . Mutants lacking beta-ketoacyl-ACP synthase II activity (fabF1 or fabF3) possessed a different molecular species of ACP (F-ACP) that was separated from the normal form of the protein by conformationally sensitive gel electrophoresis . Synthase I mutants contained the normal protein . Complementation of fabF1 mutants with an F' factor harboring the wild-type synthase II allele resulted in the appearance of normal ACP, whereas complementation with an F' possessing the fabF2 allele (a mutation that produces a synthase II enzyme with altered catalytic activity) resulted in the production of both forms of ACP . The structural difference between F-ACP and ACP persisted after the removal of the 4'-phosphopantetheine prosthetic group, and both forms of the protein had identical properties in an in vitro fatty acid synthase assay . Both ACP and F-ACP were purified to homogeneity, and their primary amino acid sequences were determined . The two ACP species were identical but differed from the sequence reported for E . coli E-15 ACP in that an Asn instead of an Asp was at position 24 and an Ile instead of a Val was at position 43 . Therefore, F-ACP appears to be a modification of ACP that is detected when beta-ketoacyl-ACP synthase II activity is impaired.

J Bacteriol, 1987 Apr, 169(4), 1386 - 90
Conditions leading to secretion of a normally periplasmic protein in Escherichia coli; Pages JM et al.; The phosphate-binding protein (PhoS) is a periplasmic protein which is part of the high-affinity phosphate transport system of Escherichia coli . Hyperproduction of PhoS in strains carrying a multicopy plasmid containing phoS led to partial secretion of the protein . By 6 h after transfer to phosphate-limiting medium, about 13% of the total newly synthesized PhoS was secreted to the medium . Kinetic studies demonstrated that this secretion consists of newly synthesized PhoS . This secretion occurs in PhoS-hyperproducer strains but not in a PhoS-overproducer strain . Another type of secretion concerning periplasmic PhoS was observed in both PhoS-hyperproducer and PhoS-overproducer strains . This mode of secretion depended upon the addition of phosphate to cells previously grown in phosphate-limiting medium.

J Bacteriol, 1987 Apr, 169(4), 1365 - 71
Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D; Radolf JD et al.; Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a protease-resistant ordered ring structure composed of 19,000-dalton subunits . On gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring . Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer . A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T . pallidum after reduction at room temperature . Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent . Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction . Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K . The properties of 4D suggest that it may fulfill an important structural role in the T . pallidum outer membrane.

Infect Immun, 1987 Apr, 55(4), 963 - 7
Expression and immunological properties of the five subunits of pertussis toxin; Nicosia A et al.; Pertussis toxin, a protein composed of five different subunits, is responsible for the pathogenicity of Bordetella pertussis and is the main component of a new vaccine against whooping cough . The genes coding for the five subunits, recently cloned and sequenced, are organized as an operon . We approached the problem of expression of the five genes in Escherichia coli and, although we obtained high levels of transcription of the native pertussis toxin genes, the amount of proteins produced was very low or undetectable . To obtain suitable expression of each of the five subunits, we fused their genes to the gene coding for the DNA polymerase of MS2 in the expression vector pEx31 . A total of 5 to 30 mg of purified fusion proteins could be obtained from 1 liter of culture . The purified fusion proteins were used to immunize rabbits to obtain sera against each of the five subunits . These sera, although able to recognize the toxin in an enzyme-linked immunosorbent assay and the corresponding subunits in Western blots, were not able to protect CHO cells from the action of pertussis toxin . Mice immunized with the five subunits were not protected from an intracerebral challenge with B . pertussis . Subunits S2 and S3, which are 67% homologous, were shown to cross-react immunologically . The fused subunit S1 was able to ADP-ribosylate transducin as efficiently as the native pertussis toxin.

Eur J Biochem, 1987 Apr 1, 164(1), 205 - 11
Early increases in the frequency of DNA initiations and of phospholipid synthesis discontinuities after nutritional shift-up in Escherichia coli; Kepes F et al.; Cultures of Escherichia coli (strains ML30 and K12 AB1157), synchronized by repeated phosphate starvation, were submitted to nutritional shifts-up at various cell ages . The progression of the replication forks was assessed by DNA-DNA hybridization of pulse-labelled chromosomal DNA with plasmid DNA probes containing specific chromosomal sequences . The rate of phospholipid synthesis and its cyclic discontinuities were measured by continuous and pulse labelling with palmitate . The DNA-DNA hybridization experiments showed that a shift-up induces a burst of initiation from the oriC region . These hybridization results, taken together with older data from the literature, suggest that most DNA initiations belonging to this burst are not followed by complete replication . Following a shift-up, the rate of phospholipid synthesis is maintained for 13-20 min, depending on cell age at shift-up, then doubles . The new steady-state rate of phospholipid synthesis is reached through a series of three doublings, while the cell mass doubles approximately twice . This discrepancy brings the rate of phospholipid synthesis per mass unit to its steady-state postshift value.

Eur J Biochem, 1987 Apr 1, 164(1), 123 - 8
Trypanothione reductase from Trypanosoma cruzi . Purification and characterization of the crystalline enzyme; Krauth-Siegel RL et al.; The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease . As a first step in the analysis of the parasite enzyme we report its purification and characterization . 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells . The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45% . The enzyme is a dimer with a subunit Mr of 50,000 . Using NADPH (Km = 5 microM) and trypanothione disulfide (Km = 45 microM) as substrates, a turnover number of 14,200 min-1 was estimated . Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates . When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used . A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay . Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions Eox + NADPH + H+----EH2 + NADP+, and EH2 + trypanothione disulfide----Eox + dihydrotrypanothione . This is also true for the crystallized enzyme in the presence of 2 M (NH4)2SO4 . The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electron-reduced form (EH2) closely resemble those of human glutathione reductase . Both proteins contain a flavin and a redox-active disulfide at the catalytic site . After reduction of Eox to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.

J Infect Dis, 1987 Apr, 155(4), 707 - 15
Phage-associated cytotoxin production by and enteroadhesiveness of enteropathogenic Escherichia coli isolated from infants with diarrhea in West Germany; Karch H et al.; We assessed the frequency of isolating enteropathogenic Escherichia coli (EPEC) from the stools of infants with diarrhea, the enteroadhesiveness of the EPEC, their production of cytotoxin, and the association of cytotoxin synthesis with lysogenic phages . One hundred twenty-five isolates of EPEC obtained from 1,674 children with diarrhea; three were isolated from 868 controls . Thirty EPEC made elevated levels (greater than or equal to 10(4) 50% cytotoxic doses/mg of cell lysate protein) of a cytotoxin for HeLa cells, and cell-associated cytotoxicity for 27 of these isolates was neutralized by antibody to Shiga toxin . Cell lysates of these isolates were paralytic and lethal for mice . Phages from cytotoxin-producing strains were tested for toxin-converting capacity . Fifteen of 30 such strains harbored toxin-converting phages, and the cytotoxicity of 12 isolates of E . coli K12 transduced with these phages was neutralized by antibody to Shiga toxin . Fifty-seven EPEC exhibited either localized or diffuse adherence to HEp-2 cells, but only nine producers of elevated levels of cytotoxin were adherent.

Genes Dev, 1987 Apr, 1(2), 179 - 84
Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli; Grossman AD et al.; The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response . We present experiments indicating a direct role for sigma 32 in controlling the heat shock response . Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32 . Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32 . These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.

Baillieres Clin Gastroenterol, 1987 Apr, 1(2), 361 - 76
Travellers' diarrhoea; Steffen R et al.; Travellers' diarrhoea each year affects six million persons . At highest risk are those originating in an industrialized country for a visit in the Third World; their incidence of diarrhoea is 20-56% for the first 14 days of the stay abroad . Younger travellers, those who care less, and those with a lack of nonspecific gastrointestinal immune factors are more susceptible . The ailment mostly takes a mild and short course . Travellers' diarrhoea is usually due to faecally contaminated food and beverages, the predominant agent being enterotoxigenic E . coli . Therefore, the traditional rules of nutritional prophylaxis play the main role in prevention; drug prophylaxis can hardly ever be recommended.

J Biochem (Tokyo), 1987 Apr, 101(4), 1033 - 9
Characteristics of phospholipid transacylase of Escherichia coli; Homma H et al.; We have previously demonstrated the activity(ies) of phospholipid transacylase in Escherichia coli extract (Homma & Nojima (1982) J . Biochem . 91, 1093-1101), which catalyzed a new type of reaction of acyl transfer from diacylphospholipids to lysophospholipids . In this communication we report the specificities and characteristics of this enzyme activity . The activity catalyzed a reversible transfer of an acyl group between diacylphospholipids and lysophospholipids . The acyl group in the 1-position of the glycerol backbone was selectively transferred, and palmitic acid was the only fatty acid species transferred . Presumably, neutral lipids do not serve as substrates . The transacylase was firmly associated with the envelope fraction of E . coli . Neither potassium chloride nor urea was effective in solubilization of the activity and only about half of the activity was solubilized with Triton X-100 . This observation was consistent with the equal distribution of the activity between the outer membrane and the inner membrane of E . coli . Functional aspects of this phospholipid transacylase are also discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Apr, (4), 11 - 4
{Significance of rec A-gene activity in altering the ultrastructure of Escherichia coli}; Rybal'chenko OV et al.; The pathogenic E . coli strain, serovar 0124 studied in this investigation and its mutants rec A 56 and rec A 441 differ in their ultrastructural organization . In strains having defects in gene rec A, the appearance of intracytoplasmic membranous structures and a great number of extracellular membranous vesicles, as well as the formation of the filamentous forms of cells in the colonies, can be observed . These data indicate that the product of gene rec A plays an active role in the metabolism of bacterial membranes.

Mol Gen Genet, 1987 Apr, 207(1), 24 - 8
The use of operon fusions in studies of the heat-shock response: effects of altered sigma 32 on heat-shock promoter function in Escherichia coli; Yano R et al.; Derivatives of lambda pF13 phage in which lacZ expression (beta-galactosidase synthesis) is directed by transcription initiated at a heat-shock promoter (PrpoDhs or PgroE) were constructed and used for analysis of the heat-shock response in Escherichia coli . A wild-type strain (MC4100) lysogenic for either of these phages exhibited typical transient induction of beta-galactosidase synthesis upon a temperature shift from 30 degrees to 42 degrees C or after addition of ethanol to the medium (4% to 5%) at 30 degrees C . In contrast, most amber rpoH (htpR) mutants tested (in a Su- background) failed to respond to a temperature shift, though some mutants affected in the carboxy-terminal region exhibited a partial response . All rpoH mutants tested showed a weak but significant response to ethanol . F' plasmids carrying each of six known nonsense suppressors were then introduced into each of four rpoH amber mutants lysogenic for lambda pF13-(Phs-lacZ), creating a set of F' strains that produce sigma 32 protein with a specific amino acid substitution at a known site . Some of these strains showed an essentially normal heat-shock response while others showed little response with either or both of the promoters . In some instances, the response was significantly delayed . These results point to the usefulness of the lambda pF13-derivative phages for quantitative and systematic analysis of heat-shock response in E . coli.

J Appl Physiol, 1987 Apr, 62(4), 1368 - 76
Effects of norepinephrine and fluid administration on diaphragmatic O2 consumption in septic shock; Hussain SN et al.; The effects of norepinephrine infusion and fluid administration on diaphragmatic O2 consumption during endotoxic shock were assessed in spontaneously breathing anesthetized dogs . Blood flow was measured with the microsphere technique, and diaphragmatic venous blood was obtained via a catheter inserted into the left inferior phrenic vein . One group of dogs (n = 6) received 10 mg/kg Escherichia coli endotoxin intravenously (E group) . In the second and third groups, blood pressure after endotoxin injection was restored by continuous infusion of norepinephrine tartrate (N group) or by infusion of normal saline and dextran infusion (F group) . The animals were observed for 2 h after endotoxin injection . Cardiac output fell significantly in the E and N group, whereas it was restored in the F group . Minute ventilation and diaphragmatic pressure-time index rose twofold in the three groups of dogs . Diaphragmatic O2 consumption (VO2 di) increased substantially in the E group to a mean value of 3.46 ml X 100 g-1 X min-1, which was achieved by higher blood flow and by an increase in O2 extraction . In the N group, VO2 di was higher than control but was lower than that of the E group (mean value of 1.43 ml X 100 g-1 X min-1), which was achieved solely by increasing O2 extraction . In the F group, VO2 di was also lower than that of the E group (mean value of 1.51 ml X 100 g-1 X min-1), which was achieved by high diaphragmatic blood flow . Thus, at any given diaphragmatic task, the diaphragm consumed less O2 in the N and F group than in the E group.

J Appl Bacteriol, 1987 Apr, 62(4), 309 - 14
Influence of specific growth rate and nutrient limitation upon the sensitivity of Escherichia coli towards chlorhexidine diacetate; Wright NE et al.; The sensitivity of Escherichia coli to chlorhexidine has been assessed for cells grown in a chemostat at a variety of specific growth rates, under conditions of carbon, nitrogen, phosphorus and magnesium limitation . At slow rates of growth (ca 0.08/h) little difference in sensitivity was observed . As growth rate was increased, however, the sensitivity of nitrogen- and carbon-limited cells increased whilst that of magnesium- and phosphate-limited cells decreased . It was not possible to correlate the observed patterns of chlorhexidine sensitivity with any single measure of cell envelope composition (phospholipid content, lipopolysaccharide, envelope proteins, etc.) . The results presented are not consistent, therefore, with any simple model for chlorhexidine binding or action and more probably reflect subtle interaction between chlorhexidine, phospholipid-lipopolysaccharide complexes and cations within the envelope.

Eur J Cell Biol, 1987 Apr, 43(2), 243 - 6
Direct and mediated Escherichia coli lipopolysaccharide action in primary hepatocyte cultures; Pagani R et al.; The biphasic behaviour observed in endotoxin-induced shock attributed to a direct interaction of bacterial lipopolysaccharides with the cell membrane and an indirect activation of multiple homeostatic regulatory mechanisms, cannot be completely elucidated with in vivo studies . In primary cultures of adult rat hepatocytes, lipopolysaccharide from Escherichia coli 0111:B4 affects the cytochrome P450 levels directly; however, albumin and aspartate aminotransferase secretion are induced by some mediators present in the sera of animals in acute-phase shock.

EMBO J, 1987 Apr, 6(4), 1129 - 35
Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations; Lee SJ et al.; The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r . and photo CIDNP . Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs . By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer . lambda-cro itself also undergoes a conformational change including loosening of the dimeric form . In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part . However, there is only a slight conformational change in the cro dimer . In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.

EMBO J, 1987 Apr, 6(4), 1107 - 14
Three-dimensional structure of the large ribosomal subunit from Escherichia coli; Radermacher M et al.; The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens . A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle . A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit . This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.

EMBO J, 1987 Apr, 6(4), 1011 - 6
The ATP requiring step in assembly of M13 procoat protein into microsomes is related to preservation of transport competence of the precursor protein; Wiech H et al.; M13 procoat protein is processed to transmembrane coat protein by dog pancreas microsomes after completion of synthesis and in the absence of the signal recognition particle (SRP)/docking protein system . ATP is required for fast and efficient processing of procoat protein by microsomes in a reticulocyte lysate . Requirement for ATP is also observed in the absence of ribosomes or docking protein . This indicates the existence of a unique assembly pathway for procoat protein into microsomes which depends on ATP but does not depend on the SRP/docking protein and ribosome/ribosome receptor systems . We suggest that the ATP requirement is linked to a so far unknown component of the reticulocyte lysate, acting on transport competence of precursor proteins.

Biotechnol Appl Biochem, 1987 Apr, 9(2), 181 - 93
Folding and activation of recombinant human prorenin; Sharma SK et al.; In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin . These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium . It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.

Am J Vet Res, 1987 Apr, 48(4), 558 - 61
Effect of jejunal loop location on the activity of Escherichia coli heat-stable enterotoxin in 4- to 5-week-old pigs; Dove CR et al.; Heat-stable enterotoxin (STa) from Escherichia coli strain 431 was injected into the jejunum of 4 pigs from each of 3 litters, using a ligated intestinal loop assay (with loops beginning 1 m caudad to the pylorus) . The jejunum was divided into 4 contiguous areas, with 4 loops in each area . Doses of 0, 10, 100, or 1,000 ng of purified toxin (10 ng/mouse unit) were injected into the loops within an area, using a 4 X 4 Latin square design . Fluid accumulation in the loops increased (P less than 0.05) with increasing concentrations of STa in pigs in all litters, but the magnitude of the response varied across litters . Fluid responses to the STa varied in the different areas of the jejunum, with pigs in 2 litters having a decrease (P less than 0.05) in the response to STa in the caudal areas . These data quantitate the variability within the different areas of the jejunum of the young pig.

Acta Pathol Microbiol Immunol Scand {B}, 1987 Apr, 95(2), 123 - 30
Colonization, diarrhoea and protective immunogenicity of a CFA-deficient, enterotoxin-producing Escherichia coli mutant in a non-ligated intestine experimental model; Lopez-Vidal Y et al.; A colonization factor antigen (CFA)-deficient mutant has been isolated that is as efficient as the CFA/I-carrying, enterotoxin-producing E . coli strain it was derived from in producing diarrhoea and colonizing the intestine in a rabbit non-ligated intestine model, the RITARD model . Infection with 10(11) mutant bacteria induced diarrhoea in 15 of the 17 rabbits challenged; corresponding diarrhoeal attack rates after challenge with similar doses of the original CFA/I-positive strain and of a non-enterotoxinogenic, non-CFA control strain were 23/24 and 0/15, respectively . Whereas the mean time of excreting the challenge strain in faeces did not differ between the mutant and the CFA/I-positive strain (4.5 and 4.3 days, respectively), it was significantly longer (p less than 0.05) than for the control strain (2.5 days) . Similarly, the magnitude of the serum antibody responses to homologous O-antigen was equally high after infection with the mutant and the CFA/I-positive strain and considerably higher than after challenge with the negative control strain . An initial infection in the RITARD model with the CFA-deficient mutant offered highly significant protection against diarrhoea (p less than 0.001) as well as colonization (p less than 0.01) on subsequent challenge with the original CFA/I-positive strain . No significant protection, either against colonization or disease, was induced by initial infection with a similar dose of the control strain.

Z Kinderchir, 1987 Apr, 42(2), 120 - 2
Xanthogranulomatous pyelonephritis in childhood: report on an unusual case; Esposito G et al.; A case of localised xanthogranulomatous pyelonephritis (XPN) in a 4-year-old female with an unusual radiological picture is reported . The young girl was suspected to have a liver tumour because of a hypervascularised area found by arteriography in the hepato-diaphragmatic region . Laparotomy disclosed a large mass in the upper pole of the right kidney which was adherent to the liver and to the diaphragm . Excision of the large mass including upper pole of the kidney was carried out . Recovery was uneventful . Pathological examination revealed the XPN disease pattern.

Parasitology, 1987 Apr, 94 ( Pt 2), 199 - 208
Processing of the precursor to the major merozoite surface antigens of Plasmodium falciparum; Holder AA et al.; Specific sequences derived from the gene for the precursor to the major merozoite surface antigens (PMMSA) of Plasmodium falciparum have been expressed in Escherichia coli and the products have been used to produce antibodies . These antibodies, together with monoclonal antibodies, have been used to investigate the form of the PMMSA protein associated with merozoites . Polypeptide fragments derived by processing from the PMMSA protein have been detected in extracts of merozoites and assigned to locations within the PMMSA coding sequence.

Microbiol Sci, 1987 Apr, 4(4), 125 - 7
An efflux system for cationic dyes and related compounds in Escherichia coli; Midgley M; Escherichia coli possesses an efflux system of broad specificity for complex organic cations . The operation of this system interferes with the use of lipophilic cations such as phosphonium ions for membrane potential measurement.

J Interferon Res, 1987 Apr, 7(2), 145 - 54
Differential purification by immunoaffinity chromatography of two carboxy-terminal portion-deleted derivatives of recombinant human interferon-gamma from Escherichia coli; Honda S et al.; Two lower-molecular-weight derivatives of recombinant human interferon-gamma (rIFN-gamma) were purified concurrently from a lysozyme-EDTA extract of Escherichia coli cells by immunoaffinity chromatography using a monoclonal antibody (MAb) against a synthetic carboxy-terminal peptide (Lys-131-Gln-146) . The two derivatives, 15K rIFN-gamma and 17K rIFN-gamma, were regarded to have been generated at the extraction step . They were successfully separated from each other by using another MAb against the same synthetic peptide with higher binding affinity than the first . The results of protein-chemical analyses indicate that 15K rIFN-gamma and 17K rIFN-gamma lack 15 (Arg 132-Gln-146) and 4 (Arg-143-Gln-146) carboxy-terminal amino acid residues, respectively . All the data suggest that the two derivatives form a noncovalent dimer and that 15K rIFN-gamma binds indirectly to the MAb column via 17K rIFN-gamma.

Mol Cell Biol, 1987 Apr, 7(4), 1459 - 64
Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation; Gebara MM et al.; Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line . Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated . These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4) . We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants . DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences . In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene . In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced . Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations . Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold . The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.

Arch Biochem Biophys, 1987 Apr, 254(1), 353 - 67
Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli; Porter TD et al.; A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones . This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection . Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme . Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography . Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E . coli proteins . The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded . Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase . High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000 . Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C) . HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver . When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo{a}pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2189 - 93
Mutations in the 2-microns circle site-specific recombinase that abolish recombination without affecting substrate recognition; Prasad PV et al.; The site-specific recombinase encoded by the yeast plasmid 2-microns circle (FLP) forms a transient covalent linkage with its substrate DNA via a tyrosine residue, which appears to be located near its COOH terminus . The homology of the COOH terminus of FLP with that of the Int family of recombinases suggests that tyrosine-343 of FLP could be involved in forming the DNA-protein bridge . We have mutated tyrosine-343 to a phenylalanine or serine . We demonstrate that the binding of each of the two mutant proteins to its substrate is indistinguishable from that of wild-type FLP . However, both mutant proteins are incapable of catalyzing strand cleavage and recombination.

J Interferon Res, 1987 Apr, 7(2), 203 - 11
Potency stability of recombinant (Serine-17) human interferon-beta; Geigert J et al.; The antiviral activity of Escherichia coli-derived (Serine-17) human interferon-beta, formulated with human serum albumin, is stable for 2 years when lyophilized and stored under refrigeration . This product shows an Arrhenius line fit for the stability of its activity when tested at multiple isothermal temperatures (25-80 degrees C) . In both isothermal and nonisothermal elevated temperature studies, increasing the level of human serum albumin in the formulation results in increased thermal stability.

J Interferon Res, 1987 Apr, 7(2), 173 - 83
Structure and expression in Escherichia coli of canine interferon-alpha genes; Himmler A et al.; Using a human interferon-alpha (IFN-alpha) cDNA probe, several recombinant phages containing type I IFN genes were isolated from a canine genomic library . One of these phages contains two complete CaIFN-alpha genes with identical coding sequences, and a second one a slightly different IFN-alpha gene . The IFN-alpha protein sequences contain six cysteine residues as well as two or three potential N-glycosylation sites . Expression of mature CaIFN-alpha 1 in E . coli results in antiviral activity on dog cells . Genomic analysis using an equine IFN-omega probe and DNA sequencing suggests the deletion of IFN-omega genes from canine genome.

J Biochem (Tokyo), 1987 Apr, 101(4), 889 - 95
E-F hand structure-domain of calcium-activated neutral protease (CANP) can bind Ca2+ ions; Minami Y et al.; The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18) . The resulting plasmids were used to transform E . coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed . The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay . Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures . The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.

Tsitologiia, 1987 Apr, 29(4), 478 - 83
{Genetic transformation of somatic cells . XIII . Limited replication of DNA containing a fragment of human satellite DNA III in mouse cells}; Vikhanskaia FL et al.; The plasmids containing the variant sequence of human satellite III "rescued" after replication in Chinese hamster cells were transfected into Chinese hamster, mouse and human cells by DEAE-dextran method . Several days after transfection extrachromosomal fractions were isolated, treated with DpnI, transformed into E . coli . In mouse cells, transformed with oncogene v-myc, after transient transfection of HS3-containing plasmids the appearance of rearranged and non-rearranged DpnI-resistant plasmids has been found . At the same time MboI-sensitive plasmids were not found in this material . The data suggest a limited replication (1 round) of HS3-containing plasmids in mouse cells transformed with oncogene v-myc.

Mol Gen Mikrobiol Virusol, 1987 Apr, (4), 43 - 6
{Directed mutagenesis of chromosomal genes of Escherichia coli in vivo: construction of RecA- and HtpR- mutants}; Kiselev VI et al.; The deletions in Escherichia coli chromosomal genes recA and htpR were constructed using the site-directed mutagenesis techniques . The obtained RecA- mutants are UV-sensitive and have a phenotype defective for the homologous DNA recombination . HtpR- mutant is temperature sensitive for growth and deficient in intracellular proteolysis . As a result a HtpR- mutant seems to be a preferable candidate for attempting to synthesize efficiently any alien protein in Escherichia coli cells.

EMBO J, 1987 Apr, 6(4), 1115 - 9
Transcriptional termination at a fully rho-independent site in Escherichia coli is prevented by uninterrupted translation of the nascent RNA; Wright JJ et al.; We have examined the possibility that translation reading through a fully rho-independent transcriptional terminator in Escherichia coli might prevent termination, as already established for rho-dependent terminators . Plasmids were constructed with and without interposition of the rho-independent coliphage T7 'early' terminator between a promoter and galK . Our constructions ensured either that there was no upstream translation, or that translation (initiated at the galE ribosome binding site) stopped upstream of, or at the normal position (the T7 gene 1.3 stop codon) with respect to, the transcriptional terminator; or else downstream of both this stop codon and the terminator . Our galactokinase enzyme and mRNA measurements on strains harbouring these plasmids indicate that 'readthrough translation' eliminates transcriptional termination at the T7 site . This effect is suppressed if the rate of ribosome movement is reduced with fusidic acid.

Acta Physiol Scand, 1987 Apr, 129(4), 451 - 8
ACTH-mediated aldosterone hypersecretion during endotoxin-induced fever with apparent influence upon renal sodium excretion; Andersson B et al.; Febrile, endocrine, and renal responses to i.v . injection of endotoxin (E . coli lipopolysaccharide, 0.25 microgram kg-1) were studied in hyperhydrated goats without, and after dexamethasone pre-treatment, performed with the aim of inhibiting the adenohypophyseal secretion of ACTH . As expected from previous investigations, the administration solely of endotoxin induced biphasic fever, pronounced and long-lasting (less than 4 h) elevation of plasma cortisol (PC), and a prompt inhibition of the water diuresis . Apparently the observation that endotoxin also induced a pronounced biphasic elevation of plasma aldosterone (PA) where the two rising phases coincided with the early, and respectively the second elevation in rectal temperature is original . The endotoxin had no obvious influence upon the renal Na excretion for 3 h post-injection, and did not affect plasma renin activity (PRA) . After dexamethasone pre-treatment (0.02 mg kg-1, i.v . 75 min prior to endotoxin) the endotoxin-induced rise in rectal temperature initially was less steep and no biphasic pattern of the fever was observed . The PC and antidiuretic responses became delayed for about 2 h and were then much attenuated . Endotoxin-induced rise in PA was no longer observed, and a conspicuous natriuresis developed within 90 min post-endotoxin . It is concluded that endotoxin at the dose used causes liberation of ACTH to such an extent that adrenocortical hypersecretion not only of glucocorticoids, but also of aldosterone occurs . The observed differences in Na excretion suggest that this aldosterone hypersecretion may be of pathophysiological importance as a protection against inappropriate renal waste of Na during the early phase of endotoxin-induced fever.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Apr, 51(4), 573 - 89
Radiation-induced DNA damage and its repair; Teoule R; Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair . An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented . The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes . A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation . Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used . Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed . Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned . Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence . Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions . Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.

Biochem Pharmacol, 1987 Apr 1, 36(7), 1069 - 76
Photosensitization of SV 40 DNA mediated by promazine derivatives and 4'-hydroxymethyl-4,5',8-trimethylpsoralen . Inhibition of the in vitro transcription; Decuyper J et al.; In vitro transcription by E . coli RNA polymerase was carried out on SV40 DNA photoreacted with various promazine derivatives . Inhibition of the template activity was recorded with increasing irradiation times in the presence of promazine derivatives . Promazine covalent adducts on guanine did not terminate RNA synthesis and seemed to be bypassed by the enzyme . HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) photoreaction with DNA was carried out under two conditions: irradiation with lambda greater than 395 nm favouring monoadduction on pyrimidine residues and irradiation at 360 nm inducing a maximum of interstrand diadducts . Both adducts were able to terminate RNA synthesis on the phototreated SV40 DNA and using the O-methyl-nucleotide sequencing procedure, the termination sites were precisely mapped . Monoadducts on the coding strand and cross-links induced termination two bases away from the covalent adduct, but monoadducts on the noncoding strand did not half RNA polymerase.

Am Rev Respir Dis, 1987 Apr, 135(4), 854 - 9
Apparent effect of catalase on airway edema in guinea pigs . Role of endotoxin contamination; Gordon T et al.; The airway edema that develops in guinea pigs after exposure to toluene diisocyanate (TDI) requires the presence of polymorphonuclear leukocytes (PMN) . To determine whether this airway edema is mediated by the release of hydrogen peroxide from PMN, we treated animals intravenously with catalase bound to polyethylene glycol and examined the extravasation of Evans blue dye into the tracheal wall after exposure to air or 3 ppm TDI for 1 h . Catalase (25,000, 100,000, and 300,000 IU/kg) caused a dose-dependent inhibition of the TDI-induced increase in dye extravasation . However, treatment with catalase, inactivated at the peroxide binding site with 3-aminotriazole, inhibited dye extravasation after exposure to TDI as effectively as the equimolar 100,000 IU/kg dose of active catalase . Injection of polyethylene glycol alone was without effect . Dose-dependent decreases in extravascular migration of PMN and in circulating PMN also were noted after catalase treatment . These results suggest that the catalase preparations used in these studies inhibited the PMN-dependent airway edema by an effect other than hydrogen peroxide scavenging . Examination of this and other commercially available catalase preparations revealed trace concentrations of endotoxin at levels that could be responsible for the observed effects on PMN function . Treatment of animals with doses of Escherichia coli endotoxin similar to those inadvertantly administered to the catalase-treated groups (0.1 ng/kg to 100 ng/kg, intravenously) inhibited TDI-induced extravasation of Evans blue dye in a dose-dependent manner . These results suggest that contaminating endotoxin may contribute to some of the protective effects of preparations of catalase observed in previous studies of vascular permeability.

Am J Physiol, 1987 Apr, 252(4 Pt 1), C436 - 40
Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells; Shiki Y et al.; Manganous (Mn) and copper zinc (CuZn) superoxide dismutase (SOD) concentrations and glutathione peroxidase (GSH-Px) and catalase (CAT) activities were measured in cultured bovine pulmonary endothelial cells with and without exposure to Escherichia coli endotoxin (10(-1) micrograms/ml) over intervals of 0.5-24 h . The activities of two mitochondrial marker enzymes, fumarase and cytochrome-c oxidase, were also measured . Endotoxin exposure caused a marked increase (9-fold) in endothelial cell Mn SOD content without significant effects on GSH-Px, CAT, fumarase, or cytochrome-c oxidase activities . Endotoxin induced a slight decrease in CuZn SOD content over 24 h . This is the first report of a selective effect of endotoxin on Mn SOD in pulmonary endothelial cells . The response appears to be independent of an increase in mitochondrial activity (no change was observed in cytochrome-c oxidase or fumarase activities) . These findings support the notion that endotoxin increases generation of toxic oxygen metabolites within pulmonary endothelial cells . An endotoxin-induced increase in Mn SOD could contribute to the reported protective effect of endotoxin against oxygen toxicity in these cells.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2130 - 4
Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon; Fornwald JA et al.; Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose . Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified . galP1 is located immediately upstream of the operon and is induced in the presence of galactose . This promoter directs transcription of the galT, galE, and galK genes . The second promoter, galP2, is located within the operon just upstream of the galE gene . This promoter is responsible for constitutive transcription of the galE and galK genes . Comparison of the S . lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons . We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

Proc Natl Acad Sci U S A, 1987 Apr, 84(7), 1809 - 13
Structural domains in phage Mu transposase: identification of the site-specific DNA-binding domain; Nakayama C et al.; Limited proteolysis of phage Mu transposase with three proteases of differing specificities produced a common pattern of fragmentation . The fragments were mapped by using a combination of immunoblotting and amino acid sequence analysis . Our results suggest that the transposase molecule is organized principally into three domains: an amino-terminal domain of molecular mass 30 kDa, a core region of approximately 35 kDa, and a carboxyl-terminal domain of approximately 10 kDa . The amino-terminal domain has at least two additional sites that are partially accessible to proteases . Filter binding and nuclease protection studies were done to determine the functions of the isolated domains . Site-specific binding to Mu DNA was localized to the amino-terminal domain . The core domain showed nonspecific DNA-binding activity.

Mutat Res, 1987 Apr, 177(2), 219 - 28
Mutagenicity of a series of N-alkyl-, N-hydroxyalkyl-, N-haloalkyl- and N-carboxyalkyl-N-nitrosoureas in Escherichia coli tester strains: dependence on the uvrA DNA-repair system; Kohda KH et al.; A series of N-alkyl-, N-hydroxyalkyl-, N-haloalkyl- and N-carboxyalkyl-N-nitrosoureas and some related derivatives were tested for mutagenicity in E . coli B (Arg-) H/r30R (wild-type) and its isogenic Hs30R (uvrA) tester strains . Mutagenic potency in Hs30R, in general, appears to depend on the substituent (-OH, -OCH3, -halogen, -COO- or -COOCH3) on the alkyl group, rather than the chain length or branching of the alkyl group . On the other hand, mutagenic potency in the wild-type H/r30R strain depends on buliness of the substituent and alkyl moiety . The term "uvrA-dependence" of mutation frequency is then defined as the ratio of the mutation frequency in Hs30R versus that in H/r30R at 1 mM dose of mutagens . Its dependence on structure is also discussed . A good correlation was found with the van der Waals volume of the substituted alkyl group, except for compounds having a carboxyalkyl or a branched alkyl group . The carboxyalkyl derivatives are the most weakly mutagenic and most seriously "uvrA-dependent", probably due to the negative charge of the molecule . The possibility of forming epoxides and lactones from N-hydroxyalkyl- and N-carboxyalkyl-N-nitrosoureas, respectively, and their participation in mutagenic potency are discussed . An attempt to correlate the partition property and activation rate of the N-nitrosoureas with mutagenic characteristics proved unsuccessful.

J Clin Invest, 1987 Apr, 79(4), 1210 - 6
Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells; Zafriri D et al.; This study was undertaken to examine whether Escherichia coli adherent to tissue cells gain advantages over nonadherent bacteria due to their proximity to the cells . We used tissue culture cells and isogenic derivatives of a proline auxotrophic strain of E . coli that were fimbriated (Fim+) or nonfimbriated (Fim-), and were heat-labile enterotoxin producing (Tox+) or toxin nonproducing (Tox-) . We found that the Fim+ bacteria; which were capable of adhering to tissue culture cells, initiated growth much sooner than did nonadherent Fim- bacteria; the adherent bacteria used tissue cell-derived proline, which was available at high concentrations only in the zone of bacterial adherence . Likewise, cyclic AMP secreted by adherent (Fim+) bacteria was maintained at high concentration on the tissue cell surfaces . As few as 2 X 10(5) adherent Fim+ Tox+ bacteria exert toxic activity upon Y1 adrenal cells, whereas toxin secreted in the medium by 6 X 10(6) Fim- Tox+ bacteria was undetectable . The results suggest that the growth advantage and enhanced toxicity of adherent E . coli is due to restricted diffusion of products secreted by the tissue culture and bacterial cells, respectively.

J Bacteriol, 1987 Apr, 169(4), 1757 - 9
Localization of a genetic region involved in McrB restriction by Escherichia coli K-12; Ross TK et al.; A 5,500-base-pair BglII-EcoRI fragment proximal to the hsd genes of Escherichia coli K-12 has been cloned in the plasmid vector pUC9 . The resultant hybrid plasmid was shown to complement the mcrB mutation of E . coli K802 . The presence of the hybrid plasmid in strain K802 caused an 18.3-fold drop in transformation efficiency with AluI-methylated pACYC184 relative to unmethylated pACYC184 . These results indicate that the cloned DNA is involved in the McrB system restriction of 5-methylcytosine DNA.

J Bacteriol, 1987 Apr, 169(4), 1731 - 6
Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant; Thoms B et al.; We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene . This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation . By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined . Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents . The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5 . The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation . The results suggest that recF plays a regulatory role in the SOS response . It is proposed that this role is to optimize the signal usage by recA protein to become a protease.

J Bacteriol, 1987 Apr, 169(4), 1724 - 30
Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication; Murakami Y et al.; The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions . The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function . The dnaA mutations tested were dnaA17, dnaA5, and dnaA46 . dnaA5 and dnaA46 are missense mutations . dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6 . Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells . However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested . As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect . Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function . This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.

J Bacteriol, 1987 Apr, 169(4), 1718 - 23
Specific magnesium-dependent diadenosine 5',5'''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli; Hurtado C et al.; A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli . The enzyme had a strict requirement for Mg2+ . Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective . The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts . The Km for Ap3A was 12 +/- 5 microM . Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction . The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5 . Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect . The enzyme differs from the E . coli diadenosine 5',5'''-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.

J Bacteriol, 1987 Apr, 169(4), 1663 - 9
Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase; Boquet PL et al.; The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques . The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions . For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export . The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.

J Bacteriol, 1987 Apr, 169(4), 1603 - 10
Nascent secretory polypeptides synthesized on Escherichia coli ribosomes are not translocated across mammalian endoplasmic reticulum; Ibrahimi I et al.; Cell-free protein-synthesizing systems from Escherichia coli and wheat germ were compared for their capacity to support the translocation of secretory proteins across microsomal membranes derived from mammalian endoplasmic reticulum . Three different secretory proteins, two of bacterial and one of eucaryotic origin, were tested in this respect . In all three cases a contrast between the results in the eucaryotic and procaryotic protein-synthesizing systems was revealed . Whereas the eucaryotic system, as expected, supported the translocation of nascent secretory proteins across the microsomal membranes, the procaryotic system failed to do so . This failure was not due to the absence of a translocation-promoting activity or the presence of a translocation-blocking activity in the procaryotic system . These results demonstrate a specificity in the requirement of components of the protein-synthesizing machinery for protein translocation . These components might participate in forming a functional ribosome-membrane junction during protein translocation . The nascent secretory chain alone is not sufficient for making this junction, which might involve the postulated binding of the ribosome to the signal recognition particle or another component of the membrane.

J Bacteriol, 1987 Apr, 169(4), 1454 - 9
Molecular cloning, characterization, and chromosomal localization of dapF, the Escherichia coli gene for diaminopimelate epimerase; Richaud C et al.; The Escherichia coli dapF gene was isolated from a cosmid library as a result of screening for clones overproducing diaminopimelate epimerase . Insertional mutagenesis was performed on the cloned dapF gene with a mini-Mu transposon, leading to chloramphenicol resistance . One of these insertions was transferred onto the chromosome by a double-recombination event, allowing us to obtain a dapF mutant . This mutant accumulated large amounts of LL-diaminopimelate, confirming the blockage in the step catalyzed by the dapF product, but did not require meso-diaminopimelate for growth . The dapF gene was localized in the 85-min region of the E . coli chromosome between cya and uvrD.

J Bacteriol, 1987 Apr, 169(4), 1447 - 53
Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification; Iida S et al.; Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1 . Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host . Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene . We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified . Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA . Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.

J Bacteriol, 1987 Apr, 169(4), 1410 - 6
Specificity of mutation by UV light and delayed photoreversal in umuC-defective Escherichia coli K-12: a targeting intermediate at pyrimidine dimers; Bockrath R et al.; Prototrophic mutants produced by UV light in Escherichia coli K-12 strains with argE3(Oc) and hisG4(Oc) defects are distinguished as backmutations and specific nonsense suppressor mutations . In strains carrying a umuC defect, mutants are not produced unless irradiated cells are incubated and then exposed to photoreversing light (delayed photoreversal mutagenesis) . The mutants thus produced are found to be specifically suppressor mutations and not backmutations . The suppressor mutations are primarily glutamine tRNA ochre suppressor mutations, which have been attributed previously to mutation targeted at T = C pyrimidine dimers . In a lexA51 recA441 strain, where the SOS mutagenesis functions are constitutive, targeting at dimers is confirmed by demonstrating that the induction of glutamine tRNA suppressor mutations is susceptible to photoreversal . In the same strain induction of backmutations is not susceptible to photoreversal . Thus delayed photoreversal mutagenesis produces suppressor mutations that can be targeted at pyrimidine dimers and does not produce backmutations that are not targeted at pyrimidine dimers . This correlation supports the idea that delayed photoreversal mutagenesis in umuC defective cells reflects a mutation process arrested at a targeting pyrimidine dimer photoproduct, which is the immediate cause of both the alteration in DNA sequence and the obstruction (unless repaired) to mutation fixation and ultimate expression.

J Bacteriol, 1987 Apr, 169(4), 1379 - 85
Isolation of mutations in the alpha operon of Escherichia coli that suppress the transcriptional defect conferred by a mutation in the porin regulatory gene envZ; Garrett S et al.; One class of mutations in the envZ gene of Escherichia coli K-12 confers a pleiotropic defect on the expression of several genes, including ompF, lamB, and phoA, that are otherwise not commonly regulated . Four second-site mutations that suppress this transcriptional defect have been isolated by using a procedure that circumvented the problem of intragenic suppressors, including true revertants . All four mutations have been mapped to the genes of the alpha operon and have been assigned tentatively to the gene rpoA, which specifies the alpha subunit of RNA polymerase . The mutations, referred to as sez (for suppressor of envZ), did not appear to confer a phenotype on an otherwise wild-type strain and did not suppress the transcriptional defects conferred by several other phenotypic classes of envZ mutations, including amber mutations . Our results led us to postulate that the alpha subunit or some other component of the alpha operon plays a role in determining the specificity of gene expression.

Infect Immun, 1987 Apr, 55(4), 974 - 8
Dephosphorylation of the lipid A moiety of Escherichia coli lipopolysaccharide by mouse macrophages; Peterson AA et al.; An Escherichia coli deep rough lipopolysaccharide (LPS), biosynthetically labeled with 32PO4 and {3H}glucosamine, was used to study dephosphorylation of the lipid A moiety by murine macrophages . Over a 48-h incubation period, the macrophages removed approximately two-thirds of the 32P from {3H32P}LPS that was added to the culture medium . The LPS-derived phosphate was incorporated into cell components (e.g., phospholipids), as well as released from the cells . Cell lysates were also able to remove phosphate from {3H32P}LPS . The phosphatase activity was optimal at acidic pH and was greatly reduced by 10 mM sodium fluoride or heating at 80 degrees C . There was no evident difference in the LPS-dephosphorylating ability of macrophages from LPS-responsive and -hyporesponsive mice . The results indicate that murine macrophages dephosphorylate the lipid A moiety of deep rough E . coli LPS and raise the possibility that enzymatic dephosphorylation may modify LPS bioactivity.

Eur J Biochem, 1987 Apr 1, 164(1), 141 - 5
New pore protein produced in cells lysogenic for Escherichia coli phage HK253hrk; Verhoef C et al.; Outer membrane pore protein OmpC was identified as the receptor for the temperate Escherichia coli phage HK253hrk . The part of OmpC protein recognized by the phage was identified by using hybrid proteins in which parts of OmpC protein are replaced by the corresponding parts of the related PhoE protein . In contrast to other OmpC-specific phages, HK253hrk recognizes a part of OmpC within the C-terminal 50 amino acids of the protein . E . coli strains lysogenic for HK253hrk produce reduced amounts of OmpC protein, and produce a new pore protein instead . Expression of this new protein was temperature-dependent, i.e . low at 30 degrees C . The functioning of this new pore protein was characterized both in vivo by studying the uptake of beta-lactam antibodies and in vitro after reconstitution of the protein in black lipid films . Its effective pore size was larger than that of the OmpF pores of E . coli B . The new porin appears to be cation-selective . A comparison with the selectivity of the known OmpC and OmpF pores of E . coli showed that the new pore has a higher selectivity than OmpF but is less selective than OmpC . The new pore protein appears to function in E . coli K12 lysogens as the receptor for the phages HK187, HK189 and HK332.

Eur J Biochem, 1987 Apr 1, 164(1), 111 - 5
The primary structure of cytochrome c1 from Neurospora crassa; Romisch J et al.; The primary structure of the cytochrome c1 subunit of ubiquinol-cytochrome-c reductase from mitochondria of Neurospora crassa was determined by sequencing the cDNA of a bank cloned in Escherichia coli . From the coding region the sequence of 332 amino acids, corresponding to the molecular mass of 36,496 Da, was derived for the precursor protein . The mature protein, the N terminus of which was previously sequenced {Tsugita et al . (1979) in Cytochrome oxidase (King, T . E . et al., eds) pp . 67-77, Elsevier, New York}, consists of 262 amino acids and has the molecular mass of 29,908 Da including the heme . The sequence contains an N-terminal hydrophilic part of 211 residues, which carries the heme, a hydrophobic stretch of 15 residues, which is assumed to anchor the protein to the membrane, and a C-terminal hydrophilic part of 36 residues . The N-terminal presequence of 70 amino acids contains 9 positive charges but only 1 negative charge and is characterized by a stretch of 20 uncharged residues.

J Virol, 1987 Apr, 61(4), 1171 - 9
Characterization of Rous sarcoma virus sequences essential for viral gene expression; Norton PA et al.; Using the Escherichia coli lacZ gene product beta-galactosidase as an indicator of gene expression, we analyzed sequences that are required for expression of the Rous sarcoma virus (RSV) genome in avian cells . The RSV long terminal repeat (LTR) and leader region were sufficient to direct the synthesis of high levels of enzymatically active gag-lacZ fusion proteins . A portion of U3 greater than 140 nucleotides upstream from the cap site was essential for gene expression . This element functioned in either orientation, but its activity was attenuated when it was relocated further away from the cap site . The insertion of exogenous LTRs 3' of lacZ augmented the expression of that gene by increasing the level of stable gag-lacZ transcripts . Furthermore, 3' LTRs could partially compensate for certain defects within the 5' LTR . Insertion of various fragmentary LTRs allowed the identification of at least three synergistically acting domains within the 3' LTR that influence gene expression . Interestingly, the gag-lacZ expression was only stimulated by a 3' LTR when the exogenous 3'-untranslated region was adjacent . Our results imply that the two LTRs of a provirus interact in a complex manner to promote high levels of stable transcripts . It was also found that gag-lacZ expression was independent of viral gene products, suggesting that trans-activation is not a key mechanism regulating RSV expression in avian cells.

Anal Biochem, 1987 Apr, 162(1), 242 - 50
Filter transfer of genomic libraries in a state accessible to DNA-binding proteins; Beebee TJ; I have developed a method for transferring plaque DNA of lambda genomic libraries onto 3MM filters in a state accessible to DNA-binding proteins . DNA bound to 3MM is available to proteins as large as Escherichia coli RNA polymerase and maintains template activity similar to that in free solution . Lambda Plaques can be lifted onto 3MM filter disks, deproteinized, and used for transcription assays in vitro . The RNA synthesized is complementary to phage rather than to E . coli DNA and plaques can be identified by autoradiography . Furthermore, the filters can subsequently be probed with radioactive nucleic acids under standard hybridization conditions . Finally, colorimetric assays can be employed with lactate dehydrogenase (LDH) A in which plaques are identified by the localized reduction of nitroblue tetrazolium.

Genetics, 1987 Apr, 115(4), 585 - 90
Mutations that affect the efficiency of translation of mRNA for the cII gene of coliphage lambda; Dul E et al.; Starting with the lambda pRE-strain lambda ctr1 cy3008, which forms clear plaques, we have isolated two mutant strains, lambda dya2 ctr1 cy3008 and lambda dya3 ctr1 cy3008, that form plaques with very slightly turbid centers . The dya2 and dya3 mutations lie in the region of overlap between the PRE promoter and the ribosome recognition region of the cII gene, and have nucleotide alterations at positions -1 and +5 of pRE, and alterations in cII mRNA at -16 and -21 nucleotides before the initial AUG codon of the gene . Both mutations destabilize a stem structure that may be formed by cII mRNA, and dya2 also changes the sequence on cII mRNA that is complementary to the 3'-end of 16 S rRNA from 5'-UAAGGA-3' to 5'-UGAGGA-3' . --The dya2 and dya3 mutations, along with the ctr1 mutation, which destabilizes either of two alternate stem structures which may be formed by cII mRNA (these being more stable stem structures than the one affected by dya2 and dya3), were tested for their ability to reverse two cII-mutations that are characterized by inefficient translation of cII mRNA . These are cII3088, an A----G mutation four bases before the initial AUG codon, and cII3059, a GUU----GAU (Val2----Asp) second codon mutation . It was found that ctr1 completely reverses the translation defects of these two mutations, while dya2 partially reverses these translation defects . The dya3 mutation has no effect on translation efficiency under any condition tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Neurol Scand, 1987 Apr, 75(4), 231 - 3
Serum antibodies to HTLV-I in human demyelinating disease; Epstein L et al.; Serum HTLV-I antibodies were measured in 17 MS patients and 2 closely matched control groups by 2 different independent assays . A highly sensitive particle agglutination test was used to detect HTLV-I core protein antigens and an immunoblot assay with E . coli-expressed P21E transmembrane protein was employed to detect antibodies to HTLV-I evelope antigens . No serologic evidence of prior HTLV-I infection in our population of multiple sclerosis (MS) or Guillain-Barre Syndrome (GBS) patients was found.

Arch Biochem Biophys, 1987 Apr, 254(1), 313 - 8
Binding of Mg2+ to the beta subunit or F1 of H+-ATPase from Escherichia coli; Futai M et al.; The bindings of Mg2+ to the F1 portion of Escherichia coli H+-ATPase and its isolated alpha and beta subunits were studied with 8-anilinonaphthalene-1-sulfonate (ANS) . The fluorescence of ANS increased upon addition of F1 or its alpha subunit or beta subunit, as reported previously (M . Hirano, K . Takeda, H . Kanazawa, and M . Futai (1984) Biochemistry 23, 1652-1656) . The fluorescence of ANS bound to F1 or its beta subunit increased significantly with further addition of Mg2+, whereas that of the alpha subunit increased only slightly . Ca2+ and Mn2+ had similar effects on the fluorescence of ANS with F1 and its beta subunit . The Mg2+-induced fluorescence enhancement (delta F) was high at an alkaline pH and was lowered by addition of ethylenediaminetetraacetic acid . Dicyclohexylcarbodiimide and azide had no effect on the delta F . Binding analysis showed that the concentration dependence of Mg2+ on the fluorescence enhancement of the beta subunit is similar to that of F1 . These results suggest that both the beta subunit and F1 have binding sites for Mg2+ and that the delta F observed with F1 may be due to the binding of Mg2+ to the beta subunit.

J Bacteriol, 1987 Apr, 169(4), 1678 - 83
Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V; Yeung MK et al.; The type 1 fimbriae of Actinomyces viscosus mediate the adherence of this organism to saliva-treated hydroxyapatite . The gene encoding a putative subunit of this fimbrial adhesin was cloned in Escherichia coli, and its product was examined . A . viscosus T14V chromosomal DNA was partially restricted with Sau3AI and cloned into E . coli JM109 by using the plasmid vector pUC13 . Two clones, each containing a different DNA insert with a common 4.1-kilobase region, reacted in colony immunoassays with specific polyclonal as well as monoclonal antibodies directed against A . viscosus T14V type 1 fimbriae . Western blot analysis revealed the expression of a 65-kilodalton protein that migrated slightly behind an antigenically similar protein from native type 1 fimbriae . Deletion analysis showed that the gene encoding the cloned protein was localized on a 1.9-kilobase PstI-BamHI fragment and that transcription was dependent on the lac promoter of the vector . The cloned fimbrial protein was purified from the E . coli cytoplasmic fraction by ion-exchange, immunoaffinity, and gel permeation chromatography . Rabbit antibodies prepared against the cloned protein and against purified A . viscosus type 1 fimbriae gave similar patterns with partially dissociated type 1 fimbriae after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting . The data therefore provide evidence that the gene cloned encodes a subunit of this fimbrial adhesin.

Infect Immun, 1987 Apr, 55(4), 968 - 73
Membrane glycoproteins of human polymorphonuclear leukocytes that act as receptors for mannose-specific Escherichia coli; Rodriguez-Ortega M et al.; Type 1 fimbriated (mannose-specific) Escherichia coli cells bind to mannose residues on human polymorphonuclear leukocytes (PMN); this leads to phagocytosis of the bacteria . To identify the mannose-containing receptors on the PMN, the cells were surface labeled with 125I and lysed in 0.5% Nonidet P-40, and the lysate was fractionated by affinity chromatography on a column of Sepharose-bound fimbriae . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the material eluted from the column with 500 mM methyl-alpha-mannoside revealed two radioactive bands of Mr 70,000 to 80,000 (gp70-80) and 100,000 (gp100) . Another weak band of Mr 150,000 (gp150) was observed after prolonged exposure of the gel . Upon blotting of the glycoproteins separated by polyacrylamide gel electrophoresis and overlaying of the blots with concanavalin A, gp150 appeared as the major band . Membrane preparations of the PMN were enriched in gp70-80, gp100, and gp150, in comparison with the cell homogenates, further suggesting that these glycoproteins are surface components . Fractionation of the membrane preparations on the immobilized fimbriae followed by concanavalin A overlay of blots of the methyl-alpha-mannoside-eluted material revealed that gp150 was the major component in this fraction . The eluted fraction, obtained from a cell lysate (4.4 micrograms/ml), inhibited by 70% the agglutination of yeasts by the intact bacteria . Our results suggest that the three surface glycoproteins isolated by us serve as receptors for mannose-specific E . coli on PMN and may be involved in the lectin-mediated phagocytosis of the bacteria.

Infect Immun, 1987 Apr, 55(4), 923 - 30
Use of monoclonal antibodies to probe subunit- and polymer-specific epitopes of 987P fimbriae of Escherichia coli; Schifferli DM et al.; The relationship between the structure and biological function of 987P fimbriae of a strain of enterotoxigenic Escherichia coli (O9:K103:H-) from piglets was investigated . A set of four monoclonal antibodies was prepared from the spleen cells of mice immunized with isolated 987P fimbriae . Antibodies E11, D5, and C3, but not G10, reacted in enzyme-linked immunosorbent assays with 987P fimbriae-bearing E . coli . Electron microscopy showed that E11 and D5 reacted in a discrete periodic pattern forming a spiral motif along the length of the fimbriae . The results of enzyme-linked immunosorbent assays were in agreement with these results; antibodies E11 and D5 reacted at a high dilution (1:12,000) with native fimbriae on the surface of E . coli, whereas antibody C3 reacted at an intermediate dilution (1:3,000) and G10 failed to react at all (less than 1:250) . In contrast, C3 and G10 reacted at a dilution of 1:3,276,000 with the fimbrial subunits derived by treating the isolated fimbriae with 6 M guanidine hydrochloride, whereas E11 and D5 reacted with the subunits at much lower dilutions of 1:800 and 1:6,400, respectively . Moreover, fimbriae reassembled from the subunits regained reactivity with antibodies D5 and E11, indicating that these antibodies are directed against quaternary conformational epitopes . Only the three antibodies (D5, E11, and C3) that recognized epitopes accessible on intact fimbriae were able to efficiently block the adhesion of 987P fimbriated E . coli to piglet enterocytes . These results indicate that certain epitopes of 987P fimbriae are dependent on quaternary structural conformation, whereas others are present on monomeric subunits; some of the latter appear to remain accessible on fully assembled fimbriae.

Genes Dev, 1987 Apr, 1(2), 185 - 96
The first 28 amino acids of mature LamB are required for rapid and efficient export from the cytoplasm; Rasmussen BA et al.; Our laboratory has been utilizing the Escherichia coli outer membrane protein LamB to study the mechanism of protein localization . Various lines of evidence suggest that, in addition to a signal sequence, regions within the mature protein are required for efficient localization . In particular, studies using LamB-LacZ hybrid proteins have identified regions between amino acids 27 and 49 of mature LamB, which may play an important role in localization . To elucidate further the function of these regions, a series of in-frame deletions that remove varying lengths of early lamB sequences was constructed . The effects of these deletions on export of a large LamB-LacZ hybrid protein, 42-1, and on export of an otherwise wild-type LamB protein were determined . We find a strong correlation between the sequences deleted and the export phenotypes these deletions impart to both LamB and the LamB-LacZ42-1 hybrid protein . On the basis of these findings, the deletions can be divided into several distinct classes that define a region within mature LamB that participates in localization . This region extends amino terminally from amino acid 28 of the mature protein and functions in the rapid and efficient localization of LamB from the cytoplasm.

Mol Gen Genet, 1987 Apr, 207(1), 1 - 8
Genetic analysis of UV mutagenesis of the Escherichia coli glyU gene; Ciesla Z et al.; By genetic analysis we examined UV mutagenesis of the Escherichia coli glyU gene . When carried by M13 phage mp9, glyU is subject to induced UV mutagenesis which is dependent on the umuC+ and recF+ genes . When carried by M13 phage mp8, glyU is not subject to induced UV mutagenesis . This difference is correlated with the nature of the target nucleotides: CTC in the mp9 derivative and GAG in the mp8 derivative . Thus, we conclude that the induced (umuC and recF dependent) mutagenesis is locally targeted on pyrimidine cyclobutane or 6-4 dimers . glyU carried by M13 is equally subject to uninduced UV mutagenesis whether carried by mp8 or mp9 . This uninduced mutagenesis is independent of the umuC+, recF+ and recA+ genes and we hypothesize that it is regionally targeted on pyrimidine cyclobutane or 6-4 dimers in the vicinity of the target CTC and GAG nucleotides . The role of recF in UV mutagenesis was tested in two ways . First, mutagenesis of glyU carried by M13 mp9 in a recA730 genetic background was found to be recF dependent . Because recA730 renders induced UV mutagenesis partially constitutive, we conclude that the RecF product plays a direct role in UV mutagenesis rather than, or in addition to, any indirect regulatory role it may play . Second, UV mutagenesis of E . coli chromosomal glyU was found to be recF independent while UV mutagenesis of M13-bourne glyU was recF dependent . We conclude that the mechanism of induced UV mutagenesis of the E . coli chromosome is at least partly different from that of M13 phage and we discuss the biochemical basis for such a difference.

Calcif Tissue Int, 1987 Apr, 40(4), 231 - 7
Amelogenin antigenic domain defined by clonal epitope selection; Lau EC et al.; To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required . For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected into E . coli . The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes . An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3' noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody . This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins . Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus . Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain . These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.

Arthritis Rheum, 1987 Apr, 30(4), 439 - 42
An antiserum to a disease-associated factor from the cells of an HLA-B27 positive patient with ankylosing spondylitis specifically recognizes an HLA-B27 associated determinant; Sullivan JS et al.; Antiserum raised to a factor elaborated by a lymphoblastoid cell line derived from the peripheral blood cells of an HLA-B27 positive patient with ankylosing spondylitis specifically lyses the B27 positive, but not the B27 negative, cells of ankylosing spondylitis patients . The cells of B27 positive and B27 negative normal controls are not lysed . This serum has similar specificity to antisera against cross-reactive bacteria.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2297 - 301
Pressure-sensitive ion channel in Escherichia coli; Martinac B et al.; We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels . The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions . We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.

J Bacteriol, 1987 Apr, 169(4), 1644 - 50
Expression of arg genes of Escherichia coli during arginine limitation dependent upon stringent control of translation; Williams MG et al.; The transcription and translation of operons for arginine biosynthetic enzymes after arginine removal (arginine down shift) were studied in relA and relA+ strains of Escherichia coli . After arginine down shift, derepression of synthesis of the arginine biosynthetic enzymes ornithine carbamoyltransferase (argF) and argininosuccinate lyase (argH) began at about 15 min in relA+ cells but was delayed in relA cells for more than 2 h . However, both relA+ and relA cells accumulated high levels of argCBH mRNA, as shown by dot blot hybridization, after arginine down shift . After 15 min of arginine limitation, the proportion of ribosome-bound argCBH mRNA was equivalent in both relA+ and relA cells . During the 15 min after the arginine down shift, relA+ cells produced a significant burst of argF and argH enzyme synthesis when arginine was added back to the culture, whereas relA cells did not produce this burst of enzyme synthesis . The relA cells regained the ability to produce a burst of argF and argH enzyme synthesis when alpha-methylglucose-induced glucose starvation was combined with arginine limitation . Significant guanosine 5'-diphosphate 3'-diphosphate accumulated in relA cells under this condition . Our results support the view that during periods of severe amino acid limitation guanosine 5'-diphosphate 3'-diphosphate acts in some way to ensure the translation of argCBH mRNA.

Infect Immun, 1987 Apr, 55(4), 1000 - 3
Characterization of antibody-reactive epitopes on the 65-kilodalton protein of Mycobacterium leprae; Buchanan TM et al.; Twenty-three monoclonal antibodies (MAbs) prepared in seven different laboratories were studied, all of which recognized the 65-kilodalton (kDa) protein of Mycobacterium leprae as determined by Western blotting or gel radioimmunoassay or both . Fourteen of the MAbs recognized different epitopes, as evaluated by cross-competition studies using radiolabeled MAb and unlabeled inhibitors; the species specificity of these epitopes was defined by nitrocellulose dot blot immunoassays with bacterial sonic extract antigen preparations from 23 species of mycobacteria . Each of the 14 distinct MAbs recognized a 65-kDa protein produced by a lysogenized Escherichia coli Y1089 host containing cloned rDNA which included the gene for the M . leprae 65-kDa protein . Of the 14 distinct MAbs, 1 recognized an epitope found only on M . leprae, and the others recognized epitopes present on as few as 8 or as many as all 23 of the mycobacterial species studied . Identification of these distinct 65-kDa protein epitopes and use of the MAbs which recognize them should assist future structural studies of this protein and characterization of the T-cell reactive and serodiagnostically useful portions of the molecule.

Eur J Biochem, 1987 Apr 1, 164(1), 103 - 9
Epitope localization in antigen-monoclonal-antibody complexes by small-angle X-ray scattering . An approach to domain organization in the beta 2 subunit of Escherichia coli tryptophan synthase; Wilhelm P et al.; Each polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.2.1.20) is made of two domains, F1 (N-terminal) and F2 (C-terminal) . To determine the relative position of these domains in the native protein, complexes between beta 2 and Fab fragments from two monoclonal antibodies, one specific for F1 (68-1) and the other for F2 (93-6), have been prepared and purified . Small-angle X-ray scattering measurements have been made on solutions of each complex . From the experimental scattering curves obtained, computer modeling leads to structural models of the two beta 2-Fab complexes . Though relatively low, the resolution of these models allows the localization on beta 2 of the antigenic sites recognized by the two antibodies, to show that the C-terminal F2 domains lie at the distal ends of the elongated beta 2 protein, and to show how steric hindrance prevents beta 2, though structurally and functionally dimeric, from binding more than one Fab 93-6 fragment per dimer.

Clin Chim Acta, 1987 Mar 30, 163(3), 289 - 99
Evidence for endotoxin binding capacity of human Gc-globulin and transferrin; Berger D et al.; In the present paper the ability of Gc-globulin and transferrin to bind endotoxin of Escherichia coli 0 111: B 4 is demonstrated . This conclusion is based on four lines of evidence . By affinity chromatography using lipopolysaccharide of E . coli 0 111: B 4 two endotoxin-binding proteins of serum were identified, showing an apparent molecular weight of 77,000 and 51,000, respectively . If serum samples preincubated with the tritiated endotoxin form have undergone isoelectric focusing under non-denaturing conditions one radioactive peak appears which coincides with the precipitate obtained by immunoelectrophoresis against anti-human Gc-globulin and anti-human transferrin . Radioimmunoprecipitation experiments of serum showed that tritiated endotoxin of E . coli 0 111: B 4 was only found in the precipitate obtained with anti-Gc-globulin, antitransferrin, and polyvalent antiserum against human serum . By isoelectric focusing of purified proteins 3H-lipopolysaccharide of E . coli 0 111: B 4 was only found associated with human Gc-globulin and transferrin.

Biochem Biophys Res Commun, 1987 Mar 30, 143(3), 923 - 7
The effect of polymyxin B nonapeptide (PMBN) on transformation; Viljanen P; The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E . coli HB101 with pBR322 plasmid DNA was investigated . Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse . In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.

Cell, 1987 Mar 27, 48(6), 945 - 52
Transcription termination factor rho is an RNA-DNA helicase; Brennan CA et al.; E . coli rho factor can unwind a short RNA-DNA duplex in vitro . The duplex is formed between a polylinker sequence at the 3' end of RNA derived from the rho-dependent terminator trp t' and the complementary sequence in a single-strand DNA molecule . Release of trp t' RNA from the duplex requires nucleoside triphosphate hydrolysis by rho's NTPase activity and is dependent on rho recognition of the RNA that is 5' to the RNA-DNA duplex region . The direction of helix unwinding appears to be 5' to 3' along the RNA molecule . These characteristics now account for how the RNA-binding and RNA-dependent NTP hydrolysis activities of rho may participate directly in transcription termination . Our results suggest that NTP hydrolysis is utilized to help unwind the RNA-DNA duplex at the 3' end of a nascent transcript, facilitating RNA release from the DNA template.

Science, 1987 Mar 27, 235(4796), 1651 - 3
Polypeptide sequences essential for RNA recognition by an enzyme; Regan L et al.; Many RNAs are complex, globular molecules formed from elements of secondary and tertiary structure analogous to those found in proteins . Little is known about recognition of RNAs by proteins . In the case of transfer RNAs (tRNAs), considerable evidence suggests that elements dispersed in both the one- and three-dimensional structure are important for recognition by aminoacyl tRNA synthetases . Fragments of alanine tRNA synthetase were created by in vitro manipulations of the cloned alaS gene and examined for their interaction with alanine-specific tRNA . Sequences essential for recognition were located near the middle of the polypeptide, juxtaposed to the carboxyl-terminal side of the domain for aminoacyl adenylate synthesis . The most essential part of the tRNA interaction strength and specificity was dependent on a sequence of fewer than 100 amino acids . Within this sequence, and in the context of the proper conformation, a segment of no more than 17 amino acids was responsible for 25% or more of the total synthetase-tRNA free energy of association . The results raise the possibility that an important part of specific RNA recognition by an aminoacyl tRNA synthetase involves a polypeptide segment that is short relative to the total size of the protein.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2611 - 26
Mono- through hexanucleotide composition of the Escherichia coli genome: a Markov chain analysis; Phillips GJ et al.; Several statistical methods were tested for accuracy in predicting observed frequencies of di- through hexanucleotides in 74,444 bp of E . coli DNA . A Markov chain was most accurate overall, whereas other methods, including a random model based on mononucleotide frequencies, were very inaccurate . When ranked highest to lowest abundance, the observed frequencies of oligonucleotides up to six bases in length in E . coli DNA were highly asymmetric . All ordered abundance plots had a wide linear range containing the majority of the oligomers which deviated sharply at the high and low ends of the curves . In general, values predicted by a Markov chain closely followed the overall shape of the ordered abundance curves . A simple equation was derived by which the frequency of any nucleotide longer than four bases in the E . coli genome (or any genome) can be relatively accurately estimated from the nested set of component tri- and tetranucleotides by serial application of a 3rd order Markov chain . The equation yielded a mean ratio of 1.03 +/- 0.94 for the observed-to-expected frequencies of the 4,096 hexanucleotides . Hence, the method is a relatively accurate but not perfect predictor of the length in nucleotides between hexanucleotide sites . Higher accuracy can be achieved using a 4th order Markov chain and larger data sets . The high asymmetry in oligonucleotide abundance means that in the E . coli genome of 4.2 X 10(6) bp many relatively short sequences of 7-9 bp are very rare or absent.

J Biol Chem, 1987 Mar 25, 262(9), 4382 - 6
In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase; Rubino SD et al.; The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2 . The plasmid, pHN12, carries a functional carB gene . A mutant E . coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein) . The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis . The large subunit has been purified from the overexpressing strain . The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP . The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.

J Biol Chem, 1987 Mar 25, 262(9), 4190 - 4
Characterization of 2'(3')-trinitrophenyl-ATP as an inhibitor of ATP-dependent initiation complex formation between the DNA polymerase III holoenzyme and primed DNA; Oberfelder R et al.; We have identified 2'(3')-trinitrophenyl-ATP to be an inhibitor of the ATP-dependent initiation complex formation reaction between the Escherichia coli DNA polymerase III holoenzyme and primed DNA . The inhibitor is specific for the initiation stage; once initiation complexes are formed the subsequent elongation reaction is unaffected . Three ATP-dependent DNA polymerase III holoenzyme reactions can be independently assayed: the ATP-dependent formation of initiation complexes, ATP binding, and the primed DNA-dependent hydrolysis of ATP . Trinitrophenyl ATP inhibits all three reactions to a similar extent with an apparent Ki between 6 and 15 microM in the presence of 5 microM ATP . This suggests all of these reactions are related and that they proceed through a common ATP-binding site . We include an improved purification of the DNA polymerase III holoenzyme in this report.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2677 - 98
Charons 36 to 40: multi enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers; Dunn IS et al.; New phage lambda based cloning vectors, Charons 36-40, have been constructed which allow cloning of large (up to 24 kb) DNA fragments with up to sixteen cloning enzymes . Several of these could not be used previously with lambda vectors . Clones produced with these vectors can be propagated under recombination deficient conditions . A novel polystuffer method has been developed that permits vector arms to be purified by simple precipitation and which allows reliable identification of clones that have reincorporated any part of the stuffer . Three of the vectors are available with amber mutations in essential genes.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2665 - 75
Activity of a Streptomyces transcriptional terminator in Escherichia coli; Deng ZX et al.; A 205bp DNA fragment from the Streptomyces multi-copy plasmid pIJ101 has in vivo terminator activity both in Streptomyces lividans and in Escherichia coli . Termination of RNA synthesis, detected by high-resolution S1 nuclease mapping, occurs at precisely the same nucleotides in both organisms . This suggests that the E . coli RNA polymerase recognizes the same sequence elements and chooses the point(s) of termination in the same way as the corresponding S . lividans enzyme.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2563 - 80
Aggregation of DNA by analogs of spermidine; enzymatic and structural studies; Srivenugopal KS et al.; A homologous series of spermidine analogs, with defined abilities to replace the natural polyamine in supporting cell growth, was examined for its influence on the structure of supercoiled, aggregated DNA and on the ability of the DNA aggregates to act as substrates for various enzymes . The concentration of amine necessary to aggregate negatively supercoiled Col E1 DNA was progressively increased as the diaminobutane moiety of spermidine was extended beyond 5 methylene groups . 1H- and 31P-NMR spectroscopy suggested that less rigid DNA aggregates were formed by spermidine analogs than by spermidine itself . Spermidine and its analogs differentially modulated the activities of bacterial and mammalian type I topoisomerases and EcoRI restriction endonuclease on aggregated DNA in a manner reminiscent of the abilities of the amines to stimulate cell growth . When DNA was not aggregated, the influence of the various amines on these reactions was almost identical . These results are discussed in relation to the structures of the DNA aggregates in the presence of the various triamines.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2479 - 97
Transcripts within the replication origin, oriC, of Escherichia coli; Schauzu MA et al.; Transcription start and termination sites were mapped in the E . coli replication origin, oriC . Outward transcription from within oriC (promoters Pori-r and Pori-l) was found to start in vivo at position 178 for Pori-l and at positions 294 and 304 for Pori-r, respectively . These transcripts were terminated after 100-150 bases, at terminators designated Tori-l and Tori-r . Transcription from the 16 kd promoter, which lies clockwise adjacent to oriC and promotes transcription toward oriC, started at position 757 and gave transcripts with 3' ends at several positions within and to the left of the minimal replication origin . However, the majority of transcripts traversed the whole oriC region, and were not terminated within the DNA segment tested . Transcription of the chromosomal 16 kd gene was negatively regulated by DnaA protein and positively affected by dam methylation . The possible function of these transcripts is discussed.

J Biol Chem, 1987 Mar 25, 262(9), 4252 - 9
Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase; O'Donnell ME et al.; The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease . The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide) . Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM) . The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers . Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle . Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles . ATP (or dATP) is not required to initiate or sustain highly processive synthesis . The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.

J Biol Chem, 1987 Mar 25, 262(9), 4231 - 40
Complementation of mutations and nucleotide sequence of FAS1 gene encoding beta subunit of yeast fatty acid synthase; Chirala SS et al.; The yeast fatty acid synthase is a complex (alpha 6 beta 6) of two multifunctional proteins alpha and beta . The alpha subunit (Mr 212,000) contains two of the seven enzymatic activities required for the synthesis of fatty acids and the site for attachment of the prosthetic group 4'-phosphopantetheine . The beta subunit (Mr 203,000) contains the remaining five activities . Cloning of the genes encoding the alpha and beta proteins has been reported (Kuziora, M . A., Chalmers, J . H., Jr., Hitzeman, R . A., Douglas, M . G., and Wakil, S . J . (1983) J . Biol . Chem . 258, 11648-11653) . In the present study it is shown that two of the clones containing the beta subunit gene, YEpFAS1 and YEp33F1, are not identical . The clone YEp33F1 contains the gene that codes for the entire beta subunit while YEpFAS1 is missing approximately half of the gene at the 3' end . Despite this loss, YEpFAS1 is still able to complement a fas1 mutation at the enoyl reductase domain . This complementation does not occur by recombination, rather a small mRNA is produced in cells transformed with YEpFAS1 and is translated into a protein of molecular weight of approximately 125,000 which is immunologically reactive with yeast fatty acid synthase antibodies . The data suggest that this truncated beta subunit interacts with the mutant alpha 6 beta 6 complex to restore fatty acid synthesis to the cell . The nucleotide sequence of the FAS1 gene cloned in YEp33F1 DNA, which encodes the beta subunit of fatty acid synthase, was determined . The coding region consists of 5940 base pairs (bp) and could encode a protein of 1980 amino acids with a calculated molecular weight of 220,077 . A major transcriptional start point was mapped to a position of about 330 bp upstream from the first ATG codon . The termination of transcription was mapped at about 300 bp downstream from the first TGA stop codon . The sequence of the beta subunit protein does not appear to be similar to any other sequenced protein . The sites of the active seryl groups for the acetyltransacylase and malonyl/palmitoyl transacylase were identified from known amino acid sequences to be residues 274 and 1808, respectively . Putative binding sites for FMN and NADPH were suggested based on similarities with amino acid sequences of known flavin and pyridine nucleotide enzymes, respectively.

J Biol Chem, 1987 Mar 25, 262(9), 4011 - 6
recA protein-promoted ATP hydrolysis occurs throughout recA nucleoprotein filaments; Brenner SL et al.; When recA protein binds cooperatively to single-stranded DNA to form filamentous nucleoprotein complexes, it becomes competent to hydrolyze ATP . No correlation exists between the ends of such complexes and the rate of ATP hydrolysis . ATP hydrolysis is not, therefore, restricted to the terminal subunits on cooperatively bound recA oligomers, but occurs throughout the complex . Similarly, during recA protein-promoted branch migration (during DNA strand exchange), ATP hydrolysis is not restricted to recA protein monomers at the branch point . DNA cofactors of lengths varying from 16 bases to over 12,000 bases support ATP hydrolysis . The maximum value of kcat at infinite DNA concentration is about 29/min independent of the length of the DNA cofactor . The apparent dissociation constant, however, is a strong function of DNA length, providing evidence for a minimum site size of 30-50 bases for efficient binding of recA protein.

J Biol Chem, 1987 Mar 25, 262(9), 3940 - 3
Stringent control in Escherichia coli applies also to transcription by T7 RNA polymerase; Yamagishi M et al.; During amino acid starvation the synthesis of rRNA and tRNA is specifically inhibited (stringently controlled) in wild type Escherichia coli but not in relaxed strains carrying the relA mutation . We have found that the in vivo transcription of a hybrid rrnB rRNA operon, in which the normal promoter region has been replaced by the lambda PL promoter, is under stringent control even though this promoter lacks the "stringent discriminator" sequence postulated to be required for stringent control . Furthermore, we have found that transcription of the rrnB operon from a phage T7 promoter, as well as T7 genes in general, by phage T7 RNA polymerase is also subject to stringent control in vivo . These results are consistent with the idea that stringent control acts in a relatively nonspecific manner to inhibit some step(s) in transcription that are often rate-limiting for very active transcription . The relative simplicity of transcription by phage T7 RNA polymerase should offer a good system to study the molecular mechanisms of stringent control.

Biochemistry, 1987 Mar 24, 26(6), 1586 - 91
A preferential role for lysyl-tRNA4 in the synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by an arginyl-tRNA synthetase-lysyl-tRNA synthetase complex from rat liver; Hilderman RH et al.; The synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) can be catalyzed in vitro by a tetrameric tRNA synthetase complex from rat liver containing two lysyl-tRNA synthetase and two arginyl-tRNA synthetase subunits . This reaction required ATP, AMP, 50-100 microM zinc, and inorganic pyrophosphatase . We show here that AMP can be omitted from the reaction and that the zinc levels can be markedly reduced provided catalytic amounts of tRNA(Lys) are added to the reaction mixture . Ap4A synthesis with purified tRNA(Lys) isoacceptors showed that the minor species, tRNA(4Lys), was 3-fold more active than either of the two major tRNA(Lys) species, tRNA(2Lys) and tRNA(5Lys) . No activity could be demonstrated with tRNA(Lys) from Escherichia coli or with tRNA(Lys) or tRNA(Phe) from yeast . Aminoacylation of tRNA(4Lys) was strictly required as determined by the fact that Ap4A synthesis was not observed until aminoacylation was nearly complete, inhibitors of aminoacylation blocked Ap4A synthesis, and there was a strict requirement for added lysine . None of the above observations could be demonstrated, however, when lysyl-tRNA(Lys) was directly supplied to the reaction mixture . Optimum Ap4A synthesis was obtained by the addition of 1 mol of tRNA(Lys)/mol of the synthetase complex . This reaction is unique because it does not require the prior formation of an aminoacyl-AMP intermediate and because it can actively synthesize Ap4A at physiological zinc concentrations . The preferential role for tRNA(4Lys) in Ap4A synthesis is consistent with its prior implication in cell division.

Biochemistry, 1987 Mar 24, 26(6), 1704 - 9
Identification and amino acid sequence of the deoxynucleoside triphosphate binding site in Escherichia coli DNA polymerase I; Basu A et al.; We have labeled the large fragment of Escherichia coli DNA polymerase I (Pol I) with pyridoxal 5'-phosphate, a substrate binding site directed reagent for DNA polymerases {Modak, M . J . (1976) Biochemistry 15, 3620-3626} . A covalent attachment of pyridoxal phosphate to Pol I results in the loss of substrate binding as well as the polymerase activity . The inactivation was found to be strictly dependent on the presence of a divalent metal ion . Four moles of pyridoxal phosphate was found to react per mole of the enzyme, while in the presence of substrate deoxynucleoside triphosphate only 3 mol of pyridoxal phosphate was bound . To identify the substrate-protected site on the enzyme, tryptic peptides from enzyme labeled with pyridoxal phosphate and tritiated borohydride, in the presence and absence of substrate, were resolved on a C-18 reverse-phase column . A single peptide containing the substrate-protected site was identified and further purified . The amino acid composition and sequence analysis of this peptide revealed it to span residues 756-775 in the primary acid sequence of Pol I . Lys-758 of this sequence was found to be the site of the pyridoxal phosphate reaction . It is therefore concluded that Lys-758 is the site of binding for the metal chelate form of nucleotide substrates in E . coli DNA polymerase I.

Biochemistry, 1987 Mar 24, 26(6), 1683 - 8
Definitive characterization of human thymine glycol N-glycosylase activity; Higgins SA et al.; An N-glycosylase activity that released cis-{3H}-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol, TG) from chemically oxidized poly(dA-{3H}dT) was unambiguously characterized both in extracts of HeLa cells and in purified Escherichia coli endonuclease III . This was accomplished by use of microderivatization procedure that quantitatively converted cis-TG to 5-hydroxy-5-methylhydantoin (HMH) . The reaction products were analyzed by high-pressure liquid chromatography before and after derivatization by using cis-{14C}TG and {14C}HMH, which had been independently synthesized, as reference compounds . This technique facilitated construction of a v/{E}t plot for the enzyme activity in HeLa cells, permitting estimation of its specific activity . The results obtained prove the existence of both human and bacterial N-glycosylase activities that effect removal of TG from DNA.

Biochemistry, 1987 Mar 24, 26(6), 1563 - 8
RNA binding site of R17 coat protein; Romaniuk PJ et al.; The specific interaction between R17 coat protein and its target of translational repression at the initiation site of the R17 replicase gene was studied by synthesizing variants of the RNA binding site and measuring their affinity to the coat protein by using a nitrocellulose filter binding assay . Substitution of two of the seven single-stranded residues by other nucleotides greatly reduced the Ka, indicating that they are essential for the RNA-protein interaction . In contrast, three other single-stranded residues can be substituted without altering the Ka . When several of the base-paired residues in the binding site are altered in such a way that pairing is maintained, little change in Ka is observed . However, when the base pairs are disrupted, coat protein does not bind . These data suggest that while the hairpin loop structure is essential for protein binding, the base-paired residues do not contact the protein directly . On the basis of these and previous data, a model for the structural requirements of the R17 coat protein binding site is proposed . The model was successfully tested by demonstrating that oligomers with sequences quite different from the replicase initiator were able to bind coat protein.

Biochemistry, 1987 Mar 24, 26(6), 1592 - 7
Ribosome protection by tRNA derivatives against inactivation by virginiamycin M: evidence for two types of interaction of tRNA with the donor site of peptidyl transferase; Chinali G et al.; Virginiamycin M (VM) was previously shown to interfere with the function of both the A and P sites of ribosomes and to inactivate tRNA-free ribosomes but not particles bearing peptidyl-tRNA . To explain these findings, the shielding ability afforded by tRNA derivatives positioned at the A and P sites against VM-produced inactivation was explored . Unacylated tRNA(Phe) was ineffective, irrespective of its position on the ribosome . Phe-tRNA and Ac-Phe-tRNA provided little protection when bound directly to the P site but were active when present at the A site . Protection by these tRNA derivatives was markedly enhanced by the formation of the first peptide bond and increased further upon elongation of peptide chains . Most of the shielding ability of Ac-Phe-tRNA and Phe-tRNA positioned at the A site was conserved when these tRNAs were translocated to the P site by the action of elongation factor G and GTP . Thus, a 5-10-fold difference in the protection afforded by these tRNAs was observed, depending on their mode of entry to the P site . This indicates the occurrence of two types of interaction of tRNA derivatives with the donor site of peptidyl transferase: one shared by acylated tRNAs directly bound to the ribosomal P site (no protection against VM) and the other characteristic of aminoacyl- or peptidyl-tRNA translocated from the A site (protection of peptidyl transferase against VM) . To explain these data and previous observations with other protein synthesis inhibitors, a new model of peptidyl transferase is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Mar 24, 26(6), 1526 - 31
Strong inhibitory effect of furanoses and sugar lactones on beta-galactosidase Escherichia coli; Huber RE et al.; Various sugars and their lactones were tested for their inhibition of beta-galactosidase (Escherichia coli) . L-Ribose, which in the furanose form has a hydroxyl configuration similar to that of D-galactose at positions equivalent to the 3- and 4-positions of D-galactose, was a very strong inhibitor, and D-lyxose, which in the furanose form also resembles D-galactose, was a much better inhibitor than expected . Structural comparisons prelude the pyranose forms of these sugars from being significant contributors to the inhibition, and inhibition at different temperatures (at which there are different furanose concentrations) strongly supported the conclusion that the furanose form is inhibitory . Studies with sugar derivatives that can only be in the furanose form also supported the conclusion . This is the first report of the inhibitory effect of furanose on beta-galactosidase . Lactones were also inhibitory . Every lactone tested was much more inhibitory than was its parent sugar . D-Galactonolactone was especially good . Experiments indicated that it was D-galactono-1,5-lactone rather than D-galactono-1,4-lactone which was inhibitory . Inhibition of beta-galactosidases from mammalian sources by lactones has been reported previously, but this is the first report of the effect of beta-galactosidase from E . coli . Since furanoses in the envelope form are analogous (in some ways) to half-chair or sofa conformations and since lactones with six-membered rings probably have half-chair or sofa conformations, the results indicate that beta-galactosidase probably destabilizes its substrate into a planar conformation of some type and that the galactose in the transition state may, therefore, also be quite planar.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Mar 24, 26(6), 1723 - 7
Inactivation of Escherichia coli glycerol kinase by 5'-{p-(fluorosulfonyl)benzoyl}adenosine: protection by the hydrolyzed reagent; Pettigrew DW; Incubation of Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) with 5'-{p-(fluorosulfonyl)benzoyl}adenosine (FSO2BzAdo) at pH 8.0 and 25 degrees C results in the loss of enzyme activity, which is not restored by the addition of beta-mercaptoethanol or dithiothreitol . The FSO2BzAdo concentration dependence of the inactivation kinetics is described by a mechanism that includes the equilibrium binding of the reagent to the enzyme prior to a first-order inactivation reaction in addition to effects of reagent hydrolysis . The hydrolysis of the reagent has two effects on the observed kinetics . The first effect is deviation from pseudo-first-order kinetic behavior due to depletion of the reagent . The second effect is the novel protection of the enzyme from inactivation due to binding of the sulfonate hydrolysis product . The rate constant for the hydrolysis reaction, determined independently from the kinetics of F- release, is 0.021 min-1 under these conditions . Determinations of the reaction stoichiometry with 3H-labeled FSO2BzAdo show that the inactivation is associated with the covalent incorporation of 1.08 mol of reagent/mol of enzyme subunit . Ligand protection experiments show that ATP, AMP, dAMP, NADH, 5'-adenylyl imidodiphosphate, and the sulfonate hydrolysis product of FSO2BzAdo provide protection from inactivation . The protection obtained with ATP is not dependent on Mg2+ . Less protection is obtained with glycerol, GMP, etheno-AMP, and cAMP . No protection is obtained with CMP, UMP, TMP, etheno-CMP, GTP, or fructose 1,6-bisphosphate . The results are consistent with modification by FSO2BzAdo of a single adenine nucleotide binding site per enzyme subunit.

Biochemistry, 1987 Mar 24, 26(6), 1539 - 46
Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II); Gunderson SI et al.; The interactions of T7 RNA polymerase with T7 late promoters were studied by using quantitative footprinting with methidiumpropyl-EDTA X Fe(II) {MPE-Fe(II)} as the DNA cleaving agent . Class II and class III T7 promoters have a highly conserved 23 base pair sequence from -17 to +6 . Among class III promoters the -22 to -18 region is also highly conserved . For a class II promoter, T7 RNA polymerase protects the -17 to -4 region from MPE-Fe(II) cleavage; when GTP is present, protection extends from -17 to +5 (noncoding strand) . For a class III promoter, protection extends from -20 to -4 and in the presence of GTP from -20 to +5 (noncoding strand) . The protected regions for the coding strands of both promoters were nearly identical with that seen for the noncoding strands . The binding constant for the class III promoter is (4 +/- 1.5) X 10(7) M-1 and in the presence of GTP increases to (10 +/- 1.7) X 10(7) M-1 . These binding constants are about 1000 and 200 times greater, respectively, than values reported previously {Ikeda, R . A., & Richardson, C . C . (1986) Proc . Natl . Acad . Sci . U.S.A . 83, 3614-3618} . The differences in binding constants are probably due to tRNA and high salt used in those earlier experiments . Both tRNA and high salt (greater than 50 mM NaCl and greater than 10 mM MgCl2) inhibit the binding of the polymerase to the promoter . Optimal binding conditions occur at 2-5 mM MgCl2 and 0-10 mM NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1987 Mar 23, 213(2), 381 - 4
Mode of inhibition of sodium azide on H+-ATPase of Escherichia coli; Noumi T et al.; Sodium azide inhibited multi-site (steady-state) ATPase activity of E . coli F1 more than 90%, but did not affect uni-site (single-site) ATPase activity . Thus azide inhibited multi-site ATPase activity by lowering catalytic cooperativity . Consistent with this observation, azide changed the ligand-induced fluorescence response of aurovertin bound to F1.

Science, 1987 Mar 20, 235(4795), 1489 - 92
Molecular cloning and expression of a human B-cell growth factor gene in Escherichia coli; Sharma S et al.; A human B-cell growth factor (BCGF) (12 kilodaltons) supports the clonal proliferation of B lymphocytes . A clone was isolated that contained the proper structural sequence to encode biologically active, 12-kilodalton BCGF in Escherichia coli and to hybridize to a specific messenger RNA, identified by in vitro translation in Xenopus laevis oocytes . A relatively hydrophobic region of 18 amino acids was found at the amino terminal of the 124-amino acid-long polypeptide . The carboxyl terminal is composed of at least 32 amino acids that are derived from nucleotide sequences bearing significant homology to the Alu repeat family.

Eur J Pharmacol, 1987 Mar 17, 135(2), 117 - 22
Protective effect of WEB 2086, a novel antagonist of platelet activating factor, in endotoxin shock; Casals-Stenzel J; WEB 2086, a novel specific platelet activating factor (PAF) antagonist derived from triazolodiazepines, inhibited in a dose-related manner the hypotensive and lethal effect of PAF as well as of E . coli endotoxin in the rat . The hypotension induced by endotoxin (15 mg/kg i.v.) or PAF (30 ng/(kg X min) i.v.) in anaesthetized rats was prevented by oral (1-10 mg/kg) and inhibited or reversed by i.v . (0.1-5.0 mg/kg or 0.1-1.0 mg/kg) doses of WEB 2086 . Similar oral and i.v . doses of WEB 2086 protected conscious rats from PAF (15 micrograms/kg i.v.)- and endotoxin (7.5 mg/kg i.v.)-induced death . The results obtained with WEB 2086 confirm that PAF has an important role in the pathophysiology of endotoxin shock . This compound may have a therapeutic effect in human septic shock.

Eur J Biochem, 1987 Mar 16, 163(3), 653 - 8
The N-terminal region of Escherichia coli lactose permease mediates membrane contact of the nascent polypeptide chain; Stochaj U et al.; Plasmids encoding N-terminal segments of the Escherichia coli lactose permease (also referred to as lactose carrier) have been used to analyze the biosynthesis and membrane insertion of this complex integral protein of the cytoplasmic membrane . Such truncated polypeptides were found to be stably associated with the membrane and to resemble the full-length protein with respect to their solubilization characteristics . Membrane-bound and free cytoplasmic polysomes were prepared from plasmid-bearing cells and incubated in the presence of {35S}methionine to permit completion of polypeptides initiated in vivo . Under these conditions, lactose permease was found to be radiolabeled in the fraction of membrane-bound polysomes; beta-galactosidase, used as a control, was translated almost exclusively by free polysomes . From similar experiments with N-terminal segments of lactose permease, we estimate that at most a polypeptide of 120 amino acid residues emerging from the ribosome is needed to target the nascent chain to the lipid bilayer and to mediate attachment of the ribosome to the membrane during elongation . Additional data support the idea that even shorter N-terminal sequences of 50 and 71 amino acid residues contain sufficient 'information' to provide contact with the membrane.

Eur J Biochem, 1987 Mar 16, 163(3), 443 - 8
Characterization of an artificial bifunctional enzyme, beta-galactosidase/galactokinase, prepared by gene fusion; Bulow L; An artificial bifunctional enzyme, beta-galactosidase/galactokinase, has been prepared by gene fusion . The hybrid protein catalyzes the hydrolysis of lactose followed by phosphorylation of galactose . The protein has been purified using DEAE-Sephacel chromatography and gel filtration on Sephacryl S-400 Superfine . The configuration of the hybrid protein is mainly tetrameric but also higher aggregates can be detected . The monomer Mr is 160,000 as judged from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and the native Mr has been calculated to be 600,000-650,000 from gel filtration experiments . beta-Galactosidase/galactokinase has different thermostability curves, pH/activity profiles and Km values as compared with the native enzymes . By using a third enzyme, galactose dehydrogenase, which competes with galactokinase for the galactose formed by beta-galactosidase, substrate channeling can be detected . This proximity effect becomes even more pronounced in an assay mixture containing poly(ethylene glycol).

Eur J Biochem, 1987 Mar 16, 163(3), 591 - 8
Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans; Voordouw G et al.; Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant . Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present . The wild-type and mutant forms of Rubisco have all been purified to homogeneity . The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A . nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM) . The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b) . However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme . The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant . Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.

J Biol Chem, 1987 Mar 15, 262(8), 3788 - 9
Crystallization and preliminary x-ray investigation of thymidine phosphorylase from Escherichia coli; Cook WJ et al.; Crystals of thymidine phosphorylase from Escherichia coli have been grown from solutions of ammonium sulfate . The crystals are tetragonal, space group P4(1)2(1)2 or P4(3)2(1)2; the axes are a = 132.0 (1) and c = 67.2 (1) A . The crystals are quite stable to x-rays and diffract beyond 2.6-A resolution . The molecule is a dimer and utilizes the 2-fold symmetry of the space group, resulting in one subunit per asymmetric unit.

J Biol Chem, 1987 Mar 15, 262(8), 3685 - 9
Purification and NMR characterization of acyl carrier protein; Holak TA et al.; The acyl carrier protein preparation obtained using the 2-propanol method of Rock and Cronan (Rock, C . O., and Cronan, J . E., Jr . (1981) Methods Enzymol . 71, 341-351) can be further purified with reversed-phase high-performance liquid chromatography . A homogeneous sample of acyl carrier protein is obtained as determined by NMR and reversed-phase high-performance liquid chromatography.

Biochem J, 1987 Mar 15, 242(3), 661 - 5
Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308; el-Mansi EM et al.; A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed . This plasmid complements an aceA mutation . The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro . Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.

J Biol Chem, 1987 Mar 15, 262(8), 3548 - 52
Isolation of a rat mitochondrial release factor . Accommodation of the changed genetic code for termination; Lee CC et al.; A single release factor has been isolated and partially purified from rat mitochondria . It requires ethanol in addition to the specific termination codon when assayed in a heterologous system with Escherichia coli ribosomes . The factor recognizes the codons UAA and UAG but not UGA, and therefore it has been designated mtRF-1 . A factor of the bacterial RF-2 type, which in E . coli recognizes UGA, or of the mammalian type, which recognizes all three termination codons, has not been detected in mitochondria . The absence of a factor responding to UGA accommodates the use of this codon as a signal for tryptophan in the rat mitochondrial genetic code . The mtRF-1 could translate all of the known termination codons in the rat mitochondrial genome . It does not respond to AGG and AGA which in bovine and human mitochondrial DNA code for termination but which in rat mitochondria may not code for either an amino acid or for termination.

J Biol Chem, 1987 Mar 15, 262(8), 3462 - 71
Isolation and structure elucidation of an epoxide derivative of the hypermodified nucleoside queuosine from Escherichia coli transfer RNA; Phillipson DW et al.; A new nucleoside has been identified in tRNATyr from Escherichia coli MRE 600, where it replaces the highly modified nucleoside queuosine . The nucleoside is also present in a large amount relative to queuosine in mixed tRNA from E . coli strains MRE 600 and W (from which it was isolated for characterization) . The new nucleoside has been characterized as an epoxy derivative of queuosine: 7-(5-{(2,3-epoxy-4,5-dihydroxycyclopent-1-yl)amino}methyl)-7-de azaguanosine, oQ, based on data from directly combined liquid chromatography/mass spectrometry, high resolution mass spectrometry, and proton NMR spectroscopy . Nucleoside oQ is also present in small amounts in mixed tRNA from E . coli B . Isomerization of oQ occurs readily under alkaline conditions to give a rearranged product, oQ', characterized as 7-(5-{(3,4-epoxy-2,5-dihydroxycyclopent-1-yl)amino}methyl)-7-deaza guanosine . The present finding constitutes the first report of epoxide formation during post-transcriptional processing of RNA.

J Immunol, 1987 Mar 15, 138(6), 1871 - 6
Antibody-independent activation of C1 . II . Evidence for two classes of nonimmune activators of the classical pathway of complement; Peitsch MC et al.; Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes . CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s . Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s . Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH . However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s . The same pattern was observed in the case of smooth E . coli and a semi-rough E . coli strain . DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r . Thus, nonimmune activators can be classified into two distinct categories . "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E . coli strain J5 can activate C1 in the presence of C1-INH . By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s . C1s-binding to C1q is a critical factor for the activation process in this group . In the case of "weak" activators, such as E . coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed . As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.

J Biol Chem, 1987 Mar 15, 262(8), 3730 - 8
The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31; Muller UR et al.; The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix . Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome . A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31 . The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C . They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure . Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state . Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable . Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.

J Biol Chem, 1987 Mar 15, 262(8), 3718 - 25
cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta; Ye RD et al.; Two nearly full-length cDNAs for placental plasminogen activator inhibitor (PAI) have been isolated from a human placenta lambda gt11 cDNA library . One positive (lambda PAI-75.1) expressed a protein that could adsorb and purify anti-PAI antibodies . The expressed protein inhibited the activity of human urokinase in a fibrin autography assay, and formed a 79-kDa (reduced) covalent complex with 125I-urokinase that could be immunoprecipitated with anti-PAI . The cDNA insert of the longer isolate (lambda PAI-75.15) consisted of 1909 base pairs, including a 5'-noncoding region of 55 base pairs, an open reading frame of 1245 base pairs, a stop codon, a 3'-noncoding region of 581 base pairs, and a poly(A) tail . The size of the mRNA was estimated to be 2.0 kilobases by Northern blot analysis . The translated amino acid sequence consisted of 415 amino acids, corresponding to a 46.6-kDa protein . The sequence was related to members of the serpin gene family, particularly ovalbumin and the chicken gene Y protein . Like these avian proteins, placental PAI appears to lack a cleavable NH2-terminal signal peptide . Residues 347-376 were identical to the partial sequence reported recently for a PAI isolated from the human monocytic U-937 cell line . Placental PAI mRNA was apparently expressed at low levels in human umbilical vein endothelial cells, but was not detectable in HepG2 hepatoma cells . It was present in U-937 cells and was inducible at least 10-fold by phorbol 12-myristate 13-acetate . Thus placental PAI is a unique member of the serpin gene family, distinct from endothelial-type PAI . It is probably identical to monocyte-macrophage PAI.

J Biol Chem, 1987 Mar 15, 262(8), 3690 - 6
Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase; Koerner TJ et al.; The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA . A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group . The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined . The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase . Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase . Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene . Although the E . coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.

J Biol Chem, 1987 Mar 15, 262(8), 3524 - 7
Chemical synthesis and expression of a cassette adapted ubiquitin gene; Ecker DJ et al.; A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter . The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides . To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons . In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels . The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.

Biochem J, 1987 Mar 15, 242(3), 809 - 15
Modulation of monocyte complement synthesis by interferons; Hamilton AO et al.; Recombinant Escherichia coli-derived gamma-interferon has been shown to stimulate synthesis of the second component of complement (C2), factor B and C1 inhibitor, but to inhibit synthesis of the third component (C3) . alpha- and beta-interferons stimulate synthesis of factor B and C3 inhibitor, inhibit C5 synthesis but do not alter synthesis of C2 . alpha- and beta-interferons act synergistically with gamma-interferon to enhance both factor B and C1-inhibitor synthesis.

Vet Rec, 1987 Mar 14, 120(11), 250 - 2
Effect of vaccination of the dam on rotavirus infection in young calves; McNulty MS et al.; Vaccination of cows with a combined, inactivated, adjuvanted rotavirus and Escherichia coli vaccine resulted in increased neutralising antibody titres to rotavirus in serum and colostral whey . Evidence was obtained that vaccination resulted in a decreased incidence of rotavirus shedding and of abnormal faeces or diarrhoea in young calves fed colostrum and milk from the vaccinated dams . The E coli component of the vaccine was not evaluated because no natural challenge was evident.

JAMA, 1987 Mar 13, 257(10), 1347 - 50
Prevention of travelers' diarrhea by the tablet formulation of bismuth subsalicylate; DuPont HL et al.; Within 48 hours of arrival in Mexico, 182 US students participated in a study to compare the efficacy of two dosages of bismuth subsalicylate (262 mg per tablet) as a prophylactic agent against diarrhea . The students were randomly assigned to receive two tablets (high dose) or one tablet (low dose) of bismuth subsalicylate four times daily or a placebo four times daily during a three-week period . Among these completing the trial, diarrhea (four or more unformed stools in 24 hours or three in eight hours, plus one other symptom) occurred in seven (14%) of 51 receiving the high-dose regimen compared with 15 (24%) of 63 receiving the low-dose regimen and 23 (40%) of 58 in the placebo group . Protection rates were 65% for high-dose and 40% for low-dose bismuth subsalicylate . Diarrhea caused by enterotoxigenic Escherichia coli was found in one student receiving the high-dose regimen, in no students receiving the low-dose regimen, and in seven placebo-treated subjects . Bismuth subsalicylate was well tolerated; the most common side effects were blackening of tongues and stools . Bismuth subsalicylate use in both dosages was associated with tinnitus at a low, clinically insignificant frequency of 1.2 days per 100 days of treatment . The dosage of two tablets of bismuth subsalicylate four times daily (2.1 g/d) appears to be a safe and effective means of reducing the occurrence of travelers' diarrhea among persons at risk for periods up to three weeks.

Biochem Biophys Res Commun, 1987 Mar 13, 143(2), 638 - 44
The distinct role of catalase and DNA repair systems in protection against hydrogen peroxide in Escherichia coli; Yonei S et al.; The katEkatG mutant of E . coli, UM1, had no assayable catalase activities in the extract and showed increased (about 20 fold) sensitivity to killing by H2O2 when compared with its parental strain CSH7 . The mutant strain was able to reactivate H2O2-damaged lambda phage . On the other hand, recA and polA mutants were also highly sensitive to H2O2, but they had normal level of catalase activities . RecA derivatives of UM1 were much more sensitive to H2O2 than UM1 and recA strains . The induction of umu operon occurred in UM1 at lower (1/10-1/20) doses of H2O2 than in CSH7 . From the results it is concluded that the lethal effect of H2O2 is due to DNA damage induced by it and that catalase and DNA repair systems have a distinct role in protection against H2O2 in E . coli.

Cell, 1987 Mar 13, 48(5), 855 - 65
Chi-stimulated patches are heteroduplex, with recombinant information on the phage lambda r chain; Rosenberg SM; Generalized recombination in Escherichia coli is elevated near Chi sites . In vitro, RecBCD enzyme can nick Chi a few nucleotides 3' of the terminal GG of the Chi sequence (5'-GCTGGTGG) . The simplest model in which this nick at Chi participates in Chi function predicts that in phage lambda, Chi-stimulated recombinants not crossed-over for flanking markers (patches) should be heteroduplex, with recombinant information on the lambda I chain . I report here that patches are heteroduplex, but that recombinant information occurs primarily on the lambda r chain . This result rules out the simplest model in which the nick at Chi promotes initiation of recombination, forces reconsideration of Chi's role in recombination, and bears on molecular models for Rec-mediated recombination.

Science, 1987 Mar 13, 235(4794), 1370 - 3
Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly; Marks CB et al.; It is a generally accepted principle of biology that a protein's primary sequence is the main determinant of its tertiary structure . However, the mechanism by which a protein proceeds from an unfolded, disordered state to a folded, relatively well-ordered, native conformation is obscure . Studies have been initiated to examine the "genetics" of protein folding, with mutants of bovine pancreatic trypsin inhibitor (BPTI) being used to explore the nature of the specific intramolecular interactions that direct this process . Previous work with BPTI chemically modified at cysteines 14 and 38 indicated that transient disulfide bond formation by these residues contributed to efficient folding at 25 degrees C . In the present work, mutants of BPTI in which these cysteines were replaced by alanines or threonines were made and the mutant proteins were produced by a heterologous Escherichia coli expression system . At 25 degrees C in vitro, the refolding behavior of these mutants was characterized by a pronounced lag . However, when expressed at 37 degrees C in E . coli, or when refolded at 37 degrees or 52 degrees C in vitro, the mutant proteins folded readily into the native conformation, albeit at a rate somewhat slower than that exhibited by wild-type BPTI . These results indicate that, at physiological temperatures, BPTI lacking cysteines 14 and 38 can refold quantitatively.

J Immunol Methods, 1987 Mar 12, 97(2), 275 - 9
A simple method for the production of specific antiserum to protein encoded in cloned genes . Immunization with precipitin lines; Shapiro SZ et al.; A simple technique for raising specific antisera to protein encoded by cloned genes is described . The procedure involves preparation of an antiserum to Escherichia coli beta-galactosidase and the use of that serum to immunoprecipitate a fusion protein in a crossed immunoelectrophoresis gel followed by immunization with fusion protein precipitin arcs . An antiserum was prepared against protein encoded by an open reading frame in a dispersed repeated DNA sequence found in the protozoan Trypanosoma brucei . This serum recognized a polypeptide doublet of 33.5 and 32.5 kDa on immunoblots prepared from extracts of T . brucei . The method described should be applicable to other investigations where an immunochemical reagent against protein encoded by a cloned gene is desired.

Nature, 1987 Mar 12-18, 326(6109), 149 - 53
Molecular cloning of a protective antigen of schistosomes; Balloul JM et al.; The complementary DNA sequence encoding the Mr 28,000 antigen of Schistosoma mansoni has been isolated and expressed in Escherichia coli . Experimental vaccination of rats, hamsters and monkeys with a recombinant fusion protein induces a strongly cytotoxic antibody response . Immunization of rats and hamsters with this protein leads to significant protection against a natural challenge infection with live cercariae.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2137 - 55
The complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli K-12; Lawther RP et al.; In this report we present the complete nucleotide sequence of the ilvGMEDA operon of Escherichia coli . This operon contains five genes encoding four of the five enzymes required for the biosynthesis of isoleucine and valine . We identify and describe the coding regions for these five structural genes and the structural and functional features of the flanking and internal regulatory regions of this operon . This new information contributes to a more complete understanding of the overall control of the biosynthesis of isoleucine and valine.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2213 - 20
Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12; Liljestrom PL et al.; Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively . We report the cloning and sequencing of the melA gene . The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd . The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position . A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related . However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2013 - 28
Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species; Johnson KR et al.; Myeloperoxidase is a component of the microbicidal network of polymorphonuclear leukocytes . The enzyme is a tetramer consisting of two heavy and two light subunits . A large proportion of humans demonstrate genetic deficiencies in the production of myeloperoxidase . As a first step in analyzing these deficiencies in more detail, we have isolated cDNA clones for myeloperoxidase from an expression library of the HL-60 human promyelocytic leukemia cell line . Two overlapping plasmids (pMP02 and pMP062) were identified as myeloperoxidase cDNA clones based on the detection with myeloperoxidase antiserum of 70 kDa protein expressed in pMP02-containing bacteria and a 75 kDa polypeptide produced by hybridization selection and translation using pMP062 and HL-60 RNA . Formal identification of the clones was made by matching the predicted amino acid sequences with the amino terminal sequences of the heavy and light subunits . Both subunits are encoded by one mRNA in the following order: pre-pro-sequences--light subunit--heavy subunit . The molecular weight of the predicted primary translation product is 83.7 kDa . Northern blots reveal two size classes of hybridizing RNAs (approximately 3.0-3.3 and 3.5-4.0 kilobases) whose expression is restricted to cells of the granulocytic lineage and parallels the changes in enzymatic activity observed during differentiation.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2029 - 42
Fertility inhibition gene of plasmid R100; McIntire SA et al.; The fin0 gene of R100 was isolated from the Fin0+ transducing phage VA lambda 57 . The limits of the gene were determined by BAL31 digestions and by analysis of deletion mutations derived from an internal restriction site . The DNA sequence contained an open reading frame of 558 nucleotides that would encode a protein of 21,268 daltons . Synthesis of such a protein was observed only when the fragment was cloned in front of the TAC promoter . Deletions entering the large open reading frame from either end were Fin0-, while internal frame shift mutations retained high Fin0 activity . One such strain had a 13 bp internal deletion that would produce a protein of 63 amino acid residues of which 21 were basic . We were consequently unable to rigorously establish that the 558 base orf encoded a fin0 product . The strand opposite the large open reading frame contained several transcription termination signals, and it is possible that the active gene product is one or two small RNAs from this strand.

Nucleic Acids Res, 1987 Mar 11, 15(5), 2089 - 101
The isolation and characterization of RNA coded by the micF gene in Escherichia coli; Andersen J et al.; A new species of micF RNA, which contains 93 nucleotides (a 4.5S size), was isolated from Escherichia coli . The sequence of the 4.5S micF RNA corresponds to positions G82 through U174 of the micF gene . The 5' terminal end of this smaller micF RNA is triphosphorylated signifying that it is a primary transcript . Its promoter region, which is situated within the greater micF structural gene, has been identified and characterized by lacZ fusion analysis . A 6S micF RNA species, which has a base composition predicted for a transcript from the full length gene has also been detected; however, the 4.5S micF RNA is the predominant species . The work clearly shows by biochemical identification the presence of chromosomally encoded micF RNA.

Biochemistry, 1987 Mar 10, 26(5), 1406 - 11
Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin; Kelley RF et al.; The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 degrees C . For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues . The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements . Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol . The kinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 degrees C . Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and the transient accumulation of a compact nativelike intermediate during refolding . The intermediate can incorporate the nonnative configuration and can accommodate its isomerization . No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein.

Biochemistry, 1987 Mar 10, 26(5), 1322 - 6
Structure of human tumor necrosis factor alpha derived from recombinant DNA; Davis JM et al.; Recombinant DNA derived tumor necrosis factor alpha, when expressed at a high level in Escherichia coli, appeared in the pellet and soluble fractions of disrupted cells . The protein was purified from the pellet fraction by solubilizing it in urea and reducing agent and was refolded into a buffer without these additives . The structure of the protein was identical with that purified from the soluble fraction without exposure to both reducing and denaturing agents, as demonstrated by circular dichroism, gel filtration, and sulfhydryl titration . As a reflection of the structural similarity, both purified proteins showed identical cytolytic activity on mouse L929 cells . The protein was characterized as an essentially nonhelical and beta-sheet-rich structure and possibly as a noncovalently associating oligomer . Two cysteine residues form an intrapolypeptide disulfide bond.

Biochemistry, 1987 Mar 10, 26(5), 1258 - 64
Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme; Casal JI et al.; We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme . The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography . Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate . The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.

Biochemistry, 1987 Mar 10, 26(5), 1251 - 7
Effect of hydrogen peroxide on the iron-containing superoxide dismutase of Escherichia coli; Beyer WF Jr et al.; The iron-containing superoxide dismutase from Escherichia coli is inactivated by H2O2 to a limit of approximately 90% . When corrected for the H2O2-resistant portion, this inactivation was first order with respect to residual activity and exhibited a pseudo-first-order rate constant of 0.066 min-1 at 25 degrees C in 0.24 mM H2O2 at pH 7.8 . The superoxide dismutase activity remaining after treatment with H2O2 differed from the activity of the native enzyme with respect to heat stability, inhibition by azide, and inactivation by light in the presence of rose bengal and by N-bromosuccinimide . The native and the H2O2-modified enzymes were indistinguishable by electrophoresis on polyacrylamide gels . Inactivation of the enzyme by H2O2 was accompanied by loss of tryptophan and some loss of iron, but there was no detectable loss of histidine or of other amino acids . H2O2 treatment caused changes in the optical spectrum of the enzyme . Inactivation of the enzyme by H2O2 depends upon the iron at the active site . Thus, the apoenzyme and the manganese-substituted enzyme were unaffected by H2O2 . We conclude that reaction of H2O2 with the iron at the active site generates a potent oxidant capable of attacking tryptophan residues . A mechanism is proposed.

Biochemistry, 1987 Mar 10, 26(5), 1326 - 32
Sequence comparisons of complementary DNAs encoding aequorin isotypes; Prasher DC et al.; Aequorin is the Ca2+-activated photoprotein which participates in the bioluminescence from the circumoral ring of the hydromedusa Aequorea victoria . The nucleotide sequences of five aequorin cDNAs have been compared and shown to code for three aequorin isoforms . The cDNA AEQ1 contains the entire protein coding region of 196 amino acids . The other four cDNAs contain only 70-90% of the coding region and apparently code for at least two other isoforms whose amino acid sequences differ significantly from that encoded by AEQ1 . The nucleotide sequences coding for the three isotypes differ at a minimum of 54 positions out of a total of 588 nucleotides necessary to code for apoaequorin . Of these nucleotide differences, 24 account for 23 amino acid replacements, substantiating the microheterogeneity observed during sequencing of purified native aequorin {Charbonneau, H., Walsh, K.A., McCann, R.O., Prendergast, F.G., Cormier, M.J., & Vanaman, T.C . (1985) Biochemistry 24, 6762-6771} . Comparison of the deduced cDNA translations with the native protein sequences suggests the loss of seven residues from the amino terminus during purification of aequorin from Aequorea . Aequorin rapidly extracted from the jellyfish using conditions to minimize proteolysis is shown to have a larger molecular weight than that of purified native aequorin . Escherichia coli expressed aequorin encoded by AEQ1 is shown to have the same molecular weight and isoelectric point as those of one of the isotypes rapidly extracted from Aequorea.

J Mol Biol, 1987 Mar 5, 194(1), 119 - 26
Translocation makes the ribosome less compact; Spirin AS et al.; Translating ribosomes of Escherichia coli were prepared either in the pre-translocation or in the post-translocation states by a special technique based on the use of poly(U)-Sepharose columns where the template was coupled to the matrix through splittable -S-S- bridges . Elongation factors were absent from the final preparations . A neutron scattering study of the translating ribosomes in the two functional states was performed at different contrasts (various 1H2O/2H2O mixtures) . Under conditions of a high contrast for the protein constituent the radius of gyration of the post-translocation-state ribosomes was found to be slightly greater than that of the pre-translocation-state ribosomes . Using the results of this study the conclusion can be drawn that translocation is accompanied by a spatial displacement of some parts of the ribosome with a magnitude of several angstrom units.

J Biol Chem, 1987 Mar 5, 262(7), 2937 - 40
Structure of the gene encoding the circumsporozoite protein of Plasmodium yoelii . A rodent model for examining antimalarial sporozoite vaccines; Lal AA et al.; The gene encoding the circumsporozoite protein (CSP) from the rodent malaria parasite, Plasmodium yoelii, has been cloned and the nucleotide sequence has been determined . The gene encodes a protein of 367 amino acids as deduced from the nucleotide sequence . This gene is structurally similar to other Plasmodium spp . CSP genes in that it contains putative hydrophobic signal and anchor sequences at the NH2 and COOH termini, respectively, two small regions (Regions I and II) that are conserved in all CSP genes analyzed to date, and a central region containing the immunodominant repeating peptide sequence . Unlike other CSP genes, however, the immunodominant repeat region of the gene is composed of two distinctly different types of tandem repeats . One repeating unit is six amino acids (Gln-Gly-Pro-Gly-Ala-Pro) in length while the other is only four (Gln-Gln-Pro-Pro) residues long . A synthetic peptide, Gln-Gly-Pro-Gly-Ala-Pro X 3, strongly inhibits the binding of anti-CSP monoclonal antibody to sporozoite antigens while another peptide, Gln-Gln-Pro-Pro X 4, weakly inhibits the binding of this same antibody to sporozoite antigens . This work should allow the construction of a mouse model system to parallel human vaccine trials.

J Biol Chem, 1987 Mar 5, 262(7), 3060 - 4
A mutation of the c subunit of the Escherichia coli proton-translocating ATPase that suppresses the effects of a mutant b subunit; Kumamoto CA et al.; A mutation of the b subunit of the Escherichia coli proton-translocating ATPase and mutations in the gene for the a subunit that suppress its effects have been previously described (Kumamoto, C., and Simoni, R . D . (1986) J . Biol . Chem . 261, 10037-10042) . In this paper, we describe the characterization of a new mutation that partially suppresses the effects of the original b mutation . The new suppressor mutation causes the substitution of serine for alanine at position 62 of the c subunit . Biochemical studies of double mutants, carrying both b and c mutations, demonstrate that the c mutation partially restores the function of the enzyme complex.

J Biol Chem, 1987 Mar 5, 262(7), 3037 - 43
Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding; Colanduoni J et al.; The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K . Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2 . The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction . Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine . These data indicate that the amine substrate must be deprotonated for binding . Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values . Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding . Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue . In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding . A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein . A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate . The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release . A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed . Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.

Eur J Biochem, 1987 Mar 2, 163(2), 323 - 30
Recombinant-DNA-derived bovine growth hormone from Escherichia coli . 2 . Biochemical, biophysical, immunological and biological comparison with the pituitary hormone; Langley KE et al.; Bacterially synthesized, recombinant-DNA-derived bovine growth hormone (r-bGH), prepared as described in the preceding paper in this journal, has been characterized in comparison with pituitary bovine growth hormone (pit-bGH) . The characterization criteria include sodium dodecyl sulfate/polyacrylamide gel electrophoresis, automated N-terminal sequence analysis, amino acid composition, isoelectric focusing, reverse-phase high-performance liquid chromatography, ultraviolet absorbance, analysis for free protein thiol, sizing by gel filtration, circular dichroism, radioimmunoassay and biological activity in the hypophysectomized rat weight-gain assay . In every respect the r-bGH appears to be virtually identical to pit-bGH.

Eur J Biochem, 1987 Mar 2, 163(2), 231 - 8
Phenobarbital-mediated modulation of gene expression in rat liver . Analysis of cDNA clones; Lechner MC et al.; Phenobarbital evokes a pleiotypic response in the liver characterized by cell hypertrophy and mono-oxygenase induction . These phenomena arise through complex modulation mechanisms changing the pattern of protein synthesis, distinct from those triggered by other well known inducers, like steroid hormones or polycyclic hydrocarbons . To investigate the mechanisms involved in regulating the expression of the phenobarbital-inducible tissue-specific genes, we constructed two libraries of recombinant bacterial plasmids pBR322 in Escherichia coli . Each library contains cDNA copies of polysomal poly(A)-rich RNA obtained from control and 16-h phenobarbital-induced rat liver . A thousand cloned sequences from each library were screened by double-cross colony hybridization using {32P}cDNA prepared from homologous and heterologous poly(A)-rich RNAs as the probes . The statistical analysis of the results revealed that phenobarbital treatment significantly changes the relative abundance of different polysomal mRNA classes in rat liver . Clones corresponding to mRNAs clearly induced following phenobarbital treatment have been further selected by plasmid DNA dot hybridization, and used as probes for measuring the changes in each mRNA concentration in the whole cell and in the polysomal RNAs from rat livers, at different times after phenobarbital treatment . The fact that changes in the concentration of each specific mRNA in the polysomes does not parallel the variation of its total concentration in the cell indicates that the induced modulation of protein synthesis in the liver is brought about by mechanisms involving both transcriptional and translational regulation, since besides the increases in whole cellular mRNA concentration a marked mobilization of mRNA into active polysomes could be demonstrated during the onset of the adaptive response to phenobarbital.

Eur J Biochem, 1987 Mar 2, 163(2), 313 - 21
Recombinant-DNA-derived bovine growth hormone from Escherichia coli . 1 . Demonstration that the hormone is expressed in reduced form, and isolation of the hormone in oxidized, native form; Langley KE et al.; The isolation of bacterially synthesized, recombinant-DNA-derived, bovine growth hormone (r-bGH) with native structure is described . The r-bGH is found in insoluble form, in a pellet fraction, after cell breakage and centrifugation . Cell envelope components (protein, lipid, endotoxin) and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction . We demonstrate that the r-bGH is largely reduced until solubilized using 6 M guanidine/HCl . Air oxidation is then carried out, in the presence of the guanidine/HCl . The oxidation results in a mixture of about one-third disulfide-linked oligomers and two-thirds oxidized monomer . The latter may include some incorrectly oxidized material, but appears to be mostly correctly oxidized . The oxidized monomer is isolated by gel filtration in the presence of guanidine/HCl . Subsequent guanidine/HCl removal leads to refolded, oxidized r-bGH . All steps in the procedure, in particular the oxidation and refolding steps, can be carried out at relatively high protein concentrations.

Proc Soc Exp Biol Med, 1987 Mar, 184(3), 267 - 77
Naloxone pretreatment prevents the bloody diarrhea of canine endotoxic shock; Ganes E et al.; We examined the importance of timing with endorphin involvement in shock by giving the opiate receptor antagonist naloxone as a pretreatment in canine endotoxic shock . Dogs anesthetized with pentobarbital (30 mg/kg iv) were given Escherichia coli endotoxin at LD80 doses iv . Naloxone (2 mg/kg plus 2 mg/kg/hr iv, N = 10) started 15 min before endotoxin attenuated the fall in mean arterial pressure, cardiac index, and the first derivative of left ventricular pressure due to endotoxin in comparison with control animals given 0.9% NaCl (N = 10) . Naloxone attenuated the endotoxin-induced decrease in superior mesenteric arterial blood flow and the increases in portal venous pressure and pulmonary arterial pressures . Moreover, naloxone pretreatment prevented the characteristic bloody diarrhea and reduced mortality . Our findings implicate endorphins acting on opiate receptors as important mediators of endotoxin-induced cardiovascular failure and bloody diarrhea in canine endotoxemia . These are early manifestations and dictate expeditious use of naloxone in endotoxic shock.

Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 422 - 7
{Study of conformation characteristics of polydeoxyribonucleotides with non-random base sequence by slow 1H----3H exchange}; Lesnik EA et al.; The rate constants of 1H----3H exchange between water and C8H-groups of purinic residues of alternating polynucleotides: poly{d(A-T)}.poly{d(A-T)} (I), poly{d(G-C)}.poly{d(G-C)} (II), poly{d(A-C)}.poly{d(G-T)} (III) and homopolynucleotides: poly(dA).poly(dt) (IV), poly(dG).poly(dC) (V), as well as DNA E . coli, was determined in 0.15 M NaCl at 25 degrees C . The retardation of exchange observed at these conditions (compared to that of the B-form DNA) is in agreement with the model of B-alternating structure for the (I) and is attributed to the co-existence of B- and A-conformers for the (V) in solution . Absence of distinguishable differences in exchange rate constants for purinic residues of the (II), (III) and (IV) (compared to that of the B-form DNA) evidences that conformations of these polynucleotides in solution are similar to "canonical" B-form DNA and don't correlate with the model of "heteronomous" DNA which was proposed for (IV).

Eur J Immunol, 1987 Mar, 17(3), 413 - 6
Isolation of rat immunoglobulin class switch variants of rat-mouse hybridomas by enzyme-linked immunosorbent assay and sequential sublining; Pluschke G et al.; The use of sequential sublining in combination with highly specific and sensitive enzyme-linked immunosorbent assays for the isolation of spontaneous rat Ig heavy chain class switch variants is described . These methods allowed us to isolate switch variants from mouse-rat hybridoma lines secreting monoclonal rat antibodies . Switch variants from IgM to IgG2a, from IgG2a or IgG2b to IgE and from IgE to IgA were obtained . Members of the BA1.2 family, which consists of IgG2b, IgE and IgA antibodies are shown to exhibit identical rhamnose-inhibitable binding to the O18A antigen of Escherichia coli and to the paratope-associated anti-idiotypic antibody BA114.

Appl Environ Microbiol, 1987 Mar, 53(3), 606 - 9
Sensitivity of genetically engineered organisms to selective media; Lechevallier MW et al.; Eighteen strains of Escherichia coli used in genetic studies were tested for their ability to grow on several selective media . Highest recoveries were obtained with m-T7 agar . The SOS system, particularly the recA gene, may play some role in the sensitivity of E . coli to selective agents . These results may be important in the selection of media used to detect genetically engineered organisms released into the environment.

Anal Biochem, 1987 Mar, 161(2), 501 - 7
Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase; Wood KV et al.; The gene for firefly luciferase (luc) can be used as a generalized genetic probe . A method that aids in the analysis of shuttle vectors containing luc by allowing verification in Escherichia coli of a functional coding sequence is presented here . Colonies containing a functional form of luc are detected on film after luciferin is added to initiate the luminescent reaction . Two conditions, lowering the pH of the environment and maintaining aerobic conditions, were found to greatly improve the sensitivity of the assay . This technique may be useful in the development of genetic constructs that alter the natural coding sequence of luc, such as in gene fusions.

Anal Biochem, 1987 Mar, 161(2), 487 - 93
Enzymatic synthesis of novel glutathione analogs; Moore WR et al.; A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K . Murata, W . A . Abbott, R . J . Bridges, and A . Meister (1985) Anal . Biochem . 150, 235-237) . In the present work, this E . coli matrix was used as the basis of a method for the synthesis of glutathione analogs . Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures . Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine . This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids . The analogs are purified by gel-filtration and ion-exchange chromatography . The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function . Fluorine-containing analogs may be used for NMR studies . The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.

Prikl Biokhim Mikrobiol, 1987 Mar-Apr, 23(2), 260 - 5
{Nephelometric parameters of plasmolytic reactions in Escherichia coli K-12}; Govorunov IG et al.; Nephelometric parameters of plasmolysis in Escherichia coli K-12 are presented . The relationship between these parameters and intracellular osmotic pressure, barrier properties of the cytoplasmic membranes and transport of inorganic ions was investigated in the present work . The nephelometric parameters can be used for quantitative estimation of the physiological state of the bacteria.

J Appl Physiol, 1987 Mar, 62(3), 1006 - 9
Elevation of superior vena caval pressure increases extravascular lung water after endotoxemia; Allen SJ et al.; In many sheep Escherichia coli endotoxin results in pulmonary hypertension, increased microvascular permeability, pulmonary edema, and increased central venous pressure . Since lung lymph drains into the systemic veins, increases in venous pressure may impair lymph flow sufficiently to enhance the accumulation of extravascular fluid . We tested the hypothesis that, following endotoxin, elevating the venous pressure would increase extravascular fluid . Thirteen sheep were chronically instrumented with catheters to monitor left atrial pressure (LAP), pulmonary arterial pressure (PAP), and superior vena caval pressure (SVCP) as well as balloons to elevate LAP and SVCP . These sheep received 4 micrograms/kg endotoxin, and following the pulmonary hypertensive spike the left atrial balloon was inflated so that (PAP + LAP)/2 = colloid osmotic pressure . It was necessary to control PAP + LAP in this way to minimize the sheep-to-sheep differences in the pulmonary hypertension . We elevated the SVCP to 10 or 17 mmHg or allowed it to stay low (3.2 mmHg) . After a 3-h period, we killed the sheep and removed the right lungs for determination of the extravascular fluid-to-blood-free dry weight ratio (EVF) . Sheep with SVCP elevated to 10 or 17 mmHg had significant increases in EVF (5.2 +/- 0.1 and 5.6 +/- 1.2) compared with the sheep in which we did not elevate SVCP (EVF = 4.5 +/- 0.4) . These results indicate that sustained elevation in central venous pressure in patients contributes to the amount of pulmonary edema associated with endotoxemia.

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Mar, 51(3), 493 - 503
Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains; Coombs AM et al.; The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E . coli strains, AB1157 and B/r . Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157 . However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2 . Pretreatment also induced resistance to broad-band NUV radiation in these strains . The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains . The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E . coli UM1, is more resistant to NUV radiation than B/r . Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect . Two hypotheses are put forward to account for the sensitivity of exponential phase B/r . Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.

Genetics, 1987 Mar, 115(3), 431 - 9
Cryptic genes for cellobiose utilization in natural isolates of Escherichia coli; Hall BG et al.; The ECOR collection of natural Escherichia coli isolates was screened to determine the proportion of strains that carried functional, cryptic and nonfunctional genes for utilization of the three beta-glucoside sugars, arbutin, salicin and cellobiose . None of the 71 natural isolates utilized any of the beta-glucosides . Each strain was subjected to selection for utilization of each of the sugars . Only five of the isolates were incapable of yielding spontaneous beta-glucoside-utilizing mutants . Forty-five strains yielded cellobiose+ mutants, 62 yielded arbutin+ mutants, and 58 strains yielded salicin+ mutants . A subset of the mutants was screen by mRNA hybridization to determine whether they were expressing either the cel or the bgl beta-glucoside utilization operons of E . coli K12 . Two cellobiose+ and two arbutin+-salicin+ strains failed to express either of these known operons . It is concluded that there are at least four gene clusters specifying beta-glucoside utilization functions in E . coli populations, and that all of these are normally cryptic . It is estimated that in any random isolate the probability of any particular cluster having been irreversibly inactivated by the accumulation of random mutations is about 0.5.

Chem Biol Interact, 1987 Mar, 61(3), 265 - 75
Toxicity, mutagenicity, intracellular drug concentration and DNA binding in Escherichia coli treated with cis-platinum(II) complexes; Razaka H et al.; The genotoxic effects of six cis-platinum(II)chloramine complexes with different alkyl substituents on their amine ligands have been measured using Escherichia coli . The toxicity and mutagenicity of these compounds were compared, after exposure of bacteria, to drug concentrations which gave known quantities of platinum-DNA lesions . The results permit several observations concerning structure-activity relations of platinum(II) complexes . Firstly, methyl substitution on the amine ligands of cis-diamminedichloro-platinum(II) (DDP) is reported to reduce its antitumor activity . The methyl group did not exert an effect in bacteria where the toxicity and mutagenicity of cis-bis(methylamine)dichloroplatinum(II) and DDP were equivalent . In fact, at equal levels of DNA binding, complexes with substituted amines were generally more toxic toward bacteria than DDP . Secondly, replacement of the chloro groups of DDP by nitrato ligands increased its toxicity and mutagenicity at a given level of DNA binding . Hence, although DDP and its dinitrato derivative have identical ammine ligands, they may form different platinum-DNA lesions in bacteria . Finally, cis-bis(cyclohexylamine)-dichloroplatinum(II) was unique among the compounds studied since it did not cause bacterial filamentation or mutagenesis . These results suggest that, although this compound binds to the bacterial genome, it may not induce the SOS response.

Biophys J, 1987 Mar, 51(3), 407 - 12
Theoretical analysis of single-round transcription experiments on trp leader region; Suzuki H et al.; A kinetic model is proposed to reproduce the time courses of the concentration change in paused leader RNA, terminated leader RNA, and readthrough RNA in the single-round transcription experiments on trp leader region of Escherichia coli and its mutants, L132, L75, and L75L135 (Winkler, M . E., and C . Yanofsky, 1981, Biochemistry, 20:3738-3744; Fisher, R., and C . Yanofsky, 1983, J . Biol . Chem., 258:9208-9212) . This model fits the experimental results well and also captures the essential aspects of the processes of transcriptional pausing and termination . In the wild type template, under optimal conditions, it is found that the transcription rate at the pause and attenuation sites is of the same order of magnitude, 10,000-fold lower than the transcription rate at the other sites, and the high termination level at the attenuation site is attributable to a higher dissociation rate . This analysis also provides a clue as to how the template base change, various concentrations of ribonucleoside triphosphates, and the presence or absence of L-factor affect the transcription and dissociation rates to yield different termination levels at the pause or attenuation site . It also discusses the molecular mechanism of the transcriptional pausing and termination.

Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1482 - 6
Requirement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction; Lahue RS et al.; The involvement of d(GATC) sequences in Escherichia coli DNA mismatch correction was ascertained by analyzing in vitro repair efficiencies of a series of related, covalently closed circular DNA heteroduplexes that contained from zero to four d(GATC) sites . A heteroduplex with four d(GATC) sites was repaired with high efficiency by extracts of E . coli, whereas no significant correction occurred on a closely related molecule lacking such sequences . Heteroduplexes containing one or two d(GATC) sites were corrected at rates between 10% and 93% of that observed for the four-site molecule, but repair efficiency did not correlate in a simple way with the number of sites present . The methylation state at a single d(GATC) sequence was sufficient to direct strandedness of repair, and correction of heteroduplexes containing one or more d(GATC) sites required functional mutH, mutL, and mutS gene products . In addition, DNA repair synthesis dependent on mutH and mutS also required the presence of at least one d(GATC) site . Although mismatch correction was not observed on a covalently closed circular heteroduplex lacking a d(GATC) sequence, such molecules were subject to strand-specific repair if they contained a strand-specific single-strand break . However, this correction reaction did not require mutH, mutL, mutS, or uvrD gene products . Consequently, we have concluded that d(GATC) sequences are directly involved in mismatch correction mediated by the mutHLS system.

Pediatr Res, 1987 Mar, 21(3), 252 - 6
Neutrophil function in an experimental model of hemolytic uremic syndrome; Vedanarayanan VV et al.; To understand the role of neutrophil leukocytosis in hemolytic uremic syndrome, we studied the changes in neutrophil function in the modified generalized Shwartzman reaction in rabbits . This model resembles hemolytic uremic syndrome associated with endotoxemia . At the end of an endotoxin infusion, we observed leukopenia, thrombocytopenia, and a decrease in hematocrit associated with schistocytosis . Plasma B-glucuronidase levels increased and this was associated with a decrease in neutrophil content of the enzyme . The chemotactic index and neutrophil aggregation to zymosan-activated serum were impaired compared to controls . The neutrophil procoagulant content increased after endotoxin infusion . The serum creatinine concentration and proteinuria increased in the endotoxin-treated animals . The changes returned to normal by 48 h . Renal cortical malondialdehyde, a reflection of lipid peroxidation, was higher in the endotoxin-treated animals than in the controls . We have shown enzyme release by neutrophils, impairment of chemotaxis and aggregation, increased procoagulant content in neutrophils, and evidence of lipid peroxidation in renal cortical tissue in this model . These observations raise the possibility that leukocytes may have a role in the pathogenesis of the hemolytic uremic syndrome.

Am Surg, 1987 Mar, 53(3), 164 - 6
Shortened endotoxin-activated clotting times in patients with carcinoma; Spillert CR et al.; Isolated human monocytes generate tissue factor when stimulated with endotoxins . This tissue factor generation provides a convenient marker of monocyte activation . Furthermore, the only circulating blood cell capable of generating large quantities of tissue factor is the monocyte . Therefore, the addition of endotoxin to citrated blood, and the determination of the recalcification times after incubation, yields a measure of monocyte activation . In order to determine whether monocyte activation as measured by this technique varies between patients with carcinoma and healthy volunteers, recalcification times were evaluated . The recalcification time and range for 19 healthy volunteers (controls) was 6.55 min (5.3-8.5) for the saline incubated sample and 5.69 min (4.6-7.2) for the endotoxin-activated sample . The results for 13 patients with carcinoma are 4.81 min (3.3-6.3) for the former and 3.17 min (2.0-4.3) for the latter . These results show that the longest recalcification time with endotoxin incubation for patients with carcinoma was lower than the lowest recalcification time in the control group . Whether this simple test can be of use in the diagnosis and monitoring of patients with carcinomas is currently being investigated.

Am Rev Respir Dis, 1987 Mar, 135(3), 665 - 70
The effect of human antiendotoxin monoclonal antibodies on endotoxin-induced lung injury in the rat; Feeley TW et al.; A model of endotoxin-induced lung injury was developed in the rat . We found that 24 h after intravenously administered endotoxin (3 mg/kg) there was increased clearance of the isotope 99mTcDTPA from the lung to blood, increased neutrophils in the lung in bronchoalveolar lavage, and increased levels of products of peroxidation of lipids and nucleic acid in the serum . Using this model, we evaluated the effect of pretreatment of rats with a human monoclonal antibody specific to the core glycolipid that is common to all endotoxins . We found that pretreatment prevented the increased clearance of 99mTcDTPA from the lung, as well as the increase in lipid peroxidation products in the serum . The antibody did not prevent increased neutrophil accumulation in the lung . The findings suggest that the administration of human antiendotoxin monoclonal antibodies prior to endotoxemia may prevent some of the changes in the lung associated with endotoxin.

Am Rev Respir Dis, 1987 Mar, 135(3), 643 - 50
Proteolytic activity in sheep lung lymph as marker of lung capillary injury; Lesser M et al.; Intravenous infusions of Escherichia coli endotoxin into sheep caused the appearance in lung lymph of high levels of an enzyme with trypsinlike activity . The time course of appearance of the enzyme and the extent of its increase corresponded to the known events of endotoxin-induced capillary injury . Accordingly, activity was low in the first phase of endotoxin-induced increased lung lymph flow caused by increased pressure filtration but was high in the second phase of increased lung lymph flow caused by increased permeability filtration . Recovery was associated with a decrease of activity to preinfusion levels . Capillary damage and increased permeability filtration induced by air emboli or oleic acid led to a similar increase in lung lymph proteolytic activity . By contrast lung lymph proteolytic activity remained virtually unchanged during increased pressure filtration induced by inflation of a balloon in the left atrium . Activity also remained unchanged in thoracic duct lymph, indicating that the increased activity in lung lymph is not an expression of a generalized response to endotoxin . The enzyme, a serine protease with a molecular weight of about 70,000 to 75,000 and a pH optimum between 7.3 and 7.6, was not related to lymph clotting and was not capable of correcting the clotting defects of plasmas deficient in enzymes of the clotting cascade . These results together with specificity studies indicate that the enzyme represents a new, hitherto unidentified, protease . Measurements of its activity in lung lymph represent a sensitive marker of lung capillary injury.

Virology, 1987 Mar, 157(1), 127 - 36
The spleen necrosis virus int gene product expressed in Escherichia coli has DNA binding activity and mediates att and U5-specific DNA multimer formation in vitro; Luk KC et al.; To facilitate the in vitro study of the spleen necrosis virus (SNV) int gene product, we expressed the viral int locus in an Escherichia coli expression vector . Antiserum made against the protein produced in bacteria precipitated a 44-kDa polypeptide from virus-infected chicken embryo fibroblasts . This result is consistent with the expected size of the SNV int polypeptide . In a protein blotting assay, the expressed protein binds strongly to DNA and was able to complex nonspecifically with both single- and double-stranded DNAs containing or lacking viral sequences . However, under solution conditions favoring transient DNA unwinding, DNA binding was confined to supercoiled molecules containing either the SNV att sequence (the viral cis-acting region required for integration) or the U5 region of the long terminal repeat alone . Under these conditions of specific binding, multimeric DNA species were formed by apparent intermolecular interaction between protein-DNA complexes . These data indicate that retroviral integration may require local DNA unwinding at the att site for complex formation between the int gene product and DNA . This complex may be an intermediate in the viral DNA insertion process.

Mutat Res, 1987 Mar, 183(2), 129 - 37
Repair of plasmid DNA damaged in vitro with cis- or trans-diamminedichloroplatinum(II) in Escherichia coli; Popoff SC et al.; Plasmid pBR322 was modified in vitro with the antitumor compound cis-diamminedichloroplatinum(II) (cis-DDP) or the isomeric trans-DDP . The numbers of platinum adducts were determined by atomic absorption spectrophotometry . DNA-repair-proficient and various DNA-repair-deficient (uvrB, uvrD, recB and recA) strains of Escherichia coli were transformed by the damaged plasmids and the ratios of the transformation frequencies of cells by damaged plasmids relative to those by untreated plasmids were determined . Results of transformation assays indicated that the uvrB gene function was essential for repair of plasmid DNA damaged with cis-DDP . A functional recA gene product seemed to be of minor importance for repair of plasmids damaged with cis-DDP . trans-DDP had a different effect on plasmid DNA . trans-DDP-modified DNA was better able to transform cells than cis-DDP-modified DNA, and the DNAs appeared to be repaired differently . Prior induction of SOS functions increased the survival of plasmids treated with cis-DDP in wild-type and uvrD mutants, but did not increase the survival of plasmids damaged with trans-DDP in these strains . In in vitro repair experiments, plasmid DNA modified with cis-DDP was more readily incised by the UVRABC excinuclease than that modified with trans-DDP.

Mutat Res, 1987 Mar, 183(2), 117 - 21
Purification of the T4 endonuclease V; Higgins KM et al.; A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V . The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter . The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels . Milligram quantities of the enzyme have been purified by the following procedure . After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column . Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column . The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step . The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.

Mutat Res, 1987 Mar, 183(2), 109 - 15
T4 endonuclease V promotes the formation of multimeric DNA structures; Lloyd RS et al.; Electron microscopy of UV-irradiated circular DNA molecules which had been treated with T4 endonuclease V revealed the formation of multimeric DNA structures in addition to the expected conversion of the superhelical DNA molecules into nicked circular and linear forms . The multimeric DNA molecules could be distinguished in electron micrographs from catenated molecules which were present in the original DNA preparation by a combination of rotary and single angle heavy metal shadowing . The complexity and frequency of these structures increased with time of reaction with endonuclease V . Their formation, as well as the endonuclease activity of enzyme, was dependent on UV irradiation of the DNA, and the complexes could be disrupted by prior phenol extraction and ethanol precipitation . Preparations of endonuclease V estimated to be 98% pure by mass promoted the same complex formation between DNA molecules as did preparations estimated to be only 5-10% pure . In addition to these intermolecular structures, the formation of complexes between regions on the same DNA molecules was manifest as discrete double-stranded 'loops' 200-300 base pairs in length . DNA 'bubble structures' were also observed and may represent folding of the 'loops' onto adjacent segments of DNA . These results suggest that at least one active form of T4 endonuclease V may be a multimeric complex of enzyme molecules in association with DNA.

J Urol, 1987 Mar, 137(3), 489 - 90
Psoas abscess in chronic dialysis patients; Tillman BF et al.; We report 4 cases of nontuberculous psoas abscess occurring in patients with end stage renal disease . Fever and pain were the presenting symptoms but diagnosis was delayed . A computerized tomography scan of the abdomen was the critical test that led to the correct diagnosis . Therapy involved drainage and antibiotics, and was successful in 3 of the 4 patients.

J Clin Invest, 1987 Mar, 79(3), 721 - 30
Comparative effects in vivo of recombinant murine interleukin 3, natural murine colony-stimulating factor-1, and recombinant murine granulocyte-macrophage colony-stimulating factor on myelopoiesis in mice; Broxmeyer HE et al.; Purified murine colony-stimulating factors (CSF) recombinant interleukin 3 (IL-3), natural CSF-1, and recombinant granulocyte-macrophage (GM) CSF were assessed in vivo for their effects on BDF1 mouse bone marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in untreated mice and in mice pretreated with purified iron-saturated human lactoferrin (LF) . The CSF and LF preparations did not contain detectable endotoxin (less than 0.1 ng) . Mice pretreated with LF were more sensitive to the effects of CSF . In mice pretreated with LF, 2,000 U IL-3 or 20,000 U CSF-1 significantly enhanced the cycling status and absolute numbers of all progenitors, whereas 20,000 U GM-CSF significantly increased the cycling status of CFU-GM and CFU-GEMM, but had no effect on cycling of BFU-E or on numbers of any of the progenitors . The effects of CSF in mice pretreated with LF were not mimicked by 0.1-100 ng E . coli lipopolysaccharide.

J Bacteriol, 1987 Mar, 169(3), 1331 - 4
Roles of the Pribnow box in positive regulation of the ompC and ompF genes in Escherichia coli; Ozawa Y et al.; The roles of the first base of the Pribnow box in positive regulation of the ompC and ompF genes were studied . G- and A-to-T substitutions of the first base of the ompC and ompF Pribnow boxes, respectively, resulted in a high-level functioning of the promoters in the ompR background . The level was further enhanced significantly in the ompR+ background . The effects of other substitutions were also studied . Based on these observations, the roles of the Pribnow box in the positive regulation are discussed.

J Bacteriol, 1987 Mar, 169(3), 1315 - 20
Control of the kilA gene of the broad-host-range plasmid RK2: involvement of korA, korB, and a new gene, korE; Young C et al.; Broad-host-range plasmid RK2 encodes several different kil genes which are potentially lethal to an Escherichia coli host . The kil genes and the essential RK2 replication gene trfA are regulated by the products of kor genes . We have shown previously that kilA can be controlled by a constitutively expressed korA gene . In this study, we have found that the wild-type, autoregulated korA gene is insufficient for control of kilA cloned on high-copy-number plasmids . One of two other genes must also be present with korA . One gene is korB, originally discovered by its ability to control the determinants in the kilB region and later found to affect expression of both trfA and korA . The other is a new gene, korE, which has been cloned from the 2.2' to 4.1' region located between korC and kilA . Studies with a kilA-cat fusion suggest that korA, korB, and korE all participate in the control of kilA gene expression.

J Bacteriol, 1987 Mar, 169(3), 1267 - 71
Isolation and genetic analysis of mutations allowing the degradation of furans and thiophenes by Escherichia coli; Abdulrashid N et al.; Successive mutations of Escherichia coli yielded a strain that was able to degrade a variety of heterocyclic oxygen- and sulfur-containing ring compounds . In particular, this strain could use both furan-2-carboxylic acid and thiophene-2-carboxylic acid as sole carbon and energy sources . Nitrogen-containing heterocyclic compounds were not degraded . This mutant was isolated by selecting first for oxidation of furan derivatives and then for thiophene degradation . Genetic analysis revealed that mutations in three novel genes, thdA (12 min), thdC (92 min), and thdD (98 min), were required for thiophene degradation . In addition, constitutively at both of the previously characterized fadR and atoC loci was required for efficient thiophene breakdown . The pathway of furan and thiophene degradation remains obscure, but the inability of our mutants to degrade 5-nitro- or 5-bromo-substituted furan derivatives suggests that hydroxylation at position 5 may be involved . Thiophene derivatives were toxic when they were present at concentrations of 0.1% or greater; however, addition of trace amounts of phenylalanine plus tyrosine greatly reduced this effect.

J Bacteriol, 1987 Mar, 169(3), 1153 - 60
Essential DNA sequence for the replication of Rts1; Itoh Y et al.; The promoter sequence of the mini-Rts1 repA gene encoding the 33,000-dalton RepA protein that is essential for replication was defined by RNA polymerase protection experiments and by analyzing RepA protein synthesized in maxicells harboring mini-Rts1 derivatives deleted upstream of or within the presumptive promoter region . The -10 region of the promoter which shows homology to the incII repeat sequences overlaps two inverted repeats . One of the repeats forms a pair with a sequence in the -35 region, and the other forms a pair with the translation initiation region . The replication origin region, ori(Rts1), which was determined by supplying RepA protein in trans, was localized within 188 base pairs in a region containing three incII repeats and four GATC sequences . Dyad dnaA boxes that exist upstream from the GATC sequences appeared to be dispensable for the origin function, but deletion of both dnaA boxes from ori(Rts1) resulted in reduced replication frequency, suggesting that host-encoded DnaA protein is involved in the replication of Rts1 as a stimulatory element . Combination of the minimal repA and ori(Rts1) segments, even in the reverse orientation compared with the natural sequence, resulted in reconstitution of an autonomously replicating molecule.

J Bacteriol, 1987 Mar, 169(3), 1102 - 6
Genetic suppression of a temperature-sensitive groES mutation by an altered subunit of RNA polymerase of Escherichia coli K-12; Wada M et al.; Temperature-resistant suppressor mutants were isolated from Escherichia coli mutant strain groES131(Ts) . Phage P1-mediated transduction and a two-dimensional gel electrophoretic analysis of cellular proteins indicated that these suppressor mutants carry an additional mutation in either the groEL gene or the rpoA gene.

Clin Sci (Lond), 1987 Mar, 72(3), 383 - 5
Effect of dietary linoleate content on the metabolic response of rats to Escherichia coli endotoxin; Wan JM et al.; Dietary fat influences many aspects of immune function . Escherichia coli endotoxin is a potent stimulator of interleukin 1 production from macrophages . The present study examines the effect of feeding with fat diets rich (corn oil) and poor (coconut oil) in linoleate at high and low concentrations on responses to endotoxin . Spleen phosphatidylcholine linoleate contents were higher in the corn oil than in the coconut oil group and arachidonate concentrations were highest in the group fed a high concentration of corn oil . Coconut oil completely abolished the responses to endotoxin . The inhibitory effects of coconut oil could largely be due to reduced prostaglandin and leukotriene synthesis.

Blood, 1987 Mar, 69(3), 913 - 8
The influence in vivo of murine colony-stimulating factor-1 on myeloid progenitor cells in mice recovering from sublethal dosages of cyclophosphamide; Broxmeyer HE et al.; Pure murine colony-stimulating factor-1 (CSF-1) was assessed for its effects in vivo in mice pretreated seven days earlier with a sublethal dosage of cyclophosphamide . The multipotential (CFU-GEMM), erythroid (BFU-E), and granulocyte-macrophage (CFU-GM) progenitor cells in these mice were in a slowly cycling or noncycling state . Intravenous administration of 20,000 units of CSF-1 to these mice stimulated the hematopoietic progenitors into a rapidly cycling state in the marrow and spleen within three hours . Significant increases in absolute numbers of marrow and spleen CFU-GM and spleen BFU-E and CFU-GEMM were also detected . No endotoxin was detected in the CSF-1 preparation by Limulus lysate assay, and treatment of CSF-1 at 100 degrees C for 20 to 30 minutes completely inactivated the in vitro and in vivo stimulating effects . The effects of CSF-1 were not mimicked by the in vivo administration of 0.1 to 10 ng Escherichia coli lipopolysaccharide . These results suggest that the effects of CSF-1 in vivo were not due to contaminating endotoxin or to a nonspecific protein effect . CSF-1 did not enhance colony formation by BFU-E or stimulate colony formation by CFU-GEMM in vitro, thus suggesting that at least some of the effects of CSF-1 noted in vivo are probably indirect and mediated by accessory cells.

Obstet Gynecol, 1987 Mar, 69(3 Pt 2), 444 - 6
Pyelonephritis associated with respiratory distress; Pruett K et al.; We present a case of pyelonephritis associated with respiratory distress and elevated liver enzymes in a pregnant patient.

J Infect Dis, 1987 Mar, 155(3), 377 - 89
Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent; Levine MM; There are four major categories of diarrheagenic Escherichia coli: enterotoxigenic (a major cause of travelers' diarrhea and infant diarrhea in less-developed countries), enteroinvasive (a cause of dysentery), enteropathogenic (an important cause of infant diarrhea), and enterohemorrhagic (a cause of hemorrhagic colitis and hemolytic uremic syndrome) . Besides manifesting distinct clinical patterns, these categories of E . coli differ in their epidemiology and pathogenesis and in their O:H serotypes . Common features (albeit distinct for each category) include plasmid-encoded virulence properties, characteristic interactions with intestinal mucosa, and elaboration of various types of enterotoxins or cytotoxins . A less-well-defined fifth category of diarrheagenic E . coli is that of enteroadherent E . coli, so far identifiable only by their pattern of adherence to Hep-2 cells in tissue culture.

Allergol Immunopathol (Madr), 1987 Mar-Apr, 15(2), 93 - 9
Autoantibody generation by cross-reactive antigens: possible homeostatic mechanism; Vidal J; Inbred (C57BL6 X CBA) F1 mice or outbred Ico: OF1 (Caw) mice received a weekly injection of rat erythrocytes for 2-3 months and the (IgM and IgG) antibody levels to autologous erythrocytes were measured by ELISA . No autoantibodies were found in serum but the erythrocyte-bound IgM and IgG antibodies rose initially and levelled off afterwards (usually from day 14 onwards) . The anti-rat erythrocyte antibody response also rose sharply at the beginning and eventually levelled off . This suggests the existence of one or several homeostatic mechanisms . Ico: OF1 (Caw) mice received a weekly injection of E . coli lipopolysaccharide (LPS), 75 micrograms/mouse, for 7 weeks, and the autoantibody levels to autologous erythrocytes and to ssDNA were measured by ELISA . While LPS readily increased the anti-ssDNA autoantibody level, it failed to elicit detectable concentrations of erythrocyte-bound antibodies . Nevertheless, LPS enhanced the response to autologous erythrocytes triggered by a suboptimal dose of rat erythrocytes . This suggests that LPS induces autoantibodies to those antigens that have already initiated an immune response.

Anal Biochem, 1987 Mar, 161(2), 370 - 9
Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents; Shoeman RL et al.; Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes . We have expressed at high levels in E . coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120 . The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea . These components were tested for reactivity with antisera to HIV proteins and peptides . We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41 . The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera . The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody . Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA) . Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.

Proc Natl Acad Sci U S A, 1987 Mar, 84(6), 1487 - 91
Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model; Katre NV et al.; Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life . This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol . The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and isoelectric focusing . This conjugate was compared to unmodified recombinant interleukin 2 in vitro and in vivo . Covalent attachment of the hydrophilic polymer polyethylene glycol enhanced the solubility of interleukin 2, decreased its plasma clearance, and increased its antitumor potency in the Meth A murine sarcoma model.

Infect Immun, 1987 Mar, 55(3), 547 - 54
Antipyretic activity of a human immunoglobulin preparation for intravenous use in an experimental model of fever in rabbits; Iwata M et al.; In an effort to elucidate the reason that fever in patients with severe bacterial infections subsided in some cases after the administration of human immunoglobulin preparations for intravenous use (IGIVs), we focused our attention on the antipyretic activity of IGIVs by investigating experimentally produced pyrexia in rabbits with Escherichia coli-derived lipopolysaccharide (LPS) . Although little difference in antibody titers against the antigens composing molecules of LPS was found among the IGIVs that were used, IGIVs treated at pH 4 were demonstrated to inhibit a strongly LPS-induced second-phase febrile response, whereas the inhibitory effect of sulfonated and pepsin-treated IGIVs was weak . In vitro experiments on interleukin-1 production by rabbit macrophages stimulated with LPS, silica gel or latex beads and on rosette formation showed that these functions of the cells were also inhibited by IGIVs . The in vivo antipyretic activity and the results of the two in vitro experiments correlated closely . The inhibitory potency decreased in the following order: immunoglobulin G (IgG) treated at pH4, sulfonated IgG, and pepsin-treated IgG . Thus, it is possible that the subsidence of LPS-induced fever by IGIVs was mediated by inhibition of interleukin 1 production by means of binding of IgG to macrophages via an Fc receptor . Results of this study also indicated the importance of the structural integrity of the Fc portion of the IgG contained in the IGIVs to bind with its receptor on the macrophage so as to influence the various functions carried out by the cell.

Mol Cell Probes, 1987 Mar, 1(1), 109 - 20
5-Bromodeoxyuridine in vivo labelling of M13 DNA, and its use as a non-radioactive probe for hybridization experiments; Sakamoto H et al.; We describe the in vivo production of 5-bromodeoxyuridine- (5-BUdR) labelled M13 DNA by a thymine-requiring Escherichia coli strain . We show that the 5-BUdR-labelled M13 single-stranded DNA is not extruded into the culture medium, but accumulates inside the bacterial cells . On the basis of this observation, a procedure involving FPLC gel filtration already reported and used for the isolation of plasmid DNA has been adapted for the isolation of at least 90% pure 5-BUdR-labelled single-stranded DNA . An M13 probe, containing part of the Hepatitis B Virus (HBV) genome was constructed, and the corresponding 5-BUdR-labelled single-stranded DNA was used in hybridization experiments to detect homologous HBV target DNA . Picogram amounts (10(-19) moles) of the probe itself or the target DNA could be detected, by monoclonal anti-5-BUdR antibodies in an immunoenzymatic assay.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 721 - 5
Cloning of the galactokinase gene (galK) from Streptomyces coelicolor A3(2); Kendall K et al.; Streptomyces coelicolor A3(2) and Streptomyces lividans 66 strains were shown to be sensitive to the galactose analogue 2-deoxy-D-galactose . Spontaneous resistant mutants were isolated that were Gal- and lacked the enzyme galactokinase . The galK gene (structural gene for galactokinase) from S . coelicolor was cloned into S . lividans using the low copy number vector pIJ922 . The resulting plasmid (pMT650), which contained a 14 kb insert, complemented gal mutations in both species . The presence of the galK gene on a 2.8 kb EcoRI fragment was confirmed by expressing it in Escherichia coli where it complemented a well characterized galK mutation.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 655 - 65
Cyanate specifically inhibits arginine biosynthesis in Escherichia coli K12: a case of by-product inhibition?
Guilloton M, Karst F.
Growth of Escherichia coli K12 cultivated in minimal medium was strongly inhibited by 2 mM-cyanate . This inhibition could be specifically reversed by arginine . Citrulline (but not ornithine, N-alpha-acetylornithine or N-acetylglutamate) could also restore a normal growth rate . Since growth inhibition by cyanate was followed by an accumulation of ornithine within the cell it was concluded that cyanate specifically inhibits the formation of citrulline from ornithine . The effect of cyanate on the growth of defined strains was consistent with a specific inhibition of carbamoylphosphate synthase . A kinetic study of carbamoylphosphate synthase and ornithine carbamoyltransferase in vitro supported this conclusion . Since carbamoylphosphate is probably the only source of endogenous cyanate it is postulated that carbamoylphosphate synthase activity can be regulated by cyanate resulting from the dissociation of carbamoylphosphate in metabolic circumstances leading to its overproduction.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 645 - 53
Isolation and characterization of Escherichia coli mutants lacking inducible cyanase; Guilloton M et al.; To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated . Five independent mutations were localized next to lac; three of them lay between lacY and codA . Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt . Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains . This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM) . Higher concentrations inhibited growth of both wild-type and mutant strains . Urea in aqueous solutions dissociates slowly into ammonium cyanate . Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not . We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 587 - 96
Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12; Hindersson P et al.; A library of Treponema pallidum DNA was constructed using a cosmid cloning system . Sixteen hundred Escherichia coli recombinant clones were generated covering the T . pallidum genome with a probability of 99% . Three hundred of the clones were screened for expression of T . pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T . pallidum . One clone was identified which produced the 'common antigen' (CA) of T . pallidum (Tp-4) . CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization . The recombinant E . coli clone will be of value for a structural analysis of the CA gene.

J Gen Microbiol, 1987 Mar, 133 ( Pt 3), 563 - 73
Nucleotide sequence of bglC, the gene specifying enzymeIIbgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product; Bramley HF et al.; The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC . The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined . DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB . From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa) . The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62) . Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent . A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.

Plasmid, 1987 Mar, 17(2), 149 - 56
Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1; Shindoh Y et al.; The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1 . Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli . The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S . lividans 66 was determined . All except one of the inserts in the 1.52-kb essential region inactivated replication . A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment . The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.

Mikrobiologiia, 1987 Mar-Apr, 56(2), 205 - 8
{n-Chloroaniline transformation by Escherichia coli under anaerobic conditions}; Mogilevich NF et al.; In a medium without oxygen in the presence of nitrates, E . coli transforms p-chloranilin (p-CA) to yield a more hydrophilic compound which cannot be extracted with an organic solvent from water . The conditions for consecutive transformation of p-nitro-chlorobenzene (p-NCB) and p-CA have been determined: the reaction p-NCB leads to p-CA is inhibited by nitrates, p-CA transformation occurs in the presence of nitrates in the medium and depends on their concentration.

Acta Chir Scand, 1987 Mar, 153(3), 165 - 70
Volume substitution and treatment with prostaglandin E1 in a porcine model of endotoxaemia-induced pulmonary and cardiovascular failure; Modig J et al.; The effects of prostaglandin E1 (PGE1) and volume substitution (5% albumin) on pulmonary haemodynamics and oxygen transport were evaluated in a porcine model of pulmonary and cardiovascular failure . Albumin was infused i.v . to maintain baseline mean left atrial pressure throughout the experiments . Ten pigs received endotoxin + albumin and showed ten-fold increase in venous admixture, 90% increased extravascular lung water and 25% fall in cardiac output . Oxygen delivery and base excess decreased significantly and four pigs died . In ten other endotoxin-albumin-treated pigs PGE1 infusion (0.25 micrograms X kg-1 X min-1) was begun after established pulmonary and cardiovascular dysfunction, for closer mimicking of clinical use . The raised pulmonary vascular resistance thereafter normalized and extravascular lung water increased by only 15%, but the increase in venous admixture was not influenced . Cardiac output was maintained at baseline . PGE1 prevented further derangement of oxygen delivery and base excess . All the animals survived . The clinical implications may be that volume infusion, even optimally titrated, is not optimum treatment in endotoxemic pulmonary and cardiovascular failure . PGE1 added to optimum volume treatment may be a useful adjuvant in endotoxaemia.

Acta Chir Scand, 1987 Mar, 153(3), 161 - 4
Septic shock in the rat: activation of plasma proteolytic systems and effects of a kallikrein inhibitor/bradykinin antagonist (S-2441); Hogstrom H et al.; Septic shock was induced in rats by intraperitoneal injection of live Escherichia coli . Plasma prekallikrein, antithrombin III and plasminogen levels were studied with chromogenic peptide substrate assays . Decrease of all the studied plasma components occurred in all rats, but not until late in shock . S-2441, a kallikrein inhibitor/kinin antagonist, slightly delayed the fall in plasma prekallikrein, but no other effects were found . Rat survival was neither enhanced nor prolonged.

Z Geburtshilfe Perinatol, 1987 Mar-Apr, 191(2), 73 - 5
{Effect of uterotonic agents on the pathophysiology of endotoxin-induced shock}; Schmidt EH et al.; Different uterus contracting compounds (methylergonovine, oxytocin and prostaglandin F2 alpha) were evaluated with respect to their effect on the pathophysiology of endotoxin-induced shock . We used the model developed by Beller and Theiss of a continuous endotoxin-infusion in female Sprague-Dawley rats . In each group fibrinogen, plasma hemoglobin, hematocrit and thrombocytes were measured . The body weight of the rats was determined before and after the infusions . After the end of the experiment the intravasal fibrin deposits in the kidneys were evaluated quantitatively . Methylergonovine showed a slight protective effect regarding the clotting system and the shock events . This effect was more pronounced with oxytocin . In contrast prostaglandin F2 alpha enhanced the shock events . Based on our results we feel that the use of prostaglandin F2 alpha under septic conditions can be associated with significant risks.

Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 515 - 28
{Comparison of the conformation of RNA from phage MS2 and 16S rRNA . Interaction with dyes specific for the secondary structure of native RNA and RNA subjected to hydrolysis by nuclease S1}; Borisova OF et al.; The interaction of ethidium bromide (EtBr) with double-stranded (ds), and acridine orange (AO) with single-stranded (ss) fragments of 16S rRNA Escherichia coli in a wide range of ionic strength, at various pH, Zn2+ ion concentrations and partial hydrolysis by nuclease S1 was investigated . It was shown that about 90% of the RNA molecule is accessible to both dyes, when the ionic strength is near of 0.01 (pH 7) . Approximately half of the RNA becomes inaccessible to dyes, when the ionic strength was increased up to 0.08-0.24 (pH 4.7-7), independent on the presence of Zn2+ ions (10(-3) M) . About a half of the ds-, and a quarter of the ss-segments of the RNA, deduced from the secondary structure model were protected from the interaction with EtBr and AO . The hydrolysis of about a half of ss-segments upon addition of the Zn2+ (10(-3) M) ions did not affect the RNA tertiary structure . The experimental data obtained confirm the idea of the existence of some "nucleus" (or "nuclei") within the 16S rRNA molecule . The "nucleus" seems to be inaccessible to the dyes and is very stable to heat denaturation . It was supposed that this structure is organized by means of interaction of some of the parallelly oriented ds-segments, as it was suggested earlier for the phage MS2 RNA structure.

Mol Biol (Mosk), 1987 Mar-Apr, 21(2), 462 - 71
{Kinetics of inhibition by 8-oxy-ATP of the dinucleotide pppApU synthesis catalyzed by Escherichia coli RNA-polymerase on the promoter A1 of phage T7 delta D111 DNA during coupled synthesis of di- and trinucleotides and a limited set of substrates}; Kuriavyi VV et al.; A kinetic analysis of inhibition of synthesis of dinucleotide pppApU catalyzed by Escherichia coli RNA-polymerase on A1 promoter of the DNA from T7 delta DIII phage mutant by 8-oxy-ATP under the conditions of the coupled synthesis of pppApU and pppApUpC and in the presence of an incomplete set of substrates, namely ATP, UTP, CTP, has been performed . It was found that 8-oxy-ATP is an unproductive analog of both ATP and CTP . A comparative analysis of the dissociation constants shows that 8-oxy-ATP binds at ATP center 3.3 . times and at CTP center 540 times weaker than natural substrates . At the UTP center 8-oxy-ATP does not bind at all.

Nippon Geka Gakkai Zasshi, 1987 Mar, 88(3), 327 - 39
{Experimental study on the development of endotoxemia in peritonitis with special reference to route of absorption of endotoxin}; Takesue Y; As endotoxemia develops and presents marked symptoms in severe peritonitis, the absorption route of endotoxin from the peritoneal cavity was studied in this study . Twenty-four adult mongrel dogs were divided into 4 groups as follows . Group 1: Physiological saline solution was injected intraperitoneally with thoracic duct (TD) lymph drainage . Group 2: Peritonitis was induced without lymph drainage . Group 3: Peritonitis was induced with TD lymph drainage . Group 4: Peritonitis was induced with right lymph duct (RLD) and TD lymph drainage . Peritonitis was induced by intraperitoneal injection of endotoxin (Difco 055 B5 LPS) at 0.5 mg/kg . In Group 4, the endotoxin level in lymph from RLD reached a maximum value of 4.19 X 10(7) +/- 2.32 X 10(7) pg/ml (mean +/- S.E.), while that from TD was only 7203 +/- 5022 pg/ml . The endotoxin level in the portal venous blood three hours after administration was almost within normal limits throughout the experiment . In conclusion the route of endotoxin absorption in peritonitis is considered to be via the lymphatic system and in particular RLD is assumed to play an important role.

J Comp Pathol, 1987 Mar, 97(2), 207 - 15
The effects of Escherichia coli endotoxin as a trigger for hepatic infection of rabbits with Fusobacterium necrophorum; Nakajima Y et al.; To evaluate the effects of endotoxin in hepatic infection with Fusobacterium necrophorum, rabbits were given several combinations of F . necrophorum and Escherichia coli endotoxin . Severe hepatic necrosis with multiplication of F . necrophorum was induced by inoculation of endotoxin via the bile duct and following inoculation of both endotoxin and F . necrophorum . Inoculation with F . necrophorum alone, preceded by inoculation of endotoxin via the bile duct, also induced hepatic necrosis, whereas rabbits which received a single inoculation of endotoxin or F . necrophorum had only slight necrotic lesions . The pathogenetic mechanism of experimental hepatic infection with F . necrophorum is discussed.

J Biochem (Tokyo), 1987 Mar, 101(3), 817 - 20
Primary structures of and genes for new ribosomal proteins A and B in Escherichia coli; Wada A et al.; We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method . The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon . The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT . The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984 . We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.

J Biochem (Tokyo), 1987 Mar, 101(3), 813 - 6
Overproduction and preliminary X-ray characterization of aspartate aminotransferase from Escherichia coli; Kamitori S et al.; The aspartate aminotransferase of Escherichia coli was overproduced in cells after genetic manipulation, and was crystallized from a polyethylene glycol solution, pH 7.0 . The crystals obtained were of good quality and had diffractions extending beyond 2.4 A . The space group and unit cell dimensions were determined with a precession camera and a four-circle diffractometer to be C222(1), and a = 157.1 A, b = 85.5 A, and c = 79.7 A, respectively . Only one protein subunit is contained in an asymmetric unit.

J Biochem (Tokyo), 1987 Mar, 101(3), 545 - 51
Purification of a lectin from the hemolymph of Chinese oak silk moth (Antheraea pernyi) pupae; Qu XM et al.; A lectin with affinity to galactose was purified to homogeneity from the hemolymph of diapausing pupae of the Chinese oak silk moth, Anteraea pernyi . The molecular mass of this lectin was 380,000 and it formed an oligomeric structure of a subunit with a molecular mass of 38,000 . The hemagglutinating activity in the hemolymph was found to increase with time after immunization with E . coli . Studies with antibody against the purified lectin showed that increase in the hemagglutinating activity was due to the same lectin, suggesting that the amount of the lectin increased in response to intrusion of foreign substances . The function of this lectin in the defence mechanism is discussed.

J Appl Bacteriol, 1987 Mar, 62(3), 261 - 8
Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes; Shimada K et al.; Phosphate buffer suspensions of resting Escherichia coli B cells at pH 7.0 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 +/- 60 mA/cm2 and 34 degrees +/- 3 degrees C . The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes . The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells . These results suggested that a.c . exposure may serve as an agent which renders E . coli cells susceptible to the basic dyes.

Biol Chem Hoppe Seyler, 1987 Mar, 368(3), 229 - 37
Isoleucyl-tRNA synthetase from Escherichia coli MRE 600: discrimination between isoleucine and valine with modulated accuracy; Freist W et al.; Discrimination between isoleucine and valine is achieved with different accuracies by isoleucyl-tRNA synthetase from E . coli MRE 600 . The recognition process consists of two initial discrimination steps and a pretransfer and a posttransfer proofreading event . The overall discrimination factors D were determined from kcat and Km values observed in aminoacylation of tRNA(Ile)-C-C-A with isoleucine and valine . From aminoacylation of the modified tRNA species tRNA(Ile)-C-C-A(3'NH2) initial discrimination factors I1 and pretransfer proofreading factors II1 were calculated . Factors I1 were computed from ATP consumption and D1, the overall discrimination in aminoacylation of the modified tRNA; factors II1 were calculated as quotient of AMP formation rates . Initial discrimination factors I2 and posttransfer proofreading factors II2 were determined from AMP formation rates observed in aminoacylation of tRNA(Ile)-C-C-A . The observed overall discrimination varies up to a factor of about four according to conditions . Under standard assay conditions 72,000, under optimal conditions 144,000 correct aminoacyl-tRNAs are produced per one error while 1.1 or 1.7 ATPs are consumed . A comparison with isoleucyl-tRNA synthetase from yeast shows that both enzymes act principally with the same recognition mechanism, but the enzyme from E . coli MRE 600 exhibits higher specificity and lower energy dissipation and does not show such high variation of accuracy as observed with the enzyme from yeast.

Biochem Int, 1987 Mar, 14(3), 511 - 6
pHmetrical determination of the glucosamine-6-phosphate isomerase deaminase reverse reaction; Mulliert G et al.; In the reverse direction, the reaction catalyzed by glucosamine 6-phosphate isomerase deaminase consumes ammonia and forms GlcN6P . As a consequence of the formation of a product with a lower pK than the substrates, a measurable pH drop in the reaction medium is produced . This property can be used to follow potentiometrically the course of the reaction . This property can be used to follow potentiometrically the course of the reaction . The usefulness of the method is demonstrated obtaining the inhibition pattern by GlcN6P when Fru6P is the varied substrate.

Isr J Med Sci, 1987 Mar, 23(3), 202 - 4
Infected renal hematoma complicating anticoagulant therapy; Morduchowicz G et al.; We describe a case of spontaneous infection of a renal hematoma complicating warfarin sodium anticoagulant therapy . The infected hematoma was successfully drained by sonar-guided fine-needle aspiration . All reported cases of renal hematomas complicating anticoagulant therapy are reviewed.

Mol Gen Genet, 1987 Mar, 206(3), 401 - 7
Separate regulatory systems for the repression of metE and btuB by vitamin B12 in Escherichia coli; Lundrigan MD et al.; Synthesis of the btuB-encoded outer membrane receptor for vitamin B12 and the metE-encoded homocysteine methyltransferase is repressed by growth of Escherichia coli in the presence of vitamin B12 . The regulation by vitamin B12 of the production of beta-galactosidase in strains carrying btuB-lac or metE-lac operon fusions indicated that repression of both genes operates at the transcriptional level . Selection for expression of these fusions under repressive conditions allowed isolation of second-site mutations in which repressibility by vitamin B12 had been lost . Mutations in metH and metF prevented vitamin B12-dependent regulation of metE, but not that of btuB . Mutations in btuB and other genes involved in uptake of the vitamin eliminated or reduced repression . Mutations in the newly identified gene, btuR, controlled the repressibility of btuB, but had no effect on metE regulation . The btuR gene resides at 27.9 min on the genetic map in the gene order cysB-topA-btuR-trp; it acts in a trans-dominant manner and appears to encode a repressor of btuB transcription.

Am J Vet Res, 1987 Mar, 48(3), 444 - 50
BW755C modifies endotoxin-induced respiratory failure in pigs; Olson NC; The porcine pulmonary response to endotoxemia was evaluated before and after 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C), a dual inhibitor of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism . Escherichia coli endotoxin (055-B5) was infused IV into anesthetized 10- to 14-week-old pigs at 5 micrograms/kg the first hour, followed by 2 micrograms/kg/hr for 3.5 hours . The BW755C was infused at 20 mg/kg before endotoxin was administered and at 2.2 mg/kg during endotoxemia . During phase 1 (ie, 0 to 2 hours), the endotoxin-induced pulmonary hypertension, increased pulmonary vascular resistance and alveolar-arterial oxygen gradient, and decreased cardiac index and lung dynamic compliance were blocked or modified by BW755C . During phase 2 endotoxemia (ie, 2 to 4.5 hours), BW755C modified or blocked the increases in pulmonary vascular pressures, pulmonary vascular resistance, alveolar dead space ventilation, alveolar-arterial oxygen gradient, lung water, and bronchoalveolar lavage albumin concentration . The BW755C also modified the phase 2 decreases in cardiac index, lung dynamic compliance, and aortic platelet count . With regard to the endotoxin-induced pulmonary vasoconstriction, bronchoconstriction, and impairment of gas exchange, the data do not support a role for lipoxygenase metabolites, because the modified blockade (provided by BW755C) was of no greater magnitude than that reported for indomethacin (cyclooxygenase blocker) . However, the data supports a possible role for lipoxygenase metabolites with regard to altering vascular permeability, cardiac index, and aortic platelet count.

Mol Cell Biol, 1987 Mar, 7(3), 1267 - 70
Kinds of mutations formed when a shuttle vector containing adducts of benzo{a}pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells; Yang JL et al.; We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (BPDE) replicates in the monkey kidney cell line COS7 . The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol . Cell . Biol . 4:2227-2230, 1984) . When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host . Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid . Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene . The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained . Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions . In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations . Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.

What Is Yeast?, What Is Botulism?, What Is Dna?, What Is Bioremediation?, What Is Staphylococcus Aureus?, i, Microorganism, n, Microbes, r, Bacterium, r, Microbiology, n, Microorganisms, i, S. cerevisiae, s, Water treatment, s, Erwinia, s, Streptococci, s, Fermentations, n, Rhizobacter, c, Staphylococcus, i, Yeasts, i, Prokaryotes, s, Microorganisms, a, Staphylococcus aureus, n, Lactococci, c, Escherichia coli, o, Thermophile, e, Cryptococci, e, Botulin, e, Neisseria, n, Cholera, n, Culture medium, a, Culture medium, c, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005