Salmonella typhimurium

Zentralbl Bakteriol, 1993 Apr, 278(2-3), 383 - 95
The cellular immune response against Yersinia enterocolitica in different inbred strains of mice: evidence for an important role of T lymphocytes; Autenrieth IB et al.; Resistance of mice against infection with Yersinia enterocolitica has been shown to be related neither to the Ity locus coding for resistance against infection with Salmonella typhimurium and other pathogens nor to the H-2 locus . From other mouse infection models, e.g., murine leishmaniasis, there is evidence that a different T cell-dependent regulation of the host immune response in various inbred strains of mice determines the susceptibility to the infectious agent . However, until recently, little was known about the cellular immune response against Y . enterocolitica . Thus, in a first approach we used the highly virulent Y . enterocolitica strain WA of serotype O:8 and different inbred strains of mice (C57 BL/6, Balb/c and athymic T cell-deficient C57 BL/6 nude mice) to investigate the cell-mediated immunity against parenteral infection . Comparison of the median lethal dose and of the net-bacterial growth in the spleens of infected mice indicated that Balb/c mice could be considered as Yersinia-susceptible whereas C57 BL/6 mice were relatively resistant . However, in contrast to normal C57 BL/6, athymic T cell-deficient C57 BL/6 nude mice have proved to be highly susceptible to Yersinia infection suggesting that T cells are required for the elimination of the pathogen . This conclusion was supported by histomorphological and immunohistological results indicating that T lymphocytes were present in Yersinia-induced tissue lesions . Moreover, the adoptive transfer of Yersinia-specific T cell lines and clones into naive animals mediated significant protection against the pathogen in both Yersinia-resistant C57 BL/6 and in Yersinia-susceptible Balb/c mice . These findings emphasize an important role of T lymphocytes in the host response against Y . enterocolitica infection.

Infect Agents Dis, 1993 Apr, 2(2), 55 - 73
New perspectives in vaccine development: mucosal immunity to infections; McGhee JR et al.; In this review, we focus on six key areas currently receiving attention in the mucosal immune system . These six areas are of considerable importance for development of vaccines . They are (a) the necessity to understand the unique features of the mucosal immune system; (b) the possibility that the common mucosal immune system may contain distinct compartments; (c) differences in antigen uptake and in types of antigen-presenting cells in mucosal inductive and effector sites; (d) more careful consideration of mucosal memory in vaccine development; (e) recent studies, which show that oral vaccines induce T-helper (Th)-cell subsets that regulate mucosal IgA responses; and (f) the mechanisms whereby mucosal S-IgA and T cells provide mucosal immune protection . An example of the above suffices to illustrate why the selected areas are of importance in vaccine development . Oral immunization preferentially induces type 2 Th (Th2) cell responses that directly correlate with antigen-specific IgA responses in mucosal effector sites . It is likely that activated, antigen-specific Th2 cells that are induced in Peyer's patches are continuously supplied to mucosal effector sites for regulation of IgA responses . These Th2 cells are producing cytokines such as interleukin (IL)-5 and IL-6 and these cytokines may direct antigen-specific surface IgA-positive B cells to become IgA-producing plasma cells . Nevertheless, additional studies will be required to establish that IgA responses to T-cell-dependent antigens depend on Th2 cell-derived help . What are the implications of these studies for current oral vaccines, including novel antigen delivery systems? The most obvious would be that vaccines should be optimized for induction of Th2-cell responses in IgA inductive sites such as the gut-associated lymphoreticular tissues . It is now clear that induction of Th2-type responses in both mucosal inductive and mucosal effector sites are essential for oral vaccines to induce S-IgA responses . However, antigens delivered by live vectors such as Salmonella typhimurium in the murine system and S . typhi in humans must consider T-cell responses induced against a live vector in addition to the inserted recombinant antigen . In this regard, it has been shown that these microorganisms induce cell-mediated immunity responses that largely result from Th1-type cells.(ABSTRACT TRUNCATED AT 400 WORDS)

J Immunol, 1993 Apr 1, 150(7), 2869 - 84
Comparison of antigen presentation of influenza A nucleoprotein expressed in attenuated AroA- Salmonella typhimurium with that of live virus; Brett SJ et al.; Rationally attenuated strains of Salmonella expressing foreign proteins represent a potentially important vaccine delivery system . The characteristics of Ag presentation of influenza nucleoprotein expressed in an AroA- strain of Salmonella typhimurium (SL3262-pNP-2) have therefore been compared with those of soluble purified nucleoprotein (NP) and infectious influenza virus . This represents three distinct modes of internalization of the same protein into APC . Human monocytes and the monocytic leukemia cell line THP-1 infected with SL3261-pNP-2 were found to present several different epitopes from NP to human CD4+ class II-restricted T lymphocytes . Ag presentation to these T cell clones was enhanced by pretreatment of THP-1 cells with IFN-gamma but not TNF-alpha . Bacterial phagocytosis and Ag presentation of NP were increased after opsonization of Salmonella with immune serum . Macrophages infected with SL3261-pNP-2 were unable to present NP to class I-restricted T cells . In contrast, cells infected with live influenza virus, although recognized by NP-specific class I-restricted CTL, were inefficiently recognized by NP-specific class II-restricted T cells . Ag presentation to CD4+ T cell clones by monocytes of SL3261-pNP-2, purified recombinant NP, and live influenza virus, but not the synthetic peptide 206-229, was inhibited by chloroquine and the protease inhibitors pepstatin A and leupeptin, suggesting that the major route of processing in each case was via the exogenous pathway . T cell recognition of NP via all of these Ag delivery systems was also abrogated by cycloheximide and brefeldin A treatment, indicating a requirement for recently synthesized MHC class II molecules in presentation of whole NP after processing but not for the corresponding synthetic peptide.

J Immunol, 1993 Apr 1, 150(7), 2737 - 45
Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells; Snapper CM et al.; We have previously demonstrated that activation of murine B cells by dextran-conjugated anti-IgD antibodies may serve as a polyclonal, in vitro model system for studying immune responses to T cell-independent type 2 (TI-2) Ag, as exemplified by the bacterial polysaccharides . Because in vivo Ig responses to TI-2 Ag are mediated primarily by B cells resident in the splenic marginal zone, we wished to determine whether this reflected an intrinsic difference in the responsiveness of marginal zone B cells (MZB) compared with follicular B cells (FB) to this class of Ag . In this report we demonstrate that highly purified MZB, isolated by electronic cell sorting, exhibit a lower proliferative response in vitro in response to unconjugated anti-Ig antibody as well as to dextran- or Sepharose-conjugated anti-IgM or anti-IgD antibodies, whereas they proliferate equal to or better than FB when stimulated by other B cell mitogens including LPS, Salmonella typhimurium mitogen, or anti-CD3-activated CD4+ Th2 cell clone . Despite the different proliferative responses of MZB and FB induced by anti-Ig, Ag receptor cross-linkage stimulates comparable increases in intracellular free calcium concentrations in both of these B cell populations . Furthermore, MZB secrete Ig and undergo Ig isotype switching to a comparable degree, relative to FB, in response to both T cell-dependent and T cell-independent stimuli . This suggests that the compartmentalization of TI-2 responses to the splenic marginal zone rather than the follicular zone reflects something other than the intrinsic responsiveness of the B cells from these two sites.

Infect Immun, 1993 Apr, 61(4), 1222 - 31
Oral vaccination of calves with an aromatic-dependent Salmonella dublin (O9,12) hybrid expressing O4,12 protects against S . dublin (O9,12) but not against Salmonella typhimurium (O4,5,12); Segall T et al.; Three groups of six calves each, 5 to 7 weeks old, were orally vaccinated with the live aromatic-dependent delta aroA Salmonella dublin (O9,12) hybrid strain SL7103 with the O4,12-specifying rfb gene cluster from Salmonella typhimurium . SL7103 was given in three weekly doses, increasing from 2 x 10(9) to 1 x 10(11) bacteria per ml, was well tolerated, and caused mild, short-term temperature increases which diminished with each immunization . The strain was shed for up to 1 week . Strain SL7103 elicited significant (P < 0.001) and equal anti-S . dublin and -S . typhimurium lipopolysaccharide serum antibody responses and skin delayed-type hypersensitivity immune responses . Six vaccinated calves orally challenged with 10(10) CFU (equivalent to 1,000 50% lethal doses) of the virulent parent strain S . dublin SVA47 were protected and experienced only transient fever and mild mucoid diarrhea . However, six vaccinated calves orally challenged with 3 x 10(9) CFU and another six challenged with 3 x 10(8) CFU (equivalent to 1,000 50% lethal doses) of the virulent S . typhimurium SVA44 became bacteremic with a profuse hemorrhagic diarrhea and had to be sacrificed within 2 to 7 days . The results suggest that the S . typhimurium antilipopolysaccharide immunity was insufficient to provide a solid protective efficacy against oral S . typhimurium infection . The immunohistopathological examination revealed that S . typhimurium SVA44 could be found in all layers of the intestinal mucosa and the lymphatic tissues of the Peyer's patches . In contrast, S . dublin SVA47 was found predominantly in the columnar enterocytes of the jejunum and ileum and the follicle-associated epithelium over the Peyer's patches . In addition, SVA47 was found in the glandular tissues of the duodenal and tonsillar areas and in the lungs . This suggests that the S . typhimurium and S . dublin strains have different virulence traits determining their tissue localization and dissemination.

Infect Immun, 1993 Apr, 61(4), 1211 - 21
Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain; Lindberg AA et al.; An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12) . By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium . Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S . dublin and S . typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts . The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S . dublin (50% lethal dose of ca . 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S . typhimurium (50% lethal dose of ca . 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice) . Compared with the protection elicited in homologous systems (S . dublin SL5631 against S . dublin and S . typhimurium SL1479 against S . typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S . dublin challenge and 100-fold against S . typhimurium challenge . Vaccination with S . typhimurium SL1479 conferred no protection against S . dublin challenge, and vaccination with S . dublin SL5631 conferred no protection against S . typhimurium challenge . The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.

Mutat Res, 1993 Apr, 301(4), 213 - 22
Mutagenicity of alkylhydrazine oxalates in Salmonella typhimurium TA100 and TA102 demonstrated by modifying the growth conditions of the bacteria; Matsushita H Jr et al.; Alkylhydrazines are important carcinogens . However, they show generally only weak mutagenicity and the activities reported from different laboratories are contradictory . We have developed a sensitive method to detect the mutagenicity of alkylhydrazines . The method is based on a modified preculturing procedure in the Ames test, the emphasis in the modification being a change in the growth period of tester strains . The optimal growth periods were found to be 11 h in Salmonella typhimurium TA100 and 5 h in Salmonella typhimurium TA102 . We tested the mutagenic activity of 12 alkylhydrazines; 1,2-dimethylhydrazine, 1,2-diethylhydrazine, 1,2-dipropylhydrazine, 1,2-dibutylhydrazine, 1,1-dimethylhydrazine, 1,1-diethylhydrazine, 1,1-dipropylhydrazine, 1,1-dibutylhydrazine, methylhydrazine, ethylhydrazine, propylhydrazine and butylhydrazine . All 12 alkylhydrazines were clearly mutagenic in Salmonella typhimurium TA102, and 10 hydrazines were mutagenic in Salmonella typhimurium TA100, both in the absence of S9 mix . The mutagenicity was inhibited by the addition of S9 mix or bovine serum albumin . This suggests deactivation of the mutagens by proteins.

Mutat Res, 1993 Apr, 299(2), 85 - 93
Quantitative structure-activity relationships for the mutagenicity of propylene oxides with Salmonella; Hooberman BH et al.; A quantitative structure-activity relationship approach was used to investigate the mutagenicity of a series of seventeen-monosubstituted propylene oxides in Salmonella typhimurium strains TA100 and TA1535 . Mutagenicity in strain TA100, using a liquid suspension assay, was found to correlate with chemical reactivity, as measured by the rates of reaction with two model bionucleophiles, nicotinamide and 4-(4-nitrobenzyl)pyridine . However, since the reactivity of three of the epoxides did not correlate to their Taft sigma * values, as a measure of the electronic effects of substituent groups, neither was their mutagenicity predicted by this substituent constant . The relative mutagenicity for the propylene oxides was different in the liquid suspension assay than that determined by the standard plate incorporation assay and also differed between the two bacterial strains . The assay differences were attributed to epoxide stability . The differences between strains was observed to be due to the response of the error-prone repair system, found only in TA100, to the stronger alkylating agents.

Mutat Res, 1993 Apr, 299(2), 111 - 20
Characterization of stable high molecular weight mutagenic product(s) of plant-activated m-phenylenediamine; Seo KY et al.; Monocyclic aromatic amines are environmental contaminants and many are promutagens and procarcinogens . Cultured tobacco cells, strain TX1, activated m-phenylenediamine into a frameshift mutagen that reverted the hisD3052 allele in Salmonella typhimurium strains TA98 and YG1024 . However, the plant-activated products were refractory in strain TA98/1,8-DNP6 . This indicated that these plant-activated products were substrates for bacterial acetyl-CoA: N-hydroxyarylamine O-acetyltransferase . A stable, high molecular weight (> 300 kDa) proximal mutagen was isolated by molecular ultrafiltration membranes . No parent compound was associated with the isolated mutagenic fraction . The high molecular weight fraction induced mutation in S . typhimurium strains TA98, YG1021 and YG1024 . From these data we propose a model for the plant-activation of aromatic amine promutagens.

Genetika, 1993 Mar, 29(3), 423 - 9
{The nature of the emergence of His+-revertants in Salmonella typhimurium}; Gizatullin FSh et al.; The His(-)-->His+ spontaneous reversion of the Salmonella typhimurium alleles hisD3052 and hisG46 was studied . The expected jackpot distribution of His+ revertants was not observed in the fluctuation tests . The experimental distributions were close to Poisson . It was also shown that the mean number of His+ reversion events and the mean number of revertants per plate were similar . These data demonstrate that the His+ reversion occurred under the influence of histidine starvation . At the same time, kanamycin resistant mutants had the jackpot distribution . Selection for His+ revertants did not increase the Kans-->Kanr mutations.

Chem Biol Interact, 1993 Mar, 86(3), 229 - 54
Mechanism of genotoxicity and electron density distribution by NMR of 5-nitro-3-thiophenecarboxamides, a novel group of direct-acting mutagens in Salmonella typhimurium; Hrelia P et al.; The mutagenic activity of 23 5-nitro-3-thiophenecarboxanilides and of 5-nitro-3-thiophenecarboxamide, the prototype, (NTCAs) have been evaluated in the Ames test on Salmonella typhimurium strains TA100 ad TA98 with and without metabolic activation . Effects of different substituents (electron-donating and electron-withdrawing) were studied to evaluate structural features that affect the metabolism and the bacterial mutagenic potency . All the derivatives were direct-acting mutagens, the mutagenic potency ranging from 0.7 to 142 revertants (rev.)/nmol in TA100 and from 0.09 to 68 rev./nmol in TA98 strain . Results obtained with strains TA98NR and TA98/1,8-DNP6 indicated that the mutagenic activity was largely dependent on bacterial nitroreductase, whereas the O-acetylation step was not critical for mutagenic potency . Superoxide (O2-.) and hydroxyl (OH.) scavengers as well as other radical scavengers and enzymes inhibited NTCAs mutagenicity to different extents . In particular, O2- . seemed to be involved in NTCAs mutagenicity, showing a free radical pathway for NTCA metabolism . {1H}- and {13C}NMR data indicated that the effects of different substituents on genotoxicity are probably not exerted on the electron density distribution . The importance of factors such as extent of nitration, reduction potential, orientation of nitrosubstituent and planarity of the molecule are discussed.

Mol Gen Genet, 1993 Mar, 237(3), 429 - 38
Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii; Prieto R et al.; Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wild-type strains . Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not . Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci . Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines . Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin) . The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited . Chlorate was also able to induce reversion of nit- mutants of C . reinhardtii, but failed to produce His+ revertants or Arar mutants in the BA-13 strain of Salmonella typhimurium . In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus . Genetic analyses of nitrate reductase-deficient CR mutants of C . reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations . These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci . Three hcr loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified . Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC . In both nit+ and nit- chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate . Our data indicate that in C . reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme . At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport . In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.

Mol Microbiol, 1993 Mar, 7(6), 883 - 91
Organization of Lrp-binding sites upstream of ilvIH in Salmonella typhimurium; Wang Q et al.; Lrp, a major regulatory protein in Escherichia coli, controls the expression of numerous operons, including ilvIH . Lrp binds to six sites upstream of ilvIH, and Lrp binding is required for ilvIH expression . We show here that an Lrp-like protein is also present in Salmonella typhimurium . This protein can bind both E . coli and S . typhimurium ilvIH DNA, as can E . coli Lrp . Methidiumpropyl-EDTA footprinting studies were performed with purified E . coli Lrp and S . typhimurium ilvIH DNA . Six binding sites were defined, three of them being similar to corresponding sites in E . coli, and three being organized differently . A consensus derived from six S . typhimurium sites is compatible with that derived from a similar analysis of E . coli sequences.

Mol Microbiol, 1993 Mar, 7(6), 825 - 30
Molecular analysis of spv virulence genes of the Salmonella virulence plasmids; Gulig PA et al.; Genes on an 8 kb region common to the virulence plasmids of several serovars of Salmonella are sufficient to replace the entire plasmid in enabling systemic infection in animal models . This virulence region encompasses five genes which previously have been designated with different names from each investigating laboratory . A common nomenclature has been devised for the five genes, i.e . spv for salmonella plasmid virulence . The first gene, spvR, encodes a positive activator for the following four genes, spvABCD . DNA sequence analysis of the spv genes from Salmonella typhimurium, Salmonella dublin, and Salmonella choleraesuis demonstrated extremely high conservation of the DNA and amino acid sequences . The spv genes are induced at stationary phase and in carbon-poor media, and optimal expression is dependent on the katF locus . The virulence functions of the spv genes are not known, but these genes may increase the growth rate of salmonellae in host cells and affect the interaction of salmonellae with the host immune system.

Mutagenesis, 1993 Mar, 8(2), 127 - 31
Mutagenic and lethal effects of halogenated methanes in the Ara test of Salmonella typhimurium: quantitative relationship with chemical reactivity; Roldan-Arjona T et al.; The mutagenic and lethal effects of nine halogenated methanes (CCl4, carbon tetrachloride; CHCl3, chloroform; CH2Cl2, dichloromethane; CBr4, carbon tetrachloride; CHBr3, bromoform; CH2Br2, dibromomethane; CI4, carbon tetraiodide; CHI3, iodoform; and CH2I2, diiodomethane) have been investigated with the Ara forward-mutation assay of Salmonella typhimurium . Five substances (CH2Cl2, CBr4, CH2Br2, CHI3 and CH2I2) gave clear mutagenic responses . In all these cases the mutagenicity diminished in the presence of mammalian metabolic activation (S9 mixture) . Two halomethanes (CCl4 and CHBr3) were classified as 'questionable' mutagens since they did not double the spontaneous value although a dose-response curve was obtained . All halomethanes tested exerted a lethal effect . A high concordance was found between lethality and chemical reactivity, as expected from the type and number of halogenated substituents . Compounds with equal numbers of substituents were lethal in the order I > Br > Cl . Additionally, the lethality of compounds with identical halogen substituents increased by successive halogenations . No such concordances were observed with respect to mutagenic activity . Structure-activity quantitative relationships were investigated by using the previously reported values of the polarographic half-wave reduction potential (-E1/2), a physicochemical parameter related to the energy required to put an electron into the lowest unoccupied molecular orbital . The lethality, quantified as LD37, correlated highly with the -E1/2 values for the nine halogenated compounds (r = -0.955 P < 0.001) . These data suggest that halomethanes which are reduced easily will induce higher lethality than those with low reduction potential, in agreement with the predicted effects on lipid peroxidation and hepatotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenesis, 1993 Mar, 14(3), 441 - 50
Vinyl carbamate epoxide, a major strong electrophilic, mutagenic and carcinogenic metabolite of vinyl carbamate and ethyl carbamate (urethane); Park KK et al.; Vinyl carbamate epoxide (VCO) was found to possess strong electrophilic, mutagenic and carcinogenic activities . It reacted with water at 37 degrees C and pH 7.4 (phosphate buffer) to form glycolaldehyde and several related reducing compounds; none of these products were mutagenic for Salmonella typhimurium TA1535 . Under these conditions VCO had a half-life (determined chemically and mutagenically) of approximately 10.5 min . This half-life was progressively lowered by increasing concentrations of chloride ion (liver, serum and isotonic levels) . This ion reacted with VCO to form chloroacetaldehyde . VCO also reacted with other nucleophiles such as glutathione, DNA and its constituent guanine and adenine bases . The purine adducts formed by VCO in DNA in vitro and in vivo were released by weak acid treatment and consisted of 7-(2'-oxoethyl)guanine and N2,3-ethenoguanine as major products with 1,N6-ethenoadenine as a minor product . VCO was a strong direct mutagen in Salmonella typhimurium TA1535 and TA100 but was only weakly active in the TA98 mutant . VCO was a stronger initiator of carcinogenesis in the skin of CD-1 mice and in the liver of infant male B6C3F1 mice than its metabolic precursors vinyl carbamate (VC) and ethyl carbamate (EC) . Unlike VC and EC, VCO was a strong complete carcinogen in the skin of CD-1 mice and induced papillomas and carcinomas following repetitive administration of sub-ulcerogenic doses . VCO also exhibited some carcinogenic activity in the lungs of mice and in the s.c . and mammary tissue of female Sprague-Dawley rats . These data and those from other recent studies support the conclusion that VCO is a major strong electrophilic, mutagenic and carcinogenic metabolite of EC and VC in the mouse.

J Gen Virol, 1993 Mar, 74 ( Pt 3), 453 - 8
Expression of the G glycoprotein gene of human respiratory syncytial virus in Salmonella typhimurium; Martin-Gallardo A et al.; The attachment protein, G, of human respiratory syncytial virus (RSV) is an M(r) 84K to 90K species which has a high content of N-linked and O-linked carbohydrates . The unglycosylated form of this protein was expressed by inserting a full-length cDNA copy of the mRNA from the A2 strain of RSV into a prokaryotic expression vector under the control of the lambda PL promoter . Salmonella typhimurium cells transformed with the G-containing plasmid synthesized a protein of M(r) 40,000 that specifically reacted with polyclonal and two neutralizing monoclonal antibodies raised against the native RSV G glycoprotein . Recombinant G protein was purified by immunoaffinity chromatography using a neutralizing monoclonal antibody . Cotton rats immunized with the recombinant G protein produced serum antibodies to the G glycoprotein that neutralized RSV in vitro . The study demonstrates that the G protein of RSV can be expressed in bacteria and that at least one neutralizing epitope is not structurally dependent on carbohydrates.

J Bacteriol, 1993 Mar, 175(5), 1524 - 7
The rfaS gene, which is involved in production of a rough form of lipopolysaccharide core in Escherichia coli K-12, is not present in the rfa cluster of Salmonella typhimurium LT2; Klena JD et al.; Partial sequencing of the rfa cluster of Salmonella typhimurium LT2 indicated a region of 336 bp between rfaP and rfaB in the site occupied by the rfaS gene in Escherichia coli K-12 . This region does not contain a functional rfaS gene, although DNA analysis suggests that the region may have contained an ancestral gene . This conclusion that S . typhimurium LT2 lacks rfaS is supported by its lipopolysaccharide (LPS) gel phenotype, since LT2 does not make the lipooligosaccharide band characteristic of LPS from smooth strains of E . coli K-12.

Eur J Biochem, 1993 Mar 1, 212(2), 431 - 40
Purification and characterization of anthranilate synthase from Catharanthus roseus; Poulsen C et al.; Anthranilate synthase (EC 4.1.3.27) has been purified from cell cultures of Catharanthus roseus by poly(ethylene glycol) precipitation/fractionation and subsequent separation by anion exchange on Q-Sepharose, Orange A dye chromatography, Mono Q anion-exchange chromatography and Superose 6 gel filtration . By analogy to anthranilate synthases from other sources it does look like the enzyme is a tetramer composed of two large and two small subunits, with molecular mass 67 and 25.5 +/- 0.5 kDa, respectively . The molecular mass determined by gel filtration was 143 +/- 5 kDa . The enzyme had a pI of 5.1 determined by chromatofocusing . The pH optimum was between pH 7.5 and pH 8.3, but the type of buffer used affected the results . The enzyme could utilize NH4+ as ammonium donor instead of glutamine . The enzyme showed normal Michaelis-Menten kinetics with respect to the substrates L-glutamine and chorismate, and the cofactor Mg2+, Km values for L-glutamine was determined to be 0.37 +/- 0.05 mM, for chorismate 67 +/- 3 microM, and for MgCl2 0.26 +/- 0.03 mM respectively . Anthranilate synthase was inhibited by L-tryptophan, tryptamine and D-tryptophan (with L-tryptophan being the best inhibitor) . The enzyme was allosterically regulated showing positive cooperatively of chorismate binding at higher concentrations of tryptophan . For a tryptophan concentration of 20 microM the Hill coefficient was determined to be 2 . The tryptophan binding sites showed positive cooperatively for higher concentrations of chorismate . The purified enzyme did not contain anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase activity and is thus not of the same type as the well characterized Salmonella typhimurium anthranilate synthase/phosphoribosyl pyrophosphate transferase bifunctional type.

Am Rev Respir Dis, 1993 Mar, 147(3), 658 - 63
Pulmonary complications of human immunodeficiency virus infection in Bujumbura, Burundi; Kamanfu G et al.; To determine the types of pulmonary disease associated with human immunodeficiency virus (HIV) infection, we conducted a prospective study of 302 consecutive patients admitted for acute respiratory disease to a university hospital in Bujumbura, Burundi . Diagnoses were made according to well-defined criteria . Of the total, 222 patients (73.5%) were HIV seropositive, with women younger than men . Features suggestive of underlying HIV infection were the clinical findings of oral thrush, peripheral lymphadenopathy, or herpes zoster and the radiographic abnormalities of hilar-mediastinal adenopathy or a reticulonodular infiltrate . Tuberculosis and community-acquired pneumonia occurred with approximately equal frequency in the HIV-seropositive and seronegative groups . Pneumocystis carinii pneumonia was diagnosed in 11 patients, all seropositive . Gram-negative bacteremia, especially Salmonella typhimurium, occurred in 23 seropositive patients (10.4%) . A total of 24 seropositive patients died during the initial hospitalization, and 11 others required readmission; no seronegative patients died or were rehospitalized . We conclude that HIV infection is a major risk factor for the development of acute respiratory diseases in adults of sufficient severity to require hospitalization in Bujumbura . In this Central African country, where exposure to virulent bacterial pathogens is ubiquitous, tuberculosis, pneumonia, and salmonellosis occur with much greater frequency than classic AIDS-defining opportunistic infections or malignancies.

Infect Immun, 1993 Mar, 61(3), 823 - 9
T-cell-independent resistance to infection and generation of immunity to Francisella tularensis; Elkins KL et al.; The intraperitoneal 50% lethal dose (LD50) for Francisella tularensis LVS in both normal control heterozygote BALB/c nu/+ mice and BALB/c nu/nu mice was 2 x 10(0) . Both nu/+ and nu/nu mice given 10(7) LVS bacteria or more intradermally (i.d.) died, with a mean time to death of about 7 to 8 days . On the other hand, nu/+ mice given 10(6) LVS bacteria or less survived for more than 60 days and cleared systemic bacteria, while nu/nu mice given 10(6) LVS bacteria or less survived for more than 10 days but died between days 25 and 30 . Thus, the short-term (i.e., < 10-day) i.d . LD50 of both nu/nu and nu/+ mice was 3 x 10(6), but the long-term (i.e., > 10-day) i.d . LD50 of nu/nu mice was less than 7 x 10(0) . The short-term survival of i.d . infection was dependent on tumor necrosis factor and gamma interferon: treatment of nu/nu mice with anti-tumor necrosis factor or anti-gamma interferon at the time of i.d . infection resulted in death from infection 7 to 8 days later, whereas control infected nu/nu mice survived for 26 days . nu/nu mice infected with LVS i.d . generated LVS-specific serum antibodies, which were predominantly immunoglobulin M: titers peaked 7 days after i.d . infection but declined sharply by day 21, after which mice died . Surprisingly, nu/nu mice given 10(3) LVS bacteria i.d . became resistant to a lethal challenge (5,000 LD50s) of LVS intraperitoneally within 2 days after i.d . infection; nu/nu mice similarly infected with LVS i.d . and challenged with Salmonella typhimurium (10 LD50s) were not protected . nu/nu mice given nu/+ spleen cells intravenously as a source of mature T cells survived i.d . infection for more than 60 days and cleared bacteria . Taken together, these studies demonstrate that i.d . infection of nu/nu mice with LVS rapidly generates T-cell-independent, short-term, specific protective immunity against lethal challenge, but T lymphocytes are essential for long-term survival.

Infect Immun, 1993 Mar, 61(3), 1004 - 15
Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins; Jagusztyn-Krynicka EK et al.; A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed . These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B . Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments . Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene . Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides . Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues . Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized . LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses . The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.

Mol Microbiol, 1993 Mar, 7(6), 933 - 6
Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages; Buchmeier NA et al.; Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s . Both the recA and recBC mutants were attenuated in mice . The mutants were also sensitive to killing by macrophages in vitro . The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage-like cells that fail to generate superoxide . This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.

Genetics, 1993 Mar, 133(3), 449 - 54
Natural populations of Escherichia coli and Salmonella typhimurium harbor the same classes of insertion sequences; Bisercic M et al.; Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange . In this study, genetically diverse collections of E . coli and S . typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction . A majority of salmonellae contained IS1 or IS3, elements originally recovered from E . coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E . coli . Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E . coli and S . typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa . This suggests that copies of IS200 have not been recently transferred between E . coli and S . typhimurium and that the element was present in the common ancestor to both species . IS200 is polymorphic within E . coli but homogeneous among isolates of S . typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.

J Bacteriol, 1993 Mar, 175(5), 1457 - 66
Genetic structure and regulation of the cysG gene in Salmonella typhimurium; Goldman BS et al.; Siroheme, a cofactor of both sulfite and nitrite reductase in Salmonella typhimurium, requires the cysG gene for its synthesis . Three steps are required to synthesize siroheme from uroporphyrinogen III, the last common intermediate in the heme and siroheme pathways . All previously characterized cysG mutants were shown to be defective for the synthesis of cobalamin (B12), which shares a common precursor with siroheme . Since few cysG auxotrophs had been previously analyzed and since there is no evidence of siroheme mutants outside of the cysG region, we sought to expand the analysis of the region by isolating more mutations and studying the transcriptional regulation of the cysG gene using lacZ fusions . We isolated and analyzed 66 cysG auxotrophs . All were defective for both siroheme and cobalamin synthesis . Five exceptional mutants were partially defective for the synthesis of both and appear to be leaky . Complementation tests with tandem duplications suggest that the mutations causing the Cys auxotrophy affect only one cistron . The cysG gene is transcribed in a clockwise direction; this was demonstrated by a method that permits determining the orientation of two genes of unknown orientation provided their relative map order is known . The cysG gene was not part of the cysteine regulon, but had a substantial basal level of expression which was induced fivefold when cells were grown anaerobically on nitrite . Finally, we used Mud-generated duplications to genetically determine the organization of the cysG and nirB genes.

Infect Immun, 1993 Mar, 61(3), 940 - 6
Isolation and characterization of a cloned Porphyromonas gingivalis hemagglutinin from an avirulent strain of Salmonella typhimurium; Dusek DM et al.; Identification of surface macromolecules of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications for studying host-parasite interactions as well as for potential vaccine development . The objective of this study was to determine whether a cloned, P . gingivalis hemagglutinin gene could be expressed in an intact form in an avirulent Salmonella typhimurium vaccine construct and to characterize the recombinant protein . The recombinant protein was purified from the vaccine strain, characterized, and tested for biological activity as a competitive inhibitor of hemagglutination . Cells of S . typhimurium SL3261/pST7 grown in Luria broth were broken by sonic disruption and fractionated . The purified recombinant protein was found to inhibit hemagglutination of erythrocytes by whole P . gingivalis cells . The same purified protein was analyzed for its N-terminal amino acid sequence and amino acid composition and found to match that predicted from the nucleotide sequence of the cloned gene . These results indicate that a surface macromolecule of P . gingivalis can be expressed in an intact and biologically active form in a Salmonella carrier strain.

Microb Pathog, 1993 Mar, 14(3), 217 - 27
Tumor necrosis factor-alpha mediates the early pathology in Salmonella infection of the gastrointestinal tract; Arnold JW et al.; Salmonella infection of the intestinal tract results in damage to the gut epithelium . While it is generally believed that bacteria and/or bacterial products account for this pathology, the role of host factors has not been explored . Using a ligated intestinal loop model, we investigated whether tumor necrosis factor-alpha (TNF-alpha) could contribute to the tissue pathology associated with Salmonella infection . Intestinal segments infected with Salmonella typhimurium had high levels of fluid secretion as early as 6 h post-bacterial infection . At this time point, low levels of TNF activity were also present in the fluid obtained from infected segments . At 20 h post-infection, high levels of TNF activity were present in fluids obtained from infected intestinal segments and was characterized as TNF-alpha by neutralization experiments using rabbit antisera to TNF-alpha . TNF-alpha production was further verified by Northern blot analysis using RNA obtained from cells eluted from the infected intestinal segments . In contrast, no TNF activity was found in fluid obtained from intestinal segments challenged with cholera toxin, which induces fluid secretion with little to no inflammatory response . Double labeling by in situ hybridization and immunocytochemistry revealed that macrophages in the lamina propria were producing the TNF-alpha mRNA . To investigate what role TNF-alpha might play in Salmonella-induced inflammation, intestinal segments were injected with recombinant mouse TNF-alpha (rTNF-alpha) or mice were pretreated with antibody to TNF-alpha or a control antibody prior to Salmonella infection . The histological profile of intestinal segments injected with rTNF-alpha appeared identical to segments infected with S . typhimurium . Further, pathology was completely eliminated in infected mice pretreated with antibody to TNF-alpha . These results document the production of TNF-alpha in the intestinal tract following S . typhimurium infection and show that the early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms.

Zentralbl Veterinarmed B, 1993 Mar, 40(2), 105 - 12
{Electron microscopic studies of fimbriae and lectin phagocytosis of Salmonella typhimurium variety copenhagen (STMVC)}; Grund S et al.; On Salmonella typhimurium variatio copenhagen (STMVC) strains, isolated from pigeons with acute Salmonellosis, two types of fimbriae can be identified, depending on different growth conditions . In addition to the common 7 nm fimbriae we were able to demonstrate in the electron microscope, thin, curled fimbriae 3 nm in diameter which are characterized by a mannose-resistant hemagglutination . The simultaneous expression of both types of fimbriae on a single bacterial cell can be induced by transferring a microcolony from agar medium (3 nm fimbriae), to the surface of a nutrient broth (7 nm fimbriae) and continuing the incubation . One selected strain expresses only the thin fimbriae on nearly all the bacteria on an agar medium . These thin fimbriae seem to play a role in the lectinophagocytosis in macrophage cultures . The attachment of Salmonellae mediated by these fimbriae as well as the internalization of fimbriated cells by macrophages is shown in the electron microscope and discussed in respect to infection and immunization.

Invest Ophthalmol Vis Sci, 1993 Mar, 34(3), 673 - 81
Monoclonal antibody against CD11b/CD18 inhibits endotoxin-induced uveitis; Whitcup SM et al.; PURPOSE . To examine the serial expression of cell adhesion molecules in C3H/HeN mice with endotoxin-induced uveitis, and to study the effect of treatment with a monoclonal antibody against Mac-1 on the development of endotoxin-induced uveitis . METHODS . Immunohistochemical staining was used to document the serial expression of Mac-1, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated antigen-1 (LFA-1) in eyes of C3H/HeN mice with endotoxin-induced uveitis . Then, in two separate experiments, endotoxin-induced uveitis was produced in a total of 48 mice by injecting Salmonella typhimurium endotoxin into one hind footpad . At the time of endotoxin injection, 24 mice received an intraperitoneal injection of anti-Mac-1 antibody and 24 control mice received an intraperitoneal injection of rat IgG . Histopathologic sections of eyes taken 24 hours after endotoxin injection were graded by two masked observers on a scale of 0 to 4 . RESULTS . Intercellular adhesion molecule-1 was first expressed on the ciliary body epithelium 6 hr after endotoxin injection and Mac-1 and LFA-1 were expressed on infiltrating inflammatory cells 12 hr after endotoxin injection . Treatment with anti-MAC-1 antibody significantly inhibited the development of ocular inflammation when compared with treatment with control IgG (P < 0.001) . CONCLUSION . Intercellular adhesion molecule-1 is expressed in the eye before clinical or histologic signs of inflammation . Furthermore, treatment with antibody against Mac-1 significantly inhibits the development of endotoxin-induced uveitis in mice and suggests that anti-Mac-1 antibody may be useful for treating acute ocular inflammation.

Mutagenesis, 1993 Mar, 8(2), 93 - 100
Report of the Association of British Pharmaceutical Industries Collaborative Study Group . Collaborative study to evaluate the inter/intra laboratory reproducibility and phenotypic stability of Salmonella typhimurium TA97a and TA102; Wilcox P et al.; A collaborative trial was carried out to determine the intra/interlaboratory variability of Salmonella typhimurium strains TA102 and TA97a with regard to spontaneous revertant frequency and in response to four model mutagens (cumene hydroperoxide and bleomycin for strain TA102, and 4-nitrophenylenediamine and 4-aminoantipyrine for strain TA97a) . A secondary objective of the trial was to monitor the stability of the strains after storage for up to 8 months and identify any technical problems associated with their use . Thirteen different laboratories participated in the trial, all receiving identical stock cultures of the bacterial strains and samples from the same batch of mutagenic compound . A standard protocol was followed and two independent experiments were carried out within 1 month of receipt of the strains/compounds (phase I), and again after a period of 6-8 months (phase II) . Comparative studies with the standard strain TA100 after treatment with 4-nitrophenylenediamine were carried out as part of phase II . Overall, both strains gave acceptably consistent results in different laboratories and are considered useful for screening purposes when used under standardized conditions . One major source of interlaboratory variability identified for TA102 appears to be the sensitivity of different types of automatic colony counter for detecting the micro-colony revertants that this strain produces.

Mutagenesis, 1993 Mar, 8(2), 113 - 20
Mutagenic activation of 4-aminobiphenyl and its N-hydroxy derivatives by microsomes from cultured human uroepithelial cells; Hatcher JF et al.; Activation of the human bladder carcinogen 4-aminobiphenyl (ABP) and its N-hydroxy derivatives was investigated using lysates and subcellular enzyme preparations from cultured human uroepithelial cells (HUC) . Mutagenic activation was determined using Salmonella typhimurium strains TA98; TA98/1,8-DNP6, a derivative deficient in acetyl coenzyme A:N-hydroxyarylamine O-acetyltransferase (OAT); and YG1024, a derivative of TA98 with elevated OAT activity and enhanced sensitivity to mutation by N-hydroxyarylamines . Mutagenicity of ABP catalyzed by HUC microsomes was detected in YG1024 but not in the parent strain TA98 . HUC microsomes also catalyzed the mutagenic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and the relative sensitivity of the tester strains was YG1024 > TA98 > TA98/1,8-DNP6, indicating N-hydroxy-4-aminobiphenyl (N-OH-ABP) as the mutagenic intermediate . In contrast, the mutagenic activity of N-acetoxy-4-acetylaminobiphenyl incubated with HUC microsomes was approximately equal in TA98 and YG1024, and may involve N-acetoxy-4-aminobiphenyl (N-OAc-ABP) as the intermediate . High pressure liquid chromatography (HPLC) of the DNA hydrolysate obtained after incubation of {3H}N-OH-ABP with YG1024, showed N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-ABP) as the primary adduct, based on mobility of the radioactivity in comparison with the synthetic standard . Additionally, HUC microsomes catalyzed the binding of {3H}N-OH-ABP to RNA in the presence of 4-acetylaminobiphenyl (AABP), N-OH-AABP and acetyl coenzyme A as acetyl donors, and this binding was blocked by paraoxon . The hydrolysate obtained from incubation of DNA with {3H}N-OH-ABP and HUC microsomes, with AABP as acetyl donor, revealed the formation of dG-ABP adduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1993 Mar, 293(3), 197 - 204
Acidification of Escherichia coli and Salmonella typhimurium cytoplasm reduces the mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine; Oktyabrsky ON et al.; Preliminary acidification of the cytoplasm of E . coli cells growing at pH 6.9 by adding to the medium 50 mM of sodium acetate or propionate reduced the mutagenic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to almost the spontaneous level . In experiments with S . typhimurium the protective effects of cytoplasm acidification against the mutagenic effect of MNNG was observed at pH 5.5 and was absent at a medium pH of 6.9 . Alkalinization of the cytoplasm by adding 80 mM of methylamine to the growth medium at pH 8.1 did not modify the effect of MNNG on the cells of E . coli and S . typhimurium . Alkalinization of the cytoplasm of E . coli B/r and K12 was followed by a reduction of the intracellular non-protein SH group level by 25 and 50%, respectively . It is supposed that the protective effect of acidification may be due to a decline in the productivity of mutagenically more active intermediates of MNNG when the pH is reduced and the associated fall of the level of intracellular non-protein thiols occurs . The above situation may serve as a model for studying the effects of MNNG and other alkylating agents on cells differing in physiological status.

Mutat Res, 1993 Mar, 299(1), 63 - 73
Mutagenicity of nitro- and amino-substituted carbazoles in Salmonella typhimurium . I . Monosubstituted derivatives of 9H-carbazole; Andre V et al.; Mononitro, monoamino and monoacetamido carbazoles were assayed for mutagenicity in Salmonella typhimurium strains TA1535, TA1538, TA1537, TA1977, TA98 and TA100, with and without the addition of S9 from phenobarbital-induced rat liver . The role of bacterial metabolism of the nitro group was also studied using the additional strains TA98NR and TA98/1,8DNP6 . None of the compounds was active in TA1535, while only 2-nitrocarbazole and 3-nitrocarbazole presented a weak mutagenicity towards its pKM101 derivative, TA100 . All four nitrocarbazole isomers were mutagenic for TA1538 and TA98, their activities decreasing in the order: 2-nitrocarbazole approximately 3-nitrocarbazole > 1-nitrocarbazole > 4-nitrocarbazole . Direct-acting mutagenicities for TA1537 were lower than for TA1538, but varied in the same order . Nitro reduction was an important step of metabolic activation of nitrocarbazoles, as indicated by the dramatic reduction of activity with TA98NR, as compared to TA98 . Results obtained with TA98/1,8DNP6 showed that O-acetyltransferase was only partly required for the expression of mutagenic potency of these compounds . 2-Aminocarbazole was a weak direct-acting mutagen for TA1538 and TA98 . Its activity was strongly increased in the presence of S9 mix, while 3-aminocarbazole became active in these conditions . The acetamido derivatives were consistently less mutagenic than their parent amines . These results show that nitrocarbazoles and aminocarbazoles behave as reactive frameshift mutagens, acting mainly through the formation of esterified hydroxylamines . The very low activity of 4-nitrocarbazole might be related to an orientation of the nitro group perpendicular to the aromatic ring.

Mutat Res, 1993 Mar, 299(1), 45 - 53
Detection of mutagenic activity in textiles with Salmonella typhimurium; Knasmuller S et al.; A hundred and ninety-six textile samples were tested in a modified version of the Salmonella/microsome assay for release of mutagenic contaminants . As heat sterilization of the samples can result in reduction of mutagenic activity, tests were performed with streptomycin resistant derivatives of Salmonella tester strains TA98 and TA100 . Textile samples were preincubated in buffered saline (PBS), DMSO or ethanol . Subsequently, the fabrics were placed on streptomycin supplemented selective agar plates . In total, 18 samples (9.2%) exerted mutagenic activity . DMSO was the most effective solvent (15 positives) followed by ethanol (9 positive samples) and PBS (7 positives) . Most fabrics (16) caused mutagenic effects only upon metabolic activation with liver S9 mix . Chemical analysis indicates that the positive results obtained with PBS are not due to release of histidine or formaldehyde . Three directly active samples gave negative results in strain TA98NR which is devoid of classical nitroreductase . With one exception all other textiles were negative in strain TA98/1,8-DNP6 (which lacks O-acetyltransferase) . These findings indicate that nitroaromatics and amines might be responsible for the mutagenic effects of the textiles.

J Toxicol Sci, 1993 Feb, 18 Suppl 1, 177 - 81
{Reverse mutation test of trandolapril (RU44570) with bacteria}; Aruga F et al.; Mutagenicity of trandolapril (RU44570) was evaluated by the reverse mutation test with bacteria (test strains used: Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2uvrA) . The test was conducted by the preincubation method without metabolic activation (in the absence of S9 mix) and with metabolic activation (in the presence of S9 mix) . The number of revertant colonies in the bacterial strains did not exceed twice that obtained in the solvent control either in the presence or absence of S9 mix . There was no dose-dependent increase in the number of revertant colonies observed . Based on the above results, it is concluded that RU44570 has no mutagenicity (gene mutation) under the conditions of the present study.

Microb Pathog, 1993 Feb, 14(2), 95 - 102
Immunization of mice with Salmonella typhimurium C5 aroA expressing a genetically toxoided derivative of the pneumococcal toxin pneumolysin; Paton JC et al.; An attenuated Salmonella strain expressing a genetically toxoided derivative of the pneumococcal toxin pneumolysin was constructed by first transforming a methylation-positive, restriction-negative Salmonella with plasmid pJCP20M, a derivative of pBR322 containing the modified pneumolysin gene . Plasmid DNA was then extracted and transformed into Salmonella typhimurium C5 aroA . The transformant (denoted JM8) was capable of constitutively expressing the modified pneumolysin gene in vitro and stably maintained the recombinant plasmid containing the pneumococcal DNA, even in the absence of antibiotic selection . When JM8, or the parental Salmonella C5 aroA carrying pBR322 (denoted JM6), were administered orally to mice, both strains were capable of at least transient colonization of the Peyer's patches . Sera from JM8 mice (but not those fed JM6) had significant anti-pneumolysin IgG and IgA ELISA titres . Intraperitoneal administration of JM8 resulted in higher anti-pneumolysin IgG titres, but lower specific IgA levels.

Enferm Infecc Microbiol Clin, 1993 Feb, 11(2), 93 - 6
{Salmonella typhimurium as the causal agent of pulmonary cavitations}; Saballs P et al.; BACKGROUND: Relation between patients with immunosuppression (malignancy, renal transplant) and bacteremia by Salmonella non-typhi, specially by Salmonella typhimurium, is known . This relation has been published for patients who suffer from AIDS, and so Salmonella bacteremia could even begin the clinical disease . But even though the relation between the infection by Salmonella and AIDS is well known, and the pulmonary involvement in them has been told, this etiology as a producing agent of lung cavitation has not much documentation . METHODS: We examined three patients (two women and one man) suffering from fever of one week of duration, cough, expectoration and thoracic pain in two of them . The third patient had fever and cachexia without clinical symptoms . The man and the two women had a blood count with neutrophilia and leucocytosis in two cases and leucopenia in the other one . All of them suffered a very important depression of cell immunity (CD4 of 140, 70 and 4, respectively) and positive blood cultures for Salmonella typhimurium . RESULTS: Chest X-Ray showed, in all the cases, pulmonary cavities . CONCLUSIONS: Salmonella typhimurium must be included among the agents that can produce pulmonary cavities like Staphylococcus aureus, mycobacteria, fungus and other gram-negative bacilli.

Zentralbl Hyg Umweltmed, 1993 Feb, 193(5), 471 - 80
The influence of culture conditions on the susceptibility of Salmonella typhimurium in the Salmonella mutagenicity test; Mersch-Sundermann V et al.; To determine the variability of tester strain susceptibility in the Salmonella mutagenicity assay and to optimize the culturing procedure we examined the influences of the overnight culture period (12-16 h), the use of an additional short-term culturing procedure (1-4 h) and the Salmonella density (cfu/plate) on the rate of revertants per plate . As tester strains we used Salmonella typhimurium TA97, TA98, TA100 and TA102 . As shown previously for other microbial genotoxicity short-term tests (i.e . with Escherichia coli PQ37), we observed the highest susceptibility of the Salmonella strains, i.e . the highest amounts of revertants per plate, when using a 12 h overnight culture followed by a 2 h short-term culturing procedure . We calibrated the bacterial count to 100 x 10(6) cfu per assay by photometric measurement (600 nm) . Initially, we used 0-1 nmole 2,4,7-trinitro-9-fluorenone with S . typhimurium TA97, 0-15 nmole daunomycin with strain TA98, 0-25 nmole sodium azide with strain TA100 and 0-15 nmole methylmethanesulfonate with strain TA102 as reference compounds in the standard plate incorporation test . Subsequently, to evaluate the results of the culturing procedure variations we examined 22 well-known mutagenic and direct-acting (-S9-mix) compounds out of different chemical classes using both the standard and a modified culturing procedure . The comparison of these results showed a 64% (for 4-nitroquinoline-N-oxide) to 421% (for sodium azide) increased amount of revertants for the modified test protocol.

Ultramicroscopy, 1993 Feb, 49(1-4), 417 - 25
Size of the export channel in the flagellar filament of Salmonella typhimurium; Ruiz T et al.; The size of the putative export channel in the bacterial flagellar filament appears small (25 A) in studies done by electron microscopy but large (60 A) in studies done by X-ray diffraction . We have undertaken additional studies by electron microscopy to examine some of the possible causes of the difference . A comparison of three-dimensional image reconstructions of native and reconstituted filaments rules out the presence or absence of flagellin monomers in the export channel as the source of the variation in apparent channel size . The channel seen in reconstructions from both kinds of filaments is 25 A in diameter . The difference in the previous studies is more probably a result of artifacts introduced in either the X-ray or the electron microscopical methodology . Comparisons of three-dimensional reconstructions from images of filaments embedded in various stains (anionic, cationic and neutral) and in ice, taken at a range of defocuses, rule out the two most likely sources of artifact in electron microscopy (i.e., staining artifacts and defocus phase contrast) . Based on these studies we suggest that the channel seen in the image reconstructions is free of exported flagellin monomers, that its true diameter is about 25 A, and, therefore, that the flagellin monomer must be unfolded to pass along it.

Mol Gen Genet, 1993 Feb, 237(1-2), 273 - 86
Integration host factor (IHF) modulates the expression of the pyrimidine-specific promoter of the carAB operons of Escherichia coli K12 and Salmonella typhimurium LT2; Charlier D et al.; We report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons . Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 bp upstream of the transcription initiation site of P1 in both organisms . This was confirmed by deletion analysis of the target site . DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A+T-rich stretch, centred around nucleotide -305 upstream of the transcription initiation site . Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix . Measurements of carbamoyl-phosphate synthetase (CPSase) and beta-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH+/-), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines . IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines . These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P1 by pyrimidines in the absence of IHF binding . IHF thus appears to be required for maximal expression as well as for establishment of full repression . IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.

J Appl Bacteriol, 1993 Feb, 74(2), 181 - 90
An ion-exchange based extraction method for the detection of salmonellas in soil; Turpin PE et al.; A method that uses a cation-exchange resin (Chelex 100) and differential centrifugation for the extraction and detection of salmonellas in soil was developed . The extraction efficiencies of a range of materials were examined and Chelex plus polyethylene glycol was identified as the best combination . Shake speeds, shake times and differential centrifugation speeds were selected to give an optimum salmonella recovery . The Chelex method accurately enumerated 1 cell per 10 g of nonsterile soil within 24 h . Addition of glycerol to soil samples enabled storage at -70 degrees C for 85 d without significant decreases in salmonella numbers . The Oxoid Salmonella Rapid Test (SRT) could be used to pre-screen large numbers of soil samples for the presence of salmonellas, prior to analysis by the Chelex method . The SRT method detected Salmonella typhimurium at levels as low as 2.5 cells per 10 g of nonsterile soil.

Poult Sci, 1993 Feb, 72(2), 259 - 66
Influence of a novel oxy-halogen compound on early growth and nitrogen retention of broiler chickens challenged with Salmonella; Pardue SL et al.; The potential of a novel oxy-halogen compound (OHC) to alter early growth and nitrogen retention of broiler chickens challenged with Salmonella was evaluated . Three hundred and twenty female broiler chicks (Arbor Acres x Arbor Acres) were weighed and distributed randomly within a 2 x 4 factorial arrangement of treatments . Main effects examined were the presence or absence of Salmonella typhimurium (ST) inoculation and OHC treatment . At hatching, 80 chicks were placed in electrically heated brooder batteries in each of four identical isolation rooms . Chicks designated to receive 100 microL of an oral inoculum containing 10(5) ST cfu at 3 days of age were in two of the rooms, and uninoculated chicks were raised in the other two rooms . Four replicates of 10 chicks each received drinking water containing either 0, .05, .1, or .5% OHC for each level of ST . Chicks administered .05% OHC exhibited enhanced (P < or = .01) growth at 7 and 14 days of age when compared with control values . A significant OHC by ST interaction was observed at 7 (P < or = .0001) and 14 (P < or = .03) days of age . Feed utilization was improved (P < or = .01) by OHC administration (.05 and .1%) from hatching to 7 days of age . The administration of OHC reduced (P < or = .01) nitrogen excretion and enhanced (P < or = .01) nitrogen retention by chicks at Day 7 . Cecal ST log10 counts at 7 days of age for chicks given water containing 0, .05, or .1% OHC were 4.72, 3.93, and 3.74, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1993 Feb, 175(4), 1032 - 7
Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of proteinase and proteinase-defective Escherichia coli mutants; Ichihara S et al.; Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using a chromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine beta-naphthyl ester . The three clones isolated, pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region . A 0.9-kb Sau3AI fragment within this region was found to be responsible for the overproduction of the proteinase, and the nucleotide sequence of the region was then determined . The proteinase was purified to homogeneity from the soluble fraction of an overproducing strain possessing the cloned gene . N-terminal amino acid sequencing of the purified protein revealed that the cloned gene is the structural gene for the protein, with the protein being synthesized in precursor form with a signal peptide . On the basis of its molecular mass (20 kDa), periplasmic localization, and substrate specificity, we conclude this protein to be protease I . By using the gene cloned on a plasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene (Kmr) on the chromosome . The Kmr gene was mapped at 11.8 min, the gene order being dnaZ-adk-ush-Kmr-purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonella typhimurium . Therefore, the gene was named apeA . Deletion of the apeA gene, either with or without deletion of other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the various media tested.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1033 - 7
Molecular, functional, and evolutionary analysis of sequences specific to Salmonella; Groisman EA et al.; In that salmonellae have been implicated in an unprecedented array of diseases, sequences found to be specific to this species are often thought to be involved in the virulence attributes not seen in other enteric bacteria . To identify the molecular, genetic, and phenotypic characteristics that differentiate bacterial species, we analyzed five cloned DNA fragments that were originally described as being confined to Salmonella . Most of these segments mapped to unique positions on the Salmonella typhimurium chromosome indicative of independent evolutionary events, and three had G+C contents considerably lower than that of the Salmonella genome, suggesting that they arose through horizontal transfer . The nucleotide sequence was determined for one of the clones exhibiting an atypical base composition . This 4.9-kb fragment contained an open reading frame with structural similarity to the LysR family of transcriptional regulators . Strains harboring deletions in this region were tested for > 120 phenotypic characteristics including the effects on a collection of environmentally regulated lac gene fusions . In addition, all deletion strains behaved like the wild-type parent when tested for virulence in mice.

Pediatr Infect Dis J, 1993 Feb, 12(2), 139 - 45
Multiply resistant nontyphoidal Salmonella gastroenteritis in children; Maiorini E et al.; From January, 1990, to December 31, 1990, 75 children with multiply resistant Salmonella gastroenteritis were studied at the Children's Hospital "Ricardo Gutierrez" of Buenos Aires . These children ranged from 1 month to 15 years of age . Infection was community-acquired in 20 (26.6%), nosocomially acquired in 50 (66.7%) and undetermined in 5 . Thirty-nine (52%) had grossly bloody stools . Fever occurred at some point in the clinical course in 61 children (81.3%) with a duration of 1 to 33 days (mean, 6.7 days) . The duration of diarrhea (1 to 69 days) was longer in those who developed complications (P < 0.001) . Six (8%) developed enterocolitis (2 with bowel perforation), 1 had a pulmonary abscess and 8 (11.4%) had bacteremia; 4 children died (5.3%) . Salmonella typhimurium was the most common serovar (85.3%) . Ninety percent minimum inhibitory concentration studies demonstrated that all strains were resistant to ampicillin (> 128 micrograms/ml), cephalothin (> 128 micrograms/ml), cefuroxime (> 128 micrograms/ml), nalidixic acid (> 256 micrograms/ml), rifampin (> 256 micrograms/ml), gentamicin (> 256 micrograms/ml) and tobramycin (256 micrograms/ml); 77.3% of strains were resistant to ceftazidime (32 micrograms/ml), 97.6% to netilmicin (> 256 micrograms/ml), 92.8% to amikacin (256 micrograms/ml), 24.4% to isepamicin (32 micrograms/ml), 5.3% to chloramphenicol (4 micrograms/ml) and 2.7% to cefoxitin (2 micrograms/ml) . The 90% minimum inhibitory concentration of cefotaxime and ceftazidime was reduced by the addition of clavulanate . Aggressive multiply resistant Salmonella strains are a major pediatric problem in Buenos Aires.

J Bacteriol, 1993 Feb, 175(3), 802 - 10
Salmonella typhimurium fliG and fliN mutations causing defects in assembly, rotation, and switching of the flagellar motor; Irikura VM et al.; FliG, FliM, and FliN are three proteins of Salmonella typhimurium that affect the rotation and switching of direction of the flagellar motor . An analysis of mutant alleles of FliM has been described recently (H . Sockett, S . Yamaguchi, M . Kihara, V . M . Irikura, and R . M . Macnab, J . Bacteriol . 174:793-806, 1992) . We have now analyzed a large number of mutations in the fliG and fliN genes that are responsible for four different types of defects: failure to assembly flagella (nonflagellate phenotype), failure to rotate flagella (paralyzed phenotype), and failure to display normal chemotaxis as a result of an abnormally high bias to clockwise (CW) or counterclockwise (CCW) rotation (CW-bias and CCW-bias phenotypes, respectively) . The null phenotype for fliG, caused by nonsense or frameshift mutations, was nonflagellate . However, a considerable part of the FliG amino acid sequence was not needed for flagellation, with several substantial in-frame deletions preventing motor rotation but not flagellar assembly . Missense mutations in fliG causing paralysis or abnormal switching occurred at a number of positions, almost all within the middle one-third of the gene . CW-bias and CCW-bias mutations tended to segregate into separate subclusters . The null phenotype of fliN is uncertain, since frameshift and nonsense mutations gave in some cases the nonflagellate phenotype and in other cases the paralyzed phenotype; in none of these cases was the phenotype a consequence of polar effects on downstream flagellar genes . Few positions in FliN were found to affect switching: only one gave rise to the CW mutant bias and only four gave rise to the CCW mutant bias . The different properties of the FliM, FliG, and FliN proteins with respect to the processes of assembly, rotation, and switching are discussed.

Infect Immun, 1993 Feb, 61(2), 719 - 28
Antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment; Yamauchi K et al.; Although the antimicrobial activity of lactoferrin has been well described, its mechanism of action has been poorly characterized . Recent work has indicated that in addition to binding iron, human lactoferrin damages the outer membrane of gram-negative bacteria . In this study, we determined whether bovine lactoferrin and a pepsin-derived bovine lactoferrin peptide (lactoferricin) fragment have similar activities . We found that both 20 microM bovine lactoferrin and 20 microM lactoferricin release intrinsically labeled {3H}lipopolysaccharide ({3H}LPS) from three bacterial strains, Escherichia coli CL99 1-2, Salmonella typhimurium SL696, and Salmonella montevideo SL5222 . Under most conditions, more LPS is released by the peptide fragment than by whole bovine lactoferrin . In the presence of either lactoferrin or lactoferricin there is increased killing of E . coli CL99 1-2 by lysozyme . Like human lactoferrin, bovine lactoferrin and lactoferricin have the ability to bind to free intrinsically labeled {3H}LPS molecules . In addition to these effects, whereas bovine lactoferrin was at most bacteriostatic, lactoferricin demonstrated consistent bactericidal activity against gram-negative bacteria . This bactericidal effect is modulated by the cations Ca2+, Mg2+, and Fe3+ but is independent of the osmolarity of the medium . Transmission electron microscopy of bacterial cells exposed to lactoferricin show the immediate development of electron-dense "membrane blisters." These experiments offer evidence that bovine lactoferrin and lactoferricin damage the outer membrane of gram-negative bacteria . Moreover, the peptide fragment lactoferricin has direct bactericidal activity . As lactoferrin is exposed to proteolytic factors in vivo which could cleave the lactoferricin fragment, the effects of this peptide are of both mechanistic and physiologic relevance.

Infect Immun, 1993 Feb, 61(2), 640 - 9
Light emission from a Mudlux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by interaction with the mouse macrophage cell line J774.2; Francis KP et al.; Hydrogen peroxide is known to induce a multigenic response in Salmonella typhimurium cells . We have used a Mudlux transcriptional reporter system to identify and isolate fusions in the virulent strain SL1344 which respond to hydrogen peroxide in vitro by light production, and one of these fusions, MPG203, has been further characterized . Transient light production was observed from MPG203 at levels of hydrogen peroxide as low as 10 microM . However, high levels of this toxic oxidizing agent resulted in light suppression, particularly at low bacterial densities . This fusion was also shown to produce light following adhesion to cells of the mouse macrophage cell line J774.2 . Furthermore, the response was greatly reduced in the presence of catalase, directly implicating hydrogen peroxide as the eliciting agent and suggesting the involvement of the hydrogen peroxide-induced bacterial stress response in the infection process . Chemiluminescence studies also indicated that inhibition of the respiratory burst may occur as the infection ratio is increased . In addition, the level of light produced from bacteria within individual macrophage cells was shown to vary.

Infect Immun, 1993 Feb, 61(2), 504 - 11
The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice; Gulig PA et al.; The virulence plasmids of Salmonella typhimurium and other invasive Salmonella serovars have long been associated with the ability of these bacteria to cause systemic infection beyond the intestines in orally inoculated animals . Genetic analysis of virulence genes on the high-molecular-weight plasmids has revealed that no more than five genes spanning a 6.2-kb region are sufficient to replace the entire plasmid for conferring virulence . However, the exact virulence function(s) encoded by these genes has not been elucidated . In this report, we measured the possible effect of the virulence plasmid on the growth rate of S . typhimurium in mice by two complementary procedures . The first procedure used segregation of a temperature-sensitive plasmid in vivo to provide a measure of bacterial divisions and the number of recovered marker plasmid-containing salmonellae as a measure of killing . In the second procedure, aroA deletions were transduced into virulence plasmid-containing and plasmid-cured S . typhimurium . Since AroA- salmonellae are inhibited for growth in vivo, if the virulence plasmid affected only growth rate, no difference in the recoveries of the paired AroA- strains would be seen . Virulence plasmid-containing S . typhimurium segregated the marker plasmid more rapidly than did the virulence plasmid-cured strain, and AroA- derivatives of both strains were recovered equally from mice . Therefore, the S . typhimurium virulence plasmid increased growth rate but had no detectable effect on killing or bacterial movement into deep tissues . To examine whether the plasmid accomplished this function by affecting the intracellular/extracellular location of bacteria, orally infected mice were injected with gentamicin to kill the extracellular bacteria . Wild-type and plasmid-cured S . typhimurium strains were equally resistant to gentamicin in vivo and hence most likely located intracellularly to equal degrees . When wild-type and plasmid-cured S . typhimurium strains were sequestered within peritoneal chambers in mice, the resulting extracellular growth was equal . Therefore, the virulence plasmid increases the growth rate of S . typhimurium in mice, probably within mouse cells.

J Antimicrob Chemother, 1993 Feb, 31(2), 313 - 9
Comparative efficacies of azithromycin and ciprofloxacin against experimental Salmonella typhimurium infection in mice; Butler T et al.; Azithromycin was compared with ciprofloxacin for the treatment of established intracellular infection with Salmonella typhimurium LT-2 in CF-1 mice . For studies of mortality, mice received five times the LD50 of organisms intraperitoneally and were given drugs intragastrically once daily for seven days . For studies of in-vivo antibacterial activity, splenic viable counts were measured in mice that had received 0.5 times the intraperitoneal LD50 and had been given drugs for three days . The MICs of azithromycin and ciprofloxacin, respectively, against LT-2 were 4.0 and 0.03 mg/L . The 50% protective doses of the drugs required to prevent mortality were azithromycin 24.7 mg/kg/day, and ciprofloxacin 30.2 mg/kg/day . Treatment with azithromycin and ciprofloxacin in doses of 25 and 100 mg/kg/day resulted in reduction of mean log10 cfu per spleen . Splenic concentrations of azithromycin up to 8 h after treatment exceeded its MIC against the LT-2 strain, whereas serum levels were less than the MIC . These results indicated that azithromycin given orally once daily was as effective as ciprofloxacin against established murine Salmonella infection and that the efficacy of azithromycin correlated with adequate tissue concentrations of antibiotic.

Mol Microbiol, 1993 Feb, 7(3), 351 - 8
Isolation and characterization of a topA mutant of Shigella flexneri; Bhriain NN et al.; Shigella flexneri was shown to possess a homologue of the Escherichia coli K-12 topA gene which encodes DNA topoisomerase I . The S . flexneri topA gene was replaced by a copy of the E . coli K-12 topA gene which has been insertionally inactivated by transposon Tn10 . The topoisomer distribution of reporter plasmids showed that the presence of this topA lesion in S . flexneri correlated with an increase in the level of negative DNA supercoiling in the mutant, indicating that topoisomerase I is required to relax DNA in S . flexneri as it is in E . coli and Salmonella typhimurium . The introduction of the topA mutation also resulted in repression of transcription of a thermally regulated invasion gene located on the 230 kb virulence plasmid . In addition, the topA mutant was hypersensitive to growth medium osmolarity, was unable to grow on MacConkey indicator plates and exhibited an increased doubling time under all growth conditions tested . All of these phenotypes were fully complemented in trans by a cloned copy of the E . coli topA gene carried on a recombinant plasmid . Unlike E . coli topA mutants which acquire compensatory mutations at a high frequency, such compensatory mutations were not detected in the S . flexneri topA::Tn10 mutant.

Antimicrob Agents Chemother, 1993 Feb, 37(2), 240 - 5
Relationship between antibacterial activity and porin binding of lactoferrin in Escherichia coli and Salmonella typhimurium; Naidu SS et al.; The effect of lactoferrin (Lf) on bacterial growth was tested by measuring conductance changes in the cultivation media by using a Malthus-AT system and was compared with the magnitude of 125I-labeled Lf binding in 15 clinical isolates of Escherichia coli . The binding property was inversely related to the change in bacterial metabolic rate (r = 0.91) and was directly related to the degree of bacteriostasis (r = 0.79) . The magnitude of Lf-bacterium interaction showed no correlation with the MIC of Lf . In certain strains, Lf at supraoptimal levels reduced the bacteriostatic effect . Thus, the Lf concentration in the growth media was critical for the antibacterial effect . The cell envelopes of Salmonella typhimurium 395MS with smooth lipopolysaccharide (LPS) and its five isogenic rough mutants revealed 38-kDa porin proteins as peroxidase-labeled-Lf-reactive components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (ligand blot) analysis . However, in the whole cell binding assay, parent strain 395MS demonstrated a very low interaction with 125I-Lf . On the other hand, Lf interaction gradually increased in correspondence with the decrease in LPS polysaccharide moiety in the isogenic rough mutants . Conductance measurement studies revealed that the low-level-Lf-binding (low-Lf-binding) strain 395MS with smooth LPS was relatively insusceptible to Lf, while the high-Lf-binding mutant Rd was more susceptible to Lf . These data suggested a correlation between Lf binding to porins and the Lf-mediated antimicrobial effect . The polysaccharide moiety of LPS shielded porins from the Lf interaction and concomitantly decreased the antibacterial effect.

Biol Pharm Bull, 1993 Feb, 16(2), 201 - 2
Low mitogenic activity of synthetic lipid A analog, 2,3-acyloxyacylgalactosamine-4-phosphate, in cultured murine splenocytes; Shimizu T et al.; The mitogenicity of chemically synthesized lipid A analogs, 2,3-acyloxyacylglucosamine-4-phosphate (A-103) and 2,3-acyloxyacylgalactosamine-4-phosphate (A-113), was compared . Synthetic lipid A analogs of the disaccharide-type (506), the monosaccharide-type (A-103) suspended in RPMI-1640 medium supplemented with triethylamine, and Salmonella typhimurium LT-2 lipopolysaccharide (LPS) were capable of increasing the incorporation of {3H}thymidine into splenocytes of C57BL/6 mice at various doses ranging from 1.0 to 100 micrograms/ml . However, the mitogenic activity of A-113 at these doses was markedly weaker than the activity of the above materials . When the A-103 and A-113 were suspended in RPMI-1640 medium supplemented with ethanol, the mitogenic activity of A-113 also showed lower activity than that of A-103 in the splenocytes of C57BL/6 mice . These findings indicate that the mitogenic activity of synthetic lipid A of the monosaccharide-type is affected by a kind of composed sugar.

J Gen Microbiol, 1993 Feb, 139 ( Pt 2), 259 - 66
Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression; Post DA et al.; The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium . A potential open reading frame (ORF) extends across the prs promoter and into the leader . The region between the prs coding region and an upstream gene (hemA) in E . coli and S . typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function . ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs . Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader . Northern blot analysis showed that the prs message in E . coli was on 1.3 and 2.7 kb transcripts . The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs . Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only . The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined . This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts . Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.

Mutat Res, 1993 Feb, 301(2), 113 - 9
Activation of benzo{a}pyrene and 2-aminoanthracene to bacteria mutagens by mussel digestive gland postmitochondrial fraction; Michel XR et al.; The ability of the mussel postmitochondrial fraction (S9) to activate benzo{a}pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested . The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP . This activation was improved by treating the mussel with 4,5,4',5'-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by alpha-naphthoflavone (ANF), a cytochrome P-450 inhibitor . However, both BaP activation and cytochrome P-450-related metabolic activities are much weaker in mussels than in vertebrates . Mussel S9 activates aromatic amines more effectively than BaP . Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation . As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent.

Mutat Res, 1993 Feb, 298(4), 269 - 75
Mutagenic activity of some platinum and palladium complexes; Uno Y et al.; The mutagenic properties of 16 platinum compounds were studied using Salmonella typhimurium TA98 and TA100 . Eight of the compounds were considered direct mutagens, as their mutagenicity was not dependent on metabolic activation by liver extracts . Potent mutagenicity and high toxicity were exhibited by cis-Pt(NH3)2Br2, cis-Pt(NH3)2Cl2, Pt(C5H12N2)Cl2 and Pt(en)Cl2 for both bacterial strains . When distilled water was used as the carrier solvent, these compounds were strongly mutagenic and toxic, but much less so when dimethyl sulfoxide was the solvent.

Mutat Res, 1993 Feb, 298(4), 261 - 7
Comparative study on the mutagenicity of three structurally related substituted aniline mustards in the Salmonella/microsome assay; Voutsinas G et al.; The ortho, meta and para isomers of N,N-bis(2-chloroethyl)aminocinnamic acid were tested for their ability to mutate Salmonella typhimurium strains in the Salmonella/microsome mutagenicity test . The aim of the work was to establish a structure-activity relationship between these three isomers . The drugs were found to induce base-pair substitutions, causing dose-dependent increases in his+ revertants, in strains TA100 and TA1535 . The study showed that the position of the substituent groups influenced the mutagenic activity of the compounds . The ortho isomer exhibited a poorer mutagenic effect than meta and this was found to be a weaker mutagen than para . The presence of metabolic activation enzymes in the test system induced a further increase in his+ revertants, in strains TA100 and TA1535, which is consistent with the findings for melphalan, a cancer chemotherapeutic agent with a chemical structure similar to that of the isomers tested.

Science, 1993 Jan 29, 259(5095), 686 - 8
Selection of bacterial virulence genes that are specifically induced in host tissues; Mahan MJ et al.; A genetic system was devised that positively selects for bacterial genes that are specifically induced when bacteria infect their host . With the pathogen Salmonella typhimurium, the genes identified by this selection show a marked induction in bacteria recovered from mouse spleen . Mutations in all ivi (in vivo-induced) genes that were tested conferred a defect in virulence . This genetic system was designed to be of general use in a wide variety of bacterial-host systems and has several applications in both vaccine and antimicrobial drug development.

Biochemistry, 1993 Jan 26, 32(3), 806 - 11
Inhibition of tryptophan synthase by (1-fluorovinyl)glycine; Xu Y et al.; Tryptophan synthase (alpha 2 beta 2 complex) from Salmonella typhimurium catalyzes the formation of tryptophan from serine and indole . The enzyme is inactivated by (1-fluorovinyl)glycine . Concomitant with enzyme inactivation, the absorbance at 485 nm increases, indicating covalent modification of pyridoxal 5'-phosphate . It is proposed that inactivation involves elimination of HF to form an allene, which reacts with a nucleophile at the active site . The inactivation reaction involves an alpha,beta-elimination, as does the formation of tryptophan from indole and serine . The inactivation occurs with k(in) > 1.3 s-1, which is very close to k(cat) (6.4 s-1) for the formation of tryptophan from indole and serine . The inactive enzyme (alpha 2 beta 2) regains activity with k(off) = 0.005 min-1 . Aminoacetone is formed during reaction, and pyridoxal 5'-phosphate is regenerated . Tryptophan synthase also catalyzes the dehydration of serine, or 3-fluoroalanine, to pyruvate in the absence of indole . This reaction involves an alpha,beta-elimination and the intermediate formation of an aminoacrylate adduct with pyridoxal 5'-phosphate, as does the formation of tryptophan . Pyruvate formation proceeds at less than 5% the rate of tryptophan formation . With {2-2H}serine an isotope effect (DVmax = 1.5) is observed . We propose that pyruvate formation is limited by the rate of hydration of the aminoacrylate intermediate and the rate of the abstraction of the serine alpha-hydrogen.

Gene, 1993 Jan 15, 123(1), 143 - 4
Complete sequence of the Salmonella typhimurium gene encoding malate dehydrogenase; Lu CD et al.; The nucleotide (nt) sequence of mdh (encoding malate dehydrogenase) in Salmonella typhimurium is presented . The relative positions of argR (encoding arginine repressor) and mdh on the chromosome of S . typhimurium were determined from analysis of the nt sequence . The homology of the 3'-terminal end with only one of two published mdh sequences in Escherichia coli identifies the correct sequence in this organism.

Gene, 1993 Jan 15, 123(1), 121 - 5
Characterization of the gene coding for the Rickettsia prowazekii DNA primase analogue; Marks GL et al.; The gene (dnaG) coding for DNA primase in the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated and characterized . An open reading frame (ORF) of 1848 bp capable of encoding 616 amino acids (aa) is located 18 bp upstream from the gene coding for the major sigma factor of R . prowazekii, sigma 73 . Based on aa sequence comparisons of DNA primase from R . prowazekii, Escherichia coli, Salmonella typhimurium and Bacillus subtilis, we propose that R . prowazekii dnaG begins 69 bp into the ORF and encodes 593 aa with a calculated M(r) of 68,683 . An upstream ORF overlaps 66 of the first 69 bp of the larger R . prowazekii dnaG ORF, suggesting either an overlapping gene structure or the generation of the smaller protein product of 593 aa . Predicted aa sequence of R . prowazekii primase compared to E . coli, S . typhimurium and B . subtilis primases reveals 30.5%, 30.5% and 29.7% aa identity, respectively . The R . prowazekii dnaG gene failed to complement an E . coli dnaG temperature sensitive mutation perhaps due to poor expression of the gene or inability to function properly in E . coli . The gene organization of an ORF followed by DNA primase (dnaG) and then the major sigma factor (rpoD) is consistent with the major macromolecular synthesis operons of E . coli, S . typhimurium and B . subtilis.

Cancer Lett, 1993 Jan 15, 68(1), 75 - 82
Modulation of cytochrome P-450IA1-mediated mutagenicity, DNA binding and metabolism of benzo{a}pyrene by Chinese medicinal herbs; Wong BY et al.; Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors . We previously showed that they inhibited mutagenesis, DNA binding and metabolism of benzo{a}pyrene (BaP) and aflatoxin B1 (AFB1) bioactivated by Aroclor 1254-induced rat hepatic S9 . The purpose of this study was to investigate the effects of OD and SB on the cytochrome P-450IA1-mediated mutagenicity of BaP in Salmonella typhimurium TA100 using beta-naphthoflavone (beta NF)-induced rat hepatic S9 . We also determined the effects of OD and SB on cytochrome P-450IA1-linked ethoxyresorufin O-deethylase (EROD) activity in beta NF-induced hepatic microsomes . In addition, we studied the effects of these two herbs on BaP metabolite binding to calf thymus DNA and using high performance liquid chromatography (HPLC) we investigated the effects of OD and SB on the metabolism of BaP by beta NF-induced S9 . Our experimental results showed that OD and SB inhibited the mutagenicity of BaP in the presence of either non-induced or beta NF-induced S9 . SB significantly inhibited BaP binding to DNA . These effects correlated with the inhibition of cytochrome P-450IA1-linked EROD activity in beta NF-induced microsomes and with an inhibition of beta NF-induced S9 mediated metabolism of {3H}BaP as determined by HPLC . These results suggest that OD and SB may possess antimutagenic activity by inhibiting P-450IA-mediated metabolism of BaP.

J Mol Biol, 1993 Jan 5, 229(1), 79 - 84
Domain organization of the subunit of the Salmonella typhimurium flagellar hook; Morgan DG et al.; The deduced amino acid sequences of the family of axial proteins of the bacterial flagellum possess N and C-terminal heptad repeats of hydrophobic amino acid residues, which suggests that these proteins all fold to form bundles of alpha-helices (e.g . coiled coils) . There is evidence that flagellin, which is one of the axial proteins, has an axially oriented bundle of alpha-helices that gives rise to the inner, rod-shaped domains seen in electron density maps . We present evidence that a second member of the family, the hook subunit, also has such an axially oriented, rod-shaped domain . In three-dimensional reconstructions from electron micrographs of the helical hook of Salmonella typhimurium, the rod-shaped domain has a diameter of 18 A, which is that expected for a coiled coil . The corresponding domain in the flagellin subunit of the filament, however, is larger, having a diameter of 24 A suggesting a bundle of three or more alpha-helices . In addition to the rod-shaped domain, the hook has two other domains . At a radius of 55 A is the middle spheroidal domain about 25 A in diameter and at a radius of 75 A is the outer ellipsoidal domain about 20 A by 30 A by 40 A . The flagellin subunit also has a middle and an outer domain although they appear different from those of the hook . This is no doubt a result of the lack of any sequence similarity of the hook and flagellin subunits, apart from the N and C-terminal heptad repeats . Along the hook axis, there is a 25 A wide channel, which presumably serves in the export of hook and flagellin subunits in the assembly of the filament . There is a comparably sized channel in the filaments as deduced from electron micrographs . Thus, electron microscopy consistently finds a small channel, whereas in X-ray diffraction studies of the filament, the channel size appeared to be about 60 A . At a diameter of 60 A, the channel could pass the flagellin or hook subunit in its completely folded state, but if the channel is only 25 A in diameter, the subunit would have to be at least partially unfolded in order to pass through the channel.

J Mol Biol, 1993 Jan 5, 229(1), 258 - 62
Crystallization and preliminary X-ray diffraction analysis of ScrY, a specific bacterial outer membrane porin; Forst D et al.; The sucrose-specific outer membrane porin ScrY of Salmonella typhimurium was isolated from Escherichia coli K-12 strain KS 26 containing the plasmid pPSO112 . The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO) . The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE . The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO) . X-ray diffraction of the crystals showed reflections to 2.3 A . The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112.85 A and c = 149.9 A . The crystal volume per unit of protein molecular weight was 3.47 A3/Da.

Cell Biol Toxicol, 1993 Jan-Mar, 9(1), 33 - 47
Mutagenicity of structurally related phenylazo-3-pyridines in Salmonella typhimurium; Shahin MM; Studies were conducted to explore structure-activity relationships for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and nine structurally related compounds in Salmonella typhimurium tester strains TA1535, TA100, TA1537, TA1538, TA98 . Each compound was tested for mutagenicity at five or more concentrations that varied from 10-5000 micrograms/plate . We used the standard plate test and the investigations were carried out both in the absence and presence of Aroclor-1254-induced rat-liver homogenate and the components of the NADPH-generating system . Negative response was observed for 4'-N,N-dimethylamino-1'-phenylazo-3-pyridine and five of its analogues (4'-N,N-diethylamino-1' phenylazo-3-pyridine; 4'-N,N-di-(beta-hydroxyethylamino)-1' phenylazo-3-pyridine; 4'-N-methylamino sulfonic acid-1'-phenylazo-3-pyridine; 4'-N,N-dimethylamino-6'-acetamido-1' phenylazo-3-pyridine, and 4'-N,N-di-(beta-hydroxyethylamino)-6'-methyl-l' phenylazo-3-pyridine) . When S9 induced by Aroclor-1254 was present, the compound 4'-N,N-dimethylamino-6-methoxy-1' phenylazo-3-pyridine exhibited mutagenic activity in the two strains TA1538 and TA98 . The compound 4',6'-diamino-3-methyl-1'-phenylazo-3-pyridine was also mutagenic, both in the presence and in the absence of S9 mix . The two compounds 4'-N,N-dimethylamino-6-butoxy-1'-phenylazo-3-pyridine and 4'N,N-di-(beta-hydroxyethylamino)-1'-phenylazo-3-{6-N,N-di-(beta- hydroxyethylamino) pyridine were either weakly mutagenic or nonmutagenic . On the basis of these data, it is concluded that the mutagenicity of phenylazo-3-pyridines, like monocyclic aromatic amines and azo dyes, is influenced by the nature of the substituent chemical groups and their positions in the molecular structure of the compounds.

Environ Mol Mutagen, 1993, 21(4), 357 - 64
Highly sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM3009 having high O-acetyltransferase and nitroreductase activities; Oda Y et al.; A highly sensitive umu test system for the detection of genotoxic activities of a variety of mutagenic nitroarenes has been developed using a new tester strain, Salmonella typhimurium NM3009 having high O-acetyltransferase (O-AT) and nitroreductase (NR) activities . The NM3009 was constructed by subcloning both the O-AT and NR genes into plasmid vector pACYC184, and the resulting plasmid was introduced into the parent tester strain S . typhimurium TA1535/pSK1002 harboring an umuC'-'lacZ fusion gene . The induction of umuC gene expression could be monitored by measuring the cellular beta-galactosidase activity produced by fusion gene . The purpose of the study was to evaluate whether the newly developed strain NM3009 is highly sensitive toward nitroarene compounds . The sensitivity of the strain NM3009 was compared with those of the parent TA1535/pSK1002 strain, the NR-overexpressing strain NM1011, the NR-deficient strain NM1000, the O-AT-overexpression strain NM2009, and the O-AT-defective strain NM2000 . The newly developed NM3009 strain had about 13-fold and 3-fold higher activities for N-AT and NR, respectively, than the original S . typhimurium TA1535/pSK1002 strain . Among six strains tested, NM3009 showed the highest sensitivity toward such chemicals as 1-nitronaphthalene, 2-nitrofluorene, 3,7-dinitrofluoranthene, 3-nitrofluoranthene, 5-nitroacenaphthene, 2-nitronaphthalene, 1-nitropyrene, 1,6-dinitropyrene, 3,9-dinitrofluoranthene, 4,4'-dinitrobiphenyl, 1,8-dinitropyrene, m-dinitrobenzene, 2,4-dinitrotoluene, and 1,3-dinitropyrene . We have also found that the order of sensitivities to induce umuC gene expression toward a variety of nitroarenes was NM3009 > NM2009 > NM1011 > TA1535/pSK1002 > NM2000 > NM1000 . These results suggest that the newly developed tester strain NM3009 is of great use for the detection of genotoxic activities of numerous carcinogenic and mutagenic chemicals including nitroarenes, which require NR and/or O-AT for the activation.

Int Arch Occup Environ Health, 1993, 64(7), 473 - 7
Increases in polycyclic aromatic hydrocarbon content and mutagenicity in a cutting fluid as a consequence of its use; Apostoli P et al.; The aim of this study was to evaluate the relationships between the length of time a cutting fluid was used, its content in polycyclic aromatic hydrocarbons (PAHs) and its mutagenic potential . The PAH concentrations were determined by means of a high-resolution gas chromatograph-mass spectrometer in samples of new cutting fluid and in samples used for 3, 6 and 9 months . The following PAHs were measured: phenanthrene, anthracene, fluoranthene, pyrene, benzo{a}anthracene, chrysene+triphenylene, benzo{e}pyrene, benzo{a}pyrene and perylene . Mutagenicity assays were carried out on the aforementioned samples using the Ames test . Salmonella typhimurium TA98 was used as an indicator to show up mutagens capable of inducing frame-shift genetic changes, and Escherichia coli WP2 uvrA was used as an indicator to detect mutagens capable of inducing base pair genetic changes . The mutagenic tests were carried out with and without microsomal activation, using 1:1, 1:10, 1:20 and 1:50 dilutions of cutting fluid samples . An increase in the concentrations of total PAHs over time was observed in the samples of cutting fluid used for 3, 6 and 9 months . The highest percentage increase in PAH concentrations was observed in the 6-month-old sample (10 times the initial concentration, from 45 to 411.8 micrograms of oil) . None of the samples were mutagenic to S . typhimurium without metabolic activation or to E . coli with and without metabolic activation . All samples except for the 1:1 diluted sample showed moderate but significant mutagenic activity in the S . typhimurium test with metabolic activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Environ Mol Mutagen, 1993, 21(3), 253 - 7
Base-pair mutations caused by six aliphatic epoxides in Salmonella typhimurium TA100, TA104, TA4001, and TA4006; Einisto P et al.; Salmonella typhimurium strains TA100, TA104, TA4001, and TA4006 were used to detect the base-pair mutations caused by six aliphatic epoxides: chloropropylene oxide, glycidyl 1-naphthyl ether, glycidyl 4-nitrophenyl ether, 1-naphthyl-propylene oxide, styrene oxide, and trichloropropylene oxide . Dose-mutagenicity relationships could be established for all six epoxides in strains TA100 and TA104 but not in strains TA4001 and TA4006 . These results, together with the lack of sensitivity of the TA100 revertants to DL-1,2,4-triazole-3-alanine, indicate CG-->TA transitions and/or CG-->AT transversions are of major importance for mutations induced by these epoxides in Salmonella TA100 and possibly TA104 . In addition, since the reproducibility of the effect of the triazole on TA104 reversions was poor, TA-->AT transversions were not eliminated as also contributing to the mutagenicity of these epoxides in this Salmonella strain.

Environ Mol Mutagen, 1993, 21(3), 247 - 52
Mutagenicity of iso-butyl nitrite vapor in the Ames test and some relevant chemical properties, including the reaction of iso-butyl nitrite with phosphate; Mirvish SS et al.; We examined the mutagenicity of iso-butyl nitrite (IBN) vapor and aqueous IBN solution in the Ames test to help evaluate the hazard of sniffing this vapor, a habit which might play a role in the induction of Kaposi's sarcoma associated with acquired immune deficiency syndrome . Chemical analysis showed that the saturated vapor contained 190 micrograms IBN/ml at 25 degrees C, and saturated aqueous solution, 2.6 mg IBN/ml at 21-23 degrees C . When agar plates containing Salmonella typhimurium TA-1535 and rat liver S-9 were exposed to IBN vapor, the number of mutants reached a maximum after 40 min . A mean of 307 mutants/plate (22 x background) was observed when the plates were exposed to IBN vapor for 30 min . Addition of 0.2 ml saturated IBN solution in water to similar plates gave a mean of 179 mutants/plate (7.9 x background) in the absence of S-9, confirming published results . The S-9 did not affect the results . Based on the IBN level in medium exposed to IBN vapor, the vapor was apparently 11 times more mutagenic than IBN solution . This was attributed to continuous replenishment of unstable IBN in the medium by the vapor . The half-life of IBN at 21-23 degrees C was > 1 hr for solutions in water and < 3 min for solutions in the assay medium . This instability was traced to a reaction with phosphate, presumably hydrolysis to nitrite and iso-butanol . IBN in solution was 2.8 times more mutagenic than sodium nitrite, suggesting that IBN was not mutagenic because of its conversion to nitrite . Iso-butanol was not mutagenic . The results demonstrate the potential hazard of sniffing IBN vapor.

Environ Mol Mutagen, 1993, 21(3), 237 - 46
Formation and characterization of bacterial mutagens from reaction of the alternative disinfectant monochloramine with model aqueous solutions of fulvic acid; Cozzie DA et al.; Monochloramine has been suggested as an alternative disinfectant to chlorine to reduce levels of trihalomethanes in treated drinking water, but little is known of the toxicological properties and potential health implications of by-products specific to the chloramination process . Model aqueous fulvic acid solutions (200-400 mg C/liter), serving as surrogates for humic surface waters, were chloraminated over a range of molar Cl:C ratios from 1:40 to 1:2 . The resulting by-products were extracted into diethyl ether at pH 2 and investigated with the Ames plate incorporation assay . Extractable mutagenicity increased with increasing chlorine and carbon dose up to about 30,000 revertants/liter at Cl:C ratios of 1:2 . Mutagenicity was higher in Salmonella typhimurium strain TA100 than in strain TA98, and was decreased in the presence of S9, indicating that the mutagens formed were direct-acting and induced predominantly base-pair substitutions . Bovine serum albumin decreased slightly, and glutathione reduced greatly, the mutagenic activity detected in extracts . HPLC fractionation of the by-products indicated that most of the mutagenic activity was found in the earliest-eluting (most polar) fraction . The mutagenic by-products appeared to be qualitatively similar to 3-chloro-4-dichloromethyl-5-hydroxy-2-(5H)-furanone (MX) in their chromatographic behavior and responses to glutathione and bovine serum albumin, but were less readily detoxified by S9 than was MX.

Environ Mol Mutagen, 1993, 21(3), 219 - 28
Genotoxicity of volatile and secondary reactive oxygen species generated by photosensitization; Camoirano A et al.; Reactive oxygen species were generated in the gas phase by photosensitization involving illumination of Rose Bengal . Depending on whether the chromophore is dry or solubilized, this system produces either energy-transfer reactions leading to generation of singlet oxygen specifically, or a combination of energy-transfer and electron-transfer reactions, providing both singlet oxygen and reduced forms of oxygen, such as superoxide anion and hydrogen peroxide . In neither case were the reactive species mutagenic in strain TA104 of Salmonella typhimurium, which had been previously shown to be reverted by oxygen species generated by the hypoxanthine-xanthine oxidase system in aqueous medium . However, mixed oxygen species induced an increased lethality in a variety of DNA repair-deficient Escherichia coli strains . This genotoxic effect, mainly reparable by the uvrA and recA mechanisms, was efficiently prevented by the thiol N-acetyl-L-cysteine . Singlet oxygen itself failed to exert direct genotoxic effects, although secondary reactants produced by its reaction with cell components enhanced lethality in some repair-deficient bacteria . Distance-dependence analyses provided measurements of the lifetimes of the oxygen species generated in the gas phase.

Avian Dis, 1993 Jan-Mar, 37(1), 183 - 8
Control of established Salmonella typhimurium intestinal colonization with in vivo-passaged anaerobes; Ziprin RL et al.; Broiler chickens were inoculated orally with 10(6) Salmonella typhimurium on the day of hatch . Twenty-four to 72 hr after challenge, the chicks were inoculated orally with cecal microflora that had been repeatedly passed through lactose-fed broiler chicks . In vivo passage proved to be a convenient and practical method for preserving protective anaerobic flora . These organisms effectively reduced S . typhimurium concentrations in the cecal contents by 4-5 orders of magnitude, even when given 24 to 72 hr after Salmonella challenge inoculation.

Mutagenesis, 1993 Jan, 8(1), 51 - 5
Quercetin and the mutagenicity of wines; Gaspar J et al.; Various studies have shown the mutagenicity of red wine . The major mutagens identified in red wine have been flavonoids, i.e . rutin and its aglycone quercetin . Besides flavonoids, however, it has recently been reported that H2O2 may account for the mutagenicity of red wine in the L-Arabinose resistance test . In the present study we report on the role of flavonoids in the mutagenicity of red wine in the Ames assay . Different wines from Portugal and Spain have been tested after concentration in XAD-2 columns in strains TA98 and TA104 of Salmonella typhimurium concurrently with the determination of the respective content of quercetin by HPLC . A similar approach was used for pilot scale productions of red wines . In all cases quercetin could be demonstrated as the major mutagen in red wines . The levels of quercetin in finished wines and during the wine-making process showed a good fit with the levels of mutagenicity detected . Catalase had no effect whatsoever on the mutagenicity of wines in both TA98 and TA104 . These results do not rule out a role for H2O2 in the mutagenicity of wines, detected in other genetic end-points, because H2O2 can be formed from the auto-oxidation of quercetin.

Mutagenesis, 1993 Jan, 8(1), 17 - 22
Mutagenicity of aristolochic acids (I, II) and aristolic acid I in new YG strains in Salmonella typhimurium highly sensitive to certain mutagenic nitroarenes; Gotzl E et al.; Aristolochic acid I and II, two naturally occurring nitroaromatics, were studied for their mutagenicity in Salmonella typhimurium tester strains YG1020, 1021, 1024, 1025, 1026 and 1029 without exogenous metabolic activation . These strains contain multicopy plasmids which carry the genes for the classical bacterial nitroreductase or O-acetyltransferase . The strains TA98, TA100 and TA1537 were included in the study for comparison . Aristolic acid I, the analogue lacking the nitro group, and its sodium salt were also tested . Both aristolochic acids revealed mutagenicity in the respective YG strains derived from TA100, but the effect was no stronger than with the parent strain . Only weak activity was observed in TA98 and some YG strains derived from it . Aristolochic acid II was generally the more active compound . Aristolic acid I and its sodium salt did not exhibit any mutagenicity in any tester strain . From the results the following conclusions were drawn . (i) Only the nitro group is important for the mutagenicity of the aristolochic acids in S . typhimurium . (ii) It is suggested that aristolochic acid II is so efficiently metabolized by the classical bacterial nitroreductase that the additional activity produced from the YG strains no longer affects the metabolic activation . (iii) The methoxy group is probably responsible for the lower activity of aristolochic acid I, producing steric hindrance for binding of the genetically active intermediate to DNA or for binding of the substrate to the active site of the enzyme(s).

Chem Res Toxicol, 1993 Jan-Feb, 6(1), 91 - 6
Isolation, structural identification, and characterization of a mutagen from Fusarium moniliforme; Lu FX et al.; Strains of Fusarium moniliforme produce a variety of toxins, including several uncharacterized mutagens that act directly in the Ames assay using Salmonella typhimurium, strain TA 100 . The Ames assay was used to monitor isolation of the direct-acting mutagens from the F . moniliforme culture extracts . Seven strains were tested, of which strains F07 and F84 contained the highest levels of direct-acting mutagens . Extracts of strain F84 were fractionated on a silica gel column, eluted with methanol-chloroform (1:9) . This fraction was then separated on a reverse-phase, C-18 column with 50% methanol in water as eluant and further purified by TLC . One compound was isolated and given the trivial name fusarin X (FX) . Its structure was determined from its UV (lambda max 357 nm), 500-MHz NMR, and mass spectra, and those of its diacetate, to be the 1-hydroxy analog of the previously characterized fusarin C . FX was present in culture extracts of strains F07 and F84 at 83 and 8 micrograms/g, respectively, which was proportional to their relative mutagenicities . It was not detected in the other strains tested . Since exposure of FX most likely occurs in cooked corn, its thermal stability was measured; it, like FC, decomposes at 100 degrees C, especially at high pH . Again, in common with FC, it was decomposed by GSH . The possible role of these Fusarium metabolites in the etiology of human cancers has still to be resolved.

Vaccine, 1993, 11(2), 122 - 5
The PhoP virulence regulon and live oral Salmonella vaccines; Miller SI et al.; The PhoP virulence regulon is essential to Salmonella typhimurium mouse typhoid fever pathogenesis and survival within macrophages . This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor) proteins and the genetic loci they positively (pags for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate . Three regulated loci pagC, pagD, and prgH, when singly mutated, affect the virulence of S . typhimurium for mice . Strains with phoP locus mutations are effective as live vaccines in mice, and strains with a constitutive regulatory mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very few organisms are administered . The addition of various PhoP regulon mutations further attenuates aroA mutants of S . typhimurium, suggesting that these mutations would be useful in further attenuating vaccine strains with metabolic pathway mutations . The phoP, phoQ, pagC, and pagD genes are highly conserved between S . typhimurium and S . typhi and may be valuable as mutations in live vaccines for human typhoid fever . A plasmid suicide vector that allows deletion of the pagC gene and stable insertion of heterologous antigen genes within the deleted pagC locus has been constructed and used successfully in S . typhimurium and S . typhi.

Arch Dis Child, 1993 Jan, 68(1), 138 - 9
Transient protein S deficiency with deep venous thrombosis during Salmonella typhimurium infection; Ceyhan M et al.; A patient with deep venous thrombosis and low protein S activity during the course of Salmonella typhimurium infection is presented . Although protein S deficiency has been reported in patients with disseminated intravascular coagulation, it was not present in this patient and his protein S activity was normal after the findings of infection and deep venous thrombosis disappeared.

Can J Vet Res, 1993 Jan, 57(1), 14 - 8
The influence of the swine major histocompatibility genes on antibody and cell-mediated immune responses to immunization with an aromatic-dependent mutant of Salmonella typhimurium; Lumsden JS et al.; Eighty-two major histocompatibility complex (MHC) swine leukocyte antigen (SLA) defined miniature pigs from 16 litters were examined for serum agglutinating antibody titer and O-polysaccharide (O-ps) specific peripheral blood lymphocyte blastogenesis following two parenteral vaccinations with 1 x 10(8) aromatic-dependent (aroA) Salmonella typhimurium and following oral challenge with 1 x 10(12) virulent parent S . typhimurium . Least mean squares analysis allowed separate determinations of the effects of MHC genotype, dam, sire and litter . In most cases only litter significantly influenced both lymphocyte blastogenesis and antibody titer before and after vaccination and following challenge . However, pig SLA haplotype significantly influenced the degree of O-ps specific lymphocyte proliferation six days after the second vaccination (p < 0.004) . Lymphocyte proliferation and serum agglutinating antibody response six days after primary vaccination were negatively correlated (r2 = -0.68, p < 0.001) . In most cases, "dd" and "gg" homozygous and "dg" heterozygous pigs, having the same MHC class II region, behaved immunologically as a group distinct from the other genotypes.

Toxicol Appl Pharmacol, 1993 Jan, 118(1), 135 - 8
Mutagenicity of hydralazine in mammalian cells and bacteria; McQueen CA et al.; The genotoxicity of hydralazine (HDZ), an antihypertensive agent, was evaluated in mammalian cells and bacteria . The formation of mutants at the hypoxanthine guanine phosphoribosyl transferase locus in an adult rat liver cell line ARL 18 was determined . Bacterial mutagenicity was assessed in Salmonella typhimurium strains TA100 and TA102 . The latter strain was chosen because it has A:T bases at the reversion site and HDZ has been reported to interact with thymidine . HDZ was mutagenic to ARL 18 cells with a concentration-dependent increase in mutants observed at 5 x 10(-6) to 5 x 10(-4) M . At 5 x 10(-4) M HDZ, there were 110 mutants/10(6) colony-forming cells compared to 129 for cells exposed to 10(-4) M benzo(a)pyrene, a known genotoxin . Bacterial mutants were observed with HDZ in both strains in the absence of an activating system . HDZ also induced mutants in the presence of S-9 from Aroclor-induced rat liver or uninduced rabbit liver . These results were consistent with previous reports of the mutagenicity of HDZ in TA100 and extend the observations to TA102, a strain designed to detect oxidative damage . This study also provides the first evidence of the mutagenicity of HDZ in mammalian cells . These data support the genotoxicity of HDZ in in vitro systems.

Carcinogenesis, 1993 Jan, 14(1), 11 - 9
Genotoxicity characteristics of reverse diol-epoxides of chrysene; Glatt H et al.; Trans-3,4-dihydroxy-3,4-dihydrochrysene (chrysene-3,4-diol), a major metabolite of chrysene, is further metabolized by rat liver enzymes to products which effectively revert the his- Salmonella typhimurium strain TA98 to histidine prototrophy, but are only weakly mutagenic in strain TA100 and in Chinese hamster V79 cells (acquisition of resistance to 6-thioguanine) . The liver enzyme mediated mutagenicity of chrysene-3,4-diol is substantially enhanced in the presence of 1,1,1-trichloropropene 2,3-oxide, an inhibitor of microsomal epoxide hydrolase . The predominant metabolites of chrysene-3,4-diol, namely the anti- and syn-isomers of its 1,2-oxide (termed reverse diol-epoxides), proved to be extraordinarily effective mutagens in S.typhimurium strain TA98, but were only moderately active in strains TA100 and TA104, and in the SOS induction in Escherichia coli PQ37 . These genotoxicity spectra in bacteria are completely different from those observed with the bay-region diol-epoxides of chrysene and 3-hydroxychrysene . In V79 cells, the reverse diol-epoxides formed low levels of DNA adducts and were very weak inducers of gene mutations . In M2 mouse prostate cells, however, high numbers of transformed foci were induced by chrysene-3,4-diol and its diastereomeric 1,2-oxides . Chrysene-3,4-diol was somewhat more potent than chrysene-1,2-diol . The potency of both reverse diol-epoxides was similar to that of the syn-diastereomers of the bay-region diol-epoxides of chrysene and 3-hydroxychrysene, but lower than that of their anti-diastereomers . The reverse diol-epoxides of chrysene, unlike the bay-region diol-epoxides, were inactivated by purified microsomal epoxide hydrolase . Noteworthy findings were also made with regard to the chemical stability of the diol-epoxides in buffer, determined from the decline in mutagenicity after preincubation in the absence of the target cells . Despite its lower delta Edeloc/beta value for the formation of the benzylic carbocation, anti-chrysene-3,4-diol 1,2-oxide was shorter-lived (t1/2 = 46 min) than anti-chrysene-1,2-diol 3,4-oxide (t1/2 = 74 min) . Unlike other investigated diastereomeric pairs of diol-epoxides, it was also shorter-lived than its syn-diastereomer (t1/2 = 340 min).

Plant Mol Biol, 1993 Jan, 21(2), 409 - 12
Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp . PCC 6803; Kohn C et al.; The genome of Synechocystis sp . PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes . We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp . PCC 7942, Escherichia coli, Salmonella typhimurium and Marchantia polymorpha . It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.

Proc Natl Acad Sci U S A, 1993 Jan 1, 90(1), 217 - 21
Cloning and characterization of the Salmonella typhimurium-specific chemoreceptor Tcp for taxis to citrate and from phenol; Yamamoto K et al.; Salmonella typhimurium shows an attractant response to citrate and a repellent response to phenol, and a chemoreceptor mediating these responses has been identified and named Tcp (taxis to citrate and away from phenol) . Tcp is one of the methyl-accepting chemotaxis proteins that have a molecular mass of approximately 60 kDa estimated by SDS/PAGE, and its methylation level is increased by citrate and decreased by phenol . Tcp also mediates an attractant response to metal-citrate complexes . The complete nucleotide sequence of the tcp coding region has been determined . The deduced amino acid sequence of Tcp, consisting of 547-amino acid residues, is homologous with that of the aspartate chemoreceptor of S . typhimurium . Thus, Tcp is another member of the bacterial transmembrane chemoreceptor family . Because citrate is a good carbon source for S . typhimurium but is not a carbon source for the closely related species Escherichia coli and because citrate utilization is used as a key diagnostic character to distinguish these species, it is reasonable to assume that Tcp is specific to S . typhimurium.

J Bacteriol, 1993 Jan, 175(2), 541 - 3
Nucleotide sequence of the Salmonella typhimurium mutB gene, the homolog of Escherichia coli mutY; Desiraju V et al.; The mutB gene of Salmonella typhimurium is involved in a methylation-independent repair pathway specific for A/G or A/C mismatches and is the homolog of the Escherichia coli mutY gene . The mutB gene of S . typhimurium was cloned and sequenced . The isolated mutB clone reduced the mutation rate of the mutB mutant to wild-type levels and also restored A/G mismatch-specific nicking activity, which is defective in mutB extracts . The amino acid sequence encoded by the mutB gene is 91% homologous to that encoded by the E . coli mutY gene.

J Bacteriol, 1993 Jan, 175(2), 479 - 86
The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase; Hughes KT et al.; The Salmonella typhimurium nadC gene and its product, quinolinic acid phosphoribosyltransferase (QAPRTase), were characterized at the molecular and biochemical levels . Fusions of Mud-lac elements isolated in the nadC gene were converted to Mud-P22 insertions . Starting with six original Mud-lac fusions, the entire sequence of the nadC gene was readily obtained . The sequence shows a long open reading frame with two potential initiator methionines, one of which is preceded by the Shine-Dalgarno sequence GGAG-7-nucleotide-ATG . The protein predicted from this second open reading frame is 297 residues in length . The nadC gene was subcloned into a T7-based expression system, allowing for facile purification of the QAPRTase (EC 2.4.2.19) protein to homogeneity . Upon gel filtration, the protein gave an M(r) of 72,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a subunit M(r) of 35,000 . Automated Edman degradation of several tryptic peptides confirmed the amino acid sequence predicted from the DNA sequence . Chromatography of the apparently homogeneous enzyme on reverse-phase high-performance liquid chromatography resolved two protein species . One of these species failed to give an amino-terminal sequence, while the other yielded the amino-terminal sequence predicted by the second open reading frame and lacked the initiator methionine . The mass of the mature protein, predicted from its DNA sequence, was 32,428 Da . Electrospray mass spectrometry gave masses of 32,501 and 32,581 Da for the two peptides . Steady-state kinetics on the purified QAPRTase indicated Km values of 32 microM for 5-phosphoribosyl-1-pyrophosphate and 20 microM for quinolinate . Vmax was 0.9 U/mg, similar to values reported for this enzyme by other sources.

J Infect Dis, 1993 Jan, 167(1), 107 - 11
Role of egg consumption in sporadic Salmonella enteritidis and Salmonella typhimurium infections in Minnesota; Hedberg CW et al.; The incidence of Salmonella enteritidis infections has increased in the eastern United States, and consumption of undercooked eggs has been associated with outbreaks of S . enteritidis . In Minnesota, the incidence of S . enteritidis infections doubled from 1980 to 1990; however, no egg-associated outbreaks were identified . A case-control study was conducted to examine potential exposures for S . enteritidis and Salmonella typhimurium infections in Minnesota adults . Sporadic cases of S . enteritidis (odds ratio {OR}, 5.2; 95% confidence interval {CI}, 1.9-14.2; P = .003) and S . typhimurium infection (OR, 2.4; CI, 1.1-5.5; P = .03) were more likely to have consumed undercooked eggs or egg-containing foods during the 3 days before onset of illness compared to a similar reference period for controls . In addition, the extent to which eggs were cooked was directly associated with illness (chi 2 test for trend, P < .001) . These findings demonstrate that eggs are important vehicles for S . enteritidis and S . typhimurium, even in the absence of recognized outbreaks.

Genetics, 1993 Jan, 133(1), 17 - 28
Lethal transposition of Mud phages in Rec- strains of Salmonella typhimurium; Sonti RV et al.; Under several circumstances, the frequency with which Mud prophages form lysogens is apparently reduced in rec strains of Salmonella typhimurium . Lysogen formation by a MudI genome (37 kb) injected by a Mu virion is unaffected by a host rec mutation . However when the same MudI phage is injected by a phage P22 virion, lysogeny is reduced in a recA or recB mutant host . A host rec mutation reduces the lysogenization of mini-Mu phages injected by either Mu or P22 virions . When lysogen frequency is reduced by a host rec mutation, the surviving lysogens show an increased probability of carrying a deletion adjacent to the Mud insertion site . We propose that the rec effects seen are due to a failure of conservative Mu transposition . Replicative Mud transposition from a linear fragment causes a break in the host chromosome with a Mu prophage at both broken ends . These breaks are lethal unless repaired; repair can be achieved by Rec functions acting on the repeated Mu sequences or by secondary transposition events . In a normal Mu infection, the initial transposition from the injected fragment is conservative and does not break the chromosome . To account for the conditions under which rec effects are seen, we propose that conservative transposition of Mu depends on a protein that must be injected with the DNA . This protein can be injected by Mu but not by P22 virions . Injection or function of the protein may depend on its association with a particular Mu DNA sequence that is present and properly positioned in Mu capsids containing full-sized Mu or MudI genomes; this sequence may be lacking or abnormally positioned in the mini-Mud phages tested.

J Bacteriol, 1993 Jan, 175(1), 240 - 50
A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome; Tubulekas I et al.; Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome . Very little is known about the elements in EF-Tu involved in this interaction . We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome . The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu . The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated . Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth . We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells . In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant . Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA . However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex . In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu . Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis . Models involving either a direct or an indirect effect of the mutation are discussed.

Environ Mol Mutagen, 1993, 22(3), 188 - 90
Screening of azo dyes for mutagenicity with Ames/Salmonella assay; Kaur A et al.; Azo dyes, the largest portion of manufactured dyestuffs, are primarily used as colouring substances in food, textiles, and the plastic industry . It has been estimated that 128 tonnes per annum of dyes are released into the environment worldwide {Anliker, 1977} . Certain azo compounds are known to be mutagenic in bacterial tests {Yahagi et al., 1975; Venitt and Bushell, 1976; Brown et al., 1978} . Watersoluble dyes are biotransformed by intestinal micro-organisms in the gastro intestinal tract, and the toxicity, mutagenicity, and carcinogenicity of these dyes in the gut or liver may be attributed to their metabolites . Since it is desirable to have a genotoxic evaluation of a chemical being released into the environment in order to check their indiscriminate use, a project has been initiated to determine the mutagenicity of the azo dyes being used commercially . The present report deals with the results of 13 dyes tested in Salmonella typhimurium with and without metabolic activation.

Environ Mol Mutagen, 1993, 22(3), 181 - 7
Test of chiral recognition in the Salmonella typhimurium (TA100) mutagenicity of mucochloric acid-cysteine adducts; LaLonde RT et al.; A difference in biological response to enantiomers is not an uncommon observation and is, therefore, to be expected in various manifestations of genotoxicity . The bacterial mutagen mucochloric acid (2,3-dichloro-5-hydroxy-2(5H)-furanone) has one chiral center, at C-5, but this mutagen exists in racemic form because of the facile stereoisomerization occurring by the mechanism of ring-chain tautomerism . Two readily synthesized enantiomeric analogs of mucochloric acid, as well as the racemic form of the two, were prepared from mucochloric acid and (R)-(+)-, (S)-(-)-, and (R,S)-(+/-)-cysteine . Using Salmonella typhimurium (TA100), the enantiomeric compounds were assayed together in four dose/response assays along with mucochloric acid, the reference mutagen . In three of the same four assays, the racemic form was also assayed . Neither statistically significant differences in mutagenicity, as determined in slope responses, nor distinctions from the plotted curves were observed among the two enantiomers and their racemic form . Therefore, no enantiospecific interaction between enantiomers and chiral DNA or enzymes involved in repair or replication could be concluded.

Protein Sci, 1993 Jan, 2(1), 31 - 40
Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases; Bork P et al.; Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families . The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium . The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family . The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase . The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases . Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families . Yet each catalyzes chemically equivalent reactions on similar or identical substrates . The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution . In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family . These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times . The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Vaccine, 1993, 11(2), 126 - 35
Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors; Cardenas L et al.; The use of attenuated strains of Salmonella as vaccine vectors frequently involves the introduction of heterologous antigens on recombinant plasmids . To overcome the problem of plasmid instability, we have integrated the gene that codes for a potential immunogen into the chromosome of a galE mutant of Salmonella typhimurium . Comparative in vitro and in vivo studies were conducted between the strain carrying the gene chromosomally integrated and an isogenic strain carrying the same gene on a multicopy plasmid . Levels of expression of the foreign antigen were significantly lower when the antigen was expressed from the chromosome than when it was expressed from the plasmid . The in vivo maintenance of the genes coding for antigen expression was determined on organisms recovered from spleen, liver and Peyer's patches of orally inoculated mice . By 24 h postinoculation, the majority of tissue isolates from the plasmid-containing strain had lost the plasmid and the ability to synthesize the antigen . By contrast, 100% of the recovered cointegrate isolates retained the ability to express the antigen throughout the 21 days of the experiment . Significantly, humoral and mucosal antibody levels against the antigen were greater when the antigen was expressed from the plasmid stabilized by the presence of the antibiotic than when the antigen was expressed from the chromosome . These observations indicate that the most important event for the development of an immune response against a foreign antigen delivered by these vectors may be the initial amount of antigen that primes the gut-associated lymphoid tissue and not persistence of the vector in tissues.

Mol Gen Genet, 1993 Jan, 236(2-3), 387 - 94
Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: lethality of rodA and mre mutations; Costa CS et al.; Thirty-three insertions of transposon Tn10 delta 16 delta 17 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells . Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21%) were mre mutants, and 1 (3%) was a divD mutant . No insertion in the pbpA gene were found . Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by beta-lactamase production) were employed . Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam . On the other hand, the divD insertion could be efficiently transduced to any strain . It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.

J Bacteriol, 1993 Jan, 175(2), 325 - 31
Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium; Elliott T; In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found . Here, I describe the characterization of these mutations . The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise . Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon . The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus . Unlabeled L-leucyl-glycine competes with labeled ALA for uptake . In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA . The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Infect Immun, 1993 Jan, 61(1), 155 - 61
Release of cytokines induced by Salmonella typhimurium porins; Galdiero F et al.; Salmonella typhimurium SH5014 porins induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), and IL-6 by human monocytes and of gamma interferon (IFN-gamma) and IL-4 by human lymphocytes . Porins at 1 microgram/ml induce the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and of IL-4 by lymphocytes, while porins at 5 micrograms/ml induce the greatest release of IFN-gamma by lymphocytes . The R form of lipopolysaccharide (LPS-R) induces the greatest release of TNF-alpha and IL-1 alpha by monocytes when used at a low concentration (1 microgram/ml) . At higher concentrations (5 and 10 micrograms/ml, respectively), LPS-R induces the maximal release of IL-6 from monocytes and the maximal release of IL-4 from lymphocytes . The S form of LPS (LPS-S) induces the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and that of IL-4 by lymphocytes when used at a concentration of 1 microgram/ml . After stimulation with LPS-S, the largest quantity of TNF-alpha and IL-1 alpha released was less than that obtained after stimulation with LPS-R at the same concentration, while the quantity of IL-6 released was found to be slightly higher than that obtained after stimulation with porins or LPS-R . LPS-S (1 microgram/ml) induces IFN-gamma release from lymphocytes in notably smaller quantities than that obtained with LPS-R and slightly larger quantities than that obtained with porins . The preparation of porins used was found to be contaminated with 10 pg of LPS per 10 micrograms of porins, a quantity which was found to have no biological effect; furthermore, porin preparations with the addition of polymyxin B gave the same results.

Biol Pharm Bull, 1993 Jan, 16(1), 96 - 8
Autooxidation of alkylhydrazones and mutagenicity of the resulting hydroperoxides; Mochizuki M et al.; Acetone alkylhydrazones were readily autooxidized to 2-alkylazo-2-propyl hydroperoxides, which were directly mutagenic in Salmonella typhimurium TA1535, TA100, TA102 and Escherichia coli WP2hcr . The mechanism of this mutagenicity presumes that the hydroperoxides in aqueous solution decompose to alkyl diazonium ions which were observed in the alkylation of 4-(p-nitrobenzyl)pyridine, and also to hydroxyl radical which was detected by ESR.

Folia Microbiol (Praha), 1993, 38(3), 177 - 80
Elimination of plasmid pKM101 from Salmonella typhimurium by monoammonium salts; Belicova A et al.; The frequency of elimination of plasmid pKM101 from Salmonella typhimurium TA92 exposed to the action of 1-alkyl-1-ethylpiperidinium bromides and N-alkyl-N-{5-(benzoyloxy)-3-oxapentyl}-N,N-dimethylammonium bromides was non-linear in the homologous series . Change in the length of the alkyl chain markedly affected the elimination properties of the piperidine derivatives but had no effect on the elimination of benzoyl derivatives . Piperidines exhibited a weaker elimination capacity than the benzoyl derivatives . The most potent eliminator was the octylbenzoyl derivative, which causes the elimination of the plasmid in 80-85% cells.

Life Sci, 1993, 53(12), 981 - 9
Prolactin protection against lethal effects of Salmonella typhimurium; Di Carlo R et al.; The immunoregulatory role of prolactin (PRL) has been well established . In order to clarify if the hormone is also able to stimulate a protective activity against pathogens-induced infections we have studied the modifications of the infective capacity of Salmonella typhimurium induced in mice by repeated treatments with ovine PRL . A significant dose-dependent reduction in the mortality rate was observed in comparison to controls . This activity is probably related to the observed increases in phagocytosis and intracellular killing of the peritoneal macrophages and chemotaxis of the peritoneal granulocytes induced by the hormonal treatment . On the contrary, the number of leukocytes in blood was not modified by PRL treatment excluding a mobilization of cells from other districts . Our findings confirm the existence of a linkage between the neuroendocrine and immune systems suggesting a possible role for PRL in the regulation of non-specific immune response.

Environ Mol Mutagen, 1993, 22(2), 115 - 22
Genotoxic activity detected in soils from a hazardous waste site by the Ames test and an SOS colorimetric test; McDaniels AE et al.; Ten soil samples from a hazardous waste site were compared for their genotoxic activity by the Ames test (Salmonella reverse mutation assay) and a modified SOS colorimetric test . Polynuclear aromatic hydrocarbons known to produce frameshift mutations were found in high levels in the soils . Salmonella typhimurium TA98, sensitive to frameshift mutations, was selected as the Ames tester strain . Escherichia coli K12 PQ37 (sulA::lacZ) was the SOS tester strain . Organic extracts were prepared from the soil samples by Soxhlet extraction . One set of the soil samples was extracted with methylene chloride and a second set with cyclohexane . Two criteria from reproducible dose-related increases in response to the soil were used to compare the positive responses: 1) the concentrations required for doubling responses and 2) a minimum concentration required to produce statistically significant increases from background controls . Analysis of variance indicated that with S9 mix, Ames and SOS results were similar for the same soils and solvent extractions . However, without S9 mix, the SOS test was significantly more sensitive than the Ames test to the genotoxins extracted from the soils . Both the Ames and SOS tests detected lower concentrations of genotoxins in methylene chloride than in cyclohexane extracts . The simplicity of the method, reduction in expenses, and results within 1 working day all contribute to the advantages of the SOS test.

Indian J Pathol Microbiol, 1993 Jan, 36(1), 13 - 20
Multiple drug resistance and genetic characterization of Salmonella typhimurium isolated at Chandigarh; Garg RK et al.; A total of 1072 strains of S . typhimurium were isolated in Chandigarh during 1985-1988 . Genetic characterization of 'R' plasmids was attempted in 21 multiple drug resistant strains (ACKSSu TT p-19, ACKSSu, TP-I, AKSSu Tp-1) . All the 21 strains were sensitive to gentamicin, furoxone and nalidixic acid . Level of resistance to different antimicrobial drugs varied from 200 to 1000 mg/L . Majority of the strains were untypable by the present set of 31 typing phages (Ut-17, Phage type 21-2, Phase type 99-2) . All the plasmids belonged to class I transfer system . All the plasmids restricted some or all the typing phages of S . typhimurium and 39 also restricted phage phi 2 . These plasmids belonged to F1me or I1 incompatibility groups and did not inhibit the function of fertility factor (fi-).

Vaccine, 1993, 11(7), 773 - 6
Expression of a recombinant Entamoeba histolytica antigen in a Salmonella typhimurium vaccine strain; Cieslak PR et al.; The expression of a major surface antigen of the intestinal protozoal parasite Entamoeba histolytica in an attenuated Salmonella typhimurium vaccine strain is described . A polymerase chain reaction fragment derived from cDNA encoding the serine-rich Entamoeba histolytica protein, SREHP, was introduced into S . typhimurium chi 3987 (delta cya delta crp delta asd) using a plasmid expression vector (pYA292) containing the aspartate semialdehyde (asd) gene . S . typhimurium expressing recombinant SREHP as a SREHP/maltose binding protein fusion protein was administered orally to mice and gerbils (an important animal model for E . histolytica infection) and was recovered from splenic tissue in both species . Our study demonstrates the feasibility of expressing recombinant amoebic proteins in attenuated S . typhimurium strains, and shows that vaccine strains of S . typhimurium can successfully infect the gerbil, a widely used model for amoebic liver abscess and intestinal amoebiasis.

Vaccine, 1993, 11(7), 725 - 9
Correlation of antibody titres induced by vaccination with protection in mouse typhoid; Xu HR et al.; The ELISA method was used to titrate the humoral immune response in vaccinated mice . When mice were given two doses of a heat-killed salmonella vaccine 6 days apart, there was a steady but low-level increase of antibody synthesis . In contrast, if a booster vaccination was administered 21 days after the primary inoculation, the anamnestic response produced a significantly greater antibody titre, rapidly reaching its peak within 10 days . Such a heightened humoral response induced in the genetically susceptible C57BL/6J mice also coincided with effective protection against an otherwise lethal challenge with Salmonella typhimurium.

Environ Mol Mutagen, 1993, 22(1), 34 - 45
Mutagenic activity of the 4,5- and 9,10-dihydrodiols of benzo{j}fluoranthene and their syn- and anti-dihydrodiol epoxides in Salmonella typhimurium; Marshall MV et al.; The objective of this study was to determine the relative mutagenic activities of the major dihydrodiol metabolites of benzo{j}fluoranthene (B{j}F) and their corresponding syn- and anti-dihydrodiol epoxides . Salmonella typhimurium tester strains TA97a, TA98, and TA100 were used to evaluate the mutagenic potencies of the parent hydrocarbon and these suspect proximate and ultimate mutagenic metabolites . B{j}F and the trans-dihydrodiol metabolites were active only in the presence of an external metabolic activation system (S9) with the exception of the B{j}F-4,5-diol, which was weakly active in TA98 and TA100 in the absence of S9 . The B{j}F-4,5-diol was more mutagenic than the B{j}F-9,10-diol in tester strains TA98 and TA100, whereas the opposite effect was observed in TA97a . In the absence of S9, the anti-B{j}F-4,5-diol epoxide was more mutagenic than the syn-B{j}F-4,5-diol epoxide and the syn- and anti-B{j}F-9,10-diol epoxides in tester strains TA97a and TA100 . The exceptional mutagenic potency of the anti-B{j}F-4,5-diol epoxide in TA100 resembles that observed by epoxides located within a fjord, or by the anti-diol epoxides of bay region methylated polycyclic aromatic hydrocarbons . In contrast, the mutagenicity of the pseudo bay region dihydrodiol epoxides arising from the B{j}F-9,10-diol more closely resembles that observed with the classical bay region dihydrodiol epoxides of chrysene . In summary, both dihydrodiol metabolites of B{j}F are mutagenic in S . typhimurium, and the relative potency varies among the tester strains . The highest mutagenic response was achieved in tester strain TA100, which detects base-pair substitutions . The most potent direct-acting dihydrodiol epoxide in this tester strain was the anti-B{j}F-4,5-diol epoxide, which agrees with the results of mouse skin painting studies that indicate that the B{j}F-4,5-diol is more tumorigenic that the parent hydrocarbon or the B{j}F-9,10-diol . A covalent DNA adduct formed between the anti-B{j}F-4,5-diol epoxide and deoxyguanosine was the major species of DNA adduct formed in S . typhimurium . This adduct corresponds to the major DNA adduct formed in mouse skin following application B{j}F.

Br J Neurosurg, 1993, 7(3), 311 - 3
Wound infection with meningitis caused by Salmonella typhimurium; Singh RV et al.; We report a case of postoperative wound infection and meningitis with Salmonella typhimurium in a 66-year-old woman who had been operated on for a cerebral meningioma . The diagnostic, therapeutic and prognostic implications are discussed.

Microbiol Immunol, 1993, 37(10), 793 - 9
Lymphocytic proliferative response to outer-membrane proteins isolated from Salmonella; Gonzalez CR et al.; Porins isolated from Salmonella typhi have been demonstrated to protect against the challenge with this bacteria in mice . The mechanism has not been clarified, but could be associated with activation of both humoral and cellular immunity . In order to evaluate the induction of specific T cell responses, the lymphocytic proliferation to porins isolated from Salmonella typhimurium, Salmonella typhi and Escherichia coli was examined by 3H-thymidine incorporation assay in mice immunized with three different antigens: acetone-killed S . typhimurium, its porins, or outer-membrane proteins (OMPs) isolated from S . typhi . Higher proliferative responses were observed in mice immunized with porins and OMPs compared with those which received the acetone-killed bacteria . Although cross-reactivity was observed between porins, they were not mitogenic . Moreover, porins were able to activate T lymphocytes isolated from mice immunized with S . typhi OMPs . These results suggest that T cell activation, through the release of lymphokines, may play a role in the induction of protective immunity with porins.

Miner Electrolyte Metab, 1993, 19(4-5), 266 - 76
Molecular aspects of Mg2+ transport systems; Smith DL et al.; The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the CorA, MgtA, and MgtB loci . The CorA transport system is the constitutive Mg2+ influx system but can also mediate efflux at high extracellular Mg2+ concentrations . The CorA protein lacks homology to any known protein, is an integral membrane protein containing 28% percent-charged amino acids, and has three carboxyl terminal membrane-spanning segments . Its properties indicate that it is a new class of membrane transport protein, likely found in all gram-negative bacteria and possibly other organisms . In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations and can mediate only the influx of Mg2+ . Both MgtA and MgtB system are P-type ATPases; they have relatively poor homology to other known prokaryotic P-type ATPases but are highly homologous to mammalian P-type ATPases, particularly reticular Ca(2+)-ATPases . Expression of both MgtA and MgtB is highly regulated by the concentration of extracellular Mg2+ . Transcription of MgtB is increased about 1,000-fold by lowering Mg2+ from 1 mM to 1 microM, and, under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system . However, it is unclear why the cells require the use of ATP to mediate the influx of Mg2+ down its electrochemical gradient . Study of these Mg2+ transport systems should lead to further understanding of cellular Mg2+ homeostasis and eventually to characterization of eukaryotic Mg2+ transport systems.

Neuropsychobiology, 1993, 28(1-2), 9 - 16
Cytokine production during sleep and wakefulness and its relationship to cortisol in healthy humans; Hohagen F et al.; A growing body of evidence indicates that cytokines, especially interleukin-1 beta, are involved in the regulation of sleep and wakefulness . The aim of the present pilot study was to investigate the relationship between interleukin-1 beta (IL-1 beta) and gamma-interferon (gamma-IFN) production and the regulation of sleep and wakefulness . Four healthy male volunteers were investigated . After one adaptation night, beginning at 8 a.m . in the morning, the EEG was recorded by means of a mobile long-term EEG and blood samples were drawn every 45 min for the analysis of IL-1 beta, gamma-IFN and cortisol for 24 h . For the analysis of cytokines whole blood cultures were established . After 48 h of incubation in the presence of endotoxin Salmonella typhimurium, IL-1 beta and gamma-IFN levels were measured in the culture supernatants using specific immunodetection assays . Methods of stochastic time series analysis were adopted to evaluate the biochemical data . Our results show the capability of cultured blood cells to produce cytokines upon endotoxin challenge to be at a maximum around the time of sleep onset and during the first hours of sleep, declining during the night to a minimum level in the morning hours . The opposite was observed for cortisol . The analysis of autocorrelation functions gives evidence of a 24-hour rhythm of cortisol and cytokines . The results indicate that the cytokines IL-1 beta and gamma-IFN may play a role in sleep regulation.

Nucleic Acids Symp Ser, 1993, (29), 217 - 8
Nucleotides bearing a cleavable genotoxic group on the phosphate; Hayatsu H et al.; A new class of nucleotide derivatives in which N-nitroso-pyrrolidine (NPYR) is linked to the phosphate group through an ester linkage at the alpha-carbon of the nitrosamine were prepared by a reaction between a nucleoside monophosphate and alpha-acetoxy-NPYR . The NPYR derivatives were prepared from dpT, dpC, dpA, dpG, pA and dpCpT . Treatment of the NPYR-pX with acid, snake venom phosphodiesterase, or near ultraviolet light caused cleavage of the NPYR group, regenerating pX . All of these nucleotide derivatives were directly mutagenic towards Salmonella typhimurium TA1535 . When phage M13mp2 double-stranded covalently closed circular DNA was treated with NPYR-dpT with near ultraviolet irradiation, single strand breaks of the DNA took place.

Vet Med (Praha), 1993, 38(9), 553 - 8
{The effect of experimental aerobic stabilization of swine slurry on survival of Salmonella typhimurium and Ascaris suum}; Juris P et al.; Laboratory aerobic mesophilic stabilisation (fermentation) of pig slurry reduced the survival time of S . typhimurium, compared with their prevalence in anaerobic excrements . The decimation time T90 for S . typhimurium, was 32 hours . The effect of aerobic stabilisation on the survival of nonembryoed eggs of the model helminth A . suum was lower than that observed in Salmonella . After 54 hours of aerobic stabilization of slurry only 32% of eggs were devitalized . Stabilized control group did not develop 13% of eggs into their embryonated stage.

Autoimmunity, 1993, 15(1), 49 - 54
Bacterium-induced autoimmune reactivity; Hol C et al.; Evidence is growing that autoimmune reactivity results from a combination of endogenous (e.g . MHC type) and environmental factors . Our experimental study focuses on the induction of autoimmune reactivity by microbial factors . Splenic formation and serum levels of anti-erythrocyte antibodies and circulating immune complexes were taken as parameters . It was found that experimental infection of mice with Escherichia coli and Salmonella typhimurium was accompanied by clear signs of autoimmune reactivity, smooth bacteria being almost ten times as potent as rough mutant strains . An attempt was made to correlate the data obtained with live bacteria to their corresponding endotoxins . It was concluded that the induction of more prominent autoimmune reactivity by smooth bacteria must be ascribed to a longer survival time in vivo . Our data support the view that bacterium-derived factors are involved in the etiology (and possibly also the course) of autoimmune diseases.

Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove Suppl, 1993, 36(1-2), 5 - 10
{The Ames test in genetic toxicology . I}; Mrackova G; The present study deals with the basic principles of the genetic toxicology, the use of detection methods to determine substances with genotoxic effects and emphasizes the advantage of using tests on prokaryotic-bacterial level . The so-called Ames test, i.e . the testing system with Salmonella typhimurium strains, is also detailed.

Yi Chuan Xue Bao, 1993, 20(5), 473 - 80
{Regulation of purine biosynthetic genes expression in Salmonella typhimurium . II . Isolation and characterization of Oc mutants}; Wang A et al.; Purine repressor protein, encoded by purR has shown to effect expression of purine structure genes except purB in de novo pathway in Salmonella typhimurium . However, up to date there is no direct evidence of the repressor binding to operator DNA of these genes . Report here is the isolation and characterization of purine Oc mutants in Salmonella typhimurium . purD:: MudJ (lacZ Kanr) and purG:: MudJ (lacZ Kanr) were used as starting strains Both strains were mutagenized by NTG, and derepressed mutants were selected on the MacConkey plate containing an excess of adenosine (2mmol/L) . 8 independent depressed mutants were obtained from purD:: MudJ (lacZ Kanr), 9 were obtained from purG:: MudJ (lacZ Kanr) . Transduction analysis and trans-acting or cis-acting test of the genes with merochromosome DNA tandem genetic duplication strains proved that 1 mutant for each group exert the constitute expression by cis-acting . This is first purine Oc mutant obtained from Salmonella typhimurium.

Acta Vet Scand, 1993, 34(4), 363 - 70
Mutagenicity, creatine and nutrient contents of pan fried meat from various animal species; Vikse R et al.; The mutagenic activity in extracts of fried meat from 16 different animal species was studied in Salmonella typhimurium TA98 . In each experiment, 1 meat sample together with a standard beef sample was fried, and the mutagenicity was expressed relative to the beef sample . All meat samples showed less mutagenic activity than beef . The contents of creatine, creatinine, water, protein, carbohydrate and fat in the meat samples were analyzed, but mutagenicity was not correlated with the concentration of any of these constituents . Beef meat treated with creatinase to remove creatine produced reduced mutagenic activity . Possibly a threshold concentration of creatine is necessary to give a high mutagenic response.

Acta Biochim Pol, 1993, 40(4), 549 - 54
A study of the genotoxic potential of flavonoids using short-term bacterial assays; Czeczot H et al.; Genotoxic activities of flavonoids (quercetin, rhamnetin, isorhamnetin, apigenin, luteolin) were investigated using two short-term bacterial assays . In the "repair test" in Salmonella typhimurium (strains TA1538 uvrB- and TA1978 uvrB+) the flavonoids studied did not introduce any damage into the DNA recognized by UvrABC nuclease (correndonuclease II) . The results of the SOS-Chromotest in Escherichia coli K-12 strains PQ37 (tag+, alk+) and PQ243 (tagA, alkA) indicated that flavonoids only weakly induced the SOS system . The addition of a liver activation system (S9 mix) did not increase the mutagenic effect of the flavonoids tested . Two compounds: rhamnetin, isorhamnetin and their putative metabolites formed in the presence of the S9 mix did not alkylate DNA at N-3 of adenine.

Wien Med Wochenschr, 1993, 143(19-20), 522 - 6
{Calcium antagonists as amplifiers of the mutagenicity of cytostatic drugs}; Scheid W et al.; Some calcium antagonists enhance synergistically the mutagenic efficiency of cytostatics . The results so far obtained concerning this phenomenon are described and discussed: (1) chromosome aberrations induced (in vitro) in human lymphocytes (cytostatics; bleomycin and peplomycin; calcium antagonists: verapamil and fendiline), (2) chromosome and chromatid aberrations induced (in vitro) in Chinese hamster ovary (CHO) cells (cytostatic: mitomycin C; calcium antagonist: verapamil), (3) gene mutations induced in the bacterium Salmonella typhimurium (cytostatics: mainly various anilinoacridine drugs; calcium antagonist: verapamil) . In all 3 studies neither verapamil nor fendiline proved to be mutagenic when applied alone . The "comutagenicity" of calcium antagonists is compared with the enhancement of the cytotoxic (cell killing) efficiency of cytostatics by calcium antagonists . Both phenomena--potentiation of mutagenicity and of cytotoxicity--are interpreted on the basis of the "accumulation hypothesis" . According to this hypothesis those calcium antagonists would enhance the mutagenic and cytotoxic efficiency of the cytostatics by inhibiting their extrusion from the cell . Consequently, the cytostatics would be accumulated in the cell and this would increase their mutagenic and cytotoxic efficiency . Since not all calcium antagonists investigated so far enhance the mutagenicity and cytotoxicity of cytostatics, this potentiation seems not to be caused by their calcium antagonistic action per se . Molecular, evolutionary, and medical aspects of the "accumulation hypothesis" are shortly discussed.

Vaccine, 1993, 11(2), 140 - 2
Immune responses to hybrid maltose-binding proteins; O'Callaghan D et al.; The Escherichia coli maltose-binding protein is a highly versatile carrier protein allowing the construction of genetically engineered hybrid proteins . It accepts large fusions to both C- and N-termini as well as the insertion of shorter peptides at 'permissive sites' within the continuity of the protein . We have genetically inserted immunogenic peptides corresponding to defined viral B- and T-cell epitopes into two permissive sites: one at amino acid site 133, the other at site 303 . The hybrid proteins are easily purifiable and immunogenic, inducing peptide-specific B- and T-cell responses . When delivered by live bacteria (E . coli K12 and aroA Salmonella typhimurium) antibody responses can be induced against both the MalE carrier and the inserted B-cell epitope . We discuss the induction of T-cell responses by bacterial delivery systems.

Clin Exp Immunol, 1993 Jan, 91(1), 73 - 7
Comparative antibody response to Salmonella antigens in genetically resistant and susceptible mice; Xu HR et al.; The ELISA was used to titrate the antibody response in mice inoculated with salmonella antigens . The genetically resistant A/J and susceptible C57BL/6J mice were either infected with the virulent or the avirulent Salmonella typhimurium . Alternatively, they were inoculated either once or twice with the heat-killed salmonella vaccine . No appreciable difference could be detected in the relative ability of these two strains of mice to produce antibodies against the lipopolysaccharide antigens of this pathogen under these four conditions.

Mutat Res, 1993 Jan, 298(3), 207 - 14
Mutagenic activity of the methyl and phenyl derivatives of the food mutagen 2-amino-3-methylimidazo{4,5-f}quinoxaline (IQx) in the Ames test; Vikse R et al.; The mutagenic activity of 15 different mono-, di-, tri-, and tetramethyl derivatives of the food mutagen IQx (2-amino-3-methylimidazo{4,5-f}quinoxaline), one diphenyl derivative of IQx and two phenyl derivatives of 5-MeIQx (2-amino-3,5-dimethylimidazo{4,5-f}quinoxaline) were studied in the Ames test with Salmonella typhimurium TA98 and enzymatic activation (S9) . The number and positioning of the methyl groups strongly affected the mutagenic activity . The phenylated compounds showed weak mutagenic potency . It seems that both resonance stabilization of the nitrenium ion and steric effects are important in determining mutagenic potency.

Mutat Res, 1993 Jan, 298(3), 187 - 95
Mutagenicity of glyceryl trinitrate (nitroglycerin) in Salmonella typhimurium; Maragos CM et al.; The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action . GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate . Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate . GTN did not cause significant reversion in any of the six other S . typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed . Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes . Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter . A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide . This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action . The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion . The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.

Mutat Res, 1993 Jan, 285(1), 101 - 8
Glucose inhibition of mutagenesis by 9-aminoacridine in Salmonella typhimurium; Kopsidas G et al.; Back mutation to prototrophy of the hisC3076 marker of Salmonella typhimurium has been measured following treatment with 9-aminoacridine (9AA) under conditions in which growth is either permitted or not permitted . Cells treated with 9AA in buffer (i.e . under conditions in which little or no replication was possible) were found to respond well to 9AA-induced mutagenesis . This remained true whether or not the plating medium contained trace amounts of histidine to allow residual replication of His- cells (as for example in the Ames test); yields of His+ mutants were also about the same regardless of whether the plating medium contained glucose or glycerol as the sole carbon source . By contrast, 9AA-induced mutagenesis was essentially abolished when cells were treated in buffer containing 1% glucose (i.e . under conditions which strongly favoured replication) . Glucose inhibition of 9AA-induced mutagenesis began to be apparent at glucose concentrations of about 0.02%, whilst total abolition was observed at about 0.2% . In addition, glucose inhibition was found to be dependent on the concentration of 9AA, the induced mutation rate increasing at levels of up to 100-150 micrograms/ml of 9AA and then declining steeply at higher levels . Further kinetic studies indicated that glucose could depress the 9AA-induced mutation rate significantly even in cells exposed to the mutagen for up to 18 min prior to the addition of glucose, whilst inhibition of 9AA mutagenesis by glucose was both temporary and reversible.

Mutat Res, 1993 Jan, 301(1), 7 - 12
Comparison of the sensitivity of Salmonella typhimurium strains YG1024 and YG1012 for detecting the mutagenicity of aromatic amines and nitroarenes; Watanabe M et al.; Salmonella typhimurium YG1024 is a derivative of S . typhimurium TA98 with a high level of N-hydroxyarylamine O-acetyltransferase (OAT) activity . We have demonstrated that this strain is highly sensitive to the mutagenic actions of N-hydroxyarylamines derived from aromatic amines and nitroarenes . In this paper, we compared the sensitivities of YG1024 with those of S . typhimurium YG1012, which has about 4 times higher OAT activity than YG1024 but lacks plasmid pKM101 . It turned out that YG1024 was more sensitive to the mutagenic actions of 1-aminonaphthalene, 1-nitropyrene, 1,8-dinitropyrene and 2-nitronaphthalene than YG1012 and showed comparable sensitivity to 2-hydroxy-acetylaminofluorene, 2-aminoanthracene and 2-amino-6-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1) to YG1012 . These results suggested that YG1024 is more suitable than YG1012 for the efficient detection of mutagenic aromatic amines and nitroarenes.

Mutat Res, 1993 Jan, 301(1), 1 - 5
Mutability by polycyclic hydrocarbons is improved in derivatives of Escherichia coli WP2 uvrA with increased permeability; Herrera G et al.; Escherichia coli B, unlike both E . coli K12 and Salmonella typhimurium, is sensitive to the rough-specific phage C21 . This sensitivity is probably due to the incomplete lipopolysaccharide core of the E . coli B cells, which confers on them a partial permeability to large molecules . Derivatives of WP2 uvrA, a tryptophan-requiring E . coli B strain, were rendered still more permeable by selecting for C21-resistant clones . The new permeable strains, when tested for mutagenesis induced by polycyclic hydrocarbons, showed a mutagenic response higher than that of the parental strains.

J Mol Biol, 1992 Dec 20, 228(4), 1147 - 62
Organization and ordered expression of Caulobacter genes encoding flagellar basal body rod and ring proteins; Dingwall A et al.; The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell . The basal body is the first part of the flagellum to be assembled . In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation . The genes in this cluster form an operon whose expression is controlled temporally . The first two genes encode homologs of FlgF and FlgG, which are the proximal and distal rod proteins, respectively . The sequences of the N and C termini of the Salmonella typhimurium flagellar axial proteins, rod, hook and HAP-1, known to be highly conserved, share a high degree of sequence identity with the FlgF and FlgG rod proteins of the distantly related, C . crescentus . Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences . The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter . Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter . The internal promoter and its activator site reside within the C-terminal coding sequence of the upstream flaD gene . This type of gene overlap is also observed in bacterial genes involved in cell division . Flagellum biogenesis, like cell division, is a morphogenic event that requires the orderly assembly of component proteins and the overlapping gene organization may affect this "ordering" of assembly . The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription . The use of sigma 54 promoters, known to require cognate binding proteins, could allow the fine-tuning that provides the temporal ordering of flagellar gene transcription . In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site . The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum.

Mutat Res, 1992 Dec 16, 284(2), 233 - 41
Rat pulmonary microsomal cytochrome P-450 enzymes involved in the activation of procarcinogens; Shimada T et al.; Rat lung microsomal cytochrome P-450 (P-450) enzymes have been characterized with regard to their catalytic specificities towards activation of several procarcinogens to genotoxic metabolites in Salmonella typhimurium TA1535/pSK1002 . We first examined the roles of rat liver microsomal P-450 enzymes in the activation of benzo{a}pyrene and its 7,8-diol enantiomers to genotoxic products, and found that P-450 1A1 is a major catalyst for the activation of these potential procarcinogens in rat livers . Using lung microsomes isolated from rats treated with various P-450 inducers we obtained evidence that at least three P-450 enzymes are involved in the activation of several procarcinogens . Immunoinhibition studies support the view that benzo{a}pyrene and its 7,8-diol derivatives, other dihydrodiol derivatives of polycyclic aromatic hydrocarbons, and 3-amino-1-methyl-5H-pyrido{4,3-b}indole are activated to genotoxins mainly by rat P-450 1A1, which is inducible in rat lungs by 5,6-benzoflavone and the polychlorinated biphenyl mixture Aroclor 1254 . Activation of 2-amino-3,5-dimethylimidazo{4,5-f}quinoline and 2-amino-3-methylimidazo{4,5-f}quinoline may be catalyzed by another P-450 enzyme because the activities were not induced by treatment with 5,6-benzoflavone or Aroclor 1254 . The observation that both activities were inhibited by antibodies raised against P-450 1A2 and by 7,8-benzoflavone suggests a role for an enzyme of P-450 1A family, probably P-450 1A2, in rat lung microsomes . The activation of aflatoxin B1 and sterigmatocystin appears to be catalyzed by other P-450 enzyme(s) rather than the P-450 1A family as judged by the different responses of activities to the P-450 inducers and the specific antibodies in rat lung microsomes . Interestingly, lung microsomal activation of several procarcinogens was found to be suppressed in rats treated with isosafrole and pregnenolone 16 alpha-carbonitrile . Thus, the results support the roles of different P-450 enzymes in the activation of procarcinogens in rat lung microsomes.

Mutat Res, 1992 Dec 16, 284(2), 205 - 13
Inhibitory activity of heat treated vegetables and indigestible polysaccharides on mutagenicity; Yamaguchi T; The effects of heat treated vegetables on mutagenicity were studied using the Salmonella typhimurium system . The mutagens used were 3-amino-1-methyl-5H-pyrido{4,3-b}indole, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 4-nitroquinoline-1-oxide, acridine yellow and 2-aminoanthracene . Most of the heated vegetables unexpectedly showed greater inhibitory activity against the mutagenicity than unheated samples . The activity was increased markedly by heat treatment of water soluble indigestible polysaccharides (IPS) . The increase in inhibitory activity due to heat treatment of IPS coincided well with the decrease in their viscosity . Incubating mixtures of mutagens with heated water soluble IPS decreased their affinity for XAD-2 resin . Heating seems to increase the detoxification ability of dietary fibers.

Biochem J, 1992 Dec 15, 288 ( Pt 3), 865 - 74
Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli; Dumas R et al.; Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids . Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria . To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme . A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase . Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium . Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate) . Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site . The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses . Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light . As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11939 - 43
Resistance to host antimicrobial peptides is necessary for Salmonella virulence; Groisman EA et al.; The production of antibacterial peptides is a host defense strategy used by various species, including mammals, amphibians, and insects . Successful pathogens, such as the facultative intracellular bacterium Salmonella typhimurium, have evolved resistance mechanisms to this ubiquitous type of host defense . To identify the genes required for resistance to host peptides, we isolated a library of 20,000 MudJ transposon insertion mutants of a virulent peptide-resistant S . typhimurium strain and screened it for hypersensitivity to the antimicrobial peptide protamine . Eighteen mutants had heightened susceptibility to protamine and 12 of them were characterized in detail . Eleven mutants were attenuated for virulence in vivo when inoculated into BALB/c mice by the intragastric route, and 8 of them were also avirulent following intraperitoneal inoculation . The mutants fell into different phenotypic classes with respect to their susceptibility to rabbit defensin NP-1, frog magainin 2, pig cecropin P1, and the insect venom-derived peptides mastoparan and melittin . The resistance loci mapped to eight distinct locations in the genome . Characterization of the mutants showed that one had a defective lipopolysaccharide and another mutant harbored a mutation in phoP, a locus previously shown to control expression of Salmonella virulence genes . Our data indicate that the ability to resist the killing effect of host antimicrobial peptides is a virulence property and that several resistance mechanisms operate in S . typhimurium.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 331 - 6
Bacterial flagellar diversity and significance in pathogenesis; Penn CW et al.; Bacterial flagella are structurally diverse, ranging from the thoroughly investigated model examples found in Escherichia coli and Salmonella typhimurium to the more exotic sheathed flagella of, for example, Helicobacter pylori, and the complex multi-flagellin endoflagella found in many spirochaetes . We summarize some of the emerging structural and genetic findings relating to these more novel flagellar types, and outline their possible significance in the pathogenicity of some medically important bacteria.

Poult Sci, 1992 Dec, 71(12), 2022 - 6
Effect of anaerobic cecal microflora and dietary lactose on Salmonella colonization in bobwhite quail (Colinus virginianus); Corrier DE et al.; The effect of oral inoculation with anaerobic cultures of cecal microflora from adult broiler chickens and dietary lactose on Salmonella typhimurium cecal colonization was evaluated in bobwhite quail chicks . One-day-old chicks were divided into four groups and provided 1) no anaerobic cultures, no lactose (control); 2) anaerobic cultures; 3) 2.5% lactose (wt/vol) in drinking water; or 4) anaerobic cultures and lactose . All groups were challenged orally with 10(4) S . typhimurium at 2 days of age . Salmonella growth in the cecal contents was significantly decreased (P < .05) at 15 days of age in each of the three treatment groups as compared with controls . Protection against Salmonella colonization was highest in the treatment group provided anaerobic cultures only . The results indicated that cecal flora from adult chickens enhances Salmonella colonization resistance in quail chicks.

FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 271 - 6
The putative sigma factor KatF (RpoS) is required for the transcription of the Salmonella typhimurium virulence gene spvB in Escherichia coli; Norel F et al.; The virulence of Salmonella typhimurium for mice is dependent on a plasmid-borne gene cluster termed spv . We previously determined that both S . typhimurium and Escherichia coli bacteria grown in a rich medium preferentially express the spv genes during the stationary phase of growth . In this study we evaluated the role of KatF, a putative sigma factor for starvation- and stationary phase-induced genes, in the expression of the spvB gene . The transcription of spvB in E . coli was compared in katF and wild-type backgrounds, using cloned spvB-lacZ and spvB-cat fusions . Expression of spvB was found to be greatly affected in katF mutants . Complementation experiments performed with the cloned katF gene confirmed that KatF is required for the expression of the S . typhimurium virulence gene spvB in E . coli.

Am J Epidemiol, 1992 Dec 1, 136(11), 1369 - 77
Incubation period, severity of disease, and infecting dose: evidence from a Salmonella outbreak; Glynn JR et al.; The associations between infecting dose, incubation period, and the severity of disease were examined in a large outbreak of Salmonella typhimurium which occurred at a medical conference in Wales in 1986 . Persons who had eaten two or more pieces of the chicken vehicle had, on average, shorter geometric mean incubation periods than those who had only eaten one piece: 16.6 hours (95% confidence interval (CI) 13.5-20.5) compared with 20.7 hours (95% CI 19.0-22.6) (t = 1.97, p < 0.05) . Incubation period was negatively correlated with the maximum frequency of diarrheal stools (r = -0.46, 95% CI -0.56 to -0.33), the maximum temperature reached (r = -0.34, 95% CI -0.50 to -0.16), the duration of symptoms (r = -0.41, 95% CI -0.53 to -0.26), and the amount of time taken off from work (r = -0.54, 95% CI -0.65 to -0.41) . Those with shorter incubation periods were more likely to have been hospitalized . There was no association between chicken consumption and any of the measures of severity . The authors discuss the evidence that incubation period is inversely related to dose, the use of incubation period as a marker for dose, and the role that individual differences in susceptibility play in determining both the incubation period and the outcome.

Carcinogenesis, 1992 Dec, 13(12), 2221 - 6
N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver; Rich KJ et al.; 2-Amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents . MeIQx requires metabolic activation by P450 before it can exert these effects . Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction . A mixture of unlabelled MeIQx, {13C,15N2}MeIQx and {14C}MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry . Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ({M+H}+) from the parent compound, MeIQx and at 230/233 ({M+H}+) from N-OHMeIQx . The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 {M+H+} provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines . Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines . Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry . In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected . MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes . The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90% . These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.

Epidemiol Infect, 1992 Dec, 109(3), 371 - 88
The relationship between infecting dose and severity of disease in reported outbreaks of Salmonella infections; Glynn JR et al.; The relationship between size of the infecting dose and severity of the resulting disease has been investigated for salmonella infections by reanalysis of data within epidemics for 32 outbreaks, and comparing data between outbreaks for 68 typhoid epidemics and 49 food-poisoning outbreaks due to salmonellas . Attack rate, incubation period, amount of infected food consumed and type of vehicle are used as proxy measures of infecting dose, while case fatality rates for typhoid and case hospitalization rates for food poisoning salmonellas were used to assess severity . Limitations of the data are discussed . Both unweighted and logit analysis models are used . There is no evidence for a dose-severity relationship for Salmonella typhi, but evidence of a correlation between dose and severity is available from within-epidemic or between-epidemic analysis, or both, for Salmonella typhimurium, S . enteritidis, S . infantis, S . newport, and S . thompson . The presence of such a relationship affects the way in which control interventions should be assessed.

Genetics, 1992 Dec, 132(4), 963 - 73
Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins; Reenan RA et al.; Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms . Degenerate oligonucleotide primers based on conserved regions of E . coli MutS protein and its homologs from Salmonella typhimurium, S . pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae . Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS . These sequences were then used to clone the full-length genes from a yeast genomic library . Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp . The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively . The overall amino acid sequence identity with the E . coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2 . Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs . Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair.

J Nutr, 1992 Dec, 122(12), 2383 - 90
Prevention of immunologic stress contributes to the growth-permitting ability of dietary antibiotics in chicks; Roura E et al.; The growth-permitting ability of antibiotics fed to broiler chicks was studied as it relates to the state of activation of the immune system . In Experiment 1, chicks were fed two levels of antibiotics (0 or 100 mg streptomycin + 100 mg penicillin/kg diet) and were raised either in an environment with poor sanitation to create a chronic immune stress or in a clean environment . Chicks raised in the unsanitary environment and not fed antibiotics had significantly lower (P < 0.05) rates of weight gain and efficiencies of feed utilization, and higher levels of plasma interleukin-1, compared with chicks raised in the clean environment or chicks raised in the unsanitary environment and fed antibiotics . Adding antibiotics to the diet of birds in the clean environment did not affect any variable . In Experiment 2, chicks were raised in a conventional environment and fed two levels of an antibiotic (0 or 100 mg tetracycline/kg diet) . After a 15-d feeding period, half of the chicks were injected with Salmonella typhimurium lipopolysaccharide to create an acute immunologic stress . Feeding antibiotic resulted in improved weight gain, feed consumption and efficiency of feed utilization . Lipopolysaccharide-injected birds developed heavier livers, spleens and intestines relative to body weights and higher rectal temperatures and hepatic metallothionein concentrations, presumably due to an immunologic stress . Omitting antibiotic from the diet resulted in similar changes . These results indicate that feeding antibiotics may permit growth by preventing immunologic stress and associated metabolic changes brought about by monokines including interleukin-1.

Infect Immun, 1992 Dec, 60(12), 5164 - 71
Early pathogenesis of infection in the liver with the facultative intracellular bacteria Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium involves lysis of infected hepatocytes by leukocytes; Conlan JW et al.; The results show that Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium are facultative intracellular bacteria with a capacity to invade and grow in nonphagocytic cells in vivo . In the liver, all of these pathogens were seen to invade and to multiply extensively in hepatocytes . In all three cases, inflammatory phagocytes were rapidly marshalled to foci of infection where they appeared to cause the destruction of infected hepatocytes, thereby releasing bacteria into the extracellular space, in which presumably they could be ingested and destroyed by the phagocytes . If phagocytic cells were prevented from accumulating at foci of liver infection by treatment of the mice with a monoclonal antibody (NIMP-R10) directed against the type 3 complement receptor of myelomonocytic cells, then lysis of hepatocytes failed to occur and bacteria proliferated unrestrictedly within them . Under these circumstances, otherwise sublethal infections became rapidly lethal . These findings strongly suggest that lysis of infected hepatocytes by phagocytic cells is an important general early-defense strategy against liver infection with at least three different intracellular bacteria.

Infect Immun, 1992 Dec, 60(12), 5091 - 8
Identification of p60 antibodies in human sera and presentation of this listerial antigen on the surface of attenuated salmonellae by the HlyB-HlyD secretion system; Gentschev I et al.; Antibodies directed against the major secreted protein of Listeria monocytogenes, termed p60, were found more frequently than antilisteriolysin antibodies in sera of listeriosis patients . Anti-p60 antibodies were also identified in all tested sera from healthy individuals . To test whether p60 provides protection against L . monocytogenes, we constructed an attenuated Salmonella typhimurium aroA strain which secretes p60 via the Escherichia coli hemolysin secretion pathway . Application of this Salmonella strain to BALB/c mice prior to an L . monocytogenes infection induced p60 antibodies in these mice and led to a significantly reduced number of viable bacteria in the spleen compared with that in control animals which were primed with the S . typhimurium aroA strain alone.

Microb Pathog, 1992 Dec, 13(6), 477 - 91
Role of T cells, TNF alpha and IFN gamma in recall of immunity to oral challenge with virulent salmonellae in mice vaccinated with live attenuated aro- Salmonella vaccines; Mastroeni P et al.; The SL3261 Salmonella typhimurium aroA live vaccine strain confers solid protection against oral challenge with virulent salmonellae, immunity persisting long after the vaccine has been cleared from the tissues . BALB/c mice immunized with SL3261 and later subjected to in vivo depletion of both CD4+ and CD8+ T cells had impaired recall of immunity to oral challenge with the virulent S . typhimurium C5, with increased mortality and higher bacterial loads in the reticuloendothelial system (RES) . Selective depletion of CD4+ cells alone significantly impaired resistance both 8 and 14 weeks after vaccination as determined by estimation of bacterial numbers in organ homogenates . Depletion of CD8+ cells alone had less effect on immunity when performed at 8 weeks than at 14 weeks after immunization . Administration of anti-IFN gamma or anti-TNF alpha antibodies also impaired recall of immunity, exacerbating a secondary infection in vaccinated mice . Challenge of T cell-depleted immune mice with virulent salmonellae caused hepatosplenomegaly with minute grossly visible focal lesions, and a marked increase in the number and severity of necrotic foci in spleen, liver and lymph nodes . A widespread mononuclear cell infiltrate was present . The histopathology in anti-IFN gamma-treated mice was qualitatively similar to that seen in T-cell depleted mice . In contrast, in the anti-TNF alpha-treated mice splenomegaly was much less than in T cell-depleted mice . Granulomas were absent, no mononuclear infiltration was observed and there was severe necrosis; the lesions appeared similar to or worse than those seen in naive mice . Surprisingly, IFN gamma was detectable in sera of both controls and T cell-depleted mice on day 8 of the secondary infection, as well as in sera of anti-TNF alpha-treated mice on day 6 of infection . The results indicate that T cells, IFN gamma and TNF alpha are all important in the specific recall of immunity to virulent salmonellae conferred by immunization with live vaccines, with the effect of T cell and IFN gamma depletion (marked macrophage infiltration) being qualitatively very different from that of TNF alpha neutralization (no mononuclear infiltrate or granuloma formation).

J Bacteriol, 1992 Dec, 174(23), 7697 - 704
Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium; Swenson DL et al.; Regulation of the gene, fimA, encoding the major fimbrial subunit of S . typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen . Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production . One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide . Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high . In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.

J Chemother, 1992 Dec, 4(6), 353 - 7
Effects of rufloxacin in Salmonella typhimurium infection in mice; Bonina L et al.; This study was undertaken to investigate the efficacy of rufloxacin, a new quinolone which is interesting due to its pharmacokinetics characterized by a long plasma half-life, in the treatment of systemic salmonella infections in the mouse typhoid model . Innately susceptible BALB/c and resistant CBA mice were used to investigate the efficacy of rufloxacin in controlling systemic salmonella infections when given for brief or prolonged periods . The present study shows that rufloxacin is not only very effective on both mouse strains, but can completely eradicate the salmonellae from livers and spleens when given early in the infection of CBA resistant mice.

Microb Pathog, 1992 Dec, 13(6), 465 - 76
Extracellular export of Shiga toxin B-subunit/haemolysin A (C-terminus) fusion protein expressed in Salmonella typhimurium aroA-mutant and stimulation of B-subunit specific antibody responses in mice; Su GF et al.; The Shiga toxin B-subunit has been fused to the 23-kD C-terminus of Escherichia coli haemolysin A (HlyA) and exported from attenuated antigen carrier strain of Salmonella typhimurium aroA (SL3261) . The expression of the gene fusion under the control of a synthetic modified beta-lactamase promoter (constitutive expression) and under the iron-regulated aerobactin promoter showed that the fusion protein could be stably expressed and exported out of the bacterial cell in significant amounts so long as high copy number plasmids were not used . Oral and i.p . immunization of mice with the hybrid salmonellae resulted in significant B-subunit specific mucosal and serum antibody responses . A comparative analysis of the location of hybrid proteins in the antigen carrier bacterial cell (i.e . cytoplasmic expression and extracellular export) has shown that both modes of expression result in antigen-specific immune responses . This is the first report demonstrating that foreign polypeptides fused to the 23-kD C-terminus of E . coli haemolysin A can be exported from attenuated Salmonella vaccine strains and that such exported polypeptides can result in antigen-specific immune responses.

Dtsch Tierarztl Wochenschr, 1992 Dec, 99(12), 492 - 4
{The survival ability of salmonella, coccidia oocysts and ascarid eggs in laying hen feces from different housing systems}; Roesicke E et al.; The time of survival of Salmonella typhimurium, coccidia oocysts and ascaris eggs in manure of layer was determined in 5 different housing systems and 2 storing places for litter . The experiments were carried out in a stable of experimental station Frankenforst of the university of Bonn with a flock of 2200 hens . The effects of the environment conditions temperature, dry matter content, pH-value and intestinal microflora of the manure have also been studied . The time of survival was different depending on the housing system . A recovery of viable coccidia oocysts was possible after 13-370 days, ascaris eggs 53-347 days and Salmonella typhimurium 2-175 days . The tenacity of the investigated test organism mainly depend on the dry matter content of the manure . The longest period of survival of salmonellas was found in dry environment conditions, were as coccidia oocysts and ascaris eggs have been observed with the shortest period of survival . The possibility of the examined resistant parasite stages to develop was disturbed . Only few of them were able to develop and with a longer development time than those examined in the control suspension . The results of this study indicate that chicken manure, before using it in plant production, should be stored long enough to prevent men or animals from possible infections.

Anticancer Drugs, 1992 Dec, 3(6), 609 - 14
Clavine alkaloids and derivatives as mutagens detected in the Ames test; Glatt H et al.; Eight cytostatic clavines were investigated for mutagenicity in Salmonella typhimurium (reversion of the his-strains TA98, TA100, TA102 and TA1537), directly and in the presence of a mammalian xenobiotic metabolizing system, S9 (NADPH-fortified postmitochondrial fraction of liver homogenate from Aroclor 1254-treated rats) . Four compounds (festuclavine, 17-bromofestuclavine, 1-allylelymoclavine and 1-methyllysergol methyl ether) were direct mutagens, whose activity was enhanced in the presence of S9 . The other compounds (1-cyclopentylfestuclavine, 13-bromo-1-cyclopropylmethylfestuclavine, 6-cyano-1-propyl-6-norfestuclavine and 6-allyl-1-propyl-6-norfestuclavine) showed mutagenic effects only in the presence of S9, as previously observed with other clavines (agroclavine and its 1-propyl and 1-pentyl derivatives) . Thus, all investigated clavines may be metabolized to mutagenic products by mammalian enzymes . Bacteriotoxic activities did not correlate with mutagenic activities . The bacteriotoxicity of several clavines was reduced in the presence of S9 . The results are discussed with regard to the potential therapeutic use of clavine alkaloids as antimicrobial and antineoplastic agents.

Gene, 1992 Dec 1, 122(1), 45 - 52
Characterization of the Salmonella typhimurium phoE gene and development of Salmonella-specific DNA probes; Spierings G et al.; In Escherichia coli K-12, the phoE gene, encoding a phosphate-limitation-inducible outer membrane pore protein (PhoE), is closely linked to the genes proA and proB . When the corresponding fragment of the Salmonella typhimurium chromosome was transferred to E . coli K-12 using an RP4::miniMu plasmid, pULB113, no production of S . typhimurium PhoE could be detected . Nevertheless, DNA hybridization studies revealed that the corresponding plasmid did contain S . typhimurium phoE . Production of S . typhimurium PhoE in E . coli was detected only after subcloning the gene in a multicopy vector . Nucleotide (nt) sequence analysis showed extensive homology of S . typhimurium phoE to the E . coli gene and suggested possible explanations for the low expression of S . typhimurium phoE in E . coli . In addition, the sequence information was used to develop Salmonella-specific DNA probes . Two oligodeoxyribonucleotides were synthesized based on nt sequences encoding the fifth and eighth cell-surface-exposed regions of PhoE . When used in polymerase chain reactions, these probes turned out to be specific, i.e., no crossreactions occurred with the non-Salmonella strains, whereas 132 out of 133 tested Salmonella strains were recognized.

Chem Biol Interact, 1992 Nov 16, 84(3), 277 - 90
Effect of caffeic acid esters on carcinogen-induced mutagenicity and human colon adenocarcinoma cell growth; Rao CV et al.; Propolis, a honey bee hive product, is thought to exhibit a broad spectrum of activities including antibiotic, antiviral, anti-inflammatory and tumor growth inhibition; some of the observed biological activities may be due to caffeic acid (cinnamic acid) esters that are present in propolis . In the present study we synthesized three caffeic acid esters, namely methyl caffeate (MC), phenylethyl caffeate (PEC) and phenylethyl dimethylcaffeate (PEDMC) and tested them against the 3,2'-dimethyl-4-aminobiphenyl, (DMAB, a colon and mammary carcinogen)-induced mutagenicity in Salmonella typhimurium strains TA 98 and TA 100 . Also, the effect of these agents on the growth of human colon adenocarcinoma, HT-29 cells and activities of ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) was studied . Mutagenicity was induced in Salmonella typhimurium strains TA 98 and TA 100 plus S9 activation using 5 and 10 micrograms DMAB and antimutagenic activities of 0-150 microM MC, 0-60 microM PEC and 0-80 microM PEDMC were determined . The results indicate that MC, PEC and PEDMC were not mutagenic in the Salmonella tester system . DMAB-induced mutagenicity was significantly inhibited with 150 microM MC, 40-60 microM PEC and 40-80 microM PEDMC in both tester systems . Treatment of HT-29 colon adenocarcinoma cells with > 150 microM MC, 30 microM PEC and 20 microM PEDMC significantly inhibited the cell growth and syntheses of RNA, DNA and protein . ODC and PTK activities were also inhibited in HT-29 cells treated with different concentrations of MC, PEC and PEDMC . These results demonstrate that caffeic acid esters which are present in Propolis possess chemopreventive properties when tested in short-term assay systems.

J Biol Chem, 1992 Nov 15, 267(32), 22798 - 803
Sequence of the sodium ion pump oxaloacetate decarboxylase from Salmonella typhimurium; Woehlke G et al.; A genomic library of Salmonella typhimurium DNA was constructed in the lambda-phage EMBL3 and screened by immunoblotting for expression of the oxaloacetate decarboxylase alpha-subunit . After subcloning on plasmids the entire sequence of the oxaloacetate decarboxylase was determined . The genes encoding subunits gamma (oadG), alpha (oadA), and beta (oadB) of the decarboxylase are clustered on the chromosome in that order . A typical consensus sequence of a promoter is not found upstream of the oadG gene, but putative ribosome binding regions can be identified before each subunit gene . The amino acid sequences are highly homologous to those of oxaloacetate decarboxylase from Klebsiella pneumoniae with 71% identity between the gamma-subunits, 92% identity between the alpha-subunits, and 93% identity between the beta-subunits . The homology between the corresponding beta-subunits appeared to exist only between the 312 N-terminal amino acid residues . It was shown that a cloning artifact has occurred during DNA sequence determination of the beta-subunit from K . pneumoniae and has led to erroneous results . The sequence of this polypeptide is corrected in the Appendix to this paper . A plasmid encoding the three oad genes and that for the anaerobic citrate carrier (citS) was cloned from the chromosomal DNA and used for sequence determination.

Appl Environ Microbiol, 1992 Nov, 58(11), 3482 - 7
Predicting the growth of Salmonella typhimurium on beef by using the temperature function integration technique; Dickson JS et al.; Lag and generation times for the growth of Salmonella typhimurium on sterile lean beef were modeled as functions of cooling time under various carcass-chilling scenarios . Gompertz growth models were fit to the log10 colony counts over time at each of six temperatures in the range of 15 to 40 degrees C . Lag and generation times were defined as the points at which the second and first derivatives, respectively, of each growth curve attained a maximum . Generation time and lag time parameters were modeled as functions of temperature by use of exponential-decay models . The models were applied to typical beef carcass-cooling scenarios to predict the potential growth of S . typhimurium during the cooling of beef . Validation studies indicated no significant difference between the observed and predicted bacterial populations on inoculated lean and fatty beef tissues cooled at either 6 or 9 degrees C/h.

J Bacteriol, 1992 Nov, 174(21), 6965 - 73
DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III; Lifsics MR et al.; In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III {Pol III}) exhibit a severe growth defect when the genetic background is otherwise wild type . Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I . In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression . Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA . Suppression of the growth defect was associated with suppression of SOS induction . Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA . Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair . The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only) . Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III . The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III . The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.

J Immunol Methods, 1992 Nov 5, 155(2), 267 - 70
Use of the lipid emulsion system and Salmonella typhimurium mitogen adjuvant to stimulate IgG production in chickens; McCune C et al.; Chickens were immunized with the lipid emulsion system and Salmonella typhimurium mitogen adjuvant for fowl plus synthetic peptides corresponding to the divergent amino acid sequences at the carboxyl terminus of each of two different alpha-tubulin isoforms from Arabidopsis thaliana . Antibodies were extracted from egg yolks and used in immunoblot assays to determine the time course of antibody production in two chickens . Specific immunoglobulin production increased 10-14 days after the primary injection in both chickens and reached peak levels shortly thereafter . Booster injections helped maintain IgG production, which eventually dropped off after the final injection.

J Biol Chem, 1992 Nov 5, 267(31), 22392 - 400
Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector; Benamira M et al.; Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052 . The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102) . PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand . Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield . The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII . Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage . All of the mutations were deletions, and 70% occurred by deletion of CpG . Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer . These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.

JPEN J Parenter Enteral Nutr, 1992 Nov-Dec, 16(6), 561 - 5
The role of protein and calorie restriction in outcome from Salmonella infection in mice; Peck MD et al.; We studied the separate effects of protein and calorie restriction in mice challenged with Salmonella typhimurium, an intracellular pathogen eliminated by cell-mediated immunity . Female A/J mice (n = 73) were placed on one of eight solid diets for 3 weeks . Animals were weighed at the beginning and the end of the feeding period . Diets were adjusted by two factors . The total amount of protein in the diet was 1%, 5%, 20%, or 40% by weight . The diets were fed to half the mice in quantities of 3 g and to the other half at 1.5 g per mouse per day . At the end of 3 weeks, mice were injected intraperitoneally with bacteria and mortality was observed for 2 weeks . Mortality was related to protein intake and was significantly higher in the 1% and 5% groups (chi 2: p = .0021) . However, mortality was lower in the calorie-restricted groups (chi 2: p = .0242) . Although caloric intake did not affect cell-mediated immunity, the response to 2,4-dinitrofluorobenzene was greater in the low protein groups . Lymphoproliferative responses in the mixed lymphocyte response were not affected by either caloric or protein intake . Lymphoproliferative responses to both lipopolysaccharide and phytohemagglutinin were affected by dietary protein but not by caloric intake; proliferative responses were higher in the low-protein groups . We conclude that protein restriction can increase mortality in this model . On the other hand, short-term calorie restriction can improve survival.

Chem Res Toxicol, 1992 Nov-Dec, 5(6), 823 - 7
Characterization of (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo{a}pyrene-9-sulfonate; Green JL et al.; The genotoxicity of certain benzo{a}pyrene (BP) derivatives is significantly enhanced in strains of Salmonella typhimurium following addition of sulfite to the incubations . The interaction between sulfite and those BP derivatives also results in the formation of isomeric BP sulfonates . As these trihydroxy sulfonates are formed in incubations of BP derivatives and sulfite in which a marked potentiation of bacterial mutagenicity occurs, we have investigated the properties of these novel intermediates . The compound (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo{a}pyrene-9-sulfonate (BPT-9-sulfonate) was isolated and characterized in terms of its chemical and biological activity . This BPT sulfonate isomer is formed by the addition of the sulfite anion radical to the 9,10-double bond of the known promutagen, (+/-)-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene (BP-7,8-diol) . Evidence for the free radical character of this addition includes the initiation of the reaction by either peroxidase-catalyzed or chemical one-electron oxidation of sulfite, the inhibition of the reaction by phenolic antioxidants, and the isolation and characterization of the chain termination product, 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo{a}pyrene-9,10-disulfonate (BPD-disulfonate) . Analysis of incubations of S . typhimurium strain TA98 with BP-7,8-diol and sulfite, which resulted in a 10-fold increase in revertant bacterial colonies above control levels, showed that BPT-9-sulfonate and BPD-disulfonate were the only isolable products derived from BP-7,8-diol . This prompted a further investigation of the chemistry of these products . BPT-9-sulfonate was found to be quite stable in aqueous media, being refractory to acid- or base-catalyzed hydrolysis over a pH range of 3-11.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1992 Nov-Dec, 5(6), 779 - 86
Fluoranthene metabolism: human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol; Day BW et al.; The metabolism of the environmental carcinogen fluoroanthene by human liver microsomes was compared to that by liver microsomes from rats treated with Aroclor 1254 . Although the human-derived system gave primarily one product, similar metabolites were noted from each system . Enantiomers of the major metabolic product, in both cases the trans-2,3-dihydrodiol, were separated by chiral stationary-phase chromatography . Absolute configurations were assigned by application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoyl esters . Liver microsomes from Aroclor 1254-treated rats produced the R,R enantiomer of the diol in 75-78% enantiomeric excess, while human liver microsomes produced this enantiomer in only 6-12% excess . The activities of these enantiomers were compared in Salmonella typhimurium strain TM677 mutagenicity assays employing the 9000g supernatant of Aroclor 1254-induced rat liver homogenates . Both the syn- and anti-2,3-dihydrodiol 1,10b-epoxides, which had only been inferred to be metabolites in previous studies, were isolated from the microsomal incubations by preparative reverse-phase HPLC . The evident exceptional aqueous stabilities of these diol epoxides were further examined by half-life determination experiments . Their tetrahydrotetrol hydrolysis products were also noted in the metabolite HPLC profiles . The structures of the tetrahydrotetrols were confirmed by total synthesis.

Osaka City Med J, 1992 Nov, 38(2), 127 - 36
Effects of metallothionein on mutagenicity and oxidation of quercetin; Okamoto A; The effects of four types of Zn/Cd-metallothioneins on the mutagenicity of quercetin in Salmonella typhimurium TA98 were studied . The four types of Zn/Cd-metallothionein used in this experiment were metallothionein I, metallothionein I/II and metallothionein II from rabbit liver and metallothionein I/II from horse kidney . All four of the metallothioneins enhanced the mutagenicity of quercetin . Metallothionein II from rabbit liver, in which Zn content was the highest of the four metallothioneins, enhanced the mutagenicity of quercetin most effectively . Metallothioneins as well as Zn/Cu-SOD prevented quercetin oxidation under aerobic conditions . The action of these metallothioneins, which enhances the mutagenicity of quercetin, was fairly proportional to the activity which prevents quercetin from being oxidized . Reduced glutathione did not enhance the mutagenicity of quercetin although it did inhibit the quercetin oxidation . These results suggest that metallothioneins have superoxide (O2-) scavenging ability and act as "SOD-like" proteins in vivo . A simple and reliable system to detect biological substances which have antioxidant activity for the superoxide (O2-) was devised.

Mol Microbiol, 1992 Nov, 6(22), 3289 - 97
Characterization of the micro-environment of Salmonella typhimurium-containing vacuoles within MDCK epithelial cells; Garcia-del Portillo F et al.; Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells . During the entire intracellular stage, bacteria are enclosed within a vacuole . To characterize the micro-environment of the bacteria-containing vacuoles, we have used a new method to measure the expression levels of several S . typhimurium genes in intracellular bacteria within Madin-Darby canine kidney (MDCK) epithelial cells . Our study was based on the determination of beta-galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein-di-beta-D-galactoside (FDG) . Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post-infection times . High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low . cadA activity was detected only at early post-infection times (4 h), suggesting that the vacuole may have a mild-acidic pH, and oxygen and lysine present at this time . Globally, the results reported indicate that the use of a highly sensitive beta-galactosidase substrate can provide information about the micro-environment within which an intracellular pathogen, such as S . typhimurium, resides.

J Nat Prod, 1992 Nov, 55(11), 1561 - 8
Antimutagenic agents from natural products; Wall ME; Certain secondary metabolites found in terrestrial and marine plants and organisms have evinced the capability for inhibiting the mutagenicity toward Salmonella typhimurium of a number of mutagens . These include 2-aminoanthracene (2AN), ethylmethanesulfonate (EMS), and benzo-{a}pyrene(B{alpha}P) . The sensitivity of the antimutagenicity assay is such that crude extracts can be evaluated and purification of extracts readily followed . Major classes of antimutagenic compounds that have been isolated include flavonoids, coumarins, and cymopols.

Mutagenesis, 1992 Nov, 7(6), 471 - 4
Clastogenicity to the mouse bone marrow of the mouse germ cell genotoxin streptozotocin; Liegibel U et al.; The methylating agent streptozotocin is active in the mouse bone marrow micronucleus assay following a single intraperitoneal injection of 150-180 mg/kg . This correlates with its previously reported toxicity to mouse germ cells when administered by the same route of exposure . The potent mutagenicity of streptozotocin to strain G46 of Salmonella typhimurium is compared with its much weaker activity in strain TA1535 . The genotoxicity of streptozotocin in vivo is reviewed.

Mutagenesis, 1992 Nov, 7(6), 427 - 31
Genotoxicity of tauromustine, a new water soluble taurine-based nitrosourea . I . Mutagenic and clastogenic activity of tauromustine in vitro; Hartley-Asp B; Tauromustine (TCNU) a new taurine-based nitrosourea in phase III clinical trials against colon cancer, has been tested and compared with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (CCNU) for mutagenic potential in Salmonella typhimurium TA98 and TA100 and, for its ability to cause chromosome aberrations and sister chromatid exchanges (SCE) in human lymphocytes . A dose dependent increase in the number of mutations in S . typhimurium TA100 but not in TA98 was found from 1.6 to 1000 micrograms/plate for both TCNU and CCNU . In addition, in the presence of Aroclor activated liver microsomes, CCNU caused a further increase in the mutation frequency which was not found for TCNU . A dose dependent increase in the chromosome aberration rate in human lymphocytes was induced by both TCNU and CCNU at concentrations ranging from 3.75 to 30 micrograms/ml after 24 h treatment during the last cell cycle . When lymphocytes were treated in G0 both TCNU and CCNU induced a higher frequency of aberrations at 15 and 30 micrograms/ml than during the last cell cycle and, unexpectedly, chromosome-type, i.e . dicentrics appeared . The difference in the frequency of aberrations in these two phases may be related to the different levels of O6-methyl guanine transferase present in resting contra cycling lymphocytes . TCNU at 0.8-8 micrograms/ml also induced a dose dependent increase in the number of SCE in human lymphocytes . Neither the number of chromosomal aberrations nor the number of SCE were affected by the addition of 1000 micrograms/ml taurine to the culture medium.

Int J Immunopharmacol, 1992 Nov, 14(8), 1415 - 20
Combined effects of synthetic lipid A analogs or bacterial lipopolysaccharide with glucosaminylmuramyl dipeptide on antitumor activity against Meth A fibrosarcoma in mice; Shimizu T et al.; The combined effects of the synthetic glucosaminylmuramyl dipeptide (GMDP) on the antitumor activity of chemically synthesized lipid A analogs, compound A-103 (glucosamine-4-phosphate with (R)-3-tetradecanoyloxytetradecanoyl group at the C-2 and C-3 positions), Escherichia coli-type lipid A (506), Salmonella typhimurium LT-2 lipopolysaccharide (LPS) against Meth A fibrosarcoma in mice were examined . Meth A fibrosarcoma cells (5 x 10(5) were inoculated intradermally into BALB/c mice on day 0, and compound A-103 and/or GMDP was administered intravenously (i.v.) on days 7 and 9 . Two i.v . injections of A-103 (50 micrograms) alone or GMDP (10 micrograms) alone induced 42.8 or 51.8% inhibition of the rate of tumor growth, however, A-103 (100 micrograms) with GMDP (10 micrograms) exhibited a high 68.7% inhibition rate 19 days after tumor inoculation . The inhibition of the tumor growth rate by the combination A-103 (100 micrograms) or 506 (50 micrograms) with GMDP (10 micrograms) was stronger than that of A-103 or 506 with MDP (10 micrograms) . The combination of LPS (1 or 10 micrograms) with GMDP (10 micrograms) exhibited a higher inhibition rate than that of LPS with MDP, and three or four tumor-free mice out of five mice were observed, suggesting that the combined effect of GMDP is more potent than that of MDP . With the addition of GMDP, A-103 did not enhance the production of tumor necrosis factor (TNF) on the basis of L929 cell lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1992 Nov 1, 77(1-3), 269 - 75
Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater; Aznar R et al.; A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity . All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds . Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth . The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.

Mol Microbiol, 1992 Nov, 6(21), 3149 - 57
A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific sigma factor, sigma F; Ohnishi K et al.; We have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium . A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino-terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine . The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (sigma F) . No DNA-binding activity was detected in FlgM . Chemical cross-linking experiments showed that the purified FlgM bound to sigma F and disturbed its ability to form a complex with RNA polymerase core enzyme . These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum-specific sigma factor through direct interaction, i.e . it is an anti-sigma factor.

Proc Natl Acad Sci U S A, 1992 Nov 1, 89(21), 10079 - 83
Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes; Alpuche Aranda CM et al.; Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products . This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S . typhimurium virulence regulators PhoP and PhoQ . Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold . In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes . The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments . Measurements of pH documented that S . typhimurium delayed and attenuated acidification of its intracellular compartment . Phagosomes containing S . typhimurium required 4-5 hr to reach pH < 5.0 . In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5 . The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression . These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.

J Bacteriol, 1992 Nov, 174(22), 7090 - 7
Mutants carrying conditionally lethal mutations in outer membrane genes omsA and firA (ssc) are phenotypically similar, and omsA is allelic to firA; Vuorio R et al.; We have previously identified the gene (the ssc gene) defective in the thermosensitive and antibiotic-supersusceptible outer membrane permeability mutant SS-C of Salmonella typhimurium and shown that this gene is analogous to the Escherichia coli gene firA (L . Hirvas, P . Koski, and M . Vaara, EMBO J . 10:1017-1023, 1991) . Others have tentatively implicated firA in a different function, mRNA synthesis . Here we report that the defect in the thermosensitive outer membrane omsA mutant of E . coli (T . Tsuruoka, M . Ito, S . Tomioka, A . Hirata, and M . Matsuhashi, J . Bacteriol . 170:5229-5235, 1988) is due to a mutation in firA; this mutation changed codon 271 from serine to asparagine . The omsA-induced phenotype was completely reverted by plasmids containing wild-type firA or ssc . Plasmids carrying the omsA allele, or an identical mutant allele prepared by localized mutagenesis, under the control of lac elicited partial complementation . Transcomplementation studies with plasmids carrying various mutant alleles of the S . typhimurium gene indicated that the ability of these plasmids to complement the omsA mutation was similar to their ability to complement the ssc mutation . The antibiotic-supersusceptible phenotype of the omsA mutant closely resembled that of the ssc mutant, i.e., the omsA mutant was supersusceptible to hydrophobic antibiotics and large-peptide antibiotics against which the intact outer membrane is an effective permeability barrier . As previously demonstrated with the omsA mutant, the outer membrane of the ssc mutant became selectively ruptured after incubation for 1 h at the growth-nonpermitting temperature; 82% of the periplasmic beta-lactamase and less than 3% of the cytoplasmic marker enzyme were released into the medium . All of these findings are consistent with our concept that firA is an essential gene involved in generation of the outer membrane.

Carcinogenesis, 1992 Nov, 13(11), 2059 - 65
Response of the ke test to NCI/NTP-screened chemicals . III . Complementary value of ke in screening for carcinogens; Ennever FK et al.; The value of using a physico-chemical carcinogen-screening test, the ke test, in conjunction with the Salmonella typhimurium/microsome assay (the Ames test) and/or structural alerts of reactivity (the S/A test), is analyzed on the basis of the response of the three tests to 171 chemicals of known rodent carcinogenicity . The Ames test is widely used to screen chemicals for potential carcinogenicity; however, its relatively low sensitivity (proportion of true positives among carcinogens tested) has prompted a search for complementary tests that increase sensitivity without an unacceptable decrease in specificity (proportion of true negatives among non-carcinogens tested) . The S/A test is a structural analysis based on recognition of chemicals groups likely to react with DNA . The S/A test does not complement the Ames test well, because of the high similarity of responses (dependence) between these two tests . The ke test measures the affinity of a test chemical for electrons, and has a sensitivity and specificity comparable to the Ames test . The ke test is shown in this work to complement both the Ames test and the S/A test . Addition of the ke test to either the Ames test or the S/A test results in a substantial decrease in false negatives and an approximately equal increase in false positives, which is a trade-off that would be desirable in all but the least risk averse situations . The S/A and ke battery has a sensitivity of > 0.9, and could be applied to untested chemicals without any biological testing . In view of these observations, it is proposed that the ke test be considered in developing future strategies to optimize the screening of potential carcinogens in the most cost-effective manner.

J Immunol, 1992 Nov 1, 149(9), 3040 - 4
Cytokine expression in vivo during murine listeriosis . Infection with live, virulent bacteria is required for monokine and lymphokine messenger RNA accumulation in the spleen; Poston RM et al.; To examine the regulation of cytokine synthesis during murine listeriosis, we have monitored IFN-gamma, TNF-alpha, and IL-1 beta mRNA levels in the spleens of C57B1/6 mice after the i.v . infusion of virulent and nonvirulent preparations of Listeria monocytogenes (LM) . Messenger RNA coding for TNF, IL-1, or IFN did not become detectable until approximately 12 to 15 h after the infusion of virulent LM . Levels of each cytokine mRNA then increased synchronously reaching peak or near peak levels around 24 h after infection . Levels gradually decreased over the next 4 to 5 days . Unlike virulent LM, neither heat-killed LM, nor nonvirulent LM variants lacking listeriolysin O, stimulated monokine or IFN mRNA accumulation even when administered in very large doses . To gain perspective concerning the response to LM, we examined the early pattern of cytokine mRNA accumulation induced by Salmonella typhimurium (ST), an intracellular pathogen expressing LPS . We noted at least three significant differences between the cytokine responses to LM and ST: 1) monokine mRNA levels increased much more rapidly (within 1 h) after ST infection; 2) unlike LM, ST retained the capacity to stimulate cytokine mRNA production when injected as heat-killed bacteria; 3) in contrast to LM, ST could not trigger the early IFN production characteristic of LM infection . Our data suggest that monokine and IFN production early in listeriosis are critically linked with the process of bacterial invasion of host cells . The timing and pattern of cytokine mRNA accumulation in this setting is qualitatively different from that induced by LPS . The pathway described in these studies may also play a role in the host cytokine response to other intracellular pathogens as well.

J Bacteriol, 1992 Nov, 174(21), 6948 - 55
Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity; Nohmi T et al.; Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli . The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S . typhimurium umuDC (umuDCST) operon resides in a chromosome . The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S . typhimurium were investigated . A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E . coli than a plasmid carrying the umuDCST operon did . When the umuDCST operon was specifically deleted from the chromosome of S . typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was . Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing . A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E . coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability . We concluded that the umuDCST operon plays a major role in UV mutagenesis in S . typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage . Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed.

Infect Immun, 1992 Nov, 60(11), 4604 - 11
Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans; Palma C et al.; Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro . Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells . LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C . albicans in vitro . Antibodies against lactoferrin effectively and specifically reduced the anti-C . albicans activity of both LPS-stimulated and unstimulated PMN . Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS . The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide . These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN . These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C . albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN . Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.

Infect Immun, 1992 Nov, 60(11), 4571 - 7
Rapid generation of specific protective immunity to Francisella tularensis; Elkins KL et al.; Mice inoculated either subcutaneously (s.c.) or intradermally (i.d.) with a sublethal dose of Francisella tularensis LVS are immune to a lethal intraperitoneal (i.p.) or intravenous (i.v.) challenge of LVS . Here, we show that this immunity developed quite rapidly: mice given a sublethal dose of live LVS s.c . or i.d . (but not i.v.) withstood lethal i.p., i.v., or i.d . challenge as early as 2 days after the initial inoculation, despite the presence of bacterial burdens already in tissues . The magnitude of this early protection was quite impressive . The i.p . 50% lethal dose (LD50) in naive C3H/HeN mice was only 2 bacteria, while the i.p . LD50 in mice given 10(4) LVS i.d . 3 days previously was 3 x 10(6) bacteria . Similarly, the i.v . LD50 in C3H/HeN mice shifted from 3 x 10(2) in naive mice to 5 x 10(6) in primed mice within 3 days after i.d . LVS infection . Comparable changes in the i.p . and i.v . LD50 were observed in C57BL/6J mice . This rapid generation of protective immunity was specific for LVS, in that mice given a sublethal i.d . inoculation of LVS did not survive a lethal challenge with either Salmonella typhimurium W118 or Escherichia coli O118 BORT at any time, nor could mice given sublethal doses of S . typhimurium, E . coli, or Mycobacterium bovis BCG survive lethal doses of LVS . Although an increase in the mean time to death from S . typhimurium infection was noted when mice were given a sublethal i.d . dose of LVS 4 to 14 days earlier, no overall increase in protection or change in the S . typhimurium LD50 was observed . Thus, sublethal infection with LVS at skin sites induced rapid and specific protective immunity.

J Bacteriol, 1992 Nov, 174(22), 7297 - 307
Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen; Klena JD et al.; The rfp gene of Shigella dysenteriae 1 and the rfa genes of Escherichia coli K-12 and Salmonella typhimurium LT2 have been studied to determine their relationship to lipopolysaccharide (LPS) core heterogeneity and their role in the attachment of O antigen to LPS . It has been inferred from the nucleotide sequence that the rfp gene encodes a protein of 41,864 Da which has a structure similar to that of RfaG protein . Expression of this gene in E . coli K-12 results in the loss of one of the three bands seen in gel analysis of the LPS and in the appearance of a new, more slowly migrating band . This is consistent with the hypothesis that Rfp is a sugar transferase which modifies a subset of core molecules so that they become substrates for attachment of S . dysenteriae O antigen . A shift in gel migration of the bands carrying S . dysenteriae O antigen and disappearance of the Rfp-modified band in strains producing O antigen suggest that the core may be trimmed or modified further before attachment of O antigen . Mutation of rfaL results in a loss of the rough LPS band which appears to be modified by Rfp and prevents the appearance of the Rfp-modified band . Thus, RfaL protein is involved in core modification and is more than just a component of the O-antigen ligase . The products of rfaK and rfaQ also appear to be involved in modification of the core prior to attachment of O antigen, and the sites of rfaK modification are different in E . coli K-12 and S . typhimurium . In contrast, mutations in rfaS and rfaZ result in changes in the LPS core but do not affect the attachment of O antigen . We propose that these genes are involved in an alternative pathway for the synthesis of rough LPS species which are similar to lipooligosaccharides of other species and which are not substrates for O-antigen attachment . All of these studies indicate that the apparent heterogeneity of E . coli K-12 LPS observed on gels is not an artifact but instead a reflection of functional differences among LPS species.

Mutat Res, 1992 Nov, 293(1), 1 - 10
The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response; Benamira M et al.; An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system . We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002 . SOS-inducing potencies varied widely among the carbonyl compounds tested . 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM . Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression . Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM . Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity . Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency . Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.

Infect Immun, 1992 Nov, 60(11), 4679 - 86
Protection of mice against Salmonella typhimurium with an O-specific polysaccharide-protein conjugate vaccine; Watson DC et al.; Serious infections with salmonellae remain a threat in many human populations . Despite extensive study of salmonella infections in animals and clinical experience with killed cellular vaccines, there are no vaccines against serotypes other than Salmonella typhi licensed for human use . Serum antibodies to the O-specific polysaccharide (O-SP) of salmonellae protect mice against invasive infection . In order to render it immunogenic, we have conjugated the O-SP of Salmonella typhimurium to carrier proteins by various schemes . O-SP conjugated to tetanus toxoid (O-SP-TT) elicited antibodies in outbred mice after three subcutaneous injections without adjuvant . The O-SP alone elicited no detectable antibody . The antibody response to O-SP-TT was boosted by successive doses and consisted of immunoglobulin G (IgG) and IgM . Most mice only produced antibodies specific for the abequose (O:4 factor) region of the O-SP . Occasional animals also produced antibodies to the core oligosaccharide . Immunized mice were protected against intraperitoneal challenge with S . typhimurium, demonstrating a 160-fold increase in the 50% lethal dose . Passive immunization with conjugate-induced IgM or IgG also protected against challenge . These results indicate that an O-SP-TT conjugate, when given by a route and formulation acceptable for human use, protects mice against challenge with S . typhimurium.

Curr Microbiol, 1992 Nov, 25(5), 257 - 60
Role of nalidixic acid in isolation of Salmonella typhimurium strains capable of growth at 48 degrees C; Droffner ML et al.; Salmonella typhimurium thermotolerant mutants dependent on the presence of nalidixic acid for growth at 48 degrees C were isolated and designated nalidixic acid-dependent, thermotolerant mutants, naldttl . Genetic mapping revealed that naldttl alleles map within the gyrA gene . When S . typhimurium strain Q was plated in the dark on nutrient agar containing nalidixic acid (20 micrograms/ml) as a photosensitizer and briefly exposed to white light or near VU light prior to incubation at 42 degrees C, nalidixic acid-resistant mutants arose in about 16 h at frequencies of 5 x 10(-8) for white light and 1 x 10(-6) for near UV light . About 10% of these nalidixic acid-resistant mutants derived from photodynamic mutagenesis exhibited the thermotolerant characteristic.

Ultramicroscopy, 1992 Nov, 45(3-4), 307 - 21
Radial mass density functions of vitrified helical specimens determined by scanning transmission electron microscopy: their potential use as substitutes for equatorial data; Trachtenberg S et al.; Using STEM dark field images, we have determined linear mass densities and radial density profiles of vitrified helical particles . The samples studied are: TMV, RNA-free helical polymers of TMV coat protein (TMV-P), Salmonella typhimurium bacterial flagellar filaments and Escherichia coli pili . The difference between the profiles obtained for TMV and TMV-P shows a maximum at a radius of about 4 nm, corresponding to the RNA in TMV . Of the peaks that are resolved in X-ray diffraction analysis we can resolve the ones for TMV at radii of approximately 4.2 and approximately 6.7 nm and a shoulder at approximately 7.8 nm . Density peaks in bacterial flagellar filaments appear at radii of approximately 4.2, approximately 6.5, approximately 8.5, and approximately 10.5 nm . Accurate mass data can be obtained if the filaments are embedded in ice layers of uniform thickness; their diameters need to be similar to that of the mass standard (TMV) when these data are measured in a comparative manner . Ice layers are often not uniform, and thickness variations are well revealed in STEM dark field . The signal-to-noise ratio and contrast for the transverse projections are lower than those measured for freeze-dried specimens: half an order and one order of magnitude, respectively . The thinnest uniformly thick ice layer still containing a single layer of particles is approximately 10-15 nm thicker than the particles . Radial mass density functions that are directly determined in STEM may have a potential use as substitutes for the unreliable equatorial data in helical reconstructions of TEM bright field images of vitrified specimens.

Mol Microbiol, 1992 Nov, 6(21), 3077 - 87
Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low-oxygen conditions; Francis CL et al.; Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection . Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence . Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S . typhimurium . Stationary-phase S . typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I . Thus, environmental cues regulate S . typhimurium invasion factors, allowing for immediate entry into host cells . Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.

J Gen Microbiol, 1992 Nov, 138 ( Pt 11), 2363 - 70
The catalase-peroxidase of Mycobacterium intracellulare: nucleotide sequence analysis and expression in Escherichia coli; Morris SL et al.; The activation of catalase genes in response to oxidative stress may contribute to the intracellular survival of mycobacteria . In this report, the nucleotide sequence of a mycobacterial catalase gene is described . The deduced protein sequence of this Mycobacterium intracellulare gene (MI85) was 60% identical to the Escherichia coli hydroperoxidase I (HPI) protein, 59% identical to the Salmonella typhimurium (HPI) catalase, and 47% identical to a Bacillus stearothermophilus peroxidase . The MI85 protein, expressed in E . coli, has also been shown to have peroxidase and catalase activities . Furthermore, Southern blot hybridizations, which demonstrated that a MI85 gene probe hybridizes with chromosomal DNA from thirteen different strains of mycobacteria, suggest that this catalase-peroxidase gene is prevalent in the mycobacterial genus . The availability of catalase gene probes should permit an evaluation, at the molecular level, of the role of catalase in mycobacterial pathogenesis.

Can J Microbiol, 1992 Nov, 38(11), 1102 - 7
Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium; Pai SR et al.; A panel of monoclonal antibodies, seven against the trimeric and seven against the monomeric forms to outer membrane protein D (OmpD) of Salmonella typhimurium were produced . The specificities of these monoclonal antibodies for the porin proteins of S . typhimurium and their cross-reactions with Salmonella porins OmpC and OmpF were determined by Western immunoblotting and enzyme-linked immunosorbent assay . We observed that OmpD shared more epitopes and had greater structural similarity with OmpC than with OmpF.

Microb Pathog, 1992 Nov, 13(5), 417 - 21
Introduction of Francisella tularensis at skin sites induces resistance to infection and generation of protective immunity; Elkins KL et al.; Mice are susceptible to systemic infection with Francisella tularensis strain LVS; thus, the intraperitoneal (i.p.) lethal dose at 50% (LD50) in C3H/HeN and C57BI/6J mice is only a single bacterium, while the intradermal (i.d.) LD50 is more than 10(4) . Here we show that the LD50 when LVS is introduced via the skin, either i.d . or subcutaneously (s.c.), ranges from 7 x 10(4) to 2 x 10(6) . Sublethal i.d . or s.c . infection (priming) invariably leads to the generation of systemic and specific protective immunity: primed mice survive lethal i.p., intravenous (i.v.), or i.d . challenges of LVS but not Salmonella typhimurium W118 or Escherichia coli 018:K1:H7 strain BORT.

J Toxicol Sci, 1992 Nov, 17 Suppl 3, 269 - 81
{Mutagenicity studies of prednisolone farnesylate (PNF)}; Otsuka M et al.; Prednisolone farnesylate (PNF) was tested for mutagenicity by Ames test using Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA), for clastogenic activity in vitro by the chromosomal aberration test in a Chinese hamster fibroblast cell line (CHL), and for induction of micronuclei by the micronucleus test in male ICR mice . 1) In Ames test, PNF with and without metabolic activation showed no mutagenicity in any strains at any dose levels (312-5,000 micrograms/plate) . 2) In the chromosomal aberration test, PNF with metabolic activation produced a slight increase in the incidence of structural chromosomal aberrations in CHL cells at 1,500 micrograms/ml . 3) In the micronucleus test, a single administration of PNF caused no significant increase of micronucleated polychromatic erythrocytes at any doses (250-2,000 mg/kg).

Vet Microbiol, 1992 Nov, 33(1-4), 249 - 62
Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus; Enjuanes L et al.; The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure . Four antigenic sites have been defined (A, B, C, and D) . The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro . These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides . Both sites might be relevant in protection in vivo . PRCV does not have sites B and C, due to a genomic deletion . Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies . An epidemiological tree relating the TGEVs and PRCVs has been proposed . The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses . In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus . These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.

Mutat Res, 1992 Nov, 298(1), 17 - 23
Assessment of the genotoxic potential of riboflavin and lumiflavin . B . Effect of light; Kale H et al.; On exposure to visible light, riboflavin and lumiflavin produced reactive oxygen species such as singlet oxygen and superoxide radicals . The reaction was found to be time- and concentration-dependent . Both riboflavin and lumiflavin, upon illumination, showed mutagenic response in the umu test as well as in the Ames/Salmonella assay with Salmonella typhimurium TA102 . The mutagenic response was partially abolished by superoxide dismutase while sodium azide did not have any effect . No mutagenicity was observed if the compounds were not illuminated . The results suggested the involvement of superoxide radicals in light-induced mutagenicity of riboflavin as well as lumiflavin.

Eur J Biochem, 1992 Oct 15, 209(2), 589 - 95
Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins; Scocchi M et al.; Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils {Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H . and Romeo, D . (1990) J . Cell Biol . 111, 1363-1371} . These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography . While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml . Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules . Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis . Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7 . These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.

J Biol Chem, 1992 Oct 15, 267(29), 20706 - 12
Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium; Nikaido K et al.; The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized . The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively . L-Histidine was found to be a relatively good ligand (KD, 500 nM) . Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO . Liganded and unliganded LAO are shown to have distinct UV spectra . The UV spectrum also varies with the nature of the substrate . Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.

J Biol Chem, 1992 Oct 15, 267(29), 20667 - 73
Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins; Kendall RL et al.; A methionine aminopeptidase that specifically removes methionine residues from peptides with amino-terminal sequences of Met-Ala-, Met-Val-, Met-Ser-, Met-Gly-, and Met-Pro- but not Met-Leu- or Met-Lys- has been isolated to homogeneity from porcine liver by a procedure involving five chromatographic steps . The enzyme, whose specificity matches that predicted for the entity responsible for the co-translational amino-terminal processing of nascent polypeptide chains, has a measured molecular mass of 70,000 Da by SDS-polyacrylamide electrophoresis and 67,000 Da by gel chromatography (under nondenaturing conditions), suggesting the native molecule is a monomer . It is activated by Co2+ and inhibited by beta-mercaptoethanol and EDTA . With octapeptide substrates related to the amino-terminal portion of the beta-chain of human hemoglobin (with a histidine in position 3), the enzyme had a pH optimum of 6.0 . With a synthetic peptide devoid of histidine, it showed no pH dependence from 6.0 to 8.0 . This sensitivity may be due to the propensity of peptides with histidine in the third position to bind divalent cations such as Co2+ . The measured Km and kappa cat values were affected by residues in the second position . The peptide corresponding to the natural sequence (Met-Val-His-) gave a kappa cat/Km value of 260 mM-1 s-1; substitution of alanine in the second position raised the kappa cat/Km to 1523 mM-1 s-1, but substitution of proline lowered the value to 130 . The effects are primarily on the kappa cat . The substitution of proline (for histidine) in the third position, the mutation found in hemoglobin Long Island, prevents the removal of the methionine residue, as occurs with the mutant protein . The porcine liver enzyme is similar to methionine aminopeptidases isolated from Escherichia coli, Salmonella typhimurium, and yeast in that it also is stimulated by Co2+ . However, it is much larger than these enzymes and differs somewhat in specificity, particularly with the yeast enzyme.

Gene, 1992 Oct 12, 120(1), 93 - 8
Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli; Johanson U et al.; The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level . The order of conservation at the nt and aa level is rpsL greater than tufA greater than rpsG greater than f usA . A striking difference is that the rpsG-encoded ribosomal protein, S7, in E . coli K-12 is 23 aa longer than in S . typhimurium . The very low (0.18) codon adaptation index of this part of the E . coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension . A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S . typhimurium is noted . In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events . An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.

Mutat Res, 1992 Oct, 272(2), 91 - 9
A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity; Oda Y et al.; A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity . The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S . typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002) . Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells . The sensitivity of strain NM1011 was compared with that of the parent tester strain S . typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S . typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes . The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene . By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene . These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

J Mol Biol, 1992 Oct 5, 227(3), 672 - 7
M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF; Ueno T et al.; The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings . When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane . Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings . Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body . We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body.

Can J Vet Res, 1992 Oct, 56(4), 296 - 302
Immune response of pigs to parenteral vaccination with an aromatic-dependent mutant of Salmonella typhimurium; Lumsden JS et al.; Cellular and humoral immune responses to parenteral vaccination with an aromatic-defined (aroA) Salmonella typhimurium and to oral challenge with the S . typhimurium parent strain were examined in pigs . The effectiveness of aroA S . typhimurium vaccination for prevention of clinical disease following challenge was also evaluated . A split litter model was utilized and analysis of variance was by least squares . The statistical model accounted for the effects of vaccination and litter . Parenteral vaccination of pigs with the aroA mutant induced a significant O-polysaccharide (O-ps) specific lymphocyte blastogenic response as well as a significant antibody response to O-ps, lipopolysaccharide and killed bacteria . The aroA strain was avirulent in pigs, was not shed in the feces and significantly reduced the severity of diarrhea following oral challenge.

Poult Sci, 1992 Oct, 71(10), 1781 - 4
Research note: in ovo administration of a competitive exclusion culture treatment to broiler embryos; Cox NA et al.; Exposure of chicks to salmonellae in the hatchery and hatchery environment limits the effectiveness of a competitive exclusion (CE) culture treatment . Therefore, in an attempt to apply treatment before chicks are exposed to salmonellae, the CE culture was introduced in ovo to unhatched embryos . An undefined, anaerobically grown CE culture, derived from cecal contents of healthy adult chickens, was diluted 1:1,000 or 1:1,000,000 and inoculated either into the air cell or beneath the inner air cell membrane of 17-day-old incubating hatching eggs . The treated chicks were more resistant than untreated chicks to varying challenge levels of Salmonella typhimurium, indicating that it may be possible to initiate protection of chicks to salmonellae challenge prior to hatching into a contaminated environment.

J Appl Toxicol, 1992 Oct, 12(5), 377 - 84
Mutagenicity evaluation of riot control agent o-chlorobenzylidene malononitrile (CS) in the Ames Salmonella/microsome test; Meshram GP et al.; o-Chlorobenzylidene malononitrile (CS), a riot control agent, was evaluated for its possible mutagenic activity in the Ames Salmonella/mammalian microsome mutagenicity test . Five histidine-deficient (His-) mutant tester strains of Salmonella typhimurium--TA97a, TA98, TA100, TA102 and TA104--were used . The liquid preincubation procedure was used with metabolic activation (presence of S9 mixture) and without metabolic activation (absence of S9 mixture) . For the experiments with metabolic activation, three different concentrations of S9 fraction (supernatant of Aroclor 1254-induced rat liver homogenate at 9000 g)--5%, 15% and 30% in S9 mixture--were used . Along with mutagenic activity, CS was also evaluated for cytotoxic activity in all the five tester strains of Salmonella typhimurium, both in the presence and absence of S9 mixture . The mutagenic and cytotoxic activities of CS were assessed by counting the His+ revertant colonies and by counting the microcolonies (His-, auxotrophs in the background lawn), respectively, and the respective mean values were compared with the relative negative (solvent) control . A dose range of 12.5-800 micrograms plate-1 for CS did not induce a mutagenic response either in the presence or absence of S9 mix . No change in the negative mutagenic response of CS has been observed even in the presence of an elevated level of S9 fraction in the S9 mix . A dose of 200 micrograms plate-1 for CS was found to be cytotoxic by decreasing the surviving cells as well as His+ revertant colonies; however, the effect was reduced in the presence of an elevated level of S9 fraction in the S9 mix.

Mol Microbiol, 1992 Oct, 6(19), 2747 - 53
The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli; Kredich NM; Most genes required for cysteine biosynthesis in Salmonella typhimurium and Escherichia coli are positively regulated by cysB, which encodes a transcriptional activator belonging to the LysR family of regulatory proteins . CysB protein binds just upstream of the -35 region of positively regulated promoters, where in the presence of inducer it facilitates formation of a transcription initiation complex . CysB protein also autoregulates its own synthesis by binding to the cysB promoter as a repressor . Cysteine down-regulates the pathway by inhibiting synthesis of O-acetylserine, a direct cysteine precursor and possibly an inducer of gene expression . O-Acetylserine spontaneously isomerizes to N-acetylserine, which is clearly an inducer . Sulphide and thiosulphate provide additional regulation by acting as anti-inducers . Inducer stimulates CysB protein binding to sites involved in positive regulation, and inhibits binding to the negatively autoregulated cysB promoter . For three sites with unknown function, binding is stimulated at one and inhibited at the other two.

Food Chem Toxicol, 1992 Oct, 30(10), 853 - 8
Effect in vitro of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the hepatic activation of dietary genotoxins by rat post-mitochondrial fractions; Ho TA et al.; The effect of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the conversion of the heterocyclic amine 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay . The assay used Salmonella typhimurium TA98 as an indicator of the mutagenicity and hepatic post-mitochondrial fractions (S-9) from male Sprague-Dawley rats as the activating system . All three fatty acids inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA repair processes within the bacterial cell . The activation of three other food mutagens, 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by these fatty acids.

Genetics, 1992 Oct, 132(2), 303 - 10
Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium; Prival MJ et al.; We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA) . Both of these mutants can revert to His+ via intragenic and extragenic mechanisms . Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site . Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites . Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs . A/T sites as well as on intragenic sites vs . extragenic suppressor sites . Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants . Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations . The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium . Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 45 - 9
Gentisate pathway in Salmonella typhimurium: metabolism of m-hydroxybenzoate and gentisate; Goetz FE et al.; Salmonella typhimurium was shown to use the gentisate pathway to metabolize m-hydroxybenzoate and gentisate, m-Hydroxybenzoate hydroxylase and gentisate 1,2-dioxygenase were induced by growth on either gentisate or m-hydroxybenzoate . These enzymes were not detected when the bacteria were grown with glucose or glucose and either m-hydroxybenzoate or gentisate . However, both enzymes were induced when the bacteria were grown on succinate with either substrate . The maleylpyruvate isomerase required reduced glutathione and was irreversibly inhibited by N-ethylmaleimide.

Carcinogenesis, 1992 Oct, 13(10), 1789 - 94
Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes; Yamazaki H et al.; An acetyltransferase-overexpressing strain of Salmonella typhimurium (NM2009) has been used to investigate roles of human liver microsomal cytochrome P450 (P450) enzymes in the activation of carcinogenic nitrosamine derivatives, including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines, to genotoxic products . Studies employing correlation of activities with several P450-dependent monooxygenase reactions in different human liver samples, inhibition of microsomal activities by antibodies raised against human P450 enzymes and by specific P450 inhibitors, and reconstitution of activities with purified P450 enzymes suggest that the tobacco-smoke-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and N-nitrosonornicotine (NNN) as well as N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) are oxidized to genotoxic products by different P450 enzymes, particularly P450 2E1 and 2A6 . The activation of NDMA and NNN by liver microsomes was suggested to be catalyzed more actively by P450 2E1 than by other P450 enzymes because the activities were well correlated with NDMA N-demethylation and aniline p-hydroxylation in different human samples, and purified P450 2E1 had the highest activities in reconstituted monooxygenase systems . The relatively high contribution of P450 2A6 to the activation of NDEA and NNK was supported by the correlation seen with coumarin 7-hydroxylation in human liver microsomes, and antibodies raised against P450 2A6 inhibited both activities by approximately 50% . P450 3A4, 2D6 and 2C enzymes appear not to be extensively involved in the activation of these nitrosamines as judged by several criteria examined . Thus, this work indicates that several P450 enzymes, particularly P450 2E1 and 2A6, catalyze metabolic activation of nitrosamine derivatives including N-nitrosodialkylamines and tobacco-smoke-related nitrosamines in human liver microsomes.

FEMS Microbiol Immunol, 1992 Oct, 5(4), 191 - 9
Mechanism of the protective immunity against murine typhoid: persistence of Salmonella L forms in the liver after immunization with live-cell vaccines; Kita E et al.; Live-cell vaccines of Salmonella typhimurium, either a sub-lethal dose of a wild-type (strain LT2) or a high dose of its two-heptose Rd1 mutant (strain SL1004), induced acquired resistance to murine typhoid, which remained 180 days after immunization . Growth of S . typhimurium as a bacillary form ceased between days 30 and 60 of immunization, but L forms of this bacterium colonized the liver (the mean number of L forms in the liver: 600 L-forming units) even at 180 days post-immunization . In contrast, a high inoculum of either a Ra mutant (strain TV148) of strain LT2 or S . schottmulleri 8006 sharing the same O antigenic components with those of S . typhimurium induced only a short-lived protection in proportion to the number of L forms in the liver, and the protective immunity was lost before day 180 . However, there was no significant difference in the salmonella-specific T-cell responses among groups of immunized mice on day 180 of immunization . A lethal infection with strain LT2 in mice which had been immunized 75 days previously with living cells of strain SL1004 resulted in a rapid clearance of the challenge inoculum, together with a rapid elevation of anti-S . typhimurium antibody responses . Thus, the present data suggest that the long-lived immunity conferred upon live S . typhimurium vaccines is attributable to the colonization of this bacterium in the liver as L forms and the ability to colonize the liver as L forms is independent of the chain length of salmonella O-antigens.

Toxicol Lett, 1992 Oct, 63(1), 69 - 74
Pentamidine isethionate is negative in tests for microbial mutagenicity and chromosomal breakage in vitro; Connor TH et al.; Pentamidine isethionate, a drug used for the treatment of Pneumocystis carinii pneumonia in AIDS patients, was assayed for mutagenicity in five strains of Salmonella typhimurium and for clastogenicity and mutagen-induced chromosomal breakage in five human lymphoblastoid cell lines . The mutagenicity assay employed both repair-deficient and repair-positive strains without and with the addition of rat liver S-9 . There was no indication of a mutagenic response in any of the strains of Salmonella . Chromosomal breakage was measured in lymphoblastoid cell lines, both in the absence and presence of bleomycin . Following 2, 5 and 24 h of treatment, pentamidine alone did not induce clastogenicity, nor was there an increase in chromosomal breakage when the cell lines were treated with bleomycin simultaneously with, or 22 h prior to, the addition of pentamidine . From these data it can be concluded that pentamidine is not mutagenic or clastogenic in the two assays employed in this study.

Toxicol Lett, 1992 Oct, 63(1), 35 - 45
Mutagenicity of nitroxyl compounds: structure-activity relationships; Gallez B et al.; Three piperidinoxyl radicals were found to be directly mutagenic in Salmonella typhimurium TA 100, one pyrrolidinoxyl compound had weaker activity, and two other pyrrolidinoxyl derivatives did not produce an increase of the spontaneous revertants . The tester strain TA 100 was selected in preliminary tests for its higher sensitivity compared to TA 98 and TA 102 . The mutagenic activity of the three active compounds was abolished by partial reduction with ascorbic acid, suggesting that the mutagenicity was linked to the free radical nature of these compounds, and reduced in the presence of a cofactor supplemented rat liver subcellular fraction . The mutagenicity of the tested compounds was correlated to the resistance of the nitroxyl spin labels to reduction: the more reactive radicals were found to possess higher mutagenic activity.

J Bacteriol, 1992 Oct, 174(20), 6644 - 52
Escherichia coli produces a cytoplasmic alpha-amylase, AmyA; Raha M et al.; In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E . coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical . The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E . coli or S . typhimurium protein (without assuming signal peptide cleavage) . Minicell experiments identified the product of the E . coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence . The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction . We conclude that the protein is cytoplasmic, as predicted by its sequence . The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly . It was specific for the alpha-anomeric linkage, being unable to digest cellulose . The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase . The newly discovered gene has been named amyA . The natural physiological role of the AmyA protein is not yet evident.

Infect Immun, 1992 Oct, 60(10), 4260 - 8
Specific lung mucosal and systemic immune responses after oral immunization of mice with Salmonella typhimurium aroA, Salmonella typhi Ty21a, and invasive Escherichia coli expressing recombinant pertussis toxin S1 subunit; Walker MJ et al.; Pertussis toxin (PT) is considered an essential protective component for incorporation into new generation vaccines against Bordetella pertussis, the causative agent of whooping cough . Traditionally, antipertussis vaccination has employed an intramuscular route . An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunization with live vaccine carrier strains of Salmonella spp . or Escherichia coli . Recombinant S1 subunit of pertussis toxin was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, in the human typhoid vaccine strain Salmonella typhi Ty21a, and in E . coli CAG629 containing the Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness of epithelial cells . Expression of recombinant PT S1 subunit (rPT-S1) did not affect in vitro invasiveness of the tested strains, which retained the ability to adhere to and invade the embryonic human intestinal cell line HI-407 . Following oral immunization of mice with the live vaccine strains expressing rPT-S1, immunoglobulin G (IgG), IgA, and IgM responses were monitored . IgG specific to PT was detected in serum samples of mice, while IgG and IgA specific to PT were detected in lung washes after oral immunization with living Salmonella spp . or E . coli (pWR110) expressing rPT-S1 . Utilization of live oral vaccines expressing B . pertussis antigens, which stimulate both a systemic and lung mucosal response, may provide an attractive alternative to purified component vaccines against whooping cough.

Infect Immun, 1992 Oct, 60(10), 3994 - 4002
Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis; Strugnell R et al.; The P.69 Bordetella pertussis protective antigen was expressed by use of the trc promoter from the chromosome of a Salmonella typhimurium aro vaccine strain, BRD509, by integrating the prn gene, encoding the 93-kDa precursor of this protein, into the aroC locus . P.69 was detected on the cell surface of the S . typhimurium strain (BRD640) by agglutination and immunoelectron microscopy . BALB/c mice immunized orally or intravenously with BRD640 showed a significant level of protection against an aerosol challenge with virulent B . pertussis, compared with control animals . No anti-P.69 antibodies in the serum or anti-P.69 antibody-secreting cells in the lungs were detected in BRD640-vaccinated animals, although cells isolated from spleens showed a P.69-dependent cell proliferative response . In contrast, low levels of anti-P.69 antibodies in the serum and anti-P.69 antibody-secreting cells in the lungs were detected in immunized mice following a B . pertussis challenge.

Mutat Res, 1992 Oct, 272(2), 183 - 92
Use of a newly developed tester strain Salmonella typhimurium NM2009 for the study of metabolic activation of carcinogenic aromatic amines by rat liver microsomal cytochrome P-450 enzymes; Yamazaki H et al.; Using an O-acetyltransferase-overexpressing strain Salmonella typhimurium NM2009 we measured the activities for metabolic activation of several carcinogenic arylamines to genotoxic products by rat liver microsomal cytochrome P-450 enzymes, and compared them with the activities obtained in the original tester strain Salmonella typhimurium TA1535/pSK1002 or the O-acetyltransferase-defective strain Salmonella typhimurium NM2000 . Since all of the tester strains had introduced the umuC'-'lacZ gene, we could detect the genotoxic activities by measuring bacterial beta-galactosidase activity resulting from the DNA damage . In the O-acetyltransferase-defective strain NM2000 most of the arylamines tested showed weak responses in inducing umu gene expression after metabolic activation by liver microsomes . The strain NM2009, on the other hand, was found to be highly sensitive towards a variety of aromatic amines, and these activities were greater than those seen in the original tester strain S . typhimurium TA1535/pSK1002 . The chemicals which marked responses in strain NM2009 include 2-aminoanthracene, 6-aminochrysene, 2-aminofluorene, 2-acetylaminofluorene, 3-methoxy-4-aminoazobenzene, O-aminoazotoluene, Glu-P-1, Trp-P-2, A alpha C, MeA alpha C, MeIQ, MeIQx and IQ . Of these procarcinogens tested MeIQ, MeIQx and IQ also showed strong cytotoxic effects in S . typhimurium NM2009 after metabolic activation by liver microsomes . Only PhIP was the substrate showing similar responses in strains TA1535/pSK1002 and NM2009 . The results with the reconstituted monooxygenase system containing purified cytochrome P-450 enzymes support the above findings obtained with the liver microsomal enzyme system . Thus, the usefulness of the newly developed strain NM2009 for the detection of reactive metabolites of several carcinogenic aromatic amines after metabolism by the liver microsomal cytochrome P-450-linked monooxygenase system has been ascertained.

Mutat Res, 1992 Oct, 269(2), 279 - 84
Inhibition of the metabolism of mutagens occurring in food by arachidonic acid; Ho TA et al.; Hepatic microsomal fractions (microsomes) were prepared from male Sprague-Dawley rats . The effect of arachidonic acid on the conversion of the heterocyclic aromatic amine 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay (indicator: Salmonella typhimurium TA98) . Arachidonic acid inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA-repair processes within the bacterial cell . The activation of 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by this polyunsaturated fatty acid.

Mutat Res, 1992 Oct, 269(2), 269 - 78
Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1; Decoudu S et al.; The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear . In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis . We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo . Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes . Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks . The AFB1-treated rats were injected i.p . with 1 mg/kg body weight . In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups . However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals . For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively) . DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test . These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo . Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo . However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism . Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.

Mutat Res, 1992 Oct, 269(2), 231 - 6
Mutagenic activation of aflatoxin B1 by pulmonary, renal, and hepatic cytochrome P450s from rats; Imaoka S et al.; The genotoxic and mutagenic activation of aflatoxin B1 (AFB1) by hepatic, renal, and pulmonary microsomes and purified cytochrome P450s was investigated in Salmonella typhimurium TA1535/pSK1002 cells in which an umu response shows DNA damage . The activity of the hepatic microsomes was greatest . Pulmonary microsomes had moderate activity and renal microsomes had low activity . P450 2C11, 2B1, 3A2, 4A2, 4B1, K-2, and K-4 were assayed in a reconstituted system with dilauroylphosphatidylcholine (DLPC) . P450 2C11 (a major hepatic cytochrome P450 in male rats) had high activity . P450 2B1 (a major form as well as P450 4B1 in pulmonary microsomes) and K-2 (a minor form in renal microsomes) had moderate activity . P450 4A2 (a major form in renal microsomes), P450 K-4 (a renal form), and P450 4B1 had low activity . P450 3A2 did not have high activity in these conditions but it had high activity toward AFB1 in a modified reconstituted system with a lipid mixture and sodium cholate instead of DLPC only . The activities of other forms were not enhanced by the modification of reconstituted system . Anti-P450 2C11 or 3A2 antibodies inhibited the bioactivation of AFB1 by hepatic microsomes to 50% . These results suggest that the greater ability of hepatic microsomes as compared with pulmonary and renal microsomes to metabolize AFB1 to mutagenic products is a function of the relative proportions of the highly active cytochrome P450s, P450 2C11 and 3A2, in the liver.

Mutat Res, 1992 Oct, 269(2), 217 - 24
Enhancement of the mutagenicity of polyphenols by chlorination and nitrosation in Salmonella typhimurium; Lin JK et al.; The hydrolytic products of lignins, humic acids and industrial waste including hydroquinone, catechol, resorcinol, pyrogallol and 1,2,4-benzenetriol are widely distributed in water sources . These polyphenols can interact with chlorine or nitrite to yield new derivatives . Generally, these new products possess more mutagenic potential than their original compounds . Furthermore, the mutagenicity of these polyphenols and their derivatives can be dramatically reduced by rodent liver microsomal enzymes (S9) . The mutagenicity of polyphenols is in this order: hydroquinone greater than 1,2,4-benzenetriol greater than pyrogallol, while catechol, resorcinol and phloroglucinol are non-mutagenic . The ultimate product of chlorination or nitrosation of hydroquinone has been identified to be p-benzoquinone . The formation of active oxygen species including superoxide anion and hydrogen peroxide by polyphenols has been demonstrated and this may contribute partly to the molecular mechanisms of polyphenol mutagenicity.

Clin Exp Immunol, 1992 Oct, 90(1), 63 - 7
The role of O-antigen polysaccharide in the activation of neutrophils by lipopolysaccharides of Salmonella species; Rasool O et al.; Activation of neutrophils by lipid A, O-antigen polysaccharides (PS) and smooth lipopolysaccharides (LPS) isolated from Salmonella choleraesuis (O-6,7) and Salmonella typhimurium (O-4,5,12) was investigated . The methods used were assays for lysozyme release and for nitroblue tetrazolium (NBT) reduction which measures the level of oxidative metabolism of neutrophils . LPS from both species stimulated neutrophils to the same extent in the presence of autologous plasma . In the absence of plasma only the O-6,7 LPS activated neutrophils . Lipid A or PS isolated from both LPS either did not activate neutrophils or did so only at very high concentrations when tested in the presence of plasma; in the absence of plasma no activation occurred . The data indicate that both PS and lipid A segments of LPS are required for activation of neutrophils by LPS . We also deduce that plasma, probably complement, is required for the interaction of some LPS, e.g . O-4,5,12 with neutrophils whereas other LPS, e.g . O-6,7 can interact directly and activate neutrophils.

Mutat Res, 1992 Oct, 280(4), 233 - 44
Mutagenic activities of azido analogues of amsacrine and other 9-anilinoacridines in Salmonella typhimurium and their enhancement by photoirradiation; Iwamoto Y et al.; Analogues of amsacrine and other 9-anilinoacridines, bearing azide groups at various positions, were tested for their mutagenic activity in five strains of Salmonella typhimurium, both before and after photoirradiation . Azido substitution at the 2- or 3-position of the acridine or the 1'-position of the aniline led to compounds which were active frameshift mutagens, as detected in strain TA1537 . Photoirradiation enhanced both the mutagenicity and the cytotoxicity of the azido compounds . Analogues bearing two azido groups at either the 2,6- or 3,6-positions were less strongly mutagenic in the dark, and light activation led to a toxic but only weakly mutagenic product . The effects of photoirradiation were decreased by aniline ring substitution, and were essentially eliminated by additional methyl substitution in the acridine ring . Comparison of events in TA1537 and TA98 suggested that photoirradiation of the 2- or 3-azido compounds gave a product which was capable of forming covalent bonds with DNA . The azide-containing analogues readily formed single strand DNA breaks on irradiation in the presence of DNA, but the efficiency of this reaction varied considerably.

Mutat Res, 1992 Oct, 280(4), 225 - 31
Structure-mutagenicity relationships in a series of indolo{3,2-c}quinoline-1,4-diones that have shown cytotoxic properties on leukemia cells; Min S et al.; A series of seven 6-methylindolo{3,2-c}quinoline-1,4-diones substituted either in the 2 position or in 3 position by various groups were examined for their ability to induce mutation in the Ames test at several concentrations in four strains of Salmonella typhimurium (TA97, TA98, TA100, and TA102) . First, relationships were established between their mutagenic activities and either the nature or the position of the substituent on the quinonic nucleus . Compounds substituted in the 2 position were less mutagenic than the 3 isomers . In the second study, the mutagenic properties were compared to the in vitro antitumor activity . Interestingly, some very cytotoxic quinones were only weak mutagens . So where the cytotoxicity is similar, the less mutagenic compounds may be suitable for clinical use as antitumor drugs, in order to avoid important side effects; the Ames test can then be used guide the selection of molecules for further in vivo antitumor screening . It can also be very helpful in selecting the best candidate molecules to be synthesized.

Mutat Res, 1992 Oct, 283(2), 145 - 56
Mechanism of mutagenicity by 5-hydroperoxymethyl-2'-deoxyuridine, an intermediate product of ionizing radiation, in bacteria . HPMdU bacterial mutagenicity and oxidation of DNA bases; Patel U et al.; The specific objective was to find what processes are responsible for the mutagenicity of 5-hydroperoxymethyl-2'-deoxyuridine (HPMdU), which is a product of ionizing radiation, and what role transition metal ions play in those processes . We found that HPMdU is a more potent mutagen than its decomposition products 5-hydroxymethyl-2'-deoxyuridine (HMdU) and 5-formyl-2'-deoxyuridine (FdU) in the Salmonella typhimurium strains tested, with the TA100 strain being the most sensitive . HMdU exerted intermediate mutagenicity and FdU was the weakest of the three compounds . At 50 nmoles/plate, HPMdU increased the number of revertants by 4-fold, whereas 1000 nmoles HMdU was required to enhance the number of revertants by 5-fold . Pretreatment of TA100 with o-phenanthroline, a membrane-permeable Fe and Cu chelator, caused an increase in mutagenicity of the low HPMdU doses but inhibited that of the 50 nmoles HPMdU/plate, while desferal, a membrane-impermeable Fe chelator, had virtually no effect . Azide (a catalase inhibitor) enhanced HPMdU mutagenicity, whereas 3-amino-1,2,4-triazole (a catalase and peroxidase inhibitor) and ammonium formate (a hydroxyl radical scavenger) were protective . Preincubation of TA100 cells with 20 and 40 nM HPMdU caused dose-dependent formation of the oxidized DNA base derivatives HMdU, thymidine glycol and 8-hydroxyl-2'-deoxyguanosine (8-OHdG), known hydroxyl radical-mediated oxidation products . Cumulatively, these results suggest that the genetic effects of HPMdU are due to its hydroperoxide moiety, which upon reacting with Fe generates hydroxyl radicals that in turn oxidize neighboring bases in cellular DNA . This also may be a mechanism by which ionizing radiation exerts its long-term effects.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2093 - 100
Sequences of the envM gene and of two mutated alleles in Escherichia coli; Bergler H et al.; The nucleotide sequence of the Escherichia coli envM gene was determined . It codes for a protein of 262 amino acids . The sequences of the E . coli and Salmonella typhimurium EnvM proteins are 98% identical . Gene envM is preceded in E . coli by a 43-nucleotide-long structural element, termed 'box c', which occurs in several E . coli operons between structural genes . This sequence element is totally absent in S . typhimurium . Gene envM was mapped at coordinate position 1366.8 kb of the physical map of Kohara et al . (Cell, 1987, 50, 495-508) . As in S . typhimurium, a Gly for Ser exchange at position 93 of the amino acid sequence leads to a diazaborine-resistant E . coli phenotype . A Ser for Phe exchange at position 241 of the EnvM protein results in a temperature-sensitive growth phenotype . Comparison of the EnvM amino acid sequence with sequences available in databases showed significant homology with the family of short-chain alcohol dehydrogenases.

Indian J Pathol Microbiol, 1992 Oct, 35(4), 345 - 50
Present phage types and antibiotic susceptibility of salmonellae; Chopra GS et al.; A total of 168 strains of Salmonella were isolated in the Command Pathology Laboratory (WC) Delhi Cantt during the year 1990 . Out of this, 143 were Salmonella typhi, 17 Salmonella paratyphi A, 7 Salmonella typhimurium and 1 Salmonella manhattan . The commonest phage type and biotype of Salmonella typhi was type E1 and type 1 respectively . The dominant biotype of Salmonella paratyphi A was type I . There was a very high degree of multidrug resistance of most of the strains . But all the strains were sensitive to ciprofloxacin and norfloxacin.

Microb Pathog, 1992 Oct, 13(4), 317 - 23
Mutant of Salmonella typhimurium lacking the inhibitory function for phagosome-lysosome fusion in murine macrophages; Ishibashi Y et al.; It has recently been described that Salmonella typhimurium is capable of inhibiting phagosome-lysosome fusion in murine macrophages after ingestion . We selected a mutant of S . typhimurium lacking the phagosome-lysosome fusion inhibitory function from a collection of Tn5-insertion mutants and examined its relevance to the pathogenesis in mice . The Tn5 insertion mutant which has a defect in fusion inhibitory function was found to be significantly sensitive to the intracellular killing by murine macrophages in vitro . However, the loss of the fusion inhibitory function did not reduce the level of virulence for mice in vivo . These results demonstrated that fusion inhibition did not play a critical role in the pathogenesis of S . typhimurium although it might contribute to at least a part of the resistance against macrophage killing mechanisms.

J Bacteriol, 1992 Oct, 174(20), 6634 - 43
Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium; Roof DM et al.; The genes required for use of ethanolamine as a carbon and nitrogen source are encoded by a single operon (eut) whose expression is induced by the simultaneous presence of both ethanolamine and cobalamin (vitamin B12) . The action of B12 as an inducer of this operon reflects the fact that this cofactor is required by the degradative enzyme ethanolamine lyase (eutBC) . The eutR gene encodes a protein that activates transcription of the eut operon in response to the simultaneous presence of B12 and ethanolamine . The eutR gene is expressed by a weak constitutive promoter activity (PII) and by the main regulated promoter (PI) . Because it is encoded within the operon that it activates, the EutR protein controls its own production . Initial induction of the eut operon by ethanolamine plus B12 causes an increase in expression of the eutR gene; this increase acts as part of a positive feedback loop that is required for maximal operon expression . Because of this mode of regulation, constitutive regulatory mutations, described here, include mutations that generate new internal promoters and thereby increase the basal level of eutR gene expression . In mutants with an increased level of activator protein, each inducer (B12 or ethanolamine), presented singly, is sufficient for partial operon induction.

Tohoku J Exp Med, 1992 Oct, 168(2), 119 - 22
Role of intestinal microflora in metabolism of glutathione conjugates of 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide; Kinouchi T et al.; DNA adduct formation in the liver of B6C3F1 mice after administration of 1-nitropyrene (1-NP) was shown by the 32P-postlabeling technique . The major adduct was not N-(deoxyguanosin-8-yl)-1-aminopyrene, which was easily formed in in vitro nitroreduction of 1-NP in the presence of DNA, but the major spots migrated to the same position as the in vitro DNA adduct spots of K-region epoxides of 1-NP (1-NP 4,5- and 9,10-oxide) . 1-NP oxides formed by the oxidative activation of 1-NP in the liver were excreted into the bile as detoxified glutathione conjugates which were changed to cysteine conjugates in the upper intestinal tract . The cysteine conjugates were degraded by cysteine conjugate beta-lyase (beta-lyase) of intestinal microflora in the lower intestinal tract . The mutagenicity of cysteine conjugates of 1-NP oxides for Salmonella typhimurium was enhanced by addition of beta-lyase and was decreased by addition of aminooxyacetic acid, a beta-lyase inhibitor . The in vitro binding of the cysteine conjugates to calf thymus DNA was increased by addition of beta-lyase and xanthine oxidase . We administered glutathione conjugates of 1-NP oxides to two groups of mice that had been treated with antibiotics or saline by gavage and analyzed the DNA adducts in the lower intestinal mucosa . The specific DNA adducts were detected in the saline-treated group but not in the antibiotics-treated group . These results suggest that intestinal microflora play an important role in activation of glutathione conjugates of 1-NP oxides.

Microb Pathog, 1992 Oct, 13(4), 305 - 15
Proliferative and T-cell specific interleukin (IL-2/IL-4) production responses in spleen cells from mice vaccinated with aroA live attenuated Salmonella vaccines; Villarreal B et al.; T-cell responses were studied in mice immunized with the Salmonella typhimurium aroA SL3261 live attenuated vaccine strain . T-cell responses in the spleen, both in whole cell populations and in nylon wool non-adherent (T-cell enriched) cells, were studied in vitro as proliferation by incorporation of tritiated thymidine and production of T-cell specific cytokines {IL-2 (interleukin-2)/IL-4} . Stimulating antigens included whole Salmonella lysates and purified lipopolysaccharide (LPS), both untreated and after alkaline hydrolysis to prevent the non-specific mitogenic effect of LPS . Strong proliferative responses were obtained with untreated whole cell extract and LPS, which were decreased by polymyxin B (PB) . Alkaline detoxification of the antigens decreased the proliferative response of nylon-wool non-adherent populations to LPS, but greatly increased their response to the Salmonella extract . Surprisingly, PB also reduced proliferation to detoxified LPS . Little or no IL-2/IL-4 production was seen in response to LPS or purified polysaccharide antigens, while there was a strong IL-2/IL-4 response to whole cell lysate, again markedly increasing after alkaline treatment . The results suggest that the T-cell response elicited by immunization with live Salmonella aroA vaccines in mice recognizes antigens other than LPS determinants, and that estimation of T-cell responses to Salmonella antigens by proliferation alone may yield misleading results.

Cancer Lett, 1992 Sep 30, 66(2), 107 - 13
Antimutagenic effects of polyphenolic compounds; Teel RW et al.; Smokers expose themselves to potent carcinogens daily . One of them is the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) . Since estimates are that humans consume 1 g of phenolic compounds/day, we investigated the inhibitory effects of five structurally related polyphenolic compounds on the mutagenicity of NNK in Salmonella typhimurium TA1535 . NNK at a concentration of 80 mM was activated by hamster liver microsomes . The antimutagenic efficacies were dose-related between the non-toxic concentrations of 0.1 and 0.5 mmol/dish in the following order: esculetin > ellagic acid > (+)-catechin > propyl gallate > (-)esculin . At the highest non-toxic dose tested (0.5 mmol/dish), these polyphenolics inhibited mutagenesis in TA1535 by 77%, 67%, 62%, 59% and 53%, respectively . The results of this study demonstrated that polyphenolic compounds may inhibit the activation of NNK.

J Mol Biol, 1992 Sep 20, 227(2), 418 - 40
Ribose and glucose-galactose receptors . Competitors in bacterial chemotaxis; Mowbray SL; The periplasmic ribose and glucose-galactose receptors (binding proteins) of Gram-negative bacteria compete for a common inner membrane receptor in bacterial chemotaxis, as well as being the essential primary receptors for their respective membrane transport systems . The high-resolution structures of the periplasmic receptors for ribose (from Escherichia coli) and glucose or galactose (from both Salmonella typhimurium and E . coli) are compared here to outline some features that may be important in their dual functions . The overall structure of each protein consists of two similar domains, both of which are made up of two non-contiguous segments of amino acid chain . Each domain is composed of a core of beta-sheet flanked on both sides with alpha-helices . The two domains are related to each other by an almost perfect intramolecular axis of symmetry . The ribose receptor is smaller as a result of a number of deletions in its sequence relative to the glucose-galactose receptor, mostly occurring in the loop regions; as a result, this protein is also more symmetrical . Many structural features, including some hydrophobic core interactions, a buried aspartate residue and several unusual turns, are conserved between the two proteins . The binding sites for ligand are in similar locations, and built along similar principles, although none of the specific interactions with the sugars is conserved . A comparison shows further that slightly different rotations relate the domains to each other in the three proteins, with the ribose receptor being the most closed, and the Salmonella glucose-galactose receptor the most open . The primary axis of relative rotation is almost perpendicular to that which describes the intramolecular symmetry in each case . These relative rotations of the domains are accompanied by the sliding of some helices as the structures adjust themselves to relieve strain . The hinges which are responsible for most of these relative domain rotations are very similar in the three proteins, consisting of a symmetrical arrangement of beta-strands and alpha-helices and two conserved water molecules that are critical to the hydrogen bonding in the important interdomain region . A region of high sequence and structural similarity between the ribose and glucose-galactose receptors is also located around the intramolecular symmetry axis, on the opposite side of the proteins from the hinge region . This region is that which is altered most by the relative rotations, and is the location of most of the known mutations which affect chemotaxis and transport in the ribose receptor.

J Biol Chem, 1992 Sep 15, 267(26), 18874 - 84
The rfaC gene of Salmonella typhimurium . Cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide; Sirisena DM et al.; We have cloned a gene from a Salmonella typhimurium with the ability to complement the rfaC mutation (heptose-deficient lipopolysaccharide, sensitivity to rough-specific bacteriophages, and susceptibility to hydrophobic antibiotics) . A 1018-base pair EcoRV-Tth111I fragment, subcloned into the pBluescriptKS+ vector to yield pKZ103, retains complementing activity . Nucleotide sequencing revealed an open reading frame corresponding to a protein of 317 amino acids (M(r) approximately 35,100) . The plasmid pKZ103, which has a properly aligned T7 promoter, can overexpress a protein of M(r) = 31,000 when T7 RNA polymerase is supplied . An in vitro system was established for analysis of heptose addition to the precursor {4'-32P}(KDO)2-IVA (Brozek, K . A., Hosaka, K., Robertson, A . D., and Raetz, C . R . H . (1989) J . Biol . Chem, 264, 6956-6966) . Soluble fractions from wild-type or heptose-deficient rfa mutants were tested for their ability to convert {4'-32P}(KDO)2-IVA to more polar substances . In wild-type extracts, these conversions required addition of ATP or ADP-heptose . In extracts of rfaC-, rfaD-, or rfaE-deficient strains, no polar products were observed with ATP . ADP-heptose restored synthesis in rfaD and rfaE but not rfaC extracts, indicating that rfaD and rfaE are involved in ADP-heptose formation . When the cloned rfaC gene was introduced into an rfaC-deficient mutant, extracts from such cells regained the ability to metabolize {4'-32P}(KDO)2-IVA, showing that rfaC encodes the enzyme that attaches the proximal heptose to lipopolysaccharide.

J Biol Chem, 1992 Sep 15, 267(26), 18336 - 41
Resistance of the melibiose carrier to inhibition by the phosphotransferase system due to substitutions of amino acid residues in the carrier of Salmonella typhimurium; Kuroda M et al.; The melibiose carrier of Salmonella typhimurium is under the control of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) . We isolated mutants of the melibiose carrier that showed resistance to inhibition via the PTS . Growth of the mutants on melibiose was not inhibited by 2-deoxyglucose, a non-metabolizable substrate of the PTS, although growth of the parent strain was inhibited . Transport activity of the melibiose carrier in the mutants was fairly resistant to inhibition by 2-deoxyglucose, although the activity in the parent was sensitive to inhibition . We cloned the mutated melB gene that encodes the melibiose carrier, determined the nucleotide sequences, and identified replaced nucleotides . The mutations resulted in substitutions of Asp-438 with Tyr, Arg-441 with Ser, or Ile-445 with Asn . All of these residues are in the COOH-terminal region of the carrier . The secondary structure of this region is predicted to be an alpha-helix, and the mutated residues were on the same side of the helix . This region showed sequence similarity to a region of the MalK protein, in which substitution of amino acid residues also resulted in PTS-resistant mutants . Thus the COOH-terminal portion of the melibiose carrier is important for the interaction of dephosphorylated IIIGlc, which is an entity causing reversible inactivation of the carrier.

Biochim Biophys Acta, 1992 Sep 15, 1117(2), 216 - 22
Biological inactivation by singlet oxygen: distinguishing O2(1 delta g) and O2(1 sigma g+)
Midden WR, Dahl TA.
Experiments were performed to determine whether bacterial inactivation in the separated-surface-sensitizer system for singlet oxygen generation is due to O2(1 delta g) or O2(1 sigma g+) . The rates of inactivation of Gram-negative Salmonella typhimurium LT-2 and a nonpigmented strain of Gram-positive Sarcina lutea were found to increase linearly with the concentration of 1 delta g . The gas phase lifetime of the inactivating agent was found to be within the range of values expected for the gas phase lifetime of 1 delta g rather than 1 sigma g+ . These measurements conclusively demonstrate that bacterial inactivation in this system is due predominantly to 1 delta g . Therefore, studies of bacterial inactivation with this singlet oxygen generating system can be used to assess the role of singlet oxygen in various biological and medically relevant situations.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 149 - 54
Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli; Lamark T et al.; We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli . Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake . Our data suggest that E . coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites . The ProU system of E . coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8784 - 8
Activity of a plasmid-borne leu-500 promoter depends on the transcription and translation of an adjacent gene; Chen D et al.; leu-500 is a chromosomal promoter mutation in Salmonella typhimurium that normally causes the promoter to be inactive in the initiation of RNA synthesis . But in a strain that has mutations in topA, the gene encoding DNA topoisomerase I, the mutant promoter becomes active . We show that the leu-500 promoter can function on a plasmid when it is adjacent to the tetracycline-resistance gene tetA . Activation of the leu-500 promoter requires that the tetA gene is transcribed and translated and that the host cell is topA . We propose that the A----G mutation in the -10 region of the leu-500 promoter is compensated by local negative supercoiling arising from transcription of the tetA gene, which may reach elevated levels in a topA background, provided that diffusional dissipation is reduced due to anchoring of the TetA peptide in the membrane . This is a clear example of the modulation of the activity of a promoter by the activity of another promoter in cis, when they can be coupled through the topology of the template.

Eur J Immunol, 1992 Sep, 22(9), 2249 - 54
Immunosuppression induced by nitric oxide and its inhibition by interleukin-4; al-Ramadi BK et al.; Mice immunized with attenuated Salmonella typhimurium, strain SL3235, while protected against virulent challenge, are unable to mount in vivo and in vitro antibody responses to non-Salmonella antigens, such as tetanus toxoid and sheep red blood cells, and exhibit profoundly suppressed responses to B and T cell mitogens . Suppression of antibody responses is mediated by macrophage (M phi)-released soluble factors, and is completely reversed by treatment with interleukin (IL)-4 . The present report identifies the suppressor factor as nitric oxide (NO), and provides evidence for a mechanism by which IL-4 abrogates suppression . Suppressed antibody responses correlated with high levels of NO secretion by splenocytes of SL3235-immunized mice . NO production was observed only in cultures consisting of the adherent cell fraction of immune splenocytes . Further, immunosuppression was reversed by NG-monomethyl-L-arginine (NMLA), a competitive inhibitor of NO synthesis, and was completely blocked by the addition of excess L-arginine . Treatment with IL-4, or anti-interferon (IFN)-gamma monoclonal antibody (mAb), also abrogated suppression . Optimal reversal of suppression was observed only when NMLA, IL-4, or anti-IFN-gamma mAb, was added at day 0 of the 5-day plaque-forming cell assay . Treatment with either IL-4 or anti-IFN-gamma mAb also lead to a sharp inhibition of NO production by immune spleen cells . Moreover, the addition of IL-4 to splenic adherent M phi inhibited their ability to generate NO . Our data characterize an immunoregulatory pathway, involving IFN-gamma and NO, by which M phi mediate immunosuppression and identify IL-4 as a potent inhibitor of this pathway.

Poult Sci, 1992 Sep, 71(9), 1464 - 70
Fermentation of {14C}lactose in broiler chicks by cecal anaerobes; Hume ME et al.; Volatile fatty acids and lactic acid were products of {14C}lactose metabolism in 10-day-old broiler chicks . The {14C}lactose and unlabeled lactose were given to chicks in combination with cultures of cecal anaerobes (direct fed microbials) from adult broiler chickens . Aliquots of these cecal anaerobes had been used previously to reduce Salmonella typhimurium colonization in broiler chicks . In the current study, chicks were treated with anaerobes and anaerobes plus lactose on the day of hatch . Chicks given lactose plus anaerobes also received unlabeled lactose in the drinking water . Chicks in all treatment groups received {14C}lactose on Day 10 posthatch . After 5 h, most of the radiolabel was detected in the foregut and intestine . Significantly lower concentrations of {14C}lactic acid were detected in the serum and foregut of chicks given anaerobes and anaerobes plus lactose than in the similar samples from control chicks . Additionally, significantly less {14C}lactic acid was detected in the feces of chicks treated with anaerobes plus lactose than in feces from control chicks and chicks receiving only anaerobes . Significant differences in the total content of volatile fatty acids and lactic acid were detected in the serum, liver, intestine, and ceca of chicks treated with anaerobes or anaerobes plus lactose . These data indicate that cecal flora utilize lactose to produce volatile fatty acids and lactic acid.

Microb Pathog, 1992 Sep, 13(3), 181 - 90
A 'safe-site' for Salmonella typhimurium is within splenic polymorphonuclear cells; Dunlap NE et al.; Following oral or systemic infection with Salmonella typhimurium, the focus of infection is in the liver and spleen . The majority of Salmonella surviving in the liver and spleen by 4 h post infection are already in an environment where they are largely protected from subsequent killing . Previous studies have shown that the majority of surviving Salmonella are intracellular . In the present study we sought to determine the cell type containing most of the cell-associated Salmonella liberated from the spleen . We enriched for Salmonella-containing cells by Ficoll-Hypaque separation followed by fluorescence-activated cell sorting . Approximately 85% of the total intracellular Salmonella were found in Mac-1+/J-11d+ cell fractions of the Ficoll-Hypaque band and pellet . By microscopic examination of stained cells from the sorted cell populations, it was evident that virtually all of the Salmonella were in polymorphonuclear cells (PMN) . The numbers of Salmonella observed microscopically were similar in numbers to Salmonella colony forming units detected by plating . Salmonella containing PMN in the Ficoll band generally contained a single bacterium, while those from the probably less healthy cells in the Ficoll pellet generally contained several Salmonella.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4423 - 8
Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure; Pel HJ et al.; We have recently reported the cloning and sequencing of the gene for the mitochondrial release factor mRF-1 . mRF-1 displays high sequence similarity to the bacterial release factors RF-1 and RF-2 . A database search for proteins resembling these three factors revealed high similarities to two amino acid sequences deduced from unassigned genomic reading frames in Escherichia coli and Bacillus subtilis . The amino acid sequence derived from the Bacillus reading frame is 47% identical to E.coli and Salmonella typhimurium RF-2, strongly suggesting that it represents B.subtilis RF-2 . Our comparison suggests that the expression of the B.subtilis gene is, like that of the E.coli and S . typhimurium RF-2 genes, autoregulated by a stop codon dependent +1 frameshift . A comparison of prokaryotic and mitochondrial release factor sequences, including the putative B.subtilis RF-2, leads us to propose a five-domain model for release factor structure . Possible functions of the various domains are discussed.

Biochem Pharmacol, 1992 Sep 1, 44(5), 913 - 20
Activation of 6-aminochrysene to genotoxic products by different forms of rat liver cytochrome P450 in an O-acetyltransferase-overexpressing Salmonella typhimurium strain (NM2009); Yamazaki H et al.; Metabolic activation of a potent mutagen, 6-aminochrysene, to genotoxic products in a newly developed tester strain, Salmonella typhimurium NM2009, was studied in a rat liver microsomal monooxygenase system containing cytochrome P450 (P450) . Since the tester strain was constructed by introducing an O-acetyltransferase gene into the original strain S . typhimurium TA1535/pSK1002, it is highly sensitive toward the reactive metabolites of carcinogenic arylamines . DNA-damaging activities of 6-aminochrysene were detected at very low concentrations of substrate (between 0.01 and 0.2 microM) and liver microsomes (from 0.2 to 2 micrograms protein/mL) in the S . typhimurium NM2009 strain . Thus, the potency of genotoxic activities induced by 6-aminochrysene was about 10- to 20-times greater than those induced by the well-known mutagens 2-aminoanthracene and 2-amino-3,5-dimethylimidazo{4,5-f}quinoline . Liver microsomes isolated from rats treated with phenobarbital (PB) and a polychlorinated biphenyl mixture, Kanechlor 500, catalyzed very efficiently the activation of 6-aminochrysene to genotoxic metabolites . Treatment of rats with beta-naphthoflavone (BNF) and with dexamethasone also caused moderate induction of the microsomal activation of 6-aminochrysene . Studies employing immunoinhibition of microsomal catalytic activities and reconstitution with purified P450 enzymes suggested that the most important enzymes involved in the activation of 6-aminochrysene were P450 2B1 and 2B2; other enzymes including P450 1A1 and 1A2 participated to some extent . We also found that the microsomal activation of 6-aminochrysene was catalyzed more effectively in an acetyltransferase-overexpressing strain (NM2009) than in the original TA1535/pSK1002 strain and that these activities could be inhibited by an acetyltransferase inhibitor, pentachlorophenol, in liver microsomes from PB-treated rats, but not in those from BNF-treated rats . These results suggest that the P450/acetyltransferase system is one of the most important catalysts for the activation of 6-aminochrysene in liver microsomes of PB-treated rats, and that activation by BNF-induced P450 enzymes occurs by different mechanisms, probably through the ring oxidation pathway.

J Clin Invest, 1992 Sep, 90(3), 953 - 64
Mechanism of resistance to complement-mediated killing of bacteria encoded by the Salmonella typhimurium virulence plasmid gene rck; Heffernan EJ et al.; We find that pADEO16, a recombinant cosmid carrying the rck gene of the Salmonella typhimurium virulence plasmid, when cloned into either rough or smooth Escherichia coli and Salmonella strains, confers high level resistance to the bactericidal activity of pooled normal human serum . The rck gene encodes a 17-kD outer membrane protein that is homologous to a family of virulence-associated outer membrane proteins, including pagC and Ail . Complement depletion, C3 and C5 binding, and membrane-bound C3 cleavage products are similar in strains with and without rck . Although a large difference in C9 binding was not seen, trypsin cleaved 55.7% of bound 125I-C9 counts from rough S . typhimurium with pADEO16, whereas only 26.4% were released from S . typhimurium with K2011, containing a mutation in rck . The majority of C9 extracted from rck strain membranes sediments at a lower molecular weight than in strains without rck, suggesting less C9 polymerization . Furthermore, SDS-PAGE analysis of gradient peak fractions indicated that the slower sedimenting C9-containing complexes in rck strains did not contain polymerized C9 typical of the tubular membrane attack complex . These results indicate that complement resistance mediated by Rck is associated with a failure to form fully polymerized tubular membrane attack complexes.

J Bacteriol, 1992 Sep, 174(18), 5869 - 80
Oligopeptidase A is required for normal phage P22 development; Conlin CA et al.; The opdA gene of Salmonella typhimurium encodes an endoprotease, oligopeptidase A (OpdA) . Strains carrying opdA mutations were deficient as hosts for phage P22 . P22 and the closely related phages L and A3 formed tiny plaques on an opdA host . Salmonella phages 9NA, KB1, and ES18.h1 were not affected by opdA mutations . Although opdA strains displayed normal doubling times and were infected by P22 as efficiently as opdA+ strains, the burst size of infectious particles from an opdA host was less than 1/10 of that from an opdA+ host . This decrease resulted from a reduced efficiency of plating of particles from an opdA infection . In the absence of a functional opdA gene, most of the P22 particles are defective . To identify the target of OpdA action, P22 mutants which formed plaques larger than wild-type plaques on an opdA mutant lawn were isolated . Marker rescue experiments using cloned fragments of P22 DNA localized these mutations to a 1-kb fragment . The nucleotide sequence of this fragment and a contiguous region (including all of both P22 gene 7 and gene 14) was determined . The mutations leading to opdA independence affected the region of gene 7 coding for the amino terminus of gp7, a protein required for DNA injection by the phage . Comparison of the nucleotide sequence with the N-terminal amino acid sequence of gp7 suggested that a 20-amino-acid peptide is removed from gp7 during phage development . Further experiments showed that this processing was opdA dependent and rapid (half-life, less than 2 min) and occurred in the absence of other phage proteins . The opdA-independent mutations lead to mutant forms of gp7 which function without processing.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8078 - 82
Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase; Saito K et al.; Cysteine synthase (CSase) {O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8} catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide . We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.) . Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase . A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach . Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones . Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da . Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium . Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450 . A bacterial expression vector was constructed and could genetically complement an E . coli auxotroph that lacks CSases . The accumulation of functionally active spinach CSase in E . coli was also demonstrated by immunoblotting and assaying enzymatic activity . Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach . RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.

Gene, 1992 Sep 1, 118(1), 13 - 9
Integration host factor facilitates repression of the put operon in Salmonella typhimurium; O'Brien K et al.; Transcriptional regulation of the put operon is mediated by a unique mechanism involving autogenous regulation by the PutA protein, a membrane-associated dehydrogenase . The 420-bp put control region contains the putP and putA promoters, multiple operator sites, multiple catabolite repression protein binding sites, and several potential integration host factor (IHF)-binding sites (ihf) . In this study, we show that IHF facilitates repression of the put operon in vivo, and IHF binds specifically to two ihf sites in the put control region in vitro . DNA gyrase mutants that alter the degree of chromosomal supercoiling do not affect put regulation, indicating that the effect of IHF on put expression is in this case independent of supercoiling.

Biochemistry, 1992 Sep 1, 31(34), 7807 - 14
Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium; Haeffner-Gormley L et al.; Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site {Haeffner-Gormley et al . (1991) J . Biol . Chem . 266, 5388-5394} . The present study was undertaken to evaluate the role of lysine-286 . The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified . The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes . As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes . The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced . The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH . Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations . A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1992 Sep, 60(9), 3780 - 9
Recombinant Salmonella typhimurium strains that invade nonphagocytic cells are resistant to recognition by antigen-specific cytotoxic T lymphocytes; Gao XM et al.; To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made . The bacteria were shown to infect Chinese hamster ovary (CHO) cells . Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S . typhimurium and used as targets for NP-specific CTL . The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used . Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition . In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses . The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells.

Infect Immun, 1992 Sep, 60(9), 3556 - 65
Cryptdins: antimicrobial defensins of the murine small intestine; Eisenhauer PB et al.; Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria . Recent studies by Ouellette and associates (A . J . Ouellette, R . M . Greco, M . James, D . Frederick, J . Naftilan, and J . T . Fallon, J . Cell Biol . 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported . We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells . Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines . Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J . Cell Biol . 108:1687-1695, 1989) . Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro . Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages . Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S . typhimurium 14028S . Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties . The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L . monocytogenes and Salmonella species.

Infect Immun, 1992 Sep, 60(9), 3518 - 22
Elimination of the vitamin B12 uptake or synthesis pathway does not diminish the virulence of Escherichia coli K1 or Salmonella typhimurium in three model systems; Sampson BA et al.; The role of iron in infection is of great importance and is well understood . During infection, both the host and the pathogen go through many complicated changes to regulate iron levels . Iron and vitamin B12 share certain features . For example, Escherichia coli has similar transport systems for both nutrients, and binding proteins for both are located in gastric juice, liver, saliva, granulocytes, and milk . It is because of such parallels between iron and B12 that we have explored the role of B12 in virulence . A btuB::Tn10 insertion which disrupts the gene encoding the vitamin B12 receptor from E . coli K-12 was P1 transduced into a virulent E . coli K1 strain . In both an infant-rat model and a chicken embryo model, no difference in virulence between the wild-type and the mutant strains was found . Strains of Salmonella typhimurium with mutations in the cobalamin synthesis pathway (Cob) and in btuB were used in a mouse model of virulence . Mutation of the Cob locus or of btuB does not decrease virulence . Interestingly, the inability to synthesize vitamin B12 actually increases virulence compared with the wild type in the S . typhimurium model . This effect is independent of the B12 intake of the mice.

Res Microbiol, 1992 Sep, 143(7), 711 - 9
The origin of His+ revertants of Salmonella typhimurium obtained on selective medium; Gizatullin FS et al.; The spontaneous reversion to His+ of the Salmonella typhimurium alleles hisD3052 and hisG46 was investigated . In fluctuation tests, the expected "jackpot" distribution of His+ revertants was not observed . The experimental distributions were close to Poisson distribution . The redistribution test showed no significant differences in the His+ colony counts between spread and unspread plates . An attempt at indirect selection of His+ revertants in fluid medium failed . It was also shown that the mean number of His+ reversion events and the mean number of revertants per plate were similar . At the same time, kanamycin-resistant mutants had jackpot distribution . Selection for His+ revertants (histidine starvation) did not increase mutation to Kanr.

Res Microbiol, 1992 Sep, 143(7), 683 - 93
Further characterization of the histidine gene cluster of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis of hisD; Limauro D et al.; We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine . A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced . Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products . Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank . S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.

Mutagenesis, 1992 Sep, 7(5), 343 - 7
Metsovo-tremolite asbestos fibres: in vitro effects on mutation, chromosome aberration, cell transformation and intercellular communication; Athanasiou K et al.; Samples of Metsovo-tremolite asbestos, previously found to be the causative agent of endemic pleural calcification and an increased level of malignant pleural mesothelioma in a rural area of north-western Greece (Metsovo area), were tested in various in vitro toxicity test systems . It was found that asbestos fibres of this type were strong inducers of micronuclei and numerical chromosomal abnormalities while they induced low levels of chromosomal aberrations in mammalian cells in culture . Furthermore, this type of asbestos can induce a low level of in vitro transformation of Syrian hamster embryo cells . The fibres had no effect on gap-junctional cell-cell communication (followed by the dye-transfer method) and did not induce any mutations in the Salmonella typhimurium strain TA102 which is known to be sensitive to the action of various oxidative agents . These results support the hypothesis generated from studies on other types of asbestos that such fibres induce tumours by causing chromosomal mutations.

Mutagenesis, 1992 Sep, 7(5), 335 - 41
Relationships between in vitro mutagenicity assays; Benigni R; This paper analyzes the mutagenicity results reported by the US National Toxicology Program (NTP), relative to 41 chemicals assayed with four in vitro short-term tests {Salmonella typhimurium (STY), Chromosomal aberrations in Chinese hamster ovary (CHO) cells (CHA), Sister chromatid exchange in CHO cells (SCE), mutation in L5178Y mouse lymphoma cells (MLY)} and puts this database in perspective with respect to other databases . It is shown that the test relationships pointed out by the experiments on the 41 chemicals are in substantial agreement with those indicated by a previous NTP report on 73 chemicals, and that the same test relationships were also indicated by the results on the International Program for the Evaluation of Short-Term Tests for Carcinogens (IPESTTC) . The NTP and IPESTTC databases consistently indicated that there is a gradual increase in the sensitivity to the genotoxins in the following order: STY < CHA < SCE < MLY . On this scale, SCE and MLY show a great degree of similarity of responses to the chemicals, as does STY with CHA . The overall evidence provided by these results, and by the general pattern of IPESTTC and NTP genotoxicity profiles, does not support the notion that the genotoxic chemicals have genetic end point specificity . Moreover, a mathematical simulation analysis demonstrated that MLY and SCE--the two most sensitive assays of those studied by NTP--are not more subject to erratic results than other assays, and that they form--together with STY and CHA--a consistent family of genotoxicity assays.

In Vivo, 1992 Sep-Oct, 6(5), 487 - 90
Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom; Toth B et al.; Aqueous extracts of baked and raw Agaricus bisporus (AB) mushroom were tested for mutagenic activity in Salmonella typhimurium strains TA1535 and TA1537 . The extracts were studied with and without metabolic activation by Aroclor-induced rat liver S9 mix . The extracts of the baked and raw AB exhibited a dose related mutagenic activity with and without activation in strain TA1535 . Similar findings were obtained in strain TA1537, although the net revertant values were of lower magnitude . Because humans mainly consume the cultivated mushroom AB in baked form, the implications of the findings are self-evident.

J Dairy Sci, 1992 Sep, 75(9), 2364 - 9
Production of an electrolyte beverage from milk permeate; Geilman WG et al.; The objective of this study was to prepare shelf-stable electrolyte beverages from milk permeate . The average composition of permeate was 4.59% total solids and .40% ash . Lactose was hydrolyzed (approximately 80%) with a commercial fungal lactase enzyme . Additional sweetness was provided by sucrose . The pH was reduced to 3.5 to 3.8 by the addition of citric acid . Shelf-stable products were made using four processes: 1) UHT followed by aseptic filling, 2) heating of filled bottles to 85 degrees C for 30 min, 3) addition of .05% benzoate, and 4) nanopore filtration . The mineral composition of the finished product, expressed in parts per million, was calcium, 150; phosphorus, 157; magnesium, 43; potassium, 1166; sodium, 286; iron, 17; copper, 8; and zinc, 3.4 . Listeria monocytogenes, Salmonella dublin, Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae grew well when they were inoculated into unacidified, hydrolyzed permeate . None of these organisms were isolated from properly processed products . A shelf-stable electrolyte beverage high in minerals was made from whole milk permeate . This beverage could be used to replace electrolytes lost from the human body . The production of a permeate beverage would help alleviate disposal problems of permeate.

Mol Microbiol, 1992 Sep, 6(18), 2715 - 23
Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex; Bischoff DS et al.; The Bacillus subtilis gene encoding FliY has been cloned and sequenced . The gene encodes a 379-amino-acid protein with a predicted molecular mass of 41,054 daltons . FliY is partly homologous to the Escherichia coli and Salmonella typhimurium switch proteins FliM and FliN . The N-terminus of FliY has 33% identity with the first 122 amino acids of FliM, whereas the C-terminus of FliY has 52% identity with the last 30 amino acids of FliN . The middle 60% of FliY is not significantly homologous to either of the proteins . A fliY::cat null mutant has no flagella . Motility can be restored to the mutant by expression of fliY from a plasmid, although chemotaxis is still defective since the strain exhibits smooth swimming behaviour . fliY::cat is in the cheD complementation group . One of the cheD point mutants does not switch although the population grown from a single cell has both smooth swimming and tumbling bacteria, implying that the switch is locked . Expression of fliY in wild-type B . subtilis makes the cells more smooth-swimming but does not appear to affect chemotaxis . Expression of fliY in wild-type S . typhimurium severely inhibits chemotaxis and also makes the cells smooth swimming . Expression in a non-motile S . typhimurium fliN mutant restores motility but not chemotaxis, although expression in a non-motile E . coli fliM mutant does not restore motility . The homology, multiple phenotypes, and interspecies complementation suggest that FliY forms part of the B . subtilis switch complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1992 Sep, 6(18), 2673 - 81
Identification and sequence of a Na(+)-linked gene from the marine bacterium Alteromonas haloplanktis which functionally complements the dagA gene of Escherichia coli; MacLeod PR et al.; A 4.0 kb fragment from a plasmid genomic DNA library of the marine bacterium Alteromonas haloplanktis ATCC 19855 was found in the presence of Na+ to complement the dagA gene of Escherichia coli . We have completely sequenced this fragment and the position of the Na(+)-linked D-alanine glycine permease gene (dagA) on the fragment has been determined by complementation . The predicted carrier protein consists of 542 amino acid residues (M(r) 58,955) . Its hydropathy profile suggests it is composed of eight transmembrane segments with a long hydrophilic region between segments six and seven . Significant similarity has been found between this Na(+)-linked permease and the Na+/proline permeases of E . coli and Salmonella typhimurium and the human and rabbit intestinal Na+/glucose cotransporters.

Chem Res Toxicol, 1992 Sep-Oct, 5(5), 618 - 24
A study of inactivation reactions of N-acetylcysteine with mucochloric acid, a mutagenic product of the chlorination of humic substances in water; LaLonde RT et al.; The Salmonella typhimurium (TA100) mutagenic compound, mucochloric acid {3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA)}, was inactivated by in vitro N-acetylcysteine (NAC) . The reaction of MCA with NAC at pH7 was second order and gave products 4, 5, and 6a that resulted from the displacement of chlorine from C-3 or C-4 of MCA . The sodium borohydride treatment of product 4 gave the same product (7) as was obtained by treating 3,4-dichloro-2(5H)-furanone with NAC . The treatment of MCA with (R)-(+)-cysteine gave the bicyclic product 9a, in which the two chlorine atoms of MCA were still present . This product was slightly more mutagenic than MCA, whereas product 5 was less mutagenic than MCA and product 4 was nonmutagenic in the Salmonella typhimurium (TA100) assay.

Pharmacol Toxicol, 1992 Sep, 71(3 Pt 1), 165 - 72
Mutagenicity of crude senna and senna glycosides in Salmonella typhimurium; Sandnes D et al.; The mutagenicity of senna glycosides and extracts of senna folium and senna fructus was investigated in the Salmonella typhimurium reversion assay . Senna glycosides were inactive in all strains, except for a slight, but significant increase in mutant frequency in TA102 in the absence and presence of liver microsomes . Extracts of senna fructus and senna folium demonstrated weak activity in TA97a, TA100 and TA102 in the presence of liver microsomes, and in TA97a and TA102 in the absence of liver microsomes . A strong increase in mutant frequency (3- to 5-fold above background frequency) was observed with all extracts in TA98 in the presence of liver microsomes . This activity increased further following enzymatic hydrolysis with hesperidinase of extracts of senna fructus from one source, and could be correlated to the release of the flavonol aglycones kaempferol and quercetin . The weak or lacking activity of anthraquinone aglycones in the tested strains of Salmonella typhimurium indicates that mutagenicity can not be attributed solely to the anthraquinone content of these plant materials . The chemical nature of other mutagenic components has not been elucidated.

Jpn J Cancer Res, 1992 Sep, 83(9), 919 - 22
Detection of 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo{4,5-b}pyridine in broiled beef; Kurosaka R et al.; 2-Amino-1-methyl-6-(4-hydroxyphenyl)imidazo{4,5-b}pyridine (4'-OH-PhIP) showed mutagenicity in Salmonella typhimurium TA98 in the presence of S9 mix, inducing 180 revertants per 100 micrograms . Since creatinine, tyrosine and glucose, which are present in meat, are expected to be involved in the formation of 4'-OH-PhIP, its presence in broiled beef was examined . 4'-OH-PhIP was detected in broiled beef by high-performance liquid chromatography and UV-spectrometry after extraction with 0.1 N HCl and purification by blue cotton treatment and ion exchange column chromatography . Its level was estimated to be 21.0 ng per g of broiled beef, which is comparable to that of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP).

Mol Microbiol, 1992 Sep, 6(17), 2467 - 76
The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity; O'Byrne CP et al.; Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity . It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA . Here, we confirm that a his::MudJ operon fusion in S . typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures . Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA . Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically . Both environmental repression and novobiocin induction of his require the his attenuator . In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.

Mutat Res, 1992 Sep, 269(1), 9 - 26
Structure-activity relationships of aromatic diamines in the Ames Salmonella typhimurium assay . Part II; Kalopissis G; Structure-activity relationships in the case of aromatic monoamines, diversely substituted on the ring, using the mutagenic activity in the Ames test were studied in part I . This part II is based on the same general principles but applied to phenylene diamines (ortho, para and meta) diversely substituted on the ring.

Mutat Res, 1992 Sep, 283(1), 83 - 6
A straight correlation between mutagenic activity and beta-galactosidase activity induced by monofunctional alkylating agents; Ohtsuka M et al.; Eight monofunctional alkylating agents were examined for their ability to induce mutation in Salmonella typhimurium . The assay was carried out in S . typhimurium TA100 with the preincubation method . The SN1-type agents were more mutagenic than the SN2-type ones; besides, methylating agents exerted more mutagenic activity than ethylating ones . Those responses in the reversion assay were quite similar to the results obtained previously with the beta-galactosidase assay in Escherichia coli CSH26/pMCP1000 (alkA'-lacZ') as to the induction of the adaptive response . A good correlation was found between mutagenic potency in the reverse mutation assay and inducing potency in the beta-galactosidase assay.

Mutat Res, 1992 Sep, 283(1), 7 - 11
Evaluation of amitrole mutagenicity in Salmonella typhimurium using prostaglandin synthase activation; Croker P et al.; Amitrole is a herbicide which has been found to induce thyroid and liver tumours in rodents, yet demonstrates limited genotoxic activity . The lack of mutagenicity of this compound in Salmonella typhimurium when employing a standard liver microsomal fraction, combined with evidence of activation of amitrole by peroxidases, warranted an investigation employing this other pathway of metabolic activation . Using prostaglandin H synthase as the activating system, the aromatic amine 2-aminofluorene provided a convenient positive control for optimisation of the metabolising system . Under such conditions, amitrole did not induce elevated numbers of revertant colonies in Salmonella typhimurium TA98, neither did it display evidence of interference with histidine biosynthesis as had been reported . Amitrole also remained nonmutagenic when preincubated at varying pHs . Thus, it has been shown that the alternative activation system, prostaglandin H synthase, does not produce metabolites which are mutagenic in the Ames test.

Mutat Res, 1992 Sep, 283(1), 35 - 43
Mutagenicity of nitro derivatives produced by exposure of dibenzofuran to nitrogen oxides; Watanabe T et al.; Dibenzofuran (DF) was reacted with various concentrations of nitrogen oxides (NOx) under light irradiation . The mutagenicities of the reaction mixtures were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix) . DF-Nox (molar ratios 1:3, 1:6 and 1:18) reaction mixtures exhibited mutagenic potency in strain TA98 without S9 mix, and their direct-acting mutagenicity was reduced in strains TA98NR and TA98/1,8-DNP6 . Four mononitrodibenzofurans and 2 dinitrodibenzofurans, i.e., 1-nitrodibenzofuran (NDF), 2-NDF, 3-NDF, 4-NDF, 2,7-dinitrodibenzofuran (DNDF) and 2,8-DNDF, were identified with authentic samples in the DF-NOx (1:18) reaction mixture by HPLC cochromatography and a gas chromatography/mass spectrometry study . The order of mutagenicity of nitrodibenzofurans in strain TA98 without S9 mix was as follows: 2,7-DNDF greater than 2,8-DNDF greater than 3-NDF greater than 2-NDF greater than 4-NDF greater than 1-NDF . The mutagenic potency of 2,7-DNDF in strains TA98 and TA100 was enhanced by the addition of S9 mix . Since these nitrodibenzofurans were less mutagenic in strains TA98NR and TA98/1,8-DNP6 than in strain TA98 without S9 mix, it was presumed that their mutagenicity was dependent on their activation by the 'classical' bacterial nitroreductase and/or transacetylase, which are absent in strains TA98NR and TA98/1,8-DNP6 but present in strain TA98, respectively . 3-NDF and 4-NDF were mutagenic in strain TA100 without S9 mix . 2-NDF and 3-NDF were determined as corresponding amino derivatives in DF-NOx (1:3), (1:6) and (1:18) reaction mixtures . They contributed about 30-65% of the direct-acting mutagenicity of reaction mixtures in strain TA98.

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