Mutat Res, 1992 Sep, 283(1), 29 - 33 Preferential induction of AT-TA transversion, but not deletions, by chlorambucil at the hisG428 site of Salmonella typhimurium TA102; Yamada M et al.; The chemotherapeutic agent chlorambucil effectively induces deletion mutations in mouse germ cells . The possibility that this chemical also effectively induces deletion mutations in bacterial DNA was examined using Ames Salmonella tester strains . Chlorambucil was mutagenic only to strains TA102 (hisG428, rfa/pKM101) and YG2975 (hisG46, rfa/pKM101) when S9 mix was absent . Since strain TA102 can detect short deletions, the mutational changes of TA102 induced by this agent without S9 mix were directly determined by the DNA sequencing technique . It turned out that chlorambucil did not induce deletion mutations but preferentially induced AT-TA transversions at the hisG428 site of plasmid pAQ1 of strain TA102 . These results caution that the positive results induced by chlorambucil in mutagenicity tests do not necessarily mean the occurrence of deletions. J Bacteriol, 1992 Sep, 174(18), 5881 - 7 Escherichia coli prlC encodes an endopeptidase and is homologous to the Salmonella typhimurium opdA gene; Conlin CA et al.; Mutations at the Escherichia coli prlC locus suppress the export defect of certain lamB signal sequence mutations . The Salmonella typhimurium opdA gene encodes an endoprotease that can participate in the catabolism of certain peptides and is required for normal development of phage P22 . Plasmids carrying either the wild-type (pTC100 prlC+) or suppressor alleles of prlC complemented all phenotypes associated with an S . typhimurium opdA mutation . A plasmid carrying an amber mutation in prlC {prlC31(AM)} was unable to complement except in an amber suppressor background . Tn1000 insertions which eliminated the ability of pTC100 (prlC+) to complement opdA mapped to the region of the plasmid shown by deletion analysis and subcloning to contain prlC . The nucleotide sequence of a 2.7-kb fragment including this region was determined, revealing an open reading frame encoding a 77-kDa protein . The sequences of this open reading frame and its putative promoter region were very similar (84% nucleotide sequence identity and 95% amino acid identity) to those of S . typhimurium opdA, showing that these genes are homologs . The nucleotide sequence of the prlC1 suppressor allele was determined and predicts an in-frame duplication of seven amino acids, providing further confirmation that the prlC suppressor phenotype results from changes in the endopeptidase OpdA. Infect Immun, 1992 Sep, 60(9), 3763 - 70 An unusual pagC::TnphoA mutation leads to an invasion- and virulence-defective phenotype in Salmonellae; Miller VL et al.; Two phenotypes believed to contribute to the pathogenesis of Salmonella infections are macrophage survival and invasion of epithelial cells . It was recently observed that the Salmonella macrophage survival factor PagC has significant amino acid similarity to the Yersinia invasion factor Ail . This observation raised the possibilities that macrophage survival is in part determined by the pathway of entry and that PagC confers an entry mechanism that does not trigger the microbicidal activities of the macrophage . Thus, we sought to investigate the role of PagC in invasion by examining (i) the invasion phenotype of pagC mutants and (ii) the invasion phenotype of Escherichia coli carrying pagC . A previously identified invasion-defective TnphoA insertion mutant of Salmonella enteritidis was found to have TnphoA inserted into the signal sequence-encoding region of pagC; the pagC allele from this mutant, SM5T, was designated pagC64 . In contrast, Salmonella typhimurium carrying the pagC1 allele (a TnphoA insertion mutation, downstream of the region encoding the signal sequence) was not defective for invasion . Further analysis of these two pagC alleles suggested that the invasion-defective phenotype associated with pagC64 is not due to the loss of PagC function but rather is due to the synthesis of a hybrid PagC-alkaline phosphatase protein that is aberrantly localized, most likely to the inner membrane, and thus may prevent proper localization or function of a factor(s) required for efficient invasion . The observation that pagC did not confer an invasive phenotype to E . coli further suggests that PagC is not an invasion factor . A cloned pagC gene complemented the macrophage survival defect of S . typhimurium pagC1 mutants, but the cloned ail gene did not . Together these results suggest that the structural similarity between PagC and Ail may not extend to a similarity in function . Interestingly, S . enteritidis carrying the pagC64 allele that results in both an invasion defect and a macrophage survival defect was less virulent for mice infected intragastrically or intraperitoneally than was S . enteritidis carrying the pagC1 allele that results only in a macrophage survival defect. J Nihon Univ Sch Dent, 1992 Sep, 34(3), 183 - 95 Mutagenicity of analgesics, their derivatives, and anti-inflammatory drugs with S-9 mix of several animal species; Kuboyama N et al.; An investigation was undertaken to determine whether analgesics and their derivatives (13 compounds), and anti-inflammatory drugs (4 compounds) had mutagenicity . Rec-assay was used to clarify specific DNA-damaging properties, and the Ames test was used to find back-mutations, using S-9 fractions obtained from the liver of 4 animal species pretreated with polychlorobiphenyl . In the Rec-assay, salicylic acid (2 mg), aspirin (5 mg), benzoic acid (4 mg), sulpyrine (0.4 mg), indomethacin (0.1 mg), oxyphenbutazone (0.1 mg) and diclofenac sodium (0.1 mg) showed a DNA-damaging tendency . In the Ames test, mutagenicity of methyl salicylate was demonstrated using the Salmonella typhimurium TA98 strain upon addition of hamster S-9 mixture . Weak mutagenicity was also found using the TA100 strain with rat S-9 mixture for salicylic acid, sulpyrine, indomethacin and oxyphenbutazone, and with hamster S-9 mixture for methyl salicylate, acetaminophen and phenacetine. Biochemistry, 1992 Aug 25, 31(33), 7535 - 42 Conformational changes and subunit communication in tryptophan synthase: effect of substrates and substrate analogs; Strambini GB et al.; The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phosphate, the alpha- and beta-subunit substrate indole, and the beta-subunit substrate analog L-histidine . The beta-subunit contains as intrinsic probes Trp-177 and pyridoxal 5'-phosphate, whereas the alpha-subunit has been mutagenized by replacing Ala-129 with a Trp residue . In contrast to the inertness of L-histidine, DL-alpha-glycerol 3-phosphate was found (i) to alter the phosphorescence spectrum of Trp-129, (ii) to shift the fluorescence thermal quenching profile of both Trp-177 and coenzyme to higher temperature, (iii) to slow down the triplet decay kinetics of Trp-177 in fluid solution, and (iv) to affect the equilibrium between different conformations of the enzyme . These findings provide direct evidence that DL-alpha-glycerol 3-phosphate binding affects the structure of the alpha-subunit and, in the presence of coenzyme, induces a conformational change in the beta-subunit that leads to a considerably more rigid structure . As opposed to DL-alpha-glycerol 3-phosphate, the shortening of the phosphorescence lifetime upon indole binding suggests that this substrate increases structural fluctuations in the beta-subunit . Implications for the mechanism of the allosteric regulation between alpha- and beta-subunits are discussed. Biochemistry, 1992 Aug 25, 31(33), 7527 - 34 Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium; Strambini GB et al.; Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan . We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site . The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band . Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme . The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges . The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer . The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay . In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1992 Aug 20, 226(4), 1287 - 90 Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2; Taylor G et al.; The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified . Single crystals of the V . cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2 . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit . Diffraction extends to at least 2.5 A . Single crystals of the S . typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2 . The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit . Diffraction extends to at least 1.8 A. FEMS Microbiol Lett, 1992 Aug 15, 74(2-3), 121 - 6 Salmonella typhimurium induces an inositol phosphate flux in infected epithelial cells; Ruschkowski S et al.; Salmonella typhimurium, like many other intracellular pathogens, is capable of inducing its own uptake into non-phagocytic cells by a process termed invasion, and residing within a membrane-bound inclusion . During invasion it causes significant rearrangement of the host cytoskeleton, indicating that signals are transduced between the bacterium and the host cell cytoplasm, across the eukaryotic cell membrane . We found that intracellular inositol phosphate concentrations in HeLa cells increased during S . typhimurium entry and returned to normal levels after bacterial internalization . A chelator of intracellular calcium (BAPTA/AM) blocked S . typhimurium uptake into HeLa epithelial cells, but extracellular calcium chelators (BAPTA, EGTA, EDTA) had no effect on bacterial invasion . These results indicate that S . typhimurium may activate host cell phospholipase C activity to form inositol phosphates which in turn stimulate release of intracellular calcium stores to facilitate bacterial uptake. Cancer Lett, 1992 Aug 14, 65(2), 107 - 13 A study of tobacco carcinogenesis . XLVII . Bioassays of vinylpyridines for genotoxicity and for tumorigenicity in A/J mice; Brunnemann KD et al.; 3-Vinylpyridine is formed from nicotine during the smoking of tobacco products . Consequently it is found in mainstream and side-stream smoke of cigarettes and cigars and in environmental tobacco smoke . In this study, 3-vinylpyridine and its isomers 2- and 4-vinylpyridine as well as styrene (vinylbenzene) were bioassayed for mutagenicity in Salmonella typhimurium strains TA 1535, TA 1538, TA 98 and TA 100 and for their genotoxicity in the rat hepatocyte DNA-repair test . In both in vitro assays-all three vinylpyridines and styrene were inactive . In a test for tumorigenicity in which the test compounds were injected intraperitoneally into A/J mice (total dose 200 mumol/animal) there was no significant incidence of lung adenoma nor of any other type of tumors. J Bacteriol, 1992 Aug, 174(15), 5117 - 22 Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription; Sanders DA et al.; The NtrC transcription factor is a member of a family of homologous prokaryotic regulatory proteins that participate in the transduction of extracellular and nutritional signals . It has been demonstrated that the phosphate group from a histidine residue of the phosphorylated NtrB protein autokinase is transferred to the NtrC protein . Phosphorylation of the NtrC protein is transient and activates its transcriptional enhancement activity . We have investigated the site of phosphorylation of the Salmonella typhimurium NtrC protein and find that it is an aspartate residue (Asp-54) that is found within a sequence conserved in all of the members of the family of regulatory proteins . We propose that this phosphorylation is an NtrC protein histidine phosphatase catalytic intermediate . This conclusion suggests that the NtrC family should be viewed not as kinase substrates but as enzymes that can catalyze the hydrolysis of their activated forms in a concentration-independent fashion . They are similar in this sense to eukaryotic signal-transducing GTPases. FEBS Lett, 1992 Jul 27, 307(1), 3 - 9 The fifth Datta Lecture . Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E . coli . Implications for transmembrane signaling; Lynch BA et al.; A high resolution structure of the N-terminal ligand-binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported . A least-squares superposition of the alpha-amino nitrogen, alpha-carbon, beta-carbon, and alpha-carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the alpha-amino and alpha-carboxylates of the respective ligands . Because the N-terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand-induced conformational changes at the active sites of these proteins . This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit. Gene, 1992 Aug 1, 117(1), 31 - 7 Mutations in the araC gene of Salmonella typhimurium LT2 which affect both activator and auto-regulatory functions of the AraC protein; Clarke P et al.; The araC gene encodes a regulatory protein, AraC, that acts as both an activator and a repressor of transcription of the genes involved in the transport and catabolism of L-arabinose in Salmonella typhimurium LT2 . Five araC mutants which have altered regulatory properties were characterized . All are point mutations which would result in amino acid substitutions near the C terminus of AraC . Each mutation results in altered activator and auto-regulatory AraC function in vivo . In vitro DNA-binding assays showed that three mutant AraC have measurable lowered affinity for ara controlling site DNA . The data are consistent with a model in which there is a DNA-binding domain in the C terminus of AraC which functions in both activation and repression. Infect Immun, 1992 Aug, 60(8), 3452 - 5 Infection of macrophages with Legionella pneumophila induces phosphorylation of a 76-kilodalton protein; Yamamoto Y et al.; Infection of peritoneal macrophages from susceptible A/J mice with Legionella pneumophila induced phosphorylation of a 76-kDa protein . The phosphorylation occurred when macrophages were infected with a virulent strain of L . pneumophila but did not occur when they were infected with an avirulent strain or with other bacteria such as either Pseudomonas aeruginosa or Salmonella typhimurium . Also, no phosphorylation of this protein was observed when macrophages were stimulated with either lipopolysaccharide or phorbol myristate acetate . However, phosphorylation did occur in macrophages infected with a virulent strain of L . pneumophila and treated with either erythromycin to inhibit growth or with cytochalasin D to inhibit uptake of L . pneumophila by macrophages . These results support the view that phosphorylation of this protein occurs during the early phases of interaction between L . pneumophila and macrophages . The role of this specific protein in the recognition, intracellular uptake, and growth of L . pneumophila in permissive macrophages remains to be clarified. Infect Immun, 1992 Aug, 60(8), 3345 - 59 Construction of stable LamB-Shiga toxin B subunit hybrids: analysis of expression in Salmonella typhimurium aroA strains and stimulation of B subunit-specific mucosal and serum antibody responses; Su GF et al.; The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified beta-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, Salmonella typhimurium, and attenuated antigen carrier strains of S . typhimurium (aroA mutants) . The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes . S . typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane . High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities . The lower levels of gene expression observed with the beta-lactamase and aerobactin promoters did not have this effect . Immunization of mice with S . typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses . This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxin-or Shiga-like toxin-producing pathogens. Infect Immun, 1992 Aug, 60(8), 3193 - 200 Adhesion of Salmonella typhimurium to porcine intestinal epithelial surfaces: identification and characterization of two phenotypes; Isaacson RE et al.; Salmonella typhimurium 798 is known to persistently colonize swine . A key step required to initiate colonization of intestines is adhesion of the organism to the intestinal epithelium . However, S . typhimurium 798 initially failed to attach to porcine enterocytes in vitro . An enrichment procedure was used to select adhesive S . typhimurium, and when cells of one colony type were grown in tryptone phosphate broth they were adhesive . Cells from a colony with a different morphology were not adhesive . Adhesion was time dependent, with maximal adhesion occurring at 1 h . As determined by electron microscopy, cells of the adhesive phenotype had pili while none of the cells with the nonadhesive phenotype produced pili . The pili on the adhesive cells were morphologically similar to type 1 pili . Mannose (0.5%) did not affect adhesion, suggesting that the adhesin on strain 798 did not recognize mannose as a receptor . An analysis of envelope proteins from cells of both phenotypes showed that the adhesive-phenotype cells expressed at least 10 unique proteins ranging in size from 20 to 60 kDa . Absorbed antiserum against cells of the adhesive phenotype agglutinated adhesive cells and was used to detect unique surface antigens on the cells of the adhesive phenotype by Western blots (immunoblots) . These antigens were in the range of 30 kDa in size . An envelope extract competitively inhibited the binding of S . typhimurium to enterocytes, as did Fab fragments prepared from the absorbed serum . Cells of both phenotypes contained two plasmids, and each had identical restriction digestion patterns . Cells of the adhesive phenotype consistently were found to be more readily phagocytosed by pig leukocytes, and once in the phagocytes they survived better than cells of the nonadhesive phenotype. Infect Immun, 1992 Aug, 60(8), 3072 - 8 A 66-kilodalton heat shock protein of Salmonella typhimurium is responsible for binding of the bacterium to intestinal mucus; Ensgraber M et al.; Salmonella typhimurium infections have increased during the last few years . However, the interplay of virulence factors in S . typhimurium pathogenesis is still poorly understood, particularly with regard to the mechanisms and components of the bacterium which are involved in its interaction with the intestinal mucus . We have observed that S . typhimurium is aggregated by incubation with colonic mucus (guinea pig model) . To quantify this phenomenon, an aggregation assay was established . By using this assay, it was found that the aggregation profile of S . typhimurium strains freshly isolated from patients (age 9 and older) with salmonellosis correlated with the severity of the disease . An isolate with high aggregation behavior was chosen for characterization of the bacterial component involved in binding to colonic mucus material . The component of S . typhimurium responsible for aggregation was purified and characterized as a 66-kDa protein which was able to completely inhibit mucus-mediated bacterial aggregation . This protein was recognized by monoclonal antibodies against the 65-kDa heat shock protein (HSP) of Mycobacterium leprae . The 66-kDa protein of S . typhimurium was inducible by incubating the bacteria at 50 degrees C and was secreted into the supernatant, from which it could be isolated in both dimeric and polymeric forms . The monoclonal anti-HSP 65, as well as a polyclonal antibody against the 66-kDa protein of S . typhimurium, caused dose-dependent inhibition of the aggregation of S . typhimurium by crude mucus preparations . This is the first report showing that a bacterial HSP is involved in mucus-mediated interaction of pathogens with the host. EMBO J, 1992 Aug, 11(8), 2819 - 28 Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases; van Dijl JM et al.; Signal peptidases (SPases) remove signal peptides from secretory proteins . The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli . Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor . The SipS protein consisted of 184 amino acids (mol . wt 21 kDa) . The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae . Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex . Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases . The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases. J Med Microbiol, 1992 Aug, 37(2), 104 - 8 Epidemiological markers of Salmonella typhimurium isolates in Rome; Martini A et al.; The phage type, antimicrobial resistance pattern, colicinogenic activity and DNA plasmid content of 172 strains of Salmonella typhimurium isolated in Rome from 1984 to 1986 were determined; 142 isolates were from patients with enteritis, 30 were from asymptomatic subjects . Most of the phage types identified were isolated during 2 or 3 of the study years; others, e.g., PT141, PT 204c and PT 194 were isolated during 1 year only, and only the last of these was isolated in large numbers . Phage typing proved most valuable in identifying epidemiologically related strains; DNA plasmid analysis was most useful in characterising further cultures of the same phage type, especially those isolated during suspected epidemics. Mol Cell Probes, 1992 Aug, 6(4), 271 - 9 Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella; Rahn K et al.; Amplification of nucleotide sequences within the invA gene of Salmonella typhimurium was evaluated as a means of detecting Salmonella . A collection of 630 strains of Salmonella comprising over 100 serovars, including the 20 most prevalent serovars isolated from animals and humans in Canada, was examined . Controls consisted of 142 non-Salmonella strains comprising 21 genera of bacteria . Cultures were screened by inoculating a single colony of bacteria directly into a polymerase chain reaction (PCR) mixture which contained a pair of primers specific for the invA gene . The specific PCR product was a 284 bp DNA fragment which was visualized in 2% agarose gels . With the exception of two S . litchfield and two S . senftenberg strains, all Salmonella strains were detected . In contrast, none of the non-Salmonella strains yielded the specific amplification product . Non-specific amplification of a few non-Salmonella strains resulted in a product that was distinctly different in size from the specific 284 bp product . Specificity of amplification was further confirmed by demonstration of hybridization of a 32P-labelled invA gene fragment only to the specific 284 bp product . The detection of 99.4% of Salmonella strains tested and the failure to specifically amplify DNA from non-Salmonella strains confirm that the invA gene contains sequences unique to Salmonella and demonstrate that this gene is a suitable PCR target, with potential diagnostic applications. Toxicology, 1992 Aug, 74(1), 57 - 67 Differences between rats and mice in the immunosuppressive activity of 2-methoxyethanol and 2-methoxyacetic acid; Smialowicz RJ et al.; Previous studies from this laboratory have demonstrated that 2-methoxyethanol (ME) and its principal metabolite 2-methoxyacetic acid (MAA) are immunosuppressive in young adult male Fischer 344 rats . In the present study, the immunosuppressive potential of ME and MAA was evaluated in young adult female Fischer 344 rats and C57BL/6J mice . Rats and mice were dosed by gavage with either ME or MAA in water, at dosages ranging from 50-400 mg/kg/day, for 10 consecutive days . Rats and mice were examined for alterations in body, spleen and thymus weights and mitogen-induced proliferation of splenic lymphocytes in vitro; separate groups were employed for the antibody plaque-forming cell (PFC) response to trinitrophenyl-lipopolysaccharide (TNP-LPS) . Rats dosed at 100-400 mg/kg/day ME and rats dosed at 50-400 mg/kg/day MAA had decreased thymus weights in the absence of decreased body or spleen weights . Lymphoproliferative (LP) responses to concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and Salmonella typhimurium mitogen (STM) were all reduced in rats treated with all dosages of ME . Rats treated with MAA displayed similar reductions in these LP responses except that the responses to PWM and STM in rats dosed at 50 mg/kg/day were not reduced . In contrast to the effects of ME and MAA on these end points in the rat, no thymic involution or suppression of LP responses were observed in mice dosed at 50-400 mg/kg/day . The PFC response to TNP-LPS was suppressed in rats dosed with either ME or MAA at dosages of 100-400 mg/kg/day . ME and MAA, however, failed to suppress the PFC response in mice immunized with TNP-LPS . These results indicate that unlike Fischer 344 rats, C57BL/6J mice are insensitive to the immunosuppressive effects of ME and MAA at the dosages employed in this study . Whether the different sensitivities of these two rodent species to ME- and MAA-induced immunosuppression are due to immunologic, pharmacokinetic or metabolic differences within each species remains to be determined. Mol Gen Genet, 1992 Aug, 234(2), 325 - 8 Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis; Zagorec M et al.; The ptsG gene of Bacillus subtilis encodes Enzyme IIGlc of the phosphoenolpyruvate: glucose phosphotransferase system . The 3' end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes IIIGlc of Escherichia coli and Salmonella typhimurium . We report here cloning of the complete ptsG gene of B . subtilis and determination of the nucleotide sequence of the 5' end . These results, combined with the sequence of the 3' end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II . The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes IIGlc and IINag of E . coli but which are present in the IIGlc-like protein encoded by the E . coli malX gene. J Cell Biol, 1992 Aug, 118(4), 929 - 36 Enteric defensins: antibiotic peptide components of intestinal host defense; Selsted ME et al.; Five intestinal defensins, termed cryptdins 1-5, have been purified from mouse small bowel, sequenced, and localized to the epithelium by immunohistochemistry . Although identified as members of the defensin peptide family by peptide sequencing, enteric defensins are novel in that four cryptdins have amino termini which are three to six residues longer than those of leukocyte-derived defensins . A fifth cryptdin is the first defensin to diverge from the previously invariant spacing of cysteines in the peptide structure . The most abundant enteric defensin, cryptdin-1, had antimicrobial activity against an attenuated phoP mutant of Salmonella typhimurium but was not active against the virulent wild-type parent . Immunohistochemical localization demonstrated that cryptdin-1, and probably cryptdins 2 and 3, occur exclusively in Paneth cells, where the peptides appear to be associated with cytoplasmic granules . Biochemical and immunologic analysis of the luminal contents of the small intestine suggest that cryptdin peptides are secreted into the lumen, similar to Paneth cell secretion of lysozyme . The presence of several enteric defensins in the intestinal epithelium, evidence of their presence in the lumen, and the antibacterial activity of cryptdin-1 suggest that these peptides contribute to the antimicrobial barrier function of the small bowel mucosa. Carcinogenesis, 1992 Aug, 13(8), 1345 - 50 Bioactivation of aflatoxin B1 in the bovine olfactory mucosa: DNA binding, mutagenicity and induction of sister chromatid exchanges; Tjalve H et al.; Nasal olfactory tumours occur in cattle in relatively high frequencies in several developing countries . Since affected animals sometimes show signs of severe aflatoxicosis, a role of aflatoxin B1 (AFB1) in tumorigenesis can be proposed . The results of the present study show that microsomal preparations of the bovine olfactory mucosa have a much higher ability than liver microsomes to induce covalent binding of AFB1 to calf thymus DNA and to microsomal proteins . The major DNA adduct formed was 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 . Incubations of microsomal preparations of the bovine nasal olfactory mucosa with glutathione (GSH) and cytosolic fractions of the nasal mucosa resulted in decreased AFB1 DNA binding . A more pronounced decrease was observed when cytosolic fractions of mouse liver were added to the incubations . Mouse liver is known to contain a glutathione-S-transferase with a high ability to scavenge the reactive AFB1-epoxide via conjugation to GSH . Our results indicate that AFB1-GSH conjugation occurs less efficiently in the bovine nasal olfactory mucosa than in the mouse liver . Supernatant preparations (9000 g) of the bovine nasal olfactory mucosa incubated with AFB1 were shown to have the capacity to induce a strong genotoxic response both as regards induction of gene mutations in Salmonella typhimurium TA100 and the induction of sister chromatid exchanges in Chinese hamster ovary cells . Preparations of the bovine liver (9000 g) has a much lower ability to induce these effects . The results of the present study show that the bovine nasal olfactory mucosa has a high AFB1-bioactivating capacity, which can be related to the potent DNA damaging and mutagenic effects observed . It is considered that our results support the assumption that AFB1 plays a role in the aetiology of nasal tumours in cattle. Can J Microbiol, 1992 Aug, 38(8), 852 - 7 Identification of Salmonella typhimurium invasiveness loci; Betts J et al.; Salmonella typhimurium is capable of entering into (invading) nonphagocytic host cells . To systematically identify the bacterial genes necessary for this process, 15,000 Tn10dCm random transposon mutants of S . typhimurium were individually screened for invasiveness, using the human colonic epithelial Caco-2 cell line . Four hundred and eighty-eight mutants had decreased levels of invasiveness; most were nonmotile . However, five mutants, representing four loci, were completely motile . Further characterization of these five mutants showed that they were also unable to enter the dog kidney epithelial cell line MDCK and the mouse macrophage line J774.A1 . In contrast to the parental strain, they were unable to disrupt the transepithelial resistance of polarized epithelial monolayers, nor were they able to penetrate across these epithelial barriers . Three of the four classes of mutants remained virulent in mice . The results confirm several aspects of S . typhimurium invasiveness: (i) intact motility enhances invasiveness of cultured cells; (ii) S . typhimurium invasiveness is multifactorial, and at least six distinct genetic loci are involved; and (iii) invasion loci involved in uptake into epithelial cells are also needed for uptake into cultured phagocytic cells . The results also emphasize that decreased levels of invasiveness eliminate bacterial penetration of polarized epithelial barriers and invasiveness loci mutants are not necessarily avirulent. Microb Pathog, 1992 Aug, 13(2), 133 - 43 Growth phase and SpvR regulation of transcription of Salmonella typhimurium spvABC virulence genes; Coynault C et al.; The 90 kb virulence plasmid of Salmonella typhimurium is required for bacterial growth beyond the small intestine to deeper tissues such as the spleen and liver of orally inoculated mice . We constructed transcriptional lacZ fusions within the cloned plasmid-borne virulence genes spvA, spvB and spvC of S . typhimurium to demonstrate that spvR encodes a trans-acting positive regulator for the transcription of spvA, spvB and spvC . Data suggesting that the activation of spvABC transcription is dependent on the growth phase of both S . typhimurium and Escherichia coli grown in Luria Broth (LB) are also presented . Complementation experiments for virulence in mice confirmed that at least spvR and spvC are virulence genes and further suggested that the spvRABC gene cluster consists of at least three transcriptional units containing spvR, spvC and spvABC, respectively . Reinitiation of transcription at spvC was confirmed in vitro, using a lacZ fusion, and was shown to be independent of SpvR-mediated control in LB. Microb Pathog, 1992 Aug, 13(2), 109 - 21 Expression and characterization of the cloned Salmonella typhimurium enterotoxin; Prasad R et al.; Earlier, our laboratory reported the cloning of a chromosomally encoded cholera toxin (CT)-like enterotoxin gene from Salmonella typhimurium Q1 into pBR322 . Cell lysates from the plasmid clone pC1, containing a 4.8 kb EcoR1 DNA fragment from Salmonella, caused elongation of Chinese hamster ovary (CHO) cells and this biologic activity was neutralized by anti-CT . However, this cloned gene product did not elicit fluid secretion in the rabbit intestinal loop (RIL) model, because of poor expression . We report here, subcloning of a 4.8 kb EcoRI and a 2.7 kb HindIII/Eco Rl fragment into a high expression T7 RNA polymerase/promoter system . Cell lysates from these clones elicited fluid secretion in the RIL model, caused firm induration in rabbit skin and elongated CHO cells . These biological activities were neutralized by anti-CT . SDS-PAGE and subsequent fluorographic analysis of Escherichia coli, harboring recombinant plasmids in a T7 expression system, revealed the presence of two prominent 35S-labeled polypeptides of 25 and 12 kDa, which were immunoprecipitated with anti-CT . The enterotoxin appeared to be 125 kDa in size, based on chromatography on a P-300 column, had a pl of 6.6 to 6.8, and was heat-labile (60 degrees C/5 min) . Unlike cloned CT and heat-labile enterotoxin (LT-l), which were localized in the periplasm, the Salmonella enterotoxin was cytoplasmic in nature. Genet Res, 1992 Aug, 60(1), 1 - 6 Fine structure mapping and complementation studies of the metD methionine transport system in Salmonella typhimurium; Grundy CE et al.; A fine structure deletion map of the metD region of the chromosome of Salmonella typhimurium responsible for a high-affinity methionine transport system has been constructed . Complementation tests involving the introduction of metD+DNA contained in a pUC8 vector into metD strains indicated the presence of four complementation groups in the metD region . This suggested that the methionine system belongs to the osmotic shock-sensitive class of transport system, and therefore should possess a periplasmic methionine-binding protein and several membrane proteins . But a deletion mutation covering all known metD point mutations did not affect the level of a methionine binding activity in osmotic shock fluids, suggesting either that the deletion did not extend into the gene encoding the binding protein, or that the binding activity is not associated with the metD system . Possible reasons for the failure to isolate mutations in the gene for the binding protein are discussed. Genetika, 1992 Aug, 28(8), 52 - 9 {Dependence of mutagenic activity of heterocyclic analogs of pyrene on their chemical structure}; Abilev SK et al.; Comparative mutagenic activity of 6 heterocyclic analogs of pyrene was studied . The highest activity was revealed for 2,7-dinitro-4,9-dioxy-5,10-dioxo-4,5,9,10-tetrahydro-4,9- diazapyrene (2,7-DN-DDTDP) and 2,7-dinitro-5,10-dioxo-4,9-dioxapyrene (2,7-DN-DDP) which induced mutations in the tester strain Salmonella typhimurium TA98 and TA100 . High mutagenicity of 2,7-DN-DDTDP and 2,7-DN-DDP is conditioned by reduction of nitro groups in the 2,7 position . The carbonyl groups in the 5,10 position were believed to cause bifunctional activity of 2,7-DN-DDTDP and 2,7-DN-DDP and to promote deletion of two G-C in the D3052 site. Yakugaku Zasshi, 1992 Aug, 112(8), 577 - 84 {Antimutagenic activities of hexane extracts of the fruit extract and the kernels of Prunus mume Sieb . et Zucc.}; Dogasaki C et al.; Antimutagenic activities of hexane extracts obtained from the fruit extract (UE) and the Kernels (KE) of P . mume were examined . These extracts showed inhibitory activities to known mutagens, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, benzo{alpha}pyrene and aflatoxin B1 in the Ames assay using Salmonella typhimurium TA100 and TA98 strains . The UE and KE fractions were then separated by silicic acid column chromatography with a stepwise elution method using ether-hexane . The location of the active substances in the fractions was also determined by thin-layer chromatography . Consequently, it was found that the effective substances for the desmutagenicity were fatty acids, and identified by gas liquid chromatography, mainly as oleic acid, linoleic acid and linolenic acid in UE, and mainly as oleic acid and linoleic acid in KE, respectively. J Interferon Res, 1992 Aug, 12(4), 307 - 10 The effects of interferon-gamma and bacterial lipopolysaccharide on CD14 expression in human monocytes; Payne JB et al.; CD14 has been reported to be the lipopolysaccharide (LPS)-LPS binding protein receptor . The effects of interferon-gamma (IFN-gamma) on CD14 expression have not been clearly established . The purpose of this investigation was to examine the effects of IFN-gamma alone and IFN-gamma followed by bacterial LPS on CD14 expression . Human peripheral blood monocytes were isolated by counterflow centrifugal elutriation (CCE) . Monocytes were cultured for 48 h with IFN-gamma alone or for 24 h with IFN-gamma followed by LPS for a second 24 h . IFN-gamma alone caused a down-regulation of CD14 expression, as assessed by flow cytometry, relative to CD14 expression in untreated monocytes . In addition, CD14 expression was even more significantly down-regulated after IFN-gamma pretreatment followed by either Prevotella intermedia or Salmonella typhimurium LPS . Likewise, the percentage of CD14+ monocytes decreased after IFN-gamma alone and even more dramatically after IFN-gamma treatment followed by either LPS . This study clearly demonstrated that IFN-gamma down-regulates CD14 expression and that LPS following IFN-gamma pretreatment potentiates this effect. Protein Expr Purif, 1992 Aug, 3(4), 347 - 54 Overexpression and purification of the separate tryptophan synthase alpha and beta subunits from Salmonella typhimurium; Yang XJ et al.; To obtain high levels of expression of the free alpha and beta subunits of tryptophan synthase from Salmonella typhimurium, we have used two plasmids (pStrpA and pStrpB) that carry the genes encoding the alpha and beta subunits, respectively . The expression of each plasmid in Escherichia coli CB149 results in overproduction of each subunit . We also report new and efficient methods for purifying the individual alpha and beta subunits . Microcrystals of the beta subunit are obtained by addition of polyethylene glycol 8000 and spermine to crude bacterial extracts . This crystallization procedure is similar to methods used previously to grow crystals of the S . typhimurium tryptophan synthase alpha 2 beta 2 complex for X-ray crystallography and to purify this complex by crystallization from bacterial extracts . The results suggest that purification by crystallization may be useful for other overexpressed enzymes and multienzymes complexes . Purification of the alpha subunit utilizes ammonium sulfate fractionation, chromatography on diethylaminoethyl-Sephacel, and high-performance liquid chromatography on a Mono Q column . The purified alpha and beta subunits are more than 95% pure by the criterion of sodium dodecyl sulfate gel electrophoresis . The procedures developed can be applied to the expression and purification of mutant forms of the separate alpha and beta subunits . The purified alpha and beta subunits provide useful materials for studies of subunit association and for investigations of other properties of the separate subunits. Mol Microbiol, 1992 Aug, 6(16), 2327 - 37 Expression and mutational analysis of the nucleoid-associated protein H-NS of Salmonella typhimurium; Hinton JC et al.; The H-NS (H1) protein is a major component of bacterial chromatin . Mutations in the hns (osmZ) gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes in an allele-specific manner . H-NS expression was found not to vary with growth phase or growth medium osmolarity . Additionally, 10 independent hns mutations were isolated and characterized . Five of these mutations were the result of an IS10 insertion, each generating a truncated polypeptide . The other five mutations were the same specific deletion of one amino acid, delta Ala46 . The various hns mutations exhibited different phenotypes and influenced DNA topology to variable extents . Implications for the mechanism by which H-NS influences gene expression are discussed. Clin Infect Dis, 1992 Aug, 15(2), 346 - 61 Hypothesis for vaccine development: protective immunity to enteric diseases caused by nontyphoidal salmonellae and shigellae may be conferred by serum IgG antibodies to the O-specific polysaccharide of their lipopolysaccharides; Robbins JB et al.; Immunoprophylaxis for bacterial enteric diseases is hindered because the protective immune mechanism(s) against nontyphoidal salmonellae or shigellae in humans are not established . On the basis of the similarities between the clinical signs, epidemiology, pathogenesis, and pathology of as well as protective immunity to salmonellae and shigellae, we propose that serum IgG antibodies to the O-specific polysaccharide (O-SP) of their lipopolysaccharides (LPSs) will confer protective immunity to these two pathogens . Critical to this notion is that (1) the virulence of these two pathogens requires full expression of their LPS; (2) active or passive immunization with serum IgG O-SP antibodies confers protection of mice against Salmonella typhimurium (there are no comparable data for humans); and (3) in humans, convalescence from shigellosis confers type (O-SP) -specific protective immunity, and indirect evidence shows a correlation between the level of serum LPS antibodies and resistance to shigellosis . We designed conjugate vaccines to elicit high levels of long-lived serum IgG O-SP antibodies to nontyphoidal salmonellae and shigellae to test this hypothesis. Mutat Res, 1992 Aug, 282(4), 273 - 81 Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium; Sarkar FH et al.; Prostaglandin H synthase (PHS) is widely distributed in mammalian tissues and has the ability to oxidize a variety of mutagens and carcinogens . It may therefore play a key role in the metabolic activation of xenobiotics . The present study documents that highly purified PHS can be used in conjunction with 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP), a relatively stable and non-mutagenic hydroperoxide substrate, for the metabolic activation of aromatic amines to mutagenic derivatives that can be detected in short-term Salmonella typhimurium mutagenesis assays . The PHS-based activation system alone was not mutagenic for these tester strains, nor were the test compounds significantly toxic for the bacteria over the concentration range tested . When used in conjunction with Salmonella strains TA98 and TA100 in a modified Ames assay, this system should prove useful for screening of a wide range of compounds for metabolic activation by this mammalian peroxidase . The potential broad utility of this purified PHS-dependent metabolic activation system was investigated by evaluating the activation of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), which are representative of a group of mutagenic and carcinogenic heterocyclic arylamines to which humans are exposed via their diet . Both IQ and MeIQ were activated by PHS to potent mutagens and confirm the utility of the PPHP/PHS system for the activation of premutagens . Whereas the extent of activation of aromatic amines by S9-based systems is significantly greater than for the PHS activation system described herein, PHS may play a significant role in target tissues in which it is present at significantly greater levels than P450 isoenzymes . Moreover, it is likely that the substrate specificity of PHS differs sufficiently from that of P450 isoenzymes so that PHS may activate some compounds that are not efficiently activated by mixed-function oxidase based systems. Mutat Res, 1992 Aug, 268(2), 199 - 210 In vitro and in vivo mutagenicity studies on sporidesmin, the toxin associated with facial eczema in ruminants; Ferguson LR et al.; Sporidesmin, a fungal toxin with widespread distribution within New Zealand, is thought to exert toxic effects through oxidative damage . The purified chemical was tested for its ability to cause point mutations in four strains of Salmonella typhimurium (TA98, TA100, TA102 and TA1537), in the presence and absence of exogenous metabolic activation . Although toxic effects were seen at concentrations exceeding 400 mu gl/plate, there were no significant increases in revertant colonies . In strain TA102, these results were not modified by the presence of glutathione . In AA8 Chinese hamster cells, sporidesmin acted as a potent clastogen, causing chromosomal breaks at concentrations as low as 3 ng/ml, where there was very little reduction in cell viability . Effects were primarily at the chromatid level, but some chromosomal events were also seen . Following low doses, the most common events were chromatid deletions and induction of double minute chromosomes . Interchange events occurred at concentrations of 10 ng/ml and above . The most common of these events was an incomplete chromatid interchange, although some examples of complete chromatid and chromosomal interchange were seen . These in vitro experiments were subsequently extended to an in vivo study of sporidesmin-induced lymphocytic micronuclei (MN) in sheep . In a double blind experiment, 5 sheep were treated with a single high dose of sporidesmin . Blood samples were taken from these, and from 5 untreated sheep, at various intervals before and after treatment . Peripheral blood lymphocytes cultures were harvested and scored for MN in cytokinesis-blocked cells, as a measure of clastogenic activity of sporidesmin in vivo . Following decoding, statistical analysis of the data revealed no significant differences between the MN levels in peripheral blood lymphocytes of sporidesmin-treated and untreated sheep . Although the possibility still exists that clastogenic effects could occur in other species, the data indicate that sporidesmin is not a clastogen in sheep, even though this species is highly susceptible to the toxic effects of sporidesmin. Invest Ophthalmol Vis Sci, 1992 Aug, 33(9), 2626 - 30 Endothelial leukocyte adhesion molecule-1 in endotoxin-induced uveitis; Whitcup SM et al.; Expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells leads to the attachment of polymorphonuclear leukocytes . The sequential expression of ELAM-1 and major histocompatibility complex (MHC) class II antigen was examined in the eyes of 59 Lewis rats with endotoxin-induced uveitis (EIU) after the injection of Salmonella typhimurium endotoxin . The eyes were enucleated at 2-hr intervals . Hematoxylin and eosin-stained paraffin-embedded sections and immunohistochemically stained cryostat sections were graded by two masked observers . The MHC class II antigen was expressed on cells in the iris and ciliary body 4 hr after injection of endotoxin and on the corneal endothelium, 8 hr postinjection . It was found that ELAM-1 was expressed first on cells of the ciliary body and iris 10 hr after the injection of endotoxin and on the corneal endothelium, 22 hr postinjection . Clinical and histopathologic disease developed 16 hr postinjection . Adherence of polymorphonuclear cells to the corneal endothelium was observed at the time of ELAM-1 expression . In conclusion, expression of ELAM-1 on ocular tissue occurred in EIU and appeared to promote polymorphonuclear cell accumulation in the anterior segment of the eye. Mutat Res, 1992 Aug, 280(2), 103 - 7 Mutagenic potency of heterocyclic amines towards Salmonella typhimurium; possible causes of variability in the results observed; Brams A et al.; The potent food mutagens and carcinogens 2-amino-3-methylimidazol{4,5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MEIQ) and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) are probably the most active bacterial mutagens so far discovered . Important discrepancies were found, however, in the specific mutagenicities published for these compounds . This paper analyzes a number of experimental factors that could explain these differences: purity of the compounds, stability under the experimental conditions employed, solvents used, bacterial toxicity, testing procedure, amount and age of the S9 fraction, dose-effect relationships, day-to-day variability, origin of the compounds investigated or of the bacterial strain and age of the strain culture used . None of these factors was found to play a critical role, when the other experimental conditions were strictly standardized . The in-house testing procedure used probably explains the interlaboratory variations observed. J Bacteriol, 1992 Aug, 174(15), 4997 - 5004 Identification of formate dehydrogenase-specific mRNA species and nucleotide sequence of the fdhC gene of Methanobacterium formicicum; White WB et al.; The overlapping fdhA and fdhB genes of Methanobacterium formicicum, which encode the alpha and beta subunits, respectively, of formate dehydrogenase, were cotranscribed as part of an approximately 4.5-kb transcript . An additional gene (fdhC) upstream of fdhA was cotranscribed with fdhA and fdhB . The deduced amino acid sequence suggested that fdhC has the potential to encode a hydrophobic polypeptide with a calculated molecular weight of 29,417 . A hydropathy plot of the hypothetical polypeptide indicated several potential membrane-spanning regions . The putative fdhC gene product had 28% identity with the deduced amino acid sequence of the nirC gene from Salmonella typhimurium . Northern (RNA) blot analyses and primer extension assays located a transcription start site 268 bp upstream of the initiation codon of fdhC . A sequence identical to the consensus promoter sequence for methanogenic organisms was situated between -35 and -25 bp from the proposed transcription start site . In addition to the 4.5-kb transcript, Northern blot analyses detected a 1.1-kb transcript with an fdhC-specific probe and a 3.4-kb transcript with either an fdhA- or fdhB-specific probe . The levels of all three transcripts were significantly greater in cells grown in media supplemented with molybdate. Biotechnology (N Y), 1992 Aug, 10(8), 888 - 92 Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine; Chatfield SN et al.; Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509 . The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge . pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable . Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge . Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies . After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge . Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains. J Pharm Pharmacol, 1992 Aug, 44(8), 704 - 6 The effect of thymidine on the antibacterial and antiviral activity of zidovudine; Shepherd AJ et al.; The effect of thymidine and deoxyadenosine on the antiviral and antibacterial effect of zidovudine was studied in human immunodeficiency virus type 1 (HIV-1) Escherichia coli and Salmonella typhimurium . In quantitative assays, 10 micrograms mL-1 thymidine was shown to increase the 50% inhibitory concentration (IC50) of zidovudine for HIV-1 by approximately 100-fold and to reduce zidovudine (1 microM)-induced protection of C8166 cells from 2.04 to 0.18 log syncytial-forming units . Thymidine also antagonized the antibacterial effect of zidovudine for two E . coli and three S . typhimurium species in a dose-dependent manner; 10 micrograms mL-1 of thymidine increased the minimum inhibitory concentration of zidovudine for E . coli strains by 10-40-fold and for S . typhimurium strains by three-fold . Deoxyadenosine reduced the minimum inhibitory concentration of zidovudine against all five bacterial strains but had no effect on the IC50 of zidovudine for HIV-1, nor did it significantly reverse the antagonism of the antibacterial and antiviral activity of thymidine . The induction of the SOS response in E . coli was reversed in a dose-dependent manner by thymidine while the presence of deoxyadenosine increased induction of the SOS response by zidovudine at suboptimal concentrations. J Immunol, 1992 Aug 1, 149(3), 1023 - 30 Salmonella typhimurium porins stimulate platelet-activating factor synthesis by human polymorphonuclear neutrophils; Tufano MA et al.; Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils . PAF synthesis was independent either from contamination by LPS or generation of TNF . Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity . Porins, indeed, induced a sustained PLA2-dependent mobilization of {14C}arachidonic acid that was inhibited by p-bromodiphenacylbromide . p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase . The addition of 2-lyso-PAF restored PAF synthesis . The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the {3H}acetyl group was incorporated in the synthetized PAF after cell preincubation with {3H}acetyl CoA . The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+ . Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol . As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis. Biochem J, 1992 Aug 1, 285 ( Pt 3), 957 - 64 Photolabelling of Salmonella typhimurium LT2 sialidase . Identification of a peptide with a predicted structural similarity to the active sites of influenza-virus sialidases; Warner TG et al.; The sialidase from Salmonella typhimurium LT2 was characterized by using photoaffinity-labelling techniques . The well-known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non- 2-enonic acid (Neu5Ac2en) was modified to contain an amino group at C-9, which permitted the incorporation of 4-azidosalicylic acid in amide linkage at this position . Labelling of the purified protein with the radioactive (125I) photoprobe was determined to be highly specific for a region within the active-site cavity . This conclusion was based on the observation that the competitive inhibitor Neu5Ac2en in the photolysis mixture prevented labelling of the protein . In contrast, compounds with structural and chemical features similar to the probe and Neu5Ac2en, but which were not competitive enzyme inhibitors, did not affect the photolabelling of the protein . The peptide interacting with the probe was identified by CNBr treatment of the labelled protein, followed by N-terminal sequence analysis . Inspection of the primary structure of the protein, predicted from the cloned structural gene for the sialidase {Hoyer, Hamilton, Steenbergen & Vimr (1992) Mol . Microbiol . 6, 873-884} revealed that the label was incorporated into a 9.6 kDa fragment situated within the terminal third of the molecule near the C-terminal end . Secondary-structural predictions using the Garnier-Robson algorithm {Garnier, Osguthorpe & Robson (1978) J . Mol . Biol . 120, 97-120} of the labelled peptide revealed a structural similarity to the active site of influenza-A- and Sendai-HN-virus sialidases with a repetitive series of alternating beta-sheets connected with loops. J Mol Biol, 1992 Jul 20, 226(2), 447 - 54 Conformational switching in the flagellar filament of Salmonella typhimurium; Trachtenberg S et al.; The flagellar filament of the mutant Salmonella typhimurium strain SJW814 is straight, and has a right-handed twist like the filament of SJW1655 . Three-dimensional reconstructions from electron micrographs of ice-embedded filaments reveal a flagellin subunit that has the same domain organization as that of SJW1655 . Both show slight changes from the domain organization of the subunits from SJW1660, which possesses a straight, left-handed filament . This points to the possible role of changes in subunit conformation in the left-to-right-handed structural transition in filaments . Comparison of the left and right-handed filaments shows that the subunit's orientation and intersubunit bonding appear to change . The orientation of the subunit in the SJW814 filament is intermediate between that of SJW1655 and SJW1660 . Its intermediate orientation may explain why the filaments of SJW1655 and SJW1660 are locked in one conformation, whereas the filament of SJW814 can be induced to switch by, for example, changes in pH and ionic strength. J Mol Biol, 1992 Jul 20, 226(2), 433 - 46 Morphological pathway of flagellar assembly in Salmonella typhimurium; Kubori T et al.; The process of flagellar assembly was investigated in Salmonella typhimurium . Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol . They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis . The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus . Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure . The hook is formed in two distinct steps; formation of its proximal part and elongation . Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring . FlgD is necessary for hook formation, but not for LP-ring formation . A revised pathway of flagellar assembly is proposed based on these and other results. Proc Natl Acad Sci U S A, 1992 Jul 15, 89(14), 6304 - 8 Localization of the Salmonella typhimurium flagellar switch protein FliG to the cytoplasmic M-ring face of the basal body; Francis NR et al.; The direction of rotation of the bacterial flagellum is determined by the flagellar switch . We have localized FliG, one of the switch proteins of Salmonella typhimurium, to the cytoplasmic face of the M ring of the flagellar basal body . This localization was made possible by the discovery of two spontaneous mutants in which the fliF (M ring) and fliG (switch) genes were fused in-frame . In the first mutant, a deletion of 7 base pairs at the 3' end of fliF resulted in an essentially full-length fusion protein . In the second mutant, a larger deletion resulted in a fusion in which 56 amino acids from the carboxyl terminus of FliF and 94 amino acids from the amino terminus of FliG were lost . Both strains were motile and underwent switching; the first strain had a clockwise bias, and the second strain had a counterclockwise bias . Gel electrophoresis and immunoblotting of isolated hook-basal-body complexes verified that they contained the fusion proteins . Electron microscopy revealed additional mass at the cytoplasmic face of the M ring, which could be decorated with anti-FliG antibody . We conclude that the natural location for FliG is at the cytoplasmic face of the M ring and that the stoichiometric ratio between FliF and FliG in wild-type cells is probably 1:1. Cancer Res, 1992 Jul 15, 52(14), 3961 - 4 Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines; Grant DM et al.; Epidemiological studies have established the carcinogenic risk of occupational exposure to aromatic amines such as benzidine, beta-naphthylamine, and 4-aminobiphenyl . Metabolic activation of these chemicals to reactive, genotoxic electrophiles, via enzymatic N-oxidation and subsequent conjugation reactions, is necessary for their carcinogenic potential to be realized . Many aromatic amines are mutagenic in prokaryotic test systems, in the presence of exogenous mammalian activating enzymes such as those contained in hepatic 9000 x g supernatant . However, in the Ames (Salmonella typhimurium) assay, induction of mutations by aromatic amines and nitroarenes is also almost completely dependent upon the activity of the endogenous bacterial enzyme, N-acetyltransferase/O-acetyltransferase . The relevance of this assay to the prediction of the carcinogenic potential of aromatic amines in humans is thus restricted by the likelihood that the bacterial and human enzymes possess different substrate specificities . In this paper we report the construction and use of new tester strains of S . typhimurium that express high levels of functional human arylamine N-acetyltransferases, NAT1 and NAT2, retaining characteristic arylamine substrate specificities that are distinct from those of the bacterial enzyme . These new strains support the mutagenic activation of benzidine, 2-aminofluorene and 2-amino-3,4-dimethylimidazo{4,5-f}quinoline in the Ames test and may provide a new tool for evaluating the carcinogenic potential of aromatic amines. J Biol Chem, 1992 Jul 15, 267(20), 14388 - 97 Characterization of the transcription activator protein C1 of bacteriophage P22; Ho YS et al.; We cloned, expressed, and purified the positive regulatory protein C1 of the temperate phage P22 of Salmonella typhimurium . The purified protein was characterized as to its amino acid composition, protein sequence, molecular weight, and antigenicity . P22 C1 was shown to be a tetrameric protein composed of four identical subunits with M(r) = 10,000 . Moreover, we identified and characterized two P22 C1-dependent phage promoters, P(RE) and Pa23, whose function was completely dependent on C1 both in vitro and in vivo . These two promoters share a common TTGCN6TTGC/T motif in their -35 regions, the same motif recognized by the analogous phage lambda transcription activator protein cII . P22 C1 protein bound selectively to this region and centered on the TTGC repeat motif . Binding and transcription experiments demonstrated that the two promoters respond coordinately to C1 activation . Last, in contrast to lambda cII, the C1 protein exhibited little cooperativity with Escherichia coli RNA polymerase for DNA binding, but because of its stronger inherent binding ability, achieved an overall promoter affinity similar to that observed for cII at its cognate promoter signals. Nucleic Acids Res, 1992 Jul 11, 20(13), 3463 - 9 Insertion (sufB) in the anticodon loop or base substitution (sufC) in the anticodon stem of tRNA(Pro)2 from Salmonella typhimurium induces suppression of frameshift mutations; Sroga GE et al.; The dominant +1 frameshift suppressors sufA6, sufB1 and sufB2, in Salmonella typhimurium act at runs of C and affect tRNA(Pro)1, tRNA(Pro)2 and tRNA(Pro)2, respectively . A recessive +1 frameshift suppressor, sufC, has a similar suppressor specificity (Riddle, D.L., and Roth, J.R., Mol . Biol . 66, 483 and 495, 1972) . We show that sufC strains harbour two frameshift suppressors of which one, sufX201, is allelic to sufB . We cloned the sufB+ wild type allele and by recombination in vivo the mutations sufB1, sufB2 and sufX201 . Determination of the DNA sequence revealed that the sufB1 and sufB2 mutations result in an extra G in the anticodon loop of the minor tRNA(Pro)2 . The sufX201 mutation results in a base substitution (G43 to A43) in the anticodon stem of this tRNA . Although the sufB1 and sufB2 mutations were earlier shown to be dominant, the sufB+ wild type allele on multi copy plasmid inhibited the chromosomal sufB1, sufB2 and sufX201 mediated frameshift suppression but not that mediated by the dominant sufA6 mutation . These results are discussed in view of the possible coding specificity of these mutated tRNAs . The DNA sequence showed a potential consensus promoter sequence upstream of the structural gene for tRNA(Pro)2 and downstream a dyad symmetrical structure followed by a T cluster, a possible rho-independent termination signal . The Salmonella tRNA(Pro)2 gene is identical to the Escherichia coli counterpart reported by Komine, Y . et al . (J . Mol . Biol . 212, 579-598, 1990) . While the 5' flanking sequence similarity between the two species is about 83%, the similarity of the 3' flanking sequence is only 42% . Still, the Salmonella tRNA(Pro)2 gene has a rho-independent transcriptional termination signal similar to the one present in E . coli tRNA(Pro)2 gene. J Biol Chem, 1992 Jul 5, 267(19), 13302 - 5 Identification of 5,6-dimethylbenzimidazole as the Co alpha ligand of the cobamide synthesized by Salmonella typhimurium . Nutritional characterization of mutants defective in biosynthesis of the imidazole ring; Johnson MG et al.; The Co beta-cyano derivative of the cobamide isolated from Salmonella typhimurium was identified as Co alpha-(alpha-5,6-dimethylbenzimidazolyl)-Co beta-cyanocobamide, indicating that this bacterium synthesizes 5,6-dimethylbenzimidazole (DMB) de novo . We found that mutants deficient in the synthesis of DMB can incorporate benzimidazole without modification to form Co alpha-(alpha-benzimidazolyl)cobamide, a cobamide that is physiologically active . The analysis of the nutritional requirements of mutants deficient in DMB synthesis identified 4,5-dimethylphenylenediamine as a putative intermediate in the synthesis of the imidazole ring of DMB . Our results suggest that the CobII region of the cob operon of S . typhimurium only encodes functions involved in the synthesis of the imidazole ring of DMB. JPEN J Parenter Enteral Nutr, 1992 Jul-Aug, 16(4), 343 - 7 Short-term dietary lipid manipulation does not affect survival in two models of murine sepsis; Clouva-Molyvdas P et al.; Dietary lipid manipulation has been shown to have various effects on the immune system, depending on the amount of fat, degree of saturation, and type of fat used . In this study we investigated the role of different sources of fat at different levels on the survival of mice in two models of peritonitis, one with Pseudomonas aeruginosa and the other with Salmonella typhimurium . CF1 mice were pair-fed diets with 5% or 40% of total calories as fat . The source of fat used was coconut oil, oleic acid, safflower oil, or fish oil . Three other diets were tested as well, one with no fat, one with only 0.5% of total calories linoleic acid as the only source of fat, and a control diet that had 12% of total calories as corn oil . At the end of 2 weeks of feeding the experimental diets, mice were challenged with Ps aeruginosa intraperitoneally and mortality was recorded over 1 week . After 3 weeks of feeding the experimental diets, mice were challenged with S typhimurium and mortality was recorded over 2 weeks . No significant differences were seen on survival among groups fed different levels of fat, as well as different sources of fat . We conclude that, overall, 2- and 3-week manipulation of dietary fat does not affect outcome from infection in these models. Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5976 - 80 Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells; Ginocchio C et al.; Penetration of intestinal epithelial cells is an important step in the pathogenesis of Salmonella infections . We have characterized a gene, invE, that is necessary for Salmonella invasion of cultured epithelial cells . The predicted amino acid sequence of InvE showed significant homology to the Yersinia outer membrane protein YopN (LcrE) . Strains of Salmonella carrying mutations in invE were unable to penetrate Henle-407 human intestinal cells and Madin-Darby canine kidney cells, although they were fully capable of attaching to the same cells . Unlike wild-type Salmonella typhimurium, invE mutants failed to change the intracellular free calcium levels or the distribution of polymerized actin in cultured epithelial cells; neither did they alter the normal architecture of the microvilli of polarized Madin-Darby canine kidney cells . Wild-type S . typhimurium was able to rescue the invasive phenotype of the invE mutants in simultaneous infections of cultured epithelial cells although it did not rescue the Escherichia coli strain RDEC-1 . We hypothesize that invE mutants are deficient in triggering the intracellular events that lead to bacterial internalization. Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5956 - 60 The cytoplasmic component of the bacterial flagellar motor; Khan IH et al.; We have used electron microscopy to examine freshly isolated Salmonella typhimurium and Escherichia coli basal flagellar fragments, purified without resort to extremes of pH or ionic strength . Such fragments contain the large bell-like basal structures visualized recently in freeze-substituted or fixed preparations . We have found mot (non-motile) mutants produced by lesions in fli genes (G, M, N) in which the bell structures do not coisolate with the flagellar basal body . The coisolation of the bell with the flagellar basal body was unaffected in strains lacking the genes for the motility-associated Mot proteins or for the Che family of proteins, which are necessary for chemotaxis . Proper assembly and interaction of the cytoplasmically located bell with the membrane-associated flagellar basal structures appears to be necessary for motor function . The FliG, FliM, and FliN proteins are thought to form a structural complex responsible for energization and switching of the flagellar motor . Our findings are consistent with the existence of such a complex and imply that it forms part of the flagellar bell. Mol Biol Evol, 1992 Jul, 9(4), 688 - 706 Nucleotide sequence, function, activation, and evolution of the cryptic asc operon of Escherichia coli K12; Hall BG et al.; The cryptic asc (previous called "SAC") operon of Escherichia coli K12 has been completely sequenced . It encodes a repressor (ascG); a PTS enzyme IIasc for the transport of arbutin, salicin, and cellobiose (ascF); and a phospho-beta-glucosidase that hydrolyzes the sugars which are phosphorylated during transport (ascB) . ascG and ascFB are transcribed from divergent promoters . The cryptic operon is activated by the insertion of IS186 into the ascG (repressor) gene . The ascFB genes are paralogous to the cryptic bglFB genes, and ascG is paralogous to galR . The duplications that gave rise to these paralogous genes are estimated to have occurred approximately 320 Mya, a time that predates the divergence of E . coli and Salmonella typhimurium. J Toxicol Environ Health, 1992 Jul, 36(3), 165 - 75 Mutagenicity of trinitrotoluene and its metabolites formed during composting; Tan EL et al.; TNT was mutagenic for Salmonella typhimurium without the need of a rat liver metabolic activation system (S9) . The mutagenic potency of TNT decreased in proportion to the number of nitro groups that were reduced to the amino form . The presence of a nitro group on the 4 position of the diamino congener is necessary for mutagenicity . Among the active congeners, mutagenicity was generally greater for TA100 than TA98, except that for the 4-amino congener the reverse was true . In cases when S9 was included in the assay, there was always a decrease in the number of mutants induced as compared with those without S9 . Tetryl behaved like TNT, except that it was approximately three times more potent . RDX and HMX were not mutagenic under the conditions of the assay . When TNT was composed, the major metabolites identified in organic extracts of compost samples were the 2-amino and 4-amino congeners . An acetonitrile extract of compost was tested and found to be more mutagenic for TA98 than TA100, much like the authentic 4-amino congener, but the amount of this congener in the extract did not account for the degree of mutagenicity. J Bacteriol, 1992 Jul, 174(14), 4798 - 806 Molecular modelling of the three-dimensional structure and conformational flexibility of bacterial lipopolysaccharide; Kastowsky M et al.; Molecular modelling techniques have been applied to calculate the three-dimensional architecture and the conformational flexibility of a complete bacterial S-form lipopolysaccharide (LPS) consisting of a hexaacyl lipid A identical to Escherichia coli lipid A, a complete Salmonella typhimurium core oligosaccharide portion, and four repeating units of the Salmonella serogroup B O-specific chain . X-ray powder diffraction experiments on dried samples of LPS were carried out to obtain information on the dimensions of the various LPS partial structures . Up to the Ra-LPS structure, the calculated model dimensions were in good agreement with experimental data and were 2.4 nm for lipid A, 2.8 nm for Re-LPS, 3.5 nm for Rd-LPS, and 4.4 nm for Ra-LPS . The maximum length of a stretched S-form LPS model bearing four repeating units was evaluated to be 9.6 nm; however, energetically favored LPS conformations showed the O-specific chain bent with respect to the Ra-LPS portion and significantly smaller dimensions (about 5.0 to 5.5 nm) . According to the calculations, the Ra-LPS moiety has an approximately cylindrical shape and is conformationally well defined, in contrast to the O-specific chain, which was found to be the most flexible portion within the molecule. J Bacteriol, 1992 Jul, 174(14), 4746 - 52 Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium; Klena JD et al.; Analysis of the sequence of a 4.3-kb region downstream of rfaJ revealed four genes . The first two of these, which encode proteins of 27,441 and 32,890 Da, were identified as rfaY and rfaZ by homology of the derived protein sequences of their products to the products of similar genes of Salmonella typhimurium . The amino acid sequences of proteins RfaY and RfaZ showed, respectively, 70 and 72% identity . Genes 3 and 4 were identified as rfaK and rfaL on the basis of size and position, but the derived amino acid sequences of the products of these genes showed very little similarity (about 12% identity) between Escherichia coli K-12 and S . typhimurium . The next gene in the cluster, rfaC, encodes a product which also shows strong protein sequence homology between E . coli K-12 and S . typhimurium, as do the rfaF and rfaD genes which lie beyond it . Thus, the rfa gene cluster appears to consist of two blocks of genes which are conserved flanking a central region of two genes which are not conserved between these species . Although the RfaL protein sequence is not conserved, hydropathy plots of the two RfaL species are nearly identical and indicate that this is a typical integral membrane protein with 10 or more potential transmembrane domains . We noted the similarity of the structure of the rfa gene cluster to that of the rfb gene cluster, which has now been sequenced in several Salmonella serovars . The rfb cluster also contains a gene which lies within a central nonconserved region and encodes an integral membrane protein similar to protein RfaL . We speculate that protein RfaL may interact in a strain- or species-specific way with one or more Rfb proteins in the expression of surface O antigen. J Bacteriol, 1992 Jul, 174(14), 4736 - 45 Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core; Pradel E et al.; Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes . The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene . It has been designated rfaS . The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein . The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S . typhimurium . Escherichia coli K-12 restriction fragments carrying these genes complement an S . typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant . The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E . coli K-12 have sugar and acceptor specificities different from those of S . typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms . Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome . The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules . Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene. J Bacteriol, 1992 Jul, 174(13), 4338 - 49 Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family; Galan JE et al.; One of the earliest steps in the pathogenic cycle of the facultative intracellular pathogen Salmonella spp . is the invasion of the cells of the intestinal epithelium . We have previously identified a genetic locus, inv, that allows Salmonella spp . to enter cultured epithelial cells . invA is a member of this locus, and it is the first gene of an operon consisting of at least two additional invasion genes . We have constructed strains carrying nonpolar mutations in invA and examined the individual contribution of this gene to the invasion phenotype of Salmonella typhimurium . Nonpolar S . typhimurium invA mutants were deficient in invasion of cultured epithelial cells although they were fully capable of attaching to the same cells . In addition, unlike wild-type S . typhimurium, invA mutants did not alter the normal architecture of the microvilli of polarized epithelial cells nor did they cause any alterations in the distribution of actin microfilaments of infected cells . The invasion phenotype of invA mutants was readily rescued by wild-type S . typhimurium when cultured epithelial cells were simultaneously infected with both strains . On the contrary, in a similar experiment, the adherent Escherichia coli strain RDEC-1 was not internalized into cultured cells when coinfected with wild-type S . typhimurium . The invA locus was found to be located at about 59 min on the Salmonella chromosome, 7% linked to mutS . The nucleotide sequence of invA showed an open reading frame capable of encoding a polypeptide of 686 amino acids with eight possible membrane-spanning regions and a predicted molecular weight of 75,974 . A protein of this size was visualized when invA was expressed in a bacteriophage T7 RNA polymerase-based expression system . The predicted sequence of InvA was found to be homologous to Caulobacter crescentus FlbF, Yersinia LcrD, Shigella flexneri VirH, and E . coli FlhA proteins . These proteins may form part of a family of proteins with a common function, quite possibly the translocation of specific proteins across the bacterial cell membrane. J Bacteriol, 1992 Jul, 174(13), 4317 - 23 Effect of Salmonella typhimurium ferric uptake regulator (fur) mutations on iron- and pH-regulated protein synthesis; Foster JW et al.; Fur is an important regulatory protein known to function in the presence of iron as a repressor of iron-controlled genes . It was recently discovered that Fur is also essential to Salmonella typhimurium for mounting an adaptive acid tolerance response (J . W . Foster, J . Bacteriol 173:6896-6902, 1991) . Because little is known about the effect of Fur on the physiology of this enteric pathogen, a systematic two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis was conducted to identify proteins whose synthesis is linked to iron levels . Mutations in the fur locus were identified and used to classify which proteins are controlled by Fur . Thirty-six proteins were overtly affected by iron availability, most of which were clearly under the control of Fur . Although most of the Fur-dependent proteins were under negative control, a significant portion (15 of 34) appeared to be under a form of positive control . Nine of the positively controlled proteins required Fur and iron for expression . However, Fur lacking iron was also required for the induction of six gene products . Surprisingly, not all iron-regulated proteins were controlled by Fur and not all Fur-dependent proteins were obviously regulated by iron status . Because fur mutants fail to mount an effective acid tolerance response, we made a comparative two-dimensional PAGE analysis of 100 total acid- and iron-regulated gene products . Production of most of these proteins was regulated by only one of the two stresses, yet a clear subset of seven genes were influenced by both acid and iron and were also controlled by fur . These proteins were also members of the acid tolerance response modulon . Consistent with the fur effect on pH-regulated protein synthesis, fur mutants lacked the inducible pH homeostasis system associated with the acid tolerance response . The results provide further evidence that Fur has an extensive impact on gene expression and cellular physiology and suggest an explanation for the acid-sensitive nature of fur mutants. J Bacteriol, 1992 Jul, 174(13), 4197 - 204 An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase; Mirel DB et al.; Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase . Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro . Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site . Direct evidence linking these genes to motility functions in B . subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al . (A . R . Zuberi, C . Ying, H . M . Parker, and G . W . Ordal, J . Bacteriol . 172:6841-6848, 1990), to within this mot . operon . Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B . subtilis . These current findings support our earlier suggestions that sigma D in B . subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions . Sigma F, the enteric homolog of sigma D, controls similar functions in E . coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family. Infect Immun, 1992 Jul, 60(7), 2855 - 62 Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium; Sjostedt A et al.; A 17-kDa lipoprotein, TUL4, of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F . tularensis-primed humans . A DNA fragment of the live vaccine strain F . tularensis LVS encoding TUL4 was cloned into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant . Expression of the protein by the recombinant S . typhimurium chi 4072 (pTUL4-15) was maintained after passage in BALB/cJ mice . When mice were immunized with S . typhimurium chi 4072(pTUL4-15), some animals showed an antibody response and a T-cell response to TUL4 . When the immunized mice were challenged with the live vaccine strain F . tularensis LVS, bacterial counts in the liver and spleen were lower than in animals immunized with S . typhimurium chi 4072 . Immunization with F . tularensis LVS caused a much stronger protection against the challenge than did immunization with S . typhimurium chi 4072(pTUL4-15) . The present study demonstrated that the 17-kDa lipoprotein TUL4 of F . tularensis is involved in a protective immunity to tularemia . Possibly, several T-cell-reactive proteins of the organism have to contribute for optimal protection to be achieved. Infect Immun, 1992 Jul, 60(7), 2823 - 7 Construction of a recombinant oral vaccine against Salmonella typhi and Salmonella typhimurium; Cao Y et al.; The viaB gene coding for the Vi antigen of Salmonella typhi Ty2 was subcloned into expression vector pYA248 . The recombinant plasmid was termed SMM202 and transformed into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant . Recombinant S . typimurium Vi4072 had the ability to produce Vi capsular polysaccharide and also to invade and colonize the small intestine, mesenteric lymph nodes, and spleen of BALB/c mice . Mice orally immunized with Vi4072 developed serum and secretory antibody responses to the Vi antigen, as measured by a passive hemagglutination assay . Mice developed a delayed-type hypersensitivity following a footpad injection with Vi antigen after being sensitized orally with a suitable dose of Vi4072 . Immunization of mice with Vi4072 afforded complete protection against fatal infection with virulent S . typhi Ty2 . All data indicate that this route of antigen delivery is effective for stimulating antibody-mediated immunity and for inducing a cell-mediated immune response in BALB/c mice . Thus, S . typhimurium Vi4072 may serve as a vaccine for protection against typhoid fever and salmonellosis caused by S . typhimurium. Infect Immun, 1992 Jul, 60(7), 2758 - 68 Salmonella choleraesuis and Salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Kupffer cells and multiply intracellularly; Nnalue NA et al.; Male Sprague-Dawley rats were inoculated intravenously with Salmonella choleraesuis or Salmonella typhimurium and used over 3 consecutive days to produce highly enriched (greater than 95% homogenous) preparations of Kupffer and mononuclear cells (KC), liver endothelial cells (LEC), and hepatocytes . The methods involved collagenase perfusion of the liver in situ, differential centrifugation of liver cells over a Percoll gradient, and selective attachment of the cells to plastic or to culture dishes coated with collagen . The different cell preparations were then assayed for the number and location, intracellular or extracellular, of associated viable bacteria . Most of the viable bacteria recovered were associated with KC and were mainly intracellular . The intracellular bacteria in KC from rats infected with either bacterial strain increased about 20- to 50-fold over 2 days . Some of the bacteria associated with LEC and in some experiments with hepatocytes also survived treatment with gentamicin and increased in number with time . Intracellular bacteria were readily visualized in KC by light microscopy and transmission electron microscopy . On rare occasions, bacteria were seen within LEC from rats infected with S . choleraesuis but not from those infected with S . typhimurium . Microcolonies of S . typhimurium but not of S . choleraesuis were occasionally found on the surface of some LEC . Bacteria were not seen within or on the surface of hepatocytes by transmission or scanning electron microscopy . The integration of microscopic and viability data suggested that most intracellular S . choleraesuis organisms in KC had been killed whereas most intracellular S . typhimurium organisms were viable. Antimicrob Agents Chemother, 1992 Jul, 36(7), 1460 - 5 Mechanisms of antibacterial action of tachyplesins and polyphemusins, a group of antimicrobial peptides isolated from horseshoe crab hemocytes; Ohta M et al.; Tachyplesins I and II and polyphemusins I and II, cationic peptides isolated from the hemocytes of horseshoe crabs, show bactericidal activities with similar efficiencies for both gram-negative and gram-positive bacteria . Tachyplesin I inhibited bacterial growth irreversibly within 40 min . A subinhibitory concentration of tachyplesin I sensitized gram-negative bacteria to the bactericidal actions of novobiocin and nalidixic acid, although polymyxin B-resistant strains which have altered lipopolysaccharides were susceptible to tachyplesin I . This implies that tachyplesin permeabilizes the outer membrane and that the likely target of its action is outer membrane constituents other than lipopolysaccharides . On the other hand, a defensin-susceptible phoP strain of Salmonella typhimurium was also susceptible to tachyplesin I . Tachyplesin I rapidly depolarized the inverted inner-membrane vesicles of Escherichia coli . These results suggest that depolarization of the cytoplasmic membrane, preceded by the permeabilization of the outer membrane for gram-negative bacteria, is associated with tachyplesin-mediated bactericidal activity . The similarity between the actions of tachyplesin and those of defensin was discussed. Anticancer Res, 1992 Jul-Aug, 12(4), 1287 - 90 Effects of different inducers of cytochrome P450 on the mutagenesis of the tobacco-specific nitrosamine 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) in Salmonella typhimurium TA1535; Teel RW; The effects of six inducers of isoenzymes of cytochrome P450 on the mutagenicity of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Salmonella typhimurium strain TA1535 by hamster liver S9 and microsomes were investigated . Comparisons of the effects of dimethylsulfoxide (DMSO) as solvent for NNK were also made . The inducing agents were Aroclor 1254 (AROC), 3-methylcholanthrene (MC), phenobarbital (PB), dexamethasone (DXM), ethanol (ETOH) and isosafrol (ISF) . The number of histidine-independent colonies induced by NNK in saline mediated by S9 from the inducing agents was as follows: ISF = AROC greater than PB greater than MC greater than DXM greater than ETOH . AROC-induced microsomes produced the most revertants by NNK (saline) greater than MC greater than PB = DXM and ISF greater than ETOH . The number of revertant colonies induced by NNK in DMSO was significantly less than that by NNK in saline for both hamster liver S9 and microsomes irrespective of the inducing agent . The greatest inhibitory effect of DMSO was observed with ISF-induced S9. J Dairy Sci, 1992 Jul, 75(7), 1821 - 5 Antigenic homology of endotoxin with a coliform mastitis vaccine strain, Escherichia coli O111:B4 (J5); Tyler JW et al.; This study examined recognition of heterologous Gram-negative endotoxin by antibodies recognizing common lipopolysaccharide core antigens . Gram-negative endotoxins from 11 heterologous bacterial strains were tested for recognition by antibodies against common lipopolysaccharide core antigens . Serum was harvested from a calf immunized with the Rc mutant, Escherichia coli O111:B4 (J5), and affinity purified against endotoxin derived from an Ra mutant, Salmonella typhimurium, producing an antibody reagent recognizing homologous Gram-negative core antigens present in the Rc mutant vaccinal antigen . This reagent demonstrated reactivity against 11 chemically purified Gram-negative endotoxins . Included were endotoxins derived from 3 smooth E . coli species, 2 Salmonella spp., Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and lipid A . Endotoxin derived from K . pneumoniae had significantly higher ELISA reactivity with core antigen specific antibodies than did endotoxin derived from either E . coli O111:B4 (J5) or P . aeruginosa . These results suggest immunization with R mutant bacterins may have utility in the prevention of Gram-negative mastitis even when whole bacteria react poorly with antibodies recognizing common core antigens. Gaoxiong Yi Xue Ke Xue Za Zhi, 1992 Jul, 8(7), 349 - 57 Molecular characterization of R-plasmids pST1 and pST2 from Salmonella typhimurium S24; Peng CF; Salmonella typhimurium S24 was isolated in September 1986 at Kaohsiung Medical College Hospital, Kaohsiung, Taiwan, from a patient suffering from gastroenteritis during an outbreak of salmonellosis . Two conjugative R-plasmids have been isolated from Escherichia coli K-12 14R525, which was mated with S . typhimurium S24 . The two R-plasmids found in S . typhimurium S24 belong to two different incompatibility (Inc) groups: the 130-kilobase IncFI plasmid pST1 and the 56-kb IncN plasmid pST2 . These two R-plasmids of pST1 and pST2 together mediate resistance to multiple antibiotics in S . typhimurium S24 . By DNA probes hybridization, plasmid pST1 was shown to carry an enteric type II chloramphenicol acetyltransferase (CAT) gene, a class C tetracycline resistance (TetR) gene and a type III dihydrofolate reductase (DHFR) gene, all of which confer resistance to chloramphenicol, tetracycline, and trimethoprim respectively . A Richmond's type III beta-lactamase gene was located on each plasmid of pST1 and pST2 . beta-lactamases specified by both plasmids pST1 and pST2 conferred high level resistance to amoxicillin, carbenicillin, piperacillin, sulbenicillin, ticarcillin in addition to ampicillin . A novel aminoglycoside 6'-N-acetyltransferase {AAC(6')} was demonstrated on plasmid pST2 . This AAC(6') enzyme modified kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, sisomicin, butirosin and ribostamycin. J Biochem (Tokyo), 1992 Jul, 112(1), 93 - 101 Nucleotide sequences and characterization of liv genes encoding components of the high-affinity branched-chain amino acid transport system in Salmonella typhimurium; Matsubara K et al.; A 7.6-kb fragment of Salmonella typhimurium LT2 containing the liv gene cluster, which specifies the high-affinity branched-chain amino acid transport system (LIV-I), has been isolated . The upstream region contains the livB and livC genes encoding the leucine-isoleucine-valine-threonine and leucine-specific binding proteins, respectively . In this study, the nucleotide sequence of the 4-kb downstream segment was determined and found to contain four reading frames, designated as livA, livE, livF, and livG, that encode putative membrane-associated proteins . The livA and livE genes encode hydrophobic proteins composed of 308 and 425 amino acid residues, respectively . The livF and livG genes encode hydrophilic proteins of 255 and 237 amino acids, respectively; both the proteins contain consensus amino acid sequences found in proteins with ATP-binding sites . These four genes linked together have a potential rho-independent transcriptional terminator adjacent to the 3'-end of livG . No promoter sequence was found in the immediate upstream region of the livAEFG cluster . The livA, livE, livF, and livG gene products were identified as proteins with apparent M(r)s of 25,500, 34,500, 28,000, and 26,000, respectively, by SDS-polyacryl-amide gel electrophoresis . The deduced amino acid sequences of these four proteins showed strong homology to those of the corresponding membrane-associated proteins required for the high-affinity branched-chain amino acid transport systems from both Escherichia coli and Pseudomonas aeruginosa. Avian Dis, 1992 Jul-Sep, 36(3), 813 - 5 Survey of clinical psittacine bird sera for Salmonella typhimurium agglutinins; Grimes JE et al.; Of 2407 serum samples from various kinds of psittacine birds submitted for Chlamydia serology, 2343 (97.4%) were negative, 25 (1.0%) were equivocal, and 39 (1.6%) were positive for Salmonella typhimurium agglutinins . In additional serum samples from two groups of African gray parrots, the prevalence of agglutinins was 0.0% (0/38) in the Timneh variety and 24.0% (6/25) in the Congo variety . In sera from one macaw, one cockatoo, and one Amazon parrot, which were negative for chlamydial antibody activity, there were strongly reactive agglutinins for S . typhimurium . Two Amazon parrots had antibody activity against Salmonella and Chlamydia antigens. J Bacteriol, 1992 Jul, 174(14), 4833 - 7 Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene; Wu WF et al.; The vitamin B12 (B12)-mediated repression of the metE gene in Escherichia coli and Salmonella typhimurium requires the B12-dependent transmethylase, the metH gene product . It has been proposed that the MetH-B12 holoenzyme complex is involved directly in the repression mechanism . Using Escherichia coli strains lysogenized with a lambda phage carrying a metE-lacZ gene fusion, we examined B12-mediated repression of the metE-lacZ gene fusion . Although B12 supplementation results in a 10-fold repression of metE-lacZ expression, homocysteine addition to the growth medium overrides the B12-mediated repression . In addition, B12-mediated repression of the metE-lacZ fusion is dependent on a functional MetR protein . When a metB mutant was transformed with a high-copy-number plasmid carrying the metE gene, which would be expected to reduce intracellular levels of homocysteine, metE-lacZ expression was reduced and B12 supplementation had no further effect . In a metJ mutant, B12 represses metE-lacZ expression less than twofold . When the metJ mutant was transformed with a high-copy-number plasmid carrying the metH gene, which would be expected to reduce intracellular levels of homocysteine, B12 repression of the metE-lacZ fusion was partially restored . The results indicate that B12-mediated repression of the metE gene is primarily a loss of MetR-mediated activation due to depletion of the coactivator homocysteine, rather than a direct repression by the MetH-B12 holoenzyme. Mutat Res, 1992 Jul, 282(3), 219 - 25 The presence of genotoxic and bioactive components in indigo dyed fabrics--a possible health risk? Rannug U, Bramstedt H, Nilsson U. Extracts of pure cotton and jeans fabrics were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 . The vat dye indigo, technical grade as well as 98% and greater than 99.5% pure, was also tested for mutagenicity . Synthetic indigo, indirubin and isatin were tested for TCDD receptor affinity in competition experiments in vitro . The mutagenicity of the extracts was associated with the cotton denim and nondyed cotton gave only marginal effects . The mutagenicity of the indigo dyed fabrics was dependent on type and treatment of the fabrics . Extracts of both bleached and nonbleached jeans gave mutagenic effects on TA98 +/- S9 and TA100 +/- S9 . The greatest effects were seen in the presence of S9 . Bleaching gave an additional increase in the mutagenicity in the absence of S9 . Normal washing of the fabrics after bleaching reduced the mutagenicity . Synthetic indigo of technical grade or 98% pure showed mutagenic effects, especially on TA98 + S9 . Further purification to 99.5% reduced the mutagenicity to 24 revertants/mg (6.2 rev/mu mole) . Considering the amount of indigo in the extracts and its low mutagenicity, the genotoxicity of jeans extracts must be caused by other unknown components . However, indigo showed a high (Kd = 1.9 nM) affinity for the Ah or TCDD receptor . Indigo can therefore still be a potential health risk either by eliciting toxic effects of other compounds or by being a nongenotoxic carcinogen . The worldwide use of jeans with a possible exposure of a large population to genotoxic and biologically active components emphasizes the need for a more thorough characterization of these effects. Mutat Res, 1992 Jul, 268(1), 35 - 41 Photo-enhancement of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrine; Iwamoto Y et al.; The frameshift mutagenicity of the DNA intercalating drug proflavine is known to be enhanced by photoirradiation of bacterial cultures . To determine whether this phenomenon was also present in acridine-derived antitumour drugs, cultures of Salmonella typhimurium were exposed to the antileukaemia agent amsacrine and the experimental agent N-{2-(dimethylamino)ethyl}acridine-4-carboxamide dihydrochloride (acridine carboxamide) in the presence or absence of visible light . A small increase in mutagenicity was observed with amsacrine but not with acridine carboxamide . A series of analogues of amsacrine were then tested, and a striking relationship was found between the minimum drug concentration for mutagenicity and DNA binding affinity . In each case, photoirradiation was associated with a small increase in mutagenicity . Each of the compounds showing the photo-enhancement effect was capable of reversible one-electron oxidation . It is suggested that this oxidation occurs in bacteria, and that the DNA binding constant of the resulting acridine radical species will increase because of the extra positive charge . This increased DNA binding would be sufficient to explain the photo-enhancement of mutagenicity of these drugs. Mutat Res, 1992 Jul, 268(1), 1 - 9 Quantitative structure-mutagenic activity relationships of triazino indole derivatives; Garcia E et al.; The mutagenicity of 3-(4'-benzylidenamino)-5H-1,2,3-triazin{5,4-b}-indol-4-one derivatives, new compounds with considerable platelet antiaggregating activity, was assayed with the Ames test using the Salmonella typhimurium strains TA97, TA98, TA100 and TA102 . The adaptive least-squares method (ALS method) was used to carry out a quantitative structure-activity relationship (QSAR) analysis . Three equations, based on 10 congeners, were found for strains TA97, TA98 and TA100 . The results suggest that lipophilicity of the substituent decreases the mutagenicity of the series. J Bacteriol, 1992 Jul, 174(14), 4614 - 21 Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide; Clarke BR et al.; Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C . Whitfield, J . C . Richards, M . B . Perry, B . R . Clarke, and L . L . MacLean, J . Bacteriol . 173:1420-1431, 1991) . The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose . To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K . pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster . In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II . The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K . pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression . However, plasmids carrying rfbkpO1 did complement a mutation in K . pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers . Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes . Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism. Mutat Res, 1992 Jul, 280(1), 67 - 71 On the induction of umu gene expression in Salmonella typhimurium strain TA1535/pSK1002 by some nitrofurans; Pal AK et al.; Several nitrofurans were found to induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 as defined on the basis of at least a 2-fold increase of beta-galactosidase activity over the background level . beta-Galactosidase activity increased with increasing concentrations of the chemical, attained a maximum at a concentration which was different for different nitrofurans used, and then gradually decreased with a further increase of the nitrofuran concentration . The umu gene expression test revealed that the genotoxic activity was highest for furazolidone and lowest for 5-nitro-2-furaldehyde. Mutat Res, 1992 Jul, 280(1), 55 - 65 Mutagenicity of quinolines in Salmonella typhimurium TA100 . A QSAR study based on hydrophobicity and molecular orbital determinants; Debnath AK et al.; The mutagenicity of 33 quinolines in the Salmonella test using TA98 and TA100 cells has been reported . Significant activity was found only with TA100 cells . Quantitative structure-activity relationships (QSAR) could be formulated using molecular orbital parameters or Hammett constants and hydrophobic parameters for those compounds with substituents in the 6, 7 and 8 positions . The QSAR points to the 2-position on the quinoline ring as being the site for activation by S9 oxidation. Infect Immun, 1992 Jul, 60(7), 2969 - 75 Effects of staphylococcal enterotoxin B on rodent mast cells; Komisar J et al.; Staphylococcal enterotoxin B (SEB) was tested in rodent mast cell cultures for the release of serotonin . Both rat RBL-2H3 mast cells and murine peritoneal cells released serotonin after SEB stimulation in culture . Release of serotonin in RBL-2H3 cells depended on the concentration of SEB; an appreciable release was seen at 50 micrograms/ml . The release of serotonin was not due to cell death . Serotonin release could be enhanced by bradykinin but not by vasoactive intestinal peptide, substance P, lipopolysaccharide from Salmonella typhimurium, the calcium ionophore A23187, acetylcholine, adenosine, 5-hydroxyeicosatetraenoic acid, indomethacin, or phorbol myristate acetate . SEB bound directly to the membrane of RBL-2H3 mast cells, and the SEB-binding site, the presumptive receptor, appeared to be a protein . The SEB receptor could not be capped under membrane-capping conditions, and serotonin release could not be enhanced by attempts to cross-link the receptor . These results suggest that mast cells ma