Escherichia coli

Int J Biochem, 1985, 17(10), 1125 - 8
Studies on N-acetylneuraminic acid biosynthesis in chicken liver and hepatoma Mc-29 by using {14C}N-acetylmannosamine and {14C}glucosamine; Ivanov S et al.; The biosynthesis of free N-acetylneuraminic (sialic) acid (N-acetylneuraminic + CMP-N-acetylneuraminic acid) in chicken liver and hepatoma Mc-29 by using {14C}N-acetylmannosamine and {14C}glucosamine was studied in vivo . The specific activity (SA) of hepatoma N-acetylneuraminic acid labelled with {14C}glucosamine is lower than that of liver, showing that the rate of conversion of UDP-N-acetylglucosamine to N-acetylneuraminic acid is reduced in tumor cells . The biosynthesis rate of sialic acid in hepatoma cells is higher when {14C}N-acetylmannosamine was applied . The UDP-N-acetylglucosamine-2'-epimerase activity was nearly 3-fold lower in hepatoma compared to that in liver . The time course of {14C}N-acetylmannosamine incorporation into free and protein bound sialic acid in hepatoma and liver was also showed . The SA of hepatoma protein bound sialic acid remained lower in all time points investigated . The results agree with the assumption that the metabolic pathways leading to sialic acid synthesis and to sialylation of tumor glycoconjugates are altered after malignant transformation.

Clin Ther, 1985, 7(5), 607 - 10
Transfer of cefoperazone into human skin fluid; Sugiyama H et al.; The transfer of cefoperazone into exudates from excoriated skin wounds was studied in 13 adolescent and adult patients . Subjects were given an intravenous bolus injection of 50 mg/kg of body weight . Concentrations of cefoperazone in serum and in exudate fluids were determined by bioassay using Escherichia coli as the test organism . Mean (+/- SD) cefoperazone concentration in serum reached 333.3 +/- 103.3 micrograms/ml 30 minutes after the injection and decreased to 16.4 +/- 5.7 micrograms/ml eight hours after the injection . In exudate fluids, the peak value was 52.7 +/- 22.1 micrograms/ml at one hour . Eight hours after the injection, the mean concentration in the exudate was 10.7 +/- 7.2 micrograms/ml, which was considerably above the minimum inhibitory concentrations for important pathogens.

Prog Clin Biol Res, 1985, 189, 241 - 9
Ideal properties of a LAL reagent for pharmaceutical testing; Cooper JF; Evaluation of medical devices and pharmaceutical products for endotoxin content is the most significant biomedical application of Limulus amebocyte lysate (LAL) . This review summarizes the means by which LAL reagent suppliers have introduced different production techniques and formulation additives to uniquely optimize their products for industry . Pharmaceutical testing requires a LAL reagent that is buffered, stabilized to a sensitivity of 0.12 EU/ml, optimized to detect E . coli-derived LPS in water, formulated to produce firm opaque gels, designed specifically to detect bacterial endotoxin, and is economical . Unique LAL reagent characteristics produce nonuniformity in drug compatibility testing but does not significantly alter detection of unsafe levels of native endotoxin . Design of a clinical LAL reagent may require additional means for standardization and exclusion of additives which are only useful for pharmaceutical testing . A cooperative effort between clinical investigators and the LAL industry should resolve these issues in a reasonable period of time.

Res Exp Med (Berl), 1985, 185(2), 107 - 13
Endotoxin-induced acute renal failure in mice . Effects of indomethacin and the thromboxane-synthetase antagonist UK 38.485; Hirschberg R et al.; Functional acute renal failure (ARF) was induced within 24 h following i.p . injection of 200 micrograms E . coli endotoxins (ET) into C3H/HeHan mice . Pre- and post-treatment with either UK 38.485, a selective thromboxane (TX)-synthetase inhibitor, or with the cyclo-oxigenase inhibitor, indomethacin (IM), does not prevent acute renal failure in these mice . Histologically, only very little fibrin degradation and few microthrombi are present 24 h later in the kidneys, so that disseminated intravascular coagulation (DIC) mechanisms cannot have caused the significant azotemia . Slight histological changes are accentuated in the UK 38.485-treated group . Only the indomethacin group has a significantly increased mortality as compared to all other groups . We conclude from our study that with low dosages of endotoxins functional ARF can be induced in mice without a circulatory shock and early mortality and that both UK 38.485 and IM are of little value in preventing it.

J Cell Biol, 1985 Jan, 100(1), 258 - 64
Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria; Pavco PA et al.; The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients . The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE . Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16 . The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods . Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions . The labeling of P16 with {35S}methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol . These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria . The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function . Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA . Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

Dev Biol Stand, 1985, 59, 175 - 80
Control of recombinant DNA produced pharmaceuticals by a combination of process validation and final product specifications; Jones AJ et al.; Traditional methods of product release for human pharmaceutical use are designed and selected to ensure control of purity, potency, safety and identity . The selection of these tests depends upon the nature of the product . In addition to the control achieved by these methods, control of the efficiency of the manufacturing process in removing substances that cannot be tested for in the final product is achieved by "process validation" . Process validation allows the use of sophisticated experimental techniques that do not lend themselves to final product testing or the use of others which, when applied to the final product, are insufficiently sensitive to be used for product release . The removal of such components as DNA and process materials can be demonstrated by these studies . Demonstrations that the manufacturing process reproducibly removes these components, eliminate the need for these tests on every batch . The essential elements of a process validation study are development of a sensitive assay technique for the component of interest and quantitation of the efficiency of individual process steps in removing that component . Even when that component is undetectable in normal process samples, the efficiency of any process step can still be determined by the addition of that component during the validation studies . The values of process validation in demonstrating the removal of DNA and in the determination of E . coli protein contamination levels during the manufacture of methionyl human growth hormone is illustrated.

Jpn J Antibiot, 1985 Jan, 38(1), 95 - 101
{Fundamental studies of cefoxitin in the field of obstetrics and gynecology}; Ito K et al.; The concentration of cefoxitin (CFX, Merxin) in dead space exudate was studied in 14 patients following total extirpation of diffuse uterine cervical cancer . A two-compartment model was used for the analysis . The results obtained were as follows: Calculated maximum concentrations of CFX in the pelvic dead space exudate were 26.55 micrograms/ml at 2.11 hours, 31.07 micrograms/mg at 2.01 hours and 51.51 micrograms/ml at 2.10 hours after 1 hour intravenous drip infusions of CFX 2, 3 and 4 g, respectively . These concentrations were higher than the MIC80 of 12.5 micrograms/ml against E . coli and B . fragilis and were maintained for a sufficient period of time . Based on the results of this study, CFX is considered to be an important and valuable drug in the field of obstetrics and gynecology.

Arch Microbiol, 1985 Jan, 140(4), 343 - 6
Functional domains of colicin M; Dreher R et al.; The structure of colicin M of Escherichia coli was studied with regard to its organization into functional domains . A proteolytic fragment with an Mr of 24,000 was isolated which comprised the carboxyterminal portion of the protein . It adsorbed to the outer membrane receptor protein and inhibited killing of cells by colicin M and by phage T5 that uses the same receptor . The fragment killed cells when the outer membrane was rendered permeable to macromolecules for a short time by the osmotic shock procedure . It is concluded that the fragment contains the receptor binding site and the active center but is lacking the sequence required for transport into cells . The carboxy-terminal amino acid sequence-Lys-Arg of the fragment was identical to that obtained from colicin M . Release of lysine and arginine led to inactivation of colicin M . The sequence of the first 39 amino acids of the amino terminal end of colicin M was determined.

Mol Cell Biol, 1985 Jan, 5(1), 140 - 6
Selective transfer of individual human chromosomes to recipient cells; Saxon PJ et al.; Two hypoxanthine phosphoribosyltransferase-deficient human cell lines, D98/AH-2 and HT1080-6TG, were stably transfected with pSV2 gpt, a plasmid containing the selectable marker Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco gpt) . Hypoxanthine-aminopterin-thymidine-resistant transformants arose with a frequency of ca . 10(-6) and contained mostly single, but occasionally multiple, copies of the plasmid sequences . These transformants actively express the Eco gpt marker . Single chromosomes from two different HT1080 gpt transformants and one D98 gpt transformant, containing the integrated plasmid sequences, were transferred via microcell-mediated chromosome transfer to hypoxanthine phosphoribosyl transferase-deficient mouse A9 cells . The transferred human chromosomes were identified as 2, 4, and 22, by using a combination of G-11 staining, G-banding, isoenzyme analysis, and in situ hybridization . This system is being used to create a library of interspecies microcell hybrid clones, each clone containing a unique single human chromosome in a mouse background . The complete library will represent the entire human karyotype.

Int Arch Allergy Appl Immunol, 1985, 76(4), 324 - 30
Immune responses to polyethylene glycol modified L-asparaginase in mice; Kawamura K et al.; Suppression of anti-L-asparaginase (anti-A-ase) IgG and IgE antibody responses was achieved in Balb/c mice with polyethylene glycol (PEG, MW, 5,000) conjugated Escherichia coli A-ase . Following the administration of the mixture of A-ase and PEG-A-ase, antibody production to A-ase was reduced . PEG-A-ase administration prior to A-ase suppressed the primary and secondary responses to A-ase antibody . The suppression could be transferred to normal mice with spleen cells from A-ase tolerant mice . The cell transfer experiment showed that the suppression was caused by suppressor T cells . Since PEG-A-ase administration failed to suppress antibody response to ovalbumin, the suppression seemed to be A-ase specific . PEG-A-ase administration also suppressed the delayed type hypersensitivity reaction . IgG and IgE antibodies to PEG or PEG-A-ase were not detected in mice immunized with PEG or PEG-A-ase in the presence of Freund's complete adjuvant or A1(OH)3, respectively.

Radiobiologiia, 1985 Jan-Feb, 25(1), 95 - 9
{Stimulating effect of aminoethylisothiuronium on the immune response and interferonogenesis in irradiated mice}; Zheleznikova GF et al.; Aminoethylisothiuronium (AET) stimulated the formation of antibodies against sheep erythrocytes, not against E . coli, in X-irradiated (4 Gy) mice . The serum containing AET-induced interferon had the same effect . AET also promoted the rejection of the allogenic skin graft in mice irradiated with the same dose . In addition, AET and cystaphos stimulated the induction of interferon by the Newcastle disease virus in mice exposed to doses of 4, 5 or 6 Gy.

J Appl Physiol, 1985 Jan, 58(1), 70 - 6
Effect of outflow pressure on lung lymph flow in unanesthetized sheep; Drake R et al.; Studies in anesthetized animals have shown that the flow rate from lung lymphatics (QL) depends on the pressure at the outflow end of the vessels (Po) . We tested this in unanesthetized sheep prepared with chronic lung lymph cannula . We measured QL with the lymph cannula held at various heights above the olecranon and calculated Po as the height + QL X cannula resistance . QL decreased with increases in Po (delta QL/delta Po = -8.2 +/- 6.4 microliter X min-1 X cmH2O-1, mean +/- SD) . We increased QL by raising left atrial pressure or infusing Ringer solution or Escherichia coli endotoxin and found that QL was even more sensitive to Po (delta QL/delta Po = -32 +/- 22) . Cannula resistance caused a 9-70% reduction in QL . Changes in QL caused by increasing Po were not associated with changes in lymph protein concentration for up to 330 min . This indicates that increases in Po shunt lymph away from cannulated vessels but do not substantially effect microvascular filtration rate . The shunted lymph may flow into other vessels or collect in the lung . We conclude that QL does not accurately represent microvascular filtration rate because it depends on the cannula resistance and position at which the investigator chooses to place the cannula.

Carcinogenesis, 1985 Jan, 6(1), 105 - 8
The role of DNA-repair processes in N-nitrosopyrrolidine-induced mutagenesis; Alldrick AJ et al.; The cytotoxic and mutagenic effects of increasing concentrations of N-nitrosopyrrolidine (NPYR) were studied using various DNA repair mutants of Escherichia coli together with rat-liver S9 activation system . Irrespective of which strain was used, the cytotoxic effects of NPYR were similar to those observed in the parent strain . Mutagenicity studies revealed that the uvrA- derivative was more mutable than its repair proficient parent . These observations suggest that NPYR reacts with DNA to generate bulky lesions, which although potentially mutagenic, do not contribute significantly to cell-killing . Subsequent experiments with the metabolic inhibitor SKF 525A revealed that this compound only partially inhibited the mutagenic activity of NPYR, suggesting that although hepatic mixed function oxidase enzymes may participate in NPYR activation other pathways of metabolism are also involved.

Infect Immun, 1985 Jan, 47(1), 329 - 31
Metabolic and mitochondrial morphological changes that mimic Reye syndrome after endotoxin administration to rats; Yoder MC et al.; The administration of sublethal doses of Escherichia coli O111:B4 endotoxin to starved rats results in significant increases in plasma ammonia, free fatty acids, and serum lactate compared with starved controls . These metabolic alterations are associated with Reye syndrome-like histological findings of hepatic microvesicular fatty accumulation and hepatic ultrastructural evidence of mitochondrial pleomorphism with matrix disruption . This sublethal endotoxin model may help elucidate the relationship between the hepatic mitochondrial injury, characteristic metabolic impairment, and encephalopathy seen in patients with Reye syndrome.

J Cell Sci Suppl, 1985, 3, 29 - 38
Production of epidermal growth factor in Escherichia coli from a synthetic gene; Allen G et al.; Mouse epidermal growth factor (EGF) is under investigation as a deflecting agent for sheep . Substantial quantities of the pure protein are required for these studies and to supply this need a gene for the protein was synthesized and inserted into plasmid vectors to direct the expression of EGF polypeptide, or fusion proteins containing the EGF peptide sequence, in transformed Escherichia coli . Mature EGF was released by lysine specific proteolysis of a fusion protein consisting of part of the E . coli TrpE protein, a lysine linker and EGF polypeptide . The EGF was purified and characterized and was found to be biologically active.

Gene, 1985, 40(2-3), 291 - 9
Conservation of a 29-base-pair sequence within maxicircle ARS fragments from six species of trypanosomes; Kim R et al.; The maxicircles from Trypanosoma brucei, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas davidi were examined for the presence of a 29-bp sequence termed CF29 that has been found in the ars 189 sequence from the Crithidia fasciculata maxicircle and in Lt-ars 189 from the maxicircle of Leishmania tarentolae . The CF29 sequence also contains a yeast consensus ARS of (T/A)TTTATPuTTT(T/A) . All of the maxicircles examined contained specific fragments that hybridized to the CF29 probe . The non-replicating yeast plasmid vector YIp5 was used to clone these CF29-containing maxicircle fragments . High-frequency transformation was observed when these chimeric plasmids were used to transform Saccharomyces cerevisiae . Autonomous replication of these transforming plasmids was verified by Southern analysis of yeast-cell extracts using pBR322 as a hybridization probe . Therefore it appears that the CF29 sequence is widely conserved in kinetoplastid protozoa and is associated with ARS sequences in the maxicircles . Hybridization of the CF29 probe to a population of P . davidi minicircles was also observed . However, the YIp5 chimeric plasmid containing this CF29-hybridizing minicircle fragment failed to transform yeast.

Gene, 1985, 40(2-3), 203 - 15
Characterization of nif regulatory genes in Rhodopseudomonas capsulata using lac gene fusions; Kranz RG et al.; Translational fusions of the Escherichia coli lacZYA operon to Rhodopseudomonas capsulata nif genes were obtained by using mini-MudII1734 {Castilho et al., J . Bacteriol . 158 (1984) 488-495} inserts into cloned fragments of R . capsulata DNA . A lac fusion to the nifH gene, which encodes dinitrogenase reductase, was used to classify Nif- mutations occurring in regulatory genes . Nine mutations were unable to activate nifHDK transcription . The nine mutations define four nif regulatory genes . Three of these genes are located on the same R . capsulata 8.4-kb EcoRI fragment . Each is transcribed independently . One of these (complementing mutant J61) is partially homologous with the ntrC gene of Escherichia coli, based on Southern hybridization . The fourth nif regulatory gene (complementing mutants LJ1, AH1 and AH3) is unlinked to the others . Lac fusions to all four regulatory genes were constructed . Each regulatory gene is weakly expressed compared to derepressed nifH and partially repressed in the presence of ammonia.

Gene, 1985, 39(2-3), 269 - 74
Cloning and expression in Escherichia coli of the synthetic proenkephalin analogue gene; Hostomsky Z et al.; Enkephalins are pentapeptides with opioid activity that have been found in brain and other neural tissues . They are released by proteolytic processing of the proenkephalin, which contains several enkephalin sequences each flanked by pairs of basic amino acid (aa) residues . We have constructed an artificial variant of the proenkephalin gene by concatenation of synthetic oligodeoxynucleotides (oligo) coding for Met-enkephalin preceded by two arginines . One of the resulting structures, containing eleven enkephalin sequences separated by pairs of arginine codons, was cloned in the expression vector pRE31 . The biological activity of enkephalin was detected after the digestion of the isolated plasmid-coded protein with trypsin and carboxypeptidase B . The product of the synthetic gene may thus serve as a defined simplified substrate for the study of the not yet fully understood enzymatic mechanisms of proenkephalin processing.

Gene, 1985, 39(2-3), 147 - 53
Shuttle vectors to study somatic mutagenesis and regulation of gene expression in the immune system; Lazo PA; Somatic mutagenesis is one of the main mechanisms for generation of diversity in the immunoglobulin genes . A family of 16 shuttle vectors has been designed to identify the mutation mechanism . These vectors are based on bovine papilloma virus replicon and carry selective markers (NmR and ApR), a mutation marker (the Escherichia coli galK) as well as several segments from the immunoglobulin (Ig) heavy- and kappa light-chain genes in different orientations . They can also be used to study general mutagenesis and the relative contribution of different DNA sequences to the regulation of gene expression by competition with trans-acting factors . These vectors start replicating in mammalian cells two days after transfection . Stable transformants carry the plasmids in an extrachromosomal form for at least three months without evidence of structural alteration.

Nucleic Acids Symp Ser, 1985, (16), 287 - 90
Synthesis and expression of the native RNase T1 gene and several mutant genes; Nishikawa S et al.; RNase T1 gene and several mutant genes were constructed by joining of chemically synthesized deoxyoligonucleotides . These genes were inserted into an expression vector and expressed as fused protein in E . coli . RNase T1 and its mutant enzymes were liberated by cyanogen bromide treatment and their activities were measured.

Mol Gen Genet, 1985, 201(3), 519 - 24
Ability of base analogs to induce the SOS response: effect of a dam mutation and mismatch repair system; Bebenek K et al.; 2-Aminopurine, 2-amino-N6-hydroxyadenine, 2-amino-N6-methoxyadenine and 2-amino-N6-methyl-N6-hydroxyadenine (but not N4-hydroxycytidine), strong mutagens of base analog type, may induce the SOS response in E . coli cells . This ability is greatly enhanced in dam3 mutants and abolished in dam3mutS, dam3mutH, and dam3mutL strains, thereby suggesting that the mismatch repair system is involved in the mechanism of induction.

J Mol Evol, 1985, 22(4), 361 - 2
A change in the genetic code in Mycoplasma capricolum; Jukes TH; Mycoplasma capricolum was previously found to use UGA instead of UGG as its codon for tryptophan and to contain 75% A + T in its DNA . The codon change could have been due to mutational pressure to replace C + G by A + T, resulting in the replacement of UGA stop codons by UAA, change of the anticodon in tryptophan tRNA from CCA to UCA, and replacement of UGG tryptophan codons by UGA . None of these changes should have been deleterious.

Gene, 1985, 36(3), 375 - 9
The effects of hybrid ribosome-binding-site variants on the expression of human interferon-beta in Escherichia coli; Whitehorn EA et al.; Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence . Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters . The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp . One spacer of 9 bp length was varied at a single position . IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects . In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.

Gene, 1985, 36(1-2), 159 - 67
The use of gene fusions to study the expression of uidR, a negative regulatory gene of Escherichia coli K-12; Blanco C et al.; The uidR regulatory gene of Escherichia coli codes for a repressor molecule that negatively controls the expression of the uidA gene . The uidR gene was fused in front of the lacZ gene in vitro on plasmid cloning vectors developed by Casadaban et al . {J . Bacteriol . 143 (1980) 971-980 . The transcriptional direction of uidR was deduced from the restriction pattern and the phenotypic properties of the uidR-lacZ fusion plasmids . The gene is transcribed counterclockwise on the standard E . coli map, as is the uidA gene . The uidR-lacZ fusions were also used to examine the regulation of expression from the uidR promoter . It was observed that the uidR gene expression is repressed by its own product and is sensitive to catabolite control . The uidR-lacZ-encoded proteins of various sizes were isolated but attempts to obtain hybrid molecules possessing, in a single polypeptide, both the beta-galactosidase and UidR repressor activities were unsuccessful.

Mol Gen Genet, 1985, 201(1), 14 - 9
Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon; Peterson KR et al.; We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam-) . Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of beta-galactosidase in dam- strains than in isogenic dam+ strains . The attempted construction of dam- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein) . Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam- strain viability.

Int J Nucl Med Biol, 1985, 12(2), 135 - 44
111In-labeled eosinophils: localization of inflammatory lesions and parasitic infections in mice; Runge VM et al.; Based upon recent development of practical isolation techniques for eosinophils, labeling and in vivo imaging of eosinophils has been achieved . Isolation of cells was performed utilizing a Percoll density gradient . The eosinophils were subsequently labeled by a modified 111In-oxine method . Migration of eosinophils in response to intradermal ear-pinna injections of SEA (soluble schistosoma egg antigen), S . mansoni eggs, E . coli, and turpentine was followed with gamma-ray camera imaging from 4 to 48 h . Maximal localization, determined by Gamma 11 data processing, occurred by 4-h post-injection of radiolabel . SEA and S . mansoni eggs provided a greater stimulus for localization than E . coli or turpentine . Neutrophils did not preferentially accumulate . Tissue distribution of labeled eosinophils was greatest in the spleen, followed by liver and bone . 111In-labeled-eosinophil scans are sensitive to parasitic infections, although somewhat nonspecific.

Mol Gen Genet, 1985, 200(1), 185 - 6
DNA methylation influences trpR promoter activity in Escherichia coli K-12; Marinus MG; Methylation of adenine in the GATC-sequence of the -35 region of the trpR promoter decreases activity by 2-3 fold.

Mol Gen Genet, 1985, 199(3), 507 - 11
Genetic analysis of uxuR and exuR genes: evidence for ExuR and UxuR monomer repressors interactions; Ritzenthaler P et al.; The uxuAB operon is under the dual control of uxuR- and exuR-encoded repressors whereas the exu regulon genes are regulated by the sole ExuR repressor . Mutations affecting the two exuR and uxuR regulatory genes were selected to investigate the relationship between the two repressors . The isolation of exuR and uxuR negative dominant mutations on a multicopy plasmid indicated that the active form of the two repressors was multimeric . The introduction of a uxuR negative dominant allele into a wild-type strain resulted in a significant increase in exu gene expression . This unexpected effect may have been the consequence of the formation of hybrid repressor molecules . This protein must be composed of native ExuR+ subunits aggregated with altered UxuR subunits . The same interference was observed for the exuR negative dominant allele on uxu gene derepression . The hypothesis given here implies that the two regions of the ExuR and UxuR repressors involved in the subunit aggregation present enough homologies to allow the formation of hybrid repressor molecules.

Gene, 1985, 35(1-2), 159 - 67
Synthesis of a wheat storage protein subunit in Escherichia coli using novel expression vectors; Bartels D et al.; Useful plasmid expression vectors have been constructed which allow the synthesis of beta-galactosidase (betaG) fusion polypeptides or of polypeptides specified by cDNA clones in Escherichia coli hosts . A foreign DNA fragment can be inserted in any one of the three reading frames at the unique EcoRI, BamHI or SmaI sites immediately after the initiation codon . The cloned foreign gene is under the control of the lac promoter . Using a cDNA clone that encodes part of a wheat storage protein {a high-Mr (HMW) glutenin subunit} synthesis of a glutenin-beta G fusion protein was demonstrated . Synthesis of the glutenin polypeptide, not fused to beta G, was achieved by replacing the lacZYA genes with a stop codon.

J Immunoassay, 1985, 6(1-2), 111 - 23
A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli; Hashida S et al.; A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli . IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes . In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods . The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.

Mol Gen Genet, 1985, 198(3), 432 - 6
Generation of deletions through a cis-acting mutation in plasmid pC194; Alonso JC et al.; When plasmid pC194-1 is ligated to pBR322 to generate plasmid pHV15-1, deletions occur with high frequency within the joined pBR322 DNA . Generation of deletions is recE4 independent, and occurs in B . subtilis with a 1,000-fold higher frequency than in Escherichia coli . In the hybrid plasmid pVH15-1, deletion end-points are not at random, but at defined locations within pBR322 . We propose that the base alteration, characterizing pC194-1, has stabilized within the plasmid a stem/loop structure, which acts as a deletion generator.

Gene, 1985, 34(1), 105 - 10
Isolation and expression in Escherichia coli of a cDNA clone encoding human beta-glucuronidase; Guise KS et al.; Mucopolysaccharidosis type VII is a lysosomal storage disease resulting from a deficiency of beta-glucuronidase (BG) activity . To facilitate the investigation of mutation in the disease and provide molecular diagnostic tools for affected families, we have isolated human BG cDNA clones . The SV40-transformed human fibroblast cDNA library of Okayama and Berg {Mol . Cell . Biol . 3 (1982) 280-289} was screened with a fragment of a murine BG cDNA clone (pGUS-1) . The 17 human cDNA clones (pHUG) isolated were identical by restriction mapping, varying only in length . The pHUG clones show 80% DNA sequence homology with pGUS-1 in a 198-bp PvuII-SstI restriction fragment . Both pGUS-1 and the pHUG clones contained an open reading frame (ORF) throughout the sequenced region with a predicted amino acid sequence homology of 73% . Expression in Escherichia coli of a 1150-bp fragment of pHUG-1 subcloned in pUC9 resulted in an isopropyl-thio-beta-galactoside (IPTG)-inducible 35-kDal fusion protein which was specifically immunoprecipitated by goat anti-human BG immunoglobulin G (IgG) . This evidence provides direct confirmation that the pHUG cDNA clones correspond to human BG.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 139 - 45
Stimulation and inhibition of cell division in synchronized Escherichia coli; Nanninga N et al.; Escherichia coli was synchronized by centrifugal elutriation . When grown in a Tris-based medium, addition of EDTA resulted in division about 20 min earlier (division of control at t = 75 min) . EDTA addition caused a change in cell shape, with cells becoming narrower and longer, whereas the surface area to volume ratio increased . Irradiation with UV inhibited not only division and constriction, but also the increase in DAP incorporation found in dividing control cells . Possibly, division requires the construction of new polar caps, whereas premature division might involve remodeling of existing murein . In both cases, cell shape is presumed to be a relevant factor for division.

C R Acad Sci III, 1985, 300(7), 255 - 60
{Construction of an infectious retroviral vector for the expression of eucaryotic genes}; Yeramian P et al.; We describe here an infectious eucaryotic expression vector derived from Moloney Murine Leukemia (Mo-MuLV) provirus and recombined in plasmid pBR 322, for the expression of eucaryotic genes . Upstream of the cloning sites lie the 5' LTR and 700 bp of the gag sequences containing the splicing and encapsidation signals . Downstream of the cloning sites are situated the env gene and the 3' LTR containing the polyadenylation signal . So as to test the potential use of this vector, Herpes Simplex TK gene and E . Coli NeoR genes were cloned in the same transcriptional polarity as the viral LTRs . When DNA from the recombinant plasmid was transfected into mouse, rat, or human cell cultures, high yields of TK+ or NeoR colonies were obtained . Recombinant plasmids constructed with TK or NeoR genes in the opposite polarity failed to produce drug resistant colonies . Cotransfection with DNA of the Mo-MuLV competent helper provirus led to the rescue of chimeric virus capable of transmitting drug resistance.

Biochimie, 1985 Jan, 67(1), 75 - 82
Does a monospecific hybridoma always secrete homogeneous immunoglobulins?
Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME.
The fusion of splenocytes (from mice immunized with the beta 2 subunit of E . coli tryptophan synthase) with myeloma cells which do not produce immunoglobulins gave rise to a clone secreting immunoglobulins with two distinct isotypes : gamma 1 and gamma 2b (Djavadi-Ohaniance et al . (1984) Biochemistry, 23, 97-104) . Analysis of the immunoglobulins secreted by this clone indicates that these two isotypes are carried by two distinct heavy chains which are able to randomly associate to form hybrid molecules . In addition, two classes of light chains are able to randomly and to form heterologous associations with both the gamma 1 and gamma 2b heavy chains . Only the association between the gamma 2b heavy chains with one of the two classes of light chains leads to a combining site specific for the binding of the antigen beta 2.

Biochimie, 1985 Jan, 67(1), 101 - 8
The amino acid sequence of thiogalactoside transacetylase of Escherichia coli; Fowler AV et al.; The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined . The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671 . The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin . All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests . The sequence analysis was aided by determination of the DNA sequence of the lacA gene . The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved . Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain . Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities . Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.

Mol Cell Biol, 1985 Jan, 5(1), 197 - 203
The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster; Amin J et al.; A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed . Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D . melanogaster and COS-1 (African green monkey kidney) cells . Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types . Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D . melanogaster cells . Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D . melanogaster gene in homologous cells . A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D . melanogaster cells.

Mol Gen Genet, 1985, 198(2), 356 - 7
Identification of a gene, tir of R100, functionally homologous to the F3 gene of F in the inhibition of RP4 transfer; Tanimoto K et al.; We detected a gene of R100 functionally homologous to the F3 gene of F in the inhibition of RP4 transfer . Using in vitro recombinant DNA techniques, we located the gene, designated tir, in a 0.9 kb region, 2,392-3,293 in the nucleotide sequence coordinate of R100 . From the DNA sequence analysis of R100 (Ohtsubo unpublished results), a coding frame of polypeptides, whose molecular weight is estimated to be 24.1 kilodaltons (kd), was inferred to be the region tir . Furthermore, we showed that tir could not repress expression of the F3 gene.

Mol Gen Genet, 1985, 198(2), 315 - 22
Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12; Wild J et al.; Evidence is presented that alanine racemase activity in E . coli K12 is due to two distinct gene products . The predominant isozyme is inducible by either alanine stereoisomer and repressible by glucose . The gene dadX coding for its structure is located by the dadA gene determining the structure of D-amino acid dehydrogenase . The regulatory site for the expression of both genes, dadR, is located on the other side of dadA . The orientation of the dad operon established by multiple-point crosses and deletion mapping is as follows: fadR ...dadRAX ...hemA . The dadX alanine racemase activity is unusually refractory to changes of incubation temperature . It differs strikingly from that of the other isozyme, probably the product of the alr gene . The latter isozyme shows a typical dependence upon incubation temperature . The synthesis of alr alanine racemase is constitutive in respect of both alanine and glucose . In dadX mutants, in which alanine racemase activity equals only 15% of that in wild-type cells grown in the absence of an inducer or catabolite repressor, the dad operon cannot be induced by D-alanine . We presume, therefore, that L-alanine is involved more directly than D-alanine in dad operon regulation.

Chromosoma, 1985, 91(3-4), 259 - 66
X chromosomal organisation and dosage compensation . In situ transcription of chromatin template activity of X chromosome hyperploids of Drosophila melanogaster; Chatterjee RN; The chromatin template activity of the polytene X chromosomal DNA was assayed by in situ transcription on the fixed polytene chromosomes using E . coli RNA polymerase holoenzyme and 3H-UTP as the monitoring substrate in various 1X2A, 2X2A and 3X2A larvae and 1X2A (+ X fragments) segmental aneuploid larvae of Drosophila melanogaster . The segmental aneuploids contained duplications for the segments 15EF-20F, 11A-20F, 8C-20F and 3E-20F of the X chromosome . Results revealed that a double dose of active loci located in the X chromosome regions 15EF-20F, 11A-20F, and 8C-20F in aneuploids synthesized nearly 40%-70% more RNA than the normal single dose of this region in the wild-type males . The activity per gene dose for the two segments in the aneuploids was also significantly higher than in their male counterpart except for the duplication dp (3E-20F), where the duplicated piece extended from the centromeric heterochromatin to include 85% of the euchromatic portion of the X chromosome . In the case of dp (3E-20F), the X chromosome was transcribed at the lower, "female" level . It may also be noted that some regions of the X chromosome when present in extra copy, especially in dp (8C-20F) influenced the template activity of the X-linked genes inside or outside the duplicated segment . Metafemales (3X2A) have 50% higher template activity of the X chromosomes than their diploid sisters . In this study, metafemales behaved as females with duplication.(ABSTRACT TRUNCATED AT 250 WORDS)

Circ Shock, 1985, 15(3), 205 - 15
Metabolic and cardiovascular effects of endotoxin infusion in conscious unrestrained rats: effects of methylprednisolone and BW755C; McKechnie K et al.; Infusion of Escherichia coli endotoxin (41.7 micrograms kg-1 min-1 i.v . for 4 h; 10 mg kg-1 total) in conscious unrestrained rats produced an increase in heart rate and a gradual decrease in arterial blood pressure . Initially plasma glucose was transiently elevated but fell to hypoglycaemic values in the 1-2 h before death . There was a marked elevation in the plasma concentrations of lactate, thromboxane B2, and 6-keto prostaglandin F1 alpha, (PGF1 alpha) . Methylprednisolone treatment (two doses of 30 mg kg-1) significantly reduced mortality, provided the first dose was given before commencing the endotoxin infusion; there was no effect on mortality if it was given 2 h after starting the endotoxin infusion . Methylprednisolone pretreatment maintained arterial blood pressure and plasma glucose and prevented the increase in plasma lactate in rats given endotoxin . Methylprednisolone treatment did not modify the increase in plasma thromboxane B2 but attenuated the increase in plasma 6-keto PGF1 alpha concentrations . Pretreatment with BW755C, an inhibitor of both cyclooxygenase and lipoxygenase enzymes, completely prevented the increase in plasma prostanoid concentrations but did not improve survival or significantly modify any other detrimental effects of endotoxin . It is suggested that the beneficial effects of methylprednisolone in this model of endotoxin shock are not related to a reduction in the formation of pharmacologically active arachidonic acid metabolites.

Circ Shock, 1985, 15(3), 155 - 62
Endotoxin-induced eicosanoid production by equine vascular endothelial cells and neutrophils; Bottoms GD et al.; Dispersed equine vascular endothelial cells grown in tissue culture, and freshly isolated neutrophils were used to determine direct effects of endotoxin on cyclooxygenase and lipoxygenase products . Endothelial cells (10(7)/ml) or neutrophils (2 X 10(6)/ml) were incubated with (a) buffer, (b) endotoxin (10 micrograms/ml), (c) endotoxin + flunixin meglumine (10 micrograms/ml), or (d) calcium ionophore, A23187 (10 micrograms/ml) . Thromboxane (TxB2), prostacyclin (6-keto-PGF1 alpha), and leukotriene C4 (LTC4) were determined in the incubation fluid by radioimmunoassay . Thromboxane and prostacyclin levels increased in endothelial cells incubated with endotoxin . Treatment with flunixin meglumine prevented the endotoxin-induced release of these cyclooxygenase products to levels below those observed in control cells . Leukotriene production was increased in endothelial cells incubated with endotoxin plus flunixin meglumine . Endotoxin as well as endotoxin plus flunixin meglumine increased the production of prostacyclin and LTC4 by freshly isolated neutrophils . Cells exposed to endotoxin plus flunixin meglumine produced more LTC4 than cells exposed to endotoxin . The data revealed that endotoxin has a direct effect on arachidonic acid metabolism in endothelial cells and neutrophils . Flunixin meglumine reduced the level of cyclooxygenase products but increased the level of lipoxygenase products . Therefore, the well-established beneficial effects of cyclooxygenase inhibitors during endotoxemia may be improved even more if they are used in conjunction with lipoxygenase inhibitors or a combined cyclooxygenase-lipoxygenase inhibitor.

Circ Shock, 1985, 15(2), 89 - 103
Benoxaprofen attenuation of lethal canine endotoxic shock; Toth PD et al.; Our prior work demonstrated in a canine endotoxic shock model (LD100) that the cyclooxygenase inhibitor ibuprofen given 60 minutes after endotoxin administration could improve hemodynamics but not survival over control animals . The present study was designed to examine the effect of benoxaprofen, a dual lipoxygenase and cyclooxygenase inhibitor, in the same canine endotoxic model (LD100) and compare it to ibuprofen treatment . After thiopental anesthesia (25 mg/kg IV), animals were instrumented to measure various cardiovascular parameters . Endotoxic shock was induced by injecting Escherichia coli (0111:B4) endotoxin (1 mg/kg IV) . Benoxaprofen (10 mg/kg IV; N = 13), ibuprofen (12.5 mg/kg; N = 6), or saline (N = 12) was injected 60 minutes after endotoxin administration . During the treatment period, both benoxaprofen and ibuprofen increased mean arterial pressure, heart rate, and vascular resistance to the same degree over the control animals . Benoxaprofen did increase dP/dtmax while ibuprofen did not . Twenty-four-hour survival was 0% for the control animals (N = 12), 0% for the ibuprofen group (N = 6), and 61.5% for the benoxaprofen group (N = 13) . In an additional set of experiments, benoxaprofen (N = 8) was given 120 minutes after endotoxin administration and demonstrated similar improvements in hemodynamics and survival . These data demonstrate that benoxaprofen could improve survival in an otherwise lethal endotoxic model and suggest that the products of the lipoxygenase pathway may contribute to the lethality of an LD100 endotoxic shock model.

Circ Shock, 1985, 15(1), 49 - 59
Dramatic changes in blood gases that are unrelated to arterial pH or cerebral oxygen delivery during endotoxin shock in conscious rats; Law WR et al.; In preliminary studies we demonstrated an effect of endotoxin on arterial blood gases that appeared to be related to the dose of endotoxin used and unrelated to changes in arterial pH . In the present study we tested the hypothesis that these changes in blood gases result from decreased oxygen delivery to central respiratory control areas . PO2 significantly rose from a pre-endotoxin value of 91.4 +/- 1.5 (mean +/- SEM) to 97.5 +/- 1.7, 104.0 +/- 0.8, and 108.4 +/- 0.9 at 10, 30, and 60 minutes, respectively, after administration of 6 mg/kg endotoxin and from 93.8 +/- 3.1 to 105.2 +/- 2.6, 118.7 +/- 1.4, and 121.0 +/- 2.8, respectively, after administration of 10 mg/kg endotoxin . PCO2 fell significantly from a pre-endotoxin value of 38.3 +/- 1.2 to 28.6 +/- 0.6 and 24.5 +/- 1.9 at 30 and 60 minutes post-endotoxin, respectively, in 6-mg/kg-treated rats, and from 40.5 +/- 2.1 to 30.7 +/- 4.5, 20.1 +/- 3.9, and 19.3 +/- 1.0, respectively, at 10, 30, and 60 minutes post-10 mg/kg endotoxin . The only significant change (decrease) in pH occurred at 60 minutes after 10 mg/kg treatment . In 10-mg/kg-treated rats, serum lactate rose significantly over time, while HCO-3 decreased . Heart rate was increased significantly (472 +/- 9.6) from a pre-10 mg/kg endotoxin value of 373 +/- 11.8 by 10 minutes post-endotoxin and remained elevated throughout the experiment . Cerebral medullary/pontine blood flow, mean arterial blood pressure, and respiratory rate were not significantly altered by endotoxin administration . Hemoglobin concentration and arterial oxygen content were significantly increased after 10 mg/kg endotoxin . These findings indicate that decreased oxygen delivery to central respiratory control areas is not a cause of the observed dramatic changes in blood gases.

Am J Vet Res, 1985 Jan, 46(1), 175 - 80
Effect of Escherichia coli endotoxin and thyrotropin-releasing hormone on prolactin in lactating sows; Smith BB et al.; Primiparous gilts were given subcutaneous injections of saline solution or 8 mg of Escherichia coli endotoxin (055:B5 strain) in saline solution on postpartum days (PPD) 2 and/or 6 and saline solution at the same site on PPD 1, 3, 5, and 7 at 1000 hours . On PPD 1 to 3 and on PPD 5 to 7, pigs were given 100 micrograms of thyrotropin-releasing hormone (TRH) IV at 1300 hours to evaluate TRH-induced prolactin (PRL) release . Blood samples were analyzed for PRL, cortisol, triiodothyronine (T3), and tetraiodothyronine (T4) concentrations . Rectal temperatures were monitored at hourly intervals between 0800 and 1500 hours on PPD 2 and 6 . The PRL declined after endotoxin administration on PPD 2, but a similar decline was not seen after saline solution administration on PPD 1, 2, or 3 . The PRL concentrations remained unchanged on PPD 5, 6, and 7 in gilts exposed to endotoxin for the 1st or 2nd time on PPD 6 and to saline solution on PPD 5 and 7 . The TRH injection caused increases in PRL in all animals, but the PRL increase after TRH injection was significantly lower (P less than 0.05) in gilts treated with endotoxin on PPD 2 . Cortisol concentrations increased after endotoxin exposure on PPD 2 and 6 . Rectal temperatures increased after endotoxin exposure on PPD 2 and 6 with peak temperatures of 41.8 C and 41.6 C seen 4 and 3 hours, respectively, after endotoxin injection . The T3 and T4 response, used as an indicator of TRH perfusion of the adenohypophysis, was unchanged after endotoxin or saline solution administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 272 - 6
Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes; Maller JL et al.; Phosphorylation of ribosomal protein S6 in NIH 3T3 fibroblasts is dependent on the presence of serum, but after transformation of these cells by Abelson murine leukemia virus (Ab-MuLV), S6 remained highly phosphorylated on serine residues either in the absence or the presence of serum . To investigate whether S6 phosphorylation in this system was a consequence of the action of the Ab-MuLV tyrosine-specific protein kinase, purified Ab-MuLV kinase made in Escherichia coli was microinjected into Xenopus oocytes and was observed to cause a 7- to 15-fold increase in the phosphorylation of S6 on serine residues . Two-dimensional phosphopeptide maps of S6 phosphorylated in Ab-MuLV-transformed NIH cells in the absence of serum were identical to those of S6 isolated from normal cells grown in the presence of serum . In addition, S6 from oocytes injected with Ab-MuLV kinase yielded an S6 phosphopeptide map indistinguishable from that of serum-stimulated NIH 3T3 cells, whereas S6 from control oocytes lacked several phosphopeptides . Ab-MuLV kinase did not phosphorylate S6 directly in vitro, and microinjection of a mutant Ab-MuLV protein lacking kinase activity had no effect . These results indicate that the Ab-MuLV kinase interacts with a cellular pathway to enhance S6 phosphorylation by directly or indirectly activating an S6 protein kinase and/or inactivating an S6 protein phosphatase.

J Bacteriol, 1985 Jan, 161(1), 461 - 2
Evidence for an internal promoter in the Escherichia coli threonine operon; Saint Girons I et al.; We constructed plasmids carrying the two first genes of the threonine operon from which the major promoter was deleted in vitro by digestion with BAL 31 nuclease . These plasmids continued to express the second gene (thrB) of the operon as judged by their ability to complement a threonine auxotroph . These data indicate that, in addition to the major promoter thrP, there was an internal promoter, thrBp, which could be used for the transcription of the thrB, the second gene of the operon . Additional evidence was given by subcloning a 230-base-pair segment of the operon in a plasmid suitable for detection of translation initiation signals and promoters . The thrBp promoter was thus shown to lie within a 61-base-pair fragment at the 3' end of the first gene, thrA, of the threonine operon.

J Bacteriol, 1985 Jan, 161(1), 450 - 3
Mapping of a mutation affecting regulation of iron uptake systems in Escherichia coli K-12; Bagg A et al.; The site of a mutation resulting in constitutive derepression of iron uptake systems has been localized at 15.7 min on the genetic map of Escherichia coli K-12.

J Bacteriol, 1985 Jan, 161(1), 272 - 6
A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity; Newman EB et al.; Mutants of Escherichia coli K-12 deficient in L-serine deaminase (L-SD) activity have been isolated . These strains required thiamine and grew normally when it was provided . The decrease in L-SD activity caused no obvious metabolic deficiency . A study of revertants and transductants showed that a single mutation was responsible for the thiamine requirement and for the decrease in L-SD activity.

J Bacteriol, 1985 Jan, 161(1), 147 - 52
Posttranscriptional regulation of the inducible nonenzymatic chloramphenicol resistance determinant of IncP plasmid R26; Dorman CJ et al.; The inducible nonenzymatic chloramphenicol resistance (Cmr) determinant of the IncP plasmid R26 was cloned on a 1,900-base-pair restriction endonuclease HindIII fragment . Transposon Tn5 mutagenesis revealed that at least 1,400 base pairs is required for expression of Cmr . There was no increase in the level of Cmr when the copy number of the determinant was raised by cloning in pBR322 or pUB5572 . Expression of Cmr by cells carrying a lower-copy-number pUB5572cml+ plasmid was inducible and thus indistinguishable from those with R26 itself . However, pBR322cml+-carrying cells expressed Cmr constitutively, possibly due to the activity of vector promoters or an elevated copy number . Transcriptional and translational cml-lac fusions were constructed . The operon (transcriptional) cml-lac fusion carried by the low-copy-number plasmid pUB5572 caused a low level of constitutive beta-galactosidase activity, which could not be elevated by induction with chloramphenicol and was not affected by a coresident R26cml+ element . In contrast, the gene (translational) cml-lac fusion expressed low-level beta-galactosidase activity, which was elevated fivefold by prior exposure to chloramphenicol . We conclude that the regulation of Cmr occurs posttranscriptionally.

J Bacteriol, 1985 Jan, 161(1), 128 - 32
Internal promoter in the ilvGEDA transcription unit of Escherichia coli K-12; Calhoun DH et al.; Segments of the ilvGEDA transcription unit have been cloned into the promoter tester plasmid pMC81 . This vector contains cloning sites situated upstream of the lacZ gene coding for beta-galactosidase . Using this method we have quantitatively evaluated in vivo (i) the activity of previously described promoter, pG, preceding ilvG; (ii) the relative activity of pE promoter, previously postulated to be located between ilvG and ilvE; and (iii) the effect of the frameshift site present in the wild-type ilvG gene by comparison with mutant derivatives lacking this frameshift site . Isogenic derivatives of strain MC1000 were constructed by transduction with phage P1 grown on rho-120, delta(ilvGEDA), delta(ilvED), and ilvA538 hosts . The potential effects of these alleles that were previously postulated to affect ilvGEDA expression were assessed in vivo by monitoring beta-galactosidase production directed by ilv DNA fragments . Cloned ilv segments were also tested for activity in vitro with a DNA-directed coupled transcription and translation system . The production in vitro of ilv-directed ilv gene expression and beta-galactosidase expression with ara-ilv-lac fusions paralleled the in vivo activity.

J Bacteriol, 1985 Jan, 161(1), 105 - 12
Aberrant regulation of methylesterase activity in cheD chemotaxis mutants of Escherichia coli; Kehry MR et al.; The adaptation process in several cheD chemotaxis mutants, which carry defects in tsr, the serine transducer gene, was examined . cheD mutants are smooth swimming and generally nonchemotactic; the defect is dominant to the wild-type tsr gene (J . S . Parkinson, J . Bacteriol . 142:953-961, 1980) . All classes of methyl-accepting chemotaxis proteins synthesized in unstimulated cheD strains are overmethylated relative to the wild type . We found that the steady-state rate of demethylation in cheD mutants was low; this may explain their overmethylated phenotype . In addition, all cheD mutants showed diminished responsiveness of methylesterase activity to attractant and repellent stimuli transduced by either the Tsr or Tar protein, and they did not adapt . These results suggest that the dominant nature of the cheD mutations is manifested as a general defect in the regulation of demethylation . Some of these altered properties of methylesterase activity in cheD mutants were exhibited in wild-type cells that were treated with saturating concentrations of serine . The mutant Tsr protein thus seems to be locked into a signaling mode that suppresses tumbling and inhibits methylesterase activity in a global fashion . We found that the Tar and mutant Tsr proteins synthesized in cheD strains were methylated and deamidated at the correct sites and that the mutations were not located in the methylated peptides . Thus, the signaling properties of the transducers may be controlled at sites distinct from the methyl-accepting sites.

Am J Clin Nutr, 1985 Jan, 41(1), 61 - 5
Hyperalimentation-associated jaundice: an example of a serum factor inducing cholestasis in rats; Latham PS et al.; A patient receiving total parenteral nutrition (TPN) at home following emergency resection of the small intestine was studied over a two year interval . Cholestatic jaundice developed after 6 months . A factor in serum was found to produce cholestatic changes in the bile flow of rats on intravenous infusion . Normal human serum and saline infusion did not produce this cholestasis . Endotoxin infusion in the rat produced a similar impairment in bile flow . The hypothesis was proposed that endotoxin might be an occult factor contributing to cholestasis in this case . An antiserum prepared to an endotoxin isolated from a sequestered E . coli infection in this patient, ameliorated the cholestatic effects of the patients' serum in rats . The possible role of endotoxin in the cholestasis of the TPN-induced jaundice in this patient is presented and discussed.

J Enzyme Inhib, 1985, 1(1), 67 - 75
Inhibition of adenosine deaminase from several sources by deaza derivatives of adenosine and EHNA; Lupidi G et al.; Deaza analogues of adenosine and EHNA were tested as inhibitors of the enzyme adenosine deaminase (ADA) obtained from several sources including human erythrocytes, calf intestine, Saccaromices cerevisiae, Escherichia coli and Takadiastase . Ki values of the inhibitors suggest differences among the enzymes both at purine and erythro-nonyl binding site . Among the ribofuranosyl derivatives, 1-deazaadenosine is the best inhibitor, its Ki ranging between 3.5 x 10(-7) and 4 x 10(-5) M for ADA from erythrocytes and Takadiastase respectively . Only ADA from erythrocytes and calf intestine bind EHNA and some of deazaEHNA analogues; 3-deazaEHNA behaves very similarly to EHNA both in affinity and slow binding mechanism, whereas 1-deazaEHNA, though less potent, is a good inhibitor.

Free Radic Res Commun, 1985, 1(2), 79 - 88
Transition metals potentiate paraquat toxicity; Kohen R et al.; The involvement of transition metal ions in paraquat toxicity was studied in bacterial model system . We show that the addition of micromolar, or lower, concentrations of copper dramatically enhanced the rate of bacterial inactivation . In contrast, the addition of chelating agents totally eliminated the killing of E . coli . No inactivation was observed under anaerobic exposure to paraquat, both in the absence and presence of copper . However, in the presence of copper, the anaerobic addition of hydrogen peroxide resulted in complete restoration of inactivation as under aerobiosis . Paraquat either produces superoxide ions or directly reduces bound copper ions in a catalytic mode . The reduced cuprous complexes react with hydrogen peroxide to locally form hydroxyl radicals (OH.) which are probably responsible for the deleterious effects . This study indicates the involvement of a site-specific metal-mediated Haber-Weiss mechanism in paraquat toxicity . It is in agreement with earlier observations that copper unusually enhance biological damage induced by either superoxide or ascorbate.

Curr Genet, 1985, 9(8), 653 - 60
Efficient expression of the Escherichia coli leuB gene in yeast; McNeil JB et al.; Efficient expression of the Escherichia coli leuB (beta-isopropylmalate dehydrogenase) gene occurred in yeast after in vitro DNase digestion and religation of plasmid bound leuB and the yeast HIS3 DNA which placed the 5' end of the yeast HIS3 gene immediately adjacent to the coding region of the E . coli leuB gene . Two structurally distinct classes of gene fusions were constructed, each involved portions of the yeast HIS3 gene which contributed DNA sequences responsible for leuB expression in yeast . The first class involved fusion of the HIS3 coding region to bacterial DNA resulting in the production of a fusion protein with beta-isopropylmalate dehydrogenase activity . The second class consisted of bacterial DNA, including the leuB coding region, fused to the HIS3 promotor region with the absence of any portion of the HIS3 coding region . In both constructions the HIS3 promotor region is required for transcription, however, translation of the class two fusion is initiated at a bacterial DNA coded AUG, and the 5' end of the mRNA coded by the leuB gene mapped predominantly at bacterial DNA sequences . The DNA sequence responsible for the 5' end of the HIS3 mRNAs remain in the class two gene fusions but this did not preclude the initiation of transcription at bacterial DNA sequences . The pattern of mRNA initiation at bacterial DNA suggests that DNA sequences at, or adjacent to, the site of transcription initiation are involved in the determination of the sites of initiation, and perhaps the frequency at which initiation occurs.

Curr Genet, 1985, 9(4), 305 - 11
Cloning and characterization of the three enzyme structural genes QUTB, QUTC and QUTE from the quinic acid utilization gene cluster in Aspergillus nidulans; Hawkins AR et al.; Heterologous DNA probes from the quinic acid gene cluster (QA) in Neurospora crassa (Schweizer 1981) have been used to isolate the corresponding gene cluster (QUT) from Aspergillus nidulans cloned in a phage lambda vector . N . crassa probes for each of the three enzyme structural genes in the cluster have been used to identify the corresponding genes within the A . nidulans cloned DNA . The three genes are in the same relative sequence {dehydrogenase (1), QA-3 = QUTB; dehydratase (3), QA-4 = QUTC; dehydroquinase (2), QA-2 = QUTE} though contained within a 3.4 kb DNA sequence in Aspergillus compared to a 5.4 kb sequence in Neurospora . The A . nidulans dehydroquinase (2) gene QUTE has been shown to complement an auxotrophic mutant aroD6 of Escherichia coli lacking biosynthetic dehydroquinase when tested for growth at 30 degrees C . A mutant of A . nidulans lacking catabolic dehydroquinase (2) and designated qutE208 has been isolated and shown to be tightly linked to the gene cluster, which maps between the ornB and fwA loci in linkage group VIII.

Mol Biol Evol, 1985 Jan, 2(1), 1 - 12
The tetracycline repressor of pSC101; Brow MA et al.; We have determined the nucleotide sequence of the gene for the repressor of the pSC101 tetracycline resistance element (tetR) . The repressor gene is transcribed divergently from the gene that encodes the resistance protein and encodes a putative protein of 219 amino acids . The genetic organizations of the three major types of bacterial tetracycline resistance elements thus appear to be equivalent, even though they do not show substantial nucleic acid similarity . The pSC101 repressor protein is 80% identical with the Tn 1721 repressor over its N-terminal 150 residues, whereas the C-termini of the two species are only 35% identical . Examination of the nucleic acid sequences of the regions between the two divergent promoters suggests a model in which two dimers of the tetracycline repressor molecule interact at two adjacent dyad repeats . The dimers may interact with each other, thus strengthening their grip on the operator, and affect transcription of the repressor gene . Comparison of the tetracycline (Tet) repressor with the lambda repressor suggests that the N-terminal region of the Tet repressor forms a helix-turn-helix structure and interacts with DNA in the major groove . The region of the Tet repressor implicated in DNA binding shows significant sequence similarity to a region of histone H4, suggesting that the histone may bind to DNA by means of a similar structural motif.

Pharmacol Ther, 1985, 31(1-2), 141 - 51
Quantitation of DNA repair capacities of human tumor cells by estimation of transfer of DNA adducts to repair proteins; Yarosh DB; The repair of O6-methylguanine produced in DNA by alkylating agents is accomplished by a unique lesion reversal mechanism which recognizes the methyl group and transfers it to itself in a suicide reaction . Much of what we know about the importance of O6-methylguanine-DNA methyltransferase repair in human cells comes from the study of Mer- tumor cell strains which are deficient in transferase activity . The human transferase has a preference for repair of methyl groups, but will also act on other substrates . Assays for transferase activity detect either the loss of O6-methylguanine from DNA or the appearance of methylated protein . A new assay detects the recovery of a restriction site in a synthetic polymer following demethylation . Inhibition of transferase activity can be produced in cells by several methods.

Drugs Exp Clin Res, 1985, 11(11), 747 - 54
Dissociated effect of amphotericin B and desoxycholate on phagocytosis of Escherichia coli by human polymorphonuclear neutrophils; Lingaas E et al.; The influence of commercial amphotericin B-desoxycholate (Fungizone) and desoxycholate alone on phagocytosis of Escherichia coli by human polymorphonuclear neutrophils in vitro was studied . A dissociated effect of amphotericin B and desoxycholate was observed . Amphotericin B was shown to stimulate the ingestion of Escherichia coli through an effect on the bacteria as well as on the neutrophils . Desoxycholate inhibited phagocytosis through an effect on the neutrophils and also, at low concentrations (0.08 microgram/ml), through an effect on the bacteria . On the other hand, pretreatment of Escherichia coli with high concentrations (8.2 micrograms/ml) of desoxycholate rendered it more susceptible to phagocytosis . The elimination of bacterial breakdown products from the neutrophils after ingestion of Escherichia coli was also inhibited by desoxycholate and stimulated by amphotericin B . Most of these effects disappeared in the presence of 10% serum . An alteration of bacterial hydrophobicity could only partly explain the effect of amphotericin B.

Bull Soc Pathol Exot Filiales, 1985, 78(5 Pt 2), 921 - 9
{Escherichia coli diarrhea of children and adults at the Brazzaville General Hospital}; Yala F et al.; The high number of the strains of Escherichia coli (E . coli) isolated from faeces, demonstrated that this bacteria takes the first place in diarrhoea . These isolations had the same frequency all the year long . In infants, the enteropathogenic E . coli (E . P . E . C.) (28.57%) are still frequent contrary to developed countries . The E . coli with hemagglutination phenomenon (colonisation factors) but without toxin detected were also responsible (mortality 6%) . The E . coli without toxin and not belonging to E . P . E . C . group were also frequent (71.42%) . In adults, only the enteroinvasive E . coli (E . I . E . C.) (16.6%) and the E . coli with hemagglutination but without toxin detected (16.6%) were observed . This situation is due to bad hygienic conditions among the people and the tropical climate.

Physiol Chem Phys Med NMR, 1985, 17(4), 347 - 50
Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli; Tokushige M et al.; NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics . The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol . The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent . In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.

J Cell Sci Suppl, 1985, 3, 11 - 7
In vivo aspects of urogastrone-epidermal growth factor; Gregory H; Recent evidence indicates that human urogastrone-epidermal growth factor originates in submandibular and Brunner's glands and that serum levels are low (less than 1 ng ml-1) . It occurs in many secreted fluids to a much greater extent and many tissues are thus exposed to concentrations greater than 100 ng ml-1 . Rapid actions in vivo of URO-EGF include the ability to inhibit gastric acid secretion at low doses (250 ng kg h-1 in humans) and to provide cytoprotective effects against ulcerogenic agents (250 ng kg h-1 in cats) . More prolonged exposure of tissues shows increases in parameters related to wound healing, beneficial effects upon ulceration and also the ability to accelerate crypt cell production rate along the gastrointestinal tract . Synthetic material has been prepared with an identical structure to the natural URO-EGF thus enabling detailed studies of the biological effects to be pursued.

Aust J Biol Sci, 1985, 38(4), 383 - 92
Studies on the accumulation of putrescine and spermidine in Escherichia coli; Smigielski AJ et al.; The rate of accumulation of the polyamines spermidine and putrescine by E . coli depended on growth rate . Spermidine accumulation was faster in chemostat cultures with high dilution rates than in those with low dilution rates and was slower in bacteria that had been grown for several generations with either putrescine or spermidine, suggesting that the spermidine-uptake system was repressed by exogenous polyamines . The uptake of spermidine required metabolic energy . Thus accumulation occurred in an energy-starved unc strain only upon addition of glucose (or D-lactate to a smaller extent) . With glucose present accumulation occurred in an unc, frd strain under anaerobic conditions, suggesting that ATP drives uptake . However, accumulation was generally sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that the proton motive force was involved in uptake . Unlike spermidine, putrescine accumulation was faster in slow-growing than in fast-growing cultures . This may have been due to greater efflux of putrescine at faster growth rates . Accumulation of putrescine was faster following prolonged growth with either putrescine or spermidine, suggesting induction of the putrescine-uptake system by exogenous polyamines . Like spermidine accumulation, putrescine accumulation required metabolic energy . Accumulation was insensitive to CCCP and occurred only when glucose was added to energy-starved unc bacteria, suggesting that high-energy bonds may drive the uptake of putrescine.

Adv Biophys, 1985, 20, 109 - 35
Ions as regulators of protein-nucleic acid interactions in vitro and in vivo; Record MT Jr et al.; The key feature of the kinetics and equilibria of both specific and non-specific noncovalent interactions of proteins with nucleic acids is their sensitivity to the details of the ionic environment . Investigation of the effects of ion concentrations provides detailed and otherwise unobtainable information about the thermodynamics and mechanisms of these interactions . We discuss the molecular and thermodynamic basis of the contribution to these ion effects from electrolyte-nucleic acid interactions, and demonstrate that a simple ion exchange formalism, involving the stoichiometric participation of individual ions, is the appropriate basis for interpreting these profound effects at a thermodynamic level . Since the in vivo ionic environment is both complex and variable, we propose that variations in intracellular concentrations of individual ions play both global and specific roles in the control of the protein-nucleic acid interactions responsible for nucleoprotein structure and gene expression.

Arch Immunol Ther Exp (Warsz), 1985, 33(6), 755 - 61
Migration of isolated rabbit lymphocytes in the course of pharmacological inhibition of pyrogenic fever; Debowy J et al.; The studies concerned in vitro migration of peripheral blood lymphocytes of rabbits given intravenously one dose of E . coli lipopolysaccharide (LPS) and antipyretic doses of acetylsalicylic acid (ASA), indomethacin (IND) or mefenamic acid (MEFA) . In normothermic animals, ASA appeared to inhibit in vitro the spontaneous migration of lymphocytes . Contrary to ASA, MEFA and IND did not produce any significant changes in lymphocytes migration . Four hours after pyrogen injection migration of lymphocytes was slightly enhanced and after 24 h it was significantly inhibited . All non-steroid anti-inflammatory drugs examined counteracted this effect.

Arch Immunol Ther Exp (Warsz), 1985, 33(6), 727 - 34
Chlormethine in small doses as immunostimulator--LPS synergism; Garbulinski T et al.; Normothermic rabbits and rabbits with LPS induced fever were treated with chlormethine (Nitrogranulogen, Ntg) in the doses of 1 microgram/kg and 10 micrograms/kg . The blood was collected 4, 24, 48 hrs and 4, 7, 10 days after Ntg injection . Following indices of immunity were studied: T and B cells number, number of IgM producing cells after immunization with SRBC, serum IgG level, killing activity of neutrophils and number of phagocytized bacteria . It was observed that both doses of Ntg injected intravenously to normothermic rabbits, significantly increased the number of T and B lymphocytes and of IgM producing lymphocytes a well as the level of IgG in the serum, number of phagocytized bacteria and killing activity of neutrophils . Ntg in combination with LPS shortened the period of fever, and through the synergistic effect, significantly increased T lymphocytes number in the blood, IgG level in the serum, number of phagocytized bacteria and killing activity of neutrophils.

Trans R Soc Trop Med Hyg, 1985, 79(6), 840 - 2
Recovery of potential pathogens from feeding bottle contents and teats in Zaria, Nigeria; Cherian A et al.; Pathogens were recovered from the contents of 41 out of 50 feeding bottles and from 32 teats in a survey in Zaria, Nigeria . 39 bottle contents and 30 teats yielded enteric pathogens . Traditional weaning gruels were more contaminated than were commercial feeds . Koko bottles yielded more pathogens than Akamu bottles . Enteropathogenic Escherichia coli was the most common pathogen recovered, seen more in bottle contents than teats . Bottle hygiene was poor and cleaning methods and feeding practices were not satisfactory . Lack of facilities in the home prevented better hygiene . Prolonged pre-cooking preparation, storage of gruels in bulk for the whole day in thermos flasks or enamel bowls, and inadequate hygiene in the preparation of commercial feeds resulted in the large recovery of pathogens . Alternate feeding methods were suggested and the need for the practice of good hygiene by all who are involved in the preparation of infant feeds was emphasized.

Microbios, 1985, 44(177), 7 - 20
Effect of anaesthetics and dichlorodifluoromethane on the viability of the cells of Escherichia coli and the activities of some of its enzymes; Laverty DM et al.; Three anaesthetics (halothane, CF3CHClBr; Ethrane, F2 HCOF2CCHClF; cyclopropane) and one other halogenated, short-chain hydrocarbon (F-12, Cl2F2C) were tested under various conditions to determine their effects on the viability of cells of Escherichia coli and the activities of some of its enzymes . When any of the test chemicals were applied for 60 min at concentrations slightly in excess of saturation, the number of surviving cells decreased substantially, with halothane being the most biocidal of the four chemicals and F-12 the least . Three enzymes (malate dehydrogenase, MD; NADH dehydrogenase; glyceraldehyde-3-phosphate dehydrogenase, GPD) were tested for activity after treatment of E . coli with the test chemicals . In all instances, GPD was least resistant to inactivation and MD was most resistant . Halothane was most inhibitory followed in order by Ethrane, cyclopropane and F-12 . Treatment of E . coli with halothane for 60 min at 23 degrees C and a concentration slightly in excess of saturation, resulted in nearly complete inhibition of all three enzymes.

Microbios, 1985, 44(177), 45 - 9
Kinetics of dark repair inhibition in ultraviolet damaged DNA by substituted quinolines; Sideropoulos AS et al.; The effects of chloroquine on the kinetics of the dark repair process were investigated by employing a cellular system . Lineweaver-Burke plots and Dixon plots of chloroquine inhibition, respectively, showed that there was an increase in slope with increasing concentration of inhibitor which followed an enzyme-like pattern . These results are consistent with a model for excision-repair in which chloroquine may block the excision repair pathway.

Gene, 1985, 40(2-3), 191 - 201
Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene; Brawner ME et al.; A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences . The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E . coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals . Expression of the E . coli galK gene in S . lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S . lividans galK- mutant . The utility of the plasmid to select, detect and assess promoter function was examined using the S . lividans XP55 and S . fradiae aph gene promoters . The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.

J Emerg Med, 1985, 3(1), 1 - 9
Acute infectious diarrheal disease in children; Barkin RM; The management of acute diarrheal disease in children must consider potential etiologic agents and their common presentation . The workup and assessment should be tailored to the clinical condition of the patient and the most likely pathogen . Management must primarily focus on fluid therapy and dietary manipulation . Antibiotics have a very restricted role as do antidiarrheal agents.

Acta Chir Scand Suppl, 1985, 526, 94 - 103
High-dose methylprednisolone in a porcine model of ARDS induced by endotoxemia; Modig J et al.; Using a continuous i.v . infusion of E . coli endotoxin in spontaneously breathing pigs under ketamine anesthesia we have developed a lung injury model which closely mimics the pathophysiological and morphological changes of early ARDS induced by sepsis in man . Pretreatment with high doses of methylprednisolone largely prevented the pulmonary, cardiovascular and morphological features induced by the endotoxin . Methylprednisolone treatment initiated 2 h after starting the endotoxin abolished further derangements in pulmonary and cardiovascular functions and there was a restoration towards normal values . Both pretreatment and delayed treatment with methylprednisolone improved survival . Although one should be extremely cautious in extrapolating these data to the more complex clinical situation, the implications are that high doses of methylprednisolone, given early in the course of sepsis in man, may help to prevent both the pulmonary and cardiovascular derangements of this disease.

Acta Chir Scand Suppl, 1985, 526, 124 - 8
High doses of corticosteroids in the treatment of septic shock; Hellman A et al.; High doses of corticosteroids are reported to be beneficial in the treatment of septic shock in many animal species, e.g . dog, rat and rabbit . Recent findings in baboons subjected to E . coli shock indicate that early treatment with a combination of antibiotics and steroids strongly enhance survival rate . In clinical studies the protective effects of steroids are more ambiguous, however . In part this may be explained by variations in the amount of steroids used or by the fact that in some studies the steroid is administered late in shock . The dose recommended, 30 mg/kg bw of methylprednisolone or an equivalent amount of another glucocorticoid given once or twice, is based on animal as well as clinical documentation.

Acta Chir Scand, 1985, 151(6), 501 - 8
Positive effects of prophylactic ventilator treatment on gas exchange and extravascular lung water in a porcine model of adult respiratory distress syndrome induced by endotoxaemia; Borg T et al.; The influence of prophylactic ventilator treatment was evaluated in a porcine model of early adult respiratory distress syndrome (ARDS) induced by endotoxaemia . Sixteen animals, controls, under continuous i.v . ketamine anaesthesia were either mechanically ventilated using intermittent positive pressure ventilation (IPPV; n = 6) with air or breathed air spontaneously (n = 10) . Twenty animals under continuous i.v . ketamine anaesthesia and spontaneously breathing air were infused i.v . with E . coli endotoxin (10 micrograms X kg-1 X h-1) over 6 h . Fifteen animals under continuous i.v . ketamine anaesthesia were given IPPV with air and were infused i.v . with E . coli endotoxin in the same dosage regimen . In the controls, cardiac output decreased slightly . Otherwise there were no changes in pulmonary gas exchange, pulmonary haemodynamics or extravascular lung water . In spontaneously breathing and IPPV animals given endotoxin there was a profound deterioration in pulmonary gas exchange, a marked rise in pulmonary vascular resistance and a moderate increase in extravascular lung water . Animals given IPPV showed a significantly less pronounced impairment in pulmonary gas exchange and a significantly smaller increase in extravascular lung water than in animals breathing spontaneously, whereas the changes in pulmonary haemodynamics were fairly similar in both groups . Animals with IPPV also had an improved survival rate . The beneficial effects of mechanical ventilation on pulmonary gas exchange are not due to changes in extravascular lung water, but are caused by its influence in counteracting terminal airway and alveolar closure . These results indicate that mechanical ventilation, when instituted early in the course of human ARDS induced by septicaemia, might be of potential value in the prevention of severe pulmonary failure and death.

Ultrastruct Pathol, 1985, 9(3-4), 215 - 24
Electron microscopic analysis of lymphocyte nuclei in non-Hodgkin's lymphoma; Dardick I; Ultrastructural studies of normal and neoplastic lymphocytes are presented that qualitatively and quantitatively assess the central cell organelle currently used by surgical pathologists in the classification of non-Hodgkin's lymphoma, the nucleus . Events occurring during normal lymphocyte transformation can be used to appreciate essential mechanisms involved in producing the appearance of the nucleus as seen by microscopy . Quantitation of nuclear subcompartments by morphometric image analysis reveals that determination of nuclear size is primarily due to the ribonucleoprotein materials distributed between condensed chromatin masses, the interchromatinic (euchromatin or nuclear matrix) region . Furthermore, such investigations show that amounts and distribution of condensed chromatin in lymphocyte nuclei cannot be adequately assessed from routine histologic sections . Ultrastructural morphometric analysis of representative cases of the principal subtypes of NHL indicates that the atypical morphologic appearance of neoplastic lymphocytes results from a complex interplay between total amounts of condensed chromatin in nuclei and the size of individual aggregates of condensed chromatin, one or both of which may be abnormal in NHL . Abnormalities of interchromatinic materials are also likely involved in ordering the gross appearance of the nucleus . Understanding of both the dynamic capabilities of the nucleus, and the organization of and interplay between the various subcompartments of this organelle will be helpful in improving the classification of NHL by surgical pathologists.

Nucleic Acids Symp Ser, 1985, (16), 261 - 3
RNA polymerase: direct evidence for two active sites involved in transcription; Dennis D; Photoaffinity reagents (3'-azido analogues of 5' ATP and 5' GTP) were used to label two different sites at the catalytic surface of the beta subunit of the E . coli DNA dependent RNA polymerase . These sites are used alternately and successively during consecutive phosphodiester bond formation . This result is consistent with our model of rotational translocation for transcription.

Nucleic Acids Symp Ser, 1985, (16), 237 - 9
Ambiguous incorporation of N4-aminodeoxycytidine 5'-triphosphate to DNA synthesized in vitro; Negishi K et al.; Molecular mechanism of the mutation induced by N4-aminocytidine was studied . The specificity of in vitro incorporation of N4-aminodeoxycytidine 5'-triphophate catalyzed by E . coli DNA polymerase large fragment was analyzed . The results have shown that this cytosine analog can be efficiently incorporated as a substitute of cytosine, and that it can also be incorporated with a low efficiency as a substitute of thymine . We have also shown that the N4-aminocytosine incorporated opposite adenine can be excised as its monophosphate at a high frequency . The N4-aminocytosine residues in the polynucleotide templates can be read by the enzyme as efficiently as cytosines, and guanines were incorporated opposite them.

Mol Gen Genet, 1985, 201(3), 537 - 42
The sfiA11 mutation prevents filamentation in a response to cell wall damage only in a recA+ genetic background; Cegielska A et al.; N-alpha-palmitoyl-L-lysyl-L-lysine dihydrochloride ethyl ester (PLL) at sublethal doses causes filamentous growth of E . coli strains except sfiA mutants, which divide normally in its presence . PLL does not elicit the SOS responses as judged by lambda prophage induction, an increase of RecA protein synthesis or induction of the sfiA operon in a sfiA::lacZ fusion strain . Thus, it appears that filamentation caused by PLL is not an SOS function and might be the result of membrane damage by PLL, which is an amphipathic compound and at higher doses causes cell lysis . This indicates that basal levels of the sfiA gene product are sufficient to inhibit cell division in the presence of PLL . We have found further that the phenotype of the sfiA mutation in the presence of PLL requires a recA+ genetic background and does not occur in E . coli recA1 sfiA11, recA13 sfiA11, recA56 sfiA11 and recA441 sfiA11 . All these strains, but rec441 sfiA11, however, regain the ability of sfiA11 mutants to divide in the presence of PLL after transformation with the RecA overproducing-plasmid pXO2 . This supports the conclusion that the RecA protein positively affects sfiA11-mediated cell division in the presence of the cell membrane damaging compound, PLL . The basal level of the RecA protein in the recA+ sfiA11 strain is sufficient for this process . An increased level due to overproduction from the multicopy plasmid pXO2 exerts the same effect.

Mol Gen Genet, 1985, 201(3), 525 - 8
Role of Escherichia coli RecBC enzyme in SOS induction; Chaudhury AM et al.; Induction of the SOS genes is required for efficient repair of damaged DNA in Escherichia coli . SOS induction by nalidixic acid or oxolinic acid, two inhibitors of DNA gyrase, requires the RecBC enzyme of E . coli . We report here that the nuclease activity of RecBC enzyme is not needed for SOS induction by these agents . We suggest that the unwinding activity of RecBC enzyme produces single-stranded DNA which activates the RecA protein to stimulate LexA repressor cleavage and SOS induction.

Mol Gen Genet, 1985, 201(3), 433 - 6
Gene rpmF for ribosomal protein L32 and gene rimJ for a ribosomal protein acetylating enzyme are located near pyrC (23.4 min) in Escherichia coli; Janda I et al.; An Escherichia coli mutant harbouring altered ribosomal protein L32 has been isolated and genetically characterized . The mutation leading to this alteration (rpmF) and the temperature-sensitive mutation (ts-1517) present in the same strain were found to map near pyrC (23.4 min), being cotransducible not only with pyrC but also with fabD, flaT and purB in P1 phage mediated transductions . Furthermore, we found that the gene rimJ, which encodes an enzyme that acetylates the N-terminal alanine of protein S5 and the temperature-sensitive mutation, ts-386, present in the rimJ mutant strain (Cumberlidge and Isono 1979) also mapped in this region . Thus, the order of genes is deduced to be: ts-386-pyrC-ts-1517-rimJ-flaT-fabD-rpmF-purB+ ++.

Mol Gen Genet, 1985, 201(3), 387 - 92
Effect of a lexA41(Ts) mutation on DNA repair in recA(Def) derivatives of Escherichia coli K-12; Ganesan AK et al.; Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein . This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation . Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if they contained the wild-type lexA gene . Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.

Mol Gen Genet, 1985, 201(3), 379 - 86
Growth rate-dependent regulation of RNA polymerase synthesis in Escherichia coli; Ralling G et al.; The rate of synthesis of the beta and beta' subunits of RNA polymerase relative to the rate of synthesis of total protein was found to remain constant with increasing steady state growth rate . This is in contrast to the relative synthesis rates of ribosomal proteins which are known to increase with growth rate . Yet the ratio of the rate of transcription of the ribosomal protein (rplJL) and RNA polymerase (rpoBC) domains of the rplKAJLrpoBC gene cluster was found to be invariant . Fusions to lacZ were used to relate the rate of transcription of the rplKAJL genes to the rate of synthesis of total protein . No change was seen at growth rates above 0.8 doublings per hour . This indicates that the growth rate-dependent expression of these ribosomal proteins is regulated at the post-transcriptional level . However because both the relative rate of transcription of rpoBC and rate of synthesis of beta and beta' were found to remain invariant over this growth range it suggests the expression of these RNA polymerase subunits is regulated at the transcriptional level.

Mol Gen Genet, 1985, 201(2), 186 - 91
Mechanism of sbcB-suppression of the recBC-deficiency in postreplication repair in UV-irradiated Escherichia coli K-12; Wang TC et al.; The mechanism by which an sbcB mutation suppresses the deficiency in postreplication repair shown by recB recC mutants of Escherichia coli was studied . The presence of an sbcB mutation in uvrA recB recC cells increased their resistance to UV radiation . This enhanced resistance was not due to a suppression of the minor deficiency in the repair of DNA daughter-strand gaps or to an inhibition of the production of DNA double-strand breaks in UV-irradiated uvrA recB recC cells; rather, the presence of an sbcB mutation enabled uvrA recB recC cells to carry out the repair of DNA double-strand breaks . In the uvrA recB recC sbcB background, a mutation at recF produced a huge sensitization to UV radiation, and it rendered cells deficient in the repair of both DNA daughter-strand gaps and DNA double-strand breaks . Thus, an additional sbcB mutation in uvrA recB recC cells restored their ability to perform the repair of DNA double-strand breaks, but the further addition of a recF mutation blocked this repair capacity.

Mol Gen Genet, 1985, 201(2), 146 - 50
A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli; Bejar S et al.; Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome . The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions . Temperature-sensitivity conferred by mutation dicA1 in a recA backround was suppressed by the presence of hybrid plasmids carrying the wild-type gene . In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval . These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.

Mol Gen Genet, 1985, 201(2), 141 - 5
Photoreactivation reverses ultraviolet radiation induced premutagenic lesions leading to frameshift mutations in Escherichia coli; Yamamoto K; The effect of photoreactivation of the ultraviolet radiation induced reversion of a trpE9777 frameshift mutation was studied in a uvr A6 derivative of Escherichia coli K12 . Two different photoreactivation treatments were used, one providing a single flash of photoreactivating light and another providing 10 min of light from fluorescent lamps . The reversion frequency of the trpE9777 frameshift mutation was strongly reduced when subsequently exposed to visible light . The dose modification factor (the ratio of equally effective doses), for cells challenged with single-flash photoreactivation, for survival and induction of reversion to Trp+ was 3.6 and 3.4, respectively . UV induction of RecA protein synthesis was not reversed by a single flash of photoreactivation . The dose modification factor for 10 min of fluorescent lamp photoreactivation for survival and for induction of reversion to Trp+ was 6.5 and 6.3, respectively . The dose modification factor for 10 min of photoreactivation for induction of RecA protein was 1.7-2.5 . Photoreactivation decreased the reversion of trpE9777 and increased survival to the same extent . We concluded that cyclobutyl pyrimidine dimers are the premutagenic lesions of UV mutagenesis of the trpE9777 allele in a uvr A6 background.

Gene, 1985, 39(1), 85 - 7
Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors; Lorenzetti R et al.; Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS . The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1 . These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations . The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Gene, 1985, 39(1), 117 - 20
Expression of a synthetic human growth hormone gene in yeast; Tokunaga T et al.; A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae) . More than 10(6) molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.

Eur J Clin Pharmacol, 1985, 29(2), 225 - 9
High performance liquid chromatographic determination of moxalactam in human plasma and cerebrospinal fluid; Nielsen J et al.; A sensitive and reproducible method for the measurement of moxalactam in plasma and cerebrospinal fluid is described . Plasma proteins were removed by precipitation with ice-cold methanol at pH 5.6 and centrifugation . The supernatant was analysed by HPLC on a mu-Bondapack/phenyl column, with a mobile phase of acetonitrile/water/PIC Reagent A (20/80/1), and detection at 280 nm . The calibration curve was linear for plasma concentrations from 10 micrograms/ml to 60 micrograms/ml . Reproducibility was 4.7% (coefficient of variation) for within-day analysis and 13.8% for day-to-day analysis . Plasma concentrations in 9 moxalactam-treated patients with severe infections ranged from 0.9 micrograms/ml to 409 micrograms/ml . Individual pharmacokinetic parameters were calculated using a personal computer . In selected cases moxalactam concentrations were also determined in cerebrospinal fluid and tracheal aspirates.

Curr Top Cell Regul, 1985, 26, 177 - 90
On conformational changes in the regulatory enzyme aspartate transcarbamoylase; Cohen RE et al.; Our preliminary studies provide clear answers to only some of the basic questions posed above regarding the nature of conformational changes in ATCase . By exploiting the reverse reaction to develop a kinetic active site titration technique we demonstrated that symmetry-related regions of ATCase are affected equally in the allosteric transition even though the bisubstrate analog is bound to only one of the six active sites . This is a vivid illustration of a "global" change in an enzyme, and it provides powerful support for the view that active site ligands promote a concerted transition of the enzyme from the low-activity to the high-activity conformation . Kinetic experiments show that this conformational change is rapid, requiring only tenths of a second . In contrast, the studies with isolated catalytic subunits provide evidence for a much more local conformational change which occurs after the ligand is bound and which is much slower, lasting over a time period of many seconds . Although the experiments on the holoenzyme do not show directly how many atoms are involved in the conformational change or how large are the displacements, they do indicate, especially in conjunction with other studies, that many amino acid residues in both the catalytic and regulatory chains must be implicated in the T----R allosteric transition . Why PALA has such a different local effect on the catalytic subunit than does the combination of carbamoylphosphate and succinate is not clear, but this uncertainty may be resolved by further NMR studies . Determining how the local changes at the active sites are linked to the global transition affecting the quaternary structure of the enzyme remains a formidable problem . Also further work is needed in order to determine whether unliganded ATCase molecules exist in an equilibrium mixture of T and R conformations even prior to the ligand binding event.

Bull Soc Pathol Exot Filiales, 1985, 78(4), 433 - 8
{Regional characteristics of enterotoxigenic Escherichia coli serotypes isolated from children with gastroenteritis in the South Pacific (New Caledonia, Vanuatu Republic, Wallis and Futuna}; Germani Y et al.; One hundred and fifty enterotoxigenic strains of Escherichia coli (ETEC) isolated from infants with acute diarrhoeas in New Caledonia, Vanuatu and Wallis and Futuna were examined for serotypes . Twenty serotypes were identified, 5% of E . coli (7 strains) were rough and 8% (12 strains) were untypable . The serotypes 06, 025, and 078 are enterotoxigenic at 100% . The serotypes 027 and 073 classically enterotoxigenic did not produce any enterotoxin . This study opens up a possibility that the predominant enterotoxigenic serotypes in that region of the South Pacific are probably different from the serotypes proposed in the pool of sera to identify ETEC.

J Cell Biochem, 1985, 29(2), 73 - 82
Site-specific mutagenesis of dihydrofolate reductase from Escherichia coli; Chen JT et al.; Two site-specific mutations of dihydrofolate reductase from Escherichia coli based on the x-ray crystallographic structure were constructed . The first mutation (His-45----Gln) is aimed at assessing the interaction between the imidazole moiety and the pyrophosphate backbone of NADPH . The second (Thr-113----Val) is part of a hydrogen bonding network that contacts the dihydrofolate substrate and may be involved in proton delivery to the N5-C6 imine undergoing reduction . The first mutation was shown to alter both the association and dissociation rate constants for the cofactor so that the dissociation constant was increased 6-40-fold . A corresponding but smaller (fourfold) effect was noted in V/K but not in V compared to the wild-type enzyme . The second was demonstrated to increase the dissociation rate constant for methotrexate 20-30-fold, and presumably dihydrofolate also, with a corresponding 20-30-fold increase in the dissociation constant . In this case an identical effect was noted on V/K but not in V relative to the native enzyme . Thus, in both mutant enzymes the decrease in binding has not been translated into a loss of catalytic efficiency.

Gene, 1985, 38(1-3), 65 - 72
Nucleotide sequence of the ksgA gene of Escherichia coli: comparison of methyltransferases effecting dimethylation of adenosine in ribosomal RNA; van Buul CP et al.; The ksgA gene of Escherichia coli encodes a methyltransferase (MeT) that specifically dimethylates two adjacent adenosines near the 3' end of 16S RNA in the 30S particle . Its inactivation leads to kasugamycin (Ksg) resistance . Several plasmids were constructed with inserts which complemented chromosomal ksgA mutations . One of these inserts was sequenced and found to contain an open reading frame (ORF) sufficient to code for the previously identified 30-kDal MeT . We have compared the amino acid (aa) sequence of the ksgA-encoded enzyme with three published sequences of MeT involved in dimethylation of an adenosine residue in 23S RNA and rendering the organisms resistant to the MLS antibiotics . The homologous patches in the sequences of all four enzymes suggest that those might correspond to contact points for the common substrates, e.g., for the adenosine residue(s) and S-adenosylmethionine (SAM).

Gene, 1985, 38(1-3), 259 - 64
Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme; Perry LJ et al.; The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells . E . coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside . A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme . Purified strains, however, are reasonably stable in culture and under normal storage conditions.

Gene, 1985, 38(1-3), 145 - 52
Demonstration of remarkable sequence divergence in variants of a complex satellite DNA by molecular cloning; Stringfellow LA et al.; Repeat units of a complex G + C-rich satellite of the Bermuda land crab have been cloned by insertion into either the PstI or EcoRI site of pBR322 or the EcoRI site of pUC9 . While most of the recombinants contained inserts of approx . 2.1 kb, the average size of repeat units seen in cellular satellite digests, several inserts were markedly different in size . Two domains that account for major sequence differences among the satellite variants and that may be 'hotspots' for sequence modification have been subcloned to permit characterization of their secondary and tertiary structures independent of the influence of the other unusual sequences present . One of these domains is striking in its content of simple repeats; one strand is highly biased in pyrimidines which may permit the formation of unusual secondary and/or tertiary conformations . The other subcloned domain is rich in Pu/Py; preliminary data indicate a transition from B----Z DNA in this region.

Gene, 1985, 38(1-3), 1 - 8
Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli; Gil JA et al.; A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S . lividans 66 on the plasmid vector pIJ61 . The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter . This DNA fragment conferred Cm resistance, through CAT activity, on S . lividans, S . coelicolor and S . parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322 . However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance . DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity . The cat provides a potentially useful screening marker for Streptomyces cloning vectors.

Folia Biol (Praha), 1985, 31(5), 321 - 32
Studies on lipopolysaccharide-induced polyclonal B cell activity in mice tolerized by sheep red blood cells and cyclophosphamide; Fontalin LN et al.; The polyclonal immune response was induced in untreated mice and mice treated with cyclophosphamide by the administration of lipopolysaccharide from E . coli or S . marcescens . The number of cells forming antibodies to sheep red blood cells and to trinitrophenyl, and of cells producing immunoglobulins increased . The administration of LPS to mice pretreated with SRBC and CY (tolerant mice) considerably reduced the number of anti-SRBC AFC in comparison with the controls . The tolerogenic treatment did not change the number of anti-TNP AFC and IPC . Analogous results were obtained in genetically athymic (nude) mice and in B mice (thymectomized, lethally irradiated and reconstituted with embryonic liver cells) . The results suggest that a deletion or a temporary inactivation of a fraction of the antigen-specific B cells occurs in tolerant mice . This inactivation cannot be explained by the absence of expression of surface immunoglobulins on B cells nor by the activity of suppressor T cells.

Biol Neonate, 1985, 48(5), 307 - 12
Observations on the development of the febrile response to pyrogen in newborn pigs; Moraes RN et al.; The febrile response of newborn pigs to exogenous pyrogen injection was investigated . Lipopolysaccharides (LPS, E . coli) were injected intravenously into the superior vena cava of 1-30-day-old piglets . All the experiments were carried out in littermates half of which were injected with pyrogen and half with pyrogen-free saline . Newborn pigs did not develop a febrile response from 1 to 4 days of age; however, when the animals were 5 days old exogenous pyrogen determined a typical monophasic febrile response . A second intravenous injection of pyrogen into newborn pigs (1 day old) 24 h after the first did not raise body temperature . It is suggested that newborn pigs behave like the newborn of other mammalian species regarding endotoxin-induced thermogenesis.

Basic Life Sci, 1985, 34, 121 - 45
Situation-dependent repair of DNA damage in yeast; von Borstel RC et al.; The concept of channelling of lesions in DNA into defined repair systems has been used to explain many aspects of induced and spontaneous mutation . The channelling hypothesis states that lesions excluded from one repair process will be taken up by another repair process . This is a simplification . The three known modes of repair of damage induced by radiation are not equivalent modes of repair; they are, instead, different solutions to the problem of replacement of damaged molecules with new molecules which have the same informational content as those that were damaged . The mode of repair that is used is the result of the response to the situation in which the damage takes place . Thus, when the most likely mode of repair does not take place, then the situation changes with respect to the repair of the lesion; the lesion may enter the replication fork and be reparable by another route.

Acta Microbiol Hung, 1985, 32(2), 183 - 92
Virulence factors of Escherichia coli . II . Antigens O4, O6 and O18, haemolysin production and mannose resistant haemagglutinating capacity are closely associated; Czirok E; Escherichia coli strains isolated from a variety of human samples were examined for alpha haemolysin (Hly) production . A total of 1156 strains was compared for incidence of Hly positivity, and the capacity of mannose resistant haemagglutinating activity of human erythrocytes (MRHA) . Incidence of Hly production in serogroups O4, O6 and O18ac was significantly higher than in other ones (70.1% vs . 18.7%), independently of the origin of strains; 78% of Hly+ strains belonging to serogroups O4, O6, O18 was MRHA+, too . The marked correlation between Hly positivity, MRHA activity and these serogroups suggested that in serogroup O4, O18 and in a lesser degree O6, genetic informations concerning O antigen, MRHA and Hly are linked in the chromosome.

Mol Gen Genet, 1985, 201(1), 25 - 9
DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations; Nashimoto H et al.; A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc) . The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified . DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E . coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E . coli.

Mol Gen Genet, 1985, 201(1), 20 - 4
A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation; Angulo JF et al.; In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K 12 RecA protein . The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation . Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis . The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD+) strains carrying mitochondrial DNA or not (rho0) . This was also true for the following DNA-repair deficient (rad-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-delta, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.

J Basic Microbiol, 1985, 25(7), 457 - 60
Studies of colicin action on wall-less stable L-forms of Escherichia coli . III . A colicin-tolerant mutant in a wall-less stable L-form; Smarda J et al.; The conversion of a tol III Escherichia coli mutant, tolerant to colicins E2, E3, Ia and K, into protoplast-like stable L-form makes it sensitive to colicins E2, Ia and K . Its original sensitivity to colicin E1 and tolerance to E3 are preserved in this cell form . Thus, rods of this mutant are not truly tolerant to colicins E2, Ia and K, but "pseudotolerant": their wall receptors have been turned out to "nonlethal" ones by the mutation in question.

J Basic Microbiol, 1985, 25(7), 451 - 6
Studies of colicin action on wall-less stable L-forms of Escherichia coli . II . Growth inhibition of complete and wall-less (L-form) cells of Escherichia coli by basic colicin types; Smarda J et al.; The inhibitive activity of colicins of 16 types (produced by 22 colicinogenic strains) on rods and protoplast-like stable L-form cells of the strains Escherichia coli B, W1655 F+ and W1655 F- was compared . The results of 58 combinations tested fall into four groups (with three subgroups): both cell forms are sensitive: a) both cell forms are about equally sensitive, b) rods are distinctly more sensitive than L-form cells, c) L-form cells are distinctly more sensitive than rods; only rods are sensitive, L-form cells are not sensitive; only L-form cells are sensitive, rods are not sensitive; neither cell form is sensitive . Group la represents simple sensitivity; both cell wall and cytoplasmic membrane receptors are present and functioning . Groups 1b and 2 represent sensitivity, substantially or completely mediated by cell wall receptors . Groups 1c and 3 represent partial or complete "pseudoresistance" or "pseudotolerance"; cell wall receptors are absent or non-lethal, but cytoplasmic membrane ones are present and mediate the lethal effect . Group 4 represents both true resistance and true tolerance.

Infection, 1985, 13 Suppl 2, S256 - 9
Studies on immunity against Escherichia coli K13 with monoclonal anti-K13 and anti-anti-K13; Soderstrom T et al.; The structural basis for the cross-reactivity between the Escherichia coli K13, K20 and K23 capsular polysaccharides is the----)-beta-ribofuranosyl-(1----7)-beta-2-keto-3-deoxyoctonate polymer . Monoclonal antibodies against E . coli K13 which require O-acetyl-2-keto-3-deoxyoctonate for binding were further investigated . Such antibodies, of both the IgG and the IgM isotype, opsonized E . coli K13 in vitro and protected against intraperitoneal infection in mice as well as ascending pyelonephritis in rats . A monoclonal IgG1 anti-idiotype, specific for the K13 polysaccharide combining site of a protective IgM idiotype, primed for protection against intraperitoneal infection with live E . coli K13 following K13 injections at four as well as 12 weeks of age, the K13 polysaccharide alone did not immunize and protect . The monoclonal anti-K13 idiotype only primed for protection at four weeks of age . These findings suggest a strong effect of a single idiotype on the outcome of a bacterial infection.

Gene, 1985, 37(1-3), 53 - 62
Repetitive extragenic palindromic (REP) sequences in the Escherichia coli gdhA gene; Becerril B et al.; Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene . Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al . {Cell 37 (1984) 1015-1026}, as a novel and major feature of the bacterial genome . The effect of these deletions on the final GDH level in the cell, has been determined . A broader compilation, analysis and alternative functions of the REP sequences, is also presented.

Gene, 1985, 37(1-3), 267 - 9
Screening expression libraries with nonradioactive immunological probes; Wang SZ et al.; An immunological screening method employing protein A-peroxidase which does not require radiolabelled antibodies for detection of Escherichia coli colonies synthesizing foreign proteins in a cDNA expression library is described . The technique is sensitive, simpler and more rapid than the procedures that rely on radiolabelled antibodies and autoradiography.

Gene, 1985, 37(1-3), 221 - 8
Molecular cloning of the terminal hairpin of vaccinia virus DNA as an imperfect palindrome in an Escherichia coli plasmid; Winters E et al.; The genome of vaccinia virus is a linear duplex molecule of approximately 185 kb with hairpins at each end that link the complementary strands . The hairpins, which exist in two forms that are inverted and complementary in sequence, were isolated as XbaI restriction fragments and converted to a linear intermolecular duplex structure by denaturation and reannealing . The latter was then stably cloned as a 142-bp imperfect palindrome in an Escherichia coli plasmid . The insert was excised from the plasmid and the palindrome was extended on both sides by ligating it to the adjacent vaccinia virus DNA segment . The resulting fragment was cloned as a 278-bp imperfect palindrome . Restriction endonuclease analysis and DNA sequencing indicated the absence of any deletions or rearrangements . After excision from the plasmid, the palindrome was converted by heating and rapid cooling to the original two hairpin forms . In this manner, large quantities of vaccinia virus telomeres may be obtained for physical and biochemical studies.

Eur Surg Res, 1985, 17(5), 281 - 5
Decreased clearance of Escherichia coli from the bile in rats with obstructive jaundice; Tanaka N et al.; Clearance of Escherichia coli from the bile was studied in 4 normal Sprague-Dawley rats and 4 rats with 3 weeks of obstruction of the common bile duct . 125I-radiolabelled heat-killed E . coli were injected into the common bile duct and the radioactivity of the bile monitored for 2 h . The radioactivity declined exponentially during the first 10 min . Bile samples collected from 2 to 15 min after injection showed higher amounts of radioactivity in all rats with biliary obstruction than in the normal rats . However, the clearance rate was higher in normal than in obstructed rats (p less than 0.05) . It was, therefore, concluded that the bacteria were cleared off the bile rapidly in normal rats by the function of a liver and/or a biliary tree . The present data, concerning the kinetic study of bacterial clearance from the biliary tract, indicates that the impaired clearance of bacteria in chronic biliary obstruction might be crucial for the development of biliary sepsis.

Cor Vasa, 1985, 27(4), 319 - 22
Septic thrombosis of the inferior caval vein detected with the aid of computed tomography; Bures J et al.; A case is described of a 55 years old woman with septic thrombosis of the inferior caval vein, detected in time with the aid of computed tomography and cavography . The patient subsequently underwent successful surgical treatment.

Circ Shock, 1985, 17(2), 137 - 45
Evidence that prostaglandins within preoptic area (POA) may mediate the antidipsogenic effect of Escherichia coli endotoxin in the rat; Foca A et al.; Intravenous injection of Escherichia coli endotoxin (LPS; 640 micrograms/kg) produced an antidipsogenic effect in 48-h water-deprived rats, which was antagonized by acetylsalicylic acid (ASA; 0.45, 0.9 microM) injected directly into cerebral preoptic area (POA) . ASA (0.9 microM) when injected into POA did not elicit, by itself, any effect on drinking . Endotoxin (0.5, 1, and 2 micrograms), when injected directly into POA, produced a dose-dependent inhibition of drinking stimulated by water deprivation . This effect was antagonized by ASA (0.45 microM) injected into POA 15 min before LPS . Injection of LPS (2 micrograms) into brain areas not involved in drinking regulation (superior colliculus or nucleus caudatus) was without effect on 48-h water deprivation-induced thirst . PGI2 (5, 50, and 500 ng) injected ino POA showed a dose-dependent antidipsogenic effect on drinking stimulated by water deprivation . The data suggest that prostaglandins, within the preoptic area, might be involved in the antidipsogenic effect of E coli LPS, given either intravenously or directly into POA.

Circ Shock, 1985, 17(1), 85 - 94
Plasma catecholamines during endotoxin infusion in conscious unrestrained rats: effects of adrenal demedullation and/or guanethidine treatment; McKechnie K et al.; We have examined the effect of E coli endotoxin infusion (41 micrograms kg-1 min-1 iv for 4 h) on plasma concentrations of epinephrine (E), norepinephrine (NE), and dopamine (DA) in the conscious unrestrained rat . Saline infusion did not change catecholamine concentrations from the preinfusion values of 230.8 +/- 32.9 pg ml-1 (E), 456.8 +/- 104.9 pg ml-1 (NE), and 49.0 +/- 19.9 pg ml-1 (DA) . Endotoxin produced marked elevations in all three catecholamines . At 1 h, the plasma concentrations were 3,279 +/- 494.6 pg ml-1 (E), 1,670 +/- 137.0 pg ml-1 (NE), and 191.5 +/- 13.7 pg ml-1 (DA) . Thereafter, concentrations of E decreased whereas concentrations of NE and DA increased . These increases were prevented by a combination of adrenal demedullation (28 days previously) and treatment with guanethidine (25 mg kg-1 iv, -24 h) ("sympathectomy") . Guanethidine alone markedly reduced the peak NE concentrations without affecting the E concentrations or the 1-h NE concentrations . Demedullation alone prevented the increase in E and reduced the 1-h NE concentrations . Survival in such "sympathectomized" animals was markedly reduced (survival at 4 h in rats receiving endotoxin alone, 100%: in "sympathectomized" animals receiving endotoxin, 12.5%) . The tachycardia produced by endotoxin was attenuated in "sympathectomized" rats and mean arterial blood pressure fell rapidly . Endotoxin-induced hyperglycemia was prevented by "sympathectomy" and hypoglycemia was evident as early as 1 h after commencing the infusion . Endotoxin produced hypoinsulinemia in normal rats but did not change plasma insulin values in "sympathectomized" animals, although these animals showed a pre-endotoxin fasting hyperinsulinemia . An important protective role for catecholamines is suggested, especially in the early stages of shock.

Neoplasma, 1985, 32(4), 407 - 14
New cytotoxic and antitumor agents . VII . Derivatives of 1-benzylidenisoindolin-3-one and 5,6-dihydro-8H-isoquinolo(2,3-a)phthalasin-5-one; Fuska J et al.; The in vitro cytotoxic effects on P388 leukemia cells of 19 derivatives and analogs of 1-benzylidenisoindolin-3-one, 5,6-dihydro-8H-isoquinolo(2,3-a)phthalasin-5-one and 4-benzyl-1,2-dihydrophthalasin-5-one were studied . The derivatives of the first group which preferentially inhibited uridine utilization were more effective . The cytotoxic effects of these substances depended on the quality of substituents, on the presence of a nitrogen atom in the 5-membered heterocycle of the molecule as well as on the spatial arrangement of the molecule . (Z)-narceine imide-N-oxide II and (Z)-3-(6-ethyl-2-methoxy-3,4-methylenedioxy) benzyliden-6,7-dimethoxy-isoindolin-3-one III were the most active agents, both of them causing a decrease in the proliferation rate of P388 cells . After its addition to the culture medium, compound III caused irreversible damages leading to death of the cells . The removal of the inhibitor did not lead to the repair of the damages either . Of the other two groups of substances, 5-hydroxy-3,4,12-trimethoxy-8-methyl-10,11-methylenedioxy-8H-isoquino lo (2,3-a)phthalasin (XVIII) had a marked inhibitory effect which, at concentrations of up to 25 micrograms ml-1, inhibited the proliferation rate of P388 cells.

Mol Gen Genet, 1985, 200(3), 451 - 7
A coupled in vitro system for the formation and packaging of concatemeric phage T1 DNA; Liebeschuetz J et al.; Extracts derived from E . coli cells infected non-permissively with phage T1 amber mutants were used in an in vitro system to investigate the packaging of T1 DNA into phage heads . The standard extract used infections with amber mutants in genes 1 and 2 (g1- g2-) which are defective in T1 DNA synthesis but can synthesis the proteins required for particle morphogenesis . g1- g2- extracts packaged T1+ virion DNA molecules with an efficiency of 3 X 10(5) pfu/micrograms DNA . Extracts from cells infected with phage also defective in DNA synthesis but carrying additional mutations in genes 3.5 or 4 which are required for concatemer formation in vivo (g1- g3.5- and g1- g4- extracts) package T1 virion DNA at substantially lower efficiencies . Analysis of the DNA products from these in vitro reaction showed that concatemeric DNA is formed very efficiently by g1- g2- extracts but not by g1- g3.5- or g1- g4- extracts . These results are interpreted as evidence that the T1 in vitro DNA packaging system primarily operates in a similar manner to the in vivo headful mechanism . This is achieved in vitro by the highly efficient conversion of T1 virion DNA into concatemers which are then packaged with a much lower efficiency into heads to form infectious particles . A secondary pathway for packaging T1 DNA into heads and unrelated to the headful mechanism may also exist.

Gene, 1985, 35(3), 321 - 31
Nucleotide sequence of the birA gene encoding the biotin operon repressor and biotin holoenzyme synthetase functions of Escherichia coli; Howard PK et al.; A 2.2-kb region of DNA containing the birA gene of Escherichia coli has been sequenced . The birA gene sequence predicts a 35.3-kDal {321 amino acids (aa)} bifunctional protein containing biotin-operon-repressor and biotin-holoenzyme-synthetase activities . Mutations, generated by random insertion of XhoI linkers, defined the extent of the gene . Mutations affecting one or more of five discernable properties of birA {Barker, D . and Campbell, A., J . Bacteriol., 143 (1980) 789-800} were mapped . Three mutations that result in temperature-sensitive (ts) growth, birA85, birA215, and birA879 mapped in the N-terminal two-thirds of the protein . The birA352 mutation, which partially complements birA215 and birA879, maps in the N-terminal third of the protein . Finally, birA361 maps closest to the amino terminus.

Dev Biol Stand, 1985, 60, 355 - 69
Cloning of a bovine interferon-alpha gene subfamily and comparisons between genetically engineered and leukocyte bovine interferons; Velan B et al.; Bovine peripheral leukocytes were virally induced for interferon production, and an acid stable, SDS stable, antiviral activity was detected in the preparation . This bovine interferon (BoIFN) was tested for its ability to induce an antiviral state in various mammalian cells and was found to be specific to cells from bovine origin . The BoIFN cross reacts with antibodies against human IFN-alpha but these antibodies do not neutralize the bovine IFN activity . Leukocyte BoIFN exhibits polymorphism upon Affi-Gel Blue chromatography and SDS-PAGE (16k and 24K) . The virally induced leukocytes produce a 13S mRNA which upon translation in oocytes yields an active IFN molecule . Bovine genomic library was constructed and screened for BoIFN-alpha sequences, using human IFN-alpha probes . From the clones isolated, five were found to represent distinct genes . Sequence analysis indicate that these genes are closely related (94% homology) . One of these genes was expressed in E . coli under the control of trp promoter operator . The physicochemical and biological properties of the bacterial BoIFN-alpha product resemble those of a subpopulation of natural BoIFN.

Environ Mutagen, 1985, 7(3), 267 - 79
Nick translation--a new assay for monitoring DNA damage and repair in cultured human fibroblasts; Snyder RD et al.; An in vitro assay has been developed to detect DNA damage and repair following chemical treatment of human diploid fibroblasts . DNA damage is measured by following the Escherichia coli DNA polymerase I-catalyzed incorporation of radiolabeled deoxycytidine triphosphate (dCTP) into the DNA of lysolecithin-permeabilized cells . DNA strand breaks with free 3' OH termini serve as template sites for incorporation, and decrease of this incorporation with time, following removal of the test chemical, indicates loss (repair) of initial damage . Inhibition of the DNA excision repair process by the addition of the repair inhibitors arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) during the incubation period gives rise to an increased number of template sites, manifesting itself in increased incorporation and indicating the induction of long-patch excision repair . This nick translation assay, originally proposed by Nose and Okamoto {1983}, is very sensitive, allows detection and quantitation of both DNA damage and repair, distinguishes between various types of induced damage, does not require radioactive prelabeling of cells, and circumvents some of the problems inherent in unscheduled DNA synthesis (UDS) assays . The assay is also useful in detecting those agents that inhibit replicative DNA synthesis and/or the excision repair process . Results presented demonstrate that all 14 direct-acting carcinogens tested and 8 of 14 carcinogens requiring metabolic activation give positive indication of DNA damage, repair, or both . Eleven of 14 noncarcinogens tested were scored as negative, the other 3 having previously been shown to interact with cellular DNA . This assay is shown to have predictive capability at least equal to that of UDS assays but to allow a broader spectrum of genotoxic effects to be analyzed.

Vet Med Nauki, 1985, 22(5), 66 - 74
{Effect of environmental factors on the body response reactions in newborn calves}; Delev K et al.; Studied was the role of the general state and the adaptability of newborn calves under the effect of some environmental factors (temperature and relative humidity) that had no optimal values in the onset of diseases caused by occasionally pathogenic agents . It was found that under conditions that were one and the same for all calves the metabolic and functional responses were varying . Those of the animals with which the nervous-and-hormonal system was more unstable manifested a specific biochemical-and-functional response through which no'equilibration' was achieved with the environment, and they got sick . Others, exhibiting metabolic processes that were stronger and more stable as the result of the involvement of more powerful regulatory mechanisms inherent to a 'neuro-functional' animal organism could adapt successfully to the nonoptimal factors of the environment . Such animals were not shown to be susceptible to the effects of conditionally pathogenic agents.

Vet Med Nauki, 1985, 22(5), 60 - 5
{Enzyme constellation of the blood plasma in the prognosis of decreased adaptability in newborn calves}; Ivanov V et al.; The dynamic was studied of the enzyme activity of ChE, APh, Ald, GOAT, GPT, LDG, and CPh in 30 newborn calves . Fifteen of the animals developed coli bacteriosis later on . Blood was sampled at the 6th to 24th hour as well as on the 5th day following birth . At the 6th hour ChE, APh, Ald, and CPh of the diseased calves showed lower activity as against the enzyme activity of the unaffected animals . Similar trend was noticed also at the 24th hour . Contrary to the other enzymes of the constellation the changes observed in the activity of GPT were invariably connected with a considerable rise in diseased animals . The changes in the activity and dynamic of the investigated enzyme constellation can be used to forecast the incidence of coli bacteriosis.

Physiol Chem Phys Med NMR, 1985, 17(1), 45 - 51
Effect of photooxidation on catalytic and regulatory properties of NAD-linked malic enzyme from Escherichia coli; Saito R et al.; In an aim to elucidate the structure-function relationship of NAD-linked malic enzyme {EC 1.1.1.38} from Escherichia coli W, the effect of chemical modification on the catalytic and regulatory properties of the enzyme was studied . Upon photooxidation of the enzyme in the presence of methylene blue, a time-dependent inactivation occurred following pseudo-first order kinetics . The pH-dependence of the inactivation rate exhibited a pK value of 6.1 . L-Malate, NAD+, and Mn2+ markedly protected the enzyme against the inactivation . Prior masking of the catalytically essential sulfhydryl groups with p-mercuribenzoate did not result in a retardation of the rate of photoinactivation . This excluded the possibility of an involvement of sulfhydryl group modification in the photoinactivation . Although the Km values for L-malate and NAD+ were not affected by photooxidation, the S0.5 value and the Hill coefficient for Mn2+ were considerably altered, and the cooperative nature of the saturation profile for Mn2+ in the native enzyme was completely abolished . The activating effect of L-aspartate on the native enzyme was completely abolished upon photooxidation, and the inhibitory effect of CoA was also diminished to a marked extent upon the treatment . The oxaloacetate decarboxylating activity of the enzyme was lost in parallel with the loss of the activity for oxidative decarboxylation of L-malate . These results suggest a possible involvement of histidyl residue(s) in the catalytic and regulatory functions of the enzyme.

Med Microbiol Immunol (Berl), 1985, 174(3), 119 - 30
Phenotypic variations among enterotoxinogenic Escherichia coli from Swedish piglets with diarrhoea; Kuhn I et al.; Escherichia coli strains causing diarrhoea in Swedish piglets were isolated; this investigation was made over a 20-year period, from 1964 to 1984 . Many of the isolates belonged to O-groups 8, 141 and 149 . These strains were separated into different phenotypes with the aid of a new method, "biochemical fingerprinting" . This method has been especially designed to sort E . coli-isolates into various phenotypes based on a pattern of quantitatively measured biochemical reactions . It was found that most pathogenic isolates carrying O-antigen 8 belonged to the same phenotype . This phenotype was common in the 1960's, but later it disappeared from the population and was replaced by a wide variety of different phenotypes, most of them non-enterotoxinogenic, but still belonging to O-group 8 . In O-group 141 one phenotype dominated in the 1960's, but in the 1970's a new phenotype appeared, which further increased in number in the 1980's . By contrast, in the O-group 149 practically all strains isolated during the 20-year period were found to carry the relevant virulence factors and belong to the same phenotype.

Microbiol Immunol, 1985, 29(5), 383 - 93
Nucleotide sequence of the replication region of plasmid R401 and its incompatibility function; Tabuchi A; The plasmids R401 and Rtsl belong to the same incompatibility group, IncT . The nucleotide sequence of the basic replicon of R401 consisting of 1,857 base pairs was determined and compared with that of mini-Rtsl previously reported . The mini-R401 was found to be composed of two clusters of direct repeated sequences flanking a large open reading frame that could encode a 33,000 Mr protein (RepA protein) consisting of 288 amino acids . This structure of mini-R401 is quite similar to that of mini-Rtsl . Furthermore, the nucleotide sequence of mini-R401 is identical to that of mini-Rtsl except for eleven nucleotides; three are located near the carboxyl terminus portion of the RepA coding region (repA) and four are in the repeated sequences (incI) located downstream from repA . Incompatibility study showed that mini-R401 plasmid coexisted stably with the cloned incI repeats of mini-Rtsl, suggesting that mini-R401 RepA protein binds to incI repeats of mini-Rtsl less efficiently than does mini-Rtsl RepA protein.

Microbios, 1985, 42(169-170), 183 - 201
Morphological changes in Escherichia coli subjected to direct current electrical fields; Ellis HW; Rod shaped Escherichia coli grown in a liquid minimal salts medium, through which an electrical current of 20 mA direct current was passed, were found to increase up to 25% of their original length . When the current was removed, or treated cells grown in fresh medium, they reverted to their original size and distribution.

Mol Gen Genet, 1985, 200(2), 295 - 301
Transcription termination regions of coliphage T7 DNA: the effects of nusA1; Garner I et al.; We report the effects in vivo of four segments of coliphage T7 DNA upon expression, from an upstream promoter, of galK in plasmids of the pKO family . Three of the segments carry the known major or putative distal terminators of host-dependent T7 early transcription . The fourth carries a novel terminator and maps in the late region of T7 . We report the efficiencies of termination in these regions: evidence, based on studies with the E . coli nusA1 mutation, for an involvement of the transcription factor NusA in events at the major early and novel terminators: and the nature of the latter transcription signal.

Mol Gen Genet, 1985, 200(1), 145 - 54
Transcription analysis of the sucAB, aceEF and lpd genes of Escherichia coli; Spencer ME et al.; Transcript mapping of the Escherichia coli sucAB, aceEF and lpd genes, encoding the five components of the pyruvate and 2-oxoglutarate dehydrogenase complexes, was carried out using single-stranded M13 probes . The sucA and aceE genes encode the specific dehydrogenase components (E1o, E1p), and the sucB and aceF genes encode the specific dihydrolipoamide acyltransferases (E2o, E2p) . The common lipoamide dehydrogenase (E3) component is encoded by a single lpd gene adjacent to the aceEF genes . The sucAB, aceEF and lpd genes were all expressed on independent transcripts, and the promoters and terminators were identified . In addition, readthrough transcription from the sucAB genes to a downstream gene designated sucC, and from the aceEF genes to the adjacent lpd gene, was found . The relative levels of transcription of the suc, ace and lpd genes, and of the three different transcript types covering the ace-lpd region, were quantified using RNA from cells grown on different substrates . Most of the E3 components supplying the pyruvate dehydrogenase complex appear to be synthesised from approximately 6415-base aceEF-lpd readthrough transcripts, but additional approximately 4640-base aceEF transcripts terminating after the aceF gene provide a transcriptional basis for the observed stoichiometric excess of E1p and E2p relative to E3 in the assembled complex . Conversely most of the E3 components required for the 2-oxo-glutarate dehydrogenase complex appear to be synthesised from the independent 1670-base lpd transcripts.

Mol Gen Genet, 1985, 200(1), 132 - 7
Characterization of a mutation conferring radiation sensitivity, ior, located close to the gene coding for deoxycytidine deaminase in Escherichia coli; Estevenon AM et al.; The isolation and characterization of a new mutation conferring radiation sensitivity in Escherichia coli is described . This mutation is located close to the gene coding for deoxycytidine deaminase, in the chromosomal region of the gat operon . It is very sensitive to gamma rays and exhibits a decrease in recombination ability . The expression of radiation sensitivity seems to result from the additive effect of the dcd mutation and another mutation of unknown function.

Mol Gen Genet, 1985, 200(1), 118 - 22
The effect of the locus pstB on phosphate binding in the phosphate specific transport (PST) system of Escherichia coli; Levitz R et al.; The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli . The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E . coli . These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding capacity was estimated by the retention kinetics . The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type . The KD values for Pi binding to periplasmic protein were determined by equilibrium dialysis . In the pstB mutants the KD value was found to be 9-31 times higher than the values obtained for the wild type and the phoT mutant . The apparent Km values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type . Vmax of the mutant is 8.3 times lower that of the wild type . The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.

Mol Gen Genet, 1985, 200(1), 114 - 7
Mutant of Escherichia coli with unusual patterns of rpoB,C expression in response to rifampicin and acridine orange; Tittawella IP; A mutation located near rpoB (89') in E . coli is responsible for unusual patterns of beta and beta ' (but not L7/L12) synthesis in response to the drugs rifampicin and acridine orange.

Mol Gen Genet, 1985, 199(3), 543 - 6
Stability of ribosomal protein mRNA and translational feedback regulation in Escherichia coli; Singer P et al.; It was previously observed that the stability of ribosomal protein (r-protein) mRNA in Escherichia coli decreases under the conditions where its translation is feedback inhibited by repressor r-protein . We have now demonstrated that the stability of mRNA for r-proteins S13, S11 and S4 increases in a strain carrying a mutation in the gene for S4, a translational repressor regulating these r-proteins . The results confirm the previous observations that translational repression increases the decay rate of r-protein mRNA, and in addition, show that the half-life of S13-S4 r-protein mRNA in cells growing under ordinary conditions is significantly shorter than its inherent stability would predict, due to the operation of translational feedback regulation.

J Membr Biol, 1985, 84(3), 259 - 67
Effect of different phospholipids on the reconstitution of two functions of the lactose carrier of Escherichia coli; Seto-Young D et al.; The lactose carrier was extracted from membranes of Escherichia coli and transport activity reconstituted in proteoliposomes containing different phospholipids . Two different assays for carrier activity were utilized: counterflow and membrane potential-driven uptake . Proteoliposomes composed of E . coli lipid or of 50% phosphatidylethanolamine--50% phosphatidylcholine showed very high transport activity with both assays . On the other hand, proteoliposomes containing asolectin, phosphatidylcholine or 25% cholesterol/75% phosphatidylcholine showed good counterflow activity but poor membrane potential-driven uptake . The discrepancy between the two types of transport activity in the latter group of three lipids is not due to leakiness to protons, size of proteoliposomes, or carrier protein content per proteoliposome . Apparently one function of the carrier molecule shows a broad tolerance for various phospholipids, while a second facet of the membrane protein activity requires very restricted lipid environment.

J Basic Microbiol, 1985, 25(5), 341 - 7
{Synthesis of alkaline phosphatase in a stringent and a relaxed strain of Escherichia coli under amino acid and phosphate limitation}; Hecker M et al.; Synthesis of alkaline phosphatase in Escherichia coli is derepressed under phosphate starvation in the stringent strain CP78 as well as in its relaxed counterpart CP79 . During limitation of phosphate as well as of amino acids a decrease of enzyme activity is observed, especially in the relaxed strain, which can not produce ppGpp under this conditions . After phosphate limitation synthesis of ppGpp is not stimulated and the kinetics of RNA synthesis is similar in both strains . We suggest that ppGpp is not directly involved in the regulation of gene expression during phosphate starvation.

Indian J Lepr, 1985 Jan-Mar, 57(1), 107 - 14
Isoenzymes of mycobacteria . II . Relevance of LDH zymograms in taxonomy and identification; Katoch VM et al.; The cell free extracts of mycobacteria namely M . kansasii M . avium, M . tuberculosis, BCG (Glaxo), M . gastri, M . phlei, M . smegmatis, M . vaccae, M . strain w., M . scrofulaceum, M . gordonae, M . nonchromogenicum E . coli, Staph, aureus, and M . leprae infected skin have been electrophoresed and stained for LDH activity . Normal skin tissue was also taken as control . It was found that all the organisms tested showed distinct species specific LDH isoenzyme patterns . There was no extra band but an aberrant zone of LDH activity was seen in M . leprae infected human skin in comparison to LDH isoenzymes from normal skin . No strain variations was found among the different strains of species investigated . Results described in the present paper indicate that LDH isoenzyme patterns of mycobacteria could be of identification value at species level.

Gene, 1985, 35(1-2), 83 - 9
Expression of a biologically active analogue of somatomedin-C/insulin-like growth factor I; Peters MA et al.; A synthetic gene coding for an analogue of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) was synthesized by solid support phosphoramidite chemistry and subsequently cloned and expressed in Escherichia coli as a fusion protein . The gene, designed with a threonine codon substituted for a methionine codon at position 59 was expressed fused to an eight-amino acid leader peptide under the direction of the E . coli tryptophan promoter . The fusion protein, termed L0-{Thr59}-Sm-C/IGF-I was purified extensively (greater than 97%) and found to be 60% as active as native Sm-C/IGF-I in a radioimmunoassay and 50% as potent as native Sm-C/IGF-I in a radioreceptor assay . Like native Sm-C/IGF-I it was also mitogenic for Balb/c 3T3 cells . After removal of the eight amino acid leader peptide by cyanogen bromide treatment, the resulting threonine analogue, termed {Thr59}-Sm-C/IGF-I was 80% as potent as native Sm-C/IGF-I in both the RIA and the radioreceptor assays . It was also mitogenic in Balb/c 3T3 cells . These two analogues, therefore, display biological activities similar to human-derived Sm-C/IGF-I.

Gene, 1985, 35(1-2), 179 - 85
An Escherichia coli recBCsbcBrecF host permits the deletion-resistant propagation of plasmid clones containing the 5'-terminal palindrome of minute virus of mice; Boissy R et al.; The deletion events that have plagued attempts to maintain molecular clones with long palindromic DNA sequences in Escherichia coli have been shown to be less frequent in recBCsbcB hosts {Collins et al., Gene 19 (1982) 139-146} . This study sought to determine if such hosts would permit the stable propagation of plasmid clones carrying the deletion-generating, 206 nucleotide (nt) long, imperfect palindrome derived from the 5' terminus of the genome of minute virus of mice (MVM), an autonomous parvovirus {Astell et al., Nucleic Acids Res . 11 (1983) 999-1018} . To this end these hybrid plasmids were used to transform several different mutant recBCsbcB hosts, followed by the isolation and restriction mapping of plasmid DNA from selected transformants . Characterization of plasmid DNA isolated from a recBCsbcBrecF host indicated deletion-resistant propagation of the intact species . Sequence analysis of unamplified and chloramphenicol (Cm)-amplified plasmid DNA obtained from these clones confirmed the integrity of the palindromic region of the viral DNA insert.

Drugs, 1985, 29 Suppl 5, 178 - 81
Temocillin treatment of gynaecological infections with special reference to blood and tissue concentrations; Weissenbacher ER et al.; Tissue concentrations of temocillin were determined in samples from gynaecological surgical patients . Measurable concentrations of temocillin were observed during the entire time period investigated (up to 7 hours post administration) . Inhibitory concentrations for the majority of susceptible bacteria were achieved . The therapeutic results observed in 40 patients with various infections (90% fully effective, 5% partially effective) confirm the high efficacy of temocillin in the treatment of gynaecological infections.

Bull Soc Pathol Exot Filiales, 1985, 78(3), 283 - 9
{Prevalence of the enterotoxigenic Escherichia coli colonization factors CFA/I, CFA/II and E8775 isolated in the South Pacific (New Caledonia, Republic of Vanuatu and Wallis and Futuna)}; Germani Y et al.; We tested the expression of adherence properties of enterotoxigenic Escherichia coli (ETEC) strains isolated in New-Caledonia, Vanuatu and Wallis and Futuna by examining for the presence of colonization factor E8775 using an agglutination test and an immuno-diffusion technique with specific antisera . Approximately 19% of ETEC strains possessed CFA/I and 21% a CFA/II . The E8775 antigen was found on 1.8% of the strains . This last factor was found on strains of the serogroup 025 from Vanuatu . Two strains 078 usually CFA/I+ possessed a CFA/II and three strains of the serogroup 0126 possessed a CFA/I . The results of this study emphasis the need to continue the search for other mechanisms of adhesion used by ETEC strains without any of the three factors of colonization.

Bull Soc Pathol Exot Filiales, 1985, 78(2), 141 - 9
{Epidemiologic study of enterotoxigenic and enteropathogenic Escherichia coli isolated from infantile diarrhea cases on the Wallis and Futuna Islands (French Overseas Territory)}; Germani Y et al.; We have studied the incidence of enteropathogenic and enterotoxigenic Escherichia coli strains associated with infant diarrhoeal disease in Wallis and Futuna (South Pacific) during a period of 3 months . We have isolated enteropathogenic E . coli in 30,4% of children . The most frequently serotypes isolated were 0119:B14 (32%), 0111:B4 (23%) and 0126:B16 (19%) . In this last serotype 6 strains released heat-stable enterotoxin . Enterotoxigenic E . coli were isolated from 8,1% of the children with diarrhea (20 strains), 13 strains released heat-labile toxin, 6 released heat-stable toxin (serotype 0126:B16) and 1 strain produced both.

Mol Gen Genet, 1985, 199(2), 265 - 70
Unstable mutations caused by regional tandem multiplications in the gene for ribosomal protein S4 show thermosensitivity in Escherichia coli; Schnier J et al.; The nucleotide sequence of the gene rpsD for the ribosomal protein S4 of three thermosensitive mutants of Escherichia coli K12 was determined . It was found that two of them contained regional multiplications of a nucleotide sequence within the gene rpsD . In one case, it is a duplication of a 31 nucleotide stretch and in another it is a triplication of a 41 nucleotide stretch . The thermosensitive phenotype of the two mutants is unstable and reverts at the frequency of approximately 10(-4) . The revertants regain the wild-type nucleotide sequence . We postulate that the two mutant genes that contain regional multiplications possibly take an intra-strands secondary structure, which is cleaved to regenerate the wild-type sequence, probably during DNA replication.

EMBO J, 1985 Jan, 4(1), 237 - 42
The invertible P-DNA segment in the chromosome of Escherichia coli; Plasterk RH et al.; In the chromosome of many strains of Escherichia coli K12 the excisable element e14 is found, which contains an invertible DNA region . This invertible P region, and the gene responsible for the inversion (pin) were cloned, together with other e14 sequences . The element e14 contains a gene which kills the host cell . This can be repressed by a function also coded by e14 . The kil and repressor genes as well as the attachment site of the element were mapped in different regions of the element . The invertible segment and pin gene were sequenced . The invertible segment is 1794 bp long, and contains one large internal open reading frame of 879 bp and reading frames which overlap the end pont of the invertible segment . Although pin highly homologous to gin of phage Mu, neither the genetic organization of the P segment nor the sequence of the putative proteins resemble the invertible G segment of phage Mu (which codes for genes involved in tail fiber assembly) . The complete DNA sequences of both invertible segments were screened for homology . No resemblance was found . The P segment is flanked by inverted repeat sequences of 16 bp . Comparison of these with related inversion systems points out that the recombination site maps probably within a 2-bp region . This cross-over site is contained within a short palindromic sequence (AAACC AA GGTTT) which is more or less conserved in the recombination sites of all related DNA invertases.

EMBO J, 1985 Jan, 4(1), 223 - 9
Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli; Plumbridge JA et al.; The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons) . To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation . The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream . A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids . The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus . A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase . This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha . Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay . A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosoma, 1985, 92(3), 209 - 13
Analysis of inactive X chromosome structure by in situ nick translation; Dyer KA et al.; Nick translation assays of fixed interphase female fibroblasts with tritiated nucleotides demonstrated a characteristic absence of label over sex chromatin . The chromatin bodies were nearly always peripheral in location and a ribbon of nick translatable DNA was detected between the sex chromatin and the nuclear envelope . High voltage electron microscopy indicated the possibility of a special nuclear envelope attachment region . The apparent resistance of sex chromatin to nick translation did not appear to be due to resistance to DNase I attack.

Radiat Environ Biophys, 1985, 24(2), 113 - 7
Temperature dependent modification of radiosensitivity following hypoxic cytocidal action of chlorpromazine; Shenoy MA et al.; The hypoxic cytotoxic effect of chlorpromazine towards E . coli B/r was observed to be dependent on temperature of incubation and irradiation . Increasing the temperature of incubation from ambient to 37 degrees C followed by irradiation revealed that the organisms showed radiosensitivity of a magnitude which was two to three times the oxygen effect . The possible mechanisms of this effect are discussed.

Nephron, 1985, 40(3), 374 - 5
Genetic factor(s) influence scar formation in experimental pyelonephritis; Miller TE et al.; Two inbred strains of rat have been identified that show markedly different responses to experimentally induced renal infection . Experiments involving these two strains and their F1 progeny have shown that a genetic factor either protects the host from a tissue-destructive inflammatory response (autosomal dominant gene) or potentiates lesion formation (autosomal recessive gene) . The data are the first indication that genetic factors may determine the degree of renal damage in pyelonephritis.

Mol Gen Genet, 1985, 198(3), 514 - 20
Genes for gentamicin-(3)-N-acetyl-transferases III and IV . II . Nucleotide sequences of three AAC(3)-III genes and evolutionary aspects; Allmansberger R et al.; The direction of transcription, exact location of the decoding region and nucleotide sequences of the aacC3 genes cloned from R-plasmids pWP14a, pWP116a, and pWP113a were determined . The respective fragments could code for a protein of 30.5 kd molecular weight, which was in agreement with the size of the polypeptide expressed by these plasmids in minicells of Escherichia coli . The aacC3 genes, including the promoters, were completely identical in pWP14a and pWP116a . In contrast, in the respective homologous segment in pWP113a were 3.3% of the nucleotides different, leading to ten exchanges in the proposed amino acid sequence for the AAC(3)-III enzyme . Comparison of the aacC3 sequence with another functionally related aminoglycoside resistance determinant, aacC4 (Brau et al . 1984), revealed only a distant relationship . A hypothetical genealogy of the aacC genes is proposed . In pWP113a no good correlation with the consensus sequence for -35 boxes of E . coli promoters was found . However, in pWP14a and pWP116a, also expressing higher gentamicin resistance, a -35 sequence (5'TTGCAA3') was complemented by an IS140 element inserted at exactly the same position in both cases . The element bears palindromic -35 boxes at both ends, inside its inverted repeats . In pWP116a, IS140 was inverted relative to its orientation in pWP14a and most of it, together with part of a structure related to Tn3, was deleted during a process leading to fusion of the two transposable elements . Downstream from the aacC3 genes, an open reading frame separated by a possible intercistronic region of 12 bp and preceded by a translational initiation site exists in both pWP113a and pWP14a.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1985, 198(3), 473 - 5
A change of threonine 266 to isoleucine in the lac permease of Escherichia coli diminishes the transport of lactose and increases the transport of maltose; Markgraf M et al.; The DNA sequence of several functionally interesting lac permease mutants of Escherichia coli has been determined . The phenotypes of the mutant permeases were described by Mieschendahl et al . (1981) . The following exchanges are noteworthy: tyr to asp in codon 26 in Y-K MUB 7; thr to ile in codon 266 in Y-K AJ 33; gly to asp in codon 262 in Y-D 3 and in Y-D 4.

Mol Gen Genet, 1985, 198(3), 465 - 72
Molecular characterisation of the colicin E2 operon and identification of its products; Cole ST et al.; The DNA sequence of the entire colicin E2 operon was determined . The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells . A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB . A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology . These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively . Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins . The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides . Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase . The mature ColE2 lysis protein was located in the cell envelope . The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.

J Toxicol Environ Health, 1985, 15(2), 237 - 44
Inhibition of DNA synthesis by chromium compounds; Nishio A et al.; Cr(VI) irreversibly inhibited DNA synthesis in cultured mouse L cells to 50% of controls at 10 microM; 3.3 mM Cr(III) did not . At 0.3 mM, Cr(III) and Cr(VI) inhibited DNA synthesis in permeabilized L cells to 50% of control values . Cr(III) was a stronger inhibitor of DNA synthesis in the DNA-Escherichia coli DNA polymerase I system than was Cr(VI) . The inhibitory effect of Cr(VI) depended on the ratio of Cr/DNA and Cr/enzyme; on the other hand, the increase in the concentration of DNA polymerase did not affect the inhibition of Cr(III), Cr(III), below the inhibitory concentration, produced an increase in the incorporation of {3H}dTMP into DNA; this was not observed with Cr(VI).

Gene, 1985, 34(2-3), 315 - 23
Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites; Wells JA et al.; A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations . Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene . The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence . The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted . The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites . Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette . To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target . This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.

Cytobios, 1985, 42(167-168), 193 - 207
Purified colicin as cytotoxic agent of neoplasia: comparative study with crude colicin; Farkas-Himsley H et al.; Purification of a bacteriocin, colicin, from Escherichia coli HSC10, is described . A 1,800 fold purified colicin was obtained and found to be an acidic polypeptide with a molecular weight of 82,000 daltons . The effect of colicin on bacterial and mammalian cells during the transition from a crude to a pure preparation is given . A turbidimetric assay for bacterial growth inhibition and 3H-thymidine uptake inhibition for measuring the effect on mammalian cells, was used . Colicin HSC10 caused DNA loss from the bacteriocin-sensitive mammalian cells, which increased with dose and time of exposure . Therefore, flow-cytometry, which detects DNA loss from the bacteriocin-affected mammalian cells, was also used to evaluate the bacteriocin potency . Similar quantitative results were obtained to those using 3H-thymidine uptake inhibition, in terms of microgram protein of pure colicin required to affect adversely 50% of the cells . The bacteriocins were found to retain their activity following freezing and thawing, both as crude and pure preparations, against both bacterial and mammalian cells.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 51 - 8
Elongation of the murein sacculus of Escherichia coli; Park JT et al.; Elongation is an orderly multisite process in which, at each of about 90 growth points, two strands of murein are concurrently being synthesized, inserted and cross-linked to the sacculus . The enzymes involved move around the circumference of the cytoplasmic membrane at a rate of about 5 nm per second as new strands are being inserted, and this rate is sufficient to double the length of the cell during the cell division cycle.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 159 - 64
Regulation of chromosome segregation in Escherichia coli; Jaffe A et al.; Cell division is tightly coupled to DNA replication in Escherichia coli, as evidenced by the rarity of anucleate cells in steady state cultures . When DNA synthesis is arrested, cell division also comes to a halt and filamentous growth ensues, again with little formation of anucleate cells . To test the precise role of the SfiA division inhibitor during filamentous growth, we compared sfiA+ and sfiA- strains in their response to thymine starvation . More residual division was observed in the sfiA mutant culture, and autoradiographic analysis revealed that 13% of the final population consisted of cells of normal size containing no DNA compared to 0.9% in the thymine-starved sfiA+ culture . The SfiA division inhibitor is known to be synthesized massively during thymine starvation as part of the inducible SOS response . We conclude that it prevents aberrant division and formation of anucleate cells, thus assuring proper segregation when DNA synthesis is perturbed . The SfiC division inhibition mechanism, also associated with the SOS response, does not affect cell division during thymine starvation . On the other hand, an SOS-independent mechanism of division arrest clearly comes into play during thymine starvation of a sfiA sfiC mutant: although considerable aberrant division took place, the majority of the cells formed long filaments with 1 or 2 masses of DNA . Thus, E . coli assures proper chromosome segregation by two systems when DNA replication is perturbed: the rapid, efficient SfiA division inhibitor and a less stringent SOS-independent mechanism.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 147 - 57
Envelope protein changes, autoagglutination, sensitivity to hydrophobic agents and a conditional division lesion in Escherichia coli strains carrying ColV virulence plasmids; Rowbury RJ et al.; The presence of the virulence plasmids ColV,I-K94 or ColV-K30 in Escherichia coli produces a number of cell membrane and envelope changes . The most striking of these are (1) the presence of the 33K VmpA outer membrane protein and (2) the ColV-associated occurrence of autoagglutination . The VmpA protein is a plasmid-encoded outer membrane protein which is synthesized from a larger precursor . It is distinct from the chromosomally-encoded OmpA protein but resembles it in a few respects . The VmpA protein does not appear to be involved in colicin synthesis or immunity, or in plasmid transfer . This protein was found in 6 out of 8 new ColV+ isolates, but not in 2 ColIa+ strains . ColV-induced autoagglutination occurred for strains grown in static culture at 37 but not at 25 degrees C . Detergents prevented agglutination, as did the presence in a ColV+ strain of a fi+ plasmid, ColB . Autoagglutination may be a virulence phenotype . Associated with the ability of ColV+ bacteria to agglutinate was inhibition of motility . ColV+ bacteria also showed changes in envelope permeability indicated by inhibitor sensitivity and by a ColV-associated suppression of the lac Y lesion . Some ColV,I-K94+ strains showed a mucoid colonial phenotype and this ability to form mucoid colonies was efficiently transferred with ColV but apparently not without it . The mucoid ColV+ strains resembled lon mutants in UV-sensitivity, division behaviour and sensitivity to lambda phage.

Ann Inst Pasteur Microbiol, 1985 Jan-Feb, 136A(1), 131 - 8
Physiological and geometrical conditions for cell division in Escherichia coli; Woldringh CL et al.; During recovery of division in filaments of a temperature-sensitive DNA replication mutant, DNA-less cells were formed with a broad variation in cell lengths . It is argued that segregated nucleoids are necessary to indicate the site of division and that, in their absence, the cell has no additional mechanism to locate the division site . A second condition for division is based on geometrical arguments: the cell must be able to reestablish its original surface to volume ratio or its diameter, either of which may decrease during elongation . Electron microscopy and auto-radiography of radio-labelled sacculi prepared from E . coli MC4100 lysA, cultured in glucose minimal medium, showed that these cells elongate at a constant diameter and double their rate of surface synthesis during the constriction period.

Allergol Immunopathol (Madr), 1985 Jan-Feb, 13(1), 41 - 4
The lipopolysaccharide polyclonal response is a function of the background response; Vidal Gomez J; The aims of this report are: to investigate the kinetics of the E . coli lipopolysaccharide polyclonal response as a function of the background response, to test the capacity of lipopolysaccharide to activate presumably resting memory cells for IgM . Because mice do not produce a plaque-forming cell background against rat erythrocytes, the mouse-rat erythrocyte system was chosen . Random bred Swiss albino mice, aged 2-3 months, were inoculated with rat erythrocytes (5 X 10(5)/mouse) and the course of the plaque response was followed up; no response was detected on day 0, it rose sharply up to day 4, dropped abruptly between days 4 and 7 and trailed off afterwards to disappear by day 29 . As to the lipopolysaccharide-triggered anti-rat erythrocyte response, lipopolysaccharide (100 micrograms/mouse) was inoculated on days--3, 1, 4, 7, 18 and 29 . post-rat erythrocytes and the responses were measured 3 days after lipopolysaccharide inoculation . The curve paralleled that of the antigenic response, although at a higher level; thus, both the specific and the polyclonal responses followed the same kinetics . At one month post-rat erythrocyte priming, the animals displayed a background comparable to that of naive mice, although their IgM response to a booster (2 X 10(7) erythrocytes, iv) was faster and greater (p less than 0.01) than the primary response to the same dose; i.e., there was IgM memory . Nevertheless, lipopolysaccharide stimulation (100 micrograms/mouse, ip) of primed animals gave a response comparable (p greater than 0.10) to that of naive mice; i.e., lipopolysaccharide alone was unable to elicit a secondary-like response.

Microbios, 1985, 42(168), 111 - 7
Influence of the genetic background on cell division and cell lysis: behaviour of different Escherichia coli strains carrying the ts-52 or the ftsA-3 mutation; Tormo A et al.; The analysis of Escherichia coli strains harbouring division mutations, namely the ts-52 or the ftsA-3 division alleles, in different genetic backgrounds showed that treatment with chloramphenicol in cells incubated at the restrictive temperature induced either cell lysis (ts-52 and ftsA-3 in MC-6 genetic background) or cell division (ts-52 in OV-2 genetic background) . This chloramphenicol treatment of ftsA-3 filaments (previously designated at divA) does not induce cell division but does induce cell lysis.

Mol Gen Genet, 1985, 199(1), 95 - 100
Lysis protein encoded by plasmid ColA-CA31 . Gene sequence and export; Cavard D et al.; A gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion . The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A . Transcription of cal is in the same direction as caa, that is in the opposite direction to cai . Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins . The C-terminal part also contains a core of hydrophobic residues . The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13 . The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A . The cal protein undergoes slow processing with loss of the N-terminal "signal" region and the mature form is released into the medium together with colicin A.

Mol Gen Genet, 1985, 199(1), 55 - 8
In vivo transcription from deletion mutations introduced near Escherichia coli ribosomal RNA promoter P2; Thayer GC et al.; In order to characterize the tandem rrnB promoters transcribing one of the ribosomal RNA operons in E . coli we subcloned the basic promoter unit . This 185 bp fragment extends from -64 to +121 counted from the transcription start site of upstream promoter P1 . The start site of downstream promoter P2 is also included in the promoter cartridge . S1 mapping experiments show that both promoters on this fragment are active in vivo . BAL-31 deletion mutations generated at the start site for promoter P2 were also tested by S1 mapping . Transcription from P2 remained active in all cases with the exception of one construction which lacks the -10 region . This demonstrates that the sequences downstream from the -10 region of P2 are not essential for basic promoter function.

Mol Gen Genet, 1985, 199(1), 101 - 5
Nucleotide sequence of a regulatory region of the uidA gene in Escherichia coli K12; Blanco C et al.; Multiple regulatory events are involved in the expression of the uidA gene . A regulatory region of this gene has been located on a 460 base pair Sau3A-EcoRI fragment and its nucleotide sequence was determined by the dideoxy method using pEMBL plasmids . A preliminary analysis of this sequence revealed the presence of numerous palindromic structures with some overlaps.

J Membr Biol, 1985, 84(2), 157 - 64
Na+ (Li+)-proline cotransport in Escherichia coli; Chen CC et al.; Na+ and Li+ were found to stimulate the transport of L-proline by cells of Escherichia coli induced for proline utilization . The gene product of the put P gene is involved in the expression of this transport activity since the put P+ strains CSH 4 and WG 148 show activity and the put P- strain RM 2 fails to show this cation coupled transport . The addition of proline was found to stimulate the uptake of Li+ and of Na+ . Attempts to demonstrate proline stimulated H+ uptake were unsuccessful . It is concluded that the proline carrier (coded by the put P gene) is responsible for Na+ (or Li+)-proline cotransport.

J Basic Microbiol, 1985, 25(1), 57 - 68
{Cell wall proteins of Escherichia coli: a substance resembling the thermolabile enterotoxin (LT)}; Seltmann G et al.; In ultrasonic extracts of all 19 investigated non-enterotoxigenic E . coli strains a substance (LTLS) could be detected reacting positively in all tests which are commonly used to detect specifically E . coli thermolabile enterotoxin (LT) . Culture supernatants of these strains in general did not contain LTLS in detectable amounts . LTLS can be found in the whole cell, however, the membrane fraction contains the highest quantities . Released LTLS appears mainly aggregated with components of the cell wall, especially with lipopolysaccharides . This fact in combination with the very low quantities produced by the bacteria renders very difficult purification of LTLS.

Gene, 1985, 33(2), 227 - 34
In vitro stimulation of Escherichia coli RNA polymerase sigma subunit synthesis by NusA protein; Peacock S et al.; A simplified DNA-directed in vitro system which measures synthesis of the NH2-terminal dipeptides of gene products has been used to study the expression of rpoD, the gene coding for the sigma subunit of Escherichia coli RNA polymerase . The rpoD gene is part of a complex operon which also includes the genes for ribosomal protein S21 (rpsU) and primase (dnaG) . Primary promoters have been identified upstream of the structural genes, but there are secondary (internal) promoters within the dnaG gene that are involved in the expression of rpoD . Significant expression of the rpsU and rpoD genes was observed in the in vitro dipeptide system using plasmid pBS105, which contains both external and internal promoters . With plasmid pMRG-1, which contains only the internal promoters, only rpoD expression was observed . From either template, synthesis of the NH2-terminal dipeptide of sigma, fMet-Glu, is stimulated about threefold by the E . coli nusA gene product . In addition, NusA protein stimulates synthesis of the entire sigma protein in a defined in vitro system . NusA protein has no effect on the expression of the upstream gene rpsU, and the stimulation of rpoD expression by NusA protein is at the level of transcription . The results are consistent with the known role of NusA protein in modulating transcription at pause or attenuation sites.

Gene, 1985, 33(2), 197 - 206
Cloning and expression of a puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli; Vara J et al.; A gene (pac) encoding a puromycin N-acetyl transferase (PAC) of Streptomyces alboniger ATCC12461 was cloned in the Streptomyces plasmid pIJ702 and expressed in S . lividans 1326 . Several clones resistant to puromycin were isolated and shown to carry pIJ702 with different inserts of S . alboniger DNA . They were classified as of low and high activity according to the levels of enzymatic activity expressed by them . The different levels of expression were related to the two possible orientations of the S . alboniger DNA inserts in the pIJ702 vector . Six of the recombinant plasmids contain a common 1.6-kb DNA sequence which, by subcloning experiments, was shown to carry a pac gene encoding PAC activity . The pac gene was subcloned next to the lac promoter of Escherichia coli plasmid pUC19 . Only one of the two possible orientations of insertion expressed PAC activity, suggesting that it was dependent on the lac promoter . Accordingly, isopropylthio-beta-D-galactoside (IPTG) was able to stimulate the expression of the enzyme activity . These results allowed the direction of transcription of the pac gene to be determined.

Exp Biol, 1985, 43(3), 191 - 9
Interaction of lipid vesicles with an heptoseless strain of Escherichia coli; Proulx P; The conditions for uptake of lipid vesicles by the deep rough mutant of Escherichia coli, strain D21F2, and the parent strain, K12 were studied . A variety of lipids including phosphatidylcholine, phosphatidylethanolamine, diglyceride, cholesterol and palmitic acid were taken up much more readily by the deep rough mutant than the K12 strain . The uptake of lipid in the mutant strain was enhanced by Ca2+ at an optimal concentration of 2 mM, by alkaline pH and by growth of the cells up to the late exponential phase . With K12 cells, cholesterol and phosphatidylethanolamine were taken up from equimolar mixtures at similar rates thus suggesting the involvement of a fusion process . With D21F2 cells, the endogenous lipids of which had been labelled by growth in {3H} acetate, the uptake of exogenous {32P} phosphatidylethanolamine and {32P} lysophosphatidylethanolamine was not accompanied by a loss of endogenous lipid to the incubation medium . This precluded the involvement of an exchange mechanism . The absorbed exogenous lipid and the endogenous lipids of D21F2 cells displayed the same susceptibility to hydrolysis by added phospholipase C . This indicated that the exogenous lipid is inserted into lipid core of the membrane rather than adsorbed at the cell surface.

Eur Surg Res, 1985, 17(3), 160 - 6
Peritonitis and septic shock--an evaluation of two experimental models in the rat; Martinell S et al.; Two different experimental models for inducing septic shock have been characterized . In one, septic shock was induced by intraperitoneal injection of live Escherichia coli bacteria . This resulted in a dose-dependent mortality . Those animals surviving the first 24 h are considered as permanent survivors . In the other models, septic shock and peritonitis was induced by ligation and needle punctures of the cecum . This resulted in a slower development of shock which was almost invariably lethal within 96 h . Arterial blood pressure remained within the normal range in both models for up to 3 h after inducing peritonitis . Then a rapid deterioration was noticed in animals injected with live E . coli . White blood cells and platelets in arterial blood were reduced compared to controls in both groups . This reduction was more pronounced in animals injected with live E . coli . Both models are considered as useful tools in further studies of the pathophysiology of peritonitis and septic shock.

Eur Surg Res, 1985, 17(3), 140 - 9
Derangement of hepatic energy metabolism in lead-sensitized endotoxicosis; Taki Y et al.; Lead-sensitized endotoxicosis was investigated in rats in terms of hepatic energy metabolism . Lead acetate (Ld, 20 mg/kg BW) or endotoxin (Etx, 4 mg/kg BW) caused no deaths within 48 h . Ld plus Etx resulted in a lethality of 50 and 100% within 6 and 12 h respectively . Etx or Ld alone caused a slight but significant decrease in the hepatic tissue levels of total adenine nucleotides and/or ATP at 3 and 6 h after the application . The energy charge potential (ECP) remained normal . The ketone bodies acetoacetate and beta-hydroxybutyrate and the ratio AcAc/beta-OHB as well as the mitochondrial oxidative phosphorylation tended to increase; the hepatic tissue levels of pyruvate and lactate were increased after 3 h, indicative of an accelerated glycolysis . These alterations were no longer detectable after 6 h . In lead-sensitized endotoxemia (Ld + Etx), the total adenine nucleotides and ATP of the liver tissue decreased significantly to 84% (86%) and 71% (52%) of the controls within 3 and 6 h respectively, and the ECP had decreased from 0.865 to 0.684 at 6 h . The ketone bodies were increased, while the ratio AcAc/beta-OHB was significantly decreased at 6 h . The hepatic tissue lactate remained elevated . The mitochondrial activity was significantly reduced . A hyperglycemia (175 mg . dl-1) at 3 h changed into a hypoglycemia (50 mg . dl-1) at 6 h . It is suggested that Ld plus Etx causes a rapid impairment of hepatic mitochondria which leads to a drastic disturbance of the hepatic energy metabolism including hypoglycemia and contributes to an enhanced lethality in Ld-sensitized endotoxicosis.

Clin Physiol Biochem, 1985, 3(1), 29 - 42
Human insulin; Raptis S et al.; The two human insulins of clinical importance are (a) semisynthetic human insulin prepared from pork pancreas by enzymatically substituting threonine for alanine-the last amino acid in the beta chain-thereby transforming pork insulin in vitro to human insulin; and (b) biosynthetic human insulin synthesized biotechnologically in Escherichia coli-K12 . Using this latter technique, it is possible to produce mass quantities of highly purified insulin for the treatment of insulin-dependent diabetics, avoiding the problems inherent in supplies of insulin produced from animal pancreas . It has been suggested that to avoid confusion the two human insulins should be called semisynthetic human insulin of pork origin and biosynthetic human insulin of E . coli origin, respectively . These insulins have four advantages over highly purified animal insulins: (a) they induce lower titers of circulating insulin antibodies; (b) their subcutaneous injection is associated with fewer skin reactions; (c) they are absorbed more rapidly from the injection site; and (d) less degradation occurs at the site of injection . These data indicate that newly diagnosed insulin-dependent diabetes, particularly in children, should be treated with either of the two human insulins . The warranty against inadequate supplies of insulin offered by biosynthetic human insulin makes the use of pork insulins unnecessary and beef insulins totally useless.

Biochimie, 1985 Jan, 67(1), 83 - 9
Roles of the trkB and trkC gene products of Escherichia coli in K+ transport; Booth IR et al.; Mutations at the trkB and trkC loci of Escherichia coli produce an abnormal efflux of K+ . The mutations are partially dominant in diploids and revert frequently by what appears to be intragenic suppression to the null state . The mutations can be reverted by insertion of Tn10 into the mutated gene, and spontaneous revertants are fully recessive to the mutant allele in diploids . K+ efflux produced by NEM* and by DNP* persists in strains with presumed null mutations at either locus, indicating neither gene product is the primary target for the effect of these inhibitors on K+ efflux . The results are consistent with the view that trkB and trkC encode independent systems for K+ efflux . Mutations at these loci alter regulation of the process so that K+ efflux occurs inappropriately . A second mutation to the null state abolishes this abnormal K+ efflux . These genes may encode K+/H+ antiporters, an activity postulated to mediate K+ efflux and demonstrated to exist in E . coli and other bacteria.

Biochimie, 1985 Jan, 67(1), 69 - 73
{Mechanisms of efflux of a substrate accumulated by the lactose permease of Escherichia coli: theoretical and experimental study}; Kepes F; At the steady-state of accumulation of intracellular lactose by the beta-galactoside permease of Escherichia coli, the rate of efflux of the substrate is equal to its rate of influx . An original experimental method and a mathematical processing of the experimental data are proposed to evaluate the relative involvements of the permease-mediated pathway and of the diffusion component in this efflux . The method consists of inducing the lac operon of the bacteria, and then of removing the inducer and allowing the cells to grow further . The permease content and the membrane surface of diffusion are thus varying independently in such a "de-induction" experiment, along which lactose uptake has been monitored at different times . The analysis of the experimental data show that, under conditions of maximal induction, over 95% of the efflux passes through the energized permease . The relevant parameters of the efflux of lactose have been computed and their values allow the prediction of most classical observations, as well as the prediction, never checked, that under physiological conditions, the higher the external substrate concentration, the higher the permease-mediated efflux, according to a saturation kinetics.

Biochimie, 1985 Jan, 67(1), 59 - 67
The melibiose permease system of Escherichia coli K12; Burstein C et al.; The melibiose permease system of E . coli K12 has been explored using a strain deficient in lactose permease: 300 P . The accumulation of 1-S-methyl-beta-D-thiogalactopyranoside (TMG) was observed . The uptake system was inducible by melibiose and a number of analogs at 30 degrees C . At higher temperatures the differential rate of synthesis decreases until becoming negligible at 42 degrees C . The uptake tends toward a steady state which corresponds to an accumulation several hundredfold over the sugar concentration in the medium . At a given temperature the steady state level was proportional to the initial rate of uptake whatever the degree of induction and the substrate concentration . Lowering the temperature decreased the initial rate of uptake but increased the steady state level . This uptake system was pH dependent with better efficiency at pH 8 . It was also dependent on the presence of sodium or lithium ions active at 5 mM whereas potassium at 170 mM enable only about half maximal uptake . The uptake in a medium with choline chloride was less than one fifth of optimal activity . Addition of Li+ brought about half maximal activation at approximately 0.5 mM . The activation consists mainly in a decrease of apparent Km . The emphasis of this study was put on the similarities and differences with lactose permease which is able to transport the same sugar to approximately the same extent . Inducer specificities and substrate specificities were compared and a method of measuring the two activities in the same cells was devised.

Biochimie, 1985 Jan, 67(1), 45 - 58
{The Escherichia coli cell cycle}; Joseleau-Petit D; This review summarizes present knowledge of the bacterial cell cycle with particular emphasis on Escherichia coli . We discuss data coming from three different types of approaches to the study of cell extension and division: The search for discrete events occurring once per division cycle . It is generally agreed that the initiation and termination of DNA replication and cell septation are discrete events; there is less agreement on the sudden doubling in rate of cell surface extension, murein biosynthesis and the synthesis of membrane proteins and phospholipids . We discuss what is known about the temporal relationship amongst the various cyclic events studied . The search for discrete growth zones in the cell envelope layers . We discuss conflicting reports on the existence of murein growth zones and protein insertion sites in the inner and outer membranes . Elucidation of the mechanism regulating the initiation of DNA replication . The concept of "critical initiation mass" is examined . We review data suggesting that the DNA is attached to the envelope and discuss the role of the latter in the initiation of DNA replication.

Biochimie, 1985 Jan, 67(1), 141 - 4
Growth of the Escherichia coli cell envelope; Jaffe A et al.; The growth pattern of the Escherichia coli envelope was studied by immunoelectron microscopy, using the outer membrane protein LamB specifically labelled by a double antibody gold particle technique . An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of LamB synthesis . In the generation following turn-off no lamB-free regions appeared, strongly suggesting that bulk outer membrane material is not inserted in restricted growth zones.

Biochemistry, 1985 Jan 1, 24(1), 40 - 6
Kinetic analysis of respiratory nitrate reductase from Escherichia coli K12; Morpeth FF et al.; Purified respiratory nitrate reductase from Escherichia coli is able to use either reduced viologen dyes or quinols as the electron donor and nitrate, chlorate, or bromate as the electron acceptor . When reduced viologen dyes act as the electron donor, the enzyme follows a compulsory-order, "Theorell-Chance" mechanism, in which it is an enzyme-nitrate complex that is reduced rather than the free enzyme . In contrast, if quinols are used as the electron donor, then the enzyme operates by a two-site, enzyme-substitution mechanism . Partial proteolysis of the cytochrome b containing holoenzyme by trypsin results in loss of cytochrome b and in cleavage of one of the enzyme's subunits . The cytochrome-free derivative exhibits a viologen dye dependent activity that is indistinguishable from that of the holoenzyme, but it is incapable of catalyzing the quinol-dependent reaction . The quinol-dependent, but not the viologen dye dependent, activity is inhibited irreversibly by exposure to diethyl pyrocarbonate and reversibly by treatment with 2-n-heptyl-4-hydroxyquinoline N-oxide . We conclude that the holoenzyme has two independent and spatially distinct active sites, one for quinol oxidation and the other for nitrate reduction.

Biochemistry, 1985 Jan 1, 24(1), 221 - 9
Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport; Herzlinger D et al.; Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant "uncoupled" for beta-galactoside/H+ symport {Wong, P . T . S., Kashket, E . R., & Wilson, T . H . (1970) Proc . Natl . Acad . Sci . U.S.A . 65, 63}, are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H+ electrochemical gradient (interior negative and alkaline) . Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H+ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225) . Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles . When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide {Viitanen, P., Garcia, M . L., Foster, D . L., Kaczorowski, G . J., & Kaback, H . R . (1983) Biochemistry 22, 2531} . That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent Km . Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1985 Jan 1, 24(1), 158 - 62
RecA protein rapidly crystallizes in the presence of spermidine: a valuable step in its purification and physical characterization; Griffith J et al.; The RecA protein of Escherichia coli, whether pure or in a crude cell lysate, will rapidly form small crystals (microcrystals) in the presence of low concentrations of spermidine . We describe the conditions of time, pH, and polyamine concentration over which crystallization occurs . Microcrystal formation is inhibited by concentrations of chloride over 25 mM and concentrations of phosphate or sulfate ions as low as 2 mM . Crystallization is not inhibited by high concentrations of other proteins, and the RecA protein microcrystals are easily collected by brief centrifugation . This provides a powerful purification step with high yield . Using this novel property, we prepared over 200 mg of RecA protein at least 95% pure with a single-strand DNA-dependent ATPase activity of 98% from 65 g of cells in 2-3 days . Spermidine was easily removed from the RecA protein by dialysis.

Basic Life Sci, 1985, 31, 339 - 51
Mutagenesis by incorporation of alkylated nucleotides; Topal MD; The cellular DNA precursor pool was shown to be a target for N-methyl-N-nitrosourea, a potent mutagen and carcinogen . O6medGTP, a product of this interaction, was chemically synthesized and shown to be incorporated into DNA in vitro by Klenow E . coli pol I and phage T4 DNA polymerases . O6medGTP incorporated predominantly opposite T template residues and to a lower extent opposite C . At some loci incorporation of O6medGTP caused DNA synthesis arrest . The significance of the behavior of O6medGTP for mutagenesis in vivo is discussed.

Antibiot Med Biotekhnol, 1985 Jan, 30(1), 22 - 7
{Role of the conjugative transfer of R-plasmids in competitive interactions between plasmid-containing and plasmid-free strains of Escherichia coli in continuous culture}; Boronin AM et al.; Competition of the plasmid-containing strain C600 (RP4) (pBS94) of E . coli and the plasmid-free strain C600 rifr of E . coli in chemostatic and pH-static continuous cultures resulted in displacement of the plasmid-free strain in spite of its higher specific growth rate . Conjugative transfer of RP4 and pBS94 plasmids into the cells of the plasmid-free strain was observed in the experiments . Competition of strains C600 (pBS94) (RP4 Tra-) and C600 rifr in the chemostatic culture in the absence of the plasmid conjugative transfer resulted in displacement of the plasmid-containing strain from the population, while in the pH-static culture, the initial ratio of the plasmid-containing and plasmid-free bacteria was preserved . The conjugative transfer of the plasmids lowered the number of the plasmid-free cells in the population and might be considered as a factor stabilizing the plasmids in the bacterial population of the continuous cultures.

Z Naturforsch {C}, 1985 Jan-Feb, 40(1-2), 134 - 7
Enzymatic nitrate assay by a kinetic method employing Escherichia coli nitrate reductase; Schild J et al.; An enzymatic assay system for nitrate employing the membrane-bound nitrate reductase (EC 1.7.99.4) of E . coli is described . Contrary to previous enzymatic assay systems, the present method is a kinetic one, i.e . the substrate, nitrate, is assayed by measuring the reaction rate of the nitrate reductase-catalyzed reaction . Based on the observation that the nitrate reductase-catalyzed reaction obeys pseudo-first order kinetics, a test system is described allowing the assay of nitrate at a concentration as low as 1 ppm . The relatively high Michaelis-Menten constant for nitrate (0.3 mM) of the E . coli nitrate reductase favours nitrate assay by the kinetic method.

Scand J Gastroenterol, 1985 Jan, 20(1), 25 - 30
Studies of the phospholipase A2 activity of rat ileal mucosa; Tagesson C et al.; A rapid and simple procedure has been used to determine phospholipase A2 activity (EC 3.1.1.4) in rat ileal mucosa . We used 14C-oleate-labeled Escherichia coli as substrate for the phospholipase activity and a 0.45-micron Millipore filter to separate the product of hydrolysis--the 14C-oleic acid--from the unhydrolyzed substrate . The phospholipase A2 activity was optimal at pH 9.8 and at 2 mM Ca2+, but another peak of activity appeared at pH 7.2 . In addition, cell fractionation revealed yet another phospholipase A2 activity at pH 5.0 in the absence of Ca2+ . These findings suggest the presence of more than one phospholipase A2 in the ileal mucosa and points to the possible use of a simple procedure for studying their distribution and properties.

Plasmid, 1985 Jan, 13(1), 51 - 8
Transcription signals in a region essential for replication of plasmid R6K; Shafferman A et al.; A DNA fragment encoding for the enzyme chloramphenicol acetyltransferase (CAT) but lacking the CAT promoter sequence was used to probe transcription signals in a DNA fragment (640 base pairs) from plasmid R6K that plays a central role in R6K DNA replication . The R6K DNA fragment analyzed contains the seven 22-bp direct repeats which express R6K incompatibility and are required in cis for activity of all three of the R6K origins of replication . In addition to the previously identified promoter sequences for the R6K initiation protein pi (P pi), a relatively weak transcriptional promoter upstream to P pi was detected . On the opposite strand an efficient (94%) transcriptional terminator was identified . This terminator is located upstream to nucleotide sequences active in the repression of R6K DNA replication.

J Gen Microbiol, 1985 Jan, 131 ( Pt 1), 77 - 85
Energy coupling to K+ uptake via the Trk system in Escherichia coli: the role of ATP; Stewart LM et al.; The dual energy requirement (protonmotive force and ATP) of the Escherichia coli Trk potassium transport system has been investigated . Using inhibitors and unc mutants we show that Trk is not an ATPase but may be regulated by ATP . Possible mechanisms of energy coupling to Trk are discussed.

J Gen Microbiol, 1985 Jan, 131 ( Pt 1), 73 - 6
The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles; Eklund T; The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated . Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential) . Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.

J Gen Microbiol, 1985 Jan, 131 ( Pt 1), 113 - 8
Effect of adenine, cytidine and guanosine on the expression of the SOS system in Escherichia coli; Llagostera M et al.; Addition of cytidine or guanosine to UV-irradiated cells of a RecA+ strain of Escherichia coli did not produce any effect on the induction of two SOS functions: inhibition of cell division and expression of the umuC gene . Under the same conditions adenine gave a slight increase in the induction of these two responses . In a RecA441 mutant growing at 42 degrees C, both cytidine and guanosine inhibited these SOS functions, whereas adenine produced a large increase in their expression . Moreover, the ATP concentration of the RecA441 mutant at 42 degrees C showed a decrease which occurred earlier in the cells growing in the presence of cytidine or guanosine than in the absence of either compound . Adenine induced an increase of about three times the initial ATP concentration of this mutant at 42 degrees C which dropped quickly after 10 min . Neither cytidine nor guanosine increased the evolution of cellular ATP in UV-irradiated cells of the RecA+ strain, whereas adenine had only a slight positive effect . However, in UV-irradiated RecA+ cells with and without adenine, ATP levels dropped quickly to the initial value after 20 min . These data suggest that the influence of adenine, cytidine and guanosine on the expression of the RecA441 phenotype at 42 degrees C may be due to alteration of the cellular ATP concentration of this mutant.

Cardiovasc Res, 1985 Jan, 19(1), 32 - 7
Role of sympathetic nerve activity in endotoxin induced hypotension in cats; Koyama S et al.; Within 10 min of intravenous (iv) injection of E . coli endotoxin (1 mg X kg-1) in alpha-chloralose anaesthetised cats, mean blood pressure (MBP) fell significantly to 86% of the pre-endotoxin level . There followed a gradual decline in MBP, so that after 60 min MBP was depressed to 49% of the pre-endotoxin level . Preganglionic splanchnic nerve (PSN) activity decreased significantly before the rapid fall in MBP . There followed a gradual decrease in PSN activity coincident with the significant fall in MBP, so that 60 min after iv injection of E . coli endotoxin PSN activity fell to 40% of the pre-endotoxin level . Heart rate did not differ significantly from the pre-endotoxin level in the control group . However, the hypotension and the reduction of PSN activity following endotoxin were abolished by intracisternal (ic) pretreatment with phentolamine (0.5 mg X kg-1) . The heart rate in this group increased significantly, by 15% at 10 min and was maintained until the end of the experiment . These results indicate that in E . coli endotoxin hypotension the blood pressure falls together with the reduction in sympathetic outflow; and suggests that stimulation of central alpha-adrenergic receptors leads to an inhibition of activity in brainstem sympathetic pathways.

Can J Biochem Cell Biol, 1985 Jan, 63(1), 57 - 63
Chemical modification and hybrid enzyme formation as probes of the active site and subunit interactions in Escherichia coli succinyl-CoA synthetase; O'Connor-McCourt MD et al.; Succinyl-CoA synthetase from Escherichia coli has an alpha 2 beta 2 subunit structure and is known to display half-of-the-sites reactivity with respect to its phosphorylation by ATP . The studies reported herein are a component of our attempts to rationalize the heterologous tetrameric structure in terms of catalytic function . The isolated refolded beta subunit interacts specifically with an affinity column of agarose-hexane-CoA, consistent with the idea that the CoA-binding subsite of the active center is located on the beta subunit . The enzyme is inactivated by phenylglyoxal according to biphasic kinetics; saturating levels of the substrates CoA and ATP, alone or in combination, give only partial protection against such inactivation . Treatment of the enzyme with the sulfhydryl reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in rapid inactivation, accompanied by the reaction of three to four-SH groups per molecule; prolonged incubation with NBD-Cl eventually results in reaction of 16 of the 24 sulfhydryl groups of the tetramer . Hybrid enzyme preparations have been constructed by refolding mixtures containing beta and various ratios of native and NBD-Cl-modified alpha subunits . The loss of activity associated with the incorporation of chemically modified alpha is not that predicted by a simple model based on binomial distribution, but is complex and consistent with the kind of intersubunit communication that may be expected for catalytic cooperativity between the two active sites of the enzyme molecule.

Avian Dis, 1985 Jan-Mar, 29(1), 177 - 87
Surface and cloacal temperatures in chicks of different genetic stocks given an Escherichia coli inoculation; Dunnington EA et al.; The effect of Escherichia coli on surface and cloacal temperatures was assessed in pure line and hybrid chicks from both white rock and leghorn stocks . Body weights, surface temperatures, and cloacal temperatures were obtained from 2 to 30 days of age . Two groups of chicks, one 8 days and the other 30 days of age, were inoculated via the caudal thoracic air sac with E . coli, and effects were quantified as changes in body weight, surface and cloacal temperatures, lesions on heart and air sacs, and mortality . Pure line white rock chicks had the highest mortality from the E . coli inoculation and proportionally lost the most weight over the 48-hr period immediately following inoculation . Chicks inoculated at 30 days of age were affected more severely than those inoculated at 8 days . Surface and cloacal temperatures were not reliable predictors of resistance to an E . coli inoculation.

Anal Biochem, 1985 Jan, 144(1), 75 - 8
M13 clones carrying point mutations: identification by solution hybridization; Hobden AN et al.; A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described . The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis . Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate . The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography . This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.

Acta Chir Scand, 1985, 151(1), 73 - 6
Prevention of post-operative infection in appendicectomy by single dose intravenous metronidazole . An open prospective randomised study; Kling PA et al.; In an open prospective randomized study of appendicectomy, single-dose (1 g) intravenous metronidazole prophylaxis (and continued metronidazole plus nalidixic acid in perforated appendices) resulted in a lower overall post-operative infection rate (1.4%) than no prophylaxis and doxycycline therapy in the case of perforation (16%, p less than 0.025) . In patients with an unperforated appendix no infections occurred after single-dose metronidazole prophylaxis as opposed to 8.2% infections in untreated controls (p less than 0.025).

Urol Radiol, 1985, 7(1), 12 - 5
Blind-ending branch of bifid ureter: report of seven cases; Muraro GB et al.; Seven cases of bifid ureter with a blind-ending branch are presented: diagnosis was primarily made by excretory urogram . In 1 case the diagnosis was confirmed by retrograde pyelogram . The pelvicalyceal system showed ureteroureteral reflux into the blind-ending branch on photofluoroscopy and cineroentgenography . One rare case of blind-ending branch originating in the upper third of the ureter are described . The clinical aspects, roentgenographic findings, and treatment of these findings are reviewed.

Mol Biochem Parasitol, 1985 Jan, 14(1), 1 - 10
The biosynthesis of the histidine-rich protein of Plasmodium lophurae and the cloning of its gene in Escherichia coli; Kilejian A et al.; Uninucleate trophozoites and schizonts of Plasmodium lophurae were labeled metabolically with {3H}proline . Analysis of labeled parasites indicated that the histidine-rich protein (HRP) was the major polypeptide synthesized by both developmental stages; in trophozoites it represented a larger proportion of total labeled polypeptides . Polyadenylated RNA was prepared from trophozoites and translated in a rabbit reticulocyte lysate system in the presence of {3H}histidine . As compared to the approx . 50 kDa mature protein, in the cell-free system HRP was synthesized as an approx . 58 kDa precursor polypeptide . Size-selected, polyadenylated RNA was used to construct a complementary double-stranded DNA library in pBR322 and plasmids containing HRP sequences were identified by hybridization with a synthetic oligonucleotide probe.

Mol Biol (Mosk), 1985 Jan-Feb, 19(1), 267 - 77
{Genetic engineering of peptide hormones}; Rubtsov PM et al.; The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated . The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs . Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin . The gene of human calcitonin was created by combination of chemical and enzymatic synthesis . This synthetic gene was further cloned in pBR322 . The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E . coli were tested.

Mol Gen Genet, 1985, 198(2), 364 - 6
Maintenance of multicopy plasmid Clo DF13 III . Role of plasmid size and copy number in partitioning; Hakkaart MJ et al.; To elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell . From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cell population . The effect of plasmid copy number is dominant over the effect of plasmid size.

Mol Gen Genet, 1985, 198(2), 279 - 82
Cloning and nucleotide sequencing of the genes for ribosomal proteins S9 (rpsI) and L13 (rplM) of Escherichia coli; Isono S et al.; The genes for the ribosomal proteins S9 (rpsI) and L13 (rplM) of Escherichia coli have been cloned into a lambda phage vector termed L47.1 . The two genes were identified by infecting UV-light irradiated cells with the resultant phages and analyzing the protein products by two-dimensional gel electrophoresis . Suitable DNA fragments of the isolate were cloned subsequently into M13 phage vectors and their nucleotide sequence was determined by the dideoxy method . It is evident that the two genes form a transcriptional unit, the rplM gene being promoter-proximal . There is a typical signal sequence for transcriptional termination after the rpsI gene . The codon usage pattern in the two genes is similar to other ribosomal protein genes of E . coli.

Mol Gen Genet, 1985, 198(2), 263 - 9
Molecular cloning and characterization of the alkB gene of Escherichia coli; Kataoka H et al.; Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation . Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation . Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000 . Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E . coli chromosome.

Mol Gen Genet, 1985, 198(2), 207 - 12
Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction; Markham BE et al.; The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response . We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process . Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions . The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA . Reestablishment of repression from RecA commenced over 30 min earlier than from lexA . These results are fully consistent with the model that the functions result from the increased gene expression.

Folia Microbiol (Praha), 1985, 30(1), 3 - 16
The effect of inhibition of protein synthesis on UV-irradiated Escherichia coli uvrE cells; Bencova M; The uvrE (E . coli KS 114) cells carry a mutation in the gene that codes for helicase II . This is the protein responsible for replicative unwinding of double-helical DNA . The repair mode of such cells may be altered as compared with the wild type . The survival of uvrE cells during postirradiation incubation under inhibition of de novo protein synthesis was increased which indicates that this process of repair in uvrE cells is mediated by constitutive proteins and does not require any inducible products but takes a certain time . This inhibition of de novo protein synthesis causes also an inhibition of dimer excision, an increase of the parental DNA degradation and a decrease of parental and daughter DNA molar mass . On the other hand, it seems that induced proteins are formed in uvrE cells after UV irradiation but their influence is low in inducible repair and they can act only under conditions of complete protein synthesis.

Eur Surg Res, 1985, 17(2), 75 - 82
Increased plasma levels of vasoactive intestinal polypeptide in pigs during endotoxinaemia; Revhaug A et al.; Vasoactive intestinal polypeptide (VIP) is a highly potent vasodilatator . Cardiovascular changes and plasma VIP levels were studied during endotoxinaemia (Escherichia coli endotoxin 1 mg/kg) in a carefully monitored porcine model . Endotoxin infusion resulted in a profound but reversible shock and a substantial rise in plasma VIP levels . Increased levels of VIP could be demonstrated already after 30 min of endotoxin infusion and increased further during the infusion . Animals followed for a period of 60 h demonstrated slowly declining levels of VIP after endotoxin infusion but significantly elevated levels were usually found 24 h after infusion . Control animals did not show any changes in VIP during a similar procedure . Release of gastrointestinal peptides may be of importance during septic shock.

Eur Surg Res, 1985, 17(2), 128 - 32
Influence of intraperitoneal Escherichia coli with septicemia on the healing of colonic anastomoses and skin wounds . An experimental study in the rat; Hogstrom H et al.; In rats a standardized left colonic resection was performed and colonic continuity restored by an end-to-end anastomosis in one layer . The rats were subjected to an LD50 septic challenge by intraperitoneal injection of live Escherichia coli pre- or postoperatively . Controls received saline in a corresponding manner . Groups of animals were sacrificed on the 3rd or 7th postoperative day . The breaking strengths of the anastomosis and of the skin wound were measured . The collagen content of the anastomotic segments was analyzed . There were no differences in anastomotic or skin wound strength between septic animals and controls . Collagen content was unaffected . Wound healing was not influenced by sepsis in this model.

Acta Neurochir (Wien), 1985, 74(1-2), 68 - 71
Acute spinal epidural empyema . Observations from seven cases; Firsching R et al.; Seven cases with an acute epidural spinal empyema are presented . The best results are observed in cases with early treatment . Since this rare condition is not widely known and swift diagnosis and treatment is imperative, it is stressed that immediate referral of the patient with back pain and any signs of infection in addition to commencing neurological abnormalities to a neurological unit for myelography can be vital or can at least reduce the risk of permanent paraplegia.

Vet Pathol, 1985 Jan, 22(1), 54 - 9
Scanning and transmission electron microscopy of attaching effacing Escherichia coli in weanling rabbits; Peeters JE et al.; A strain of an enteropathogenic Escherichia coli, originally isolated from diarrheic weaned rabbits, produced diarrhea in five-week-old New Zealand white rabbits . Sequential examination of the intestines by scanning and transmission electron microscopy revealed that the strain attaches first to the Peyer's patch dome epithelium and later to the enterocytes of distal small intestine, cecum, and colon . Colonized cells became rounded and detached . The colibacilli were intimately associated with the apical cell membrane . Both absorptive and goblet cells were affected . The strain caused effacement of the microvillous border of colonized epithelial cells . Colibacilli were regularly seen in the partially evacuated cavities of goblet cells, but not in absorptive epithelial cells.

Reprod Nutr Dev, 1985, 25(1A), 49 - 60
Resistance of gnotobiotic large white and Chinese piglets to in vivo attachment of a K88ab enterotoxigenic Escherichia coli strain; Chappuis JP et al.; In vivo adhesion of a K88ab-positive enterotoxigenic Escherichia coli (ETEC) strain to the small intestinal wall of gnotobiotic, colostrum-deprived Chinese and Large White piglets was investigated . A non-enterotoxigenic, attachment factor-deprived E . coli strain, inoculated in association with the K88ab ETEC strain, was used as a marker to determine the content residues on the intestinal walls of gnotobiotic piglets . Both strains were selectively numerated in the luminal contents and on the washed wall from three segments of the small intestine . In vivo attachment was assessed by the ratio between the number of K88ab ETEC adherent to the wall and the number of both the E . coli strains which came from the luminal content residues and were not completely removed by washing . This ratio was first calculated in one group of 8 Large White piglets associated with the marker strain and with a K88ab antigen-deprived E . coli which derived from the parental K88ab ETEC strain . Thus, the range of the ratio was determined in those piglets where no attachment was expected . A comparison between the latter values of the ratio and the values obtained from 15 Large White and 10 Chinese piglets inoculated with the marker strain and the K88ab ETEC strain allowed us to classify the Large White piglets into adhesive (8 piglets) and non-adhesive (7 piglets) phenotypes and to show that the 10 Chinese piglets belonged to the non-adhesive phenotype.

Radiobiologiia, 1985 Jan-Feb, 25(1), 33 - 6
{Nomograms for determining the parameters for a probabilistic model of radiosensitivity}; Faisi Ch; Nomograms are introduced for the determination, from the experimental survival curves, of a and b parameters of the probabilistic model of cell radiosensitivity (proposed by Kapul'tsevich, 1978) . The parameter errors are estimated too . Some examples of using these nomograms for bacteria, yeast and mammalian cells are considered.

Radiobiologiia, 1985 Jan-Feb, 25(1), 29 - 32
{Estimation of the probability parameters for a model of radiation inactivation of cells from experimental survival curves}; Amirtaev KG et al.; A simple method is proposed for estimation of a and b parameters of the probability model of radiation inactivation of cells with a reference to the experimental survival curves . The examples of such an estimation for bacteria, yeast, and mammalian cells are discussed.

Radiobiologiia, 1985 Jan-Feb, 25(1), 20 - 3
{Effect of alpha-irradiation on sensitive and super resistant mutants of Escherichia coli}; Amirtaev KG et al.; A study was made of the sensitivity of E . coli (the wild type, rec A13 and Gamr 444 mutants) to gamma- and alpha-radiation . The most pronounced differences in radiosensitivity of the strains under study were noted with gamma-radiation . The sensitivity of the studied strains to alpha-radiation was levelled . The authors discuss the mechanisms of the effects observed in various E . coli strains exposed to ionizing radiation of different LET.

Mutat Res, 1985 Jan-Mar, 145(1-2), 49 - 53
Paradoxical behaviour of pKM101; inhibition of uvr-independent crosslink repair in Escherichia coli by muc gene products; Cupido M et al.; In strains of Escherichia coli deficient in excision repair (uvrA or uvrB), plasmid pKM101 muc+ but not pGW219 mucB::Tn5 enhanced resistance to angelicin monoadducts but reduced resistance to 8-methoxy-psoralen interstrand DNA crosslinks . Thermally induced recA-441 (= tif-1) bacteria showed an additional resistance to crosslinks that was blocked by pKM101 . Plasmid-borne muc+ genes also conferred some additional sensitivity to gamma-radiation and it is suggested that a repair step susceptible to inhibition by muc+ gene products and possibly involving double-strand breaks may be involved after both ionizing radiation damage and psoralen crosslinks.

Mutat Res, 1985 Jan-Mar, 145(1-2), 25 - 34
Mutational and recombinational events in carcinogen-modified plasmid DNA . Influence of host-cell repair genes; Abbott PJ; A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo{a}pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites . Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured . The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated . Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites . Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene . Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain . Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA . These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.

Membr Biochem, 1985, 5(4), 269 - 90
Reconstitution of the melibiose carrier of Escherichia coli; Wilson DM et al.; Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli . Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit {3H}-melibiose uptake by proteoliposomes . In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM . Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly . Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane . The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.

Am J Vet Res, 1985 Jan, 46(1), 75 - 9
Cellular and lipopolysaccharide subunit requirements for the caprine lymphoblastogenic response to endotoxin; Greenlee A et al.; Caprine B lymphocytes were established as the cell type that divided when cultured with lipopolysaccharide (LPS), and lipid A was defined as the in vitro mitogenic component of LPS . The conclusion that the caprine B lymphocyte was stimulated by LPS was based on 3 observations: (i) Numbers of B lymphocytes increased in cultures containing LPS, but not in unstimulated or concanavalin A-stimulated cultures, (ii) mixtures of T lymphocytes and monocytes did not incorporate tritiated thymidine when LPS was added, and (iii) removal of monocytes from mixtures of T and B lymphocytes did not reduce the LPS-stimulated reaction . Stimulation of B lymphocytes by LPS occurred when less than 1% monocytes were present and was augmented by greater than 5% monocytes . The lipid A subunit of LPS was most likely responsible for mitogenesis, since purified lipid A stimulated lymphocytes and the addition of polymyxin B, a specific inhibitor of lipid A activity, blocked the lymphocyte reaction.

Am J Vet Res, 1985 Jan, 46(1), 24 - 30
Endotoxin-induced hematologic and blood chemical changes in ponies: effects of flunixin meglumine, dexamethasone, and prednisolone; Ewert KM et al.; To evaluate the effect of certain drugs on hematologic changes, blood chemical values, and survival in endotoxin shock, anesthetized ponies were given (IV) endotoxin (Escherichia coli O55:B5) and then treated as follows: Group A ponies--given a saline infusion at 5 minutes and at 3 hours after they were given endotoxin; group B ponies--given flunixin meglumine at 5 minutes and at 3, 6, 9, and 24 hours after they were given endotoxin; group C ponies--treated with dexamethasone; and group D ponies--treated with prednisolone at 5 minutes and at 3, 9, and 24 hours after they were given endotoxin . Anesthesia was maintained for 4 hours, after which time the ponies were allowed to recover . Throughout the experiment, samples of blood were collected for blood gas, hematologic, and blood chemical values . The endotoxin effects were seen in the 4 groups: lactic acidosis, prolonged coagulation times, leukopenia, hemoconcentration, and elevated blood chemical values . Although none of the treatments prevented the effects of endotoxin, changes were less severe and survival times were longer in ponies treated with flunixin meglumine.

Scand J Immunol, 1985 Jan, 21(1), 11 - 20
Interleukin-1 production by a cloned line of human monocyte-like cells (CM-SM) . Correlation with state of differentiation; Revoltella RP et al.; Interleukin-1 (IL-1) production by the human monocyte-like cloned cell line CM-SM has been investigated as a function of the state of cell differentiation . CM-SM cells were induced to differentiate along the monocyte-macrophage lineage by bacterial lipopolysaccharides (LPS) or by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) . Cell differentiation was studied by various morphological, functional, cytochemical, and immunological variables, whereas IL-1 activity in the supernatants was measured by the lectin-primed thymocyte proliferation assay . Unstimulated CM-SM cells constitutively produced small amounts of IL-1, and most of the cells appeared relatively undifferentiated . LPS induced cell differentiation, but the effect was reversible, and the cells, in general, did not acquire a capacity for phagocytosis . IL-1 levels were increased about 10-fold over the controls . TPA induced further cell differentiation to macrophage-like cells capable of phagocytosis . IL-1 activity could not be measured directly in the supernatants owing to the synergistic effect of TPA in the assay system . Unequivocal removal of the phorbol was not achieved, but the data indicated that the 'real' levels of IL-1 in the TPA-induced cultures were not significantly higher than those from LPS-induced cultures.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 543 - 7
Interspersed blocks of repetitive and charged amino acids in a dominant immunogen of Plasmodium falciparum; Stahl HD et al.; We describe an antigen of Plasmodium falciparum that is a dominant immunogen in man . The corresponding cDNA clone, Ag231, expressing this antigen in Escherichia coli reacted in an in situ colony assay with sera from up to approximately equal to 93% of 65 people living in an area in which P . falciparum is endemic . Human antibodies affinity purified on immobilized Ag231 lysates identified the corresponding parasite antigen as a polypeptide of Mr approximately equal to 300,000 . It was present in schizonts and also in ring-stage trophozoites, where a speckled immunofluorescence pattern suggested an association with the erythrocyte . Its mRNA was enriched in merozoites relative to other blood stages, a distinctive property shared by a recently described antigen located on the surface of ring-infected erythrocytes, and it is encoded by a single gene having a number of allelic variants . The complete nucleotide sequence of Ag231 revealed a structural unit composed of 13 hexapeptide repeats flanked by a highly charged region containing both acidic and basic amino acids . This structural unit is itself repeated, so that blocks of repeats and charged units are interspersed along the molecule . The sequences within the repeats vary much more extensively than those in the charged units.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 488 - 92
Rapid and efficient site-specific mutagenesis without phenotypic selection; Kunkel TA; Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions . Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100% . The approach is applicable to phenotypically silent mutations in addition to those that can be selected . The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine . This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell . Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored . The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization . In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 483 - 7
Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA; Kelley RL et al.; Several classes of trp repressor mutants were selected and analyzed in vivo . Mutants that produced repressors with either enhanced or reduced activity were obtained . One class of mutants produced inactive or slightly active repressors that were trans-dominant to the wild-type repressor . The amino acid substitutions in many of these repressors were clustered in a segment of the polypeptide that is homologous to the DNA recognition domain of the lambda cro repressor . A second functionally important region of the trp repressor was identified; this region could participate in L-tryptophan binding . Observations with trpR nonsense mutants suggest that the first 67 residues of the repressor polypeptide are sufficient for subunit association.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 361 - 5
Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase; Robey EA et al.; Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains . However, the precise locations of the active sites and of the residues involved in catalysis are not known . To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue . One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis . Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer . The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers . The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers . The results constitute strong evidence for shared active sites in aspartate transcarbamoylase.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 339 - 42
Covalent enzyme-RNA complex: a tRNA modification that prevents a covalent enzyme interaction also prevents aminoacylation; Starzyk R et al.; Previous work indicates that aminoacyl-tRNA synthetases make a transient covalent adduct with cognate tRNAs, through Michael addition of an enzyme nucleophile to the carbon-6 position of uridine 8 . We report the selective reduction of the 5,6 double bond of 4-thiouridine at position 8 in Escherichia coli tyrosine tRNA, so as to prevent formation of the presumed covalent enzyme-nucleic acid adduct . The completely reduced tRNA molecules are inactivated for aminoacylation . With partial reduction, a mixed pool of active and inactive molecules is created and the degree of inactivation exactly matches the extent of 4-thiouridine reduction . The active molecules recovered from this mixed pool are specifically unaltered at position 8 . The results are consistent with the view that the covalent enzyme-RNA adduct is an obligatory intermediate for aminoacylation of this tRNA.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 282 - 6
Macrophage activation by monosaccharide precursors of Escherichia coli lipid A; Nishijima M et al.; Certain Escherichia coli mutants defective in phosphatidylglycerol biosynthesis accumulate two novel glycolipids, designated X and Y . Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3, and lipid Y has the same structure as X, except for the additional presence of a palmitoyl moiety on the N-linked beta-hydroxymyristate . We have examined the activities of X, Y, and several related compounds as activators of macrophages . Both X and Y induce morphological changes (spreading), prostaglandin E2 synthesis, and killing of tumor cells by mouse peritoneal macrophages in vitro, properties with which lipopolysaccharide and lipid A are also endowed . Both glycolipids have similar effects on the macrophage-like mouse cell line J774.1 . Selective removal from lipid X of either the ester-linked beta-hydroxymyristate at position 3 or the phosphate at position 1 abolishes activity . Our results show that the monosaccharides X and Y retain some of the properties of intact lipopolysaccharide and lipid A with respect to macrophage activation . Because the structures of X and Y are defined, our findings should facilitate the elucidation of the molecular mechanism of macrophage activation by lipid A.

Proc Natl Acad Sci U S A, 1985 Jan, 82(1), 29 - 33
Thioredoxin is required for filamentous phage assembly; Russel M et al.; Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin . Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase . The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it . The lesion lies within a highly conserved thioredoxin active site . Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production . Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system . A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly.

J Bacteriol, 1985 Jan, 161(1), 80 - 4
Conjugation-mediated genetic exchange in Legionella pneumophila; Dreyfus LA et al.; Genetic exchange mechanisms, to our knowledge, have not been reported for Legionella pneumophila, and consequently, studies on the genetic organization of L . pneumophila have not appeared in the literature . Here, we describe gene transfer mediated by broad host range conjugative plasmids in Legionella spp . Escherichia coli strains carrying plasmids RP1 and R68.45 (IncP1), S-a (IncW), and R40a (IncC), but not plasmids of incompatibility groups FI, FII, and FV, served as donors in matings with L . pneumophila Knoxville 1 (LPK-1) . Transconjugants selected by resistance to kanamycin (RP1, R68.45, and S-a) and carbenicillin (R40a) were observed at frequencies of 6.6 X 10(-3), 4.7 X 10(-3), 2.2 X 10(-4), and 5.4 X 10(-5), respectively . Plasmid transfer was not affected by DNase added to the mating medium . After plasmid transfer, LPK-1 stably maintained RP1, R68.45, and S-a, but not R40a . Plasmid-containing LPK-1 isolates also served as donors in agar plate matings with E . coli W1485-1 and naladixic acid-resistant mutants of LPK-1, Legionella micdadei, and Legionella longbeachii . Recombinational exchange of a chromosomal trait was demonstrated when a thymidine auxotroph of L . pneumophila was repaired by R68.45-mediated chromosomal mobilization of a prototrophic donor strain.

J Bacteriol, 1985 Jan, 161(1), 51 - 9
Chemotactic signaling in filamentous cells of Escherichia coli; Segall JE et al.; Video techniques were used to record chemotactic responses of filamentous cells of Escherichia coli stimulated iontophoretically with aspartate . Long, nonseptate cells were produced from polyhook strains either by introducing a cell division mutation or by growth in the presence of cephalexin . Markers indicating rotation of flagellar motors were attached with anti-hook antibodies . Aspartate was applied by iontophoretic ejection from a micropipette, and the effects on the direction of rotation of the markers were measured . Motors near the pipette responded, whereas those sufficiently far away did not, even when the pipette was near the cell surface . The response of a given motor decreased as the pipette was moved away, but it did so less steeply when the pipette remained near the cell surface than when it was moved out into the external medium . This shows that there is an internal signal, but its range is short, only a few micrometers . These experiments rule out signaling by changes in membrane potential, by simple release or binding of a small molecule, or by diffusion of the receptor-attractant complex . A likely candidate for the signal is a protein or ligand that is activated by the receptor and inactivated as it diffuses through the cytoplasm . The range of the signal was found to be substantially longer in a cheZ mutant, suggesting that the product of the cheZ gene contributes to this inactivation.

J Bacteriol, 1985 Jan, 161(1), 469 - 72
Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides; Muller ED et al.; We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides . We present evidence that the form II enzyme is encoded by a single gene in R . sphaeroides; however, this gene does hybridize to a second chromosomal locus.

J Bacteriol, 1985 Jan, 161(1), 368 - 76
Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine; Sullivan MA et al.; Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL . These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E . coli chromosome . Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes . The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations . The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALys, was impaired in asuE mutant strains, presumably at the 2-thiolation step.

J Bacteriol, 1985 Jan, 161(1), 326 - 32
Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants; Klock G et al.; Homologous and heterologous repressor-operator interactions among four different classes of tetracycline resistance determinants have been compared . These are represented by RP1/Tn1721 (class A), R222/Tn10 (class B), pSC101/pBR322 (class C), and RA1 (class D) . By the use of the purified repressor proteins of class A (TetRA) and class B (TetRB), operator sequences of all four classes are recognized by both with an identical stoichiometry of four repressor subunits per control sequence, but with different affinities . In vitro transcription has been used to demonstrate regulatory activities of TetRA and TetRB upon all four classes of tet genes . Tetracycline acted as an inducer . A functional relationship among the tet regulatory systems was also shown in vivo by complementation of a class A tetR'-galK fusion mutant with the tetR genes of classes A, B, and C . Repression of tetRA-linked galactokinase was ca . 80% in the presence of tetRA or tetRC, and ca . 50% in the presence of tetRB . Taken together, these results demonstrate heterologous repressor-operator interaction, suggesting close relationships among the four classes of Tcr determinants.

J Bacteriol, 1985 Jan, 161(1), 285 - 91
Identification of five new essential genes involved in the synthesis of a secreted protein in Escherichia coli; Oliver DB; To define additional components of the export machinery of Escherichia coli, I have isolated extragenic suppressors of a mutant {secA(Ts)} that is temperature sensitive for growth and secretion at 37 degrees C . Suppressors that restored growth at 37 degrees C, but that rendered the cell cold sensitive for growth at 28 degrees C, were obtained . The suppressor mutations fall into at least seven loci, two of which (prlA and secC) have been previously implicated in protein secretion . The five remaining loci (ssaD, ssaE, ssaF, ssaG, and ssaH) have been mapped by P1 transduction and appear to define new genes in E . coli . All of the suppressor mutations allow both enhanced growth and protein secretion of the secA(Ts) mutant at 37 degrees C, but not 42 degrees C, indicating a continued requirement for SecA protein . Strains carrying solely the cold-sensitive mutations show reduced levels of certain periplasmic proteins when grown at low temperatures . In at least one case, that of maltose-binding protein, this defect is at the level of synthesis of the protein . Since mutants in any of seven genes as well as secA amber mutants halt or reduce the synthesis of an exported protein, it appears that E . coli may possess a general and complex mechanism for coupling protein synthesis and secretion.

J Bacteriol, 1985 Jan, 161(1), 258 - 64
Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli; Rasmussen BA et al.; It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes . In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins . Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes . In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned . We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery . It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes . On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.

J Bacteriol, 1985 Jan, 161(1), 238 - 42
Structural modifications in the peptidoglycan of Escherichia coli associated with changes in the state of growth of the culture; Pisabarro AG et al.; By determining the composition in muropeptides of the murein of a number of strains of Escherichia coli, purified from cells at various states of growth, the sacculus was found to be considerably modified when cells stop active growth . Murein from resting cells becomes hypercross-linked and richer in covalently bound lipoprotein, whereas the mean length of the glycan chains is considerably reduced . The alteration of the sacculus occurs progressively during the transitions from active growth to stationary phase and vice versa.

J Bacteriol, 1985 Jan, 161(1), 189 - 98
Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships; Surin BP et al.; The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported . Along with the DNA sequence for the phoS gene reported previously (Surin et al., J . Bacteriol . 157:772-778, 1984; Magota et al., J . Bacteriol . 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region . The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG . The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU . Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele . We have confirmed that pstA was allelic to phoT32 . The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins . The pstA gene product appears to be an integral membrane protein.

J Bacteriol, 1985 Jan, 161(1), 169 - 78
Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope; Bankaitis VA et al.; An Escherichia coli mutant carrying delta malE12-18, a 21-base pair deletion confined to the coding DNA of the maltose-binding protein signal peptide, is unable to export maltose-binding protein to the periplasm efficiently . Consequently, such a strain is defective for the utilization of maltose as a sole carbon source . We obtained 16 mutants harboring extragenic delta malE12-18 suppressor mutations that exhibit partial restoration of export to the mutant maltose-binding protein . A genetic analysis of these extragenic suppressor mutations demonstrated that 15 map at prlA, at 72 min on the standard E . coli linkage map, and that 1 maps at a new locus, prlD, at 2.5 min on the linkage map . Our evidence indicates that the prlA and prlD gene products play an important role in the normal pathway for export of proteins to the cell envelope . Efficient execution of the secretory process requires that these prl gene products interact properly with each other so that a productive interaction of these gene products with the signal peptide also can occur . Our data suggest that proper assembly of a complex is required for efficient export of E . coli envelope proteins to their various extracytoplasmic compartments.

J Appl Physiol, 1985 Jan, 58(1), 274 - 84
Dexamethasone and indomethacin modify endotoxin-induced respiratory failure in pigs; Olson NC et al.; We studied the porcine pulmonary response to endotoxemia before and after administration of nonsteroidal antiinflammatory drugs (NSAID, i.e., indomethacin or flunixin meglumine) or dexamethasone (DEX) . Escherichia coli endotoxin was infused intravenously into anesthetized 10- to 12-wk old pigs for 4.5 h . In endotoxemic pigs, the phase 1 (i.e., 0-2 h) increases in pulmonary arterial pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient and the decreases in cardiac index (CI) and lung dynamic compliance (Cdyn) were blocked by NSAID . Thus phase 1 changes were cyclooxygenase dependent . Furthermore, these effects were blocked or greatly attenuated by DEX . During phase 2 of endotoxemia (i.e., 2-4.5 h), the increased PVR and decreased CI and Cdyn were not blocked by NSAID but were attenuated by DEX, suggesting the presence of cyclooxygenase-independent metabolites . Both NSAID and DEX blocked the endotoxin-induced increases in lung water, bronchoalveolar lavage (BAL) neutrophil, and BAL albumin content . The fall in plasma proteins persisted in NSAID but not DEX-treated pigs . We conclude that endotoxemia in the pig causes severe acute respiratory failure largely mediated by cyclooxygenase and possibly lipoxygenase products of arachidonic acid metabolism.

Int Arch Allergy Appl Immunol, 1985, 76(2), 116 - 9
Assessment of neutrophil chemotaxis and random migration in children and adults . Comparison between filter and agarose techniques; Hindocha PJ et al.; Polymorphonuclear leucocyte migration was studied in 16 children and 11 normal adults, both by a filter technique and under agarose . Assays were carried out in parallel . There were no significant differences (p greater than 0.5) between adult controls and children's mean neutrophil migration coefficients within methods, but highly significant differences were found between methods (p less than 0.001) both in adult and children's samples . The observed intermethod differences are shown to be the result of increased random migration under agarose in both age groups.

Hepatology, 1985 Jan-Feb, 5(1), 32 - 7
Effect of endotoxin on fibronectin and Kupffer cell activity; Richards PS et al.; The phagocytic activity of the reticuloendothelial system (RES) in the liver is important in host resistance to shock . Fibronectin is a large molecular weight glycoprotein which influences particulate uptake by phagocytic cells . This study addressed the effect of repeated low-dose endotoxin challenge on immunoreactive fibronectin and reticuloendothelial phagocytic function in rats . Intravenous Escherichia coli endotoxin increased circulating immunoreactive fibronectin by 100% within 24 hr; normalization was within 96 hr . Elevated fibronectin levels at 48 hr were associated with increased plasma opsonic activity as tested by liver slice phagocytic assay and RES stimulation, and in vitro uptake of gelatinized target particles by Kupffer cells in liver slices from endotoxin treated rats was significantly increased . Endotoxin tolerance was produced by repeated low dose challenge with endotoxin for 7 days and was associated with RES stimulation, even though the circulating fibronectin concentrations had returned to normal . By immunofluorescence, insoluble fibronectin was widely distributed in the liver in a pattern analogous to the sinusoidal vascular network . We suggest that increased RES phagocytic activity after low dose endotoxin challenge is due to early elevation of plasma fibronectin and cellular stimulation of phagocytic function followed by a sustained stimulation of Kupffer cells in the presence of normal fibronectin levels . Both cellular and humoral factors may contribute to increased Kupffer cell phagocytic activity during endotoxin tolerance.

Fed Proc, 1985 Jan, 44(1 Pt 1), 43 - 5
Metabolites of arachidonic acid in experimental lung vascular injury; Brigham KL; Several metabolites of arachidonic acid have potent effects on lung vascular and airway function . Some of these substances are released from the lungs when the lungs are diffusely injured . For example, after infusion of Escherichia coli endotoxin into unanesthetized sheep, high concentrations of both cyclooxygenase and lipoxygenase products appear in lung lymph . These products appear in sequential waves: thromboxane B2 peaks early, coincident with peak pulmonary artery pressure and maximum changes in lung mechanics; a prostacyclin metabolite peaks slightly later as pulmonary artery pressure begins to fall; both 5- and 12-lipoxygenation products peak even later, as lung vascular permeability begins to increase . Cyclooxygenase inhibitors attenuate the early changes in lung mechanics and pulmonary artery pressure caused by endotoxemia in sheep but do not prevent the later increase in lung vascular permeability . High doses of corticosteroids attenuate both the late phase increase in lung lymph lipoxygenation products and the increase in vascular permeability . These unequivocal associations between lung injury and endogenous generation of biologically active arachidonate metabolites suggest but do not prove that these substances mediate the pathophysiology.

Biull Eksp Biol Med, 1985 Jan, 99(1), 95 - 7
{Spontaneous and mitogen-induced proliferative activity of mononucleocytes in pollinosis patients}; Serov AA; The proliferative response of lymphocytes to mitogens was studied in 17 patients according to 3H-thymidine incorporation . The patients had high sensitivity to timothy pollen, confirmed by the allergological anamnesis, skin tests, and the presence of allergen-specific IgE-antibodies . Mononuclears of peripheral blood were cultivated with a lipopolysaccharide (LPS) to study the response to the polyclonal B cell activator, while with PHA to study the response of T cells over 7 days . The patients with pollinosis manifested increased spontaneous cell proliferation . The degree of the proliferative response of the cells to LPS and PHA was similar in patients and normal subjects . It is suggested that the magnitude of spontaneous proliferation influences the degree of the mitogenic response of B cells.

Arch Biochem Biophys, 1985 Jan, 236(1), 47 - 51
Extensive synthesis of poly{r(G-C)} using Escherichia coli RNA polymerase; Hall K et al.; Conditions have been found that allow for extensive synthesis of poly{r(G-C)} using Escherichia coli RNA polymerase with a poly{d(I-C) X poly{d(I-C)} template . The extensive synthesis, in which many copies of the template were produced by the enzyme, continued for 24 h . Repeated addition of the template was necessary for synthesis to continue, as the poly{r(G-C)} appeared to be binding to the template, inhibiting transcription by the polymerase . Four hundred ODU, or 20 mg of the poly{r(G-C)} has been prepared using 0.3 mg of protein.

Arch Biochem Biophys, 1985 Jan, 236(1), 252 - 9
Inhibitors of nitrofuran reduction in Escherichia coli: evidence for their existence, partial purification, binding of nitrofurantoin in vitro, and implications for nitrofuran resistance; Sastry SS et al.; Nitrofurantoin (NF)-resistant mutants of Escherichia coli were isolated as described previously (18) . One of the mutants (SSJ-2) was found to possess NF reductase activity equal to that of its parent (E . coli KL16) . Two NF-resistant transductional derivatives, SSJ-2A and SSJ-2B, were isolated using SSJ-2 as the donor . SSJ-2 was found to be a double mutant carrying two mutations, nfnA and nfnB, while SSJ-2A (nfnA) and SSJ-2B (nfnB) carried these mutations individually . Heated extracts from SSJ-2A and SSJ-2B were found to inhibit the reduction of NF by unheated extracts of the NF-sensitive strain E . coli KL16 in vitro . Unheated extracts of these mutants reduced NF poorly relative to E . coli KL16 . The poor reduction of NF by unheated extracts of SSJ-2A and SSJ-2B was greatly stimulated by heated extracts of SSJ-2B and SSJ-2A, respectively, and also by heated extracts of E . coli KL16 . When heated extracts of SSJ-2A and SSJ-2B were mixed in a particular ratio and added to unheated extracts of E . coli KL16 they lost their inhibitory activity . Two proteins, designated inhibitor A and inhibitor B, have been partially purified from heated extracts of SSJ-2B and SSJ-2A, respectively . Their respective molecular weights, as determined by gel chromatography, were 37,000 and 20,500 . The two inhibitors bound nitrofurantoin in vitro, and the NF-binding ability was lost when mixed in the molar ration of 3/1 (B/A) . These observations were rationalized in terms of a hypothesis which explains (i) maximal NF reduction in wild-type cells, (ii) maximal NF reduction of nfnA-nfnB- double mutant, and (iii) poor NF reduction in nfnA- or nfnB- single mutants . The possible role of these inhibitors in nitrofurantoin resistance is also discussed.

Pediatrics, 1985 Jan, 75(1 Pt 2), 172 - 6
Protective factors in milk and the development of the immune system; Hanson LA et al.; The neonate is immature in certain immunologic functions . The slow development of secretory immunoglobin A (IgA) seems to be compensated by selective transfer of secretory IgM into exocrine secretions on mucous membranes during the first few months of life . Secretory IgA and secretory IgM antibodies against Escherichia coli and poliovirus are already found in the neonate, possibly in response to the maternal anti-idiotypic IgG antibodies transplacentally exposing the fetus . Via such a mechanism, food antibodies could occur before direct food exposure in the infant . Human milk provides large amounts of antibodies (as a crude comparison, about 50 times the amount of antibodies given to a patient with hypogammaglobulinemia) . The milk antibodies, dominated by secretory IgA, protect especially against intestinal infections . The milk also contains oligosaccharide analogues to epithelial receptors for bacteria . They, as well as a number of milk components such as lactoferrin and lysozyme, may contribute to host defense . The food antibodies in human milk may influence the infant's immune response to foreign food proteins introduced during weaning.

J Trauma, 1985 Jan, 25(1), 53 - 9
Effect of a body burn on the lung response to endotoxin; Wong C et al.; Our purpose was, in general, to determine the effect of a body burn on the pulmonary response to endotoxemia and, specifically, to determine whether increased thromboxane (TxA2) production by the burn wound was responsible for the accentuated lung injury . Thirty-two unanesthetized sheep with lung and soft tissue lymph fistulae were studied . Twelve sheep were given a sublethal dose of intravenous E . coli endotoxin (2 micrograms/kg) . A characteristic two-phase injury was noted as evidenced by early pulmonary hypertension and hypoxia and later increased lung permeability . TxA2 was significantly increased in lung lymph as well as aortic plasma relative to venous plasma, indicating the lung to be the source . Twelve of 12 sheep survived . Five of 13 sheep died from endotoxemia when given 3-5 days after a 25% total body surface (TBS) burn and five of seven died with endotoxin (2 micrograms/kg) and a 50% burn . Physiologic parameters were at preburn levels before endotoxin . Animals died both during the early phase from hypoxia and the later phase due, in large part, to increasing pulmonary dysfunction . Absolute levels of TxA2 were not increased in the postburn animals, nor was there a clear release of TxA2 from burn tissue to explain the accentuated response . Prostacyclin levels were, however, less elevated in postburn animals in response to endotoxin, thereby altering the TxA2/PGI2 ratio in favor of TxA2 . However, a cause and effect relationship between the increased lung injury and TxA2 remains undetermined . Lymph flow or lymph protein content was not altered in burn tissue in response to endotoxin.

J Leukoc Biol, 1985 Jan, 37(1), 87 - 99
Modulation of endotoxin-induced neutrophil alveolitis by captopril and by hyperoxia; Rinaldo JE et al.; We compared survival and the intensity of bronchoalveolar inflammation reflected by lung lavage after the intraperitoneal injection of endotoxin from Escherichia coli serotype 055B5 in rats breathing air and those breathing 60% oxygen for six days after endotoxin injection . Survival following 7.5 mg/kg of endotoxin was comparable in air-breathing rats (50%) and in oxygen-breathing rats (63%) . Endotoxin caused a dose-dependent increase in the recovery of polymorphonuclear leukocytes from the lung . Oxygen breathing reduced the percentage of neutrophils recovered by lavage 24 hr after endotoxin from 17% to 9% after 2.5 mg/kg of endotoxin and from 34% to 12% after 7.5 mg/kg of endotoxin . The absolute number of neutrophils recovered was also significantly decreased in oxygen-breathing rats . The activity of pulmonary angiotensin-converting enzyme (ACE) has been reported to be affected by oxygen tension, and ACE degrades bradykinin, a proinflammatory mediator . Therefore, we questioned whether the salutary effect of increased inspired oxygen tension on the magnitude of neutrophil influx into the airspaces could be related to changes in ACE activity . We found that after 48 hr of peroral pretreatment of the rats with captopril, a specific ACE inhibitor, there was increased recovery of neutrophils by lavage 24 hr after injection of endotoxin in air-breathing rats . Captopril pretreatment also increased the chemotactic activity of bronchoalveolar lavage fluid (BALF) . There was no concomitant alteration in the accumulation of 125I albumin in the lung following captopril pretreatment either in endotoxin-treated rats or in controls . Thus, breathing 60% oxygen decreased the accumulation of neotrophils in airspaces after intraperitoneal endotoxin injection and pharmacologic inhibition of ACE had the opposite effect . Alterations in the activity of pulmonary angiotensin-converting enzyme related to alveolar oxygen tension is a potential speculative mechanism for modulation of alveolar inflammation by the inspired oxygen concentration in this model.

J Infect Dis, 1985 Jan, 151(1), 124 - 30
Identification by DNA hybridization of enterotoxigenic Escherichia coli in a longitudinal study of villages in Thailand; Echeverria P et al.; Radioactively labeled enterotoxin genes were used to study the epidemiology of enterotoxigenic Escherichia coli infections in two Thai villages . When E . coli that were isolated from 674 specimens were fixed on nitrocellulose paper and examined for hybridization with E . coli enterotoxin gene probes in Bangkok, the technique had a sensitivity of 94% (31 of 33) and a specificity of 100% (641 of 641), when compared with tests of E . coli for enterotoxin production in the Y-1-adrenal cell and suckling-mouse assays . However, when the same specimens were fixed directly onto nitrocellulose paper at a field laboratory and transported to the reference laboratory for assay with the gene probes, 27 specimens that contained enterotoxigenic E . coli did not hybridize with the E . coli gene probes . Enterotoxigenic E . coli that hybridized with the LT, ST-H, and ST-P probes were identified in 10% (17 of 177) of villagers with diarrhea, 7% (8 of 108) of contacts of individuals with diarrhea caused by enterotoxigenic E . coli, and 3% (32 of 1,199) of persons not associated with cases of diarrhea caused by enterotoxigenic E . coli . Enterotoxigenic E . coli that hybridized with the ST-II probe was not a cause of diarrhea . Alternative methods of retaining DNA on filters under field conditions are needed before this technique can be used for direct examination of specimens with enterotoxin gene probes.

Infect Immun, 1985 Jan, 47(1), 338 - 40
Enterotoxin production, presence of colonization factor antigen I, and adherence to HeLa cells by Escherichia coli O128 strains belonging to different O subgroups; Guth BE et al.; Strains of three subgroups of Escherichia coli O128 were studied . Enterotoxin production was observed in 30 (91%) O128ac strains, whereas strains of subgroups O128ab and O128ad were not toxigenic . CFA/I was only found in two serotypes of subgroup O128ac, all of them producing heat-stable enterotoxin except for one which produced both toxins . None of the strains studied produced CFA/II . In a binding test with HeLa cells, localized adherence was found only in strains of subgroup O128ab; diffuse adherence occurred in strains of subgroup O128ac . As flagellar antigens were specific in subgroups ab and ac and toxin production was observed only in subgroup ac, the present results suggest that subgroup and serotype are useful markers for O128 strains that are enterotoxigenic or enteropathogenic.

Infect Immun, 1985 Jan, 47(1), 318 - 21
Adherence of Escherichia coli in pathogenesis of endometritis and effects of estradiol examined by scanning electron microscopy; Nishikawa Y; Escherichia coli was inoculated into the uterine lumen of ovariectomized rats, and the endometrial surfaces were examined by scanning electron microscopy . Adherence of E . coli to the epithelium and destruction of the surface leading to purulent endometritis were noticed . When rats were treated previously with estradiol, adherence of E . coli was not detected.

Infect Immun, 1985 Jan, 47(1), 311 - 7
Effects of ovarian hormones on manifestation of purulent endometritis in rat uteruses infected with Escherichia coli; Nishikawa Y et al.; To assess the influence of hormones on uterine infections, Escherichia coli was infused into uterine lumens of ovariectomized or adrenoovariectomized rats receiving exogenous administration of various doses of ovarian hormones . Large numbers of E . coli were recovered from the rat uterine lumens, irrespective of hormonal influences . The number of leukocytes in the uterine flushings, representing the magnitude of purulent inflammation, differed significantly depending upon the hormonal regimen given to each host . Purulent endometritis was induced by E . coli in ovariectomized rats receiving progesterone or corn oil (hormone vehicle) . Infections were asymptomatic in rats receiving estradiol, but promethazine-treated uterine horns were susceptible to infection . When progesterone was administered along with estradiol, purulent inflammation was caused by E . coli, but the number of leukocytes in the uterine lumens was significantly less than that obtained from the rats treated with progesterone or corn oil . These effects of ovarian hormones on uterine infections were observed in adrenoovariectomized rats as well as in ovariectomized rats . It is suggested that estradiol alters the nature of endometrial epithelium and prevent manifestation of purulent endometritis; progesterone antagonizes estradiol . Adrenal hormones appear not to participate in the pathogenesis of endometritis induced by E . coli.

Differentiation, 1985, 29(3), 284 - 8
Biological effects of biosynthetic human EGF on the growth of mammalian cells in vitro; Nakagawa S et al.; The effects were examined of biosynthetic human epidermal growth factor (Bh-EGF) produced from cloned E . coli on DNA synthesis and all divisions of 13 different kinds of primary and established cell lines . Primary cultures of mammary epithelia, hepatocytes and stomach cells were strongly stimulated by EGF to undergo DNA synthesis in serum-free culture medium with concentrations of Bh-EGF as low as 0.1-10 ng/ml . In sharp contrast, 0.1-100 ng/ml of Bh-EGF failed to enhance thymidine incorporation into DNA when applied to established cell lines using the serum-free medium . Higher concentrations of Bh-EGF (30-100 ng/ml) promoted morphological changes only in hepatocytes, e.g., elongation, enlargement and projection of their cytoplasm . The above results were also obtained in mouse EGF (m-EGF) . In our binding assay, Bh-EGF competed against {125I}-m-EGF with a one-fourth to one-fifth efficacy when compared with m-EGF . It was concluded that the in vitro biological activity of Bh-EGF was similar to that of m-EGF.

Immunol Lett, 1985, 9(2-3), 105 - 8
Human B cell proliferation is stimulated by interleukin 2; Gearing A et al.; The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology . The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1 . IL-2 was found to stimulate B cell proliferation . Activation of the B cells using anti-IgM antibodies increased this response . Resting T cells from the same donors were found to be less reactive to IL-2 . The results suggest that human B cell proliferation can be stimulated by IL-2 alone.

Can J Comp Med, 1985 Jan, 49(1), 104 - 8
The effect of cadmium chloride on the immune response in mice; Blakley BR; Six week old BDF1 female mice were exposed to cadmium chloride in the drinking water at concentrations ranging from 0 to 50 micrograms/mL cadmium for three weeks . The humoral immune response against sheep red blood cells which is T-lymphocyte and macrophage dependent, was suppressed in a dose dependent fashion with the maximum suppression of 28.2% observed in the highest exposure group (P less than 0.0001) . Mitogen studies demonstrated that cadmium was a weak mitogen, producing a dose-dependent enhancement of blastogenesis (P = 0.026) . T-lymphocyte responses which were induced by concanavalin A were not affected by cadmium exposure (P = 0.284) . A dose-dependent enhancement of the B-lymphocyte activity was produced in the presence of cadmium when the lymphocytes were induced with Escherichia coli, lipopolysaccharide, a B-lymphocyte mitogen (P = 0.007) . These results suggest that the immunosuppressive effects of cadmium associated with the humoral immune response are not due to an impairment of lymphocyte proliferation, an intermediate step involved in the generation of an immune response . The immunosuppressive effects were produced at relatively low cadmium exposures as indicated by the renal cadmium concentrations suggesting that the immune systems is very vulnerable to the toxic effects of cadmium.

Mol Gen Genet, 1985, 200(3), 351 - 5
Mutagenic and error-free DNA repair in Streptomyces; Baltz RH et al.; Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light . S . fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix {i.e . UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)} but not to agents which produce small lesions {i.e . hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)} . JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli . S . fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS) . JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA . This unusual phenotype suggests that the mcr-4+ protein plays some role in error-prone repair in S . fradiae.

Eur J Respir Dis Suppl, 1985, 139, 66 - 70
Eglin c, a pharmacologically active elastase inhibitor; Schnebli HP et al.; Eglin c is an elastase/cathepsin G inhibitor from leech Hirudo medicinalis . The gene for this 70 aminoacid peptide was synthesized chemically, cloned and expressed by E . coli . Here we report biochemical and pharmacological studies . The rate of complex formation between Eglin c and human leukocyte elastase (HLE) or human cathepsin G (H . Cat . G) was determined and compared to those of a number of other proteinase/proteinase-inhibitor interactions (alpha 1 PI and alpha 2M) . The association rate constants of Eglin c with the leukocyte enzymes are of the same order of magnitude as those with the naturally occurring inhibitors alpha 1 PI and alpha 2M . The association rate constant of Eglin c (extracted from leech) and Eglin c (biotechnology product) with HLE was found to be identical . The equilibrium constants Ki of the Eglin c/HLE and the Eglin c/H . Cat . G interactions are in the order of 10(-10) M . In an experiment with the hamster emphysema model, 0.5 mg or 2 mg of Eglin c applied intratracheally one hour before an HLE-insult completely protected the animals against emphysema and no signs of toxicity due to Eglin c were observed.

Proc Natl Acad Sci U S A, 1985 Jan, 82(2), 445 - 8
Molecular approach to thermogenesis in brown adipose tissue: cDNA cloning of the mitochondrial uncoupling protein; Bouillaud F et al.; The uncoupling protein (UCP) of mammalian brown fat is a specialized and unique component responsible for energy dissipation as heat . Translation and immunoprecipitation from sucrose-fractionated mRNA indicated that the mRNA of UCP sedimented at 14-16 S . A recombinant cDNA library prepared from mRNA of thermoactive brown fat enriched for UCP mRNA has been constructed and cloned in Escherichia coli . Recombinant plasmids were screened by differential colony hybridization to a cDNA probe complementary to poly(A)+ RNA isolated from thermogenic or from weakly thermogenic brown fat . Several differentially hybridizing plasmids were shown to contain UCP cDNA sequences by their ability to select a mRNA coding for an in vitro translation product that was immunoprecipitable with antibodies against UCP . Blot hybridization of brown fat mRNA to a 32P-labeled UCP cDNA probe revealed two major species of mRNA (15S and 18S) . As compared to non-thermogenic tissue, a strikingly increased hybridization to the probe was observed with brown fat mRNA from thermoactive tissue . Moreover, hybridization was observed with RNA of brown adipose tissue from rat, hamster, or mouse but not with RNA from rat or mouse liver.

J Natl Cancer Inst, 1985 Jan, 74(1), 223 - 8
Differences in activity of N2-guanine tRNA methyltransferase II among several inbred strains of mice; Dizik M et al.; The tRNA methyltransferase activities of C57BL/6J, C57L/J, C58/J, AKR/J, and C3H/HeJ inbred mice were studied with the use of various amino acid-specific Escherichia coli tRNA's as substrates . Mice from two strains with high incidence of spontaneous leukemia (AKR/J and C58/J) exhibited levels of liver N2-guanine tRNA methyltransferase II (N2-MeGII) activity that were double those of two strains of mice with low incidence of spontaneous leukemia (C57BL/6J and C57L/J) . Activities of liver and kidney N2-MeGII of the high spontaneous hepatoma strain C3H/HeJ were also found to be twice as high as those of C57BL/6J mice . The activities of other tRNA base-specific liver tRNA methyltransferases were very similar in all strains studied . The N2-MeGII activity of the F1 progeny of a cross between C57BL/6J and C3H/HeJ showed levels of activity intermediate to those of the parental strains . Activities of liver N2-MeGII of two inbred strains of mice that differ in their H-2 haplotype (C57BL/10SnJ and the congenic strain B10.BR/SgSnJ) were also compared . Both C57BL/10SnJ and B10.BR/SgSnJ strains exhibited low levels of liver N2-MeGII activity, indicating that H-2 does not directly control the activity of this enzyme.

Nucleic Acids Symp Ser, 1985, (16), 217 - 20
A study of the interaction between tRNASer and seryl-tRNA synthetase from bovine liver; Tachibana Y et al.; Study by chemical modification of Ser, Arg, His residues and sulfhydryl groups on bovine seryl-tRNA synthetase showed that Ser residues appeared to be unnecessary for the recognition mechanism, but Arg and His residues were essential . It was considered that different sulfhydryl groups related with each recognition of tRNA and ATP . Poly-arginine inhibited the interaction between serine tRNA and SerRS . The CD spectra of a mixture of serine tRNA and poly-arginine indicated that higher-order structure of tRNA changed . Furthermore, the Km and Vmax values of bovine serine isoacceptor, yeast serine tRNA and E . coli serine tRNA for bovine SerRS examined and it was discussed the differences of those base sequences.

Comp Biochem Physiol B, 1985, 82(1), 99 - 106
Effects of an azasteroid on growth, development and reproduction of the free-living nematodes Caenorhabditis briggsae and Panagrellus redivivus; Bottjer KP et al.; The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth, development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus . The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 micrograms cholesterol per ml and/or 25 micrograms ASA-6 per ml medium . All cultures also contained 50 micrograms Tween 80 per ml medium . After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability . These effects were more apparent in the sterol-deficient medium containing ASA-6 . In the presence of cholesterol and ASA-6, growth and reproduction of C . briggsae, but not of P . redivivus, was inhibited after five generations . Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium . These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species . In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.

Circ Shock, 1985, 17(2), 147 - 61
Role of subcutaneous tissue endotoxin in the production of prostanoid-induced lung injury: comparison with intravenous endotoxin response; Demling RH et al.; Local injection of endotoxin into soft tissues of the flank results in hypoxia and pulmonary hypertension . Our purpose was to determine whether this was caused by tissue prostanoid production or production by the lung as is seen with endotoxemia . Twenty-six sheep were prepared with lung and flank tissue lymph fistulae . Thirteen sheep were given 2 micrograms/kg Escherichia coli endotoxin into the flank soft tissue, six of which were pretreated with ibuprofen, 12.5 mg/kg . Thirteen sheep were given intravenous endotoxin, 2 micrograms/kg, with six pretreated with ibuprofen . An early hypertensive phase was noted with both insults characterized by pulmonary hypertension, hypoxia, and increased lung lymph flow (QL) . With subcutaneous tissue endotoxin, there was a significant increase in tissue lymph TxB2 and 6-keto-PGF1 alpha when compared to lung lymph and increased values in venous plasma compared to arterial plasma, indicating tissue to be the source . With intravenous endotoxin, lung lymph and aortic plasma levels were significantly higher than tissue lymph and venous plasma, respectively . The hypoxia, hypertension and increased prostanoids were prevented using ibuprofen . An increased lung permeability phase was noted with intravenous endotoxin but not with tissue endotoxin . As expected, this phase was not inhibited with ibuprofen and, therefore, not prostanoid-induced.

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