Escherichia coli

J Mol Biol, 1988 Apr 5, 200(3), 439 - 47
Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei; Le Blancq SM et al.; Trypanosoma brucei contains a tandem array of three genes for phosphoglycerate kinase (PGKase), genes A, B and C, each coding for a different protein . We have compared allelic variants of this gene array and find evidence for gene conversion between the three genes . Near the 3' end, the different alleles and gene B contain a variable sequence that is similar to the corresponding sequence in either gene A or gene C . This sequence is flanked by glycine triplets that are conserved in all PGKases from bacteria to mammals . The triplets are encoded by (GGT)n, resulting in sequences that resemble the recombination-promoting chi-sites of Escherichia coli . Upstream of the variable sequence, there is an area of 800 base-pairs in which genes A, B and C are highly homologous; in all three genes this region ends with a sharp boundary at which gene B again shows segmental homology with both genes A and C . These results suggest that repeated gene conversion events partially erase the differences between genes A, B and C that arise in evolution and suggest that chi-like sequences may act as recombinational hotspots in protozoa such as T . brucei.

Biochemistry, 1988 Apr 5, 27(7), 2277 - 81
Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding; Sams CF et al.; Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding . Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue . The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function . The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity . In contrast, inducer binding was not recovered upon reversal of the histidine modification . However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity . Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine . The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss . Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29 . Results from the modification of core domain paralleled observations with intact repressor.

J Biol Chem, 1988 Apr 5, 263(10), 4740 - 4
Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli; Parsonage D et al.; The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide . This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain . Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate . Properties of the soluble purified F1-ATPase from each mutant were studied . The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively . 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis . 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site . 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity . 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.

J Biol Chem, 1988 Apr 5, 263(10), 4619 - 23
A mutation in the alpha-subunit of F1-ATPase from Escherichia coli affects the binding of F1 to the membrane; Maggio MB et al.; The mutation Gly-29----Asp in the alpha-subunit of the F1-ATPase from Escherichia coli was characterized and shown to cause the following effects . 1) Oxidative phosphorylation was markedly impaired in vivo 2) Membrane ATPase and ATP-driven proton-pumping activities were decreased markedly . 3) Membranes were proton-permeable, and membrane-bound ATPase was dicyclohexylcarbodiimide-insensitive . Therefore, it appeared that integration between F1 and F0 was abnormal . This was confirmed directly by the demonstration that the mutant F1 bound poorly to stripped membranes from a normal strain . Purified, soluble mutant F1 had normal ATPase activity . These results suggest that residue Gly-29, which is strongly conserved in alpha-subunits of F1-ATPases, lies in a region of the alpha-subunit important for membrane binding . Thus, three regions of the F1-alpha-subunit have now been recognized, specialized for membrane binding, nucleotide binding, and alpha/beta intersubunit signal transmission, respectively . The approximate locations of the three regions are described.

Eur J Biochem, 1988 Apr 5, 173(1), 227 - 31
Electrostatic potential of macromolecules measured by pKa shift of a fluorophore . 1 . The 3' terminus of 16S RNA; Friedrich K et al.; We have investigated the use of the pH-sensitive fluorescein label as a probe for electrostatic potential in macromolecules . The practicality of this technique is demonstrated by its application to the 16S RNA molecule . The dependence of the electrostatic potential upon ionic conditions and upon the presence of ribosomal proteins and the state of the RNA was studied . The combination of electrostatic and anisotropy data emphasizes the role of the 30S ribosomal proteins, rather than of the renaturation of the 16S RNA or the presence of the 50S subunit, in shaping the environment of the 3' terminus of the 16S RNA in the active ribosome.

J Biol Chem, 1988 Apr 5, 263(10), 4984 - 90
The effect of H2O2 upon thioredoxin-enriched lens epithelial cells; Spector A et al.; Thioredoxin, a dithiol polypeptide, has been examined as a potential contributor to the recovery of lens epithelial cells from oxidative insult . It is reported that Escherichia coli thioredoxin can (a) effectively reduce lens-soluble protein disulfide bonds generated by H2O2, (b) restore to its initial activity H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, (c) act as an effective source of reducing potential for lens methionine sulfoxide peptide reductase, and (d) act as a free radical quencher based on studies with a stable free radical system generated by ascorbic acid and 2,6-dimethoxy-p-benzoquinone . Thioredoxin is much more effective than dithiothreitol in restoring glyceraldehyde-3-phosphate dehydrogenase activity and as a cofactor for methionine sulfoxide peptide reductase . Upon incubation with epithelial cells, thioredoxin can be observed in the cell using rocket immunoelectrophoresis . These cells recover from H2O2 insult more rapidly than control cell preparations based upon 1) analyses of plasma membrane-related activities: leucine and 86Rb uptake and 2) analyses of parameters primarily related to the internal cell metabolism: ATP concentration and glyceraldehyde-3-phosphate dehydrogenase activity . Analysis of thioredoxin in cell preparations indicates that only about 9% is in the reduced state implying a low effective concentration of the polypeptide . The experiments suggest that low levels of thioredoxin may significantly increase the ability of lens epithelial cells to recover from exposure to H2O2.

J Biol Chem, 1988 Apr 5, 263(10), 4641 - 6
Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase; Cronan JE Jr et al.; The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique . Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo . A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences . The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800 . Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage . The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor . A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue . We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.

Biochemistry, 1988 Apr 5, 27(7), 2629 - 34
Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III; Gossett J et al.; A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae) . The enzyme has been purified by a series of column chromatography steps and cleaves OsO4-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts . The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates . Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations . The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.

Genomics, 1988 Apr, 2(3), 215 - 9
The complete nucleotide sequence of murine beta-glucuronidase mRNA and its deduced polypeptide; Gallagher PM et al.; The complete nucleotide sequence of murine beta-glucuronidase (GUS) mRNA has been compiled from three overlapping cloned cDNAs and a single GUS-specific genomic clone . The sequence is composed of 2455 nucleotides, exclusive of the poly(A) tail . The 5' and 3' untranslated regions contain 12 and 499 bases, respectively, with the open reading frame encoding a polypeptide of 648 amino acids (74.2 kDa), including a 22 amino acid signal sequence . The nucleotide and deduced amino acid sequences of murine GUS are compared to those published for rat and human GUS and the results are presented . Murine GUS also shares amino acid sequence identity with Escherichia coli GUS and beta-galactosidase . The complete sequences of murine GUS mRNA and its deduced polypeptide provide a basis from which to study the mechanisms responsible for the well-characterized variation in GUS expression among inbred mouse strains.

J Clin Microbiol, 1988 Apr, 26(4), 648 - 53
Immunoblot analysis of immunoglobulin G response to the Lyme disease agent (Borrelia burgdorferi) in experimentally and naturally exposed dogs; Greene RT et al.; Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs . Adsorption studies confirmed that the antibodies were specific for B . burgdorferi . Experimentally exposed dogs were asymptomatic . Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease . Naturally exposed dogs were from four geographic regions of the country . No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country . The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity to fewer and different protein bands . Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not . Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for the observed differences.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2568 - 72
Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I; Hovde CJ et al.; Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA . By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I . The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E . coli . Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid . As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA . Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase . Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation . We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.

Arch Biochem Biophys, 1988 Apr, 262(1), 337 - 44
6-Nitro-L-tryptophan: a novel spectroscopic probe of trp aporepressor and human serum albumin; Phillips RS et al.; The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism . 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O . In the presence of trp aporepressor, the visible absorption intensity is sharply diminished . Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C . While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1) . Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C . The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C . In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane . Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin . The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm . Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results . Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.

Virology, 1988 Apr, 163(2), 268 - 75
Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein; Bruss V et al.; The gene encoding the major core protein P22c of hepatitis B virus is preceded by a precore sequence . Expression of the core gene with the precore in Escherichia coli results in a membrane protein of HBe antigenicity . Expression in mammalian cells generates secreted HBeAg . To study the biosynthetic pathway of HBeAg and the function of precore in this process, we translated mRNAs for core proteins with and without precore using reticulocyte lysates and microsomal vesicles . The precore sequence was cleaved cotranslationally as a signal peptide, probably at alanine 19 . The processed product P23e was partially translocated to the lumen of the microsomes . The arginine-rich carboxy-terminal domain of P23e was however not translocated and susceptible to trypsin . Clusters of positive-charged amino acids seem to act as a novel type of translocation stop signal . Trypsin generated a P16e which no longer had a transmembraneous configuration . The findings may explain the biosynthesis and potential function of HBeAg in hepatitis B virus-infected hepatocytes.

Genomics, 1988 Apr, 2(3), 231 - 9
Genomic mapping by fingerprinting random clones: a mathematical analysis; Lander ES et al.; Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms . In such projects, the physical map is assembled by first "fingerprinting" a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints . Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap . In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme . Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project.

Genetika, 1988 Apr, 24(4), 622 - 7
{Suppression of dnaZ mutation during integration of factor F' into the chromosome of a mutant strain}; Oskolkova OB et al.; The phenomenon of suppression of dnaZ mutation has been revealed in the course of F' factor integration into the chromosome of the mutant strain . We have shown that under non-permissive conditions (t = 43 degrees C), chromosome replication in dnaZts strains proceeds under control of the factor F' replicon stably integrated into the chromosome . Possible mechanism of suppression effect, based on the formation of a bireplicon replication system, is discussed.

Br J Pharmacol, 1988 Apr, 93(4), 955 - 63
Modification by steroids of pulmonary oedema and prostaglandin E2 pharmacokinetics induced by endotoxin in rats; Izumi T et al.; 1 . A single i.p . injection of bacterial endotoxin in rats (3.5 mg kg-1) caused lung injury assessed as changes in lung dry:wet weight ratio and leukopaenia over the subsequent 28 h . 2 . This treatment also slowed the efflux of 14C from {14C}-prostaglandin E2 (PGE2), i.e., increased t1/2 and increased the survival of PGE2 in isolated perfused lungs over the same period . 3 . These effects of endotoxin were reversed by methylprednisolone (30 mg kg-1), given 30 min after the endotoxin . 4 . Another synthetic corticosteroid, budesonide (1.2 mg kg-1) given 1 h before endotoxin partially prevented the lung injury and leukopaenia but did not affect the increased t1/2 for PGE2 nor its survival . 5 . The reversal by methylprednisolone of both the physical signs of lung injury and the changes in PGE2 pharmacokinetics caused by endotoxin suggests that changes in PGE2 pharmacokinetics could serve as an index of acute lung injury following sepsis.

Mol Biochem Parasitol, 1988 Apr, 28(3), 235 - 47
Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in Escherichia coli; Brothers VM et al.; An antigenic surface protein of Eimeria tenella sporozoites has been identified that is the target of two neutralizing monoclonal antibodies Ptn 7.2A4/4 and Ptn 9.9D12 . The antigen as isolated from the parasite is composed of a 17 kDa polypeptide and a 8 kDa polypeptide linked by a disulfide bridge . De novo synthesis of the antigen does not begin until approximately 16-20 h after the initiation of oocyst sporulation . A cDNA library was constructed using mRNA from sporulated oocysts and a clone encoding the antigen was isolated . The Ta4 gene encodes a single polypeptide of 25 kDa which contains the 17 and 8 kDa polypeptides . The protein has been synthesized in Escherichia coli either directly or as part of a beta-galactosidase fusion protein . The products synthesized in E . coli are single polypeptides and are not cleaved to two polypeptides as is seen in the parasite . The products accumulate in bacteria in an insoluble form which can be solubilized and renatured to an immunoreactive form.

Biotechnol Appl Biochem, 1988 Apr, 10(2), 143 - 53
Characteristics and applications of adsorbents for pyrogen removal; Minobe S et al.; Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated . Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range . The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent . The apparent dissociation constant was 1.57 X 10(-9) M . The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed . The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length . Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.

Biochem Med Metab Biol, 1988 Apr, 39(2), 176 - 81
Effect of enterotoxigenic Escherichia coli heat stable toxin on intestinal lysosomal enzymes in the suckling rat; Aye-Kyaw et al.; A preliminary study on 9 suckling Wistar rats, which received E . coli stable toxin, and on 12 sham-operated controls showed that acid phosphatase, the marker enzyme for lysosome, was significantly increased in the infected group whereas alkaline phosphatase, glucose 6-phosphatase, succinic dehydrogenase, and proteinase, the marker enzymes for brush border, microsome, mitochondria, and the soluble fraction, respectively, remained unaffected . The results suggest that lysosome, the subcellular organelle responsible for intracellular digestion could be modified by E . coli stable toxin . In another set of experiments, where 7 infected suckling rats and 7 sham-operated controls were used, the maximal activities of lysosomal enzymes (released by Triton X-100) were found to be increased in the infected group confirming the results obtained in the preliminary experiment . The values of the ratio between maximal and basal activity (an expression of the degree of retention of enzymes to lysosome) of acid phosphatase and cathepsin D were also significantly increased, indicating that lysosomal membrane may also be stabilized during the infection . The increased activities of lysosomal enzymes and the increased lysosomal membrane stability suggest that intracellular digestion by lysosome could be increased during E . coli stable toxin infection.

Mol Gen Genet, 1988 Apr, 212(1), 76 - 84
Characterization of a sequence (hlyR) which enhances synthesis and secretion of hemolysin in Escherichia coli; Vogel M et al.; A sequence (hlyR) of about 600 bp which enhances the expression of hemolysin (HlyA) more than 50-fold was identified in the plasmid pHly152-specific hemolysin (hly) determinant . Deletion of this entire hlyR sequence led to the same low level of hemolysin synthesis and excretion as that expressed by the recombinant plasmid pANN202-312 . HlyR was active in cis but its activity was orientation-dependent . The enhancing sequence, hlyR, is separated from the promoter phlyI transcribing hlyC, hlyA and possibly hlyB by more than 1.5 kb including an IS2 element . Stepwise removal of the hlyR sequence from its 5' end by exonuclease III (ExoIII) digestion yielded several types of deletion mutants which expressed decreasing amounts of hemolysin . A similar observation was made when hlyR was shortened by ExoIII from its 3' end, which suggests that more than one functional region may be present in the hlyR sequence . A deletion of 717 bp within the adjacent IS2 element reduced the activity of hlyR only slightly, indicating that IS2 is not directly involved in the enhancement mechanism but that it may support an optimal positioning in hlyR relative to the hly promoter . The nucleotide sequence of hlyR is rich in A + T and does not contain an extended open reading frame, but exhibits several sequence motives that may represent sites for protein binding and DNA bending.

Mol Gen Genet, 1988 Apr, 212(1), 177 - 81
The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12; Nishi K et al.; A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA . The degradation and the synthesis rates of individual ribosomal proteins were determined . Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation . Other ribosomal proteins were either weakly or not at all degraded in both mutants . Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid . Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.

DNA, 1988 Apr, 7(3), 211 - 7
Site-specific oligonucleotide-directed mutagenesis using T4 DNA polymerase; Chang GJ et al.; A simple and efficient mutagenesis procedure is described which uses both the 3'----5' exonuclease and 5'----3' polymerase activities of T4 DNA polymerase . Different types of mutation-deletion, insertion, and substitution-can be introduced into the DNA in a single reaction . The technique uses recombinant M13 single-stranded DNA and two complementary DNA oligonucleotides to target and control the extent of deletions catalyzed by T4 DNA polymerase . The second oligonucleotide not only directs ligation, but also serves as a template for insertion or substitution of nucleotides by T4 polymerase . Mutant phages in a genetically pure form can be obtained at high efficiency, allowing their characterization directly by nucleotide sequencing without prior enrichment, plaque purification, and screening . We tested the versatility of this method by manipulating five regions of cDNA encoding the structural proteins of eastern equine encephalitis virus.

Can J Vet Res, 1988 Apr, 52(2), 280 - 2
Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy; Broes A et al.; Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea . Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy . The E . coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E . coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E . coli . A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E . coli O49 antiserum.

J Clin Microbiol, 1988 Apr, 26(4), 784 - 6
Evaluation of a nonisotopically labeled oligonucleotide probe to detect the heat-stable enterotoxin gene of Escherichia coli by the DNA colony hybridization test; Nishibuchi M et al.; A commercially available, alkaline-phosphatase-conjugated oligonucleotide probe for detecting the heat-stable enterotoxin gene of Escherichia coli was compared with cloned gene probes by examining E . coli isolates from traveler's diarrhea by DNA colony hybridization tests . The oligonucleotide probe was useful in specifically identifying the so-called STh gene . No deproteinization of sample was necessary to prepare the colony blots.

Genetics, 1988 Apr, 118(4), 593 - 600
Repair of single base-pair transversion mismatches of Escherichia coli in vitro: correction of certain A/G mismatches is independent of dam methylation and host mutHLS gene functions; Lu AL et al.; Six different base-pair transversion mismatches are repaired with different efficiencies in an in vitro mismatch repair system . In particular, the T/T and C/C mismatches appear to be less efficiently repaired than the A/A and G/G mismatches . Four A/G and four C/T mismatches at different positions are repaired to different extents . One of the A/G mismatches is repaired equally efficiently when DNA heteroduplexes are fully methylated or hemi-methylated at the d(GATC) sequences . This type of mismatch repair appears to be unidirectional with A to C conversion by acting at A/G mispairs to restore the C/G pairs . This methylation-independent correction is not controlled by the mutH, mutL, mutS, uvrE, uvrB, phr, recA, recF, and recJ gene products . The independence of the transversion mismatch repair of these genes and methylation distinguishes this from the known mismatch repair pathways.

Genetics, 1988 Apr, 118(4), 571 - 9
A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene; Riggs PD et al.; It was shown previously that the secA gene of Escherichia coli is derepressed in cells that have a defect in protein export . Here it is demonstrated that the beta-galactosidase produced by a secA-lacZ gene fusion strain is regulated in the same way . Studies on the fusion strain reveal that the promoter or a site involved in regulation of the secA gene is located considerably upstream from the structural gene . The properties of the fusion strain provide a new selection for mutants that are defective in protein export . Selection for increased lac expression of a secA-lacZ fusion strain yields mutations in three of the known sec genes, secA, secD and prlA/secY . In addition, mutations in several genes not previously known to affect secA expression were obtained . A mutation in one of these genes causes a pleiotropic defect in protein export and a cold-sensitive growth defect; this gene, which maps at approximately 90 min on the bacterial chromosome, has been named secE.

J Surg Res, 1988 Apr, 44(4), 417 - 24
Neutrophil phagocytosis during endotoxin-induced lung injury; Griswold J et al.; Depressed neutrophil (PMN) phagocytosis in patients with ARDS may contribute to the known increased incidence of pulmonary sepsis . To evaluate changes in phagocytosis, circulating PMNs from normal rats were compared to circulating and alveolar PMNs (obtained by bronchoalveolar lavage, BAL) from rats after 72 hr of endotoxin infusion (LPS-Rx)-induced acute lung injury . Since phagocytosis correlates with adherence, PMN adherence to coverslips and to a standard nylon wool column was also measured . PMN adherence to nylon wool was 65% for control, 77% for circulating LPS-Rx, and 20% for BAL PMNs . As a measure of phagocytosis the PMNs were incubated for 30 min with opsonized fluorescent (FITC) tagged yeast . Total PMN with yeast were 95.4 +/- 2.1% for control; 96.4 +/- 1.8% for circulating LPS-Rx; and 78.7 +/- 7.8% (P less than 0.05 compared to control) for BAL PMNs . Total numbers of yeast particles per 100 PMN are 270 +/- 64 for control, 300 +/- 42 for circulating LPS-Rx, and 170 +/- 45 (P less than 0.05 compared to control) for BAL PMN . Conclusions: (1) Intraalveolar (BAL) PMNs have decreased adherence; (2) nonadherent PMNs have decreased uptake of yeast; (3) BAL PMNs, overall, have a significantly decreased uptake of yeast; (4) this depression in BAL PMN phagocytosis may partially explain the known decreased rate of bacterial clearance in injured lungs and the increased risk of pulmonary sepsis with adult respiratory distress syndrome.

J Surg Res, 1988 Apr, 44(4), 397 - 403
The role of intestinal flora on the interactions between nonparenchymal cells and hepatocytes in coculture; Billiar TR et al.; Kupffer cells are exposed directly to a number of factors in the portal circulation that can modify or regulate their responses to septic stimuli . The gut represents a potential source of a number of these factors including endotoxin, lymphokines, and prostaglandins . We examined Kupffer cells from germfree rats and germfree rats exposed to endotoxin or bacteria via their GI tracts to determine the importance of the intestinal flora in maintaining or modulating Kupffer cell responses . Kupffer cells from germfree animals were reduced in numbers and failed to respond to LPS in Kupffer cell: hepatocyte coculture . When germfree rats were exposed to bacterial endotoxin or bacteria via the gastrointestinal tract their Kupffer cells increased in numbers to normal and the cells responded to LPS in culture . Intestinal overgrowth with Escherichia coli for 2 days activated the Kupffer cells and significantly increased Kupffer cell sensitivity to LPS . These data suggest that the environment of the gastrointestinal tract is important for normal Kupffer cell responses and that intestinal bacterial overgrowth can modify Kupffer cell responses to septic stimuli.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2449 - 53
An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity; Graves MC et al.; In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli . Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain . The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE . This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease . DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E . coli . This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E . coli . Extracts of E . coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro . These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation.

Epidemiol Infect, 1988 Apr, 100(2), 213 - 20
Managemental influences on the selective proliferation of two strains of haemolytic Escherichia coli in weaned pigs; Hampson DJ et al.; In an experimental study on a piggery it was found that haemolytic Escherichia coli of O-serotypes 138 or 139 proliferated in the intestinal tracts of pigs following weaning, with E . coli of the O-138 type also being occasionally recovered from unweaned pigs, and once from a sow . Organisms of the O-138 type produced heat labile enterotoxin and their presence in weaned pigs was associated with the development of severe post-weaning diarrhoea . E . coli of O-139 type produced a vero cell cytotoxin and were associated with a milder diarrhoea in weaned pigs . Under various managemental circumstances the O-138 type E . coli almost invariably proliferated after weaning . The O-139 strain of E . coli did however proliferate rather than the O-138 strain following the movement of weaned pigs to new accommodation, after weaned pigs were returned to their sow and then weaning again 5 days later, and very occasionally in pigs weaned at 5 weeks of age . In all these cases earlier proliferation of the O-138 E . coli had been detected, suggesting that this may be a prerequisite for proliferation of the O-139 strain.

Am Rev Respir Dis, 1988 Apr, 137(4), 783 - 9
Pulmonary edema after Escherichia coli peritonitis correlates with thiobarbituric-acid-reactive materials in bronchoalveolar lavage fluid; Ishizaka A et al.; We developed a new model of acute lung injury caused by live Escherichia coli peritonitis in guinea pigs . Arterial blood gas determinations, arterial blood pressure, and white blood cell counts were monitored serially for 12 h after the injection of either 2 x 10(9) E . coli J96 or saline . Lung water, albumin concentration in bronchoalveolar lavage fluid (BALF) and in lung tissue, WBC counts in BALF, and thiobarbituric-acid-reactive materials (TBARM) in plasma, lung tissue, and BALF were examined . Increased TBARM might be associated with pulmonary injury and are produced either by the generation of lipoperoxides secondary to oxygen-free radicals or as metabolic byproducts of prostanoid metabolism . Lung tissue sections were studied by light microscopy . E . coli peritonitis, as compared with control animals, caused significant peripheral neutropenia, histopathologic evidence of lung inflammation, acidosis, and hypotension . The wet-to-dry lung ratio was increased in the peritonitis group when compared with that in the control group (p less than 0.01) . Pulmonary edema in the peritonitis group was associated with significantly increased albumin concentrations in BALF and lung tissue . We report the new finding of increased TBARM concentrations in BALF after E . coli peritonitis (p less than 0.01 and p less than 0.05, respectively) . In contrast, plasma TBARM concentrations were unchanged . The levels of TBARM in the BALF correlated significantly with both lung water (p less than 0.01) and lung tissue albumin concentration (p less than 0.01) . The measurement of elevated TBARM in BALF may allow acute lung injury to be detected . We conclude that this model may be useful for further studies of acute lung injury caused by E . coli peritonitis.

Proc Soc Exp Biol Med, 1988 Apr, 187(4), 408 - 15
Enhanced production of monokines by canine alveolar macrophages in response to endotoxin-induced shock; Tabor DR et al.; The enhanced production of soluble mediators by alveolar macrophages may be responsible for promoting lung injury in canines administered endotoxin . One of the most prominent monokines, interleukin 1 (IL-1), has the potential to significantly influence the responses of host tissues . In this study we analyzed alveolar macrophages from canines that were experimentally administered endotoxin (AMEC) for their ability to produce IL-1 . When concentrated AMEC supernatants from in vitro cultures were incubated with fresh C3H/HEJ thymocytes, a threefold greater incorporation of {3H}thymidine resulted as compared to the response produced by controls . Heat treatment of the experimental preparations ablated this difference . Conversely, the activity of AMEC intracellular lysates did not significantly differ from the controls . Silver-staining the preparations separated by SDS-PAGE revealed a low-molecular-weight species (17 kD) in the AMEC supernatant lane while a similar molecular distribution was absent in all of the control preparations examined . Moreover, using the L929 cell line in a cytolytic bioassay we found that these same AMEC supernatants also contained significantly elevated levels of tumor necrosis factor . Collectively, this study suggests that during endotoxin-induced canine lung injury, the alveolar macrophages generate soluble species that can substantially regulate the hosts cellular response . This activity in the canine lung may play a critical role in the development and/or maintenance of the pathology associated with exposure to endotoxin.

Mutat Res, 1988 Apr, 203(2), 81 - 94
Evaluation of the SOS chromotest; von der Hude W et al.; In the present investigation, the SOS chromotest with E . coli PQ37 was evaluated . The potential to identify different kinds of bacterial mutagens was examined . 124 chemicals of different chemical classes were tested . Their responses in the SOS chromotests were compared to reported test results obtained with the Ames test.

Mutat Res, 1988 Apr, 198(2), 343 - 50
Mutagenic DNA repair in Escherichia coli . XVI . Mutagenesis by ultraviolet light plus delayed photoreversal in recA strains; Bridges BA; Mutagenesis was demonstrable after delayed photoreversal of UV-irradiated strains carrying a recA deletion indicating that RecA protein is not essential for the misincorporation process that is revealed by delayed photoreversal . Moreover, the data suggest that RecA protein actually depresses misincorporation to varying extents depending on the recA allele . No delayed photoreversal was demonstrable in reA1 or recA56 bacteria unless the lexA102(ind-) allele was also present . It is suggested that the level of these RecA proteins may be lower in the lexA102(ind-) strains thus minimising their depressive effect . Delayed photoreversal mutagenesis in strains carrying the recA441 allele was not affected by either adenine or guanosine plus cytidine, substances which affect the proteolytic activity of RecA441 protein.

J Bacteriol, 1988 Apr, 170(4), 1973 - 4
A progenitor of the outer membrane LamB trimer; Stader J et al.; During its localization to the outer membrane, LamB possesses distinctive biochemical properties as it passes through the cytoplasmic membrane . Because LamB entered this dynamic state with an attached signal sequence and leaves after cleavage, we call this export-related form of LamB the early-translocation form (et-LamB).

J Bacteriol, 1988 Apr, 170(4), 1582 - 8
Abnormal induction of heat shock proteins in an Escherichia coli mutant deficient in adenosylmethionine synthetase activity; Matthews RG et al.; Most prototrophic strains of Escherichia coli become restricted for methionine at 44 degrees C . A mutant strain (RG62 metK) in which the level of S-adenosylmethionine synthetase activity is only 10 to 20% of normal shows constitutive expression of one of the heat shock proteins, the lysU gene product, lysyl-tRNA synthetase form II, at 37 degrees C . These findings suggested a possible linkage between methionine metabolism and heat shock . We examined the induction of heat shock polypeptides in strain RG62 (metK) and in its parent, RG (metK+), from which it was derived by spontaneous mutation . Exponential-phase cultures of the two strains were pulse-labeled with {3H}leucine shortly after a shift from 37 to 44 degrees C, and the total cellular polypeptides were examined by two-dimensional electrophoresis . The results confirmed the constitutive production of the lysU gene product previously reported for strain RG62, but also revealed that the induction of 2 of the 17 heat shock polypeptides, C14.7 and G13.5, was markedly depressed . Otherwise the heat shock induction pattern was similar in timing and magnitude in the two strains . Transformation of the mutant strain with a plasmid, pK8, containing the metK coding sequence and promoter region as a 1.8-kilobase insert into pBR322 restored normal induction of C14.7 and G13.5, but did not prevent constitutive expression of the lysU gene product in the medium required for growth of this strain . The three heat shock polypeptides abnormally controlled in strain RG62 are the three polypeptides which are not induced when rapid synthesis of the htpR gene product is induced by isopropyl-beta-D-thiogalactopyranoside at 28 degree C (R . A . VanBogelen, M . A . Acton, and F . C . Neidhardt, Genes Dev . 1:525-531, 1987) . We postulate that induction of these three polypeptides involves metabolic signals in addition to the synthesis of the htpR gene product and that strain RG62 (metK) fails to produce the signals involved in induction of C14.7 and G13.5 on a shift-up in temperature and produces the signal related to lysU induction even at 37 degree C.

J Bacteriol, 1988 Apr, 170(4), 1505 - 10
Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity; Weiner JH et al.; Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase . The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography . The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The native molecular weight was determined by gel electrophoresis to be 155,000 . The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme . The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra . The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.

Infect Immun, 1988 Apr, 56(4), 930 - 5
Genetic control in the susceptibility of germfree inbred mice to infection by Escherichia coli O115a,c:K(B); Itoh K et al.; We studied the susceptibility of five germfree inbred strains of mice to oral infection by murine pathogenic Escherichia coli O115a,c:K(B) (MPEC), the causative agent of mouse megaenteron . Although MPEC colonized all strains of mice at 10(9)/g of feces, the mouse strains could be divided into three groups according to their intestinal lesions . In CF1 and C3H/He mice, intestinal lesions were produced in the cecum and colon with hyperplasia of epithelial cells accompanied by severe inflammatory reactions and erosion . The lesions in NC and C57BL/6 mice were restricted to the tip of the cecum, and hyperplasia of epithelial cells was more severe in these mice than in CF1 or C3H/He mice . BALB/c mice had no lesions . Analysis of F1 hybrids of CF1, NC, and BALB/c mice and offsprings from backcrosses of F1 mice to parental strains showed that susceptibility to MPEC seemed to be controlled genetically by a single locus which may be related to the receptors on epithelial cells for MPEC adherence . However, the differences in lesions between CF1 and NC mice suggest that a combination of this locus and another locus to which it may be related regulates the hyperplasia of intestinal epithelial cells.

EMBO J, 1988 Apr, 7(4), 1061 - 9
Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis; Zurawski SM et al.; We have analyzed structure--function relationships of the protein hormone murine interleukin 2 by fine structural deletion mapping . A total of 130 deletion mutant proteins, together with some substitution and insertion mutant proteins, was expressed in Escherichia coli and analyzed for their ability to sustain the proliferation of a cloned murine T cell line . This analysis has permitted a functional map of the protein to be drawn and classifies five segments of the protein, which together contain 48% of the sequence, as unessential to the biological activity of the protein . A further 26% of the protein is classified as important, but not crucial, for the activity . Three regions, consisting of amino acids 32-35, 66-77 and 119-141 contain the remaining 26% of the protein and are critical to the biological activity of the protein . The functional map is discussed in the context of the possible role of the identified critical regions in the structure of the hormone and its binding to the interleukin 2 receptor complex.

J Clin Microbiol, 1988 Apr, 26(4), 641 - 7
Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population; Tribe DE et al.; Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens . The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6% . Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only {GRO}) (0.4 to 0.5% of donors) . Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot . Most GRO sera reacted with p15/p17 bands on HIV immunoblot . Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E . coli-expressed p55gag . This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera . More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.

J Appl Physiol, 1988 Apr, 64(4), 1700 - 8
Effect of verapamil on pulmonary and eicosanoid responses to endotoxin in awake sheep; Ahmed T et al.; Leukotrienes have been suggested to play a role in the endotoxin-induced changes of the pulmonary hemodynamics and airway mechanics . Since Ca2+ is necessary for contraction of airway and vascular smooth muscle as well as for activation of phospholipase A2 and 5-lipoxygenase enzymes, we wondered whether the calcium antagonist verapamil would modify the endotoxin-mediated pulmonary effects as well as the generation of circulating eicosanoids . In twelve conscious sheep, measurements of pulmonary vascular resistance (PVR), systemic vascular resistance (SVR), lung resistance (RL), arterial PO2 (PaO2), leukocyte (WBC) count, arterial thromboxane B2 (TxB2), prostaglandin (PG) F2 alpha, and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) concentrations were obtained before and at predetermined intervals after a 10-min infusion of Escherichia coli endotoxin (0.3 microgram/kg) . On separate occasions, the sheep received a bolus injection of verapamil (150 micrograms/kg) before endotoxin, followed by a continuous infusion of verapamil {10 micrograms.kg-1.min-1 (n = 5) or 20 micrograms.kg-1.min-1 (n = 7)} for up to 4 h post-endotoxin . Endotoxin caused a biphasic response with an increase in mean PVR and RL to 326 and 276% of base line during phase I (0-1 h) and lesser increases to 177 and 157% of base line during phase II (1.5-4 h), respectively (P less than 0.05) . SVR also showed biphasic increases of 44 and 42% during phase I and II, respectively . Mean PaO2 decreased by 16 Torr and WBC count decreased from 6.4 +/- 1.5 to 3.3 +/- 1.1 thousand/mm3, associated with marked increases in plasma TxB2, PGF2 alpha, and 6-keto-PGF1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1988 Apr, 2(1), 63 - 8
A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro; Lehtovaara PM et al.; A new efficient in vitro mutagenesis method for the generation of complete random mutant libraries, containing all possible single base substitution mutations in a cloned gene is described . The method is based on controlled use of polymerases . Four populations of DNA molecules are first generated by primer elongation so that they terminate randomly, but always just before a known type of base (before A, C, G or T respectively) . Each of the four populations is then mutagenized in a separate misincorporation reaction, where the correct base can now be omitted . The regeneration of wild-type sequences can thus be efficiently avoided . Also, the misincorporating nucleotide concentrations can be optimized to give the three possible single mutations in close to equal ratio . The mutagenesis can be precisely localized within a predetermined target region of any size, and vector sequences remain intact . We have mutagenized the DNA coding for the alpha-fragment of Escherichia coli beta-galactosidase, and identified 176 different base substitution mutations by sequencing . The present method gives mutant yields of 40-60%, when the mutants contain about one amino acid change per protein molecule . All types of base substitution mutations can be generated and deletions are rare . The efficiency of this method permits the use of relatively elaborate screening systems to isolate mutants of either structural genes or regulatory regions.

Mol Gen Mikrobiol Virusol, 1988 Apr, (4), 37 - 41
{Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase}; Petrenko VA et al.; Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis . In both cases the fused protein was expressed . The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase . The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.

Nippon Geka Gakkai Zasshi, 1988 Apr, 89(4), 482 - 93
{Postoperative debasement of the host-defense system and wound healing in elderly patients, and nutritional support as a therapeutic implication}; Kuroiwa K; Plasma opsonic activity and several kinds of plasma protein levels in each pre- and post-operative days were compared between older 25 cases (70 years or older) and 31 younger cases (59 years or younger) which both underwent elective abdominal surgery . The activity to opsonize E . coli 075 as a host-defense capacity was reduced on postoperative days 1,3 and returned gradually in either young or elderly . But opsonic activity levels in elderly patients in each postoperative days were significantly lower than in younger group . Plasma albumin level and rapid turnover protein levels in elderly patients tended to be lower than those in younger group through the postoperative period but statistically not significant . Plasma levels of C3 and fibronectin overshot the each preoperative value on days 5-7, after sharp reduction on day 1 in younger patients, but those in elderly didn't . Opsonic activity levels were closely correlated with plasma protein levels . Based on these clinical results, the experiments were designed to evaluate the effect of nutritional support in the early postoperative period on opsonic activity and protein synthesis in aged rats . These studies elucidated that early nutritional support brought beneficial effects on opsonic activity and plasma protein levels on postoperative days 7, but not on days 3 in aged rats . This may be because in aged animals, protein synthetic function has not fully recovered in the early postoperative period.

Eur J Clin Microbiol Infect Dis, 1988 Apr, 7(2), 167 - 71
Identification of enterotoxigenic Escherichia coli using alkaline phosphatase-labeled synthetic oligodeoxyribonucleotide probes; Olive DM et al.; Alkaline phosphatase-conjugated synthetic oligodeoxyribonucleic acid probes were used to detect enterotoxigenic Escherichia coli strains containing either the heat stable or heat labile toxin genes . Both of the synthetic probes detected as little as 5 ng of purified plasmid DNA bearing the appropriate toxin gene . In addition, both probes could detect 5 X 10(6) toxigenic bacteria by colony hybridisation . No cross reactivity was observed between probes . When 197 clinical isolates of Escherichia coli were examined for toxigenicity using bioassays, 13 heat stable and 17 heat labile toxin strains were identified . Of the 13 heat stable toxin strains, 12 were positive using the heat stable toxin synthetic probe (sensitivity, 92%; specificity, 98%) while 16 of 17 bioassay heat labile toxin positive samples were identified using the heat labile toxin synthetic probe (sensitivity, 94%; specificity, 97%) . Alkaline phosphatase-conjugated synthetic probes with high sensitivity and specificity should provide a rapid means of identifying toxigenic Escherichia coli.

J Interferon Res, 1988 Apr, 8(2), 169 - 78
Effect of recombinant interferon-gamma on protein content, phagocytic, and cytotoxic activity of mouse peritoneal macrophages; Rollag H et al.; We have studied the effect of recombinant murine interferon-gamma (rMuIFN-gamma) on the protein content, phagocytic activity, and cytotoxicity of mouse peritoneal macrophages (MPM) . The aim of this study was to see whether rMuIFN-gamma alone could influence these parameters of MPM activity or if an additional stimulus, like elicitation or cultivation with lipopolysaccharide (LPS), was required . The MPM cultures were treated with rMuIFN-gamma for 24, 48, or 72 h . Generally, rMuIFN-gamma treatment of the cultures increased the protein content of the MPM . MPM were generally cytotoxic and they phagocytized IgG-opsonized Escherichia coli under the experimental conditions of this study . The effects of rMuIFN-gamma on phagocytic and cytotoxic activities were complex and highly dependent on the dose and length of treatment . Low or high concentrations may exert opposite effects on the same functions . Addition of a low dose of LPS to the cultures did not generally amplify the rMuIFN-gamma-induced alterations of MPM activities . However, the combination of LPS, high doses of rMuIFN-gamma, and long incubation time reduced the protein content, suppressed the phagocytic activity, and negatively influenced the viability of the MPM . Thioglycolate-elicited MPM had a higher baseline activity than resident MPM, but the rMuIFN-gamma effect on elicited MPM was parallel to the effect on resident MPM.

J Antimicrob Chemother, 1988 Apr, 21(4), 439 - 43
Standardization of inoculum size for disc susceptibility testing: a preliminary report of a spectrophotometric method; Moosdeen F et al.; Many methods of disc susceptibility testing aim at an inoculum to yield a semi-confluent growth . We have improved the means of standardizing inoculum by measuring the turbidity with a spectrophotometer . Absorbance measurements were made for various bacterial suspensions and dilutions thereof, from 1 in 2 to 1 in 1000 . Volumes of 0.1 ml of each dilution were spread on to Diagnostic Sensitivity Test agar . Viable counts of bacteria were made for each absorbance reading and for what constituted semi-confluent growth . Suspensions of bacteria having similar absorbance readings contained variable numbers of viable bacteria depending on the species . The number of bacterial cells that yielded semi-confluent growth also varied with different bacterial species . A chart was prepared to indicate the appropriate dilutions of bacterial suspensions with different absorbance readings to produce semi-confluent growth in sensitivity testing of each species.

Br J Exp Pathol, 1988 Apr, 69(2), 169 - 76
Observations on the delay in onset of the acute phase protein response; Myers MA et al.; The early time course of the acute phase protein response (APPR) and mediators involved in its control were investigated in the rat and mouse . After turpentine-induced inflammation in the rat C-reactive protein and fibrinogen increased in concentration peaking at 48 h and 18-24 h, respectively . A 9 h delay prior to elevation of these protein was observed . After injection of endotoxin into mice, a 4-6 h delay was observed prior to any increase in the concentration of the acute phase protein serum amyloid P-component . This delay was shortened to 2 h after injection of leucocytic endogenous mediators (LEM) produced from rabbit peritoneal exudate cells . It is concluded that the delay between the initiating stimulus and the increases in the acute phase proteins is due to some obligatory intermediate steps which lead to the production of the final mediators of the APPR, and that these mediators are present in LEM.

Arthritis Rheum, 1988 Apr, 31(4), 506 - 14
Quantitative immunoassay of anti-La antibodies using purified recombinant La antigen; St Clair EW et al.; A purified recombinant La fusion protein was tested in an enzyme-linked immunosorbent assay to quantitate anti-La responses . This protein contained the immunodominant region of the La molecule fused to beta-galactosidase . In solid-phase assays, recombinant La protein was solubilized in urea and bound to polystyrene wells without loss of immunoreactivity . The recombinant-based enzyme-linked immunosorbent assay proved to be a sensitive method for the detection of anti-La binding, and it accurately distinguished anti-La precipitin positive sera from normal sera.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2709 - 13
The mutY gene: a mutator locus in Escherichia coli that generates G.C----T.A transversions; Nghiem Y et al.; We have used a strain with an altered lacZ gene, which reverts to wild type via only certain transversions, to detect transversion-specific mutators in Escherichia coli . Detection relied on a papillation technique that uses a combination of beta-galactosides to reveal blue Lac+ papillae . One class of mutators is specific for the G.C----T.A transversion as determined by the reversion pattern of a set of lacZ mutations and by the distribution of forward nonsense mutations in the lacI gene . The locus responsible for the mutator phenotype is designated mutY and maps near 64 min on the genetic map of E . coli . The mutY locus may act in a similar but reciprocal fashion to the previously characterized mutT locus, which results in A.T----C.G transversions.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2603 - 7
Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ; Nolan GP et al.; We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed . To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells . Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity . This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods . We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ . These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2474 - 8
Phosphorylation of the RAS2 gene product by protein kinase A inhibits the activation of yeast adenylyl cyclase; Resnick RJ et al.; The RAS2 gene product of Saccharomyces cerevisiae expressed in Escherichia coli was phosphorylated by protein kinase A in vitro to approximately 0.5-0.7 mol of phosphate per mol of protein . Neither protein kinase C nor protein kinase P phosphorylated the RAS2 protein significantly . The RAS2 protein is known to activate, in the presence of either Mg2+ and GTP or Mn2+, a yeast membrane preparation with an overexpressed adenylyl cyclase and a deficiency in endogenous RAS1 and RAS2 proteins . When the RAS2 protein was phosphorylated by protein kinase A prior to exposure to the yeast membranes, its capacity to activate the adenylyl cyclase was diminished by 40-60%, while activation by Mn2+ remained unaffected . The phosphorylated protein retained, however, its ability to bind GTP . Incubation of protein kinase A with a specific protein kinase A inhibitor prior to phosphorylation prevented the inhibition . Furthermore, the hydrolysis of GTP was not required for the observed inhibition . These data suggest that phosphorylation of the RAS2 gene product by protein kinase A may function as one mechanism by which the intracellular level of cAMP in yeast is regulated.

J Gen Virol, 1988 Apr, 69 ( Pt 4), 765 - 76
Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene; Vlak JM et al.; The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene . The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA . Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10 . Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes . Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent . The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells . Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product . The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane . The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S . exigua larvae than was wild-type AcMNPV . The increased virulence of the recombinant is an important property for the control of insects.

J Bacteriol, 1988 Apr, 170(4), 1975 - 7
The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor; Peterson KR et al.; LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background . LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor . lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).

Biotechniques, 1988 Apr, 6(4), 332 - 9
An enrichment selection for mutants resulting from oligonucleotide-directed mutagenesis of double-stranded DNA; McCracken AA et al.; We report here a simple and rapid procedure for enrichment and selection of mutants from oligonucleotide-directed mutagenesis on double-stranded plasmid DNA . Mutagenic oligonucleotides were designed to insert or delete a unique restriction site with silent codon changes . After mutagenesis, plasmid DNA from all resulting colonies was pooled, restricted with the appropriate endonuclease, and the resulting unique form of DNA (linear or circular) was isolated and used for transformation of competent E . coli . These procedures provided an enrichment of mutant plasmid from the 4% obtained by more conventional techniques to greater than 65%.

Pathology, 1988 Apr, 20(2), 167 - 72
Enterotoxigenic Escherichia coli in Central Australia: diagnosis using cloned and synthetic nucleic acid probes; Higgins GD et al.; Feces from 169 children admitted with diarrhea to Alice Springs Hospital, were screened for enterotoxigenic E . coli (ETEC) using specific DNA probes . E . coli which hybridized with probes for ST-P, ST-H and LT were confirmed by bioassay for toxin production . Fifty children were shown to excrete ETEC . The probes for LT correlated well with bioassay; however, probes for ST-H and ST-P hybridized with more E . coli than were shown to produce toxin by bioassay . When probing for ST was repeated using a synthetic oligonucleotide probe, only those specimens which were bioassay-positive hybridized with the probe.

Bioorg Khim, 1988 Apr, 14(4), 472 - 7
{Affinity modification of Escherichia coli ribosomes with the non-hydrolyzed GTP analog, gamma-{4-N-(2-chloroethyl)-N-methylaminobenzyl}amide of GTP}; Babkina GT et al.; Substituted gamma-amides of GTP viz . GTP gamma-{4-N-(2-chloro- and gamma-{4-N-(2-hydroxyethyl)-N-methylaminobenzyl}amide (CIRCH2NHpppG and OHRCH2NHpppG, resp.) were shown to be unhydrolisable GTP analogues in the EF-Tu-dependent GTP-ase reaction of ribosomes . The reactive analogue, CIRCH2NHpppG, was used for affinity labelling within the 70S ribosome.poly(U).tRNAPhe(P-site).Phe-tRNAPhe.EF-Tu.CIR{14C}CH2.NHpppG complex . Both 50S and 30S subunits were thus labelled but 50S subunit was modified considerably more than 30C subunit . Labelled were proteins L17, L21, S16, S21, and rRNA of both subunits, 23C rRNA within 50C subunit being labelled preferentially as compared with 50C proteins . No labelling of EF-Tu within the complex was detected.

Vet Q, 1988 Apr, 10(2), 117 - 25
Pathophysiological effects of endotoxins in ruminants . 2 . Metabolic aspects; Lohuis JA et al.; Metabolic disturbances following intravenous and intramammary administration of endotoxins in ruminants are described . In contrast to the similarity in response of blood biochemical parameters after intravenous and intramammary administrations of endotoxins, responses in plasma concentrations of enzyme activities, the thyroid hormones, cortisol, and somatotropin differ markedly . Biochemical changes in blood after endotoxin administration are predominantly dose-dependent; thus some of the biochemical parameters - especially plasma concentrations of Fe and Zn - serve also to evaluate the effects of certain drugs in endotoxin models . Changes in milk composition have been documented only after intramammary infusion of endotoxins and can partly be explained by the increased permeability of the blood/milk barrier . Appearance and production of milk returns to normal within a week after intramammary endotoxin treatment, indicating that the mammary gland is only temporarily damaged by endotoxin-induced mastitis.

Vet Q, 1988 Apr, 10(2), 109 - 16
Pathophysiological effects of endotoxins in ruminants . 1 . Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration; Lohuis JA et al.; Data from the literature on the clinical effects of bacterial endotoxins in ruminants are reviewed . Special attention is paid to the effects on body temperature and reticulo-rumen motility . Furthermore, the effects of repeated intravenous injection of endotoxin are summarised . Pathophysiological disturbances after intramammary infusion of endotoxins proved to be identical to those found after intravenous injection of non-lethal doses . Strikingly, however, no marked inhibitory effect on rumen motility nor abortion was observed after intramammary infusion of endotoxins . Moreover, in cows that were made tolerant to endotoxin by daily intravenous injections, intramammary infusion of one-fifth of this daily dose produced a maximum effect on body temperature and plasma Zn concentrations . This suggests that inflammatory endogenous mediators were released in the udder and then absorbed into the blood circulation, rather than the absorption of endotoxin.

Zhonghua Liu Xing Bing Xue Za Zhi, 1988 Apr, 9(2), 84 - 7
{Serotypes of enteroinvasive Escherichia coli in China}; Yang ZS; results of identification and serotyping of enteroinvasive escherichia in china from 13 provinces in China in 1984-85 were reproted.

Behring Inst Mitt, 1988 Apr, (82), 349 - 59
A histidin alanine rich recombinant antigen protects Aotus monkeys from P . falciparum infection; Knapp B et al.; We have isolated a cDNA clone coding for 165 amino acids of a histidine alanine rich protein . An extended repeat region of this clone codes for the tripeptide Ala-His-His, which is extremely conserved, and for the tripeptide Ala-Ala-Asp, which shows only slight variability among the repeat units . This antigen exhibits high homology to the HRPII antigen described by Wellems and Howard (1986) . The coding region was expressed in E . coli as a MS2-polymerase fusion protein, which was purified and used for immunization of Aotus monkeys . The animals immunized with this fusion protein showed only low parasitemias (less than 2%) after infection with P . falciparum, while animals from the control group or animals immunized with a MS2-fusion protein carrying other malaria specific sequences were not protected . The result suggests that this antigen is a good candidate for a malaria vaccine.

Behring Inst Mitt, 1988 Apr, (82), 338 - 48
Immunoreactivity of human immunodeficiency virus (HIV-1) envelope polypeptides expressed in Escherichia coli; Broker M et al.; Defined cDNA sequences encoding segments of envelope glycoproteins of the human immunodeficiency virus were expressed in E . coli . The recombinant env fusion proteins were tested for immunoreactivity with patient sera by radio immunoprecipitation procedures . The regions of the env proteins, carrying relevant antigenic determinants in respect to diagnostic purposes, could be identified.

Behring Inst Mitt, 1988 Apr, (82), 26 - 34
Efficient expression system for human antithrombin III in baby hamster kidney cells; Zettlmeissl G et al.; A DNA fragment carrying the enhancer of an immediate early gene of human cytomegalovirus (hCMV) was tested as a transcriptional control element by fusion downstream of a transcription unit for human antithrombin III (AT III) driven by the Simian virus (SV40) enhancer and promoter . Measurement of transient AT III expression in baby hamster kidney cells (BHK) shows that by the presence of the hCMV enhancer the synthesis of AT III is increased considerably (three to fourfold) . The AT III expression vectors carrying the hCMV enhancer were used to establish stable BHK cell-lines by a new and fast G418/methotrexate selection protocol, which express up to 12 micrograms AT III/10(6) cells/24h after 40-50 days . The given system might be generally useful for the fast expression of recombinant proteins in mammalian cells, e.g . the screening of altered AT III molecules obtained by site directed mutagenesis.

Behring Inst Mitt, 1988 Apr, (82), 59 - 67
Isolation and expression of cDNA coding for a new member of the phospholipase A2 inhibitor family; Grundmann U et al.; The placental protein PP41,2 was shown to have thromboplastin-inhibitor activity . We used partial amino acid sequence information from PP4 cyanogen bromide fragments to design oligonucleotide probes for the screening of a human placental cDNA library . In addition to the PP4 cDNA we isolated a cDNA coding for a protein with considerable homology which we subsequently termed PP4-X . PP4 and PP4-X belong to the phospholipase A2 inhibitor family, as judged by their homology to lipocortin I and calpactin I3 . The full-length PP4-X cDNA encodes a protein of 321 amino acid residues including a fourfold repeat structure . Northern blot analysis using the PP4-X cDNA reveals two hybridizing RNA species of approximately 1400 nucleotides and 2500 nucleotides, respectively . The shorter one could well represent the PP4-X transcript which is in good agreement with the isolated cDNA insert of 1326 nucleotides . Expression of the PP4-X coding sequence in E . coli resulted in the appearance of a protein which crossreacts with antibodies raised against PP4.

Genetics, 1988 Apr, 118(4), 551 - 60
Changes in DNA base sequence induced by gamma-ray mutagenesis of lambda phage and prophage; Tindall KR et al.; Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced . Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation . For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites . The remaining mutations were 1 and 2 base deletions . In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage . In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.

Am J Physiol, 1988 Apr, 254(4 Pt 2), R567 - 71
Immunoreactive atrial natriuretic factor is increased in ovine model of endotoxemia; Lubbesmeyer HJ et al.; A bolus of Escherichia coli endotoxin (1.5 micrograms/kg) was administered to chronically instrumented sheep . Immunoreactive atrial natriuretic factor (IR-ANF) was measured in extracted plasma by radioimmunoassay . There was a thirteenfold increase in IR-ANF 2 h after endotoxin administration, and IR-ANF levels remained significantly elevated during the first 6 h . A marked diuresis and natriuresis occurred between 4 and 6 h . ANF not only affects renal function but is also associated with decreased cardiac output, increased peripheral resistance (in sheep), and decreased capillary absorption (in rats) . These renal and hemodynamic changes are also characteristic of the early (first 6 h) response to endotoxin . Therefore ANF should be considered as a potential mediator of renal and hemodynamic changes induced by sepsis . It is difficult to determine if ANF elevation is an epiphenomenon or a causative factor, because no antagonist of ANF is currently available.

Zh Mikrobiol Epidemiol Immunobiol, 1988 Apr, (4), 90 - 3
{Detection of the gene sequences of the leukocytosis (lymphocytosis)-stimulating factor of Bordetella pertussis in Bordetella parapertussis and Bordetella bronchiseptica by using molecular hybridization}; Rozinov MN et al.; The 4.7 Kb EcoRI-fragment of phase I B . pertussis 475 (serovar 1.2.3) chromosome DNA carrying the pertussis toxin (PT) operon was cloned on vector plasmid pUC19 in Escherichia coli . Three fragments (1.14 Kb KpnI-PstI, 1.27 Kb PstI-PstI, and 0.96 Kb PstI-PstI) were obtained from the resulting hybrid plasmid, coded pRH119, by electrophoretic techniques and used as a combined molecular probe for analysis of the EcoRI-digested and PstI-digested chromosomal DNA of B . pertussis strain 475 in phase I, B . pertussis in phase IV, B . parapertussis strains 504 and 17903, B . bronchiseptica strain 214, and B . parapertussis strain 17903 (a convertant obtained by means of B . pertussis phage 134), as well as B . pertussis phage 134 . Southern blot hybridization under the conditions of 100% DNA-DNA homology showed the presence of DNA sequences characteristic of the PT operon in all cases except the DNA of phage 134; moreover, the use of the above-mentioned probe made it possible to hybridize all EcoRI-fragments of chromosomal DNA, having the same molecular size (4.7 Kb) . Consequently, the PT genes in the above Bordetella species were mapped in identical loci.(ABSTRACT TRUNCATED AT 250 WORDS)

Zh Mikrobiol Epidemiol Immunobiol, 1988 Apr, (4), 86 - 90
{Cloning and gene expression of the leukocytosis (lymphocytosis) stimulating factor of Bordatella pertussis in Escherichia coli by bringing the PT genes close to the lactose promoter of plasmid pUC19}; Rozinov MN et al.; The 4.7 Kb EcoRI-fragment of phase I B . pertussis 475 (serovar 1.2.3.) chromosome carrying all five genes of the pertussis toxin (PT) operon was cloned on plasmid pUC19 in E . coli . The resulting hybrid plasmid pRH119 contained the PT operon in the same orientation of transcription as the lac promoter of plasmid pUC19 . Nevertheless, the expression of the PT operon was not observed even after induction with isopropyl thio-beta-D-galactopyranoside (IPTG), which suggested either the inability of the PT operon to work in E . coli, or the presence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119 . The expression was determined by the incubation of the clones harboring plasmid pRH119 with antiserum to PT and their subsequent in situ treatment with 125I-labeled protein A . Three deletion variants of plasmid pRH119 were constructed with the aim of approaching the PT genes to the lac promoter: pRH121 (the 0.45 Kb KpnI-fragment deleted), pRH122 (the 0.95 Kb SalGI-fragment deleted) and pRH123 (the 1.35 Kb XbaI-fragment deleted) . In all these cases different levels of expression were observed (but only in the presence of IPTG) . Site KpnI in the 4.7 Kb fragment was found to be localized in the -57 b . p . region in relation to the PT promoter, i . e . to lie, seemingly, in the promoter zone; for this reason, the expression of the PT genes in plasmid pRH121 proved the existence of a transcription terminator between the lac promoter and the PT operon in plasmid pRH119.(ABSTRACT TRUNCATED AT 250 WORDS)

Vaccine, 1988 Apr, 6(2), 197 - 9
Progress towards a vaccine against enterotoxigenic Escherichia coli; Kaper JB et al.; A variety of approaches are being investigated in the development of a vaccine against enterotoxigenic Escherichia coli (ETEC) . These approaches include purified fimbriae vaccines, toxoid vaccines, live attenuated E . coli vaccine strains and ETEC antigens expressed in carrier organisms . Studies of the pathogenesis and immune response to ETEC indicate that development of a vaccine against human ETEC is a realistic goal but considerable work remains before this goal is realized.

Vaccine, 1988 Apr, 6(2), 110 - 2
Tip proteins of pili associated with pyelonephritis: new candidates for vaccine development; Lund B et al.; Escherichia coli strains associated with extra-intestinal infections frequently express carbohydrate binding adhesins which are present as minor components of pili . The adhesin protein, PapG, and at least two other minor pilus subunits, PapE and PapF, are associated with the tips of Gal alpha (1-4)Gal-binding Pap pili or P fimbriae of uropathogenic E . coli . The structural and antigenic variation of these tip-associated proteins is discussed and evidence is presented showing that serologically identical pili may contain antigenically distinct adhesins each capable of binding to a specific receptor . One approach to the purification of these tip-associated proteins is presented and involves complex formation with the periplasmic transport protein PapD.

APMIS, 1988 Apr, 96(4), 337 - 41
In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes . Correlation with size of alpha-hemolysin production; Gadeberg OV et al.; The correlation of the in vitro cytotoxic effect of 107 alpha-hemolytic strains of Escherichia coli with various other bacterial characteristics was investigated . Damage to human blood granulocytes in the presence of fresh or heated autologous plasma was quantified by measuring the release of chromium-51 from labelled cells . 95 strains had a cytotoxic effect which was equal in the presence of fresh or heated plasma, whereas 12 strains showed an effect which was reduced in fresh compared with heated plasma . The cytotoxic effect increased as the number of bacteria per granulocyte was increased . The average size of the alpha-hemolysin production of the strains was 185 HU50/ml ranging from 3-2519HU50/ml . The cytotoxic effect of the strains was directly correlated with the size of the alpha-hemolysin production . The cytotoxic effect was not correlated with the O-antigen serotype or the type of infection from which the strains were derived . These results indicate that the ability to produce alpha-hemolysin is the bacterial characteristic which is of decisive importance for the cytotoxicity of alpha-hemolytic E . coli towards human blood granulocytes.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2464 - 8
On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation; Huang CS et al.; gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques . The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000 . Comparison of the E . coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers) . Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group . Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate . However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol . Thus, the rat kidney and E . coli enzymes share several catalytic features but differ in active site structure . If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.

Proc Natl Acad Sci U S A, 1988 Apr, 85(7), 2046 - 50
Identification of the second chromophore of Escherichia coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate; Johnson JL et al.; Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin . Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties . The same species, in the same distribution, were obtained from the yeast enzyme . The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis . The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid . Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate . The results were consistent with gamma linkages in the triglutamate derivative with additional glutamates linked via the alpha-carboxyl group of the preceding residue . Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate . The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure.

J Bacteriol, 1988 Apr, 170(4), 1887 - 94
Structure and antigenic properties of the tip-located P pilus proteins of uropathogenic Escherichia coli; Lund B et al.; Pyelonephritogenic Escherichia coli frequently expresses pili which bind to Gal alpha (1-4)Gal receptors present on the uroepithelium . Binding of these pili is mediated by a pilus-associated adhesin, PapG, and not by the major subunit which constitutes the bulk of the pilus structure . The adhesin and two pilinlike proteins, PapE and PapF, are present in only a few copies each at the pilus tip . Surface exposure of both PapF and PapG is required to achieve receptor-specific binding . The nucleotide sequences for the genes encoding the tip-associated proteins PapE, PapF, and PapG were determined for two E . coli clones expressing P pili of serotypes F11 and F7(2) and compared with the corresponding sequences established for proteins of F13 pili . Specific antisera were used to study the cross-reactivity between the F13 tip proteins and the equivalent proteins in F11 and F7(2) pili . We present data showing that, like the major pilus subunit, PapE varies its structure and antigenic properties among pili of different serotypes . In contrast, the PapF protein was highly conserved, and PapF-specific antisera raised against serotype F13 cross-reacted with the PapF proteins of both F11 and F7(2) serotypes . The PapG adhesin protein from F11 and F7(2) pili differed by only five amino acids out of 316 residues . However, the F13 adhesin showed only 45% amino acid homology with the other two variants.

J Bacteriol, 1988 Apr, 170(4), 1666 - 71
Regulation of fatty acid degradation in Escherichia coli: fadR superrepressor mutants are unable to utilize fatty acids as the sole carbon source; Hughes KT et al.; Localized mutagenesis of the fadR region of the Escherichia coli chromosome resulted in the isolation of two classes of fadR regulatory mutants . The first class was constitutive for the fatty acid degradative enzymes and presumably defective for fadR function . The second class was rarer and resulted in the inability to utilize fatty acids as a sole carbon source (Fad-) . These fadR superrepressor mutants {fadR(S)} had greatly reduced levels of the beta-oxidative enzymes required for growth on fatty acids . The fadR(S) mutants reverted to Fad+ at a high frequency (10(-5}, and the resulting Fad+ revertants were constitutive for expression of the fad enzymes (fadR) . Merodiploid analysis showed the fadR(S) allele to be dominant to both fadR+ and fadR alleles.

J Bacteriol, 1988 Apr, 170(4), 1610 - 6
Polymorphisms in the umuDC region of Escherichia species; Sedgwick SG et al.; The umuDC operon of Escherichia coli encodes mutagenic DNA repair . The umuDC regions of multiple isolates of E . coli, E . alkalescens, and E . dispar and a single stock of E . aurescens were mapped by nucleotide hybridization . umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long . Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract . Restriction site polymorphisms were not found around the DNA repair gene recA or polA . The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons . Possible mechanisms for the generation of these variants are discussed.

Infect Immun, 1988 Apr, 56(4), 815 - 22
Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes; Steadman R et al.; Uropathogenic strains of Escherichia coli bearing mannose-sensitive (type 1) fimbriae promote a unique pattern of degranulation from human polymorphonuclear leukocytes (PMN) . Significant quantities of the primary (1 degree) and tertiary (3 degree) granule markers, neutral protease-myeloperoxidase and N-acetyl-beta-D-glucosaminidase, respectively, were released by PMN in a dose- and time-dependent manner when stimulated by these defined bacterial strains . Organisms bearing mannose-resistant (P) fimbriae promoted release of only the secondary (2 degree) granule marker, vitamin B12-binding protein . When this pattern of degranulation was compared to that produced by PMN in response to a variety of soluble and particulate stimuli, only the calcium ionophore A23187 similarly triggered 1 degree and 3 degree granule marker release . All the other stimuli tested--zymosan, serum-treated and unopsonized; n-formylmethionyl-leucyl-phenylalanine; and phorbol myristate acetate--promoted release of only the 2 degree granule marker . These results demonstrate selectivity of PMN degranulation in response to a number of transmembrane signals . In addition, the capacity of E . coli to promote PMN degranulation is dependent on its phenotypic fimbrial expression, a surface characteristic which correlates significantly with its relative surface hydrophobicity as measured by binding to octyl Sepharose . Those bacteria demonstrating the greatest hydrophobicity were capable of triggering discharge of all three granule marker proteins . Thus, the mannose-sensitive fimbriae of uropathogenic E . coli may contribute significantly to their potential pathophysiologic role in renal scarring.

EMBO J, 1988 Apr, 7(4), 1211 - 8
Cellular factors required for multiple stages of SV40 DNA replication in vitro; Fairman MP et al.; Plasmids containing the SV40 origin replicate in the presence of SV40 T antigen and a cell free extract derived from human 293 cells . Upon fractionation of this extract, two essential replication factors have been identified . One of these is a multi-subunit DNA binding protein containing polypeptides of 70,000, 34,000 and 11,000 daltons which may function as a eukaryotic single strand DNA binding protein (SSB) . The other partially purified fraction is required with T antigen for the first stage of DNA replication, the formation of a pre-synthesis complex at the replication origin . These results, and others, define multiple stages of SV40 DNA replication in vitro which are analogous to multiple stages of Escherichia coli and phage lambda replication, and may reflect similar events in the replication of cellular chromosomes.

EMBO J, 1988 Apr, 7(4), 1191 - 6
The expression of novel antigens from the Epstein-Barr virus large internal repeat; Walls D et al.; A large Epstein-Barr virus (EBV) B95-8 genomic bank has been prepared in an Escherichia coli expression vector and screened with a pool of sera from human infectious mononucleosis patients . Four immunopositive clones which also contained sequences from the viral large internal repeat were selected . DNA sequence analysis has located them on the repeat sequence and shown that they come from three potential open reading frames and that two of them consist of overlapping reading frames . This must imply extensive intron/exon splicing or that the repeat itself encodes several different proteins . The four expressed epitopes were shown to be present simultaneously in independent cases of infectious mononucleosis . These have not been previously described and based on the experimental design, they must reflect the situation in vivo.

Mol Biol Med, 1988 Apr, 5(2), 107 - 22
Cloning and characterization of a cDNA encoding human galactose-1-phosphate uridyl transferase; Reichardt JK et al.; We report the cloning and characterization of a cDNA that encodes a functional human galactose-1-phosphate uridyl transferase (GALT) . The cDNA is 1400 bases in length and encodes a 43,000 Mr protein . The cloning strategy involved the identification of short peptide sequences conserved between the homologous enzymes from Escherichia coli and yeast, and the construction of oligonucleotide pools corresponding to the conserved patches . These patches of conserved amino acids tend to be conserved in humans as well.

Biochem J, 1988 Apr 1, 251(1), 111 - 4
Use of progress curves to estimate the co-substrate-to-substrate flow ratio of a symport mechanism . Application to the isoleucine-Na+ symport of mouse ascites-tumour cells and to the lactose-proton symport; Eddy AA et al.; The model envisages two components in the process, whereby Ht equivalents of co-substrate and St equivalents of substrate accumulate in the cellular compartment in time t . The first is the flow through the symport, n equivalents of co-substrate entering or leaving with each substrate equivalent . The second is the basal flow of co-substrate outside the symport . In certain specific circumstances n can be derived by plotting Ht/t against St/t . The principal requirement is that, whereas the ratio of the component flows must change in the interval t, the magnitude of the basal flow must either be zero or constant . The procedure is applied to published observations {West & Mitchell (1973) Biochem . J . 132, 587-592} on the lactose-proton symport of Escherichia coli {n = 1.075 +/- 0.064(7)} and to new observations on the isoleucine-Na+ symport of mouse ascites-tumour cells {n = 1.136 +/- 0.120(18)}.

Biotechnol Appl Biochem, 1988 Apr, 10(2), 107 - 17
Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration; Hoffman RC Jr et al.; Polyphosphate kinase (ATP:polyphosphate phosphotransferase; EC 2.7.4.1), partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity . The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor . Chromatographic analyses of the product mixture produced from ADP and {32P}polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase (EC 3.6.1.11), adenosine triphosphatase (EC 3.6.1.4), and adenylate kinase (EC 2.7.4.3) . Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C . It may be stored indefinitely as a lyophilized powder at -10 degrees C . Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme . A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium . The ease of purification and immobilization of E . coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration.

Mol Cell Biol, 1988 Apr, 8(4), 1509 - 17
Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence; Kumar R et al.; In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R . Kumar, T . A . Firak, C . T . Schroll, and K . N . Subramanian, Proc . Natl . Acad . Sci . USA 83:3199-3203, 1986) . Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation . In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication . A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently . Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present . Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity . Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration . Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer . These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication . Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

Mol Gen Genet, 1988 Apr, 212(1), 99 - 104
AsnC, a multifunctional regulator of genes located around the replication origin of Escherichia coli, oriC; Kolling R et al.; The expression of the gidA gene which is located immediately counterclockwise of the replication origin of Escherichia coli, oriC, was found to be negatively regulated by the AsnC protein in an in vitro transcription-translation system . This effect is not due to simple repression of transcription originating at the gidA promoter, because the AsnC protein did not change the level of gidA promoter dependent transcription as analysed by promoter-galK fusions and by S1 mapping . From these data we conclude that the AsnC protein controls gidA gene expression at a post-transcriptional level . gidA is the third gene in the oriC region, besides asnA and asnC, whose expression is under AsnC control . However, the mechanisms involved are different: regulation of transcription in the case of asnA and asnC and post-transcriptional control of gidA . The gidA promoter was mapped by deletion analysis and by S1 mapping . We defined two regions that affect promoter activity negatively . Additional transcripts, regulated by AsnC, started more than 300 bp upstream of the gidA promoter and were found to enter the gidA region . These transcripts, originating either at the mioC and/or the ansC promoter traverse the replication origin.

Mol Gen Genet, 1988 Apr, 212(1), 1 - 5
Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12; Nakamura H et al.; The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b561 and its flanking region was determined . The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20,160 . From its deduced amino acid sequence, cytochrome b561 is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions . Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane . No significant homology of the primary structure of cytochrome b561 with those of other bacterial b-type cytochromes was observed.

DNA, 1988 Apr, 7(3), 193 - 201
Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor; Shirai T et al.; The mouse tumor necrosis factor (TNF) gene was isolated from a mouse genomic library . The entire sequence of the gene was determined using both an automated DNA sequencer with improved primer extension reaction conditions as well as the standard radioisotopic method . Comparison of the nucleotide sequence of the gene with that of mouse TNF cDNA showed that the mouse gene consists of four exons, like rabbit and human TNF genes . There is strong nucleotide sequence homology in the 5'-flanking region among the mouse, rabbit, and human TNF genes, suggesting that the mechanisms regulating TNF gene expression are highly conserved . Direct expression of mature mouse TNF was achieved using a plasmid constructed by site-directed mutagenesis . Purified mouse TNF produced in Escherichia coli showed cytotoxicity to mouse L cells.

DNA, 1988 Apr, 7(3), 173 - 9
Effect of ribosome binding site on gene expression in Escherichia coli; Curry KA et al.; Using the expression of human renin gene in Escherichia coli as a model, we have observed that a distance of 7-10 nucleotides between the Shine-Dalgarno site to the initiation codon ATG is optimal for translation initiation . The sequence ATA as the triplet preceding the ATG gives the best expression among those tested . These observations agree with the statistical bias observed for the genes of E . coli and its phages . We have also compared gene expression with three different Shine-Dalgarno sites . Expression of a given gene can be increased by as much as 1000-fold with slight modifications in the Shine-Dalgarno sequence.

Genetics, 1988 Apr, 118(4), 561 - 70
Different reading frames are responsible for IS1-dependent deletions and recombination; Braedt G; Two classes of plasmids in addition to the parent become apparent when plasmids that contain direct repeats of IS1 . One class of plasmids has deleted sequences from the end of IS1 to nonrandom sites within the plasmid . The appearance of these plasmids in the population requires intact insA and insB reading frames, but not insC . The other class of plasmids has undergone an exchange within the direct repeats of IS1 on the plasmid . Their appearance requires InsC but neither InsA nor InsB . The two reactions may represent two distinguishable steps in IS1 transposition . The InsC-catalyzed exchange is independent of RecA and resembles hologous recombination since the frequency of recombinants arising from exchanges in different regions of IS1 appears to be roughly proportional to the size of the region . InsC can also catalyze an exchange between direct repeats of non-IS1 DNA.

Leukemia, 1988 Apr, 2(4), 211 - 5
Binding of iodinated recombinant human GM-CSF to the blast cells of acute myeloblastic leukemia; Kelleher CA et al.; Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML) . Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein . Normal human neutrophils and the blast cells from AML patients were examined for binding . We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils . After brief culture in suspension, receptor number increased and affinity decreased . Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM) . This difference was reflected in the increased effectiveness of the E . coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2563 - 7
A unique deoxyguanosine triphosphatase is responsible for the optA1 phenotype of Escherichia coli; Beauchamp BB et al.; Escherichia coli optA1, a mutant unable to support the growth of T7 phage containing mutations in gene 1.2, contains reduced amounts of dGTP . Extracts of E . coli optA1 catalyze the hydrolysis of dGTP at a rate 50-fold greater than do extracts of E . coli optA+ . The dGTPase responsible for the increased hydrolysis has been purified to apparent homogeneity . Purification of the protein is facilitated by its high affinity for single-stranded DNA . By using this purification scheme an identical dGTPase has been purified from E . coli optA+ . The purified proteins catalyze the hydrolysis of dGTP to yield deoxyguanosine and tripolyphosphate . The products of hydrolysis, chromatographic properties, denatured molecular mass of 56 kDa, N-terminal amino acid sequence, substrate specificity, and heat inactivation indicate that the proteins purified from optA1 and from optA+ cells are identical and identify the enzyme as the deoxyguanosine 5'-triphosphate triphosphohydrolase purified to homogeneity from wild-type E . coli {Seto, D., Bhatnagar, S . K . & Bessman, M . J . (1988) J . Biol . Chem . 263, 1494-1499} . OptA1 cells contain approximately equal to 50-fold more active molecules of the 56-kDa dGTPase than do E . coli optA+ cells.

Virology, 1988 Apr, 163(2), 330 - 40
A poliovirus mutant defective for self-cleavage at the COOH-terminus of the 3C protease exhibits secondary processing defects; Kean KM et al.; By in vitro recombination between the wild-type full-length infectious cDNA of poliovirus and a clone generated by the construction of a cDNA bank from a chemically derived temperature-sensitive plurimutant, we obtained a mutant cDNA with a T to C change at nucleotide 5658 . This mutation replaces the isoleucine at residue 74 of the viral protease 3C by a threonine . The mutant virus recovered after transfection exhibited a small-plaque phenotype, and was deficient for viral RNA synthesis . Both these defects were more marked at 39 than at 37 degrees . The mutation was introduced into a bacterial plasmid which expresses the 3C protease along with its flanking autocatalytic cleavage sites . Analysis of the cleavage products expressed in Escherichia coli provided direct evidence that the modification impaired cleavage at the COOH-terminus of 3C . Cleavage at this same site was partially defective in mutant virus-infected HeLa cells, reducing the production of mature 3C and the viral replicase, 3D . Cleavage of P1, the precursor to the capsid polypeptides, was apparently unaffected by this defect, whereas cleavage events within the P2 region of the genome occurred inefficiently . This is indicative of differential strategies for 3C-specific cleavage events in vivo.

Proc Natl Acad Sci U S A, 1988 Apr, 85(7), 2224 - 8
Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends; Makris JC et al.; Insertion sequence 50 (IS50) transposition utilizes a 19-base-pair "outside" end and a 19-base-pair "inside" end in inverted orientation relative to each other, whereas transposon 5 (Tn5) transposition utilizes two inverted outside ends . The frequency of transposition events that involve an inside end is regulated 1000-fold by the host dam methylase system . The end sequence requirements for transposition and its regulation by dam methylase were analyzed in Escherichia coli by generating random single base pair mutations in either an IS50 inside end or outside end placed in inverted orientation with respect to an unmutagenized outside end . The mutations were then isolated, assayed for transposition phenotype, and sequenced . Mutations were isolated at 15 of the 19 sites in the outside end . All of these mutations except those at position 4 decreased transposition . Mutations at position 4 (which is the only nonidentical base pair in a region of homology between the outside and inside ends) had no effect on transposition . Mutations were isolated at 11 of the 19 sites in the inside end . All of these mutations, including one at position 4, decreased transposition in dam- cells . Mutations at position 10 (within a dam recognition sequence) and 2 (not within a dam recognition sequence) reduced the magnitude of dam regulation . A mutation within a dam recognition sequence adjacent to the required 19 base pairs of the inside end did not reduce the magnitude of dam regulation.

J Bacteriol, 1988 Apr, 170(4), 1965 - 8
Transposition of IS50L activates downstream genes; Kendrick KE et al.; A transposition system constructed to detect the transposition of Tn5 to a site upstream of the lacZ gene has revealed that transposition of IS50L can activate downstream genes . Expression is apparently mediated by the NPTII promoter . Transposase produced either by IS50R or by the suppressed IS50L catalyzed transposition of IS50L.

J Bacteriol, 1988 Apr, 170(4), 1902 - 6
Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411; Ishiguro N et al.; The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined . The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence . IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase . Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells . Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.

J Bacteriol, 1988 Apr, 170(4), 1837 - 42
Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase; Farr SB et al.; Different conditions of oxidative stress were used to study their effects on membrane transport in Escherichia coli K-12 . The oxidizing conditions included H2O2, plumbagin (a redox cycling compound that generates superoxide radicals {O2-}), and increased partial pressure of oxygen . Both superoxide radical-generating conditions and H2O2 treatments were found to cause a rapid decrease in proton motive force-dependent and -independent transport . H2O2-pretreated cells had the ability to rapidly recover both proton motive force-dependent and -independent transport . The induction required transcription and translation and was dependent on oxyR+ and katG+, providing evidence that these genes play crucial roles in the rapid recovery of transport . The effects of oxidatively induced loss of proton motive force on cell growth and macromolecular synthesis were also investigated.

J Bacteriol, 1988 Apr, 170(4), 1721 - 9
narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes; Sodergren EJ et al.; In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon . From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively . Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated . The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined . The nar operon therefore contains four genes designated and ordered as narGHJI.

J Bacteriol, 1988 Apr, 170(4), 1589 - 97
Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12; Stewart V et al.; Previous studies have shown that narL+ is required for nitrate induction of nitrate reductase synthesis and for nitrate inhibition of fumarate reductase synthesis in Escherichia coli . We cloned narL on a 5.1-kilobase HindIII fragment . Our clone also contained a previously unidentified gene, which we propose to designate as narX, as well as a portion of narK . Maxicell experiments indicated that narL and narX encode proteins with approximate MrS of 28,000 and 66,000, respectively . narX insertion mutations reduced nitrate reductase structural gene expression by less than twofold . Expression of phi (narL-lacZ) operon fusions was weakly induced by nitrate but was indifferent to aerobiosis and independent of fnr . Expression of phi (narX-lacZ) operon fusions was induced by nitrate and was decreased by narL and fnr mutations . A phi (narK-lacZ) operon fusion was induced by nitrate, and its expression was fully dependent on narL+ and fnr+ . Analysis of these operon fusions indicated that narL and narX are transcribed counterclockwise with respect to the E . coli genetic map and that narK is transcribed clockwise.

J Bacteriol, 1988 Apr, 170(4), 1511 - 8
Molecular cloning and expression of the Escherichia coli dimethyl sulfoxide reductase operon; Bilous PT et al.; The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molybdoenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from an E . coli gene bank . Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO and trimethylamine N-oxide (TMAO) reductase activities 13- to 15-fold compared with wild-type cells . Amplification was highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reduced levels of enzyme amplification . Growth on nitrate or aerobic growth repressed expression of the enzyme . A 6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vector pBR322, yielding a recombinant DMSO reductase plasmid, pDMS159 . Two polypeptides were amplified and identified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E . coli HB101 harboring pDMS159: a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight 23,600 . Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 were detected by in vivo transcription/translation studies . The smallest subunit was poorly defined and not detectable by Coomassie blue staining . The DMSO reductase operon was localized to the 20.0-min position on the E . coli linkage map.

J Bacteriol, 1988 Apr, 170(4), 1461 - 6
Two genes, pemK and pemI, responsible for stable maintenance of resistance plasmid R100; Tsuchimoto S et al.; Plasmid R100 was found to have two genes, designated pemK and pemI, that were responsible for its stable inheritance during cell division . They are located near the region that is essential for autonomous replication . Under conditions that inhibit replication of R100 derivatives, the plasmid containing these pem genes gave only a few segregants in viable cells and increased the number of nonviable cells in the population, suggesting that a product from the pem region stabilized the plasmid by killing plasmid-free segregants . Inactivation of one of the two translational open reading frames in the pem region caused the loss of the killing function, and thus, the open reading frame is a gene designated pemK, which encodes the killing factor . The coexistence of the pem+ plasmid with a high-copy-number plasmid carrying the other open reading frame inhibited stabilization, and thus, the second open reading frame is a gene designated pemI, which encodes the inhibitor which might control the killing function of pemK . It is likely that the two open reading frames were transcribed from a promoter . There were no significant homologies in DNA sequences between the pem gene of R100 and the genes previously shown to be responsible for the stable inheritance of the other plasmids.

J Bacteriol, 1988 Apr, 170(4), 1417 - 22
Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations; Garges S et al.; We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP . These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein . Currently, we have isolated intragenic suppressors of the crp mutations . These crp (Sup) mutants require cAMP for activity . The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation . From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains . Cyclic GMP fails to effect the last step.

Gan To Kagaku Ryoho, 1988 Apr, 15(4 Pt 2-1), 810 - 9
{Colony-stimulating factors in cancer chemo- and radiotherapy}; Okabe T; Recombinant human granulocyte colony-stimulating factor (Re Hu G-CSF) was prepared and its stimulating effect on granulocytopoiesis was examined in mice . Human G-CSF was purified to homogeneity from conditioned media of a G-CSF-producing cell line . The amino-terminal sequence was determined . By using oligonucleotides as probes, determined by the amino acid sequence, a cDNA library prepared from human macrophages was screened . The cloned G-CSF cDNA was expressed in E . coli K12MM294, and the mature protein was purified to homogeneity . Mice were given intraperitoneal injections of Re Hu G-CSF every day for 14 days . Peripheral blood granulocyte counts were examined 4, 8, 12 and 14 days after injection . Mice were sacrificed on the 14th day for histologic examinations of the bone marrow and spleen . Granulocyte counts began to increase on the 4th day and reached about 80,000/mm3 on the 14th day . Cells of granulocyte lineage were markedly increased in the bone marrow and spleen . Granulocyte precursors (CFU-C) were remarkably increased in the spleen . When mice were treated with 5-fluorouracil, cyclophosphamide or irradiation, the period of granulocytopenia was significantly shortened by subcutaneous injection of Re Hu G-CSF . These results suggest that human G-CSF play a central role in granulocyte production in vivo . The ability of Re Hu G-CSF to stimulate granulocyte production implies that this factor will be clinically useful in neutropenic patients treated with anti-cancer agents or irradiation.

Vaccine, 1988 Apr, 6(2), 94 - 8
Preferential pairing of T-B specificities in the same antigen: the concept of directional help; Celada F et al.; It is important for an effective planning of the new generation of vaccines to consider that the eventual response is regulated by the interplay of stimulating and suppressing epitopes, resulting in dominance of certain sites over others, and that in order for a molecule to be crossimmunogenic with the outside invader both B and T epitopes must be present . However, T-B cooperation is not random but is guided by preferential pairing of sites within a single macromolecule; this situation can be explained by antigen processing by B cells and paratope mediated interference with endosomial degradation.

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2538 - 42
Structure of rho factor: an RNA-binding domain and a separate region with strong similarity to proven ATP-binding domains; Dombroski AJ et al.; The domain structure of rho protein, a transcription termination factor of Escherichia coli, was analyzed by oligonucleotide site-directed mutagenesis and chemical modification methods . The single cysteine at position 202, previously thought to be essential for rho function, was changed to serine or to glycine with no detectable effects on the protein's hexameric structure, RNA-binding ability, or ATPase, helicase, and transcription termination activities . A 151-residue amino-terminal fragment (N1), generated by hydroxylamine cleavage, and its complementary carboxyl-terminal fragment of 268 amino acids (N2) were extracted from NaDod-SO4/polyacrylamide gels and renatured . The N1 fragment binds poly(C) and mRNA corresponding to the rho-dependent terminator sequence trp t', but not RNA unrecognized by rho; hence, this small renaturable domain retains not only the binding ability but also the specificity of the native protein . Uncleaved rho renatures to regain its RNA-dependent ATPase activity, but neither N1 nor N2 exhibits any detectable ATP hydrolysis . Similarly, the two fragments, isolated separately but renatured together, are unable to hydrolyze ATP . Sequence homology to the alpha subunit of the E . coli F1 membrane ATPase, and to consensus elements of other nucleotide-binding proteins, strongly suggests a structural domain for ATP binding that begins after amino acid 164 . The implications of discrete domains for RNA and nucleotide binding are discussed in the context of requirements for specific interactions between RNA-binding and ATP-hydrolysis sites during transcription termination.

J Immunol, 1988 Apr 1, 140(7), 2422 - 30
Hybrid genes between HLA-A2 and HLA-A3 constructed by in vivo recombination allow mapping of HLA-A2 and HLA-A3 polymorphic antigenic determinants; Sire J et al.; HLA-A2 and -A3 genes have been modified in their third exon (second domain) by using in vivo recombination . In this method Escherichia coli are transfected with a plasmid which contains two highly homologous sequences (e.g., the third exons of HLA-A2 and -A3) and has been linearized by cleavage between these two sequences . Circularization takes place in the bacteria by homologous recombination leading to hybrid A2-A3 sequences . The analysis by DNA sequencing of a number of such recombinants shows that they indeed occur by homologous recombination (no insertions or deletions) and that the probability of crossing over decreases as the distance from the free end of DNA in the homologous region increases . No double recombinants were observed . These hybrid exons were reinserted into either HLA-A2 or HLA-A3 genes, thus generating a panel of functional hybrid genes containing one or several HLA-A2 specific substitutions in an HLA-A3 background or vice versa . These genes were expressed by transfection into murine P815-high transfection efficiency recipient cells . Serologic analysis leads to the conclusion that expression of polymorphic antigenic determinants specific for HLA-A2 (detected with M58, A2A28M1, and CR11.351 mAb) is linked to the presence of threonine residue (amino acid (AA) 142) and/or histidine residue (AA 145) and valine residue (AA 152) . The expression of specific HLA-A3 polymorphic determinants (recognized by GAP-A3 mAb) is correlated with the existence of a asparagine residue (AA 127) and a aspartic residue (AA 161) . But aspartic residue 161 contributes with glutamic acid residue 152 in the formation of the A3 epitope recognized by the anti-A3 mAb X1.23.2.

J Bacteriol, 1988 Apr, 170(4), 1622 - 30
Transcriptional organization of the Escherichia coli hemolysin genes; Welch RA et al.; The transcriptional organization of the Escherichia coli hemolysin genes (hlyCABD) encoded by pSF4000 was examined . The use of different hemolysin gene-specific radiolabeled probes in blots containing isolated in vivo RNA revealed 4.0-kilobase hlyCA and 8.0-kilobase hlyCABD transcripts . The treatment of cells with rifampin just before RNA isolation showed the half-lives of these mRNAs to be 10.2 and 4.4 min, respectively . The 5' ends of the hly transcripts were 462 and 464 nucleotides from the putative initiation codon of hlyC based on a primer extension method of RNA mapping . Deletion analysis of pSF4000 combined with quantification of the hemolysin structural protein HlyA by immunoblotting confirmed that major control of HlyA expression occurs within a 168-base-pair PstI fragment located 433 base pairs upstream of the start of hlyC . A second recombinant plasmid, pANN202-312, encoding an E . coli hemolysin of different origin expressed 6-fold less total HlyA and 50-fold less extracellular HlyA than pSF4000 in identical cell backgrounds . The pANN202-312 recombinant had a different hly promoter, with the hly mRNA beginning 264 nucleotides upstream from the start of hlyC . We showed by RNA blotting that cells harboring pANN202-312 compared with pSF4000 have similar steady-state levels of the hlyCA transcript but they lack a consistently detectable hlyCABD transcript . We propose that one reason for the disparate levels of extracellular hemolysin produced by hemolytic E . coli is dissimilar levels of mRNA encoding in part the transport genes hlyB and hlyD.

J Virol, 1988 Apr, 62(4), 1433 - 6
Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia coli; Mous J et al.; Expression of the human immunodeficiency virus type I pol open reading frame in Escherichia coli led to several protease-mediated processing steps of the pol precursor polyprotein . Accumulation of two polypeptides with molecular sizes of 64 and 52 kilodaltons, with which reverse transcriptase activity is associated, was observed . The protease moiety of the precursor polyprotein accumulated as a 10-kilodalton species as a result of two specific cleavages . Furthermore, a single-amino-acid substitution in the putative active site of protease totally abolished processing of the precursor polyprotein.

Gene, 1988 Mar 31, 63(2), 331 - 6
Genetic basis of hypoxanthine guanine phosphoribosyltransferase deficiency in a patient with the Lesch-Nyhan syndrome (HPRTFlint); Davidson BL et al.; The molecular basis for complete hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency has been determined in a patient with Lesch-Nyhan syndrome . A B-lymphoblastoid cell line derived from this patient expresses normal amounts of HPRT mRNA yet no detectable immunoreactive protein as determined by radioimmunoassay . These findings suggest either a decreased rate of translation or accelerated degradation due to enhanced proteolytic susceptibility . cDNAs synthesized from this patient's RNA have a single nucleotide (nt) substitution, a C----A transversion at nt 222 . RNase A cleavage analysis confirms the presence of a mutation at this position within mRNA isolated from lymphoblasts from patient A.C . This transversion predicts a phenylalanine to leucine replacement at amino acid position 73 in the translated protein . We have designated this mutant HPRTFlint . The mutation in HPRTFlint disrupts a strongly conserved region among PRTases from Escherichia coli, rodents and man, suggesting an important role for this region for the normal function of HPRT . Since it is unlikely that this amino acid substitution alters the translational rate, we hypothesize that disruption of the secondary structure within this region renders HPRTFlint more susceptible to proteolysis.

Biochim Biophys Acta, 1988 Mar 31, 949(3), 318 - 24
DNA transfection of Escherichia coli by electroporation; Taketo A; Electroporation was applied to transfection and transformation of Escherichia coli . Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor . As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E . coli . Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions . In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation) . Various aspects of the electroporation-mediated DNA uptake are discussed.

Gene, 1988 Mar 31, 63(2), 309 - 20
Transcriptional patterns for the thrS-infC-rplT operon of Escherichia coli; Wertheimer SJ et al.; The genes coding for threonyl-tRNA synthetase (thrS), translation initiation factor 3 (infC) and ribosomal protein L20 (rplT) are clustered in the Escherichia coli genome . Previous studies had suggested the possibility that the expression of these genes is coupled . The transcriptional events in this operon have now been examined by S1 nuclease mapping and promoter fusion studies . The results indicate that infC-containing mRNAs are initiated from three separate promoters . Two of these are located in the protein-coding region of thrS and one, P12, is the major promoter at all growth rates tested . In addition, there is co-transcription of thrS and infC from the thrS promoter (PT) . A single promoter for thrS has been mapped approx . 170 nucleotides upstream from its translation initiation site . Another promoter has been located within the infC-coding region . It is separated from the next downstream gene, rplT, by a transcription end point . However, termination at this region is only 50-70% efficient and transcripts starting at this promoter can read through into rplT . These findings demonstrate that the pattern of transcription in this operon is highly complex and the mRNA levels for each of the genes is determined by a variety of factors, including multiple promoters, co-transcription and readthrough of transcription termination signals.

Gene, 1988 Mar 31, 63(2), 277 - 85
Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant; Krebs MP et al.; We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition . Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media . Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition . In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements . Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain . Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions . We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R . The mutation altered the amino acid sequence of both transposase and inhibitor.

Gene, 1988 Mar 31, 63(2), 199 - 212
An amdS-lacZ fusion for studying gene regulation in Aspergillus; Davis MA et al.; A translational fusion has been constructed between the amdS gene of Aspergillus nidulans and the lacZ gene of Escherichia coli . Sequencing across the fusion junction confirmed the generation of an in-frame fusion at amino acid 34 of amdS and a novel protein has been detected in transformants carrying the fusion plasmid . Transformants of A . nidulans and Aspergillus niger carrying the fusion plasmid were obtained by co-transformation with a second selectable plasmid . These transformants were readily identified on media containing XGal . The intensity of the reaction on XGal media was indicative of the number of copies of the fusion plasmid carried by the transformants . The growth of highly expressing strains of A . nidulans was inhibited on XGal media . The fusion plasmid was used to develop a two-step gene replacement strategy in which the resident amdS gene was replaced with the fusion gene free of vector sequences . Plate tests and in vitro assays of the beta-galactosidase enzyme confirmed that expression of the fusion gene was regulated by amdS flanking sequences and trans-acting regulatory genes.

Biochim Biophys Acta, 1988 Mar 31, 949(3), 297 - 304
Macromolecular crowding extends the range of conditions under which DNA polymerase is functional; Zimmerman SB et al.; The nick-translation reaction of E . coli DNA polymerase I (Pol I) was used as a model system to demonstrate the ability of macromolecular crowding to alter the response of an enzyme to a number of basic parameters, such as pH, temperature or inhibitors . In the presence of high concentrations of non-specific polymers, nick translation occurred under a variety of otherwise strongly inhibitory conditions . The conditions tested included a range of pH values or temperatures or inhibitory concentrations of urea, formamide or ethidium bromide . These crowding effects are accentuated at higher ionic strengths, suggesting their origin in increased binding between the polymerase and its DNA template-primer under crowded conditions . Kinetic measurements were consistent with such a mechanism.

Biochim Biophys Acta, 1988 Mar 30, 933(1), 35 - 41
Electron conduction between b cytochromes of the mitochondrial respiratory chain in the presence of antimycin plus myxothiazol; West IC et al.; The b haems of the bc1 complex of bovine heart mitochondria were poised with succinate and fumarate so that only the high-potential haem (b-562) was reduced, and then isolated from further redox exchange with the ubiquinone pool by adding antimycin and myxothiazol . A transmembrane electric potential difference was then developed, either by electron flow from {Ru(NH3)6}Cl2 to oxygen or by ATP hydrolysis . The small difference spectrum, caused by the electric field, indicated 32-55% oxidation of b-562 with concomitant reduction of b-566 . No lag greater than 0.1 s was detectable between the initiation of respiration and the development of the difference spectrum, thus providing a direct demonstration of (fairly) rapid electron transfer between the b haems.

Biochem Biophys Res Commun, 1988 Mar 30, 151(3), 1033 - 8
Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli; Babul J et al.; When E . coli carrying multicopy plasmids for fructose-1,6-P2 aldolase or phosphoglycerate kinase was grown in the presence of 32Pi, there was label at the position of cognate high level polypeptide after SDS-PAGE . As tested for aldolase, the label was resistant to acetone, RNase, and hot TCA treatments, and was also observed by immunoprecipitation, which was competed for by purified aldolase . Incorporation of label also occurred in the presence of chloramphenicol . Immunoprecipitation revealed apparent aldolase labeling in the wild type strain as well.

Biochim Biophys Acta, 1988 Mar 30, 933(1), 65 - 9
Energetic consequences of two mutations in Escherichia coli K+ uptake systems for growth under potassium-limited conditions in the chemostat; Mulder MM et al.; The energetics of growth of two Escherichia coli strains (TK 2240 and TK 2242) differing in Km of the high-affinity potassium uptake system and lacking the low-affinity system were studied in the chemostat under potassium-limited conditions . The results were compared with the results obtained previously (Mulder, M.M., Teixeira de Mattos, M.J., Postma, P.W . and Van Dam, K . (1986) Biochim . Biophys . Acta 851, 223-228) with the wild-type FRAG-1, having two potassium uptake systems, and FRAG-5, a mutant which lacks the high-affinity potassium uptake system . We postulated that the high-affinity potassium uptake system was able to generate such a steep gradient across the membrane that the low-affinity system would act in reverse, thus creating a futile cycle of potassium ions at the cost of energy . As a result, FRAG-1 would show a higher ATP turnover at all growth rates tested than the mutant FRAG-5, in which strain the proposed futile cycle is interrupted because of the lack of the high-affinity system . It is shown here that the results obtained with TK 2240 and TK 2242 are in line with our hypothesis of futile potassium cycling . Under our experimental conditions, the yield on potassium was not dependent on the kinetic parameters of the uptake systems . The (thermodynamic) energy demand of the uptake systems determined the carbon substrate conversion required to achieve this yield.

Biochim Biophys Acta, 1988 Mar 30, 933(1), 179 - 83
Relationships between membrane-bound cytochrome o from Vitreoscilla and that of Escherichia coli; Georgiou CD et al.; The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related . They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly . Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups . Antibodies raised against the oxidase purified from E . coli crossreact with the Vitreoscilla oxidase.

FEBS Lett, 1988 Mar 28, 230(1-2), 201 - 4
Human interferon-gamma lacking 23 COOH-terminal amino acids is biologically active; Sakaguchi M et al.; We constructed five mutated cDNAs encoding human interferon-gamma (IFN-gamma) derivatives lacking 19-23 COOH-terminal residues and expressed them in Escherichia coli . All the derivatives were purified to homogeneity . They showed substantially the same order of antiviral activity in vitro as the intact molecule, and behaved as a dimer . The far- and near-UV circular dichroism spectra of the derivatives were quite similar to those of the intact one . These results indicate that the 23 COOH-terminal amino acids at least are not essential for achieving the full antiviral activity and constructing the higher structure of human IFN-gamma.

FEBS Lett, 1988 Mar 28, 230(1-2), 171 - 5
Bleomycin resistance conferred by a drug-binding protein; Gatignol A et al.; The protein coded by a bleomycin-resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E . coli strain and its action on bleomycin was determined by in vitro assays . The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA . The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin-binding protein.

Nucleic Acids Res, 1988 Mar 25, 16(6), 2369 - 88
Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA; Stiege W et al.; Poly(A) can be cross-linked to E . coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation . The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety . Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose . The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA . The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys . The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model.

J Biol Chem, 1988 Mar 25, 263(9), 4386 - 91
Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli; Guerra DJ et al.; Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E . coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism . The plant ACP expressed in E . coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP . We have purified and characterized the recombinant spinach ACP-I . NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine . Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I . Recombinant ACP-I was a poor substrate for E . coli fatty acid synthesis . In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E . coli ACP . Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E . coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B . napus reacted equally well with either E . coli ACP or recombinant ACP-I . E . coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E . coli ACP . Expression of spinach ACP-I in E . coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.

J Biol Chem, 1988 Mar 25, 263(9), 4293 - 301
Identification of a cross-link in the Escherichia coli ribosomal protein pair S13-S19 at the amino acid level; Pohl T et al.; Escherichia coli 30 S ribosomal subunits and 70 S ribosomes were treated with the bifunctional reagent diepoxybutane, acting as a cross-linker . One major cross-linked protein pair in the 30 S subunit was generated in relatively high yields . This cross-link was shown to consist of ribosomal proteins S13 and S19 . Purification of this complex was achieved by a series of conventional and/or high pressure liquid chromatography techniques allowing its isolation in milligram quantities . To reveal the exact position of the two amino acids involved in the cross-link formation, the purified protein pair S13-S19 was subjected to several enzymatic fragmentations, and the resulting peptides were characterized by sequence analysis, amino acid analysis, and fast atom bombardment mass spectrometry . After isolation of the cross-linked peptides, Cys84 in protein S13 and His68 in S19 could be unequivocally identified as the amino acids cross-linked by the bifunctional reagent . This result demonstrates that, despite neutron scattering data which place the centers of mass of S13 and S19 85 A apart, at least these regions of the two proteins are located within a 4-A distance in the ribosomal particle.

J Biol Chem, 1988 Mar 25, 263(9), 4274 - 9
Inductions of superoxide dismutases in Escherichia coli under anaerobic conditions . Accumulation of an inactive form of the manganese enzyme; Privalle CT et al.; Escherichia coli growing anaerobically respond to NO3- with a 3-fold induction of the iron-containing superoxide dismutase . Mutants lacking nitrate reductase do not show this response . Anaerobically grown cells also contain an inactive form of the manganese-containing superoxide dismutase (MnSOD) which can be activated by addition of Mn(II) salts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer . Direct addition of Mn(II) to a neutral solution of the inactive MnSOD does not impart activity . This inactive MnSOD thus behaves as would the apoenzyme or the enzyme bearing a metal other than Mn(II) at its active sites . Terminal electron acceptors, such as NO3- or trimethylamine N-oxide, increase the amount of inactive MnSOD produced by anaerobic E . coli . Paraquat, which is itself ineffective in this regard, markedly augments the effect of these terminal electron acceptors . It appears that flow of electrons to sinks such as NO3- or trimethylamine N-oxide, facilitated by paraquat, is sufficient to elicit biosynthesis of the MnSOD protein and that O2- is not needed for this process . Yet, oxygenation and concomitant O2- production do appear important for the insertion of manganese into the growing MnSOD polypeptide, possibly because O-2 oxidizes Mn(II) to Mn(III), and the latter is the valence state most effective in combining with the apoenzyme.

J Biol Chem, 1988 Mar 25, 263(9), 4269 - 73
The role of redox in the regulation of manganese-containing superoxide dismutase biosynthesis in Escherichia coli; Schiavone JR et al.; The manganese-containing superoxide dismutase in Escherichia coli is an inducible enzyme that protects cells against oxygen toxicity . The manganese-enzyme is induced by oxygen, nitrate, redox active compounds that react with oxygen to generate superoxide radicals, as well as iron chelators . In order to test the hypothesis that the redox state of the cell is involved in regulating manganese-superoxide dismutase biosynthesis, we studied the effects of several oxidants on growth and superoxide dismutase biosynthesis . The data showed, that under anaerobic conditions, the active manganese-enzyme is induced in the presence of potassium ferricyanide, copper-cyanide complex, ammonium persulfate, and hydrogen peroxide . Western blot analysis revealed that the induction of manganese-superoxide dismutase by the oxidants is associated with de novo protein biosynthesis . Potassium ferricyanide and hydrogen peroxide induced the enzyme under aerobic conditions as well . It is concluded that the redox state of the cell possibly influences the biosynthesis and/or activity of an iron-containing repressor protein that serves to negatively regulate manganese-superoxide dismutase biosynthesis.

J Biol Chem, 1988 Mar 25, 263(9), 4172 - 81
Effectors of Escherichia coli aspartate transcarbamoylase differentially perturb aspartate binding rather than the T-R transition; Hsuanyu YC et al.; New systematic methods developed for equilibrium isotope exchange kinetics have been used to analyze the effects of activator ATP and inhibitor CTP with Escherichia coli aspartate transcarbamoylase . This indepth approach requires (a) variation of {modifier} with fixed subsaturating levels of substrates, and (b) variation of at least three combinations of reactant-product pairs in constant ratio at equilibrium: {A,B,P,Q}, {A,P}, and {B,Q} with the co-substrates held constant, in the presence and absence of added modifier . Both ATP and CTP had much stronger effects on the {14C}Asp in equilibrium C-Asp exchange rate than on {32P}C-P in equilibrium Pi . The bisubstrate analog N-phosphonacetyl-L-aspartate activated, then inhibited, Asp in equilibrium C-Asp more strongly than C-P in equilibrium Pi . N-Phosphonacetyl-L-aspartate gave complete (100%) inhibition, whereas CTP inhibition of either exchange was only partial . Substrate saturation curves in the presence and absence of effectors indicate that ATP and CTP perturb the observed values of Rmax and S0.5 in different fashions without appreciably changing the observed Hill number . Computer simulations indicate that the primary site of ATP and CTP action is the association rate for Asp, not the allosteric T-R transition . This finding is substantiated by previous studies in which modified aspartate transcarbamoylase had lost cooperative Asp binding without loss of sensitivity to effectors, or in which sensitivity to one effector could be deleted selectively . The present results, with newly devised computer simulation and analysis methods, illustrate the usefulness of equilibrium isotope exchange kinetic probes for providing unique insights to enzyme regulatory mechanisms, to define exactly which steps are altered in a given kinetic mechanism.

J Biol Chem, 1988 Mar 25, 263(9), 4430 - 3
Functional domains and methyl acceptor sites of the Escherichia coli ada protein; Sedgwick B et al.; The ada gene of Escherichia coli encodes a 39-kDa protein which serves both as a transcriptional activator of the adaptive response to alkylating agents and as a DNA repair enzyme demethylating O6-methyl-guanine and phosphotriester residues . Here, the isolated Ada protein was found to be readily cleaved into two fragments of similar size by treatment with trypsin, chymotrypsin, subtilisin, or V8 protease . The fragments retained their respective methyltransferase activities . The Ada protein is, therefore, comprised of two stable active domains united by a central hinge region of about 10 amino acids . Post-translational modification of the Ada protein by methylation of a specific cysteine residue in the NH2-terminal domain is known to convert it to an efficient transcriptional activator . This residue has now been identified as Cys-69.

Nucleic Acids Res, 1988 Mar 25, 16(6), 2565 - 83
RNA binding proteins of the large subunit of bovine mitochondrial ribosomes; Piatyszek MA et al.; RNA binding properties of proteins from the large subunit of bovine mitochondrial ribosomes were studied using four different approaches: binding of radiolabeled RNA to western blotted proteins; disassembly of the intact 39 S ribosomal subunits with urea; binding of ribosomal proteins to RNA in the presence of urea; and binding of proteins extracted with lithium chloride to RNA . Results from these four approaches allowed us to identify a set of six proteins (L7, L13, L14, L21, L26, and L44) which appear to be strong RNA binding proteins . Seven additional proteins (L8, L11, L28, L35, L40, L49, and L50) were identified as secondary RNA binding proteins . RNA binding properties of the proteins in both of these sets were compared with the topographic disposition and susceptibility towards lithium chloride extraction of the individual proteins . Proteins from the first set are good candidates for early assembly proteins since they have a high affinity for RNA, are generally found in 4M lithium chloride core particles, and are among the most buried proteins in the 39 S subunit.

Nucleic Acids Res, 1988 Mar 25, 16(5), 2015 - 30
The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21; Xin WN et al.; The interaction of E . coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique . Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs . Of the six, one site, I1, binds IHF strongly . The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence . Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly . The 10 site does not bind IHF under the conditions used here . In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined . Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site . The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda . The significance of the results for lambda DNA packaging is discussed.

Cell, 1988 Mar 25, 52(6), 883 - 92
Copy choice illegitimate DNA recombination; Brunier D et al.; Precise excision of Tn10 and related transposons occurs by recombination between directly repeated 9 bp sequences that flank the transposon . The excision, which is a model for a class of illegitimate DNA recombination events, was stimulated 10(6) times by induction of single-stranded DNA synthesis, occurred during conversion of single-stranded DNA to double-stranded form, and entailed no transfer of physical material from parental to progeny molecules . We conclude that it occurred by copy choice DNA recombination and suggest that other illegitimate recombination events may occur by a similar mechanism.

J Biol Chem, 1988 Mar 25, 263(9), 4400 - 7
AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli; Francklyn CS et al.; We examined the recognition of the araBAD promoter by the AraC protein in the Escherichia coli arabinose operon . A mutant promoter, with base substitutions at positions contacted by AraC, was used to isolate suppressor mutations in araC by direct selection . Two hydroxylamine-induced araC mutations were isolated repeatedly; each contained a single amino acid substitution . When tested against a set of base substitution promoter mutants, one revertant, an Arg to His substitution at residue 250, displayed altered base specificity for a single position within the araBAD promoter . The other revertant, a Cys to Tyr substitution at residue 204, did not show consistent base-specific suppression . Neither demonstrated a higher affinity than the wild type protein for the mutant promoter in vitro . Both proteins suppress mutant sequences by a mechanism that does not appear to involve the formation of new net favorable contacts with the mutant base pairs of the promoter.

J Biol Chem, 1988 Mar 25, 263(9), 4202 - 7
Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE; Batenburg AM et al.; The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques . The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area . The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed . Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.

Biochemistry, 1988 Mar 22, 27(6), 2217 - 22
Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage; Brush EJ et al.; Recently, Knappe and co-workers {Knappe, J., Neugebauer, F . A., Blaschkowski, H . P., & Ganzler, M . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 1332} have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite . Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction . We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using {2H2}hypophosphite . This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation . In addition, the inactivated enzyme can be fully reactivated . We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from {2H}formate and acetyl coenzyme A . Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction . On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.

Biochemistry, 1988 Mar 22, 27(6), 1908 - 17
Biosynthesis of lipid A in Escherichia coli: identification of UDP-3-O-{(R)-3-hydroxymyristoyl}-alpha-D-glucosamine as a precursor of UDP-N2,O3-bis{(R)-3-hydroxymyristoyl}-alpha-D-glucosamine; Anderson MS et al.; The lipid A disaccharide of the Escherichia coli envelope is synthesized from the two fatty acylated glucosamine derivatives UDP-N2,O3-bis{(R)-3-hydroxytetradecanoyl}-alpha-D- glucosamine (UDP-2,3-diacyl-GlcN) and N2,O3-bis{(R)-3-hydroxytetradecanoyl}-alpha-D-glucosamine 1-phosphate (2,3-diacyl-GlcN-1-P) {Ray, B . L., Painter, G., & Raetz, C . R . H . (1984) J . Biol . Chem . 259, 4852-4859} . We have previously shown that UDP-2,3-diacyl-GlcN is generated in extracts of E . coli by fatty acylation of UDP-GlcNAc, giving UDP-3-O-{(R)-3-hydroxymyristoyl}-GlcNAc as the first intermediate, which is rapidly converted to UDP-2,3-diacyl-GlcN {Anderson, M . S., Bulawa, C . E., & Raetz, C . R . H . (1985) J . Biol . Chem . 260, 15536-15541; Anderson, M . S., & Raetz, C . R . H . (1987) J . Biol . Chem . 262, 5159-5169} . We now demonstrate a novel enzyme in the cytoplasmic fraction of E . coli, capable of deacetylating UDP-3-O-{(R)-3-hydroxymyristoyl}-GlcNAc to form UDP-3-O-{(R)-3-hydroxymyristoyl}glucosamine . The covalent structure of the previously undescribed UDP-3-O-{(R)-3-hydroxymyristoyl} glucosamine intermediate was established by 1H NMR spectroscopy and fast atom bombardment mass spectrometry . This material can be made to accumulate in E . coli extracts upon incubation of UDP-3-O-{(R)-3- hydroxymyristoyl}-GlcNAc in the absence of the fatty acyl donor {(R)-3-hydroxymyristoyl}-acyl carrier protein . However, addition of the isolated deacetylation product {UDP-3-O-{(R)-3-hydroxymyristoyl} glucosamine} back to membrane-free extracts of E . coli in the presence of {(R)-3-hydroxymyristoyl}-acyl carrier protein results in rapid conversion of this compound into the more hydrophobic products UDP-2,3-diacyl-GlcN, 2,3-diacyl-GlcN-1-P, and O-{2-amino-2-deoxy-N2,O3- bis{(R)-3-hydroxytetradecanoyl}-beta-D-glucopyranosyl}-(1----6)-2-amino- 2-deoxy-N2,O3-bis{(R)-3-hydroxytetradecanoyl}-alpha-D- glucopyranose 1-phosphate (tetra-acyldisaccharide-1-P), demonstrating its competency as a precursor . In vitro incubations using {acetyl-3H}UDP-3-O-{(R)-3-hydroxymyristoyl}-GlcNAc confirmed release of the acetyl moiety in this system as acetate, not as some other acetyl derivative . The deacetylation reaction was inhibited by 1 mM N-ethylmaleimide, while the subsequent N-acylation reaction was not . Our observations provide strong evidence that UDP-3-O-{(R)-3-hydroxymyristoyl}glucosamine is a true intermediate in the biosynthesis of UDP-2,3-diacyl-GlcN and lipid A.

Biochemistry, 1988 Mar 22, 27(6), 1839 - 43
Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding; Stump DG et al.; T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism . These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers . In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132) . Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding . The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells . The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene . The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA . Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129 . These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

Biochemistry, 1988 Mar 22, 27(6), 1869 - 80
Expression of a human alpha-tubulin: properties of the isolated subunit; Yaffe MB et al.; We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts . Both systems produce soluble, full-length human alpha-tubulin polypeptide . When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures . The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation . Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer . Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer . In contrast, alpha-tubulin expressed in E . coli lysates was incapable of coassemblying with bovine brain tubulin . Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit . These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies {Sherman, G., Rosenberry, T.L., & Sternlicht, H . (1983) J . Biol . Chem . 258, 2148-2156} which imply that this residue is in a salt-bridge interaction in the dimer.

Biochemistry, 1988 Mar 22, 27(6), 2126 - 32
Magnetization of the sulfite and nitrite complexes of oxidized sulfite and nitrite reductases: EPR silent spin S = 1/2 states; Day EP et al.; The saturation magnetizations of the sulfite complex of oxidized sulfite reductase and the nitrite complex of oxidized nitrite reductase have been measured to determine their spin state . Each shows the saturation magnetization signal of a spin S = 1/2 state with sigma g2 = 16, which is typical of low-spin ferrihemes . However, the EPR spectra of these complexes lack the expected signal intensity of a spin S = 1/2 state . Indeed, one of these complexes is EPR silent . The reasons for this unexpectedly low EPR signal intensity are considered.

Biochemistry, 1988 Mar 22, 27(6), 1972 - 81
Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs; Lau KS et al.; The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods . Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis . The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component . A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe . The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2 . A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe . Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively . Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions . In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail . Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase . The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain . Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.

Biochemistry, 1988 Mar 22, 27(6), 1964 - 72
Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis; Takano E et al.; Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed . The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues . A unique N-terminal domain is followed by four mutually homologous domains . The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0% . The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues . The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) . The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences . It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.

J Mol Biol, 1988 Mar 20, 200(2), 239 - 51
Missense mutation in the lacI gene of Escherichia coli . Inferences on the structure of the repressor protein; Gordon AJ et al.; The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown . Studies using mutational data can complement other information and provide insight into protein structure . We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation . The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor . Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions . Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain . The core domain (residues 60 to 360) is less sensitive to amino acid replacement . Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding . The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Nature, 1988 Mar 17, 332(6161), 281 - 4
Regulation of the Drosophila segmentation gene hunchback by two maternal morphogenetic centres; Tautz D; Segmentation in the inset embryo is initiated by maternally provided information, which is stored in the developing oocyte . In Drosophila, the genes necessary for this process have been genetically characterized . The anterior segmented region is organized by the bicoid (bcd) gene product . The posterior segmented region is organized by several interacting gene products, among them the oskar (osk) gene product . The first zygotic group of genes, which are thought to respond to the spatial cues provided by the maternal genes, are the gap genes, whose members include hunchback (hb), Kruppel (Kr) and knirps (kni) . To elucidate the role played by the maternal genes in expression of the gap gene hb, antibodies were raised against a fusion protein and were used for the cytological localization of the hb gene product in wild-type and mutant embryos . The hb protein is predominantly located in the nucleus . Its spatial expression includes the formation of an anterior-posterior gradient during the early cleavage stages and a strong zygotic expression in the anterior half of the embryo . Analysis of embryos mutant for the maternal genes affecting the anterior-posterior segmentation pattern shows that the formation of the early gradient is controlled by the osk group of genes, whereas efficient activation of the zygotic anterior expression domain is dependent on bcd activity.

Biochem Biophys Res Commun, 1988 Mar 15, 151(2), 844 - 50
Effect of endotoxin on the rat colon glutathione level; Chen MF et al.; The effect of endotoxin on the colon glutathione level was studied in male rats . Endotoxin (Escherichia coli) from 25 ug to 100 ug/100g body weight was administered intravenously . The Glutathione level was measured 16 hours after endotoxin was given . Results showed that endotoxin significantly enhanced the colon glutathione concentration as measured by 5,5'-dithiobis (2-nitrobenzoic acid) . The increase which ranged from 11% to 50% was dose dependent . At an endotoxin dose of 1000 ug/100g body weight, colon glutathione level was found to be enhanced from 2 hours up to 48 hours . In contrast, the duodenum and jejunum glutathione levels were found to be significantly reduced . The increase in the colon glutathione level may have a protective effect against oxidative damage to the colon.

J Biol Chem, 1988 Mar 15, 263(8), 3546 - 9
Retinol-regulated gene expression in human tracheobronchial epithelial cells . Enhanced expression of elongation factor EF-1 alpha; Ann DK et al.; Conducting airway epithelial cells requires vitamin A or its synthetic chemicals (retinoids) for their survival and for the expression of normal mucociliary functions . By using molecular cloning, we have shown that one of the effects of retinol on cultured human tracheobronchial epithelial (HTBE) cells is the enhancement (from 2- to 4-fold) of the mRNA encoding the elongation factor EF-1 alpha . Sequence analysis has shown that clone HT7, which was identified by differential hybridization procedures, contained a cDNA insert which encoded a protein closely resembling (81%) elongation factor EF-1 alpha from brine shrimp and completely identical to the published sequence of human elongation factor EF-1 alpha (Brands, H.H.G.M., Maassen, J.A., Van Hemert, F.J., Amons, R., and Moller, W . (1986) Eur . J . Biochem . 155, 167-171) . Regions of homology of HT7 to EF-Tu from yeast mitochondria, plant chloroplasts, and Escherichia coli are also evident . A single RNA band at 1700 bases was observed for both untreated and retinol-treated HTBE cells, and for mouse liver and parotid glands when Northern transfer from denaturing agarose gel was probed with a 32P-labeled HT7 insert . An enhanced amino acid incorporation and increased protein content per cell for HTBE cells grown in the presence of retinol were observed . Results presented by these studies indicate that retinol may regulate the transcription of a factor required for translation.

Biochem J, 1988 Mar 15, 250(3), 925 - 8
Interaction of the trp repressor from Escherichia coli with a constitutive trp operator; Chandler LR et al.; The interaction of the trp repressor from Escherichia coli with a 20 bp fragment of DNA (CGTACTGATT.AATCAGTACG) corresponding to a mutant trp operator was studied by c.d . in the presence and absence of the co-repressor, L-tryptophan, and as a function of the concentration of K+ and Na+ ions . The affinity of the repressor for the mutant operator in the presence of tryptophan is about three orders of magnitude lower than the wild-type sequence . Binding in the absence of tryptophan is about 100-fold weaker than to the wild-type . The dependence of the dissociation constant on the concentration of K+ or Na+ is weak {d(log Ks)/d(log{M+}) = 2.5}, and independent of the cation, indicating that electrostatic interactions are not as important for this repressor as for others.

Biochem J, 1988 Mar 15, 250(3), 789 - 96
Purification and characterization of {acyl-carrier-protein} acetyltransferase from Escherichia coli; Lowe PN et al.; A multi-step procedure has been developed for the purification of {acyl-carrier-protein} acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme . The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure . The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle . The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA . However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate . The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E . coli, yeast and vertebrates.

Eur J Biochem, 1988 Mar 15, 172(3), 565 - 72
Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector; Balland A et al.; A stable transformed cell line constitutively expressing human factor IX has been established . Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase . One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40% . The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer . The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels . N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX . Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX . The carbohydrate composition of both the natural and recombinant molecules has been determined . Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.

Biochem Biophys Res Commun, 1988 Mar 15, 151(2), 768 - 73
Electric field effects on the virus M13, detected by electro-optical measurements; Duenki RM et al.; We have carried out transient electric birefringence experiments with M13 in aqueous solution . A normal effect with a negative sign is superimposed by anomalous positive effects . These prevail more and more upon raising the field strength . They apparently reflect electrically induced structural transitions . The threshold field strength decreases when buffer and/or electrolyte is removed.

J Biol Chem, 1988 Mar 15, 263(8), 3539 - 41
Lysis induction of Escherichia coli by the cloned lysis protein of the phage MS2 depends on the presence of osmoregulatory membrane-derived oligosaccharides; Holtje JV et al.; Expression of the cloned lysis protein of phage MS2, which is sufficient to lyse wild type Escherichia coli, does not cause lysis of mutants lacking the osmoregulatory membrane-derived oligosaccharides (MDO) . The lysis gene product normally found in the membrane fraction was not stably inserted into the membranes of a mdoA mutant; rather degradation and release from the membrane occurred . Gentle plasmolysis of the MDO-lacking mutant clearly showed an increased periplasmic space as compared to wild type cells . It is concluded that the MDOs play an important role in maintaining a proper arrangement of inner and outer membrane, a prerequisite for a functional insertion of the MS2 lysis protein.

J Biol Chem, 1988 Mar 15, 263(8), 3984 - 9
Purification and biological characterization of an adenovirus type 2 E1A protein expressed in Escherichia coli; Bruner M et al.; The adenovirus 2 E1A gene encodes a multifunctional protein of 289 amino acids that can immortalize primary rodent cells and transcriptionally activate a number of viral and cellular genes . To facilitate an understanding of the molecular basis for the various actions of E1A, we have redesigned our bacterial expression vector (Ko, J.-L., and Harter, M . L . (1984) Mol . Cell . Biol . 4, 1427-1439) containing the cloned E1A gene such that a soluble authentic E1A protein now constitutes approximately 1.5% of the total Escherichia coli cellular protein . Further, we have developed a simple rapid purification scheme without the use of detergents or denaturants and show a purity of greater than 98% with a yield of approximately 53% . The E1A so purified is biologically active, stimulating cellular DNA synthesis following microinjection into quiescent NIH 3T3 and REF52 cells . In another report (Spangler, R., Bruner, M., Dalie, B., and Harter, M . L . (1987) Science 237, 1044-1046) we have also shown that our purified E1A protein activates transcription from appropriate promoters in an in vitro system.

Biochem J, 1988 Mar 15, 250(3), 897 - 902
Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase; Pinkney M et al.; Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase . Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence . Binding to core RNA polymerase under the same conditions was weak (Kdiss . approx . 80-100 microM) and independent of cyclic AMP . Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP . Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

Biochem Biophys Res Commun, 1988 Mar 15, 151(2), 701 - 8
Stabilizing basic fibroblast growth factor using protein engineering; Seno M et al.; Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine . The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein . This finding indicates that the cysteines at these positions are not essential for expressing biological activity . The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations . Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity . These facts suggest that this protein has been successfully modified by protein engineering.

J Biol Chem, 1988 Mar 15, 263(8), 3811 - 7
Stimulation of the activity of horseradish peroxidase by nitrogenous compounds; Kuo CF et al.; A variety of nitrogenous compounds broaden the activity versus pH profile for the peroxidation of dianisidine catalyzed by horseradish peroxidase (HRP), but not by myeloperoxidase, chloroperoxidase, Escherichia coli hydroperoxidase I, methemoglobin, or microperoxidases . The peroxidation of dianisidine catalyzed by cytochrome c peroxidase was affected by the nitrogenous compounds, but to a lesser extent than was the action of HRP . The peroxidations of a variety of phenols by HRP exhibited broad activity versus pH profiles and were unaffected by the nitrogenous compounds . The energy of activation for the peroxidation of dianisidine by HRP was unaffected by changes of pH in the range 6.5-8.5 and was unchanged by the presence of the nitrogenous compounds . The nitrogenous compounds markedly increased Vm for the peroxidation of dianisidine by HRP, but did not change the slope of Lineweaver-Burk plots of kinetic data . These results are accommodated by a mechanism in which nitrogenous compounds hydrogen-bond to the distal histidine of HRP and in so doing raise its pK alpha . Since the acid form of the distal histidine is thought to facilitate peroxidations catalyzed by HRP by hydrogen bonding to the ferryl oxygen of compound II, raising its pK alpha broadens the activity versus pH profile for the peroxidation of anilino substrates, such as dianisidine . We propose that phenolic substrates hydrogen-bond directly to the ferryl oxygen, thus displacing the distal histidine and eliminating the possibility of being influenced by nitrogenous compounds.

J Biol Chem, 1988 Mar 15, 263(8), 3542 - 5
Site-specific mutagenesis of human apolipoprotein E . Receptor binding activity of variants with single amino acid substitutions; Lalazar A et al.; Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor . Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor . Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia . To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken . This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3 . Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced . The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE) . In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152 . Both variants were defective, exhibiting 13 and 27% of normal binding, respectively . In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity . These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.

J Biol Chem, 1988 Mar 15, 263(8), 3772 - 7
Gliotoxin causes oxidative damage to plasmid and cellular DNA; Eichner RD et al.; The cytotoxic effects of gliotoxin (Mullbacher, A., and Eichner, R . D . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 3835-3837), a fungal secondary metabolite, and related epipolythiodioxopiperazines have been investigated using plasmid and eukaryotic DNA . Incubation of the dithiol derivative of these compounds with DNA and Fe3+ is sufficient to cause single- and double-stranded breaks as determined by neutral agarose gel electrophoresis . The disulfide form is inactive except in the presence of a suitable reducing agent, such as reduced glutathione, dithiothreitol, or reduced pyridine coenzymes . The autooxidation of these dithiols produces reducing equivalents as evidenced by (i) the production of H2O2 and (ii) the generation of thiobarbituric acid reactive products when incubated with deoxyribose . The latter process is inhibited by ethanol and desferrioxamine . The DNA damage is abrogated by metal chelators and catalase . We conclude that the antiproliferative action of gliotoxin may be caused by DNA damage effected by reactive oxygen species or other radicals generated through redox cycling.

FEBS Lett, 1988 Mar 14, 229(2), 279 - 82
Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli; Minami M et al.; The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector . The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants . The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl-modifying reagents . The results demonstrate that a protein with an Mr of 70,000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity . Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.

FEBS Lett, 1988 Mar 14, 229(2), 360 - 2
Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II; Forsman C et al.; To test the hypothesis that histidine 64 in carbonic anhydrase II has a crucial role as a 'proton shuttle group' during catalysis of CO2-HCO3- interconversion, this residue was replaced by lysine, glutamine, glutamic acid and alanine by site-directed mutagenesis . All these variants turned out to have high CO2 hydration activities . The kcat values at pH 8.8 and 25 degrees C were only reduced by 1.5-3.5-fold compared to the unmodified enzyme . These results show that intramolecular proton transfer via His 64 is not a dominating pathway in the catalytic reaction . The variants also catalyze the hydrolysis of 4-nitrophenyl acetate . The pKa values for the activity-controlling group are between 6.8 and 7.0 for all studied forms of the enzyme except the Glu 64 variant which shows a complex pH dependence with the major pKa shifted to 8.4.

Nucleic Acids Res, 1988 Mar 11, 16(5), 1683 - 91
A flexible multiple sequence alignment program; Martinez HM; The 'regions' method for multisequence alignment used in the previously reported program MALIGN has been generalized to include recursive refinement so that unaligned portions between two regions at the current level of resolution can be handled with increased resolution . Additionally, there is incorporated a limiting of the number of regions to be used at any level of resolution from which to abstract an alignment . This provides a significant increase in speed over the unlimited version . The program GENALIGN uses this improved regions method to execute fast pairwise alignments in the framework of Taylor's multisequence alignment procedure using clustered pairwise alignments . Pairwise alignments by dynamic programming are also provided in the program.

Biochemistry, 1988 Mar 8, 27(5), 1729 - 35
Effect of pH on the base-mispairing properties of 5-bromouracil during DNA synthesis; Driggers PH et al.; We have utilized an electrophoretic assay of misincorporation to investigate the possibility that ionization of 5-bromouracil (BU) may play a role in its mispairing during DNA synthesis in vitro . We examined the effects of increasing pH on the relative rates of formation of BU.G and T.G mispairs during chain elongation catalyzed by various DNA polymerases . For the Klenow fragment of Escherichia coli DNA polymerase I, increasing pH facilitated BU.G mispair formation (relative to T.G mispairing) when BU was present in the template strand . This effect showed a strong dependence on sequence context . Increasing pH had little effect on the relative rate of misincorporation of BrdUMP versus dTMP (at template G) by the Klenow polymerase . Misincorporation opposite template BU residues catalyzed by Maloney murine leukemia virus DNA polymerase and DNA polymerase beta (Novikoff hepatoma) also increased with pH, but for these two enzymes, there was no apparent dependence on sequence context . With T4 DNA polymerase and E . coli DNA polymerase III holoenzyme, a similar occurrence of BU.G and T.G mispairing during polymerization was observed, whether BU was present in the template or in the incoming nucleotide, and there was little effect of pH . The results reported here are consistent with a mispairing mechanism for template BU wherein the anionic form of the base mispairs with G.

Biochemistry, 1988 Mar 8, 27(5), 1604 - 10
Evaluation of 5-enolpyruvoylshikimate-3-phosphate synthase substrate and inhibitor binding by stopped-flow and equilibrium fluorescence measurements; Anderson KS et al.; The binding of substrates and the herbicide N-(phosphonomethyl)glycine (glyphosate) to enolpyruvoylshikimate-3-phosphate (EPSP) synthase was evaluated by stopped-flow and equilibrium fluorescence measurements . Changes in protein fluorescence were observed upon the binding of EPSP and upon the formation of the enzyme-shikimate 3-phosphate-glyphosate ternary complex; no change was seen with either shikimate 3-phosphate (S3P) or glyphosate alone . By fluorescence titrations, the dissociation constants were determined for the formation of the enzyme binary complexes with S3P (Kd,S = 7 +/- 1.2 microM) and EPSP (Kd,EPSP = 1 +/- 0.01 microM) . The dissociation constant for S3P was determined by competition with EPSP or by measurements in the presence of a low glyphosate concentration . At saturating concentrations of S3P, glyphosate bound to the enzyme--S3P binary complex with a dissociation constant of 0.16 +/- 0.02 microM . Glyphosate did not bind significantly to free enzyme, so the binding is ordered with S3P binding first: (formula; see text) where S refers to S3P, G refers to glyphosate, and E.S.G . represents the complex with altered fluorescence . The kinetics of binding were measured by stopped-flow fluorescence methods . The rate of glyphosate binding to the enzyme--S3P complex was k2 = (7.8 +/- 0.2) X 10(5) M-1 s-1, from which we calculated the dissociation rate k-2 = 0.12 +/- 0.02 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Mar 8, 27(5), 1581 - 7
Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-fit mechanism; Fersht AR et al.; Site-directed mutagenesis of the tyrosyl-tRNA synthetase followed by kinetic studies has shown that residues which are distant from the active site of the free enzyme are brought into play as the structure of the enzyme changes during catalysis . Positively charged side chains which are in mobile loops of the enzyme envelope the negatively charged pyrophosphate moiety during the transition state for the formation of tyrosyl adenylate in an induced-fit mechanism . Residues Lys-82 and Arg-86, which are on one side of the rim of the binding site pocket, and Lys-230 and Lys-233, which are on the other side, have been mutated to alanine residues and also to asparagine or glutamine . The resultant mutants still form 1 mol of tyrosyl adenylate/mol of dimer but with rate constants up to 8000 times lower . Construction of difference energy diagrams reveals that all the residues specifically interact with the transition state for the reaction and with pyrophosphate in the E.Tyr-AMP.PPi complex . Yet, the epsilon-NH3+ groups of Lys-230 and Lys-233 in the crystalline enzyme are at least 8 A too far away to interact with the pyrophosphate moiety in the transition state at the same time as do Lys-82 and Arg-86 . Binding of substrates must, therefore, induce a conformational change in the enzyme that brings these residues into range . Consistent with this proposal is the observation that all four residues are in flexible regions of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Mar 8, 27(5), 1401 - 8
Characterization of Escherichia coli-Anabaena sp . hybrid thioredoxins; Lim CJ et al.; Thioredoxin is a small redox protein with an active-site disulfide/dithiol . The protein from Escherichia coli has been well characterized . The genes encoding thioredoxin in E . coli and in the filamentous cyanobacterium Anabaena PCC 7119 have been cloned and sequenced . Anabaena thioredoxin exhibits 50% amino acid identity with the E . coli protein and interacts with E . coli enzymes . The genes encoding Anabaena and E . coli thioredoxin were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate hybrid genes, coding for two chimeric thioredoxins . These proteins are designated Anabaena-E . coli (A-E) thioredoxin for the construct with the Anabaena sequence from the N-terminus to the middle of the active site and the E . coli sequence to the C-terminus, and E . coli-Anabaena (E-A) for the opposite construct . The gene encoding the A-E thioredoxin complements all phenotypes of an E . coli thioredoxin-deficient strain, whereas the gene encoding E-A thioredoxin is only partially effective . Purified E-A thioredoxin exhibits a much lower catalytic efficiency with E . coli thioredoxin reductase and ribonucleotide reductase than either E . coli or Anabaena thioredoxin . In contrast, the A-E thioredoxin has a higher catalytic efficiency in these reactions than either parental protein . Reaction with antibodies to E . coli and Anabaena thioredoxins shows that the antigenic determinants for thioredoxin are located in the C-terminal part of the molecule and retain the native conformation in the hybrid proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1988 Mar 8, 27(5), 1771 - 7
Modes of DNA cleavage by the EcoRV restriction endonuclease; Halford SE et al.; The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods . In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate . In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA . Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover . The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions . At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time . The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.

Biochemistry, 1988 Mar 8, 27(5), 1570 - 6
Tyrosine-371 contributes to the positive cooperativity between the two cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I; Bubis J et al.; The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding . Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 {Bubis, J., & Taylor, S.S . (1987) Biochemistry 26, 3478-3486} . The site that was targeted for mutagenesis was Tyr-371 . The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes . A single base change converted Tyr-371 to Phe . This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer . The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99 . The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1988 Mar 5, 200(1), 65 - 87
Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli; Capel MS et al.; Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components . When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering . Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.

J Mol Biol, 1988 Mar 5, 200(1), 217 - 9
Crystallization of the met repressor from Escherichia coli; Rafferty JB et al.; The met repressor from Escherichia coli has been crystallized in space group P21, with unit cell dimensions a = 35.6 A, b = 62.6 A, c = 44.5 A, beta = 102.4 degrees and one aporepressor dimer per asymmetric unit . Preliminary X-ray diffraction photographs show measurable intensities to beyond 1.5 A resolution, and the crystal form is ideally suited to high-resolution crystallographic analysis (1 A = 0.1 nm).

J Biol Chem, 1988 Mar 5, 263(7), 3328 - 34
Hypoxanthine-DNA glycosylase from Escherichia coli . Partial purification and properties; Harosh I et al.; Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns . Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base {3H}hypoxanthine from {3H}dIMP-containing phi X174 DNA . In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S . Hypoxanthine-DNA glycosylase from E . coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA . Co2+ can only partially replace Mg2+ . The enzyme is inhibited by hypoxanthine which at 4 mM causes 85% inhibition . The optimal pH range of the enzymatic activity is 5.5-7.8, and the apparent Km value is 2.5 x 10(-7) M.

J Biol Chem, 1988 Mar 5, 263(7), 3208 - 15
Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction; Lahue EE et al.; Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase . The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis . All eight predominant nucleoside 5'-triphosphates can satisfy this requirement . Unwinding is unidirectional in the 5' to 3' direction . The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive . Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another . The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors . When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate . This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.

J Biol Chem, 1988 Mar 5, 263(7), 3448 - 53
Monoclonal antibodies that inhibit activation of transcription by the Escherichia coli cyclic AMP receptor protein; Li XM et al.; The properties of the two monoclonal antibodies which were found to inhibit cyclic AMP receptor protein (CRP)-stimulated abortive initiation without affecting cAMP binding (Li, X.-M., and Krakow, J . S . (1986) J . Biol . Chem . 260, 4378-4383) have been characterized . Binding of monoclonal antibody (mAb) 66C3 to CRP is stimulated by cAMP while CRP binding by mAb 63B2 is not affected by cAMP . Binding of cAMP-CRP-mAb 63B2 to the lac P+ DNA is completely inhibited . Whereas cAMP-CRP forms a stable complex only at the CRP site 1 of the lac P+ promoter fragment, cAMP-CRP-mAb 66C3 binds to both site 1 and site 2 . DNase I footprinting using a HpaII fragment carrying only the lac site 2 does not show any protection by cAMP-CRP-mAb 66C3 . With the lac L8UV5 promoter, binding is not seen at either the L8 site 1 or the unaltered site 2 . In the presence of 25% glycerol, cAMP-CRP-mAb 66C3 binds to both L8 site 1 and site 2 . RNA polymerase is unable to bind to the cAMP-CRP-mAb 66C3-lac P+ complex . In the presence of RNA polymerase, cAMP-CRP forms a stable complex at the L8 site 1, the subsequent addition of mAb 66C3 results in the release of CRP . The CRP present in the lac P+ open promoter complex is partially resistant to subsequent incubation with mAb 66C3 . The results provide further evidence regarding possible contacts between CRP and RNA polymerase involved in establishing the open promoter complex.

J Biol Chem, 1988 Mar 5, 263(7), 3067 - 70
Molecular characterization of an anion pump . The arsA gene product is an arsenite(antimonate)-stimulated ATPase; Rosen BP et al.; The products of the arsenical resistance operon of resistance plasmid R733 form an efflux system for arsenicals . Detoxification results from active efflux of the oxyanions, preventing their concentration from reaching toxic levels . The largest polypeptide encoded by the ars operon was purified . From N-terminal sequencing the purified protein, termed the ArsA protein, was shown to correspond to the product of the arsA gene . The purified protein was demonstrated to bind ATP by two methods . First, a photoadduct of the protein with {alpha-32P}ATP was formed by irradiation at 254 nm . Second, the purified protein bound a fluorescent ATP analogue, 2',3'-o-(2,4,6)trinitrophenyl ATP, with a half-maximal affinity of 2 microM . By both assays competition was observed with ATP or ADP, but not with AMP, GTP, CTP, or UTP . In both nucleotide binding assays, Mg2+ was required, but neither arsenite nor antimonate had any affect . In contrast, the ArsA protein exhibited an ATPase activity which was dependent on the presence of arsenite or antimonate . The results suggest that the ArsA protein is the catalytic subunit of an oxyanion-translocating ATPase.

Science, 1988 Mar 4, 239(4844), 1105 - 10
Insights into enzyme function from studies on mutants of dihydrofolate reductase; Benkovic SJ et al.; Kinetic analysis and protein mutagenesis allow the importance of individual amino acids in ligand binding and catalysis to be assessed . A kinetic analysis has shown that the reaction catalyzed by dihydrofolate reductase is optimized with respect to product flux, which in turn is predetermined by the active-site hydrophobic surface . Protein mutagenesis has revealed that specific hydrophobic residues contribute 2 to 5 kilocalories per mole to ligand binding and catalysis . The extent to which perturbations within this active-site ensemble may affect catalysis is discussed in terms of the constraints imposed by the energy surface for the reaction.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1432 - 6
Cloning and sequencing of the cytoplasmic precursor to the alpha subunit of rat liver mitochondrial succinyl-CoA synthetase; Henning WD et al.; Succinyl-CoA synthetase {succinate-CoA ligase (GDP-forming); EC 6.2.1.4} of rat liver, an alpha beta dimer, is a component of the enzymology of the tricarboxylic acid cycle and functions within the mitochondrial matrix . We have isolated and determined the sequence of a cDNA clone containing the coding sequence of the cytoplasmic precursor to the alpha subunit of this enzyme together with stretches of nontranslated sequence at the 5' and 3' ends . The translated amino acid sequence indicates the presence of a 27-residue N-terminal signal sequence for mitochondrial targeting . The amino acid sequence of the mature alpha subunit shows an extraordinary degree of homology to the alpha subunit of Escherichia coli succinyl-CoA synthetase, with greater than 70% of the residues identical . This suggests that the fundamental differences in the quaternary structures and catalytic functions of the mammalian and bacterial enzymes must be attributable to differences in the beta subunits . mRNA that hybridizes to the cloned DNA is approximately equal to 1800 nucleotide residues in length, confirming that each of the two subunits is encoded separately and does not arise by proteolysis of a primary gene product containing both subunits of the mature protein.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 323 - 30
{Prediction of the DNA-recognizing supersecondary protein structure, alpha helix-turn-alpha helix, using the modified Ohlendorf-Anderson-Matthews method of necessary stereochemical requirements}; Shestopalov BV; A method for prediction of DNA-recognizing supersecondary structure alpha-helix--turn--alpha-helix localization in an amino acid sequence of any protein is described . The method allows to predict this structure in segment 67-89 of E . coli ribosomal protein L7/L12 and in corresponding segments of L7/L12 analogues from other six bacteria and spinach chloroplasts.

J Pediatr Surg, 1988 Mar, 23(3), 216 - 20
Duodenal atresia: late follow-up; Kokkonen ML et al.; In this study, 41 randomly chosen patients aged 15 to 35 years (mean 22 years) were carefully examined . As primary operations there were 13 membrane excisions, five duodenoduodenostomies, 22 duodenojejunostomies, and one gastrojejunostomy . Twenty-eight patients were symptom-free, ten admitted some discomfort, three had major pains, including one with a history of duodenal ulcer . Reoperation for adhesion ileus had been performed in six patients, in the early postoperative phase in one instance . At late follow-up barium meals (N = 41) showed completely normal findings in two cases only, hiatal hernia in two, gastritis in three, duodenogastric reflux in 12, slight dilation of the duodenum with good emptying and no reflux in 16, a huge duodenal sac in nine, diminished peristalsis in eight, delayed emptying in five, slight luminal narrowing in three, duodenal diverticuli in nine, bezoars in two, and a polyp in the duodenum of one patient . Ultrasound (N = 35) revealed a gallbladder septum in one patient and a dilated common bile duct in another; in one subject the gallbladder was not visualized satisfactorily . Isotope biligraphy (N = 15) showed biliary reflux to the stomach in 12 cases . Endoscopy (N = 20) findings were: esophagitis (1), hiatal hernia (2), gastric mucosa in the lower esophagus (2), biliary reflux (9), gastritis (7), gastric polyps (2), dilated duodenum of variable degree (19), diminished peristalsis (4), marked retention (2), abnormal papilla (3), diverticuli (4), and a persistent membrane (1) . Histology showed superficial gastritis in three patients . E coli was cultured from the duodenal juice in five patients and Candida found in two.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1988 Mar 1, 250(2), 547 - 55
Rat liver beta-glucuronidase . cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells; Powell PP et al.; A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied . The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase . Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland . Only one change leads to an amino acid difference in the mature enzyme . A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region . After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase . The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme . It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling . At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing . These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.

J Bacteriol, 1988 Mar, 170(3), 1354 - 9
Inhibition of the SOS response of Escherichia coli by the Ada protein; Vericat JA et al.; Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene . On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant . The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene . Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied . This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity . However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.

J Bacteriol, 1988 Mar, 170(3), 1205 - 14
Regulation of the fixA gene and fixBC operon in Bradyrhizobium japonicum; Gubler M et al.; The transcriptional start site of the Bradyrhizobium japonicum fixBC operon was identified by nuclease S1 mapping . It was located approximately 700 base pairs upstream of fixB and was preceded by a promoter sequence that showed strong homology to the B . japonicum fixA promoter and thus to the general nif consensus promoter sequence . Further transcript mapping experiments revealed that fixA and fixBC transcription in B . japonicum strictly depended on the presence of the regulatory gene nifA and on low oxygen partial pressure . Consistent with these data, chromosomally integrated fixA- and fixB-lacZ fusions expressed beta-galactosidase activity only in the wild type but not in a nifA mutant and only under microaerobic but not aerobic growth conditions . The presence of nifA accounted for a 19-fold and 44-fold activation of the fixA and fixB promoters, respectively . These results show that the fixA and fixBC genes are regulated in a way similar to that of the nitrogenase genes nifH and nifDK . A very peculiar finding was that the fixA and fixB promoters, when they were located on plasmids, could hardly be activated by the NifA protein, irrespective of whether this was tested in Escherichia coli or B . japonicum backgrounds . This is in clear contrast to the situation with nifH and nifD promoters.

J Virol, 1988 Mar, 62(3), 998 - 1007
Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies; Hardy WR et al.; Plasmids were constructed which contained a large portion of each of the four nonstructural genes of Sindbis virus fused to the N-terminal two-thirds of the trpE gene of Escherichia coli . The large quantity of fusion protein induced from cells containing these plasmids was subsequently used as an antigen to generate polyclonal antisera in rabbits . Each antiserum was specific for the corresponding nonstructural protein and allowed ready identification of each nonstructural protein and of precursors containing the sequences of two or more nonstructural proteins . These antisera were used to determine the stability of the mature nonstructural proteins and to examine the kinetics of processing of the nonstructural proteins from their respective precursors in vivo . Pulse-chase experiments showed that the precursor P123 is cleaved with a half-life of approximately 19 min to produce P12 and nsP3; P12 is then cleaved with a half-life of approximately 9 min to produce nsP1 and nsP2 . Thus, although the rate of cleavage between nsP1 and nsP2 is faster than that between nsP2 and nsP3, the latter cleavage must occur first and is therefore the rate-limiting step . The rate at which P34 is chased suggests that the cleavage between nsP3 and nsP4 is the last to occur; however the regulation of nsP4 function in Sindbis virus-infected cells may be even more complex than was previously thought . The products nsP1 and nsP2 (and nsP4) are relatively stable; nsP3, however, is unstable, with a half-life of about 1 h, and appears to be modified to produce heterodisperse, higher-molecular-mass forms . In general, the processing schemes used by Sindbis virus and Semliki Forest virus appear very similar, the major difference being that most nsP3 in Sindbis virus results from termination at an opal condon, whereas in Semliki Forest virus cleavage of the P34 precursor is required.

J Virol, 1988 Mar, 62(3), 1046 - 54
Delineation of the viral products of recombination in vaccinia virus-infected cells; Spyropoulos DD et al.; Plasmids containing the vaccinia virus thymidine kinase gene, its flanking DNA sequences, and the Escherichia coli beta-galactosidase gene were used in conjunction with a thymidine kinase-deficient virus to examine the viral products of recombination . Progeny derived from single-crossover events could be distinguished from those generated by gene conversion or double-crossover events when the beta-galactosidase gene was separated from the thymidine kinase gene by the flanking sequences . Using methotrexate to select for recombinant virus and a chromogenic indicator to detect beta-galactosidase, the generation of viral recombinants was measured over a 48-h period . Recombinant progeny were first observed at 12 h and increased to a maximum of 2.5% at 48 h . Single-crossover products, as determined by beta-galactosidase expression, reached a maximum of 57% of the recombinant population at 24 h and thereafter declined . DNA hybridization analysis was used to examine genomic structures of the progeny of the initial viral plaques, plaques purified three times, and those subject to a 10(4)-fold amplification . These analyses confirmed that single-crossover events within either the 5'- or 3'-homologous flanking sequences generated unstable recombinant structures . These structures were shown to contain a single copy of the intact thymidine kinase gene within the corresponding copy of the duplicated thymidine kinase flanking sequences, separated by the beta-galactosidase gene and plasmid DNA . Significantly, these duplicated structures could undergo further recombination to produce repeats of either the intact or the deleted thymidine kinase sequences . These intermediate structures ultimately degenerated to produce either the parental thymidine kinase-deleted or the wild-type genome . The wild-type genome was also shown to be generated directly by gene conversion or double-crossover events.

EMBO J, 1988 Mar, 7(3), 851 - 8
Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site; Summers DK et al.; Plasmid ColE1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers . The uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability . Exonuclease III-generated deletions have been used to define the left-hand boundary of the cer site . Deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type site . Although this boundary region is essential for dimer resolution, its DNA sequence is poorly conserved among multimer resolution sites in related plasmids . We present evidence that its function is to influence the three-dimensional organization of the site and suggest that it may be required for the formation of a condensed nucleoprotein complex.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 538 - 48
{Symmetry-regulated dynamics of multi-enzyme complexes . A model of a pyruvate dehydrogenase complex from Escherichia coli}; Gol'dshtein BN et al.; A dynamic model for quaternary structure of a multienzyme complex is considered . The model is based on the supposition of simultaneously existing similar subunits in a number of different conformational states in the "core" of the multienzyme complex . It is supposed that cyclic conformational transitions of the "core" subunits conserve the symmetry of the entire complex . Such transitions drive the core dynamics as well as the suprastructural multienzyme dynamics . The dynamic model is constructed for the pyruvate dehydrogenase complex from E . coli in a supposition of three different conformers existing in its "core" which correspond to the three steps of the cyclic catalytic process . The model is in accordance with the data from the literature.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 485 - 92
{Modification of tyrosine residues of the Klenow fragment of DNA-polymerase I from Escherichia coli by acetylimidazole}; Lavrik OI et al.; The modification of tyrosine residues of DNA polymerase I Klenow fragment from E . coli by acetylimidazole has been investigated . This reagent was shown to inactivate both polymerization and 3',5'-exonuclease activities but with different velocity . The poly(dT)-template and r(pA)10-primer each added separately to the enzyme have no notable influence on the rate of enzyme inactivation . Simultaneous presence of both template and primer increases the rate of inactivation . In the presence of poly(dT).r(pA) 10 there is not effect of dCTP and dTTP (noncomplementary to the template) on the rate of inactivation of polymerization activity . However, dATP complementary to the template, provides a complete protection . A weak protective action is detected in the presence of dADP . Orthophosphate, pyrophosphate and dAMP each taken separately increase the rate and the level of the enzyme inactivation . dAMP together with either ortho- or pyrophosphate have the same protective action as ATP . All data obtained allow to suggest the functional significance for polymerization activity of tyrosine located in the dNTP binding site of DNA polymerase I.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 384 - 92
{RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters}; Ozolin' ON et al.; The formation of complexes of RNA polymerases from E . coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique . The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged . The mutation does not interfere with the affinity of the enzyme for both initiating substrates . The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors . The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.

Nippon Seikeigeka Gakkai Zasshi, 1988 Mar, 62(3), 255 - 66
{Incidence and location of synovitis in the metatarso-phalangeal joints of the fore and hindlimbs of rabbits immunized with Escherichia coli}; Goto H; Arthritis of the metatarso-phalangeal joints resembling rheumatoid arthritis was produced by immunization of rabbits with heat-killed Escherichia coli 0: 14 . In all of fifteen rabbits immunized for 4 months, hyperplasia of synovial lining cells was observed: 108/240 joints, synovial edema in 15 rabbits: 126/240, lymphoid cell infiltration in 12: 110/240 and fibrinoid deposition in 15: 115/240 . Inflammatory findings were induced in each of 14 rabbits immunized for 10 months, 14 rabbits: 113/224 joints, 14: 97/224, 12: 66/224, 14: 88/224 respectively . The lymphfollicle was noted in 7 joints of only one rabbit after 10 months immunization . Lymphoid cell infiltration of the perichondral synovium was observed in 13 joints of 11 rabbits after 4 month immunization and in 33 joints of 10 rabbits after 10 months immunization . The incidence of the inflammatory findings in the volar synovium was significantly higher than that in the dorsal one (p less than 0.01).

J Biochem (Tokyo), 1988 Mar, 103(3), 470 - 3
Purification and characterization of a signal peptide, a product of protein secretion across the cytoplasmic membrane of Escherichia coli; Suzuki T et al.; A signal peptide, a processing product of the precursor of the lipoprotein in the cytoplasmic membrane of Escherichia coli, has been purified through extractions with butanol and ethyl ether and chromatographies with a Sephadex LH-60 column and Sep-pak C18 . Analysis of the amino acid composition and sequencing of the N- and C-termini indicate that the signal peptide was intact, suggesting that the first step of the signal peptide catabolism in the cytoplasmic membrane is the cleavage of the intact signal peptide . During the purification, the signal peptide exhibited unique features, including strong interaction with phospholipids . The possible importance of such features in the process of protein translocation across membranes is discussed.

Bioorg Khim, 1988 Mar, 14(3), 408 - 11
{Cloning, expression and structure of the functionally active shortened lon gene in Escherichia coli}; Amerik AIu et al.; Lon gene of E . coli has been cloned into the plasmid pBR327 . Full nucleotide sequence of the gene has been established . It was shown that the cloned gene does not possess the terminal codon and is somewhat shortened . Nevertheless it retains full phenotypic activity and expresses the C-end modified La proteinase which retains ATP-dependent proteolytic activity.

Bioorg Khim, 1988 Mar, 14(3), 405 - 7
{Immune electron microscope localization of RNA-polymerase on the Escherichia coli chromosome using monoclonal antibodies against the beta-subunit}; Grachev MA et al.; Possibility of the immunoelectron microscopic visualization of RNA polymerase on the Escherichia coli chromosome with monoclonal antibodies against the beta-subunit labelled by {protein A.gold} complex was demonstrated . Using this method RNA polymerase molecules were revealed within nucleoid as well as on the membrane-free chromosome.

Bioorg Khim, 1988 Mar, 14(3), 321 - 32
{Affinity modification of Escherichia coli ribosomes with 2',3'-O-{4-(N-2-chloroethyl)-N-methylamino}-benzylidene derivative of AUGU3 in the 70S initiation complexes}; Ven'iaminova AG et al.; Affinity labelling of the Escherichia coli ribosomes with the 2',3'-O-{4-(N-(2-chloroethyl)-N-methylamino}benzylidene derivative of AUGU3(AUGU3{14C}CHRCl) has been studied within 70S initiation complexes ribosome.AUGU3{14C}CHRCl.fMet-tRNA(Metf) and binary complex ribosome.AUGU3{14C}CHRCl . Various ways of the 70S initiation complex formation resulted in differently labelled products . Proteins S5, S7, S9, L1, L16 were thus identified as cross-linked with AUGU3{14C}CHRCl within an initiation complex obtained in the presence of initiation factors IF-1, IF-2, IF-3, whereas only proteins S5 and S7 were cross-linked within the complex obtained with the sole factor IF-2 . Proteins S1, S3, L1 and L33 were labelled within the initiation complex obtained nonenzymatically but only protein S1 within the binary complex . In all complexes formed with use of initiation factors labelling of IF-2 factor was invariably observed.

Mol Microbiol, 1988 Mar, 2(2), 219 - 25
A replication region of the IncHI plasmid, R27, is highly homologous with the RepFIA replicon of F; Saul D et al.; A region of the IncHI plasmid R27 has been found to share very close nucleotide sequence homology with the RepFIA replicon of F . This region has been located on a 1.6 kb segment of R27 plasmid DNA, and corresponds to ori-2 and the E gene of F . The incC repeat sequence region shows reduced homology, with the F repeats being an imperfect subset of a larger repeated sequence found in R27 . The E gene homologue of R27 is able to initiate replication from the F ori-2 sequence and to repress the E gene promoter of F . The results are consistent with the observed incompatibility behaviour of R27, and have a bearing on the specificity of interaction of E protein with its DNA-binding sites.

Mol Microbiol, 1988 Mar, 2(2), 165 - 72
Functional analysis of different sequence elements in the Escherichia coli galactose operon P2 promoter; Ponnambalam S et al.; Starting with a DNA fragment containing the galactose operon P2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P2 activity . The results show that specific sequences upstream of -32 are not important . Removal of the sequence 5'-CACA-3' from -32 to -28 reduces P2 activity by 50%: longer deletions to -16 further reduce activity but do not remove the information specifying the transcription startpoint . DNA sequences between -32 and -16 at gal P2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase . The activity of gal P2 in the absence of -35 region sequences is dependent on the sequence TG just upstream of the -10 hexamer, TATACT: a mutation at -14 changing the TG sequence to TT totally inactivates P2 . However, P2 activity can be restored if the consensus -35 region sequence TTGACA is cloned 17 bp upstream of the -10 hexamer . Thus, for transcription initiation, the -10 hexamer, TATACT, must 'cooperate' with upstream sequences that may be located either around -35 or -14.

J Biochem Biophys Methods, 1988 Mar, 15(5), 235 - 40
An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli; Kumar KP et al.; DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method {(1975)Biochemistry 14, 4634} The total yield of the pure protein was 10 mg from 50 g of E.coli cells . The method was found to be very reproducible and convenient . The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.

J Cell Biochem, 1988 Mar, 36(3), 297 - 309
Mechanism of induction of class I major histocompatibility antigen expression by murine leukemia virus; Faller DV et al.; Alterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells . These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction . Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus . MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens . These viral effects on MHC antigen expression profoundly influence immune-mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing . Control of class I MHC and beta-2 microglobulin genes by MuLV takes place via a trans-acting molecular mechanism . MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene . These findings indicate that MuLV exerts its effects on MHC expression via a trans mechanism . The MuLV-responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for those genes.

An Esp Pediatr, 1988 Mar, 28(3), 191 - 5
{Enterotoxigenic Escherichia coli: description of a nosocomial diarrhea outbreak}; Escribano Montaner A et al.; A nosocomial diarrhea outbreak caused by Escherichia coli 0153: H45, which produces a thermostable enterotoxin, in five neonates admitted to the neonatology ward of the pediatric service of the Valencia University Hospital is described . The outbreak was discovered during a prospective study lasting eight months and aimed at evaluating the importance of Escherichia coli with enterotoxic capacity in acute infant diarrhea within our environment . The study involved conventional enterotoxigenicity tests applied both in vivo and in vitro . One of the patients, admitted with acute diarrhea was the source of the outbreak, with a possible person to person transmission . The diarrhea was slight to moderate . Emphasis is placed on the importance of this type of diarrhea in developed countries, and the problem is analyzed by reviewing its situation to the present.

Acta Chir Scand, 1988 Mar, 154(3), 169 - 77
Haematological, physiological and survival data in a porcine model of adult respiratory distress syndrome induced by endotoxaemia . Effects of treatment with N-acetylcysteine; Modig J et al.; The effects of N-acetylcysteine (NAC)--which is supposed to act as a free radical scavenger--were evaluated on lung function, haemodynamics and oxygen transport in a porcine model of pulmonary and cardiovascular failure induced by endotoxaemia . Three pigs, serving as controls, received NAC without endotoxin (E) for 6 h, and no notable physiological changes were found . Five pigs received a continuous infusion of E alone for 6 h and displayed a 90% decrease in leukocyte count and a 66% decrease in platelet count . Physiologically a four-fold increase in venous admixture (Qva/Qt), a nearly 2-3 fold increase in mean pulmonary arterial pressure (MPAP) and a progressive decline in cardiac output (Qt) of 60% were documented . Extravascular lung water (EVLW) increased 66%, mean arterial pressure (MAP) decreased 46% and oxygen delivery decreased 52%, leading to a metabolic acidosis . Three animals died during the observation period . Contrastingly, eight pigs, pretreated with NAC 150 mg.kg-1 which was continued at 20 mg.kg-1.h-1, showed a significantly attenuated response to E . Thus, leukocyte and platelet counts decreased 70% and 48%, respectively . Physiologically Qva/Qt increased 2.5-3 fold, MPAP increased 1.3-2 fold, and Qt decreased 32% . EVLW increased 27%, MAP decreased 27% and oxygen delivery decreased only 33% which kept the pH in the normal range . All animals survived the observation period, a significant difference from the E alone group . Thus, NAC significantly attenuated all monitored haematological and pathophysiological changes in the endotoxin model of ARDS in pigs . In addition to a reported free radical scavenger effect of NAC, our results support the assumption that NAC may counteract leukocyte and platelet aggregation in the lung thereby contributing to the beneficial outcome.

Vet Microbiol, 1988 Mar, 16(3), 273 - 81
Detection of enterotoxigenic Escherichia coli in piggeries in Victoria by DNA hybridisation using K88, K99, LT, ST1 and ST2 probes; Monckton RP et al.; Rectal swabs collected from piglets with diarrhoea from commercial pig farms were examined for the presence of enterotoxigenic Escherichia coli (ETEC) using DNA hybridisation methods . The probes specifically detected genes for the K88 and K99 fimbrial antigens and the heat-labile and heat-stable enterotoxins . DNA hybridisation methods detected more ETEC than could be detected by either enzyme-linked immunosorbent assay (ELISA) or slide agglutination methods, and also offered the opportunity to test for fimbrial antigens and toxins concurrently . The DNA hybridization method was shown to be applicable to ETEC detection in mixed growths cultured directly from rectal swabs to filters . The method eliminates the need for toxin tests using animals and enables very large numbers of samples to be investigated . The use of toxin probes has revealed large numbers of ETEC with uncharacterized fimbrial antigens.

Prostaglandins Leukot Essent Fatty Acids, 1988 Mar, 31(3), 139 - 46
Salutary effects of the prostacyclin analogue CG 4203 in lethal endotoxemia in rats; Schneider J; In pentobarbitone anesthetized rats infusion of E . coli endotoxin (0.42 mg.kg-1.min-1 for 4 hours) produced 96% lethality within 6 hours . Transient decrease in mean arterial blood pressure, thrombocytopenia, leukopenia, loss of plasma fibrinogen, fibrin deposits in renal glomeruli, hemolysis and decrease in arterial oxygen tension and pH were observed . Infusion of the prostacyclin analogue CG 4203 (0.464 and 1.0 micrograms.kg-1.min-1 for 6 hours), starting concomitantly with the endotoxin infusion, improved the survival rate to 95 and 100% . Blood pressure during endotoxemia was slightly lower in CG 4203 treated rats than in vehicle controls . CG 4203 infusion marginally attenuated thrombocytopenia and obviously inhibited leukopenia, but did not effect fibrinogen consumption in endotoxemic rats . Incidence of glomerular fibrin deposits was dose dependently and significantly reduced by CG 4203 . The lower dose reduced and the higher dose completely prevented the occurrence of hemolysis . Acidotic changes were not observed in CG 4203 treated endotoxin-shocked rats . Also with treatment starting 1 hour after the onset of endotoxemia CG 4203 in the same doses significantly inhibited the endotoxin-induced lethality . As protective mechanisms against lethal rat endotoxemia the prostacyclin-like hemodynamic, fibrinolytic, rheological and membrane stabilizing properties of CG 4203 are discussed.

Peptides, 1988 Mar-Apr, 9(2), 301 - 7
Production of a biologically active variant form of recombinant human secretin; Olson H et al.; A biologically active variant form of recombinant human secretin was produced using a gene fusion system designed to facilitate the purification of the protein . The fusion protein was recovered from the culture medium of Escherichia coli by IgG affinity chromatography, and recombinant secretin was released by cyanogen bromide treatment . A novel approach involving addition of a C-terminal Gly-Lys-Arg extension, was used to overcome the lack of amidation of recombinant proteins in Escherichia coli . The biological activity of the recombinant variant of secretin was at least 80% of the porcine secretin standard.

Mol Immunol, 1988 Mar, 25(3), 313 - 20
Antigenic competition in the immune response against protein mixtures: strain-specific non-immunogenicity of Escherichia coli antigens; Hammerl P et al.; Antigenic competition is argued to impair the immune response on the level of accessory cell-dependent antigen presentation to responsive T-cells (regulated by MHC encoded Ir gene products) . A possible influence of these mechanisms on in vivo immunization with antigen mixtures was investigated by using cytoplasmic extracts of four different strains of Escherichia coli as antigen sources for immunizing rabbits . The immune response against these antigen mixtures was tested by crossed immune electrophoresis (CIE) and immunoblotting (IB) in a homologous system (a given antigen extract of one strain against the corresponding antisera) and in the heterologous system (antigen extract of one strain against the antisera of different other strains) . Several proteins were non-immunogenic in the extract of one strain but elicited good antibody responses in other strains . One of the strain-specific non-immunogenic proteins was purified and revealed a normal immune response upon immunization . The data suggest that different antigenic competition effects are induced by different protein compositions of antigen mixtures . This strain-specific competition seems to determine the immunogenicity of certain molecules (and not only the immunogenic properties of the molecules themselves) . Furthermore this method offers a practical approach to increase the antibody production against weak immunogens in antigen mixtures.

J Dairy Sci, 1988 Mar, 71(3), 826 - 34
Relationship of milk proteins in blood with somatic cell counts in milk of dairy cows; McFadden TB et al.; Intramammary leucocytosis was induced by injection of Escherichia coli endotoxin via the teat canal in three lactating Holstein cows . Concentrations of alpha-lactalbumin and casein in blood serum were measured, and somatic cell concentration and yield and composition of milk were determined . Endotoxin injection elicited mean increases of 100-fold in somatic cell concentration and 50% in protein concentration, whereas milk yield declined 5-fold and lactose concentration was halved . Concentrations of alpha-lactalbumin and casein in blood rose from 80 to 1909 and 0 to 1231 ng/ml, respectively . By 96 h postinjection, all variables were approximately equal to those preinjection . In a second study, concentration of alpha-lactalbumin was determined in blood of lactating cows in two herds (n = 332) and related to milk somatic cell count . Concentrations of alpha-lactalbumin in blood were correlated with somatic cell counts (r = .60) . Mean concentrations of alpha-lactalbumin increased with increasing cell count even at low somatic cell concentrations (25 to 250 X 10(3)) . Concentrations of milk proteins in blood serum apparently reflect competency of the blood-milk barrier and may therefore yield an indirect measure of udder health.

Mol Cell Biol, 1988 Mar, 8(3), 1206 - 15
Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP; Clarke CF et al.; Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D . I . H . Linzer and A . J . Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P . Hinds, C . Finlay, A . Frey, and A . J . Levine, Mol . Cell . Biol . 7:2863-2869, 1987) have all been previously described . To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification . p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody . This mild and specific elution condition allows p53-protein interactions to be maintained . The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein . The maximum apparent molecular mass of such complexes is 660,000 daltons . Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components . Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex . Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E . coli protein of 70 kDa . Immunoblot analysis with specific antisera demonstrated that this E . coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein . Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex . This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

Mol Gen Genet, 1988 Mar, 211(3), 526 - 30
Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells; Vericat JA et al.; UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA . This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein . IPTE is independent of the lexA, recB recC, and recF genes . It is not related to the size or the replicon type of the plasmid . Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid . IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein . IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.

Anticancer Drug Des, 1988 Mar, 2(4), 361 - 70
Involvement of apurinic sites in the synergistic action of alkylating and intercalating drugs in Escherichia coli; Malvy C et al.; The toxicity of the intercalating compounds 9-aminoellipticine (9AE) and isopropyl-oxazolopyridocarbazole (Ipr-OPC) were studied . The inhibitory effect of non-toxic doses of 9AE, which incises DNA at apurinic (AP) sites, or Ipr-OPC, which does not cleave DNA at AP sites, with non-toxic doses of the alkylating agent dimethylsulphate (DMS) on the growth of Escherichia coli strain AB1157, is additive . The same result has been observed with an exonuclease III mutant which has only 10% of the AP endonuclease activity . However, 9AE or Ipr-OPC display a synergistic toxic effect with a DMS concentration which allows 20% of E . coli AB1157 survival . This synergy is increased for 9AE in the AP endonuclease mutant when compared to the wild-type strain . Under identical conditions 9AE and Ipr-OPC have no synergistic effect on a mutant deficient in the enzymes which generate AP sites . Therefore AP sites are involved in the synergistic toxicity of DMS and the studied intercalating agents . However, the precise role of the interaction of intercalating agents with AP sites, either without cleavage (type 1 compounds) or with cleavage (type 2 compounds), in the observed effect remains an open question.

Pediatr Emerg Care, 1988 Mar, 4(1), 9 - 11
Retropharyngeal abscesses in children: a 10-year review; Morrison JE Jr et al.; Retropharyngeal abscess is a rather rare, deep-neck infection of children and may seriously compromise the airway and mimic other diseases . A retrospective review of 17 cases of retropharyngeal abscess presenting to The Children's Hospital, Denver, from 1976 to 1986 was performed . Nine children (56%) had stridor or airway obstruction . Seven patients (41%) had perforations of their hypopharynx or esophagus, including two neonates (most likely associated with intubation attempts) . Two patients presented in the emergency department with a tentative diagnosis of "epiglottitis," while another referred to as having "persistent fever" was found to have a needle embedded in the hypopharynx . Fourteen children (81%) were brought to the operating room for examination and/or drainage of the abscess under general anesthesia . One child received an elective tracheotomy, and two others remained intubated postoperatively, pending resolution of their airway compromise . X-rays of the lateral neck were confirmatory in all these cases, with an unusually high incidence of "air/fluid levels," probably reflecting the corresponding large number of perforations of the hypopharynx or esophagus with subsequent communication into the retropharyngeal space.

DNA, 1988 Mar, 7(2), 71 - 8
A second antigenic heat shock protein of Plasmodium falciparum; Peterson MG et al.; We describe here an antigen of Plasmodium falciparum, defined by a cDNA clone designated Ag361 . The antigen is a soluble cytoplasmic 70-kD polypeptide present in all isolates analyzed and in all stages of asexual development in the blood . The antigen is a natural immunogen, although it lacks repeating epitopes of many P . falciparum antigens . Ag361 shares extensive sequence homology with the hsp70 proteins of Xenopus laevis, Drosophila melanogaster, Escherichia coli, and man, as well as a previously isolated P . falciparum hsp70 protein . The genome of P . falciparum contains at least five hsp70-like genes, located on at lest four different chromosomes.

DNA, 1988 Mar, 7(2), 127 - 34
A simple and efficient procedure for generating random point mutations and for codon replacements using mixed oligodeoxynucleotides; Ner SS et al.; A very simple and highly efficient procedure for the generation of single and multiple substitutions in segments of DNA is described which has no requirements for conveniently placed restriction sites, and allows all DNA sequences to be equally accessible . A mixed pool of oligodeoxynucleotides is synthesized by contaminating the monomeric nucleotides with low levels of the other three nucleotides such that the full-length oligonucleotide contains on the average one to two changes per molecule . This pool is used in priming in vitro synthesis of the complementary strand of cloned DNA fragments in M13 or pEMBL vectors which have previously been passed through a dut-, ung- Escherichia coli host . Strong selection for the newly synthesized strand is provided by transforming the heteroduplex into a dut+, ung+ host . Single and multiple substitutions in the carboxy-terminal coding region of the MATa1 gene of Saccharomyces cerevisiae are introduced at high efficiency (25-55%) and the changes are identified by direct sequencing alone . The same principle can be used to generate multiple sets of changes at any specified codon.

Carbohydr Res, 1988 Mar 1, 173(2), 243 - 53
Comparative structural elucidation of the K18, K22, and K100 antigens of Escherichia coli as related ribosyl-ribitol phosphates; Rodriguez ML et al.; The structures of the capsular K18, K22, and K100 antigens of E . coli O23:K18:H15, O23:K22:H15, and O75:K100:H5, respectively, were elucidated by determination of composition, 1H-, 13C-, and 31P-n.m.r . spectroscopy, periodate oxidation, alkaline hydrolysis followed by incubation with alkaline phosphatase, and methylation analysis of the polymers and their neutral fragmentation products . The polymers are poly(ribosyl-ribitol phosphates) related to the capsular antigen of H . influenzae (Hib) . The K22 antigen has the repeating unit -P-2)-beta-Rib-(1----2)-RibOH-(5-, and the K18 antigen has the same polymer chain with partial 3-O-acetylation of the ribose moiety . The K100 antigen consists of repeating units of -P-3)-beta-Rib-(1----2)-RibOH-(5- and seems to have a secondary structure different from that of the other antigens . Together with the Hib capsular antigens, the structure of which was reported as -P-3)-beta-Rib-(1----1)-RibOH-(5-, these capsular antigens represent a structurally related group of capsular polymers.

Hepatology, 1988 Mar-Apr, 8(2), 232 - 6
Endotoxin levels measured by a chromogenic assay in portal, hepatic and peripheral venous blood in patients with cirrhosis; Lumsden AB et al.; Endotoxin concentrations were measured in the portal, hepatic and peripheral venous blood of two groups of patients with cirrhosis using a limulus-based chromogenic assay . The high sensitivity of chromogenic detection allowed measurement of endotoxin as low as 10 to 15 pg per ml, an order of magnitude greater than previously possible by gelation studies . Group 1 consisted of 56 patients with cirrhosis undergoing angiographic evaluation . In this group, there was wide variability in hepatic venous concentration {73 +/- 110 pg per ml (mean +/- S.D.)} and peripheral venous concentration {31 +/- 58 pg per ml} . However, paired t test showed peripheral venous concentration was significantly (p less than 0.001) lower than hepatic venous concentration . Neither hepatic or peripheral venous endotoxin levels correlated significantly with a variety of clinical, biochemical or radiological parameters . Group 2 consisted of 21 patients with cirrhosis undergoing shunt surgery . Endotoxin levels again showed a wide range, with portal venous concentration (142 +/- 167 pg per ml) and simultaneous peripheral venous concentration (82 +/- 150 pg per ml) . Paired t test in this group showed a significant (p less than 0.001) portal to peripheral venous gradient . This study showed the feasibility of measuring endotoxin in plasma to low concentrations by a chromogenic assay technique . It supports the concept of relatively high levels of endotoxin in the portal circulation . In the presence of liver disease, systemic endotoxemia occurs, which is augmented by stressful situations.

Biochem J, 1988 Mar 1, 250(2), 429 - 34
Isolation, amino acid analyses and refolding of subunits of pig heart succinyl-CoA synthetase; Nishimura JS et al.; For the first time, pig heart succinyl-CoA synthetase has been refolded from its isolated subunits after denaturation . Amino acid analyses of pig heart succinyl-CoA synthetase and its subunits were performed . Subunits were isolated by gel filtration in neutral 6 M-urea . The amino acid composition of the native enzyme bears a strong resemblance to that of the Escherichia coli enzyme . Application of the various methods for comparing amino acid compositions {Cornish-Bowden (1983) Methods Enzymol . 91, 60-75} shows that the degree of relatedness between the alpha-subunits of the pig heart and E . coli enzymes and between the beta-subunits of the two synthetases is intermediate between 'strong' and 'weak' . As for the E . coli synthetase, it is unlikely that the alpha-subunit arises from the larger beta-subunit by post-translational modification . The pig heart enzyme contains a single tryptophan residue, which is located in the beta-subunit . Excitation of the enzyme at 295 nm resulted in a typical tryptophan emission spectrum . Refolding of enzyme denatured in 6 M-guanidine hydrochloride or of alpha- and beta-subunits isolated in this solvent required the presence of either ethylene glycol or glycerol, optimally at 20-25% (v/v) . GTP-Mg2+ did not stimulate reactivation of the enzyme, in contrast with the result obtained with ATP-Mg2+ in the reconstitution of the enzyme from E . coli . Yields of 60% and 40% were obtained in the refolding of denatured enzyme and isolated subunits respectively . The fluorescence spectrum of the refolded protein was essentially the same as that of native enzyme . Unrecovered activity could not be accounted for in the form of protein aggregates . The specific activity of refolded enzyme that had been separated from inactive protein on a Bio-Sil TSK 250 column was the same as that of native enzyme . Km values for GTP of 27 microM and 14 microM were determined for native and refolded enzyme respectively.

Mutat Res, 1988 Mar, 198(1), 37 - 43
Methyl-directed DNA mismatch repair in Escherichia coli; Lahue RS et al.; Some of the molecular aspects of methyl-directed mismatch repair in E . coli have been characterized . These include: mismatch recognition by mutS protein in which different mispairs are bound with different affinities; the direct involvement of d(GATC) sites; and strand scission by mutH protein at d(GATC) sequences with strand selection based on methylation of the DNA at those sites . In addition, communication over a distance between a mismatch and d(GATC) sites has been implicated . Analysis of mismatch correction in a defined system (Lahue et al., unpublished) should provide a direct means to further molecular aspects of this process.

J Trauma, 1988 Mar, 28(3), 312 - 8
Role of muscle microvasculature during hyperdynamic and hypodynamic phases of endotoxin shock in decerebrate rats; Cryer HM et al.; Microcirculatory derangements in skeletal muscle could act to change cardiac output during endotoxemia . To explore this idea, we measured arteriole and venule responses to low-dose and high-dose endotoxemia in the rat cremaster muscle by direct in vivo videomicroscopy . Our data indicate that cardiac output increased in the low-dose group and decreased in the high-dose group . In both animal groups, a differential arteriolar response occurred to give small arteriole dilation and large arteriole constriction while venous diameters did not change . We conclude that: 1) changes in cardiac output during endotoxemia are not related to microvascular responses in skeletal muscle, and 2) the microvascular responses in skeletal muscle could be responsible for the decreased systemic vascular resistance during high cardiac output endotoxemia, but not for the elevated systemic vascular resistance during low cardiac output endotoxemia.

Immunology, 1988 Mar, 63(3), 411 - 4
The influence of Peyer's patches on the organ-specific distribution of IgA plasma cells; Enders GA et al.; After the surgical removal of Peyer's patches (PP) in rats, the IgA-containing cells in the thoracic duct, mesenteric lymph nodes and lamina propria of the small intestine are decreased, as shown by immunohistology . The analysis of the immunoglobulin secretion in agar of single-cell suspensions confirmed these results . Using lipopolysaccharide (LPS) 055B5 as antigen, it could be demonstrated that this reduction may be the result of an inadequate presentation of antigen and/or impaired migration of locally primed antigen (AG)-specific cells . The oral application of heat-inactivated Escherichia coli 055B5 to PP-deprived rats resulted almost exclusively in anti-LPS-secreting cells in the mesenteric lymph nodes and spleen, whereas in control animals these cells were distributed along the intestine . Therefore, in rats PP have an important function in the regulation of the intestinal immune responses.

Biull Eksp Biol Med, 1988 Mar, 105(3), 374 - 6
{Ultrastructural changes in the microcirculatory bed of the lungs in endotoxin shock}; Kharlanova NG et al.; The electron microscopic study of the lungs during the initial and intermediate stages of endotoxin shock has revealed the manifestations of thrombo-hemorrhagic syndrome . Three days later interstitial fibrosis developed . Ultrastructural alterations of microcirculation become the basis for the formation of acute pulmonary failure.

Am J Physiol, 1988 Mar, 254(3 Pt 2), R463 - 9
Further evidence implicating prostaglandin E2 in the genesis of pyrogen fever; Coceani F et al.; Conscious cats were used to study the effects of endotoxin and interleukin 1 (IL 1) on levels of prostaglandin (PG) E2 and thromboxane (TX) B2 (the stable TXA2 byproduct) in cerebrospinal fluid (CSF) from the third ventricle . Pyrogens were given intravenously or intraventricularly and prostanoids were measured by radioimmunoassay . PGE2 was normally less abundant than TXB2 (mean, 37 vs . 528 pg/ml), and its level increased severalfold during the sustained fever following intravenous endotoxin (bolus) or IL 1 (bolus plus infusion) . PGE2 elevation preceded the fever and was maintained thereafter . Likewise, intraventricular pyrogens promoted PGE2 formation, and their effect was also manifest during the latent period of the fever . The PGE2 metabolite, 13,14-dihydro-15-keto-PGE2, was not measurable in CSF from either afebrile or febrile animals . Basal content of PGE2, on the other hand, was higher in animals pretreated with probenecid (30 mg/kg ip or iv; 50 or 100 micrograms ivt), confirming the importance of transport processes in removing prostanoids from brain . Unlike PGE2, TXB2 levels did not change during the fever to intravenous endotoxin . TXB2 rose instead in response to intraventricular endotoxin, although the elevation did not extend beyond fever uprise . Furthermore, a TXA2 analog (ONO-11113;2 or 4 micrograms ivt) had inconsistent effects on body temperature, while a TXA2 antagonist (ONO-11120;2 micrograms ivt) did not interfere with endotoxin fever . These findings strongly support a causative role for PGE2 in the onset and progression of pyrogen fever . No evidence of a similar role was obtained for TXA2.

Am J Med, 1988 Mar, 84(3 Pt 2), 636 - 9
Escherichia coli emphysematous endophthalmitis and pyelonephritis . Case report and review of the literature; Faraawi R et al.; Emphysematous escherichia coli endophthalmitis occurred in a 72-year-old patient as a complication of E . coli septicemia secondary to emphysematous pyelonephritis and endocarditis . This is the first reported case of endogenous emphysematous endophthalmitis secondary to E . coli septicemia.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1831 - 4
Nucleotide sequence for yeast dihydrolipoamide dehydrogenase; Browning KS et al.; Rabbit antiserum to the dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) component of the pyruvate dehydrogenase complex from bakers' yeast was used to screen plaques produced by a lambda gt11 yeast cDNA library . A 2.1-kilobase insert was isolated that also hybridized to a 17-base mixed oligonucleotide probe corresponding to the amino-terminal sequence of the yeast dihydrolipoamide dehydrogenase . The cDNA has a coding sequence of 499 amino acids that corresponds to a 21-residue signal peptide and a 478-residue mature protein (Mr = 51,558) . Computer analysis shows that yeast dihydrolipoamide dehydrogenase has about 41% amino acid identity with Escherichia coli dihydrolipoamide dehydrogenase . Particularly striking is the conservation of sequence in the active site region of the dihydrolipoamide dehydrogenases from E . coli, yeast, and pig heart.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1816 - 20
RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation; Nohmi T et al.; The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli . It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes . In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis . Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1811 - 5
UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA; Burckhardt SE et al.; The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light . This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage . To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes . The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression . Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA . The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons . Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD . We show that RecA mediates cleavage of UmuD, probably at this site . As with LexA, UmuD also undergoes a self-cleavage reaction . We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1754 - 8
Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase; Suttle DP et al.; The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase (OPRT; orotidine-5'-phosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine-5'-monophosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase; EC 4.1.1.23) . In mammals these two activities are found in a single, bifunctional protein called UMP synthase . A human T-lymphoblastic cell cDNA library constructed in lambda gt10 was screened with a UMP synthase-specific rat cDNA probe . Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments . The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments . One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA . This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA . The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199 . The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA . The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain . This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence . The NH2-terminal 214 amino acids contain the OPRT domain . There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT . The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1749 - 53
Three binding sites for AraC protein are required for autoregulation of araC in Escherichia coli; Hamilton EP et al.; Three binding sites for AraC protein were shown to be required for the autoregulation of araC: araI1, araO1, and araO2 . Selective inactivation of AraC-binding sites on the DNA demonstrated that araO1 and araO2 are required in vivo to produce repression of araC in the presence of arabinose, whereas araI1 and araO2 are required in its absence . We found that the low-affinity site araO2 is essential for araC autoregulation; araO1 and araI1 provide high-affinity AraC-binding sites, which allow cooperative binding at araO2 . Profound effects on the araBAD promoter and the araC promoter are produced by ligand-induced changes in AraC occupancy of functional sites on the DNA . We suggest that AraC exerts its multiplicity of controls through two alternative states of cooperative interactions with DNA and we illustrate this with a model . This model presents our interpretations of activation and repression of the araBAD operon and the autoregulation of the araC gene.

Mutat Res, 1988 Mar, 193(2), 131 - 7
Use of a dodecadeoxynucleotide to study repair of the O4-methylthymine lesion; Dolan ME et al.; A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with {32P} by the reaction with (gamma-32P}ATP and polynucleotide kinase . Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion . The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient . There was complete repair of O4-methylthymine by the E . coli alkyltransferase upon incubation for 4 h at 37 degrees C . There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period . There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract . However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this . The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.

J Med Microbiol, 1988 Mar, 25(3), 197 - 203
Lipopolysaccharide-induced non-specific resistance to systemic Escherichia coli infection in mice; Vuopio-Varkila J et al.; A high degree of non-specific resistance to a lethal systemic Escherichia coli infection was induced in mice by pretreatment with a small dose (less than 5 micrograms/mouse) of the homologous lipopolysaccharide (LPS) or with heterologous rough-type LPS from E . coli K-12 . The route of LPS administration, intraperitoneally or subcutaneously, did not influence the development of resistance, suggesting that a systemic cell activation was responsible for the effect . The enhanced elimination of bacteria was similar to that in mice recovering from a sublethal E . coli infection . In the LPS-treated mice, elimination of the challenge bacteria from the peritoneal cavity and the blood started 3-4 h after challenge whereas, in controls, the bacterial numbers continued to increase until the mice died . The detoxified LPS derivative, monophosphoryl lipid A (MPL), also increased the survival of mice infected with E . coli O18:K1 . However, the dose of MPL required for optimal infection resistance was 100-fold greater than that of native, E . coli K-12 LPS, corresponding to the 100-fold reduced toxicity of MPL for mice and rabbits in lethality and pyrogenicity assays.

J Immunol, 1988 Mar 1, 140(5), 1566 - 70
Antiviral action of tumor necrosis factor in human fibroblasts is not mediated by B cell stimulatory factor 2/IFN-beta 2, and is inhibited by specific antibodies to IFN-beta; Reis LF et al.; A protein termed IFN-beta 2, originally described on the basis of antiviral activity and antigenic cross-reactivity with the classical IFN-beta, is now known to be identical with the independently isolated B cell stimulatory factor (BSF-2) . Earlier it was suggested that IFN-beta 2 (i.e., BSF-2) mediates the antiviral action of TNF in human fibroblasts . We examined Escherichia coli-derived recombinant preparations of human IFN-beta and BSF-2 for antiviral activity and plasmacytoma growth factor (PCT-GF) activity . IFN-beta had antiviral activity but showed no PCT-GF activity . BSF-2 showed potent PCT-GF activity but lacked antiviral activity . Antiviral activity of IFN-beta was neutralized by polyclonal antibodies and mAb to IFN-beta, but not by antibody to rBSF-2 . PCT-GF activity of BSF-2 was neutralized by antibody to rBSF-2, but not by antibodies neutralizing the antiviral action of IFN-beta . Five mAb and a polyclonal antibody to human IFN-beta failed to react with BSF-2 in a solid phase RIA and antibody to BSF-2 did not react with IFN-beta . PCT-GF activity in supernatants of human FS-4 fibroblasts stimulated with TNF, IL-1 or poly(I).poly(C) was neutralized by antibody to rBSF-2, but not by antibodies neutralizing the antiviral activity of IFN-beta . Finally, the antiviral activity of TNF in FS-4 cultures was neutralized by antibodies to IFN-beta but not by antibodies to BSF-2 . Taken together, these results support the view that the antiviral action of TNF in human fibroblasts is mediated by IFN-beta, and not by BSF-2/IFN-beta 2 that apparently lacks significant antiviral activity.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Mar, 53(3), 477 - 88
Synergistic killing of Escherichia coli K-12 by UV (254 nm) and H2O2; Leitao AC et al.; Prior UV irradiation strongly increased the sensitivity to H2O2 of wild-type E . coli K-12 cells . This synergistic lethal interaction was also observed to a reduced extent in a polA mutant but was absent in uvrA, uvrArecA and xthA mutants . In a recA mutant an antagonist effect was observed . Prior H2O2 treatment did not sensitize the wild-type cells to UV irradiation . Alkaline and neutral sucrose gradient analysis, as well as DNA degradation studies, demonstrated that the synergism is due to the production of DNA double-strand breaks and a block of their repair . The possible mechanism of induction of such lesions is discussed.

Int J Radiat Biol Relat Stud Phys Chem Med, 1988 Mar, 53(3), 381 - 93
Dithiothreitol pretreatment and inducible repair in UV-irradiated Escherichia coli K12 cells; Claycamp HG; The UV radiation survival of several Escherichia coli K12 strains was measured after pretreatment of the cells with dithiothreitol (DTT) . In DNA repair-competent cells (AB1157), UV survival was enhanced (ER = 1.2) after pretreating cells for 1.0 h using 10 mmol dm-3 DTT and then incubating the cells for 1.5 h in buffer before UV irradiation . Similar experiments using the excision repair mutant, AB1886uvrA6, or the recombination repair and SOS-deficient mutant, AB2462recA, strains did not show enhanced UV survival . None of the E . coli strains tested were protected against UV killing by simultaneous treatment with DTT (10 mmol dm-3) . These results, and the fact that incubation in chloramphenicol removed the wild-type response in DTT-pretreated, UV-irradiated cells, suggest that the observed UV radioprotection was a result of inducible enzymatic repair processes such as recA-dependent repair . The proposed stimulus for inducible repair in these cells is DNA damage caused by intracellular hydroxyl radicals arising from thiol oxidation . The involvement of oxygen radicals in the induction pathway is supported by results that showed superoxide dismutase and catalase could inhibit a portion (one-third) of the inducible repair.

Blood, 1988 Mar, 71(3), 672 - 6
Human granulocyte-monocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor-dependent mechanism; Cannistra SA et al.; Human colony-stimulating factors (CSF) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells . In this study, the effects of three recombinant human CSFs (granulocyte-monocyte CSF {GM-CSF}, interleukin 3 {IL-3}, and granulocyte CSF {G-CSF}) on antibody-independent monocyte tumoricidal activity were investigated by using WEHI 164 fibrosarcoma cells as monocyte-sensitive targets . None of the CSFs directly induced monocyte cytotoxicity, although both GM-CSF and IL-3 were found to significantly enhance monocyte killing in response to a second stimulatory event (endotoxin) . No effect was seen with G-CSF . Antitumor necrosis factor antibody completely abolished CSF-enhanced monocyte cytotoxicity, which suggests that this effect was mediated through increased release of tumor necrosis factor (TNF) . As previously shown for GM-CSF, IL-3 was found to induce cytoplasmic accumulation of TNF messenger RNA (mRNA) after 18 hours of exposure . These results suggest that GM-CSF and IL-3 may stimulate monocyte killing indirectly by enhancing expression of TNF mRNA, thereby leading to augmented TNF protein secretion in response to a second activation signal.

Surgery, 1988 Mar, 103(3), 383 - 5
Pelvic cellulitis: a life-threatening complication of hemorrhoidal banding; Scarpa FJ et al.; Rubber band ligation is a commonly employed office procedure for the eradication of symptomatic internal hemorrhoids . Since 1980 increasingly frequent reports of an often fatal complication--"pelvic cellulitis"--have appeared . Death has been avoided for some of these patients by early recognition and treatment . One such survivor is reported here, in a case report that illustrates the value of early recognition of symptoms and appropriate diagnostic and therapeutic intervention.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1472 - 6
Functional role of cysteine-146 in Escherichia coli thymidylate synthase; Dev IK et al.; Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process . The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive . In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants . The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding . The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1467 - 71
lac permease of Escherichia coli containing a single histidine residue is fully functional; Puttner IB et al.; Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in lactose/H+ symport, possibly as components of a catalytic triad similar to that postulated for the serine proteases {Kaback, H . R . (1987) Biochemistry 26, 2071-2076} . By using restriction fragments of lacY genes harboring specific site-directed mutations, a fusion gene has been constructed that encodes a permease in which His-35 and His-39 are replaced with arginine, and His-205 with glutamine (RQHE permease) . The resultant molecule contains a single histidine residue at position 322 and exhibits all of the properties of the wild-type permease . In addition, an analogous single-histidine permease was engineered with alanine at position 325 in place of glutamic acid (RQHA permease) . This construct is defective in active transport but catalyzes exchange and counterflow normally . RQHA permease, like the single-histidine permease with Glu-325, also shows normal behavior with respect to N-ethylmaleimide inactivation, substrate protection, and binding . In addition to providing strong support for previous experiments, the engineered permease molecules should be useful for determining the apparent pK of His-322 under various conditions.

J Bacteriol, 1988 Mar, 170(3), 1405 - 7
New locus for exopolysaccharide overproduction in Escherichia coli K-12; Zinkewich-Peotti K et al.; A new locus for exopolysaccharide overproduction in Escherichia coli K-12 was mapped by insertion mutagenesis . A 66% linkage to serA, which is located at 62 min on the E . coli K-12 linkage map, was shown by P1 transduction . The polysaccharide produced by the mutant was isolated and was shown to be similar to colanic acid.

J Bacteriol, 1988 Mar, 170(3), 1380 - 3
Replication patterns of multiple plasmids coexisting in Escherichia coli; Leonard AC et al.; The replication patterns of several plasmids were measured simultaneously during the cell division cycle of Escherichia coli B/r . F plasmids harboring oriS, both oriS and oriV, pSC101, and pBR322 were found to replicate at all stages of the cell division cycle with kinetics which were indistinguishable from one another and clearly different from the periodic synthesis of the minichromosomes pAL49 and pAL70.

J Bacteriol, 1988 Mar, 170(3), 1268 - 74
First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene; Coleman J et al.; The min 4 region of the Escherichia coli genome contains genes (lpxA and lpxB) that encode proteins involved in lipid A biosynthesis . We have determined the sequence of 1,350 base pairs of DNA upstream of the lpxB gene . This fragment of DNA contains the complete coding sequence for the 28.0-kilodalton lpxA gene product and an upstream open reading frame capable of encoding a 17-kilodalton protein (ORF17) . In addition there appears to be an additional open reading frame (ORF?) immediately upstream of ORF17 . The initiation codon for lpxA is a GUG codon, and the start codon for ORF17 is apparently a UUG codon . The start and stop codons overlap between ORF? and ORF17, ORF17 and lpxA, and lpxA and lpxB . This overlap is suggestive of translational coupling and argues that the genes are cotranscribed . Crowell et al . (D.N . Crowell, W.S . Reznikoff, and C.R.H . Raetz, J . Bacteriol . 169:5727-5734, 1987) and Tomasiewicz and McHenry (H.G . Tomasiewicz and C.S . McHenry, J . Bacteriol . 169:5735-5744, 1987) have demonstrated that there are three similarly overlapping coding regions downstream of lpxB including dnaE, suggesting the existence of a complex operon of at least seven genes: 5'-ORF?-ORF17-lpxA-lpxB-ORF23-dnaE-ORF37-3 '.

J Bacteriol, 1988 Mar, 170(3), 1261 - 7
Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid; Terawaki Y et al.; RepA protein, essential for replication of plasmid Rts1, was found to bind in vivo immediately upstream of the repA promoter in studies with mini-Rts1 derivatives with deletions in the upstream region of repA . We constructed another series of repA mutants that would encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl-terminal six amino acids . These modified RepA proteins could not activate ori (Rts1) at all and showed various degrees of incompatibility, or no incompatibility, toward a mini-Rts1 plasmid . These results suggest that the carboxyl-terminal six (or fewer) amino acids of RepA are important for exerting replication and incompatibility functions . One of the RepA derivatives, which showed an evident incompatibility without initiating replication, was examined for its ability to repress the repA gene.

J Bacteriol, 1988 Mar, 170(3), 1235 - 8
Loss of the spacer loop sequence from the rrnB operon in the Escherichia coli K-12 subline that bears the relA1 mutation; Harvey S et al.; A polymorphism affecting the spacer region of the rrnB rRNA operon is described . Strains from a major Escherichia coli K-12 subbranch are missing a 106-nucleotide portion of the rrnB 16S-to-23S spacer, and a 20-nucleotide sequence is found in its place . We have called this mutant operon rrnB2 . The rrnB2 spacer was most probably derived from either rrnC or rrnE . This alteration of rrnB may have occurred by a recombinational exchange or by gene conversion . In the genealogy of E . coli K-12 strains, the appearance of rrnB2 is associated with the spontaneous occurrence of the first relaxed mutation, but attempts to show a selective relationship between the two mutational events have had negative results . The sequences of the rrnG and rrnC 16S-to-23S spacers have also been determined and their comparisons to the other rrn operons encoding tRNAGlu2 are presented.

J Bacteriol, 1988 Mar, 170(3), 1220 - 6
Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12; Stoker K et al.; A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme . By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity . As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2 . Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L . The presence of such an activity was recently suggested in various papers and was called isoenzyme 3 . Hydrogenase L might be identical or part of the latter isoenzyme . By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2 . Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity . However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay . This suggested that the fast initial activity of HB101 was due to hydrogenase L . The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8 . Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero . Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.

J Bacteriol, 1988 Mar, 170(3), 1175 - 82
Nucleotide sequence of an Rts1 fragment causing temperature-dependent instability; Tanaka M et al.; Rts1 is a multiphenotypic drug resistance plasmid which is eliminated from host bacteria at 42 degrees C but not at 32 degrees C . This phenotype has been called temperature-dependent instability (Tdi) . We determined the nucleotide sequence of the Rts1 DNA b' segment which causes this phenotype . Within this 786-base-pair segment, several open reading frames (ORFs) were found, including one which encodes a protein with a molecular weight of 16,000 . A protein approximately corresponding to this protein is expressed in Escherichia coli minicells harboring plasmids containing the b' segment . In addition, we found the chi sequence at 112 bases proximal to this ORF . Temperature-dependent elimination due to this segment was not observed in the RecA strain of E . coli, but the RecB protein was not required for expression of this phenotype . We constructed various deletion derivatives and found that three portions, the region containing the chi (nucleotides 1 to 24), ORF (nucleotides 25 to 546), and tail (nucleotides 631 to 786) sequences are necessary for Tdi activity . Site-directed mutagenesis studies indicated that ORF I is required for Tdi expression.

J Bacteriol, 1988 Mar, 170(3), 1041 - 5
Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement; Collins CM et al.; Ureolytic Escherichia coli strains are uncommon clinical isolates . The urease phenotype in a large percentage of these isolates is unstable and lost upon storage . We examined two urease-positive uropathogenic E . coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded . The urease phenotype was cloned from E . coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment . Transposon mutagenesis indicated that at least 3.2 kilobases of this fragment were necessary for production of urease . The urease recombinant plasmid pURE coded for at least four insert-specific polypeptides as determined by maxicell analysis . Disruption of the region encoding two of these polypeptides (67 and 27 kilodaltons) abolished urease activity . Analysis by Southern hybridization of urease-positive E . coli 1021 and seven independently isolated urease-negative segregants showed that a DNA rearrangement was associated with the urease-negative phenotype.

Arch Surg, 1988 Mar, 123(3), 345 - 50
Pulmonary microvascular changes following fluid resuscitation in an ovine model of endotoxemia; Lubbesmeyer HJ et al.; Fluid resuscitation is complicated in hypotensive septic patients by their susceptibility to pulmonary edema . This problem was evaluated in the ovine model of endotoxemia with a chronic lung lymph fistula . Escherichia coli endotoxin (lipopolysaccharide, 1.5 micrograms/kg) was given intravenously over 30 minutes . Group M (n = 9) continued to receive baseline fluids (2 mL/kg/h), while group R (n = 6) received 7 mL/kg/h of Ringer's lactate . After an initial drop in cardiac index, animals in both groups developed a hyperdynamic state . The fall in mean arterial pressure seen in group M was absent from group R . The higher fluid volume resulted in a rise in left atrial pressure and pulmonary microvascular pressure . The lung lymph flow and permeability index were elevated in both groups but were higher in group R . The calculated filtration coefficient showed a threefold increase in both groups . Augmented fluid resuscitation during endotoxemia resulted in an elevated interstitial fluid flux and permeability index secondary to an increase in pulmonary microvascular pressure and greater surface area of the injured microvascular beds being perfused.

Gastroenterology, 1988 Mar, 94(3), 787 - 96
Experimental portal fibrosis produced by intraportal injection of killed nonpathogenic Escherichia coli in rabbits; Kono K et al.; An attempt was made to develop an animal model for the study of the etiology of noncirrhotic portal fibrosis or idiopathic portal hypertension based on the assumption that it is related to chronic abdominal infection . Rabbits were given killed nonpathogenic Escherichia coli intraportally or intravenously . The animals to which a mixture of killed E . coli and rabbit antiserum (aggregated E . coli) was given intraportally developed remarkable histologic changes in the liver . The early inflammatory reactions in the portal area and parenchyma were followed by rapid disappearance of inflammation and development of portal fibrosis with bile duct proliferation . Three intraportal challenges with aggregated E . coli were sufficient to produce pronounced portal fibrosis, although there was considerable variation in response among individual animals . This procedure also produced splenomegaly, and in some animals marked portal hypertension . Injection of nonaggregated killed E . coli into the portal vein or aggregated E . coli into the ear vein also caused similar hepatic changes, but they were milder in degree . These histologic changes resemble portal fibrosis seen in idiopathic portal hypertension and, less closely, pericholangitis associated with inflammatory bowel disease in humans.

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1759 - 62
Repair of O-alkylpyrimidines in mammalian cells: a present consensus; Brent TP et al.; Enzymatic repair of the O-alkylpyrimidines (O2- and O4-alkylthymine, O2-alkylcytosine) and alkyl phosphotriesters has been studied in Escherichia coli, and the two proteins involved, a glycosylase (DNA-3-methyladenine glycosylase) and a methyltransferase (DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.63), have been well characterized . In mammals or mammalian cells treated with carcinogenic alkylating agents, loss of these derivatives has been demonstrated repeatedly . Nevertheless, mammalian repair proteins that are analogous to those from E . coli do not detectably act on these alkyl derivatives . A variety of techniques has been used by many investigators in the United States and Europe, who conclude here that the mode of O-alkylpyrimidine and alkyl phosphotriester repair in mammalian cells differs from that in E . coli . New approaches and methods are needed to characterize these processes at the biochemical and molecular level.

Microb Pathog, 1988 Mar, 4(3), 175 - 87
The interaction of Escherichia coli with normal human serum: factors affecting the capacity of serum to mediate lipopolysaccharide release; Tesh VL et al.; We previously demonstrated that incubation of E . coli in normal human serum (NHS) resulted in the release of a finite fraction (approximately 30%) of LPS from the bacterial outer membrane . In experiments reported here, we examined factors which may enhance or diminish the capacity of NHS to mediate this limited LPS release . Both the susceptibility to serum killing and LPS release were dependent on growth phase . Optimal killing and release coincided with the midlogarithmic growth phase . The composition of LPS subunits in the outer membrane appeared to influence serum-mediated LPS release . Serum treated E . coli enriched for Rc-chemotype LPS released less LPS from their outer membrane than the wildtype 'smooth' bacteria during exponential growth . LPS fractions released by NHS or EDTA appeared to a large degree to overlap, suggesting that NHS-mediated LPS release may involve the action of a serum chelator . A serum-resistant mutant failed to release LPS in either NHS or EDTA . This latter observation suggests that LPS release may be a relevant event in serum killing . We did not detect any modulation of LPS release when E . coli were pre-incubated with a series of antibiotics prior to treatment with NHS.

Ital J Biochem, 1988 Mar-Apr, 37(2), 78 - 84
Studies on recognition of selenahomolysine by aminoacid transport systems and aminocyl-tRNA synthetase; Di Girolamo M et al.; In E . coli, Se-3 aminopropylselenocysteine or selenahomolysine (SeHL) does not affect intracellular lysine transport, i.e . it cannot bind E . coli lysine transport systems . In CHO cells it inhibits cationic aminoacid transport system, but only in the presence of Na+, this indicating that it behaves like polar neutral aminoacids . On the other hand, it poorly affects leucine transport both in the presence and in the absence of Na+ . SeHL is not activated by aminoacyl-tRNA synthetase preparations from bacterial and mammalian sources, thus it cannot be utilized for protein synthesis.

Arzneimittelforschung, 1988 Mar, 38(3A), 474 - 8
{Structure, function and use of fibrinolysis-promoting and inhibiting factors}; Bachmann F; Our knowledge about the components of the fibrinolytic system has greatly increased during the last few years thanks to the cloning of the cDNA of the two plasminogen activators t-PA and u-PA, of the two plasminogen activator inhibitors PAI-1 and 2, and of other profibrinolytic and of inhibitory factors of this system . The two principal plasminogen activators (PA) and the two PA-inhibitors are present in infinitesimal concentrations in blood (pM to microM range) and in tissues . The expression of the t-PA and of the u-PA cDNA in mammalian cells and in E . coli bacteria has rendered it possible to produce sufficient amounts of these thrombolytic disorders for clinical studies . Over 2000 patients afflicted with acute myocardial infarction have been treated so far with recombinant t-PA . In general, the thrombolytic effect of t-PA appears to be superior to that obtained with streptokinase and the fibrin-specific-PA produces lesser fibrinogenolysis than the first generation thrombolytic agents streptokinase and two-chain urokinase.

Jpn J Cancer Res, 1988 Mar, 79(3), 384 - 9
Endogenous tumor necrosis factor induction with Bordetella pertussis vaccine as a triggering agent and its therapeutic effect on MM46 carcinoma-bearing mice; Minagawa H et al.; Induction of endogenous tumor necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as a triggering agent and its therapeutic effect against MM46 carcinoma were investigated in C3H/He mice . Test triggering agents were injected intravenously into mice after intravenous injection of 4-fold dilution of macrophage activating factor (MAF) or 10(4) units of murine interferon-gamma (Mu-IFN-gamma) . Then sera were obtained from the mice, and their TNF activities were assayed on L-929 cells by the method of Ruff and Gifford . The triggering activity of BPV was the highest among those of conventional triggers, such as lipopolysaccharide (LPS) of Escherichia coli, and OK-432 . The levels of serum TNF activity triggered by BPV (4 X 10(9) cells), LPS of E . coli (3 micrograms) and OK-432 (3 KE) were 5350, 85 and 102 units/ml, respectively . Growth of MM46, a spontaneous mammary carcinoma cell line of C3H/He was observed for 35 days after tumor inoculation and was suppressed significantly by intravenous injection of MAF and BPV (4 X 10(9) cells) . On local injection of BPV (2 X 10(9) cells) into murine tumors, complete regression was observed in 67% of the mice tested with or without MAF priming on day 25 after tumor inoculation, and intratumoral TNF activity was observed even in the case of the single injection of BPV.

Curr Eye Res, 1988 Mar, 7(3), 321 - 7
Effects of inhibitors of arachidonic acid metabolism on endotoxin-induced ocular inflammation; Fleisher LN; Rabbits were treated with the cyclooxygenase inhibitors indomethacin (2.5 and 10 mg/kg; IP) or flunixin meglumine (2.2 and 8.8 mg/kg; IM), or the dual cyclooxygenase/lipoxygenase inhibitor BW755C (10 and 30 mg/kg; IP) (administered 4 times over 25 hours) . One hour following drug administration, 10 ng of E . coli endotoxin was injected into the vitreal chamber and the subsequent inflammatory response was measured 24 h later . Indomethacin was the most effective inhibitor of prostaglandin (PG) E2 and PGF 2 alpha accumulation in the aqueous humor and their ex vivo production by the lens . It was the only agent that decreased iridal hyperemia and protein concentration in the aqueous humor . Flunixin decreased PG production, although generally to a lesser degree than indomethacin . BW755C produced modest inhibition of PG production in the aqueous humor, but did not affect PG production by the lens . The high dose of BW755C also reduced 5-hydroxyeicosatetraenoic acid (5-HETE) levels in the aqueous humor, but did not alter production by the lens . The results indicate that despite drastic reductions in PG levels in the aqueous humor and PG production by the lens, only modest decreases in aqueous humor protein concentration and iridal hyperemia were achieved . Furthermore, 5-HETE does not appear to be important in the modulation of these inflammatory signs, although it is possible that larger decreases in its production could result in more dramatic effects.

Biochem J, 1988 Mar 1, 250(2), 343 - 8
Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils . Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase; Matsumoto T et al.; The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release {1-14C}oleic acid from labelled Escherichia coli has been demonstrated . The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S . More than 80% of maximal activity is reached at 10 microM-Ca2+ . The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation . Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity . Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity . Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity . The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased . Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of {3H}arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187 . This potentiation is not due to an inhibition of the acyltransferase activity . H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA . These results suggest several points . (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C . (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis . (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Am J Physiol, 1988 Mar, 254(3 Pt 2), H509 - 16
Effects of nonhypotensive endotoxemia in conscious rats: role of prostaglandins; Burnier M et al.; A nonhypotensive dose of endotoxin was administered to normal conscious rats to evaluate the vascular and humoral effects of endotoxemia per se . Mean blood pressure and heart rate remained stable during the 45 min infusion of Escherichia coli endotoxin (0.01 mg/min) . However, a marked increase in plasma renin activity (4.2 +/- 0.48 vs . 30.2 +/- 6 ng.ml-1.h-1, mean +/- SE, P less than 0.01), plasma epinephrine (0.112 +/- 0.04 vs . 1.71 +/- 0.5 ng/ml, P less than 0.01), and plasma norepinephrine (0.269 +/- 0.028 vs . 1.3 +/- 0.2 ng/ml, P less than 0.001) was observed during infusion in endotoxin-treated rats when compared with the vehicle-treated animals . In addition, the blood pressure response to exogenous norepinephrine was significantly reduced during nonhypotensive endotoxemia . Significant changes in regional blood flow distribution, as assessed by radiolabeled microspheres, were observed in endotoxemic rats; in particular a decrease in renal blood flow (7.39 +/- 0.43 vs . 5.97 +/- 0.4 ml.min-1.g-1, P less than 0.05) and an increase in coronary blood flow (5.01 +/- 0.38 vs . 6.44 +/- 0.33 ml.min-1.g-1, P less than 0.01) were found . The role of prostaglandins in the vascular and humoral alterations induced by nonhypotensive endotoxemia was also examined . Pretreatment with indomethacin (5 mg) prevented the increase in plasma renin activity as well as plasma catecholamine levels . On the contrary, the decreased vascular reactivity and the reduction in renal blood flow observed during endotoxemia were not affected by prostaglandin synthesis inhibition . Thus significant vascular and humoral changes have been found during endotoxemia even in absence of hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1988 Mar, 85(6), 1806 - 10
RecA protein-dependent cleavage of UmuD protein and SOS mutagenesis; Shinagawa H et al.; Induction of the Escherichia coli SOS system increases the ability of the cell to perform DNA repair and mutagenesis . Products of the recA and umuD,C genes are required for mutagenesis induced by radiation and many chemicals . Transcription of the SOS genes including recA and umuD,C is repressed by a repressor, LexA protein, and is derepressed by the proteolytic cleavage of LexA facilitated by RecA protein that had been activated by inducing signals produced in the cell by agents that damage DNA . An activated form of RecA protein, RecA, seems to have roles in SOS mutagenesis other than its known role as an antirepressor . Derepression of the genes involved in SOS mutagenesis such as recA and umuD,C in defective chromosomal lexA(Def) mutants does not increase the ability of the cell to perform mutagenesis . Activation of RecA protein is essential to this ability . RecA facilitates the proteolytic cleavage of several repressors such as lambda, P22, and 434 phage repressors and LexA, and UmuD protein contains a sequence homologous to the regions surrounding the cleavage sites of these repressors; therefore, we examined the possibility that UmuD protein is cleaved by RecA . We found evidence that the intact UmuD protein was cleaved after mutagenic treatment and that the cleavage was dependent on RecA . The results suggested that UmuD protein may be proteolytically processed by RecA, and that processed UmuD may be the active form of the protein participating in mutagenesis.

J Med Microbiol, 1988 Mar, 25(3), 205 - 11
Killing of Escherichia coli in the peritoneal cavity of convalescent mice; role of specific and non-specific immune mechanisms; Vuopio-Varkila J et al.; Mice surviving a sublethal E . coli O18:K1 infection possess a greatly increased resistance to a subsequent lethal E . coli O18:K1 peritonitis . A similar increase in resistance can also be achieved by LPS pretreatment . The early course of the infection in the convalescent mice at day 1 was identical to that in LPS-pretreated mice . At this time, the convalescent mice were also able to restrict the growth of the heterologous E . coli O78(ColV) strain, suggesting that non-specific phagocyte activation was responsible for the increased destruction of the bacteria . At day 4, the kinetics of infection in convalescent mice were identical to those in mice passively immunised with specific anti-K1 capsule antiserum . A rapid decline in the numbers of viable homologous, but not heterologous, bacteria took place in the peritoneal cavity suggesting effective antibody-mediated opsonisation followed by phagocytosis and killing of the bacteria.

J Clin Invest, 1988 Mar, 81(3), 700 - 9
Metabolic fate of arachidonic acid in hepatocytes of continuously endotoxemic rats; Rodriguez de Turco EB et al.; The present experiments were designed to characterize the kinetics of {1-14C}arachidonic acid (AA) metabolism as a function of time in hepatocytes obtained from rats infused continuously for 30 h with a nonlethal dose of Escherichia coli endotoxin (ET) . Chronic endotoxemia greatly reduces the ability of hepatocytes to utilize {1-14C}AA, which is reflected from the earliest times of incubation in very low labeling of intermediates in the biosynthetic pathways of glycerolipids (phosphatidic acid and diacylglycerol) and slower removal of {1-14C}AA from the free fatty acid pool as compared with saline-infused rats . At later times of incubation, the labeling of phospholipids (especially phosphatidylethanolamine and phosphatidylinositol {PI}), but not of triacylglycerides is decreased . Analysis of fatty acid composition of individual phospholipids from cells of ET-infused rats reveals that the content of AA is significantly reduced only in PI . Hence an impairment in activation/acylation enzymatic mechanisms could affect the turnover of metabolically active phospholipid pools, i.e., PI, involved in signal transmission processes, and result in increased availability of 20:4 for eicosanoid synthesis, contributing to cellular metabolic perturbations in endotoxicosis.

Biotechniques, 1988 Mar, 6(3), 238 - 42
A novel method for rapid isolation of plasmid DNA; Zervos PH et al.; A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA . Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times . pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria . Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.

Gene Anal Tech, 1988 Mar-Apr, 5(2), 32 - 9
Optimal conditions for supercoil DNA sequencing with the Escherichia coli DNA polymerase I large fragment; Lim HM et al.; Sequencing ladders produced from supercoiled DNA templates with the Escherichia coli DNA polymerase Klenow fragment are often unreadable because of a high background and misincorporated nucleotides . This study showed that contaminating RNA molecules can interfere with template:primer hybridization . Procedures are provided for the purification of template DNA and stringent conditions for primer-template hybridization that overcome these problems.

J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 829 - 34
Vero cytotoxin production and presence of VT genes in Escherichia coli strains of animal origin; Smith HR et al.; Vero cytotoxin (VT) production has been studied in 34 Escherichia coli strains isolated from animals with enteric diseases . The strains were tested by DNA hybridization with probes for VT1 and VT2 sequences and also in toxin neutralization experiments with specific antisera . Twenty bovine strains, belonging to nine different O serogroups, produced VT1 or VT2 but not both toxins . In contrast, all 14 porcine strains of four different O serogroups produced VT2 only . Six of these porcine strains, belonging to serogroups O138, O139 and O141, were isolated from cases of oedema disease . In general, the porcine isolates produced toxin at a lower level than the bovine strains.

J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 777 - 83
Continued growth of Escherichia coli after stopping medium addition to a K+-limited chemostat culture; Mulder MM et al.; The steady-state bacterial dry wt of Escherichia coli, growing under K+-limited conditions in the chemostat, was inversely dependent on the growth rate . This phenomenon was more carefully investigated in medium-flow stop experiments . Growth did not stop immediately but continued for a time, initially at the same rate as before . The dry wt increased to a value corresponding to a steady-state growth rate near zero, independent of the initial specific growth rate . This was observed in both the wild-type strain and a mutant that lacked the high-affinity K+ uptake system . The wild-type strain maintained a low extracellular K+ concentration both in the chemostat under steady-state conditions and after stopping the medium flow . The mutant, on the other hand, maintained a much higher extracellular K+ concentration in the steady state, which decreased to much lower values after stopping the medium flow . From the increase in bacterial dry wt and the low external K+ concentration after stopping the medium flow it is concluded that the intracellular K+ is redistributed among the cells, including new cells . The growth yield on K+ was highest in the stationary growth phase of a batch culture and all steady-state cultures converged ultimately to this yield value after the medium flow had been stopped . It is proposed that the growth rate of E . coli under K+-limited conditions is determined by the intracellular K+ concentration.

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