Salmonella typhimurium

Mol Gen Genet, 1996 Oct 16, 252(5), 626 - 9
The nadB gene of Salmonella typhimurium complements the nicotinic acid auxotrophy of Shigella flexneri; Mantis NJ et al.; Shigella species are characteristically nicotinic acid (NA) auxotrophs . The invasive S . flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding the nadB gene of Salmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacks L-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway . The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.

Commun Dis Rep CDR Rev, 1996 Oct 11, 6(11), R159 - 62
An outbreak of Salmonella typhimurium DT104 food poisoning associated with eating beef; Davies A et al.; An outbreak of Salmonella typhimurium DT104 infection in Shropshire in May 1995 was identified when four isolates were noted to be from members or supporters of a local football team that had held several social functions in the same week . The subsequent investigation identified 16 people with gastrointestinal symptoms and 12 with microbiologically confirmed infection . The outbreak was complex, associated with several social functions on different days, but infection was associated with eating beef at a public house . A number of errors were detected in the cooking, storage, and handling of the implicated food . The investigation identified beef as the vehicle of infection in this outbreak but was unable to show whether it was the original source of infection or whether cross or manual contamination occurred in the kitchen.

Commun Dis Rep CDR Rev, 1996 Oct 11, 6(11), R155 - 9
Epidemiological application of differentiating multiresistant Salmonella typhimurium DT104 by plasmid profile; Threlfall EJ et al.; Human isolates of multiresistant Salmonella typhimurium definitive phage type (DT) 104 in England and Wales are currently second in number only to those of S . enteritidis phage type 4 . Differentiation of strains is essential in epidemiological investigations and the value of one method, plasmid profile typing, has been assessed in a study of 600 isolates of S . typhimurium DT 104 with multiresistant antibiograms (R-types) ACSSuT, ACSSuTCp and ACSSuTTm from humans, food animals, human food, pets, and animal feed made in England and Wales from January 1990 to April 1996 . Twenty plasmid profile (PP) types have been identified in isolates of R-type ACSSuT and ACSSuTCp . One profile type, with a single plasmid of 60 megadaltons-PP type A-has predominated, but identification of PP type has proved useful in some epidemiological investigations . A further four PP types have been identified in isolates of DT 104 R-type ACSSuTTm, in which resistance to trimethoprim is encoded by a plasmid of 4.6 megadaltons and the two commonest PP types are related to those also common in DT 104 R-type ACSSuT . Methods of differentiating within the commonest profile type are now needed.

Gene, 1996 Oct 10, 175(1-2), 23 - 7
A Shigella flexneri invasion plasmid gene, ipgH, with homology to IS629 and sequences encoding bacterial sugar phosphate transport proteins; Venkatesan MM et al.; Sequences representing the 2684 bp flanking the ipaH4.5 gene on the invasion plasmid of Shigella flexneri 5 indicate an unusual fusion gene, designated ipgH, in which the first 27 amino acids (aa) are identical to ORF2 of IS629 . The aa sequence encoded by the remainder of ipgH bears significant homology to Escherichia coli and to Salmonella typhimurium GlpT and UhpT proteins and to the S . typhimurium PgtP protein, which are involved in the uptake of high-energy sugar phosphates from an external source.

Eur J Pharmacol, 1996 Oct 10, 313(1-2), 129 - 34
Tolerance to lipopolysaccharide-induced increase in vascular permeability in mouse skin; Fujii E et al.; We investigated whether tolerance develops to the lipopolysaccharide-induced increase in vascular permeability of mouse skin on pretreatment with Salmonella typhimurium lipopolysaccharide . Lipopolysaccharide-induced plasma extravasation was assessed by determining Pontamine sky blue dye accumulation in the skin where lipopolysaccharide was injected s.c . 2 h previously . When mice were pretreated with lipopolysaccharide (0.15 mg/kg i.p.), the dye leakage induced by s.c . challenge with lipopolysaccharide (400 micrograms/site) was significantly, inhibited for 2-24 h after pretreatment, indicating the development of lipopolysaccharide tolerance . At 4 h after lipopolysaccharide (0.15 mg/kg i.p.), the dose-response curve of dye leakage against the challenge dose of lipopolysaccharide shifted about 2-fold to the higher dose . The dye leakage induced by lipopolysaccharide was inhibited by pretreatment with lipopolysaccharide in a dose-dependent manner (0.05-0.15 mg/kg i.p.) . Lipopolysaccharide tolerance was not seen in adrenalectomized mice . When mice were pretreated with lipopolysaccharide and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, at the same time, the hyporesponsiveness to lipopolysaccharide challenge disappeared . However, L-NAME was ineffective to inhibit the development of lipopolysaccharide tolerance when administered 24 h after lipopolysaccharide pretreatment or just before the lipopolysaccharide challenge . Tumor necrosis factor-alpha and interleukin-1 alpha but not interleukin-6 induced a similar hyporesponsiveness to lipopolysaccharide . These results suggest that tolerance develops to the lipopolysaccharide-induced increase in vascular permeability in mouse skin after a single lipopolysaccharide administration and that endogenous glucocorticoids and NO are necessary for induction of lipopolysaccharide tolerance . Hyporesponsiveness induced by lipopolysaccharide pretreatment may be mediated by production of some cytokines such as tumor necrosis factor-alpha or interleukin-1 alpha.

Genetika, 1996 Oct, 32(10), 1333 - 40
{Specificity and time of the appearance of His+ reversions induced by histidine starvation in Salmonella typhimurium}; Gizatullin FSh et al.; It was previously established that reversion of the hisG46 allele of Salmonella typhimurium to prototrophy occurred upon histidine starvation . In this paper, it was shown that histidine starvation does not affect the appearance of mutants resistant to L-arabinose and rifampicin . Threonine starvation did not change the frequency of His+ revertants . Analysis of His+ revertant clones did not reveal additional L-arabinose resistance mutations . Thus, these experiments allowed the conclusion that amino acid starvation does not lead to a nonspecific increase in the mutation rate . In addition, it was shown that spontaneous His+ revertants start to arise after two to three hours of histidine starvation, this process lasting for four days . Nevertheless, original His+ cells did not grow in a culture generating His+ revertants . Traces of histidine and novobiocin added to a minimal medium retarded reversion realization . However, the occurrence of revertants was not markedly inhibited by chloramphenicol . Based on the results, it is assumed that adaptive His+ reversions occurred due to a special mode of replication induced upon histidine starvation and requiring no de novo protein synthesis.

Vaccine, 1996 Oct, 14(14), 1384 - 90
Delivery of the Pertactin/P.69 polypeptide of Bordetella pertussis using an attenuated Salmonella typhimurium vaccine strain: expression levels and immune response; Anderson R et al.; Pertactin/P.69, a surface-associated polypeptide antigen of Bordetella pertussis, was expressed in a Salmonella typhimurium aroA aroD vaccine strain, BRD509, using different expression systems, and the immune response in mice against these constructs was compared . Initially, Pertactin/P.69 was expressed on the surface of BRD509 from a single copy of the gene encoding the antigen localized on the Salmonella chromosome . As previously shown, secretory and humoral antibody responses could not be detected following multiple immunization with this strain (BRD640) . However, a strong anti-Pertactin/P.69 proliferative response was observed in murine splenocytes following a single oral dose with BRD640 . The stimulated splenocytes secreted interferon-gamma but not interleukin-5, indicating that BRD640 induced a Th1 type T-helper response against Pertactin/P.69 . We wished to construct a vaccine strain that might induce secretory and humoral responses against Pertactin/P.69 as well as a cell-mediated immune response . Consequently, Pertactin/P.69 was expressed at high levels in the cytoplasm of BRD509 under the control of the inducible nirB promoter from a ColE1-based replicon . Anti-Pertactin/P.69 immune responses were not observed following immunization of BALB/c mice with this strain (BRD975) . Priming of the immune system against Pertactin/P.69 was, however, observed following oral immunization with BRD975 and boosting subcutaneously with purified Pertactin/P.69 antigen . The major anti-Pertactin/P.69 IgG subclass detected in boosted mice was IgG2a; thus, as BRD640, BRD975 appeared to induce a Th1 type T-helper response against Pertactin/P.69.

Avian Dis, 1996 Oct-Dec, 40(4), 924 - 6
Effect of ochratoxin A on Salmonella typhimurium-challenged layer chickens; Fukata T et al.; Eleven-day-old chickens received 10(8) colony-forming units Salmonella typhimurium orally for 2 consecutive days . The next day, the 13-day-old chickens were given a high dose of ochratoxin A (3 mg/kg) orally . The number of S . typhimurium in both the duodenal and cecal contents of chickens administered with high doses of ochratoxin A increased significantly when compared with control birds . Ochratoxin A was shown to be one of numerous factors that affect the susceptibility of chickens to salmonellae colonization.

APMIS, 1996 Oct, 104(10), 750 - 4
Immunity to experimental Salmonella typhimurium infections in rats . Transfer of immunity with primed CD45RC+ and CD45RC- CD4 T-cell subpopulations; Thygesen P et al.; The protective effect of primed CD4 T cells against a lethal dose of Salmonella typhimurium was studied in Lewis rats . Primed CD4 T cells were obtained by inoculating Lewis rats with a non-lethal dose of S . typhimurium . Four weeks after the infection, spleen CD4 T cells were separated by antibody-coated magnetic microspheres using an antibody against the CD4 molecule (W3/25) . The cells were separated according to their expression of the CD45RC isoform of the leukocyte common antigen by FACS . CD45RC+ and CD45RC- CD4 T-cell subpopulations were transferred to untreated rats 24 h prior to infection with S . typhimurium . Transfer of CD45RC+ and CD45RC- CD4 T cells induced a significant survival, p = 0.022 and p = 0.023 respectively, following inoculation with S . typhimurium compared to animals with no cells transferred . The infection induced an increase in CD4 T cells expressing the CD45RC isoform compared to untreated controls (p < 0.001) . It is concluded that both CD45RC+ and CD45RC- cells can induce a significant protection against S . typhimurium infections in rats . Therefore the CD45RC antigen cannot be used as a phenotypic marker for functionally distinct CD4 T-cell subpopulations . The infection-induced increase in CD45RC+ cells is most likely due to generation of antigen-specific memory T cells.

Regul Toxicol Pharmacol, 1996 Oct, 24(2 Pt 2), S261 - 3
Mutagenicity studies on erythritol in bacterial reversion assay systems and in Chinese hamster fibroblast cells; Kawamura Y et al.; The sweetener erythritol tested negative in reverse mutation assays using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and the WP2 uvrA strain of Escherichia coli . Erythritol tested negative in chromosome aberration tests using the Chinese hamster fibroblast cell line CHL/IU.

Arzneimittelforschung, 1996 Oct, 46(10), 972 - 5
Assessment of genotoxic effect of piperine using Salmonella typhimurium and somatic and somatic and germ cells of Swiss albino mice; Karekar VR et al.; Piperine (CAS 94-62-2) is a constituent of various spices and is used as a common food additive all over the world . The genotoxic potential of piperine was assessed using four different test systems, namely, Ames test using Salmonella typhimurium, micronucleus test, sperm shape abnormality test and dominant lethal test using Swiss albino mice . In the Ames test, six different doses of piperine, in the range of 0.005-10 mumol/plate, did not induce his+ revertants, with or without metabolic activation, indicating its nonmutagenic nature . In the bone narrow micronucleus test using two doses in the range of therapeutic usage (10 and 20 mg/kg body weight), piperine itself was non-mutagenic . Like in somatic cells, piperine (10 and 50 mg/kg body weight) failed to induce mutations in male germ cells of mouse as assessed by using the sperm shape abnormality and dominant lethal tests . Piperine thus appears to be a non-genotoxic chemical.

Mol Microbiol, 1996 Oct, 22(2), 367 - 78
Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction; Valdivia RH et al.; The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH . This requirement for an acidic vacuole suggests that low pH is an important environmental stimulus for the transcription of genes necessary for intracellular survival . To study the behaviour of acid-inducible genes in response to the phagosomal environment, we have applied a novel enrichment strategy, termed differential fluorescence induction (DFI), to screen an S . typhimurium library for promoters that are upregulated at pH 4.5 . DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FACS) to perform genetic selection . In the presence of an inducing stimulus, such as low pH, a FACS is used to sort highly fluorescent bacterial clones bearing random promoters fused to the mutant GFP protein (GFPmut) . This population is then amplified at neutral pH and the least fluorescent population is sorted . Sequential sorts for fluorescent and non-fluorescent bacteria in the presence or absence of inducing conditions rapidly enriches for promoter fusions that are regulated by the inducing stimulus . We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosome milieu . These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enzymes, stress proteins, and generalized efflux pumps . Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock induction . This suggests that while low pH is a relevant inducer of intracellular gene expression, additional stimuli in the macrophage can modulate the expression of acid-inducible genes.

Infect Immun, 1996 Oct, 64(10), 4363 - 8
Invasion of murine intestinal M cells by Salmonella typhimurium inv mutants severely deficient for invasion of cultured cells; Clark MA et al.; We have examined the role of the Salmonella typhimurium inv locus in invasion of the murine intestine . Previous studies have demonstrated that M cells within the lymphoid-follicle-associated epithelia are the primary site of intestinal invasion by S . typhimurium . In this study, we show that mutants possessing defects in one of two inv genes, invA or invG, which render them severely deficient for invasion of polarized epithelial MDCK cells, retain their ability to actively invade mouse Peyer's patch M cells . The interaction of these mutants with M cells was associated with apical membrane remodelling resembling that induced by wild-type strains . These data demonstrate that Salmonella invasion in vivo can proceed via mechanisms other than those previously defined in cultured cells.

Fundam Appl Toxicol, 1996 Oct, 33(2), 212 - 9
Bacterial and human cell mutagenicity and mouse lung tumorigenicity of the oxygenated polynuclear aromatic hydrocarbon phenalenone; Wang JS et al.; Phenalenone (perinaphthenone) is a major oxygenated polynuclear aromatic hydrocarbon (oxy-PAH) atmospheric pollutant formed from the combustion of fossil fuels . Mutagenicity of phenalenone was measured in quantitative forward mutation assays with Salmonella typhimurium TM677 and metabolically competent human B-lymphoblastoid cell lines (MCL-5 and h1A1v2 cells), and its tumorigenicity was also assessed in a newborn mouse assay . Phenalenone was mutagenic in Salmonella in the presence of rat liver postmitochondrial supernatant (PMS) at a minimum detectable mutagen concentration (MDMC) of 12 micrograms/ml, but was not mutagenic in the absence of PMS at concentrations up to 100 micrograms/ ml . Phenalenone was not significantly mutagenic in either human cell line after 28 hr treatment, although mutant fractions were increased by nearly fivefold in h1A1v2 cells (at the tk locus) exposed at 30 micrograms/ml . However, after 72 hr treatment, phenalenone was mutagenic at the hprt locus in h1A1v2 cells with an MDMC of 3 micrograms/ml . Phenalenone was also tumorigenic in male BLU:Ha mice with a lung tumor incidence of 33% 6 months after injection with 4.2 mg phenalenone, the highest dose tested . Lung tumor multiplicity in this treatment group was 0.5 tumor/mouse . No increase in lung tumors in female mice was observed . Indices of lung tumor incidence (ED50) and multiplicity (TM1.0) for male mice were 29.3 and 34.9 mumol, respectively . These data suggest that phenalenone does not contribute significantly to the mutagenicity or carcinogenicity of combustion emission extracts.

Mutat Res, 1996 Oct 1, 370(3-4), 203 - 8
Mutagenicity of 24-hour duplicate of Japanese diet; Mano H et al.; In order to elucidate the genotoxicological characteristics of the Japanese diet, the mutagenicity of 24-h duplicate of the diet samples were investigated . The mutagenicity of blue rayon extract was examined in the Ames Salmonella/microsome assay . Thirty-two (91.4%) of 35 samples revealed mutagenicity toward Salmonella typhimurium TA98 in the presence of S9 mix . The mutagenic activities showed significant correlations with the consumption rates of broiled fish (r = 0.517, p = 0.0021) and broiled meat (r = 0.494, p = 0.0036) . In other test conditions, 6 (17.1%), 5 (14.3%) and 8 (22.9%) samples were mutagenic to Salmonella typhimurium TA98 without S9 mix, TA100 with S9 mix and TA100 without S9 mix, respectively . Findings in the present study suggest that high consumption of broiled fish and broiled meat are important as the source of mutagens/carcinogens in the Japanese diet . In the present study, however, biological inference of these findings could not be made in relation to the occurrence of cancers, especially of the gastric cancer, which is the most prevalent form of cancer in Japan.

J Vet Med Sci, 1996 Oct, 58(10), 1021 - 3
Biochemical changes in fowl serum during infection with Salmonella typhimurium; Itoh N et al.; Two groups of White Leghorns were inoculated with Salmonella Typhimurium in the breast muscle . One group was injected with amikacin every 9 hr (8 times) from 24 to 96 hr after the bacterial inoculation and the other group was given no amikacin . In both groups, a significant increase in the levels of aspartate aminotransferase and creatine kinase, and a significant decrease in the levels of alkaline phosphatase, total cholesterol and glucose were found 96 hr after inoculation . There were, however, no significant differences in the blood chemical values between the amikacin-treated group and non-treated group . The results from the present study may provide basic data for further investigation of blood chemical values during Salmonella infection and amikacin therapy in fowls.

Microb Pathog, 1996 Oct, 21(4), 249 - 63
Immunoprotection by monoclonal antibodies to the porins and lipopolysaccharide of Salmonella typhimurium; Singh SP et al.; Monoclonal antibodies (MAbs) were raised against the outer membrane (OM) antigens of Salmonella typhimurium . Enzyme-linked immunosorbent assays and Western immunoblots indicated that 10 MAbs in the panel were specific for surface epitopes, and 10 recognized buried epitopes of OmpC or OmpD porins; three MAbs reacted with smooth lipo-polysaccharide (LPS), two bound rough LPS, and the remaining three MAbs apparently reacted with a porin-LPS complex . We screened these MAbs and immune polyclonal sera in CAF1 (Ity) mice for their relative immunoprotective potential against a challenge with 10 to 500 LD50 of the virulent S . typhimurium LT-2 strain WB600, or against two LD50 of purified OM from this organism . Polyclonal sera that contained high titers of antibodies to porin monomers and trimers, and LPS, provided significant protection (33 to 100% survivors) . Antiporin MAbs, when administered individually, did not protect or prolong the survival of mice . A mixture of MAbs with specificity for the surface, but not buried epitopes of porins, prolonged the survival of mice against endotoxemia, but none provided significant protection against mouse typhoid . MAbs specific for smooth (but not rough) LPS on the other hand, conferred significant protection against endotoxemia and mouse typhoid . Finally, MAbs that presumably recognized epitopes present in porin-LPS complexes, were also protective against endotoxemia and mouse typhoid . These results support the role of antibodies to LPS O-chains, porin-LPS complexes, and to a lesser degree, native porins in acquired resistance to infection by S . typhimurium.

Mol Microbiol, 1996 Oct, 22(1), 149 - 60
Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon; Coynault C et al.; The alternative sigma factor RpoS (sigma S) is required for Salmonella virulence in mice . We report the immunizing capacity of Salmonella typhimurium rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S . typhimurium . When administered orally or intraperitoneally, rpoS derivatives of the mouse-virulent S . typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single-dose live vaccines . rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis . Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design . Consistent with this, Salmonella typhi Ty2, a 'wild-type' strain used widely for the development of human live-vaccine candidates against typhoid fever, was shown to be defective for rpoS . In addition, our results demonstrate that rpoS not only controls the growth and persistence of S . typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.

Carcinogenesis, 1996 Oct, 17(10), 2275 - 7
Glutathione modulates the formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibres; Howden PJ et al.; Treatment of isolated DNA with crocidolite and man-made vitreous fibre-21 (MMVF-21) significantly increased the concentration of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA above background levels and co-treatment with glutathione (GSH) eliminated this effect . Crocidolite, MMVF-21 and chrysotile fibres increased the number of revertants in Salmonella typhimurium TA100 and GSH-deficient strains, TA100/NG-54 and TA100/NG-57, over background levels . This increase was small in TA100 but was greater in the GSH-deficient strains . When these bacterial strains were further depleted of GSH by co-culture with buthionine sulfoximine, all fibres tested caused a significant increase in the number of revertants over the parent strains . Pre-treatment with the GSH precursor N-acetyl-L-cysteine reduced the number of revertants to below that of the parent strain . Previous studies have shown a mechanistic role for iron-catalyzed production of oxygen radicals in the mutagenicity of fibres and this study suggests a protective role for GSH against such oxidative damage possibly by acting as a radical scavenger.

Biophys J, 1996 Oct, 71(4), 2040 - 8
Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide; Baxa U et al.; Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes . Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides . Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used . Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently . At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike . Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature . Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10584 - 8
Crystal structure of phage P22 tailspike protein complexed with Salmonella sp . O-antigen receptors; Steinbacher S et al.; The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and D1 serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus . Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two O-antigenic repeating units from Salmonella typhimurium, Salmonella enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution . The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified . Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.

J Bacteriol, 1996 Oct, 178(20), 5904 - 9
Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases; Slauch JM et al.; Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment . The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria . Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen . In Salmonella typhimurium, the O-antigen is acetylated, conferring the 05 serotype . We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes . Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene . The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit . The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties . In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.

J Bacteriol, 1996 Oct, 178(19), 5683 - 91
The role of fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition; Hall HK et al.; The response of Salmonella typhimurium to low pH includes a low-pH protection system called the acid tolerance response (ATR) . The iron-regulatory protein Fur has been implicated in the ATR since fur mutants are acid sensitive and cause altered expression of several acid shock proteins (J . W . Foster, J . Bacteriol . 173:6896-6902, 1991) . We have determined that the acid-sensitive phenotype of fur mutations is indeed due to a defect in Fur that can be complemented by a fur(+)-containing plasmid . However, changes in cellular iron status alone did not trigger the ATR . Cells clearly required exposure to low pH in order to induce acid tolerance . The role of Fur in acid tolerance was found to extend beyond regulating iron acquisition . A mutation in fur converting histidine 90 to an arginine (H90R) eliminated Fur-mediated iron regulation of enterochelin production and deregulated an iroA-lacZ fusion but had no effect on acid tolerance . The H90R iron-blind Fur protein also mediated acid shock induction of several Fur-dependent acid shock proteins and acid control of the hyd locus . In addition, a Fur superrepressor that constitutively repressed iron-regulated genes mediated normal Fur-dependent acid tolerance and pH-controlled gene expression . The results indicate the acid-sensing and iron-sensing mechanisms of Fur are separable by mutation and reinforce the concept of Fur as a major global regulator in the cell.

J Bacteriol, 1996 Oct, 178(19), 5676 - 82
Mutations in apbC (mrp) prevent function of the alternative pyrimidine biosynthetic pathway in Salmonella typhimurium; Petersen L et al.; The alternative pyrimidine biosynthetic (APB) pathway can synthesize the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine in Salmonella typhimurium independently of de novo purine biosynthesis . When mutants defective in function of the APB pathway were isolated, the predominant class (40%) were defective in a single locus we have designated apbC . Mutations in apbC block function of the APB pathway since they prevent growth of a purF mutant in the absence of thiamine . Lesions in apbC also cause a thiamine auxotrophy in strains proficient in purine biosynthesis when fructose is provided as the sole carbon and energy source . Results presented here are consistent with ApbC being involved in the conversion of aminoimidazole ribonucleotide to HMP, and we suggest that ApbC performs a redundant step in thiamine synthesis . Sequence analysis demonstrated that apbC mutations were alleles of mrp, a locus previously reported in Escherichia coli as a metG-related protein . We propose that this locus in S . typhimurium be designated apbC to reflect its involvement in thiamine synthesis.

Clin Immunol Immunopathol, 1996 Oct, 81(1), 55 - 61
Suppressive effect of transforming growth factor beta1 on the recurrence of experimental melanin protein-induced uveitis: upregulation of ocular interleukin-10; Li Q et al.; Uveitis is induced in Lewis rats by immunization with bovine melanin protein (BMP) derived from the uvea and retinal pigment epithelium . Recurrence of this experimental melanin protein-induced uveitis (EMIU) develops after footpad injection of a minimal amount of Salmonella typhimurium endotoxin (LPS) following the remission of EMIU . To investigate the effect of transforming growth factor beta1 (TGFbeta1) on the recurrence of EMIU, 5 micrograms LPS booster was given to Lewis rats by footpad injection on Day 45 after BMP immunization . Daily TGFbeta1 or phosphate-buffered saline was administered either from Day 0 to 7 (group 1) or from Day 7 to 13 (group 2) after LPS booster . Delayed-type hypersensitivity (DTH) ear test was conducted on Day 12 after LPS booster and eye and blood were collected on Day 14 . The incidence and severity of recurrent uveitis markedly decreased in both groups of TGFbeta1-treated rats . A lower level of serum BMP antibody was also observed by agglutination in these groups . There was no statistical difference in DTH responses between the treated and control groups . Ocular cytokine mRNA of group 1 and controls was analyzed by RT-PCR . Interleukin (IL)-2 and interferon-gamma were not detectable . IL-4 was identified at a similar level in both groups . A higher level of IL-10 was observed in group 1 rats . We conclude that TGFbeta1 suppresses recurrent EMIU, probably through upregulation of IL-10.

Mutat Res, 1996 Sep 26, 361(1), 41 - 8
Bacterial photomutagenicity testing: distinction between direct, enzyme-mediated and light-induced events; Utesch D et al.; A bacterial photomutagenicity test system has been established according to a predetermined protocol using a light source emitting multiple wave lengths, including UV-A and UV-B, and the tester strains used for standard bacterial mutagenicity testing . 8-Methoxypsoralen (8-MOP) was used as a photomutagenic positive control showing no mutagenicity in the absence of light and borderline (Salmonella typhimurium TA1537) or clear (Salmonella typhimurium TA102 and Escherichia coli WP2) mutagenic effects in the presence of light . Using the same experimental conditions, the UV filters p-aminobenzoic acid (PABA), octyl dimethyl PABA (Eusolex 6007), and 4-methylbenzylidene camphor (Eusolex 6300) were non-mutagenic (in the absence of light) and non-photomutagenic (in the presence of light) . Dihydroxyacetone (DHA), used as an active ingredient in self-tanning lotions, slightly increased the number of revertants in Salmonella typhimurium TA100 and TA102 in the absence of light . However, the relevance of these effects is equivocal, since they occurred at very high, cytotoxic concentrations (5000 micrograms/plate) . Furthermore, these increases were not potentiated by light, thus demonstrating the non-photomutagenicity of DHA . In contrast, 7,12-dimethylbenz{a}anthracene (DMBA), which is non-mutagenic per se and is activated by external metabolizing systems (e.g., S9-mix) in the absence of light, was clearly mutagenic in Salmonella typhimurium TA98 and TA1537 in the presence of light (and the absence of S9-mix) . Although the photomutagenic potency of DMBA, on a molar basis, was certainly lower than that of 8-MOP, the absolute mutagenic effects of DMBA in the respective bacterial strains were in a similar range under either S9-mix or photoactivation conditions . The strain specificity of the mutagenic effects were, however, different for either enzyme- or light-mediated mutagenicity . This indicates that different reactive intermediates are responsible for the mutagenicity in the tests using the two different activation systems . These results further suggest to use DMBA as a positive photomutagenic control compound alternatively to 8-MOP, since the latter is non- or only weakly photomutagenic in Salmonella typhimurium TA98 and TA1537 . Furthermore, the usefulness and application of this photomutagenicity test system could be demonstrated for the testing of different photoabsorbing chemicals and cosmetic ingredients.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10303 - 8
Highly plastic chromosomal organization in Salmonella typhi; Liu SL et al.; Gene order in the chromosomes of Escherichia coli K-12 and Salmonella typhimurium LT2, and in many other species of Salmonella, is strongly conserved, even though the genera diverged about 160 million years ago . However, partial digestion of chromosomal DNA of Salmonella typhi, the causal organism of typhoid fever, with the endonuclease I-CeuI followed by separation of the DNA fragments by pulsed-field gel electrophoresis showed that the chromosomes of independent wild-type isolates of S . typhi are rearranged due to homologous recombination between the seven rrn genes that code for ribosomal RNA . The order of genes within the I-CeuI fragments is largely conserved, but the order of the fragments on the chromosome is rearranged . Twenty-one different orders of the I-CeuI fragments were detected among the 127 wild-type strains we examined . Duplications and deletions were not found, but transpositions and inversions were common . Transpositions of I-CeuI fragments into sites that do not change their distance from the origin of replication (oriC) are frequently detected among the wild-type strains, but transpositions that move the fragments much further from oriC were rare . This supports the gene dosage hypothesis that genes at different distances from oriC have different gene dosages and, hence, different gene expression, and that during evolution genes become adapted to their specific location; thus, cells with changes in gene location due to transpositions may be less fit . Therefore, gene dosage may be one of the forces that conserves gene order, although its effects seem less strong in S . typhi than in other enteric bacteria . However, both the gene dosage and the genomic balance hypotheses, the latter of which states that the origin (oriC) and terminus (TER) of replication must be separated by 180 degrees C, need further investigation.

Biochim Biophys Acta, 1996 Sep 11, 1308(3), 189 - 92
Cloning and characterization of the tyrB gene from Salmonella typhimurium; Nakai Y et al.; We found a gene homologous to tyrB, which encodes aromatic amino acid aminotransferase (ArAT, EC2.6.1.57) in Escherichia coli, in the genome of Salmonella typhimurium IFO 13245 . The S . typhimurium tyrB product consists of 397 amino acid residues . The amino acid sequence shows 87.9% identity with that of E . coli ArAT, but shows lower identity (42.3%) with that of E . coli aspartate aminotransferase (AspAT, EC2.6.1.1) . When the S . typhimurium tyrB gene was expressed in an E . coli mutant whose intrinsic tyrB gene had been inactivated, the activity of transaminating tyrosine and phenylalanine could be recovered, indicating that the S . typhimurium tyrB gene product possesses transamination activities similar to those of the E . coli ArAT . Elucidation of the molecular features of a new ArAT may be helpful for structural and functional analyses of ArAT and AspAT with regard to the different but overlapping substrate specificity of the two enzymes.

J Immunol Methods, 1996 Sep 9, 195(1-2), 15 - 25
Enzyme-linked immunomagnetic electrochemical detection of Salmonella typhimurium; Gehring AG et al.; There is a need for rapid methods to detect pathogenic bacteria in food products as alternatives to the current laborious and time-consuming culture procedures . We report a microbial detection technique that combines the selectivity of antibody-coated superparamagnetic beads with the rapidity and sensitivity of electrochemical detection in a format termed enzyme-linked immunomagnetic electrochemistry . In it, Salmonella typhimurium were sandwiched between antibody-coated magnetic beads and an enzyme-conjugated antibody . With the aid of a magnet, the beads (with or without bound bacteria) were localized onto the surface of disposable graphite ink electrodes in a multi-well plate format . Enzyme substrate was added and conversion of substrate to an electroactive product was measured using electrochemical detection . The electrochemical response was directly proportional to the number of captured bacteria . Using this technique, a minimum detectable level of 8 x 10(3) cells/ml of Salmonella typhimurium in buffer was achieved in ca . 80 min.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9833 - 8
Salmonella typhimurium invasion induces apoptosis in infected macrophages; Monack DM et al.; Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism . Noninvasive S . typhimurium strains are unable to induce this membrane ruffling . Invasive S . typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains . Invasive S . typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages . Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation . S . typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis . Mutant S . typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis . The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages . The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

Biochemistry, 1996 Sep 3, 35(35), 11260 - 7
Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria; Napper S et al.; Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system . HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar . In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium . One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15 . In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation . A series of mutations were made at Ser46 in E . coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly . The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria . In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%) . The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values . The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation . There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure . There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr . In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11 . The consequence of this change is that there is no torsion angle strain at residue 16 . This result suggests that there is no active site torsion angle strain in wild-type E . coli HPr . The lack of substantial change at the active center of E . coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Sep-Oct, (5), 76 - 80
{The evaluation of different agglutination and adhesion tests with erythrocytic reagents in determining antibodies to the O and H antigens of Salmonella typhimurium in comparison with immunoenzyme analysis (IEA)}; Karal'nik BV et al.; The comparison of the effectiveness of EIA with that of a number of agglutination and adhesion tests with erythrocyte diagnostica in the determination of antibodies to different S.typhimurium antigens demonstrated higher sensitivity of EIA . The relative specificity of the determination of O- and H-antibodies in EIA and in agglutination and adhesion tests depended on the isotype of antibodies to be determined and the specificity of sensitins used in the production of immunoreagents.

East Afr Med J, 1996 Sep, 73(9), 575 - 8
Prevalence of human immunodeficiency virus infection: its impact on the diagnostic yields in exudative pleural effusions at the Kenyatta National Hospital, Nairobi; Owino EA et al.; This was a descriptive cross-sectional study carried out at Kenyatta National Hospital (KNH), Nairobi, among consecutively admitted adult patients with exudative pleural effusions over a one year period . The aim of the study was to determine the prevalence of human immunodeficiency virus (HIV) infection in these patients and to compare the diagnostic yields from the pleural fluid and pleural biopsy between the HIV seropositive and HIV seronegative patients . Sixty six patients were studied, with a mean age of 33.8 (+/- SD = 15.6) years and a male to female ratio of 1.6:1 . Overall, 27 patients(40.9%) were found to be HIV seropositive . The commonest cause of exudative pleural effusions, overall, was tuberculosis (78.8%) followed by neoplasms (7.6%) . Comparing the aetiology of exudative pleural effusion in HIV seropositive and HIV seronegative patients, tuberculosis was still the commonest cause accounting for 42.3% and 57.7% of the cases in each of the groups respectively . Conversely, 42.3% of patients with tuberculous pleural effusions were HIV seropositive . There was no significant difference in yields from pleural fluid, pleural biopsy culture and histology in the diagnosis of tuberculosis in the two patient groups . The only two patients with empyema were HIV seropositive and the bacterial isolates were Salmonella typhimurium and Pseudomonas aeruginosa . Kaposi's sarcoma was the cause of exudative pleural effusion in the one HIV seropositive patient with a malignant effusion . The only patient with a parapneumonic effusion was HIV seronegative . No fungi were isolatedPIP: 66 adult patients of mean age 33.8 years with exudative pleural effusions were studied to determine the prevalence of HIV infection and compare the diagnostic yields from the pleural fluid and pleural biopsy between the HIV-seropositive and HIV-seronegative patients . The patients were consecutively admitted to Kenyatta National Hospital (KNH) over a 1-year period and of male:female ratio 1.6:1 . 27 patients were found to be HIV seropositive . Tuberculosis (TB) and neoplasms were the most common causes of exudative pleural effusions, responsible for 78.8% and 7.6% of cases, respectively . Comparing the etiology of exudative pleural effusion in HIV-seropositive and HIV-seronegative patients, TB remained the most common cause, accounting for 42.3% and 57.7% of cases in each of the groups, respectively . 42.3% of the patients with TB pleural effusions were HIV seropositive . No significant difference was identified in the yields from pleural fluid, pleural biopsy culture, and histology in the diagnosis of TB in the 2 patient groups . The only 2 patients with empyema were HIV seropositive and the bacterial isolates were Salmonella typhimurium and Pseudomonas aeruginosa . Kaposi's sarcoma was the cause of exudative pleural effusion in the 1 HIV-seropositive patient with a malignant effusion . The only patient with a parapneumonic effusion was HIV seronegative . No fungi were isolated .

Mutagenesis, 1996 Sep, 11(5), 519 - 23
Role of oxidative DNA damage in hydroxychavicol-induced genotoxicity; Lee-Chen SF et al.; Chewing betel quid has been linked to the development of oral cancer . In Taiwan, fresh Piper betle inflorescence is uniquely added to betel quid, and hydroxychavicol is the major phenolic components of P.betle inflorescence . In this study, we tested the mutagenic potential of hydroxychavicol in Salmonella typhimurium TA97, TA98, TA100 and TA102 with and without Aroclor-1254 induced S9 fraction . The results showed that hydroxychavicol was positive in S.typhimurium TA102 without metabolic activation . This increase in revertants was partially inhibited by catalase and superoxide dismutase . In Chinese hamster ovary (CHO-K1) cells, hydroxychavicol induced chromosome aberrations in a dose-dependent manner (10-50 microM) and the majority were chromosome-type aberrations . Hydroxychavicol also significantly increased the frequency of micronuclei in CHO-K1 cells up to 3-fold at a concentration of 40 microM . In addition, hydroxychavicol dose-dependently (0.1-20 microM) induced copper-dependent strand breaks in plasmid DNA . We further tested the oxidative DNA damage potential of hydroxychavicol by measuring 8-hydroxydeoxyguanosine (8-OH-dG) formation in CHO-K1 cells following an 18-h incubation and found that hydroxychavicol (6.25-100 microM) induced 8-OH-dG levels dose-dependently . The increase of 8-OH-dG formation was positively correlated (r = 0.79) with the hydroxychavicol-induced cytotoxicity . In conclusion, hydroxychavicol may exert its genotoxic potential through oxidative DNA damage.

Mutagenesis, 1996 Sep, 11(5), 497 - 504
The photomutagenicity of fluoroquinolones in tests for gene mutation, chromosomal aberration, gene conversion and DNA breakage (Comet assay); Chetelat AA et al.; The ability of fluoroquinolones to cause light-induced adverse effects has been established in experimental studies and clinical observations . The formation of active oxygen species appears to be responsible for this activity . Photomutagenicity tests with bacterial, lower eukaryotic and mammalian cells were performed with three fluoroquinolones (Fleroxacin, Ciprofloxacin and Lomefloxacin) . After concomitant irradiation with simulated solar light (with a reduced UVB component), weak increases in the number of revertants were observed in Salmonella typhimurium TA104 and TA100 . No photomutagenic activity was detected in Saccharomyces cerevisiae D7 . In the chromosomal aberration (CA) test with Chinese hamster V79 cells the number of aberrant metaphases was markedly increased . In the Comet assay with mouse lymphoma cells, evidence of extensive DNA breakage was obtained . All three compounds showed similar potencies in the Comet and Ames assays while there was a clear gradation of potencies in the CA assay (Lomefloxacin > Fleroxacin > Ciprofloxacin), which conformed with reports on the relative potencies regarding phototoxicity . The oxygen radical scavengers catalase, superoxide dismutase and N, N'-dimethylurea modulated the photoclastogenicity and phototoxicity but had no influence on the effects in the Comet and Ames tests . It thus appears that different kinds of mechanism are responsible for toxicity and clastogenicity on the one side and DNA breakage and gene mutation on the other side . Pre-irradiation of the test articles did not lead to enhanced genotoxicity, indicating the involvement of very short lived genotoxic agents . The results endorse the advice to avoid excessive light exposure during antibiotic therapy with fluoroquinolones.

Cancer Immunol Immunother, 1996 Sep, 43(1), 44 - 8
Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains; Krul MR et al.; Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions . There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions . Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions . For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector . Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express . This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.

Crit Rev Toxicol, 1996 Sep, 26(5), 551 - 83
New applications of bacterial systems to problems in toxicology; Guengerich FP et al.; Bacterial systems have long been of use in toxicology . In addition to providing general models of enzymes and paradigms for biochemistry and molecular biology, they have been adapted to practical genotoxicity assays . More recently, bacteria also have been used in the production of mammalian enzymes of relevance to toxicology . Escherichia coli has been used to express cytochrome P450, NADPH-cytochrome P450 reductase, flavin-containing monooxygenase, glutathione S-transferase, quinone reductase, sulfotransferase, N-acetyltransferase, UDP-glucuronosyl transferase, and epoxide hydrolase enzymes from humans and experimental animals . The expressed enzymes have been utilized in a variety of settings, including coupling with bacterial genotoxicity assays . Another approach has involved expression of mammalian enzymes directly in bacteria for use in genotoxicity systems . Particularly with Salmonella typhimurium . Applications include both the reversion mutagenesis assay and a system using a chimera with an SOS-response indicator and a reporter.

J Appl Physiol, 1996 Sep, 81(3), 1316 - 22
Endotoxin alters hypoxic pulmonary vasoconstriction in isolated rat lungs; Frank DU et al.; Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism for maintaining oxygenation, which may be altered by endotoxin . We determined that acute endotoxemia alters the HPV response secondary to changes in endothelium-derived vasoactive products . Rats were treated with Salmonella typhimurium lipopolysaccharide (LPS; 15 mg/kg i.p.) either 1 to 6 h before lung isolation and compared with control rats (no LPS) . Additional 6-h LPS-treated and control rats were pretreated with either indomethacin (15 mg/kg i.p.), a cyclooxygenase inhibitor, or bosentan (10 mg/kg po), a nonselective endothelin-receptor antagonist . The rats lungs were isolated and challenged with 3% O2 for 10 min to elicit HPV responses before and after nitric oxide (NO) synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME; 100 microM) . LPS (6 h) significantly increased the peak HPV responses by 108% . L-NAME had no significant effect in LPS-treated lungs but increased the peak HPV response in control lungs to levels equal to LPS-treated lungs . Bosentan increased the peak HPV response in all lungs, and indomethacin increased the peak HPV in LPS-treated lungs . The HPV response was sustained in control lungs at 10 min and in additional 20-min studies . In contrast, in LPS-treated lungs the HPV response faded after 10 min to levels equal to control, and in 20-min studies it faded by 82% to levels significantly less than in control lungs . The 10-min fade in LPS-treated lungs was attenuated by indomethacin (51%) and bosentan (80%) but not by L-NAME . In conclusion, acute endotoxemia with LPS increased the peak HPV response, but this effect was not sustained and by 20 min was nearly abolished . Inhibition of endogenous NO by LPS may explain the increased peak HPV response, but NO is not involved in the fade . The fade is at least partially due to increased vasodilating cyclooxygenase products and endothelins.

J Antimicrob Chemother, 1996 Sep, 38(3), 425 - 34
Multi-drug resistant non-typhi salmonellae in Kenya; Kariuki S et al.; Two methods of plasmid characterization, restriction digest patterns and incompatibility grouping, were used to study self-transmissible multi-drug resistance among non-typhi salmonellae (NTS) . Resistance to ampicillin and other commonly applied beta-lactams was evaluated by iso-electric focusing and disc inactivation . Of the NTS isolated from blood, 75% were Salmonella typhimurium but those included several different phage types . Over 47% of isolates were resistant to three or more of the readily available drugs including ampicillin, cefuroxime, chloramphenicol, co-trimoxazole, streptomycin and tetracycline . Self-transferable resistance plasmids (c . 100 kb) were essentially of incompatibility group incFIIA, but their restriction fragment patterns revealed a diversity in relatedness . More than half of parent strains and their transconjugants produced beta-lactamases which co-electrophoresed with TEM-1 and OXA-1 . This study has observed a disturbingly high prevalence of transmissible multi-drug resistance among NTS which are an important cause of morbidity in HIV-1 seropositive individuals.

Int J Food Sci Nutr, 1996 Sep, 47(5), 391 - 8
Splenic and intestinal lymphocyte proliferation response in mice fed milk or yogurt and challenged with Salmonella typhimurium; Puri P et al.; Two groups of 4-5 week old DBA/2J Nii mice were put on either a yogurt-based (n = 33) or a milk-based (n = 32) diet for a period of 4 weeks . At the end of the feeding trial one sub group of mice each from the two dietary groups was sacrificed for assessment of immune response . The remaining mice were challenged intragastrically with 2 x 10(10) live Salmonella typhimurium organisms and continued on their respective diets for 8 days after which they were also sacrificed . The immune response was measured by tritiated thymidine uptake by splenic or intestinal lymphocytes in response to the mitogens concanavalin A (Con A), Phytohaemaggutinin (PHA), and Lipopolysaccharide from Escherichia coli (LPS) . Serum Immunoglobulin A levels were also estimated . Feed efficiency, measured as weight gain per unit energy intake, was significantly higher for the yogurt diet than for the milk diet . The mitogenic response of splenic and intestinal lymphocytes in the two groups of unchallenged mice was not different . In the Salmonella-challenged mice the stimulation index (SI) of splenic lymphocytes from yogurt-fed mice (mean +/- SD) was significantly higher (P = 0.001) in response to Con A (24.71 +/- 3.40) than that of milk-fed mice (15.85 +/- 2.09) . Further, in these mice the SI of intestinal lymphocytes from yogurt-fed mice was higher than that of milk-fed mice in response to Con A (7.35 +/- 0.61 vs 5.65 +/- 0.78, P = 0.016) and LPS (9.04 +/- 0.93 vs 6.15 +/- 1.32, P = 0.016) . Serum IgA levels in Salmonella-challenged mice were significantly higher 8 days after the challenge in the yogurt-fed group than in the milk-fed group (P < 0.001) . The experiments indicate an improvement in local gastrointestinal as well as systemic immunity on a yogurt diet as compared to a milk diet.

Mol Microbiol, 1996 Sep, 21(5), 1101 - 15
Salmonella spp . are cytotoxic for cultured macrophages; Chen LM et al.; We have shown by a variety of microscopical and biochemical techniques that Salmonella spp . are cytotoxic for cultured J774A.1 and bone marrow-derived murine macrophages . The cytotoxicity is initially manifested by inhibition of membrane ruffling and macropinocytosis in infected macrophages, and is followed by cell death . Macrophages killed by Salmonella spp . exhibited features of apoptosis such as condensation and fragmentation of chromatin, membrane blebbing, and the presence of cytoplasmic nucleosomes and apoptotic bodies . Cytotoxicity does not require bacterial internalization as cytochalasin D, a drug that prevents bacterial uptake, did not prevent Salmonella-induced macrophage cell death . However, the cytotoxic effects are strictly dependent upon the expression of the invasion-associated Type III protein-secretion system encoded at centisome 63 of the Salmonella chromosome . Wild-type Salmonella typhimurium grown under conditions that do not allow optical expression of this system or strains of Salmonella carrying mutations in genes that encode components of this protein-secretion system were devoid of macrophage cytotoxicity . In addition, mutations in invJ, spaO, sipB, sipC and sipD, which encode proteins that are secreted via this secretion apparatus and are required for bacterial entry into non-phagocytic cells, also abolished the toxicity . In contrast, mutations in sipA and sptP, which encode secreted proteins that are not required for bacterial invasion, had no effect on macrophage cytotoxicity . These results indicate a close correlation between the mechanisms of bacterial internalization into non-phagocytic cells and those that lead to macrophage cytotoxicity . Host-adapted serotypes of Salmonella such as S . typhi, S . gallinarum and S . dublin were also toxic for murine macrophages, indicating that this virulence property is probably present in most Salmonella spp . and is not associated with the mechanisms responsible for host range.

Genetics, 1996 Sep, 144(1), 15 - 26
DNA adenine methylase mutants of Salmonella typhimurium and a novel dam-regulated locus; Torreblanca J et al.; Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles . The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene . Dam- mutants of S . typhimurium resemble those of E coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair . However, differences between S . typhimurium and E . coli dam mutants are also found: (1) S . typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella . (2) S . typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation . We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S . typhimurium virulence (or "cryptic") plasmid.

Pediatr Res, 1996 Sep, 40(3), 415 - 21
Escherichia coli transcytosis in a Caco-2 cell model: implications in neonatal necrotizing enterocolitis; Panigrahi P et al.; Necrotizing enterocolitis (NEC) is a serious gastrointestinal disorder of preterm infants . Other than an association with prematurity and gastrointestinal feeding, no single factor or mechanism has been consistently linked to this disease . We have previously demonstrated that Escherichia coli isolates obtained from the stool of infants with NEC caused NEC-like injury in a weanling rabbit ileal loop model; this injury, in turn, could be blocked by coinfection with selected Gram(+) bacteria (Enterococcus faecium) isolated from asymptomatic controls . Using Caco-2 cells in a trans-well system, we now demonstrate that the same E . coli isolates can cross epithelial cell monolayers in the absence of ultrastructural change or damage . These results with E . coli contrast with those seen with Salmonella typhimurium, which passed through the monolayer at a higher rate and were associated with striking ultrastructural damage . Transcytosis of E . coli was reduced 3-5-fold in the presence of E . faecium previously shown to block NEC-like injury in the loop model . There was a mild increase in the rate of E . coli transcytosis when studies were conducted with younger, undifferentiated cells; these immature cells had no brush border, had decreased production of brush border-specific enzymes, but retained well defined tight junctions, as demonstrated by transepithelial electrical resistance and electron microscopy . A further reduction/ complete blockage of E . coli transcytosis was observed when E . faecium was used as the coinfectant in studies with these undifferentiated cells . We hypothesize that the ability of E . coli to cross epithelial cell layer is a critical initial step in the cascade of events which lead ultimately to NEC; blockage or reduction in E . coli transcytosis in the presence of certain Gram(+) organisms may play a significant role in prevention of NEC.

Carcinogenesis, 1996 Sep, 17(9), 2009 - 12
Synthesis and mutagenicity of some cyclopenta{c}phenanthrenes and indeno{c}phenanthrenes; Marrocchi A et al.; An efficient two-step synthesis of 8(H)-9,10,11,12-tetra-hydrodicyclopenta{a,c}phenanthren-7-one, based on the high pressure Diels-Alder cycloaddition of 4-acetoxy-2-cyclopenten-1-one with 1-(1-naphthyl)cyclopentene and a subsequent dehydrogenation-aromatization reaction, is reported . Further, the synthesis of two cyclopenta{c}-phenanthrenes and indeno{c}phenanthrenes is described . Structural analysis of the new products by 1H and 13C NMR spectroscopy is presented . The mutagenic activity of the compounds in Salmonella typhimurium was estimated by Ames' test . Three compounds were shown to be mutagenic for the strain TA 100 . The mutagenic activities exhibited by cyclopenta{c}phenanthrenes are compared with those shown by the related cyclopenta{a}phenanthrenes and then discussed with respect to the effect of the cyclopentane ring facing the bay region . Indeno{c}phenanthrenes are mostly inactive . The effect of benzoannulation on the mutagenic activities of cyclopenta{c}phenanthrenes is discussed.

Plant J, 1996 Sep, 10(3), 479 - 89
A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation; Tsugeki R et al.; The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation . The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development . Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation . Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR . The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST) . The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium, as well as Neurospora crassa mitochondria and higher plant plastids . Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.

FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 155 - 60
Role of ribosome degradation in the death of heat-stressed Salmonella typhimurium; Tolker-Nielsen T et al.; Heat treatment of Salmonella typhimurium results in cell death, which coincides with a significant reduction of the cellular content of 16S ribosomal RNA . It is suggested that the degradation of ribosomal RNA is a direct cause of cell death . This conclusion is based on the observation of carbon-starved and magnesium-supplemented cells, which survive heat treatment much better, and which also maintain stable levels of ribosomal RNA.

J Appl Bacteriol, 1996 Sep, 81(3), 251 - 6
Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice; Rodrigues AC et al.; Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties . We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice . Conventional animals were given daily 10 mg doses of S . boulardii, whereas germ-free animals were given a single 10 mg dose . Both groups were challenged orally 5 d later with the pathogenic bacteria (10(8) or 10(2) viable cells, respectively) . Control groups were treated with saline instead of S . boulardii . Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice . Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10(10) g-1 of faeces . In experimental and control gnotobiotic animals, Salm . typhimurium and Sh . flexneri became rapidly established at a level of about 10(10) viable cells g-1 of faeces and remained at high levels until the animals died or were sacrificed . The protection against Salm . typhimurium and Sh . flexneri obtained in conventional and/or gnotobiotic mice previously associated with S . boulardii is not due to the reduction of the bacterial populations in the intestines.

J Bacteriol, 1996 Sep, 178(17), 5112 - 20
Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein; Fang FC et al.; sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation . Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth . sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds . Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci . Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase . For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth . The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.

J Bacteriol, 1996 Sep, 178(17), 5092 - 9
Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes; Soncini FC et al.; The PhoP-PhoQ two-component system is essential for virulence in Salmonella typhimurium . This system controls expression of some 40 different proteins, yet most PhoP-regulated genes remain unknown . To identify PhoP-regulated genes, we isolated a library of 50,000 independent lac gene transcriptional fusion strains and investigated whether production of beta-galactosidase was regulated by PhoP . We recovered 47 lac gene fusions that were activated and 7 that were repressed when PhoP was expressed . Analysis of 40 such fusions defined some 30 loci, including mgtA and mgtCB, which encode two of the three Mg2+ uptake systems of S . typhimurium; ugd, encoding UDP-glucose dehydrogenase; phoP, indicative that the phoPQ operon is autoregulated; and an open reading frame encoding a protein with sequence similarity to VanX, a dipeptidase required for resistance to vancomycin . Transcription of PhoP-activated genes was regulated by the levels of Mg2+ in a PhoP-dependent manner . Strains with mutations in phoP or phoQ were defective for growth in low-Mg2+ media . The mgtA and mgtCB mutants reached lower optical densities than the wild-type strain in low-Mg2+ liquid media but displayed normal growth on low-Mg2+ solid media . Six PhoP-activated genes were identified as essential to form colonies on low-Mg'+ solid media . Cumulatively, our experiments establish that the PhoP-PhoQ system governs the adaptation to magnesium-limiting environments.

Infect Immun, 1996 Sep, 64(9), 3877 - 83
Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium; Oh YK et al.; The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages . Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes . This report describes the maturation of phagosomes containing S . typhimurium inside mouse bone marrow-derived macrophages . Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers . These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis . By all measures, phagosomes containing S . typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis . The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads . Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours . Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion . Therefore, we conclude that S . typhimurium survives within the lysosomal compartments of macrophages.

Infect Immun, 1996 Sep, 64(9), 3811 - 7
Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization; Licht TR et al.; An avirulent, streptomycin-resistant Salmonella typhimurium strain, SL5319, and its lipopolysaccharide (LPS)-deficient mutant strain, SL5325, differ in their ability to colonize the large intestines of streptomycin-treated mice . When fed to mice independently, the strains colonize equally well, but when fed together, the LPS-deficient mutant is outcompeted by the wild-type strain during establishment in the gut (J.J . Nevola, B.A.D . Stocker, D.C . Laux, and P.S . Cohen, Infect . Immun . 50:152-159, 1985) . In the present study, the spatial distribution in the intestinal mucosal layer of the two strains was visualized by specific hybridization to bacterial rRNA in histological sections of mouse colon and cecum . The first day after infection, 9.8% of the smooth SL5319 cells observed in mucus were found to be associated with the mouse epithelial cells, but three days after infection, the corresponding fraction of adhering bacteria was reduced to 2.1% . The LPS-deficient S . typhimurium strain was confined to the part of the mucosal layer closest to the colonic lumen and was not observed to adhere to the epithelium either at day 1 or 3 after infection . Quantitative determinations of the distance from the S . typhimurium cells to the epithelial wall confirmed that the average distance for the rough S . typhimurium SL5325 was much larger than for its smooth counterpart, S . typhimurium SL5319 . Quantification of the hybridization signal from bacteria isolated from the cecal mucus revealed that the two strains had the same ribosome concentration, indicating that they have the same potential for growth in the intestinal environment . On the basis of these observations, we suggest that the better colonization ability of the strain carrying wild-type LPS is due to the better abilities to penetrate the intestinal mucosal layer and to subsequently bind to the epithelial cells in vivo.

Infect Immun, 1996 Sep, 64(9), 3786 - 92
Attenuated Salmonella vaccine-induced suppression of murine spleen cell responses to mitogen is mediated by macrophage nitric oxide: quantitative aspects; Huang D et al.; Previous reports from our laboratory have shown that 7 days after infection of C3HeB/FeJ mice with an attenuated strain of Salmonella typhimurium, there is profound suppression of responses to B- and T-cell mitogens and suppression of the capacity of spleen cells to mount a primary, in vitro plaque-forming-cell (PFC) response to sheep erythrocytes . Inhibition of the PFC response was shown to be mediated by nitric oxide (NO), as NG-monomethyl-L-arginine (NMMA) gave complete reversal of suppression . The experiments reported here examined the role of NO in suppression of the response to the mitogen concanavalin A (ConA) . In contrast to the PFC system, it was found that addition of NMMA to ConA-stimulated immune spleen cells resulted in less than 20% reversal of suppression . However, addition to NMMA resulted in a 50% reversal of suppression in cocultures of immune and normal spleen cells at a ratio of 1:4 . A complete restoration of ConA-induced responses was achieved in cocultures incubated in medium containing a reduced concentration of L-arginine plus 1.25 mM NMMA . Investigation of why NMMA alone was not 100% effective in reversing suppression showed that addition of ConA significantly augmented production of nitrite and gamma interferon (IFN-gamma) in cocultures containing immune cells . Addition of anti-IFN-gamma reduced nitrite levels in the cultures, although results with the combination of anti-IFN-gamma and NMMA were not significantly better than results with NMMA alone . These findings suggest that suppression in cultures stimulated with ConA is difficult to reverse completely with NMMA alone because of an overproduction of NO, which can be offset by either reducing the L-arginine concentration or blocking IFN-gamma . The quantitative relationship between nitrite levels and suppression in cocultures was examined . It was found that suppression did not correlate directly with the nitrite concentration but rather with the log10 of the nitrite concentration . Nitrite levels above 15 microM gave almost complete suppression, and levels between 1 and 10 microM gave a wide range of suppression . These results strongly support NO as the suppressor factor in Salmonella-induced immunosuppression of responses to ConA and, by inference, suppression of responses to mitogens induced by other microbes . The results show that involvement of NO cannot always be demonstrated by simple addition of NMMA to suppressed mitogen-stimulated spleen cell cultures.

Infect Immun, 1996 Sep, 64(9), 3524 - 31
Requirement for exported proteins in secretion through the invasion-associated type III system of Salmonella typhimurium; Collazo CM et al.; The inv and spa loci of Salmonella typhimurium encode a type III protein secretion system which is essential for the ability of this microorganism to gain access to cultured epithelial cells . These loci are located at centisome 63 in the Salmonella chromosome . We have carried out a functional analysis of several genes of these loci and have found that two exported proteins encoded in this region, InvJ and SpaO, are required for secretion through the invasion-associated type III secretion system . These findings suggest the existence of a hierarchy in the export process, since mutations in other targets of this secretory system have no effect on protein secretion . We have also shown that the spaO, spaP, spaQ, and spaR genes are required for protein secretion and for the ability of S . typhimurium to gain access to cultured epithelial cells . In addition, we investigated the ability of an invJ S . typhimurium mutant strain to present the SipB protein to the bacterial surface and demonstrated that, in contrast to Spa32, its putative Shigella homolog, InvJ is not involved in the surface presentation of the Sip proteins.

Pediatr Cardiol, 1996 Sep-Oct, 17(5), 330 - 1
Fatal Salmonella typhimurium infection of a vascular graft in an infant; Juneja R et al.; A case of Salmonella typhimurium endocarditis of a Blalock-Taussig shunt in an infant is described for its rarity . Wider appreciation of such infections is warranted.

Biophys Chem, 1996 Aug 30, 61(1), 9 - 22
Time-resolved fluorescence of tryptophan synthase; Vaccari S et al.; Time-resolved and steady-state fluorescence of the tryptophan synthase alpha 2 beta 2 complex and of the beta 2 dimer from Salmonella typhimurium were measured to characterize the conformational properties of the beta subunit in the presence and in the absence of the alpha subunit when the catalytic species internal aldimine, external aldimine and alpha-aminoacrylate Schiff bases were selectively accumulated within the beta active site . The fluorescence decay of the coenzyme pyridoxal 5'-phosphate, bound via a Schiff base in the beta subunit of the alpha 2 beta 2 complex (internal aldimine species), is accounted for by two lifetimes (2.9 and 0.9 ns) of almost equal fractional intensity that are slightly affected by pH . Accordingly, both the absorption and emission spectra were found to be pH independent . The emission properties of the internal aldimine in the beta 2 dimer are pH dependent, suggesting that the alpha-subunit binding alters the microenvironment of the beta-subunit active site . This conclusion is also supported by the emission of the single tryptophanyl residue of the enzyme (Trp-177 beta) . In the reaction of L-serine with the alpha 2 beta 2 complex, the predominant catalytic intermediate is the external aldimine (lambda(max) = 422 nm) at pH 10, and the alpha-aminoacrylate (lambda(max) = 350 nm) at pH 7 . The external aldimine exhibits a high fluorescence intensity at 500 nm that decays with a single lifetime of 6.2 ns in the alpha 2 beta 2 complex, at pH 10, and at a similar value in the beta 2 dimer . The emission properties of the external aldimine with respect to the internal aldimine, and the small effects induced by alpha-subunit binding indicate a shielding of the coenzyme and a stabilization of its excited state . In contrast, the short fluorescence lifetime (0.4 ns) and the weak fluorescence emission of the alpha-aminoacrylate Schiff base indicate an increase of non-radiative processes possibly due to a more tight coupling of this intermediate with the protein matrix with respect to the external aldimine . Whereas the internal aldimine is distributed in two tautomeric forms, both the external aldimine and the alpha-aminoacrylate are present in single conformational states with distinct structural and/or dynamic properties that may modulate regulatory intersubunit signals.

J Biol Chem, 1996 Aug 30, 271(35), 21243 - 50
Ligand-dependent conformational plasticity of the periplasmic histidine-binding protein HisJ . Involvement in transport specificity; Wolf A et al.; The periplasmic histidine permease of Salmonella typhimurium is composed of a membrane-bound complex and a soluble histidine-binding protein (the periplasmic receptor), HisJ . Liganded receptor interacts with the membrane-bound complex, inducing ATP hydrolysis and substrate translocation . Preliminary evidence had shown a lack of direct correlation between the affinity of HisJ for a ligand and translocation efficiency, suggesting that the precise form of the receptor is important in determining its interaction with the membrane-bound complex . We have investigated the nature of the conformations assumed by HisJ upon binding a variety of ligands by tryptophan fluorescence enhancement, reaction with a closed form-specific monoclonal antibody, and changes in UV absorption spectra . It is demonstrated that although HisJ binds all the ligands and undergoes a conformational change, it assumes measurably different conformations . We also show that the interaction between HisJ and the membrane-bound complex depends on the nature of the ligand . Transport specificity appears to be defined, at least in part, by the conformation of the bound receptor, manifested either by the effect of a given ligand on the closed structure per se, or by the effect of ligand association on the equilibrium constant relating the open and the closed liganded forms.

J Mol Biol, 1996 Aug 16, 261(2), 195 - 208
FliN is a major structural protein of the C-ring in the Salmonella typhimurium flagellar basal body; Zhao R et al.; The Salmonella typhimurium FliN protein has been proposed to form a mutually interacting complex with FliG and FliM, the switch complex, that is required for flagellar morphogenesis and function . We have used affinity chromatography for purification of extended flagellar basal bodies sufficient for quantitative analysis of their protein composition . The belled, extended structure is predominantly comprised of the switch complex proteins; with FliN present in the most copies (111 +/- 13) . This explains why single, missense fliN, fliG or fliM mutations, found in many non-motile strains, can alter the belled morphology . Cell lysates from these strains contained the wild-type complement of FliG, FliM and FliN; but the basal bodies lacked the outer, cytoplasmic(C)-ring of the bell and were separated by sedimentation from FliM and FliN . The amount of FliG present in basal bodies from wild-type and one such mutant, FliN100LP, was comparable . These data show that: (1) the mutations define a FliG and FliMFliN multiple contact interface important for motility . (2) FliG is responsible for the increased size of the membrane-embedded MS-ring complex of belled relative to acid-treated basal bodies . (3) FliN, together with FliM, account for most of the C-ring . As a major component of the C-ring, FliN is distinct from the other proteins implicated in axial flagellar protein export . Inner, cytoplasmic rod basal substructure, seen by negative-stain and quick-freeze replica electron microscopy, may gate such export . Lack of connectivity between the cytoplasmic rod and ring substructures places contacts between FliG and FliMFliN at the periphery of the basal body, proximal to the flagellar intramembrane ring particles . This topology is consistent with models where torque results from interaction of circumferential arrays of the switch complex proteins with the ring particles.

Eur J Biochem, 1996 Aug 15, 240(1), 63 - 70
A thermodynamic analysis of conformational change due to the alpha 2 beta 2 complex formation of tryptophan synthase; Hiraga K et al.; A characteristic property of the tryptophan synthase alpha 2 beta 2 complex is the mutual activation of the alpha and beta subunit upon complex formation . It has been speculated that this mutual activation results from the conformational change due to the alpha/beta subunit interaction . To elucidate this mechanism, we investigated the thermodynamic parameters of association for the various combinations of the alpha and beta subunits from Escherichia coli and Salmonella typhimurium using isothermal titration calorimetry . The negative association enthalpy of the S . typhimurium alpha subunit with the beta subunit from E . coli (or S . typhimurium) was about 20 kJ mol-1 larger than that of the E . coli alpha subunit at 40 degrees C . However, the favorable enthalpy of the S . typhimurium alpha subunit was perfectly compensated by the unfavorable association entropy, therefore, the Gibbs energy of association was similar to that of the E . coli alpha subunit . Furthermore, the site-directed mutagenesis study revealed that a single mutation (K109N; {Asn109} alpha subunit) of the E coli alpha subunit at the subunit interface from E . coli to the S . typhimurium type could change the characteristics of the thermodynamic parameters of association to the S . typhimurium alpha subunit type . The heat-capacity changes of the association of the alpha subunit with the beta subunit were quite great, 6.37-8.21 kJ mol-1 K-1, compared with that due to a decrease in accessible surface area in the subunit interface . The analysis of the thermodynamic parameters of association suggested that the complex formation couples with the folding (rearrangements) of the alpha subunit monomer or/and beta subunit dimer.

Mutat Res, 1996 Aug 12, 369(3-4), 195 - 208
The Salmonella sulA-test: a new in vitro system to detect genotoxins; el Mzibri M et al.; The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins . This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes . A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538 . Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S . typhimurium TA1538/pEM1968 . A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay . The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships . Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test . Nineteen chemicals were genotoxic in the Salmonella sulA-test . The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin) . Sodium azide and 5-fluorouracil were not genotoxic . Frameshift, base-pair and oxidative genotoxins were detected by the tester strain . The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test . The SOSIP values of 12 compounds were the highest in this new assay . Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests . ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test . Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test . Four compounds had close MCD values in this assay and one of the two other techniques . The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191 . The umu-test was the technique of choice for 3-methylchloranthrene.

Mutat Res, 1996 Aug 12, 369(3-4), 183 - 94
Effects of beta-carotene and alpha-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen; Bianchi L et al.; The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7 . After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S . typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S . cerevisiae D7 . In S . typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage . Alpha-T was more protective than beta-C . The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage . An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed . The results obtained in S . typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition . At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective . At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP . In S . cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture . No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC . Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T . This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

Mutat Res, 1996 Aug 8, 360(3), 165 - 71
The mutagenicities of alkaloids and N-nitrosoguvacoline from betel quid; Wang CK et al.; In Taiwan, betel quid is a natural masticatory, which is composed of fresh green areca fruit, Piper betle and slaked lime paste . Areca fruit contains some alkaloids, of which arecoline is the major one . N-Nitrosoguvacoline (NG), one of the N-nitrosation products of arecoline, is the only one N-nitrosamine found in Taiwanese betel quid chewing saliva . The mutagenic studies in Ames Salmonella microsome test showed that crude alkaloid extracts of areca fruit and arecoline were active in Salmonella typhimurium TA100, and NG was weakly active in TA98 and TA100 . The activities in both arecoline and NG decreased further in the presence of rat liver S9 mix . Nitrite was significantly consumed during the N-nitrosation of arecoline and sodium nitrite at acidic condition (pH 3), whereas the formation of NG was favored at neutral condition (pH 7) . Crude phenolic extracts of leaf and inflorescence of Piper betle inhibited the formation of NG by blocking the nitrite . However, a high amount of crude phenolic extracts of areca fruit enhanced the formation of NG.

Biochemistry, 1996 Aug 6, 35(31), 10031 - 40
Identification of retained N-formylmethionine in bacterial recombinant mammalian cytochrome P450 proteins with the N-terminal sequence MALLLAVFL...: roles of residues 3-5 in retention and membrane topology; Dong MS et al.; An N-terminal block to Edman degradation was observed when any of five different mammalian cytochrome P450 (P450) proteins was expressed in Escherichia coli using the N-terminal sequence MALLLAVFL.. . This block was also seen in Salmonella typhimurium . With all proteins examined, the block could be removed by mild acid hydrolysis (0.6--6 N HCl, 23 degrees C) to expose Met as the N-terminus, suggesting N-formylMet retention . The N-terminal peptide of a modified P450 1A2 ("mutant 1", containing a thrombin-sensitive site inserted at residue 25) was released with thrombin and analyzed by electrospray mass spectrometry and found to yield the M(r) expected for the N-formyl derivative (+/- 0.8 amu) . The region of positions 3--5 was altered by random mutagenesis, and three P450 1A2-expressing clones were analyzed for nucleotide and amino acid sequences . The changes from LLL were to RER (P450 1A2a), VDS (P450 1A2b), and WRH (P450 1A2c); these all show slightly dissimilar hydropathy plots compared to the MALLLAVFL.. . sequence . Mutant P450 1A2a had the N-terminal Met removed to yield N-terminal Ala; P450 1A2b contained an unmodified Met at the N-terminus; P450 1A2c had an approximately 80% block of the N-terminal Met . Experiments with bacterial membranes containing expressed P450 1A2 mutant 1 and P450 1A2 mutant 2 (thrombin-sensitive site inserted at residue 46) suggest that thrombin site 2, but not 1, is sequestered in the membrane . Spheroplasts of bacteria expressing P450 1A2 and the mutants at positions 3--5 were treated with proteinase K; amino acid analysis indicated that no cleavage occurred . These results are interpreted in a model in which most of the mammalian P450 expressed in the bacterium is located in the cytosol, the region near residue 46 is in the inner membrane, the region near residue 25 is in the cytosol, and the N-terminus is either imbedded in the membrane or free in the cytosolic space, depending upon the sequence . However, the possibility that the differences in N-terminal processing are the result of direct changes in interactions with the deformylase and Met aminopeptidase cannot be excluded.

Front Biosci, 1996 Aug 01, 1, d131 - 45
New insights on molecular pathways utilized by salmonella species in cell binding; McCormick BA et al.; Salmonella infections are a principal source of gastroenteritis and enteric fever in a variety of animals, including humans . An essential step in the development of Salmonella pathogenesis is the entry of bacteria into non-phagocytic cells, including those that line the intestinal epithelium . As a consequence of specific cues from the host intestinal micro-environment, Salmonella entry into the intestinal epithelium is the product of a multistep process that culminates in host cell membrane ruffling, and subsequent bacterial uptake . The events that trigger the internalization event appear to require an array of bacterial secreted proteins, exemplified by the formation of bacterial surface appendages (invasomes) which are important for the induction of host-cell signal transduction pathways that lead to membrane ruffling . In addition, during intestinal disease states induced by Salmonella typhimurium, transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial membrane . Current evidence indicates that the intestinal epithelium plays a key role in orchestrating the inflammatory response to surface attached S . typhimurium . In this review, we explore current insights on the molecular pathways utilized by Salmonella spp . in cell binding that are important not only in the processes of Salmonella internalization but also in the generation of signals which lead to active states of intestinal inflammation.

Vet Med (Praha), 1996 Aug, 41(8), 255 - 9
{Use of hydrated lime for disinfection of sewage sludge containing Salmonella typhimurium and Ascaris suum as model pathogens}; Plachy P et al.; Hydrated lime was lethal to the strain of Salmonella typhimurium (Sk 14/39) after 60 min of exposure . In the control without addition of hydrated lime this strain was still viable after 168 hours, counting 6.1 x 10(5) pathogens/l ml sludge . 168-hr disinfection of primary sludges with 10 g/l hydrated lime showed no significant reduction in the viability of Ascaris suum eggs . In three experiments, the number of viable eggs was reduced only by 3.6% . Indicator microorganisms, except psychrophilic ones that survive for only 24 hr, were destroyed after 60 min of exposure . The temperature of stabilized sludges did not vary considerably during experiments, ranging between 21 and 25 degrees C . With the addition of Ca(OH)2, sludge pH increased to the values for COD, organic matters and total nitrogen were reduced throughout the experiments . The values for sludge dry residues remained unchanged.

Immunopharmacology, 1996 Aug, 34(1), 1 - 7
Recombinant human prolactin induces protection against Salmonella typhimurium infection in the mouse: role of nitric oxide; Meli R et al.; In the present study, we demonstrated that repeated treatment with recombinant human prolactin (rhPRL) protected mice against Salmonella typhimurium infection . The protective activity was statistically significant, dose-dependent and present only when rhPRL treatments were performed before the infection . This activity was probably related to the observed increases in phagocytosis and intracellular killing of peritoneal macrophages induced by the hormonal treatment . The number of peripheral leukocytes was not modified, excluding a mobilization of cells from other compartments . A decrease in the mortality rate after challenge was also observed in mice treated with the monoclonal antibody anti-PRL receptor U5, confirming that the protective activity was associated with receptor activation . Our studies also suggest that nitric oxide (NO) production was involved in the protective effect of rhPRL since pre-treatment of the animals with L-NAME, an inhibitor of NO-synthase, was able to completely revert the protective activity, whereas D-NAME, the inactive D-isomer, was without effect.

Biochem Mol Biol Int, 1996 Aug, 39(6), 1157 - 65
Purification of an alpha,beta-ketoalkene double bond reductase from Salmonella typhimurium; Ishida M et al.; An alpha, beta-ketoalkene double bond reductase was purified from the cell-free extract of Salmonella typhimurium . The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular weight of the enzyme was estimated to be 24,700 by the electrophoresis and 23,900 by HPLC gel filtration, respectively . The isoelectric point is pH 7.2 . The enzyme in the presence of NAD(P)H exhibited double bond reductase activity toward alpha, beta-ketoalkenes such as trans-phenyl-1-propenyl ketone, trans-benzylideneacetophenone and 15-ketoprostaglandins . The enzyme activity was markedly inhibited by dicumarol.

Int J Pancreatol, 1996 Aug, 20(1), 51 - 7
Mutagenic activation of N-nitrosobis(2-oxopropyl)amine by pancreatic juice and assessment of its ductal tumorigenicity following intraductal administration in dogs; Kamano T et al.; CONCLUSION: The results suggest that systemic administration (s.c . and i.p.) of BOP induces liver damage due to BOP itself and/or its metabolites which might be formed in the liver and that interaction of BOP itself in the pancreatic duct with pancreatic juice plays an important role for pancreatic duct tumorigenicity . METHODS: Mutagenic activation and pancreatic duct tumorigenicity of N-nitrosobis (2-oxopropyl)amine (BOP) administered s.c., i.p., and i.d . were studied in dogs . RESULTS: Following i.p . administration of BOP, N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP) and N-nitrosobis(2-hydroxypropyl)amine (BHP), but not BOP, were detected in pancreatic juice, while following i.d . administration, only BOP was detected . The pancreatic juice of one dog that received 100 mg of BOP i.d . showed positive mutagenicity towards Salmonella typhimurium TA100, but the pancreatic juice of two dogs that received 100 mg of BOP i.p . was not mutagenic . BOP showed clear mutagenicity in the presence of pancreatic juice from untreated dogs, but the pancreatic juice could not activate HPOP and BHP to mutagens . BOP administered sc for 2 wk (total dose: 600 mg) induced clinical toxicity, nausea, vomiting, and loss of appetite at 10 wk . BOP administered i.p . for 4 mo (total dose: 2000 mg) induced liver damage at 6 mo, but no pancreatic injury . BOP administered i.d . for 6.5 or 12 mo (total dose: 2500 or 4700 mg, respectively) induced papillary hyperplasia and dysplasia of duct epithelial cells and ductal proliferation with fibrosis.

Mol Microbiol, 1996 Aug, 21(3), 633 - 41
A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium; Kaniga K et al.; A number of bacterial pathogens have evolved sophisticated strategies to subvert host-cell signal-transduction pathways for their own benefit . These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells . In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence . Sequence analysis revealed that a carboxy-terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases . Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine . An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases . In addition, an amino-terminal region of SptP exhibited sequence similarity to the ribosyltransferase exoenzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp . The modular nature of this effector protein may allow multiple interactions with host-cell signalling functions.

Mutat Res, 1996 Aug, 370(1), 39 - 47
Genotoxicity of 4-chloro-o-toluidine in Salmonella typhimurium, human lymphocytes and V79 cells; Goggelmann W et al.; In the absence of a metabolizing system (S9 mix) 4-chloro-o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells . In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S . typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.

Mutat Res, 1996 Aug, 370(1), 11 - 7
Suppressive effects of chlorophyllin on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) and tumor promoter-dependent ornithine decarboxylase induction in BALB/c 3T3 fibroblast cells; Okai Y et al.; The potentially protective role of chlorophyllin, the sodium and copper salt of chlorophyll a against the initiation and promotion stages in carcinogenesis was studied by in vitro short-term assays . Chlorophyllin showed a dose-dependent suppressive effect on 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indol (Trp-P-1)-induced umu C gene expression of Salmonella typhimurium (TA 1535/pSK 1002) in the presence of metabolizing enzyme mixture . The similar inhibitory effect of chlorophyllin was detected in mitomycin C (MMC)-dependent umu C gene expression in the absence of metabolizing enzyme mixture . Furthermore chlorophyllin also exhibited a dose-dependent inhibition on 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity of 3T3 fibroblast cells at the same concentrations . However, when chlorophyll a isolated from Japanese tea leaves was applied on the same assay systems as a comparative experiment, chlorophyll a showed much weaker activity compared with that of chlorophyllin . The significance of this finding is discussed from the viewpoint of the protective role of chlorophyllin against carcinogenesis.

Protein Expr Purif, 1996 Aug, 8(1), 126 - 36
PCR mutagenesis and overexpression of tryptophan synthase from Salmonella typhimurium: on the roles of beta2 subunit Lys-382; Yang L et al.; We have devised convenient methods for mutagenesis and very high level expression of wild type and mutant tryptophan synthase alpha and beta2 subunits and alpha2beta2 complex from Salmonella typhimurium . The trpBA genes were modified by introduction of five new restriction sites by polymerase chain reaction (PCR) and were then cloned into the plasmid pTrc99A under trc promoter control . The recombinant plasmid pEBA-10 and three plasmids constructed from pEBA-10 were transformed into Escherichia coli CB149, which lacks tryptophan operon genes . Optimization of growth conditions of the transformed cells resulted in 10- to 40-fold higher yields of cells ( approximately 22 g/liter) than attained previously . The improved expression system gave higher yields of tryptophan synthase proteins (23-70% of the soluble protein) and led to correspondingly high yields of purified alpha and beta2 subunits or alpha2beta2 complex (200-800 mg/liter) . A plasmid containing 8 copies of the trpA gene gave the highest yield of alpha subunit . The PCR-based mutagenesis method permits mutation of any base pair in the trpBA genes, between suitable pairs of restriction sites, and requires only one new primer per mutation . The method is illustrated by construction of mutant beta2 subunits with any of five amino acid substitutions at Lys-382, the site of a previously described missense mutation . Characterization of the purified mutant alpha2beta2 complexes shows that Lys-382 in the wild type alpha2beta2 complex does not serve an essential catalytic role but may stabilize an active "closed" conformation of the enzyme by forming a salt bridge with Glu-350.

Can J Microbiol, 1996 Aug, 42(8), 855 - 8
Identification of two core types in lipopolysaccharides of Actinobacillus pleuropneumoniae representing serotypes 1 to 12; Jacques M et al.; Lipopolysaccharides (LPS) of Actinobacillus pleuropneumoniae were separated by Tricine-SDS-polyacrylamide gel electrophoresis, which has been shown to improve resolution of low-molecular-mass fast migrating bands . Strains representing the 12 serotypes of A . pleuropneumoniae can be divided in two groups according to the gel mobility of the core - lipid A region of their LPS . The first electromorphic core type (core type I), found in serotypes 1, 6, 9, and 11, had a migration slower than Salmonella typhimurium Ra LPS . The second electromorphic core type (core type II), found in the remaining serotypes (i.e., 2, 3, 4, 5, 7, 8, 10, and 12) had a migration similar to S . typhimurium Ra LPS . Furthermore, we observed that these two core types were antigenically different . Western blot analyses indicated that core - lipid A region of LPS from electromorphic core type I strains reacted when probed with serum from a pig experimentally infected with a core type I strain but not when probed with serum from a pig experimentally infected with a core type II strain . Conversely, core - lipid A region of LPS from electromorphic core type II strains reacted only when probed with serum from a pig experimentally infected with a core type II strain . Our results, based on both electrophoretic mobility and antigenicity, suggest the presence of two LPS core types in A . pleuropneumoniae.

J Appl Bacteriol, 1996 Aug, 81(2), 188 - 90
Inhibition of Salmonella typhimurium by the products of tartrate metabolism by a Veillonella species; Hinton A Jr et al.; The inhibition of the growth of Salmonella typhimurium by a Veillonella species grown on media supplemented with tartrate was examined . Growth of Salmonella typhimurium was not inhibited by the concentrations of products metabolized by Veillonella cultures on media supplemented with 0 or 50 mmol l-1 of tartrate, but was inhibited on media supplemented with 100 or 150 mmol l-1 of tartrate . Inhibition of Salm . typhimurium was correlated with the increased production of acetate and propionate from tartrate by the Veillonella species.

J Bacteriol, 1996 Aug, 178(16), 5032 - 8
Proteome of Salmonella typhimurium SL1344: identification of novel abundant cell envelope proteins and assignment to a two-dimensional reference map; Qi SY et al.; Forty-nine cell envelope proteins of Salmonella typhimurium SL1344 have been identified by microsequencing and assigned to a two-dimensional reference map . Ten of the sequenced proteins appear to be novel . Several others closely match currently hypothetical proteins or proteins found in other bacteria but not previously reported in salmonellae.

Infect Immun, 1996 Aug, 64(8), 3385 - 93
The Salmonella virulence plasmid enhances Salmonella-induced lysis of macrophages and influences inflammatory responses; Guilloteau LA et al.; The Salmonella dublin virulence plasmid mediates systemic infection in mice and cattle . Here, we analyze the interaction between wild-type and plasmid-cured Salmonella strains with phagocytes in vitro and in vivo . The intracellular recovery of S . dublin from murine peritoneal and bovine alveolar macrophages cultured in the presence of gentamicin in vitro was not related to virulence plasmid carriage . However, the virulence plasmid increased the lytic activity of S . dublin, Salmonella typhimurium, and Salmonella choleraesuis for resident or activated mouse peritoneal macrophages . Lysis was not mediated by spv genes and was abolished by cytochalasin D treatment . Peritoneal and splenic macrophages were isolated from mice 4 days after intraperitoneal infection with wild-type or plasmid-cured S . dublin strains . The wild-type strain was recovered in significantly higher numbers than the plasmid-cured strain . However, the intracellular killing rates of such cells cultured in vitro for both S . dublin strains were not significantly different . Four days after infection, there was a lower increase of phagocyte numbers in the peritoneal cavities and spleens of mice infected with the wild-type strain compared with the plasmid-cured strain . The virulence plasmid influenced the survival of macrophages in vitro following infection in vivo as assessed by microscopy . Cells from mice infected with the plasmid-cured strain survived better than those from mice infected with the wild-type strain . This is the first report demonstrating an effect of the virulence plasmid on the interaction of Salmonella strains with macrophages . Plasmid-mediated macrophage dysfunction could influence the recruitment and/or the activation of phagocytic cells and consequently the net growth of Salmonella strains during infection.

Infect Immun, 1996 Aug, 64(8), 3062 - 8
Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems; Yamamoto Y et al.; The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses . In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved . Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC . However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not . Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA . In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA . These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive . Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system . Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message . Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.

Infect Immun, 1996 Aug, 64(8), 2923 - 9
The Bcg/Ity/Lsh locus: genetic transfer of resistance to infections in C57BL/6J mice transgenic for the Nramp1 Gly169 allele; Govoni G et al.; The murine Bcg/Ity/Lsh locus determines the susceptibilities of inbred strains to infection with unrelated intracellular parasites, such as Mycobacterium bovis, Salmonella typhimurium, and Leishmania donovani . A candidate for Bcg/Ity/Lsh, designated Nramp1, has been recently identified and shown to encode a novel integral membrane protein that is expressed exclusively in professional phagocytes but whose function remains unknown . In inbred strains, the susceptibility to infection is associated with a single glycine-to-aspartic acid substitution at position 169 (G169D) in the predicted TM4 of the protein . To confirm the candidacy of Nramp1 as Bcg/Ity/Lsh and to determine the importance of the G169D mutation on Nramp1 function, we constructed transgenic mice in which the G169 allele of Nramp1 was transferred onto the background of a homozygous D169 allele . These transgenic mice were analyzed for their sensitivity to infections under the control of Bcg/Ity/Lsh . The transgene constructed for these studies contained the entire Nramp1G169 gene together with approximately 5 kb of sequences upstream of the transcription initiation site of this gene . We observed that these sequences were sufficient to direct Nramp1G169 expression in transgenic macrophages, resulting in the appearance of a mature protein of 90 to 100 kDa over a background of Nramp1G169 characterized by the complete absence of the mature Nramp1 polypeptide . The appearance of the Nramp1G169-encoded protein in transgenic macrophages was concomitant with the emergence of resistance to infection by M . bovis BCG, as measured by the extent of bacteria} replication in the spleen, and by S . typhimurium, as measured by survival after an intravenous challenge . The gain of function detected in transgenic Nramp1G169 animals establishes unambiguously that Nramp1 and Bcg/Ity/Lsh are allelic.

J Bacteriol, 1996 Aug, 178(15), 4628 - 34
Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum; Limberger RJ et al.; A locus encoding polypeptides associated with flagellar structure and function was identified, sequenced, and characterized in Treponema phagedenis and Treponema pallidum . This locus includes homologs of the FlgD, FlgE, MotA, MOB, FliL, and FliM polypeptides found in Salmonella typhimurium and Bacillus subtilis . These polypeptides are extensively conserved between the two treponemes . Several additional polypeptides or unknown function, including Tapl, located upstream of FlgD, and ORF4, located between FlgE and MotA, were also identified . Transcription analysis using RNA PCR indicated that these genes are likely transcribed as part of a single operon and comprise the 5' region of the treponemal fla operon . Primer extension analysis identified a putative promoter, preceding T . phagedenis tap1 in a region of divergent transcription . Pfla resembles the class II or class III motility-related promoters of S . typhimurium . FlgE and Tap1 were further characterized . Western blotting (immunoblotting) indicated that T . pallidum FlgE exhibited an unusual polypeptide ladder that was similar but not identical to that of T . phagedenis . Triton X-114 phase partitioning of T . phagedenis cells coupled with Western blotting revealed that Tap1 was located in the aqueous phase . Computer analysis indicated that Tap1 had no significant membrane spanning regions, suggesting that it resides primarily in the cytoplasm . The organization and expression of this operon are similar in both treponemes but different from those of previously described motility-related operons . These results indicate that despite extensive amino acid sequence conservation, the expression of spirochete flagellar polypeptides is different from that in other bacteria.

J Bacteriol, 1996 Aug, 178(15), 4582 - 9
Analysis of a FliM-FliN flagellar switch fusion mutant of Salmonella typhimurium; Kihara M et al.; In the course of an analysis of the three genes encoding the flagellar motor switch, we isolated a paralyzed mutant whose defect proved to be a 4-bp deletion of the ribosome binding sequence of the fliN switch gene (V . M . Irikura, M . Kihara, S . Yamaguchi, H . Sockett, and R . M . Macnab, J . Bacteriol . 175:802-810,1993) . This sequence lies just before the 3' end of the coding sequence of the upstream fliM switch gene, in the same operon . This mutant readily gave rise to pseudorevertants which, though much less motile than the wild type, did exhibit significant swarming . One such pseudorevertant was found to contain a compensating frameshift such that the fliM and fliN genes were placed in frame, coding for an essentially complete FliM-FliN protein fusion . Minicell analysis demonstrated that, as expected, the parental mutant synthesized an essentially full-length FliM protein but no detectable FliN . The pseudorevertant, in contrast, synthesized a protein with the predicted size for the FliM-FliN fusion protein and no detectable FliM or FliN . Immunoblotting of minicells with antibodies against FliM and FliN confirmed the identities of these various proteins . Immunoblotting of book-basal-body complexes from the wild-type strain gave a strong signal for the three switch proteins FliG, FliM, and FliN . Complexes from the FliM-FliN fusion mutant gave a strong signal for FliG but no signal for either FIiM or FliN; a moderately strong signal for the FliM-FliN fusion protein was seen with the anti-FliM antibody, and a weaker signal was seen with the anti-FliN antibody . The cytoplasmic C ring of the structure, which is seen consistently in electron microscopy of wild-type complexes and which is known to contain the FliM and FliN proteins, was much more labile in the FliM-FliN fusion mutant, giving a fragmented and variable appearance or being completely absent . Complementation data indicated that wild-type FliM had a mild dominant negative effect over the fusion protein, that wild-type FliN and the fusion protein work much better than the fusion protein alone, and that wild-type FliM and FliN together have no major positive or negative effect on the function of the fusion protein . We interpret these data to mean that the FliM-FliN fusion protein incorporates into structure but less stably than do the FliM and FliN proteins separately, that wild-type FliM tends to displace the fusion protein, and that wild-type FliN can supplement the FliN domain of the fusion protein without displacing the FliM domain . The data support, but do not prove, a model in which FliM and FliN in the wild-type switch complex are stationary with respect to each other.

J Mol Biol, 1996 Jul 26, 260(4), 506 - 22
Homologous recombination between the tuf genes of Salmonella typhimurium; Abdulkarim F et al.; The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium . The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication . Sequence comparisons between S . typhimurium and Escherichia coli suggest that within each species the two tuf genes are evolving in concert . Here we show that each of the S . typhimurium tuf genes can transfer genetic information to the other . In our genetic system the transfers are seen as non-reciprocal, i.e . as gene conversion events . However, the mechanism of recombination could be reciprocal, with sister chromosome segregation and selection leading to the isolation of a particular class of recombinant . The amount of sequence information transferred in individual recombination events varies, but can be close to the entire length of the gene . The recombination is RecABCD-dependent, and is opposed by MutSHLU mismatch repair . In the wild-type, this type of recombination occurs at a rate that is two or three orders of magnitude greater than the nucleotide substitution rate . The rate of recombination differs by six orders of magnitude between a recA and a mutS strain . Mismatch repair reduces the rate of this recombination 1000-fold . The rate of recombination also differs by one order of magnitude depending on which tuf gene is donating the sequence selected for . We discuss three classes of model that could, in principle, account for the sequence transfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocal recombination involving sister chromosomes; (3) non-allelic gene conversion involving sister chromosomes, initiated by a double-strand break close to one tuf gene . Although the mechanism remains to be determined, the effect on the bacterial cells is tuf gene sequence homogenisation . This recombination phenomenon can account for the concerted evolution of the tuf genes.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7800 - 4
Identification of a pathogenicity island required for Salmonella survival in host cells; Ochman H et al.; We have identified a region unique to the Salmonella typhimurium chromosome that is essential for virulence in mice . This region harbors at least three genes: two (spiA and spiB) encode products that are similar to proteins found in type III secretion systems, and a third (spiR) encodes a putative regulator . A strain with a mutation in spiA was unable to survive within macrophages but displayed wild-type levels of epithelial cell invasion . The culture supernatants of the spi mutants lacked a modified form of flagellin, which was present in the supernatant of the wild-type strain . This suggests that the Spi secretory apparatus exports a protease, or a protein that can alter the activity of a secreted protease . The "pathogenicity island" harboring the spi genes may encode the virulence determinants that set Salmonella apart from other enteric pathogens.

J Mol Biol, 1996 Jul 19, 260(3), 317 - 31
Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme; North AK et al.; NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes . To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP . NtrC can also repress transcription by sigma70-holoenzyme . In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme . The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation . Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal . In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers . (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity . We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription . These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity . Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

Mutat Res, 1996 Jul 10, 369(1-2), 107 - 12
Mutagenic activity of some coal-derived humic compounds evaluated by the Ames test; Bernacchi F et al.; Two coal-derived humic substances (Sulcis and South Africa, Eniricerche, Italy) have been evaluated for their mutagenic activity on TA98 and TA100 Salmonella typhimurium strains, either in presence or in absence of metabolic activation (S9) . Both compounds showed no effect on the two strains, as observed with natural humic acid (Fluka) . After chlorination, coal-derived humic acids induced a strong dose-related increase in the number of revertants on TA100 without S9, whose extent was directly proportional to the chlorination ratios . Such effect was completely suppressed when a sodium thiosulfate solution (10%) was added at the end of the chlorination period (about 90 h) . The analogies with natural humic acid mutagenicity are discussed.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7028 - 31
The N-terminal domain of Escherichia coli enzyme I of the phosphoenolpyruvate/glycose phosphotransferase system: molecular cloning and characterization; Chauvin F et al.; The bacterial phosphoenolpyruvate/glycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to sugars concomitant with their translocation . The first two steps of the phosphotransfer sequence are PEP <--> Enzyme I (EI) <--> HPr (the histidine-containing phosphocarrier protein) . We have proposed that many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition . EI monomer (63.5 kDa) comprises two major domains: a flexible C-terminal domain (EI-C) and a protease-resistant, structurally stable N-terminal domain (EI-N) containing the active site His . Trypsin treatment of Salmonella typhimurium EI yielded EI-N, designated EI-N(t) . Homogeneous recombinant Escherichia coli EI-N {i.e., EI-N(r)}, has now been prepared in quantity, shows the expected thermodynamic unfolding properties and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP . In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K/a = 1.4 x 10(5) M(-1) and delta H = +8.8 kcal x mol(-1) . Both values are comparable to those for HPr binding to intact EI . Fluorescence anisotropy {dansyl-EI-N(r)} and gel filtration of EI-N(r) show that it does not dimerize . These results emphasize the role of EI-C in dimerization and the regulation of intact EI.

Mutat Res, 1996 Jul 5, 368(3-4), 261 - 5
Lack of mutagenicity of diethylstilbestrol metabolite and analog, (+/-)-indenestrols A and B, in bacterial assays; Ishikawa S et al.; Indenestrol A (IA), one of metabolites of the indanyl group of diethylstilbestrol, has a stronger binding affinity for the estrogen receptor and also a weaker uterotropic activity than endogenous estradiol . We tested the microbial mutagenicity of structural isomers of indenestrol A and indenestrol B (IB) in Salmonella typhimurium TA100 and TA98 and in Escherichia coli WP2 uvrA to investigate whether the interaction of diethylstilbestrol or IA with genomic DNA has any part in their carcinogenicity and other biological activities . In the absence of S9 mix, (+/-)-IA was cytotoxic at higher doses (1 and 10 mumol/plate), and both (+/-)-IA and (+/-)-IB were non-mutagenic at lower doses (0.1-100 nmol/plate) . In the presence of S9 mix, (+/-)-IA was cytotoxic at higher doses (0.5 and 1 mumol/plate), and at the other doses, (+/-)-IA and (+/-)-IB did not show any distinct increase in revertants . Although (+/-)-IA and (+/-)-IB showed a slight increase in the revertants in strain TA100 by the preincubation method without S9 mix, these results were considered to be negative, because no reproducible dose-revertants relationship necessary for a chemical to be determined as mutagenic was obtained . The S9 fraction interacted with (+/-)-IA or (+/-)-IB enzymatically or non-enzymatically, and weakened its cytotoxicity, so that the toxic dose was higher in the presence of S9 mix than in its absence . Both the plate incorporation and preincubation methods were used with a wide range of concentrations of (+/-)-IA and (+/-)-IB in the present experiment . No clear positive mutagenic data were obtained . These results are the first reports on the mutation assays of (+/-)-IA and (+/-)-IB, and suggest that they were non-mutagenic towards the bacterial strains tested . The study revealed that the cytotoxic activity of (+/-)-IA and (+/-)-IB did not correlate with DNA interaction, but was the result of a direct effect on microtubule polymerization, although indenestrols are known to have strong binding affinities for estrogen receptors.

Mutat Res, 1996 Jul 5, 368(3-4), 221 - 33
Modulation of the mutagenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by phenolic compounds; Miller C et al.; NNK is a potent environmental carcinogen generated during tobacco processing and smoking . The carcinogenic response to tobacco smoking is modulated by nutritional factors . In this study, liver microsomes from phenobarbital and beta-naphthoflavone-treated or control hamsters were used to assay the mutagenicity (Salmonella typhimurium TA1535) of NNK . Western analysis of these microsomal preparations revealed an increased expression of protein recognized by polyclonal antibodies specific for P-450 1A2 in beta-naphthoflavone-induced microsomes and P-450 2B1/2B2 in phenobarbital-induced microsomes . Both inducers significantly increased the mutagenicity of NNK . Metabolism of NNK by the three microsomal preparations was compared . Metabolites formed by methyl-hydroxylation of NNK by microsomes from control animals were significantly greater than those formed by alpha-methylene hydroxylation . Phenobarbital treatment had the greatest effect on alpha-methylene hydroxylation while beta-naphthoflavone had the greatest effect on methyl hydroxylation . The antimutagenic action of the polyphenolic compounds ellagic acid, esculetin and propyl gallate correlated with an inhibition of the metabolism of NNK . There were, however, differences in the effects of these compounds on specific pathways of NNK metabolism depending upon the microsomal enzyme induction treatment . This suggests that phenolic compounds have selective affinity for specific P-450 isozymes activating NNK.

Mutat Res, 1996 Jul 5, 368(3-4), 157 - 63
Genotoxicity of dihydroabikoviromycin, a secondary metabolite of Streptomyces anulatus; Holmalahti J et al.; The potential genotoxicity of dihydroabikoviromycin was assessed in bacterial and sister-chromatid exchange (SCE) test systems . Direct cytotoxicity was also assayed using bioluminescence methods to screen for differences in cell viability among different tumour cell lines following exposure to the drug . Differential killing tests with Escherichia coli WP2 and its repair-deficient derivative CM871 indicated that a functional DNA repair system was protective against the action of dihydroabikoviromycin, implying that this compound causes some form of DNA damage and is almost certainly therefore genotoxic . Dose-dependent reversion from His- to His+ with dihydroabikoviromycin was observed in the Ames test with Salmonella typhimurium TA100, but not in frameshift tester strain TA98 . Dihydroabikoviromycin also induced the sfiA gene, as indicated by beta-galactosidase induction in an SOS-chromotest with E . coli PQ37 strain . A dose-related increase in SCEs by dihydroabikoviromycin was observed in CHO cells . Growth of tumour cells was also suppressed by dihydroabikoviromycin at a dose of 10 micrograms/ml.

East Afr Med J, 1996 Jul, 73(7), 432 - 4
Fate of Salmonella enteritidis and Salmonella typhimurium during the fermentation of siljo; Dessie G et al.; The behaviour of Salmonella enteritidis and S . typhimurium in control gruel and fermenting siljo was evaluated . S . enteritidis and S . typhimurium reached maximum levels of 10(8) cfu/ml and 10(6) cfu/ml, respectively within 48 hours in the control gruel . In fermenting "siljo", pH fell to < 5.0 within 48 hours and titratable acidity increased markedly . This resulted in the complete inhibition of S . enteritidis in 48 hours and S . typhimurium in 72 hours . Acid production by the fermenting lactic acid bacteria and components of mustard powder could have inhibited the test organisms . Since siljo is usually ready for consumption after 72 hours of fermentation, contaminating salmonellae would be eliminated before the product is consumed.

Mol Microbiol, 1996 Jul, 21(2), 247 - 56
In vitro characterization of constitutive CysB proteins from Salmonella typhimurium; Colyer TE et al.; Expression of the cysteine regulon in Salmonella typhimurium and Escherichia coli is controlled by the LysR-type transcriptional activator CysB and by the inducer N-acetyl-L-serine . Sulphide and thiosulphate are anti-inducers . Two highly purified constitutive CysB proteins, CysB(T149M) and CysB(T149P), were found to bind to the cysJIH, cysK and cysP promoters, to activate transcription from the cysJIH and cysK promoters in the absence of N-acetyl-L-serine, and to be insensitive to the effects of anti-inducers . At 10 mM MgCl2, the in vitro transcription activity of CysB(T149M) was maximal without N-acetyl-L-serine, but that of CysB(T149P) was increased by inducer . At 2 mM MgCl2, both proteins were fully active without inducer . A third mutant protein, CysB(W166R), was totally inactive at 10 mM MgCl2, but gave constitutive expression of the cysK and cysJIH promoters at 2 mM MgCl2 . Surprisingly, wild-type CysB was also constitutive for the cysK promoter at 2 mM mgCl2 but not at 10 mM MgCl2; it required inducer for cysJIH promoter activation at both concentrations . Mutagenic studies indicated that this difference between promoters is due to the distance between activation site half-sites, which are separated by 1 bp in the cysJIH promoter and by 2 bp in the cysK promoter . We speculate that inducer acts to decrease the distance between the binding domains of two CysB subunits that interact with an activation site . In vitro activities of wild-type and mutant CysB proteins correlated much better with in vivo behaviour at 2 mM than at 10 mM MgCl2, suggesting that the former is the more physiological concentration.

Mol Microbiol, 1996 Jul, 21(1), 111 - 22
A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require rec functions for viability; Gari E et al.; We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon . Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family . SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product . Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37 degrees C . Selection of suppressors that concomitantly restore growth at 37 degrees C and SOS induction in a recB- background yielded mutations that relleve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE . Such mutations also restore SOS induction in quinolone-treated gyr+ recB- strains . These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37 degrees C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria . Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of "cleavable' gyrase-DNA complexes in plasmid or chromosomal DNA . Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA-cells at restrictive temperatures . Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks . We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.

Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 697 - 702
Effect of primary structure modifications in Trypanosoma cruzi neuraminidase/trans-sialidase activities; Cremona ML et al.; Neuraminidases have been implicated in various processes involving the interaction of pathogens and their receptor cells . Trypanosoma cruzi, the agent of Chagas disease, has an unusual neuraminidase, able to transfer bound alpha(2-3)sialic acid to a suitable acceptor rather than to water: the trans-sialidase (TcTS) . This enzyme is encoded by a family of several homologous genes, some of them rendering inactive the products . We have shown that enzymatically active proteins have Tyr in position 342, whereas inactive TcTS contain a His342 . We have now mutated this Tyr residue to Phe or Thr . Both mutant proteins resulted in enzymatically inactive products . Chimeras expressing parts of Salmonella typhimurium neuraminidase (NANH) and TcTS were constructed . Only the construct containing the complete NANH molecule fused to the last 272 residues of TcTS had neuraminidase activity . However this construct did not present TcTS activity . This finding suggests that other residues present in the homology region are required for TcTS activity.

J Bacteriol, 1996 Jul, 178(14), 4200 - 7
Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium; Schoenhals GJ et al.; The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG C) for lipoylation and signal peptide cleavage . We have confirmed the previous finding (M . Homma, K . Ohnishi, T . Iino, and R . M . Macnab, J . Bacteriol . 169:3617-3624, 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca . 25 kDa . flgH alleles with an in-frame deletion or a 3' truncation still permitted processing . The deletion permitted partial restoration of motility in complementation tests, whereas the truncation did not . Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH . When cells transformed with a plasmid containing the flgH gene were grown in the presence of {3H}palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used . Hook-basal body complexes from cells grown in the presence of {3H}palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled . An in-frame fusion between the leader sequence of the periplasmic protein PeIB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form . With a low-copy-number plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type . Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated {3H}palmitate at low levels, perhaps through acylation of the N-terminal alpha-amino group . We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body . We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.

J Toxicol Sci, 1996 Jul, 21 Suppl 2, 465 - 74
{A mutagenicity study of calcipotriol (MC903)}; Kitagaki T et al.; Calcipotriol (MC903), an anti-psoriasic agent, was examined for mutagenicity in the reverse mutation test and the chromosomal aberration test in vitro, and the micronucleus test in Slc:ddY mice . 1 . In the reverse mutation test using Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2uvrA), MC903 did not significantly increase revertant colonies in any of the test strains with or without metabolic system . 2 . In the chromosomal aberration test with a Chinese hamster fibroblast cell line (CHL), MC903 did not significantly increase aberrant cells in the direct method or in the activation method . 3 . In the micronucleus test with male mice, MC903 did not significantly increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow . These results suggest that MC903 has no mutagenic as well as clastogenic effects under the present experimental condition.

Carcinogenesis, 1996 Jul, 17(7), 1499 - 503
Formation of DNA adducts by the co-mutagen norharman with aromatic amines; Mori M et al.; Norharman, widely distributed in our environment, is alone not mutagenic to Salmonella typhimurium TA98 and TA100 either with or without S9 mix, but becomes mutagenic to S.typhimurium TA98 with S9 mix when non-mutagenic aromatic amines like aniline or o- or m-toluidine are added . Thus norharman has been called a 'co-mutagen' . In the present study we examined whether or not DNA adducts are formed in DNA of S.typhimurium TA98 by treatment with norharman and aromatic amines using 32P-post-labeling analysis under modified adduct intensification conditions . When a sample of norharman (8 mg) and aniline (4 mg) was incubated with 4 ml of overnight culture of S.typhimurium TA98 in the presence of 20 ml S9 mix for 6 h at 37 degrees C, three adduct spots were detected at a total relative adduct labeling (RAL) of 10.8 +/- 2.27/10(8) nucleotides . Under the same conditions, a mixture of norharman (8 mg) and o-toluidine (4 mg) yielded three adduct spots at a RAL of 3.74 +/- 1.71/10(8) nucleotides . With a combination of norharman and m-toluidine, a single adduct spot was seen at a RAL of 0.04 +/- 0.01/10(8) nucleotides . In contrast, norharman with p-toluidine did not produce adduct spots . Furthermore, neither norharman nor the aromatic amines themselves gave any evidence of adducts . Thus DNA adduct formation by norharman with aromatic amines correlates with the co-mutagenic action of norharman in S.typhimurium TA98.

Infect Immun, 1996 Jul, 64(7), 2765 - 73
Acidification of phagosomes containing Salmonella typhimurium in murine macrophages; Rathman M et al.; Salmonella species are facultative intracellular pathogens . Following entry into mammalian host cells, they reside in membrane-bound vacuoles, resist killing, and replicate . In this work, we investigated the importance of phagosomal pH in the ability of Salmonella typhimurium to survive and replicate within macrophages . Intraphagosomal pH was measured in situ by recording the fluorescence intensity of a pH-sensitive probe, DM-NERF dextran . The majority of vacuoles containing S . typhimurium (live, heat killed, or formalin fixed) acidified from pH > or = 6.0 to between pH 4.0 and 5.0 within 60 min after formation . In contrast, Mycobacterium avium-containing vacuoles failed to acidify even at later time points . Acidification of S . typhimurium-containing vacuoles was completely blocked by treatment of host cells with bafilomycin A, a specific inhibitor of vacuolar proton-ATPases . Bafilomycin inhibition of vacuolar acidification from the onset of infection significantly decreased the survival of S . typhimurium in macrophages . Furthermore, bafilomycin treatment at 2, 4, 8, or even 12 h postinfection decreased the percentage of recoverable bacteria by up to 20-fold . Loss of bacterial viability was seen with several other reagents which, like bafilomycin, raise the pH of phagosomal compartments but are not directly lethal to the bacteria or host cells . Thus, we conclude that Salmonella-containing phagosomes acidify soon after formation and hypothesize that an acidic environment is necessary for survival and replication of the bacteria within the macrophage.

Infect Immun, 1996 Jul, 64(7), 2730 - 6
Studies of immunity and bacterial invasiveness in mice given a recombinant salmonella vector encoding murine interleukin-6; Dunstan SJ et al.; Interleukin-6 (IL-6) was expressed in Salmonella typhimurium in an attempt to increase the mucosal immune response against the bacterium . Murine IL-6 was PCR amplified from cDNA, cloned, sequenced, and found to be functionally active when expressed in S . typhimurium BRD509, the (delta)aroA (delta)aroD vaccine strain . Expression of murine IL-6 did not appear to adversely affect the growth of salmonellae, as the construct was retained in the absence of antibiotic selection and the growth rate was unaffected compared with that of the parent strain in vitro . However, IL-6 expression led to a significant reduction in bacterial invasiveness in vitro and in vivo . Splenocytes and small intestinal lamina propria lymphocytes were isolated from mice orally immunized with BRD509 expressing IL-6 (pKK233-2/IL-6), and the number of antibody-secreting cells was determined by the ELISPOT technique . No differences were observed between mice immunized with BRD509(pKK.233-2/IL-6) and those immunized with BRD509(pKK233-2) with respect to the antibody subclass-specific responses elicited despite the markedly reduced invasiveness of the former . Serum antibody responses were also examined by a kinetic enzyme-linked immunosorbent assay (ELISA), and equivalent levels of antibody response were detected in mice given BRD509(pKK233-2/IL-6) and those given BRD509(pKK233-2) . The humoral immune response against bacterial lipopolysaccharides was also examined in transgenic IL-6-deficient mice given oral inocula of BRD509 . Equivalent numbers of antibody-secreting cells (ELISPOTs) were observed in the spleens and laminae propriae of both IL-6-deficient (-/-) mice and control (+/+) mice harboring an intact IL-6 gene, whereas small, yet significant differences in the serum immunoglobulin A ELISA titers were observed . These data suggest that the immunoglobulin A response against Salmonella lipopolysaccharides is largely IL-6 independent.

J Exp Med, 1996 Jul 1, 184(1), 271 - 6
A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection; Klimpel GR et al.; Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops . Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure . In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection . Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge . Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA . Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella . Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection . Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion . Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates . Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

J Bacteriol, 1996 Jul, 178(13), 3854 - 9
Multicopy suppressors of the cold-sensitive phenotype of the pcsA68 (dinD68) mutation in Escherichia coli; Yasuda T et al.; The Escherichia coli strain cs2-68 is a cold-sensitive (c) mutant that forms a long filamentous cell at 20 degrees C with a large nucleoid mass in its central region . We have recently shown that the pcsA68 mutation causing the cs phenotype is a single-base substitution within the dinD gene, a DNA damage-inducible gene which maps at 82 min . Since null mutants of the pcsA (dinD) gene are viable, with no discernible defect in cell growth, the cs phenotype is attributed to a toxic effect by the mutant protein . In an attempt to identify a target(s) for the toxic pcsA68 mutant protein, we screened for chromosomal fragments on multicopy plasmids that could suppress the cs phenotype . Three different BamHI fragments were found to suppress cold sensitivity, and the lexA, dinG, and dinI genes were identified to be responsible for the suppression in each fragment . DinG shares multiple motifs with many DNA helicases . The complete sequence of dinI revealed that DinI is a small protein of 81 amino acids . It is similar in size and sequence to ImpC of the Salmonella typhimurium plasmid TP110 and to a protein (ORFfs) of the retronphage phi R67, both of which are also under the control of LexA.

J Bacteriol, 1996 Jul, 178(13), 3829 - 39
Mutation in the structural gene for release factor 1 (RF-1) of Salmonella typhimurium inhibits cell division; Olafsson O et al.; A temperature-sensitive mutant of Salmonella typhimurium LT2 was isolated . At the nonpermissive temperature cell division stopped and multinucleated filaments were formed . DNA, RNA, or protein synthesis was not affected until after about two generations . Different physiological conditions, such as anaerobiosis and different growth media, suppress the division deficiency at high temperatures . Certain mutations causing a reduced polypeptide chain elongation rate also suppress the division deficiency . The mutation is recessive and shown to be in the structural gene for release factor I (prfA) . DNA sequencing of both the wild-type (prfA+) and mutant (prfA101) allele revealed a GC-to-AT transition in codon 168 . Like other known prfA mutants, prfA101 can suppress amber mutations . The division defect in the prfA101 mutant strain could not be suppressed by overexpression of the ftsQAZ operon . Moreover, at the nonpermissive temperature the mutant shows a normal heat shock and SOS response and has a normal ppGpp level . We conclude that the prfA101-mediated defect in cell division is not directed through any of these metabolic pathways, which are all known to affect cell division . We speculate that the altered release factor I induces aberrant synthesis of an unidentified protein(s) involved in the elaborate process of septation.

J Bacteriol, 1996 Jul, 178(13), 3763 - 70
Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene; Brown L et al.; The RpoS transcription factor (also called sigma Sor sigma 38) is required for the expression of a number of stationary-phase and osmotically inducible genes in Escherichia coli . RpoS is also a virulence factor for several pathogenic bacteria, including Salmonella typhimurium . The activity of RpoS is regulated in response to several different signals, at the transcriptional and translational levels as well as by proteolysis . Here we report that host factor I (HF-I), the product of the hfq gene, is required for efficient expression of rpoS in S . typhimurium . HF-I is a small, heat-stable, site-specific RNA-binding protein originally characterized for its role in replication of the RNA bacteriophage Q beta of E . coli . Its role in the uninfected bacterial cell has previously been unknown . Assays of Beta-galactosidase in strains with rpoS-lac fusions, Western blot (immunoblot) analysis, and pulse-labeling and immunoprecipitation of both fusion proteins and native RpoS show that an S . typhimurium hfq mutant has a four- to sevenfold reduction in expression of rpoS that is attributable primarily to a defect in translation . These results add a new level of complexity to the regulation of RpoS activity.

J Bacteriol, 1996 Jul, 178(13), 3722 - 6
Detection and characterization of the flagellar master operon in the four Shigella subgroups; Al Mamun AA et al.; Strains in the genus Shigella are nonmotile, but they retain some cryptic flagellar operons whether functional or defective (A.Tominaga, M . A.-H . Mahmoud, T . Mukaihara, and M . Enomoto, Mol . Microbiol . 12:277-285, 1994) . To disclose the cause of motility loss in shigellae, the presence or defectiveness of the flhD and flhC genes, composing the master operon whose mutation causes inactivation of the entire flagellar regulon, was examined in the four Shigella subgroups . The flhD operon cloned from Shigella boydii and Shigella sonnei can activate, though insufficiently, the regulon in the Escherichia coli flhD or flhC mutant background . The clone from Shigella dysenteriae has a functional flhD gene and nonfunctional flhC gene, and its inactivation has been caused by the IS1 element inserted in its 5' end . The operon of Shigella flexneri is nonfunctional and has suffered an IS1-insertion mutation at the 5' end of the flhD gene . Comparison of restriction maps indicates that only the central 1.8-kb region, including part of the flhC gene and its adjacent mot operon, is conserved among the four Shigella subgroups as well as in E . coli, but in Salmonella typhimurium the whole map is quite different from the others . Motility loss in shigellae is not attributable to genetic damage in the master operon of a common ancestor, but it occurs separately in respective ancestors of the four subgroups, and in both S . dysenteriae and S.flexneri IS1 insertion in the master operon might be the primary cause of motility loss.

J Bacteriol, 1996 Jul, 178(13), 3683 - 8
Starvation- and Stationary-phase-induced resistance to the antimicrobial peptide polymyxin B in Salmonella typhimurium is RpoS (sigma(S)) independent and occurs through both phoP-dependent and -independent pathways; McLeod GI et al.; A common stress encountered by Salmonella serovars involves exposure to membrane-permeabilizing antimicrobial peptides and proteins such as defensins, cationic antibacterial proteins, and polymyxins . We wanted to determine if starvation induces cross-resistance to the membrane-permeabilizing antimicrobial peptide polymyxin B (PmB) . We report here that starved and stationary-phase (Luria-Bertani {LB} medium) cells exhibited ca . 200- to 1,500-fold-higher (cross-)resistance to a 60-min PmB challenge than log-phase cells . Genetic analysis indicates that this PmB resistance involves both phoP-dependent and -independent pathways . Furthermore, both pathways were sigma(S) independent, indicating that they are different from other known sigma(S) -dependent cross-resistance mechanisms . Additionally, both pathways were important for PmB resistance early during C starvation and for cells in stationary phase in LB medium . However, only the phoP-independent pathway was important for P-starvation-induced PmB resistance and the sustained PmB resistance seen in 24-h-C-starved (and N-starved) or stationary-phase cells in LB medium . The results indicate the presence of an rpoS- and phoP-independent pathway important to starvation- and stationary-phase-induced resistance to membrane-permeabilizing antimicrobial agents.

Lett Appl Microbiol, 1996 Jul, 23(1), 49 - 54
Pretreatment to reduce somatic Salmonella phage interference with FRNA coliphage assays : successful use in a one-year survey of vulnerable groundwaters; Stetler RE et al.; Somatic salmonella (SS) phages were commonly found in higher numbers than F-specific RNA (FRNA) coliphages in a multi-site survey of contamination-vulnerable groundwaters . The relative abundance of SS phages required that a pretreatment procedure be implemented to reduce the SS phage content of samples before FRNA coliphage assay with Salmonella typhimurium WG49 . Pretreatment involved selective SS phage removal by Salm . typhimurium WG45 cells . This pretreatment proved effective in producing interference-free samples throughout the one-year survey period and in seeded evaluation, was shown not to affect the detection of representative FRNA coliphage MS2 . During the survey, 30 groundwater sites located in the continental United States, Puerto Rico and the Virgin Islands were examined for FRNA coliphages and SS phages at monthly intervals . FRNA coliphages were detected at six of the 30 sites and in 33 of 329 monthly samples . SS phages were also detected at six sites and in 28 of 329 monthly samples . Five of the phage-positive sites were positive for both phage groups . At those five sites, 58 monthly samples were collected during the survey period . Those 58 samples yielded an average FRNA coliphage concentration of 140 pfu per 100 l of groundwater as compared to an average SS phage concentration of 565 pfu per 100 l of groundwater . Twenty of the 58 samples were positive for both the FRNA coliphages and SS phages . In those samples, FRNA coliphages were more abundant in five samples; SS phages were more abundant in 15 samples . Because these results demonstrate that SS phage levels may often exceed FRNA coliphage levels in environmental waters, it is clear that SS phage removal procedures will greatly enhance the effectiveness of the WG49-based FRNA coliphage assay.

Cancer Res, 1996 Jul 1, 56(13), 2979 - 84
Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1; Shimada T et al.; A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage . We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli . The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile . Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo{a,l}pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene, 11,12-dihydroxy-11,12-dihydrobenzo{g}chrysene, 3,4-dihydroxy-3,4-dihydrobenzo{c}phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene . P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo{4,5-f}quinoline, 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline, 2-amino-3-methylimidazo{4,5-f}quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz{a}anthracene, and 6-nitrochrysene to appreciable extents . However, P450 1B1 did not produce genotoxic products from benzo{a}pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo{a}anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo{a}anthracene, 7,12-dimethylbenz{a}anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo{c}phenanthrene, 2-amino-6-methyldipyridol{1,2-a:3',2'-d}imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system . P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models . Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.

Mutagenesis, 1996 Jul, 11(4), 341 - 8
Molecular analysis of Salmonella hisG428 ochre revertants for rapid characterization of mutational specificity; Koch WH et al.; The Salmonella typhimurium tester strains TA104 and TA102 were developed primarily to aid in the detection of oxidative mutagens and other agents that react preferentially with AT base pairs . Reversion of prototrophy of strains harboring the hisG428 ochre allele can occur by (i) any of seven single base substitutions or (ii) several tandem double base substitutions at the ochre codon, (iii) in-frame deletions removing all or part of the ochre codon or (iv) mutations at several distinct tRNA extragenic suppressor loci . We have used allele-specific oligonucleotide probes and DNA sequence analysis to characterize 625 revertants of strain TA104 (hisG428, rfa, DeltauvrB/pKM101) arising spontaneously or after treatment with methyl methane-sulfonate, glyoxal, streptonigrin or angelicin with UVA radiation . The reversion profiles obtained from these analyses distinguished readily each of the mutagen-treated populations from one another and from spontaneously derived revertants . Both GC and AT base pair-specific revertants were observed . Molecular analyses of S . typhimurium hisG428 revertants permitted rapid assessment of base pair substitution specificity of mutagens, especially the detection of AT base pair substitutions not recovered in strains carrying the complementary hisG46 allele.

J Med Microbiol, 1996 Jul, 45(1), 40 - 7
Growth hormone modulates IL-alpha and IFN-gamma release by murine splenocytes activated by LPS or porins of Salmonella typhimurium; Sommese L et al.; The effect of growth hormone (GH) on the release of IL-1alpha and IFN-gamma from murine splenocytes was investigated . Their release from splenocytes activated by Salmonella enterica serovar Typhimurium lipopolysaccharide (LPS) 0.5 microg/ml was increased by c . 65% in the presence of GH 100 pg/ml . With splenocytes activated by S . Typhimurium porins 5 microg/ml, GH increased the production of both IL-1alpha and IFN-gamma by c . 56% . Polymyxin treatment abolished the cytokine-releasing activity of LPS but had no effect on the activity of the porin preparation.

DNA Res, 1996 Jun 30, 3(3), 165 - 9
Organization and transcription of a putative gene cluster encoding ribosomal protein S14 and an oligopeptide permease-like protein in the cyanobacterium Synechococcus sp . strain PCC 6301; Fujishiro T et al.; A 2.0-kbp Pst I DNA fragment of the unicellular cyanobacterium Synechococcus sp . strain PCC 6301 genome contains two open reading frames (ORFs) . The first ORF of 100 codons potentially encodes a polypeptide having 47% amino acid identity to Escherichia coli ribosomal protein S14, suggesting it as a ribosomal protein S14 gene (rps14) . The second ORF of 351 codons is located 81 bp downstream of rps14 and its deduced amino acid sequence is in part similar to that of the Salmonella typhimurium oligopeptide permease membrane protein OppC . Northern blot analysis showed that rps14 is expressed as a 0.48-kb transcript whereas no transcript was detected from ORF351 . Pulsed-field electrophoresis and blot hybridization analysis revealed that rps14 is a single-copy gene and is found within a 165-kbp region located upstream of rrnA on the circular genome.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6527 - 31
Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium; Yan D et al.; In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate . To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis . Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH) . Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase) . By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+ . When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S . typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool . When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool . Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate . Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.

J Mol Biol, 1996 Jun 21, 259(4), 666 - 78
Rate constants of sugar transport through two LamB mutants of Escherichia coli: comparison with wild-type maltoporin and LamB of Salmonella typhimurium; Jordy M et al.; Two LamB (maltoporin) point mutants of Escherichia coli (R8H and Y118F) and wild-type LamB of Salmonella typhimurium were reconstituted into artificial lipid bilayer membranes . Ion transport through wild-type LamB of S . typhimurium and the LamB mutants was inhibited by the addition of carbohydrates of maltose and maltooligosaccharide type in a dose-dependent fashion . The sugar-induced block of the channel function could be used for the study of current noise through the different wild-type and mutant LamB-channels . The analysis of the power density spectra allowed the evaluation of the on and off-reactions (k1 and k-1) of sugar-binding to the binding site inside the channels . Wild-type LamB of S . typhimurium had approximately the same sugar-binding kinetics as has been observed for LamB of E . coli . The results suggest that the binding site inside the channel interacts with a maximum of three glucose residues within the maltooligosaccharides . The LamB mutants R8H and Y118F showed kinetics for sugar binding substantially different from that of wild-type LamB . In particular, the on-rate, k1, for the binding of different sugars of the maltooligosaccharide series to the mutant R8H was approximately 500-times smaller than for wild-type LamB, which resulted in substantially smaller stability constant of sugar binding to the channel . Similarly, the off-rate constant, k-1, for sugar binding to the mutant Y118F decreased about 20-fold, which led to a strong increase of the affinity of carbohydrates to the site . The role of the amino residues acid R8 and Y118 in the transport of maltose and maltooligosaccharides through LamB-channels is discussed on the basis of the net flux of sugars through the channels.

J Mol Biol, 1996 Jun 21, 259(4), 589 - 607
Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation; Ikeda TP et al.; Assimilation of nitrogen requires the synthesis of only two central intermediates, glutamate and glutamine, from which other compounds derive nitrogen by secondary transfers . We measured the internal pool sizes of glutamate and glutamine in Salmonella typhimurium under conditions of external nitrogen limitation or sufficiency . When growth was slowed by nitrogen limitation, the glutamine pool was lower by a factor of up to 10, whereas the glutamate pool remained high . The decrease in the glutamine pool was general in nature, being seen with various limiting nitrogen sources in batch culture and with ammonia, the optimal nitrogen source, as the limiting nutrient in continuous culture . The only nitrogen source that gave discordant results was alanine, and we present evidence that alanine has inhibitory effects which extend beyond simple nitrogen limitation . Studies with mutant strains having altered nitrogen assimilation indicated that the decreases in the glutamine pool observed in the wild-type strain under nitrogen-limiting conditions were probably sufficient to account for slow growth and were likely to be responsible for slow growth . Hence we postulate that external nitrogen limitation is first perceived by Salmonella as a drop in its internal glutamine pool.

J Biol Chem, 1996 Jun 14, 271(24), 14264 - 70
Liganded and unliganded receptors interact with equal affinity with the membrane complex of periplasmic permeases, a subfamily of traffic ATPases; Ames GF et al.; The histidine-binding protein, HisJ, is the soluble receptor for the periplasmic histidine permease of Salmonella typhimurium . The receptor binds the substrate in the periplasm, interacts with the membrane-bound complex, transmits a transmembrane signal to hydrolyze ATP, and releases the ligand for translocation . HisJ, like other periplasmic receptors, has two lobes that are apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation), burying deeply the ligand . Such receptors are postulated to interact with the membrane-bound complex with high affinity in their liganded conformation, and, upon substrate translocation, to undergo a reduction in affinity and therefore be released . Here we show that in contrast to the current postulate, liganded and unliganded receptors have equal affinity for the membrane-bound complex . The affinity is measured both by chemical cross-linking and co-sedimentation procedures . An ATPase activity assay is also used to demonstrate the interaction of unliganded receptor with the membrane-bound complex . These findings support a new model for the transport mechanism, in which the soluble receptor functions independently of the commonly accepted high-low affinity switch.

Mutat Res, 1996 Jun 12, 353(1-2), 109 - 21
Development of new molecular procedures for the detection of genetic alterations in man; Steingrimsdottir H et al.; The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus . Mutations are identified as alterations of the DNA sequence at a chosen restriction site . DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE) . Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR . We have developed this procedure using both bacterial and mammalian cells . With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium . The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU) . With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells . In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors . Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53 . These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies . In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells . This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions . We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells . As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

Mutat Res, 1996 Jun 12, 368(2), 133 - 40
Suppressive effects of retinoids, carotenoids and antioxidant vitamins on heterocyclic amine-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002); Okai Y et al.; Effects of retinoids, carotenoids and antioxidant vitamins were studied by mutagen-induced umu C gene expression system in Salmonella typhimurium (TA 1535/pSK 1002) . Retinol (vitamin A), retinol acetate and retinoic acid showed remarkable inhibitory activities, whereas retinol palmitate exhibited significant but weak activity for umu C gene expression in tester bacteria induced by 3-amino-3,4-dimethyl-5H-pyrido{4.3-b}indol (Trp-P-1) in the presence of hepatic metabolizing enzymes (S9 mixture) . Carotenoids having provitamin A activity (beta-carotene and canthaxanthin) exhibited moderate suppressive effects on the same experimental system . The ranks of suppressive activities were retinol > retinol acetate > retinoic acid > canthaxanthin > beta-carotene > retinol palmitate and their doses for inhibition by 50% (ID50) were estimated to be 1.2 x 10(-7), 3.0 x 10(-7), 5.4 x 10(-7), 1.5 x 10(-6), 4.0 x 10(-5) and 6.0 x 10(-5) M, respectively . However, they did not cause significant inhibition on umu C gene expression induced by direct-acting mutagen (adriamycin or mitomycin C) in the absence of S9 mixture . Inhibition of umu gene expression appears to be due to inhibition of P450-mediated metabolic activation of the heterocyclic amine Trp-P-1 . Ascorbic acid (vitamin C) showed weak but significant suppressive activity at high-dose concentrations (3 x 10(-6) - 10(-4)M) . However, alpha-tocopherol did not exhibit significant suppression at all dose concentrations . The significance of the experimental results is discussed from the viewpoint of the chemoprevention against genotoxicity associated with carcinogenesis.

Mutat Res, 1996 Jun 12, 368(2), 103 - 7
Induction of microsomal enzymes in liver of rats treated with cyclohexanol; Espinosa-Aguirre JJ et al.; The S9 fraction obtained from rats orally pretreated for 3 days with cyclohexanol was able to activate the pro-mutagen N-nitrosodimethylamine (NDMA) into highly mutagenic metabolite(s) detected in the TA100 strain of Salmonella typhimurium . NDMA was not mutagenic when uninduced S9 was used as metabolic source but was approximately twice more mutagenic with cyclohexanol-induced S9 compared to ethanol-induced S9 . Separation of microsomal proteins by sodium dodecylsulfate gel electrophoresis, displayed protein bands situated in the range of 50,000 to 52,000 molecular weight induced by both, ethanol and cyclohexanol . These results are evidence of the induction properties of cyclohexanol.

Gene, 1996 Jun 12, 172(1), 33 - 9
Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimurium aroA vaccine strain; d'Oliveira C et al.; The genes, Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta), have recently been cloned and characterized . Both genes encode a protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide . Another hydrophobic stretch is predicted at the C terminus which probably functions to anchor the protein in the membrane of the merozoite and piroplasm . Here, we report the successful expression of both Tams1-1 and Tams1-2 in Escherichia coli (Ec) using gene fragments lacking both hydrophobic domains . Attempts to produce high amounts of the entire recombinant (re-) protein, or a fragment containing the N terminus only, were unsuccessful . This is presumably due to the toxicity of these re-proteins . The internal part of both genes was also expressed in Salmonella typhimurium (St) aroA vaccine strain SL3261 . We employed a dual-plasmid expression system based on an invertible promoter and selected the most stable St construct in vitro using liquid cultures and a macrophage-like cell line . The re-Tams1-1 protein produced in Ec, as well as in St, was recognized by monoclonal antibody (mAb) 5E1 specific to the 30-kDa protein . Both re-Tams1-1 and re-Tams1-2 were recognized by Ta immune calf serum.

Biochemistry, 1996 Jun 11, 35(23), 7378 - 86
Evidence of a low-barrier hydrogen bond in the tryptophan synthase catalytic mechanism; Hur O et al.; In the absence of other substrates, L-Ser reacts rapidly with the tryptophan synthase alpha 2 beta 2 bienzyme from Salmonella typhimurium at pH 7.8 and 25 degrees C to give an equilibrating mixture of species dominated by comparable amounts of the L-Ser external aldimine Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base, E(A-A) . The D-isomer of Ser is unreactive toward alpha 2 beta 2, and therefore, D,L-Ser can be used in place of L-Ser for investigations of catalytic mechanism . Due to the equilibrium isotope effect, when alpha-2H-D,L-Ser is substituted for alpha-1H-D,L-Ser, the position of equilibrium is shifted in favor of E(Aex1) . On a much slower time scale, the 2H sample undergoes the exchange of enzyme bound 2H for the 1H of solvent water and is converted to a distribution of E(Aex1) and E(A-A) identical to that obtained with the 1H sample . This slow exchange indicates that the proton abstracted from the alpha-carbon of E(Aex1) is sequestered within a solvent-excluded site in E(A-A) . Analysis of the UV/vis spectra gave an isotope effect on the equilibrium distribution of E(Aex1) and E(A-A) of KH/KD = 1.80 +/- 0.18 . This large equilibrium isotope effect is the consequence of an unusual isotope fractionation factor of 0.62 for the residue which functions as the base to deprotonate and protonate the alpha-carbon proton in E(Aex1) . A fractionation factor of 0.62 qualifies as evidence for the involvement of a low-barrier H-bond (LBHB) in this equilibration . Since this effect arises from abstraction of the alpha-proton from E(Aex1), the LBHB must be associated with the E(A-A) species . In contrast to weak H-bonds with energies of 3-12 kcal/mol, LBHBs are proposed to exhibit energies in the 12-24 kcal/mol range {Frey, P.A., Whitt, S.A., & Tobin, J . B . (1994) Science 264, 1927-1930} . Possible roles for this LBHB both in the chemical mechanism and in the stabilization of the closed conformation of E(A-A) are discussed.

Mutat Res, 1996 Jun 10, 352(1-2), 135 - 42
Frameshift mutagenesis by 9-aminoacridine: antimutagenic effects of adenosine compounds; Kopsidas G et al.; It has been shown that frameshift mutagenesis by 9-aminoacridine (9AA) in Salmonella typhimurium is significantly inhibited if glucose is present while cells are being treated in liquid defined medium . We suggested that this effect might be a result of glucose-provoked alterations of cAMP levels within the cell . We therefore sought to investigate the effects of exogenous cAMP on mutagenesis by 9-aminoacridine in both Salmonella typhimurium and Escherichia coli . Contrary to expectation, we found that frameshift mutagenesis was significantly depressed when high concentrations of cAMP were added to the defined medium during liquid treatment with 9AA . Other adenosine 5'-phosphates such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) added to the liquid medium during 9AA treatment also substantially decreased the reversion rate to prototrophy in both S . typhimurium and E . coli, as did adenosine itself . Further experiments showed that neither influx nor efflux of 9-aminoacridine molecules were greatly affected by adenosine compounds, and that although cAMP and adenosine exerted similar antimutagenic effects on 9AA-treated stationary phase cells, their effects on log phase cells were quite different . The antimutagenic effect of a representative adenosine compound (ATP) was found to persist for some time after stationary phase cells had been washed, with maximal mutability being regained only after about 3 h.

J Mol Biol, 1996 Jun 7, 259(2), 264 - 80
The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution; Crennell SJ et al.; The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors . The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring . The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively . These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available . Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion . The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA) . The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid . However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.

Parasite Immunol, 1996 Jun, 18(6), 307 - 16
Heterologous expression of the cuticular-glutathione peroxidase of lymphatic filariae in an attenuated vaccine strain of Salmonella typhimurium abrogates H-2 restriction of specific antibody responses; Chacon MR et al.; The major soluble cuticular glycoprotein of lymphatic filariae, gp29, has been expressed in the Salmonella typhimurium aroA aroD live vaccine strain BRD509 . Two distinct constructs were generated: a) pgp29, in which gp29 was expressed directly via the inducible promoter nirB, or b) pTetC-gp29, in which it was expressed as a C-terminal fusion to the non-toxic immunogenic fragment C of tetanus toxin, again under the control of nirB . In both cases, plasmid stability in vivo was demonstrated by recovery of recombinant bacteria from livers and spleens of mice immunized via the intravenous route . Negligible gp29-specific antibodies were detected in animals immunized with bacteria expressing the fragment C fusion protein, but bacteria expressing the non-fused protein resulted in gp29-specific antibody production in a proportion of animals immunized . Notably, a number of BALB/c and B10.D2/n (i.e . mice of the H-2d haplotype) responded, in contrast to previously documented nonresponsiveness during infection, or immunization with parasite extracts . Presentation of gp29 by live attenuated S . typhimurium resulted in a broad spectrum of antigen-specific IgG isotypes.

Tokushima J Exp Med, 1996 Jun, 43(1-2), 47 - 54
Mutagenicity of chemicals produced from sodium dodecylbenzenesulfonate by treatment with ozone and ultraviolet irradiation; Murakami K et al.; The interaction of formaldehyde and glyoxal produced from linear dodecylbenzenesulfonate (DBS) in aqueous solution after simultaneous treatment with ozone and ultraviolet (UV) irradiation and the effect of DBS on the mutagenic activity of formaldehyde and glyoxal were investigated . The decomposition of DBS in aqueous solution resulted in the formation of the mutagens formaldehyde and glyoxal as intermediate products after the simultaneous treatment for 4 hr . Therefore, the aqueous solution containing decomposed DBS after the treatment for 4 hr was mutagenic for Salmonella typhimurium strains TA100 and TA104 in the presence and absence of S9 mix . However, the mutagenic activity was greater than the sum of the mutagenic activity of formaldehyde and glyoxal formed from DBS . The aqueous solution mixed with formaldehyde and glyoxal did not increase the mutagenicity above the sum of the mutagenic activity in the presence or absence of DBS . Furthermore, DBS at concentrations of 2.0 micrograms/plate or less did not affect the mutagenicity of the mixtures containing formaldehyde and glyoxal . In addition, there was little bacteriocidal effect of the mixed solution on the mutagenicity test strains . These results suggest that unidentified mutagenic intermediate products were produced from DBS after simultaneous treatment with ozone and UV irradiation for 4 hr.

Vaccine, 1996 Jun, 14(9), 868 - 78
Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A (SpaA) of Streptococcus sobrinus: effects of the Salmonella virulence plasmid on the induction of protective and sustained humoral responses in rats; Redman TK et al.; Recombinant strains of Salmonella typhimurium have been studied as antigen delivery systems to determine their effectiveness as multivalent vaccines . Here we compare the efficacy of two strains of S . typhimurium . chi 4072 (pYA2905) and chi 3987 (pYA2905), expressing SpaA of Streptococcus sobrinus 6715 as oral vaccines for dental caries . Both strains are attenuated delta cya delta crp delta asd mutants with their Asd phenotypes complemented by the Asd+ plasmid pYA2905, which also encodes a peptide fragment of SpaA . S . typhimurium chi 3987 (pYA2905), unlike S . typhimurium chi 4072 (pYA2905), contains the 100 kb S . typhimurium virulence plasmid . Fischer rats were orally immunized with approximately 10(9) S . typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) and then challenged with cariogenic S . sobrinus 6715 . Rats orally immunized with either strain of recombinant Salmonella developed salivary IgA anti-SpaA responses and had lower levels of S . sobrinus-induced dental caries than nonimmunized, infected animals . In a second series of experiments, the kinetics and isotype of the serum and salivary antibody responses were determined in rats orally immunized with S . typhimurium chi 3987 (pYA2905) or chi 4072 (pYA2905) on weeks 0 and 8 . IgG and IgM serum antibody responses to SpaA and S . typhimurium were detected after the primary and secondary immunizations, and the secondary immunization boosted serum IgG anti-Salmonella activity . In general, animals immunized with chi 3987 (pYA2905) had higher serum anti-SpaA, as well as serum and salivary anti-Salmonella, responses than animals immunized with chi 4072 (pYA2905) . This study demonstrates the effective use of two recombinant S . typhimurium strains as oral vaccines for inducing protective and sustained immune responses against a mucosal pathogen and suggests that the recombinant Salmonella vaccine strain carrying the virulence plasmid induced similar or higher protective immune responses than the strain lacking the virulence plasmid.

Cell Biol Toxicol, 1996 Jun, 12(3), 135 - 45
The comparative genotoxicological study of new local anesthetics, 3-(2-alkoxyphenylcarbamoyloxy)quinuclidium chlorides, on Salmonella typhimurium, Saccharromyces cerevisiae, Vicia faba, Hordeum vulgare and Drosophila melanogaster; Miadokova E et al.; Potential gentoxicity of five new local anesthetics, derivatives of phenylcarbamic acid differing in the length of the alkyl chain of the alkoxy substituent, was studied on five test systems . There was a direct relationship with increased toxic effect in bacteria and yeast as a function of the elongation of the alkyl chain of the alkoxy substituents of the phenylcarbamic acid esters . On the other hand, no structure-toxicity relationship was found after application of 3-(2-alkoxyphenylcarbamoyloxy)-quinuclidium chlorides on plants and Drosophila . All anesthetics were nonmutagenic to Salmonella typhimurium strains TA97, TA98, TA100, and TA102 in the absence and in the presence of S9 mix . Pentyloxy and heptyloxy derivatives increased rates of genetic changes in Saccharomyces cerevisiae, mainly revertants at the isoleucine locus . Pentyloxy and hexyloxy derivatives increased the frequency of chromosome aberrations in Vicia faba root-tip meristems . No chlorophyll mutations were detected after treatment of Hordeum vulgare with pentyloxy, hexyloxy and heptyloxy derivatives . No sex-linked recessive lethals were scored in Drosphila melanogaster males . The rates of aneuploids induced in their germ cells were significantly increased after treatment with butoxy and octyloxy derivatives . However, the local toxic and genotoxic effects of test anesthetics on the microorganisms of the anesthetized tissues may be of some importance . In particular, the genotoxic effect exhibited in fungi by the heptyloxy derivative, a potent local anesthetic, was remarkable.

J Vet Med Sci, 1996 Jun, 58(6), 543 - 6
Infectivity of Salmonella typhimurium isolated from the bengalee to chickens; Sato Y et al.; Due to its importance in public health, the infectivity of Salmonella Typhimurium (S . Typhimurium) originating from the bengalee, a variety of Lonchura striata, was examined in 3-day-old chickens . When chickens were inoculated orally with 10(4) CFU or 10(8) CFU of S . Typhimurium isolated from the bengalee and observed for 7 days, Salmonella was found in cloacal swabs from both groups . S.Typhimurium was also recovered from the liver, spleen and cecal of all the birds, and the counts in the cecal content were approximately 10(8) CFU per gram in each bird . Focal necrosis in the liver was observed in both groups . When chickens were inoculated with 10(4) CFU of S . Typhimurium originating from the bengalee or the chicken and observed for 21 days, more birds were positive for Salmonella in the former group than the latter group during the first week . S . Typhimurium was isolated from the liver, spleen and cecal content of both groups, and the counts of the cecal content were approximately 10(8) CFU per gram . Focal necrosis in the liver was noted in both groups . The results indicate that S . Typhimurium originating from the bengalee is infective and pathogenic to chickens, and the pathogenicity is almost similar to that of Salmonella derived from the chicken.

FEMS Immunol Med Microbiol, 1996 Jun, 14(2-3), 121 - 7
A purified protein from Salmonella typhimurium inhibits proliferation of murine splenic anti-CD3 antibody-activated T-lymphocytes; Matsui K; In previous study, we observed that the purified substance Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) had an immunosuppressive effect, demonstrated as the suppression of mitogenic lectin-induced proliferation of murine spleen cells . In the present study, we confirmed the immunosuppressive effect of STI, which suppressed the proliferation of murine splenic T-lymphocytes activated with the anti-CD3 antibody (Ab) and phorbol 12-myristate-13 acetate (PMA) and this phenomenon was accompanied by augmentation of interferon-gamma (IFN-gamma) secretion and inhibition of interleukin-2 (IL-2) secretion . Furthermore, the augmentation of IFN-gamma secretion caused IL-2 receptor alpha chain (IL-2R alpha) over expression on T-cells . However, the addition of an anti-IFN-gamma Ab and recombinant IL-2 (rIL-2) did not reverse the suppressed T-cell proliferation, although the level of IL-2R alpha expression on T-cells recovered to around normal . Furthermore, Western blotting using an anti-phosphotyrosine Ab showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in T-cells was inhibited by incubation with STI for 48 h and this inhibition was not reversed by adding the anti-IFN-gamma Ab and rIL-2 . These results suggest that STI-induced suppression of T-cell proliferation involves a defect in IL-2R function and/or IL-2 signaling pathway in T-cells.

Appl Microbiol Biotechnol, 1996 Jun, 45(5), 629 - 37
Extracellular PagC-HlyAs fusion protein for the generation and identification of Salmonella-specific antibodies; Mollenkopf HJ et al.; The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E . coli hemolysin protein (HlyAs) to the mature PagC peptide . This PagC-HlyAs fusion protein was expressed and efficiently secreted into the culture supernatant by E . coli upon complementation with the hemolysin secretion proteins HlyB and HlyD . Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested . A monoclonal antibody against the PagC-HlyAs fusion protein recognized only PagC in membrane fractions . The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains . Sera from Salmonella-infected human patients and from a rabbit infected with S . typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.

Poult Sci, 1996 Jun, 75(6), 803 - 8
Comparison between irradiated and thermally pasteurized liquid egg white on functional, physical, and microbiological properties; Wong YC et al.; A comparative study was undertaken to determine the effect of irradiation and thermal pasteurization on the functional, physical, and microbiological properties of liquid egg white (LEW) . The LEW was irradiated or thermally pasteurized then stored at 4 C for 3 mo . Both treatments destroyed the inoculum, Salmonella typhimurium . The microbial growth rate was slower in the irradiated LEW than in the thermally pasteurized treatment . Irradiated samples had 47% lower foam drainage and more stable viscosity than samples that were thermally pasteurized . Volume of angel food cake prepared with irradiated or pasteurized LEW decreased 48 and 57%, respectively, after 90 d . Color did not differ between treatments . Ionizing radiation is an alternative processing method that inhibits microbial growth and helps maintain functionality of LEW.

Antimicrob Agents Chemother, 1996 Jun, 40(6), 1422 - 5
Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense; Dykhuizen RS et al.; Dietary intake of nitrate generates salivary nitrite, which is acidified in the stomach, leading to a number of reactive intermediates of nitrogen, among which are the potentially carcinogenic N-nitrosamines . Acidified nitrite, however, also has antimicrobial activity which coincides with the formation of nitric oxide . The present study examines the antimicrobial effect in vitro of acidified nitrite on Salmonella enteritidis, Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, and Escherichia coli O157 . First-order regression plots showed a linear inverse relationship of log-transformed proton and nitrite concentrations with MICs and MBCs after 30 min, 2 h, and 24 h of exposure (P < 0.001 for all antibacterial activities) . Susceptibility to the acidified nitrate solutions ranked as follows: Y . enterocolitica > S . enteritidis > S . typhimurium = Shigella sonnei > E . coli O157 (P < 0.05) . Addition of SCN-, but not that of CI-, increased the antibacterial activity (paired t testing, P < 0.001) . Generation of salivary nitrite from dietary nitrate may provide significant protection against gut pathogens in humans.

Genetics, 1996 Jun, 143(2), 645 - 59
A search for a general phenomenon of adaptive mutability; Galitski T et al.; The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid . Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations . We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles . One-third of the mutants produced > or = 10 late revertant colonies (appearing three to seven days after plating on selective medium) . For the prolific mutants, the number of late revertants showed rank correlation with the residual beta-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests) . Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants . However, the number of late revertants was not proportional to residual growth . Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated . For several leaky mutants, the observed revertant number exceeded the expected number . We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene.

J Toxicol Sci, 1996 Jun, 21 Suppl 1, 241 - 57
{Mutagenicity studies of prulifloxacin (NM441) and the active metabolite (NM394)}; Iwakura K et al.; The mutagenicity of prulifloxacin, a new antibacterial agent, was investigated by the reverse mutation test in bacteria, the chromosomal aberration test in cultured cells, and the micronucleus test in mice . In addition, NM394, an active metabolite of prulifloxacin, was examined for mutagenicity in the chromosomal aberration test in cultured cells . The reverse mutation test was performed at dose range of 0.0078-0.25 micrograms/plate using Salmonella typhimurium strains (TA100, TA1535, TA98, and TA1537), and Escherichia coli (WP2uvrA) . Prulifloxacin did not increase revertant colonies significantly in any of the test strains with or without metabolic activation system (S9 mix) . The chromosomal aberration tests were carried out in cultured Chinese hamster lung cells (CHL/IU) . Prulifloxacin increased aberrant cells without S9 mix, and NM394 also induced chromosomal aberrations . In human lymphocytes, no significant increases of the frequencies of cells with chromosomal aberrations were observed at dose range of 5-320 micrograms/ml with or without S9 mix . The micronucleus test was conducted at doses of 625-5000 mg/kg in the bone marrow cells of Slc : ddY male mice . There were no significant increases in the frequencies of micronucleated polychromatic erythrocytes.

Mutat Res, 1996 Jun, 340(2-3), 141 - 9
Tetracycline reduces fluoroquinolones-induced bleaching of Euglena gracilis; Ebringer L et al.; Inhibitory activity of tetracycline against ofloxacin- and fleroxacin-induced bleaching of green and etiolated Euglena gracilis was examined . Tetracycline hydrochloride in concentrations of 83-2079 microM in the light partially inhibited the bleaching activity of 83 microM ofloxacin and of 162 microM fleroxacin . In the dark, the TC inhibition of the fluoroquinolones-induced bleaching activity was most obvious, the white colony counts were all decreased . The total inhibition of bleaching was observed in 43 microM ofloxacin and 81 microM fleroxacin both in light and darkness . Cell growth was not significantly influenced by ofloxacin, fleroxacin and tetracycline in the light or darkness . Cell growth was not significantly influenced by ofloxacin, fleroxacin and tetracycline in the light or darkness . Inhibition of ofloxacin-induced Euglena bleaching by tetracycline was more effective in etiolated cells . TC at 0-416 microM did not influence the growth of ofloxacin (2.15 microM)-induced Salmonella typhimurium revertants.

Food Chem Toxicol, 1996 Jun, 34(6), 559 - 62
Mutagenicity of white grape juice in the Ames test; Patrineli A et al.; The mutagenicity of commercially available white grape juice was evaluated in the Ames mutagenicity test . Grape juice elicited a positive mutagenic, response in Salmonella typhimurium strain TA104 and a weaker response in strains TA97, TA98, TA100 and TA1530 . The mutagenic response was evident in the absence of an activation system and inclusion of such a system did not influence mutagenicity . The grape juice-mediated mutagenic response was not due to histidine residues in the juice or likely treatment with sulfite . Moreover, freshly prepared grape juice displayed a similar mutagenic response . Three different brands of commercially available white grape juice were investigated in the Ames test; they all provoked a clear positive mutagenic response, but the degree of mutagenicity differed and could not be attributed to differences in the content of solids . It is concluded that grapes contain direct-acting genotoxic component(s).

Food Chem Toxicol, 1996 Jun, 34(6), 515 - 23
The effect of teas on the in vitro mutagenic potential of heterocyclic aromatic amines; Stavric B et al.; Water extracts of eight brands (five types: 'green', 'black', 'oolong', decaffeinated and instant) of common teas (derived from Camellia sinensis) and infusions of six randomly selected herbal teas were examined for inhibitory or potentiating effects on the mutagenicity of eight heterocyclic aromatic amines (HAA) using the Ames Salmonella typhimurium TA98 and S-9 assay . HAA, produced in foods during regular heat processing of meat, exhibit mutagenic/carcinogenic activities . Tea extracts from C . sinensis displayed very potent antimutagenic effects against most HAA: total or substantial inhibition of mutagenic activity of the eight HAA was obtained with extracts equivalent to 50 mg tea leaves/plate (mgEq) and potent inhibition was frequently achieved even with 10 mgEq/plate . Decaffeinated tea produced the same effect as observed for 'regular' teas . However, lower concentrations of some tea extracts enhanced mutagenic activity of 2-amino-3,4,7,8-tetramethyl-3H-imidazo{4,5-f}quinoxaline (4,7,8-TriMeIQx) and 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) . Herbal tea extracts displayed variable effects on the mutagenicity of different HAA . While some extracts had no effect, others exhibited a moderate inhibitory effect on the mutagenicity of IQ-type HAA . In contrast to common tea, herbal teas showed substantial potentiating effects on the mutagenicity of several HAA, especially Trp-P-2 and 4,7,8-TriMeIQx.

Carcinogenesis, 1996 Jun, 17(6), 1361 - 4
Activation of H-ras oncogenes in male B6C3F1 mouse liver tumors induced by vinthionine or 2-chloroethyl-methyl sulfide; Sohn YW et al.; Vinthionine (S-vinyl-DL-homocysteine) is hepatocarcinogenic in rats and mice . {Vinyl-14C}vinthionine binds covalently to rat liver DNA, RNA and protein in vivo, but not in vitro . This amino acid is directly mutagenic in Salmonella typhimurium TA100 and TA1535; the mechanism of its metabolic activation in vivo in bacteria and liver is under study . In the present study liver tumors were induced in 12-day-old male B6C3F1 mice by single i.p . injections of vinthionine or the alkylating agent 2-chloroethyl methyl sulfide (CEMS) . At 10 months the gross tumors were examined for the presence of activated H-ras oncogenes . DNA was isolated from single tumors per mouse from 37 mice treated with vinthionine and from 31 mice treated with CEMS . These DNAs were screened for codon 61 mutations by restriction fragment length polymorphism of PCR-amplified H-ras gene fragments . Thirty seven of 37 vinthionine-induced hepatomas had H-ras mutations in this codon, which consisted of seven C-->A transversions in the first base, with 29 A-->T transversions and one A-->G transition in the second base . Twenty five of 31 CEMS-induced hepatomas had mutations in the same codon, which consisted of seven C-->A transversions in the first base, with eight A-->T transversions and 10 A-->G transitions in the second base . These mutation spectra are quite different to that noted by others in spontaneous hepatomas in untreated B6C3F1 mice . These data appear to result from the covalent binding of these carcinogens to the liver DNA.

Infect Immun, 1996 Jun, 64(6), 2365 - 7
The predicted amino acid sequence of the Salmonella typhimurium virulence gene mviAA(+) strongly indicates that MviA is a regulator protein of a previously unknown S . typhimurium response regulator family; Benjamin WH Jr et al.; The Salmonella typhimurium virulence gene mviA+ has a predicted amino acid sequence with homology to the N-terminal 112-amino-acid sequence of response regulator proteins . A previously described mutant allele (mviA), which restores virulence to avirulent LT2 strains, was shown to contain a point mutation which would be predicted to cause a single amino acid change, V-102-->G (W . H . Benjamin, Jr., J . Yother, P . Hall, and D . E . Briles, J . Exp . Med . 1,74:1073-1083, 1991) . A comparison of the nucleotide sequence of mviA+ with that of the Escherichia coli and Salmonella typhi genes revealed a high degree of conservation.

Infect Immun, 1996 Jun, 64(6), 2137 - 43
Cholera toxin induces synthesis of phospholipase A2-activating protein; Peterson JW et al.; The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated . CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min . In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis . CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis . Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP) . PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids . Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells . A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides . Synthesis of PLAP protein was increased after 2 h of exposure to CT . Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels . The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 121 - 6
Salmonella typhimurium cob mutants are not hyper-virulent; Bjorkman J et al.; It was previously reported that Salmonella typhimurium LT2 cob mutants defective in the biosynthesis of vitamin B12 (cobalamin) are more virulent than the wild type in mice . Here we show that the strains used previously are non-isogenic and that the proposed increase in virulence of the cob mutant strain results from an uncharacterized mutation in the "wild type" which attenuates virulence, most likely by decreasing expression of the spv genes on the virulence plasmid . As a result the cob mutant will appear as hyper-virulent . Examination of the virulence of reconstructed wild-type and cob mutant strains showed that their growth rates were similar in mice, and we conclude that vitamin B12 does not affect the virulence of S . typhimurium LT2.

J Immunol, 1996 Jun 1, 156(11), 4137 - 45
Distinct effects of recombinant cholera toxin B subunit and holotoxin on different stages of class II MHC antigen processing and presentation by macrophages; Matousek MP et al.; Cholera toxin (CT) is a potent mucosal adjuvant with enhancing effects on Ag presentation, although the mechanisms of its adjuvanticity remain poorly understood . Using an in vitro Ag presentation assay, we found CT and recombinant B subunit (rCTB) to have distinct effects on different stages of processing and class II MHC (MHC-II)-restricted presentation of hen egg lysozyme (HEL) . CT treatment of macrophages resulted in enhanced presentation of soluble HEL(48-61) peptide to3A9 hybridoma cells . However, CT had inhibitory effects on intracellular processing of soluble native Ag . Thus, CT inhibited presentation when added prior to HEL, whereas presentation was enhanced when CT was added after HEL exposure and the generation of peptide-MHC-II complexes . Pretreatment of macrophages with CT also markedly inhibited phagocytic processing of a Crl-HEL fusion protein (containing the HEL(48-61) epitope) expressed in intact bacteria (Escherichia coli HB101.Crl-HEL or Salmonella typhimurium 14028s.Crl-HEL), whereas addition of CT to macrophages after a 2-h incubation with the bacteria again enhanced presentation . CT produced little effect on overall uptake and catabolism of radiolabeled HEL or HB101.Crl-HEL . In contrast to the holotoxin, purified rCTB subunit did not inhibit intracellular processing of soluble or bacterial Ag, although it similarly enhanced the presentation of surface HEL-(48-61)-I-Ak complexes to 3A9 cells . These data suggest that the inhibitory effects of CT on Ag processing are mediated by the A subunit.

Pflugers Arch, 1996 Jun, 432(2), 225 - 33
Evidence for a rapid, direct effect on epithelial monolayer integrity and transepithelial transport in response to Salmonella invasion; Jepson MA et al.; In cultured monolayers of high-resistance Madin-Darby Canine Kidney (MDCK) cells, infection with Salmonella typhimurium SL1344 resulted in a dose- and time-dependent increase in transepithelial conductance (Gt) and short-circuit current (Isc) . There was a direct linear relationship between the S . typhimurium-induced increments in Isc and Gt suggesting that this early change in epithelial parameters is, in part, the result of a cellular conductance change most probably at the apical membrane . An additional wild-type S . typhimurium strain, SR11, and an invasion-deficient isogenic mutant SB111 carrying a non-polar mutation in invA were used to confirm that the S . typhimurium-induced change in epithelial electrical parameters is directly linked to the invasion process . The S . typhimurium-induced change in epithelial electrical parameters was markedly attenuated in Na+-free choline medium . Addition of piretanide (10(-4) M, basal side) failed to affect the increased epithelial conductance and Isc after a 40-min incubation with S . typhimurium . NPPB (5x10(-4) M) added to the apical medium reduced the S . typhimurium-stimulated Isc by 28%, but Gt was not significantly reduced . It is unlikely that the S . typhimurium-induced Isc is due to Cl- secretion . Staining of S . typhimurium-infected MDCK I monolayers with TRITC-phalloidin revealed marked alterations of F-actin; diffuse intracellular accumulations of F-actin corresponding to the presence of invading bacteria were observed by 15 min . After 60 min, prominent extrusions of the apical membrane corresponding to previously described "membrane ruffles" were noted . Marked accumulations of perijunctional F-actin in infected cells corresponded to contraction of the perijunctional actin ring at the apical pole . In adjacent cells marked distortion and stretch of the apical surface was evident . The invasion-deficient invA mutant SB111 failed to induce these morphological changes . These data demonstrate that S . typhimurium invasion induces increased transcellular conductance which does not result from stimulation of Cl- secretion but instead appears to be predominantly due to increased Na+ permeability . The increased membrane conductance is coincident with increased transepithelial inulin permeability indicating that the increment in Gt has an additional "paracellular" component . The S . typhimurium-induced alterations in epithelial parameters may be related to "membrane ruffling" and/or to the accompanying changes in cell shape.

J Surg Res, 1996 Jun, 63(1), 193 - 8
A single endotoxin challenge induces delayed myocardial protection against infarcation; Rowland RT et al.; Sublethal endotoxemia attenuates cardiac functional injury from global ischemia but it is unknown whether endotoxemia can protect myocardium against infarction . Furthermore, increases in myocardial catalase and heat shock protein (HSP) following endotoxemia have been associated with cardiac ischemic protection . We therefore hypothesized that a 72-hr pretreatment with endotoxin (ETX) would reduce myocardial tissue necrosis in association with augmented catalase activity and stress protein expression . Rabbits were treated with normal saline or lipopolysaccharide (Salmonella typhimurium) at 10, 5, and 1 microgram/kg doses . Three days after saline or ETX injection they were subjected to 45 min of coronary artery occlusion followed by 3 hr of reperfusion . Area of necrosis (tetrazolium staining) was normalized to anatomic risk zone size (Evans blue staining) . Catalase activity was measured by a standard assay and HSP 72 was assessed by immunohistochemistry . During regional ischemia and reperfusion there were no differences in heart rate or mean arterial blood pressure between groups . ETX treated rabbits had the same risk zone size as controls . Infarct size was reduced in the ETX treated rabbits at the 10 and 5 microgram/kg doses compared with control rabbits (17.5 +/- 1.5% and 22.2 +/- 3.1% vs 45.3 +/- 2.5%; P < 0.05) but no protective effect was observed at the 1.0 micrograms/kg dose (38.0 +/- 4.6%; P > 0.05 vs control) . Catalase activity was not different between control and ETX (5 microgram/kg) treated groups (997.8 +/- 59.1 U/g vs 1099.6 +/- 69.3 U/g myocardium; P > 0.05) but endotoxin induced expression of myocardial HSP 72 . We conclude that a single challenge with endotoxin can induce delayed myocardial protection against infarction in vivo . This delayed cardioprotective response involves enhanced stress protein expression without changes in myocellular catalase activity.

J Bacteriol, 1996 Jun, 178(11), 3377 - 9
Site-directed mutational analysis of the osmotically regulated proU promoter of Salmonella typhimurium; Zhang X et al.; We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control . We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect . However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.

J Bacteriol, 1996 Jun, 178(11), 3146 - 55
Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination; Miesel L et al.; A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay . The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment . Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded . In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction . In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery . In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency . We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process . The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions . When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.

J Bacteriol, 1996 Jun, 178(11), 3113 - 8
Role of FlgM in sigma D-dependent gene expression in Bacillus subtilis; Caramori T et al.; The alternative sigma factor sigma D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis (sigmaD regulon) . The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex . We have characterized the effects of an in-frame deletion mutation of flgM . By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different sigmaD-dependent promoters, i.e., Phag, PmotAB, and PfliDST . The number of flagellar filaments was only slightly increased by the flgM mutation . Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter . On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.

Cancer Res, 1996 Jun 1, 56(11), 2550 - 5
The capability of rat colon tissue slices to metabolize the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine; Malfatti MA et al.; A major target tissue for carcinogenesis from the cooked-food carcinogen 2-amino-l-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) in rodents is the colon, yet the role of colon metabolism on the carcinogenicity of PhIP is not clearly understood . The mutagenic potency of PhIP is highly dependent upon cytochrome P450 N-hydroxylation . In the present study, the ability of rat colon tissue to activate PhIP to a mutagen was investigated in Salmonella typhimurium (strains TA98 and YGI024) and rat colon tissue slices . In the Ames/Salmonella assay, using rat colon S9 as the activating system, no mutations were evident from bacteria exposed to PhIP at any concentration tested . However, mutations were observed when bacteria were exposed to 2-aminoanthracene (2AA) and colon S9, indicating sufficient P450 activity in the S9 to activate 2AA but not PhIP . In rat colon slice preparations, the sulfotransferase and acetyltransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate DNA adduct and metabolite formation . Incubations of 3-methylcholanthrene-induced colon slices dosed with 50 microMolar {(3)H}PhIP produced no detectable metabolites . However, incubations of uninduced slices exposed to 10 microMolar of the reactive intermediate, {(3)H}2-(hydroxyamino)-l-methyl-6-phenylimidazo{4,5-b}pyridine (N-hydroxy-PhIP), produced a single detectable metabolite, a glucuronide conjugate of N-hydroxy-PhIP . This metabolite decreased when PCP or DCNP was added to the incubation medium . DNA adducts were detected in colon slices exposed to N-hydroxy-PhIP at approximately 33 adducts/10(7) nucleotides . Interestingly, when PCP was added to the incubation mixture, an increase in DNA adduct levels was detected, whereas DCNP produced a decrease in adducts . Because these inhibitors are thought to have similar mechanisms with regard to sulfotransferase inhibition, the inverse relationship in DNA adduct levels due to PCP or DCNP treatment is at present unexplainable . The formation of DNA adducts and metabolites from colon slices exposed to N-hydroxy-PhIP but not PhIP implies that there is insufficient P450 activity in the rat colon to activate PhIP to hydroxylated metabolites, suggesting that the rat colon is a site of Phase II metabolism for PhIP and that the liver is the primary source for hydroxylation.

Toxicol Lett, 1996 Jun, 85(3), 151 - 6
A comparative study of five nitro musk compounds for genotoxicity in the SOS chromotest and Salmonella mutagenicity; Emig M et al.; Five nitro musk compounds widely used in cosmetics and detergents were examined for DNA-damaging and mutation-inducing properties . For this purpose two short-time assays were used, the SOS chromotest and the Salmonella/mammalian microsome test . Musk ambrette showed mutagenicity in Salmonella typhimurium TA 100 requiring metabolic activation by rat liver postmitochondrial supernatant (S9) but it lacked mutagenicity in the absence of S9 and genotoxicity in the SOS chromotest . Musk xylene, musk ketone, musk moskene and musk tibetene showed neither mutagenicity nor genotoxicity in the presence and absence of S9.

Mol Cell Biochem, 1996 May 24, 158(2), 125 - 31
Interaction of a rat intestinal brush border membrane glycoprotein with type-1 fimbriae of Salmonella typhimurium; Ghosh S et al.; Type-1 fimbriated Salmonella typhimurium was found to adhere to rat intestinal brush border membrane in a mannose sensitive manner . The maximum binding of the purified fimbriae observed with the rat illeal enterocytes was inhibited by 69.2% in presence of D-mannose . Brush border membrane from rat illeum was isolated, delipidified, solubilised and fractionated by affinity chromatography on type-1 fimbriae coupled Sepharose CL 4B column . Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the material eluted from the column with D-mannose revealed a single band of molecular weight 60 kDa . The direct binding of this affinity eluted glycoprotein to the purified type-1 fimbriae was demonstrated by a modified Western blot experiment . Our findings suggest that the 60 kDa glycoprotein may serve as a receptor for the type-1 fimbriae in the rat intestinal brush border membrane and thereby may help in mediating bacterial adherence to the host epithelium.

J Biol Chem, 1996 May 24, 271(21), 12626 - 31
DNA binding exerted by a bacterial gene regulator with an extensive coiled-coil domain; Hurme R et al.; Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare . One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain . Herein, we demonstrate that TlpA is a novel sequence-specific DNA-binding protein . Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding . Two of the mutant proteins were shown to be deficient in DNA binding . Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins wre shown to be deficient in DNA binding . Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding . In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor . Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo . We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

Mol Gen Genet, 1996 May 23, 251(2), 225 - 35
Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in Salmonella typhimurium; Kowarz L et al.; The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of the spvABCD virulence operon of Salmonella typhimurium . An alternative sigma factor, sigma S (RpoS), in conjunction with SpvR, controls the transcription of the spvR gene . In this study, we used a combination of primer extension experiments and deletion/fusion analyses of the spvR gene to identify sequences involved in spvR transcription in S . typhimurium . When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription of spvR in S . typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of the spvR start codon . The level of spvR transcription originating at spvRp1 was 20-fold higher in the wild-type strain than in the rpoS mutant . In both strains, however, transcription at spvRp1 requires the SpvR protein . 5' Deletions up to position -86, relative to the spvR start codon, did not inhibit inducibility by sigma S and/or SPVR . In contrast, 5' deletion up to -75 abolished the activation of spvRp1 by SpvR in both the wild-type strain and rpoS mutant . Within the 11-bp sequence lying between position -86 and position -75, a 10-bp consensus motif TNTNTGCANA, present in both the spvR and spvA promoter regions, was identified and may contain the DNA recognition site for SpvR . In addition, we detected initiation of transcription within the spvR coding region . This finding may have implications for comparative studies of regulation with spvR gene fusions.

Mol Gen Genet, 1996 May 23, 251(2), 121 - 9
Analysis of chimeric UmuC proteins: identification of regions in Salmonella typhimurium UmuC important for mutagenic activity; Koch WH et al.; Unlike Escherichia coli, the closely related bacterium Salmonella typhimurium is relatively unresponsive to the mutagenic effects of DNA-damaging agents . Previous experiments have suggested that these phenotypic differences might result from reduced activity of the S . typhimurium UmuC protein . To investigate this possibility, we have taken advantage of the high degree of homology between the UmuC proteins of E . coli and S . typhimurium and have constructed a series of plasmid-encoded chimeric proteins . The possibility that the phenotypic differences might be due to differential expression of the respective UmuC proteins was eliminated by constructing chimeric proteins that retained the first 25 N-terminal amino acids of either of the UmuC proteins (and presumably the same translational signals), but substituting the remaining 397 C-terminal amino acids with the corresponding segments from the reciprocal operon . Constructs expressing mostly E . coli UmuC were moderately proficient for mutagenesis whereas those expressing mostly S . typhimurium UmuC exhibited much lower frequencies of mutation, indicating that the activity of the UmuC protein of S . typhimurium is indeed curtailed . The regions responsible for this phenotype were more precisely localized by introducing smaller segments of the S . typhimurium UmuC protein into the UmuC protein of E . coli . While some regions could be interchanged with few or no phenotypic effects, substitution of residues 212-395 and 396-422 of E . coli UmuC with those from S . typhimurium resulted in reduced mutability, while substitution of residues 26-59 caused a dramatic loss of activity . We suggest, therefore, that the primary cause for the poor mutability of S . typhimurium can be attributed to mutations located within residues 26-59 of the S . typhimurium UmuC protein.

Biochemistry, 1996 May 21, 35(20), 6358 - 65
Kinetic isotope effects as a probe of the beta-elimination reaction catalyzed by O-acetylserine sulfhydrylase; Hwang CC et al.; Primary and alpha-secondary deuterium kinetic isotope effects have been measured for the O-acetylserine sulfhydrylase from Salmonella typhimurium using both steady-state and single-wavelength stopped-flow studies . Data suggest an asymmetric transition state for alpha-proton abstraction by the active site lysine and the elimination of the acetyl group of O-acetyl-L-serine (OAS) to form the alpha-aminoacrylate intermediate . The value of D(V/KOAS) using OAS-2-d is dependent on pH from 5.8 to 7.0 with independent values of 2.8 and 1.7 estimated at low and high pH, respectively . Thus, OAS is sticky, and a value of 1.5 is calculated for the forward commitment to catalysis, indicating that the OAS external Schiff base preferentially partitions toward the alpha-aminoacrylate intermediate compared to OAS being released from enzyme . The intrinsic primary deuterium isotope effect determined from single-wavelength stopped-flow studies of alpha-proton abstraction by the active site lysine is about 2.0 . D(V/KOAS) and T(V/KOAS) were determined as 2.6 +/- 0.1 and 4.2 +/- 0.2 at pH 6.1, respectively, giving a calculated intrinsic deuterium isotope effect of 3.3 +/- 0.9, consistent with the D(V/KOAS) obtained from steady-state studies at low pH . The alpha-secondary deuterium kinetic isotope effect using OAS-3,3-d2 is 1.11 +/- 0.06 obtained by direct comparison of initial velocities and 1.2 obtained by single-wavelength stopped-flow experiments . Data can be compared to a value of 1.81 +/- 0.04 using OAS-3,3-d2 for alpha-DKeq for the first half-reaction.

Arch Biochem Biophys, 1996 May 15, 329(2), 235 - 240
Establishment of transgenic mice carrying human fetus-specific CYP3A7; Li Y et al.; CYP3A7 is a cytochrome P450 isozyme expressed prenatally in humans . Six lines of mice transgenic for human CYP3A7 were established by microinjecting a CYP3A7 cDNA downstream of a mouse metallothionein-1 promoter gene into the male pronucleus of fertilized mouse oocytes . The inserted CYP3A7 transgene was expressed at a mRNA level in a variety of tissues including the liver, kidney, lung, spleen, testis, small intestine, thymus, brain, skin, and heart of adult mice . The protein expression of the transgene was also detected in the liver and testis of line M10 mice . A significantly higher level of total testosterone in the serum was found in line M10 male mice . In addition, this transgenic line exhibited weight increases in the liver, kidney, and uterus but a decrease in the testis (P < 0.01) . The transcript of the integrated CYP3A7 gene possessed the ability to activate aflatoxin B1 in Ames test in which the his+ revertants of Salmonella typhimurium TA100 per plate were significantly higher (P < 0.01) when liver microsomes of line M10 transgenic mice were used . This result demonstrates that the CYP3A7 gene has been integrated into the mouse genome and translated into a catalytically active enzyme . These transgenic mice are expected to give useful information for studies on fetal toxicities in humans.

Int J Cancer, 1996 May 3, 66(3), 404 - 8
Effect of quercetin on the genotoxic potential of cisplatin; Cross HJ et al.; The natural product flavonoid quercetin has been shown to sensitise cells to the cytotoxic potential of cisplatin . Both cisplatin and quercetin are genotoxicants . As quercetin is currently in clinical trial as a cytotoxicant-sensitising agent, we wanted to elucidate whether it affects the genotoxicity associated with cisplatin . The genotoxic potential of both agents alone and in combination was studied in Salmonella typhimurium strains TA 98, TA 100 and TA 102 and by assessment of unscheduled DNA synthesis (UDS) in rat hepatocytes . Furthermore, effects of quercetin on levels of cisplatin-DNA adducts were studied in hepatocytes by ELISA . Cisplatin was mutagenic in all 3 bacterial strains and quercetin in strain TA 98 . The number of revertant Salmonella colonies observed with the combination did not differ significantly from that caused by the drugs on their own . In the UDS assay, cisplatin was genotoxic but quercetin was not . In combination, quercetin decreased the nuclear grain count caused by cisplatin, but quercetin did not alter the level of cisplatin-DNA adduct formation in hepatocytes . Our results suggest that the mutagenic potential of the combination cisplatin-quercetin, as judged by the bacterial short-term test, does not exceed that associated with the individual components . However, in hepatocytes, quercetin appears to inhibit repair of cisplatin-induced DNA damage . Therefore, in patients who are to be treated with a combination of cisplatin and quercetin, the risk of genotoxicity in normal tissues will have to be taken into consideration.

J Appl Bacteriol, 1996 May, 80(5), 496 - 504
The certification of a reference material for the evaluation of the ISO method for the detection of Salmonella; in 't Veld PH et al.; A reference material (RM) containing Salmonella typhimurium was certified as CRM 507 by the Standards, Measurements and Testing Programme of the European Commission . The material consists of a gelatin capsule filled with artificially contaminated milk powder . The material is certified for the evaluation of presence/absence methods based on the ISO 6579 procedure for the detection of Salmonella . In the certification study 11 laboratories determined the presence/absence of Salmonella from each of 50 capsules . They also determined the mean number of colony-forming particles (cfp) and the homogeneity of the batch of RM according to an enumeration procedure . Certified values were calculated for both procedures separately . Based on the presence/absence procedure a fraction of capsules in which no Salmonella could be detected of 2.7% (one-sided 95% confidence upper limit 4.4%) was certified, for the enumeration procedure this fraction was 0.61% (one-sided 95% confidence upper limit 1.6%) . The certified mean number of Salmonella cfp in one capsule is 5.9 (two-sided 95% confidence interval 5.3-6.4) . Data on the preparation, identification, stability (at storage and higher temperatures) and homogeneity of the material are presented.

Mikrobiol Z, 1996 May-Jun, 58(3), 49 - 53
{The mutagenic action of radioactive contaminants of feed and food products on strains TA98 and TA100 of Salmonella typhimurium}; Matseliukh BP et al.; Radionuclide-polluted slices of beef (2775 Bq/kg), pork (274.8 Bq/kg), potato (159.0-444.0 Bq/kg), carrot, garden beet, kidney bean and sorrel (44.5-460.0 Bq/kg) have been studied for their mutagenic effect on the tester strains TA98 and TA100 of Salmonella typhimurium and this index as a rule correlates with the level of the slices pollution . On the other hand the slices of cabbage and samples of rye and wheat (46.0-460.0 Bq/kg) as well as fish (2314.0 Bq/kg) had no mutagenic effect on these strains . The samples of milk (230.0-581.5 Bq/1) also did not evoke the increase of mutagenesis level while high concentrations of these preparations (20 mg/ml, and above) manifested toxic effect on the mentioned strains.

Biochem Mol Biol Int, 1996 May, 39(2), 307 - 17
A supercoil-specific endonuclease from Salmonella typhimurium cleaves both negatively and positively supercoiled DNA; Zargar MA et al.; An enzyme which specifically cleaves supercoiled DNA to linear form through nicked circular form as intermediate was isolated from rifampicin-resistant mutant of Salmonella typhimurium, rif 39 . The enzyme activity was stimulated by Mg+2, whereas Ca+2 had no effect . It does not require ATP for its activity . No activity could be detected with relaxed or single stranded circular DNAs . The molecular weight of the enzyme is approximately 34 kDa . The most characteristic feature of this enzyme is that it cleaves both positively and negatively supercoiled DNAs.

Mol Med Today, 1996 May, 2(5), 205 - 11
Structure and function of the natural-resistance-associated macrophage protein (Nramp1), a candidate protein for infectious and autoimmune disease susceptibility; Blackwell JM; The ability of macrophages to become activated is central to antimicrobial immunity . Microbial stimuli can elicit a cascade of gene-inductive events mediating inflammation, elimination of the invading organism and induction of T-cell memory against reinvasion . Nramp1, a gene originally identified as Ity/Lsh/Bcg for its role in controlling Salmonella typhimurium, Leishmania donovani and Mycobacterium bovis infections in mice, regulates this cascade . Here we examine how the structure of the Nramp1 protein might relate to its function, and how variable expression of the human homologue (NRAMP1) might mediate enhanced resistance to infection but cause susceptibility to autoimmune disease.

Zh Mikrobiol Epidemiol Immunobiol, 1996 May-Jun, (3), 50 - 3
{The immunobiological characteristics of a Salmonella typhimurium strain acquiring a plasmid that controls antibiotic resistance and antibactericidal activity}; Chainikova IN et al.; The influence of Rdf (resistance defense factor) plasmid inherited by Salmonella typhimurium recipient strain 189, previously deprived of its own virulence plasmid of 60 MD, on the immune status of F1 (CBA x C57BL/6) mice was studied . Experimental infection induced by genetically related pairs of S . typhimurium strains, differing by the presence of Rdf plasmid, revealed the stereotypical character of changes in the number of thymic, splenic, marrow and blood cells with some variations . In mice infected by transconjugant carrying Rdf plasmid immunological shifts were, on the whole, less pronounced than those in the animals infected with clone Rdf.

Zh Mikrobiol Epidemiol Immunobiol, 1996 May-Jun, (3), 16 - 8
{Genetic control of the induction of a nonculturable state in pathogenic bacteria}; Romanova IuM et al.; The laboratory model for the induction of nonculturable forms in Salmonella typhimurium has been developed . S . typhimurium mutants with disturbances at different stages of their transfer from vegetative to nonculturable state have been obtained by insertion mutagenesis with the use of TnPhoA transposon . To identify the functions of genes involved in the process of the appearance of nonculturable forms, their cloning in the sequencing vector has been carried out.

J Vet Med Sci, 1996 May, 58(5), 425 - 9
Antimicrobial effects of amikacin therapy on experimentally induced Salmonella typhimurium infection in fowls; Itoh N et al.; The antimicrobial effects of amikacin on Salmonella Typhimurium were investigated in fowls using pharmacokinetic parameters of amikacin and the minimum inhibitory concentration (MIC) of the drug for the bacteria . Pharmacokinetic parameters of amikacin after the intramuscular administration into the fowls were measured using the fluorescence polarization immunoassay . As there was no protein binding amikacin, the total concentration was identical to the free concentration . After inoculation of the bacteria, the following intramuscular dosage regimens were carried out to test the antimicrobial effects: injection with 20 mg/kg of amikacin sulfate every 9 hr for 72 hr, injection with 20 mg/kg every 18 hr for 72 hr, injection with 20 mg/kg every 36 hr for 72 hr, and injection with 10 mg/kg every 12 hr for 72 hr . The control birds were not injected with amikacin . Abdominal organs were collected from each bird after the treatment ended . The organs were cultured and the number of colonies on each plate was calculated . No significant differences were detected among the four amikacin-treated groups, whereas the number of colonies in the control group was significantly higher than that in the amikacin-treated groups . An antimicrobial drug concentration exceeding the bacterium's MIC for at least 1/4 of the administration interval might be effective for the treatment of the infection, and the degree of peak drug concentration had no effect on antibacterial activity as long as the duration of the drug concentration above the MIC value remained the same.

Mol Microbiol, 1996 May, 20(3), 605 - 11
Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium; Park YK et al.; Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values . The acid tolerance response (ATR) includes both log-phase and stationary-phase systems . The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins . We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments . The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0 . Under the conditions tested, both lysine decarboxylase and sigma s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis . The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S . typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S . typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.

Mol Microbiol, 1996 May, 20(3), 497 - 505
Essential roles of core starvation-stress response loci in carbon-starvation-inducible cross-resistance and hydrogen peroxide-inducible adaptive resistance to oxidative challenge in Salmonella typhimurium; Seymour RL et al.; The starvation-stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g . C-source . A subset of SSR genes, known as core SSR genes, are required for the long-term starvation survival of the bacteria . Four core SSR loci have been identified in S . typhimurium: rpoS, stiA, stiB, and stiC . Here we report that in S . typhimurium C-starvation induced a greater and more sustainable cross-resistance to oxidative challenge (15 mM hydrogen peroxide (H2O2) for 40 min) than either N- or P-starvation . Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C-starvation-inducible cross-resistance to H2O2 challenge . Interestingly, (unadapted) log-phase S . typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge . Based on this, we determined if these core SSR loci were important for H2O2 resistance developed during a 60 min adaptive exposure to 60 microM H2O2 (adapted cells) . Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H2O2 challenge . In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H2O2 challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild-type strain . The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C-starvation-inducible cross-resistance to oxidative challenge, and (ii) the rpoS, stiA, and, in a delayed effect, stiB loci are needed for H2O2-inducible adaptive resistance to oxidative challenge . Moreover, we found that both stiA and stiB are induced by a 60 microM H2O2 exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.

Drug Metab Dispos, 1996 May, 24(5), 515 - 22
Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal liver and adult lungs; Shimada T et al.; Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers . P450s immunoreactive with anti-human P4501A1 and anti-human P4503A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related P4501A1, 2A6, 2C9, 2E1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P4501A2 was only the 1A subfamily of P450 found in adult livers . Drug oxidation activities with the substrates ethoxyresorufin, coumarin, 7-ethoxycoumarin, bufuralol, and testosterone were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers . Activation of procarcinogens to reactive metabolites that induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 or NM2009 was also examined and it was found that activities with (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene were higher in fetal liver microsomes than adult lung or liver microsomes . The adult liver and lung activities for these two procarcinogens were similar on the basis of microsomal protein contents despite the fact that p450 contents are higher in liver than lung microsomes . alpha-Naphthoflavone, a known inhibitor of P4501A-related activities, did not affect these procarcinogen activation in fetal liver microsomes . Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, two procarcinogens known to be activated by P4503A4/7 in humans, although activation of carcinogenic arylamines that are good substrates for P4501A2 was much lower in microsomes of fetal livers and adult lungs than in adult livers . These results suggest that in human fetal livers at least two P450 enzymes, a form of P450 that is immunoreactive P4501A1 and P4503A7, are actually expressed and these enzymes are suggested as being involved in the activation of the (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene and the carcinogenic mycotoxins, respectively . The exact nature of the former enzyme in fetal livers is unknown . In adult human lungs, several P450 enzymes are expressed, although the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.

Genetics, 1996 May, 143(1), 37 - 44
Genetic analysis of metabolic crosstalk and its impact on thiamine synthesis in Salmonella typhimurium; Petersen L et al.; The first five steps in de novo purine biosynthesis are involved in the formation of the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine . We show here that the first enzyme in de novo purine biosynthesis, PurF, is required for thiamine synthesis during aerobic growth on some but not other carbon sources . We show that PurF-independent thiamine synthesis depends on the recently described alternative pyrimidine biosynthetic (APB) pathway . Null mutations in zwf (encoding glucose-6-dehydrogenase), gnd (encoding gluconate-6-P dehydrogenase), purE (encoding aminoimidazole ribonucleotide carboxylase), and purR (encoding a regulator of gene expression) were found to affect the function of the APB pathway . A model is presented to account for the involvement of these gene products in thiamine biosynthesis via the APB pathway . Results presented herein demonstrate that function of the APB pathway can be prevented either by blocking intermediate formation or by diverting intermediate(s) from the pathway . Strong genetic evidence supports the conclusion that aminoimidazole ribotide (AIR) is an intermediate in the APB pathway.

J Med Assoc Thai, 1996 May, 79(5), 333 - 6
Salmonella lung abscess and bacteraemia in an AIDS patient; Riantawan P et al.; This report describes a 28-year-old, HIV-infected man presenting with subacute onset of pyrexia, cough, dyspnoea and pleuritic pain . Chest radiograph showed bilateral multiple cavitary lesions . The diagnosis of salmonellosis was secured by isolation of salmonella typhimurium in blood, as well as in sputum . Therapy with sequential ceftriaxone/ciprofloxacin led to satisfactory improvement symptomatically and radiologically . The present report serves to heighten the awareness of AIDS-associated salmonella bacteremia and lung abscesses.

Mutagenesis, 1996 May, 11(3), 241 - 5
Mutagenicity and toxicity of the DNA alkylation carcinogens 1,2-dimethylhydrazine and azoxymethane in Escherichia coli and Salmonella typhimurium; Xiao W et al.; DNA alkylating agents such as 1,2-dimethylhydrazine (SDMH) and azoxymethane (AOM) are potent carcinogens and are widely used to induce colon tumors in experimental animals . However, standard bacterial mutagenesis assays have failed to detect the mutagenic effects of these chemicals . Using derivatives of a set of Escherichia coli test strains developed by Cupples and Miller (Proc . Natl . Acad . Sci . USA, 86, 5345, 1989), we hve demonstrated that under two conditions, SDMH and AOM induced point mutations by several-fold in a dose-dependent manner: (i) of six possible base substitutions, they only induced GC-->AT transitions; and (ii) the cells must be deficient in O6-methylguanine (O6MeG) DNA methyltransferase (MTase) activity . SDMH and AOM up to 200 microg/ml were unable to induce His+ revertants in a Salmonella Ames test strain TA1535 (GC-->AT); however, in the absence of mammalian S9 extract, His+ revertants increased up to 55-fold upon treatment of an isogenic Salmonella strain deficient in MTase activity . These results indicate that SDMH and AOM are indeed bacterial mutagens and that lesions induced by them are the target of DNA repair MTases, which probably include mutagenic and carcinogenic lesions such as O6MeG . Furthermore, variable responses of bacterial species to SDMH- and AOM-induced mutagenicity suggests a difference either in the metabolism of potential mutagens or in the repair of specific lesions . Since O6MeG is not only a mutagenic lesion but also a lethal lesion if left unrepaired, we compared the mutagenicity and toxicity of SDMH and AOM with an SN-type methylating carcinogen . N-methyl-N'-nitro-N-nitrosoguanidine, and conclude that SDMH and AOM are weak bacterial mutagens.

Mutagenesis, 1996 May, 11(3), 235 - 40
Naturally occurring diallyl disulfide inhibits the formation of carcinogenic heterocyclic aromatic amines in boiled pork juice; Tsai SJ et al.; Three heterocyclic aromatic amines, 2-amino-3-methyl-imidazo{4, 5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoxaline and 2-amino-3,4-dimethylimidazo{4,5-f}quinoline, have been found in boiled pork juice . We have investigated the effect of naturally occurring organosulfur compounds, which are present in garlic and onion, on mutagen formation in boiled pork juice . Six organosulfur compounds - diallyl disulfide (DAD), dipropyl disulfide (DPD), diallyl sulfide (DAS), allyl methyl sulfide (AMS), allyl mercaptan (AM) and cysteine - were added separately to the pork juice before reflux boiling and then the mutagenicity of each sample was examined with the Salmonella typhimurium strain TA98 in the presence of S9 mix . All six compounds were found to inhibit the mutagenicity of boiled pork juice . The greatest inhibitory effect was observed with DAD and DPD, and this was 111-fold higher than that of the lowest, cysteine . To elucidate the inhibitory effect of DAD on mutagen formation in boiled pork juice, the major mutagenic fractions were monitored after HPLC separation by their mutagenicity with S . typhimurium TA98 . By comparing the retention times of authentic IQ compounds from boiled pork juice with those following the addition of DAD, we showed that the mutagenicity of three major fractions was significantly inhibited compared with those same fractions in boiled pork juice alone . In addition, the Maillard reaction products (MRPs) in the boiled pork juice with and without the addition of DAD were quantified and identified by capillary gas chromatography and gas chromatography-mass spectrometry . The results show that the reduction in the total amount of MRPs (pyridines, pyrazines, thiophenes and thiazoles) in boiled pork juice after boiling for 12 h is correlated with their mutagenicity . Among the MRPs, tetrahydrothiophene-3-one exhibited the strongest correlation . These data suggest that the inhibition of IQ mutagen formation by DAD is mediated through the reduction of MRPs production.

Lab Invest, 1996 May, 74(5), 907 - 20
In vivo enzymatic removal of alpha 2-->6-linked sialic acid from the glomerular filtration barrier results in podocyte charge alteration and glomerular injury; Gelberg H et al.; The epithelial polyanion (podocalyxin) on the foot processes (pedicels) of podocytes plays a pivotal role in maintaining slit pore integrity and excluding proteins from the glomerular filtrate . Chromatographically purified recombinant sialidase from Vibrio cholerae, a corresponding heat-inactivated enzyme, truncated enzyme (missing the last 17 amino acids from the carboxyl terminus), and the sialidase from Salmonella typhimurium strain LT2 were inoculated intraperitoneally into mice, and the resultant renal alterations were documented by a variety of functional, morphologic, and histochemical techniques . Proteinuria and renal failure developed in a dose-dependent manner after a single inoculation of sialidase from Vibrio cholerae, but not with the corresponding heat-inactivated enzyme, truncated enzyme, or the sialidase from Salmonella typhimurium strain LT2 . Biotinylated lectins of known sialyl linkage specificity demonstrated that Vibrio cholerae sialidase primarily removed alpha 2-->6-linked sialic acids from the glomerulus . Furthermore, the use of a poly-L-lysine cationic gold ultrastructural probe confirmed a transient loss of charge from the endothelium and epithelium of the glomerular filtration barrier . Loss of the epithelial polyanion was accompanied by the effacement of pedicels and the apparent formation of tight junctions between adjacent podocytes . The anionic charge returned to endothelial and epithelial sites within 2 days of sialidase inoculation, but the foot process loss remained . This animal model, in addition to providing an opportunity to study basic mechanisms of renal physiology, seems to mimic minimal change disease in children, diabetic nephropathy, and the renal effects of some bacterial infections.

Carcinogenesis, 1996 May, 17(5), 1063 - 8
Enhancement of benzo{a}pyrene diol epoxide mutagenicity by sulfite in a mammalian test system; Reed GA et al.; Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogen for benzo{a}pyrene (BP) . We have demonstrated previously that the interaction between sulfite, the physiological form of sulfur dioxide, and (+/-) -7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (anti-BPDE), the ultimate carcinogenic form of BP, results in an enhanced mutagenic effect in Salmonella typhimurium strains TA98 and TA100 . We report here that this same co-mutagenic effect of sulfite occurs in a mammalian cell line . Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE, a concentration on the linear portion of the dose-response, resulted in a four-fold increase in mutations at the hprt locus relative to the spontaneous rate . When V79 cells were exposed to 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE, the mutation rate increased by 73% and 210%, respectively, over that elicited by anti-BPDE alone . Sulfite itself was moderately cytotoxic, but caused no increase in mutation over the spontaneous rate . Characterization of the dose- and time-dependance of this enhancement of diol epoxide mutagenicity by sulfite closely resembled the effects seen previously in the bacterial system . In particular, enhancement by sulfite was evident when sulfite was added to the cells between 60 min and 1 min prior to the addition of the diol epoxide . Concurrent addition of sulfite and the diol epoxide attenuated the enhancement, and the effect was lost altogether when sulfite was added 10 min after the diol epoxide . The specificity of this effect of sulfite was shown by comparison with sulfate, which at concentrations of either 1 or 10 mM exhibited modest cytotoxicity, but neither was directly mutagenic nor able to enhance the mutagenic effect of anti-BPDE . Binding studies with labeled anti-BPDE showed that the addition of 10 mM sulfite increased binding of anti-BPDE to DNA by over 43%, corresponding to the observed increase in mutant frequency . Interestingly, this difference in level of DNA modification was not apparent after 30 min to 2 h exposures, but only emerged at the 4 h time point . The 4 h point was routinely used for all mutagenicity studies . Binding of anti-BPDE-derived materials to cellular RNA was not altered by 10 mM sulfite . The emergence of increased DNA modification at the latest time point suggests either a more prolonged period of active DNA binding than would occur with diol epoxide, or a difference in the ability to recognize and clear specific DNA adducts . Both possibilities are discussed in regard to the observed formation of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo{a} pyrene-10c-sulfonate (BPT-10-sulfonate) in those incubations . BPT-10-sulfonate is a relatively stable BP derivative which retains the ability to covalently modify DNA . The role of this derivative in the enhancement of diol epoxide mutagenicity by sulfite is strongly suggested by these data.

Can J Microbiol, 1996 May, 42(5), 515 - 8
Negative chemotaxis in Cytophaga johnsonae; Liu ZX et al.; Chemotaxis, both positive and negative, has been extensively studied in flagellated bacteria, such as Escherichia coli and Salmonella typhimurium, but not in gliding bacteria . The rapidly motile gliding bacterium Cytophaga johnsonae has been seen to be repelled by H2O2, OCl-, and N-chlorotaurine, as well as by low pH . Its response to H2O2 was eliminated by catalase . Nalidixic acid at 200 microM, which inhibits the growth but not the motility of C . johnsonae, did not interfere with its negative chemotactic response to H2O2, whereas sodium phosphate at 10 mM, which inhibits motility, did so . Cytophaga johnsonae was not repelled by taurine, n-octanol, phenol, L-valine, or high pH . Chemotaxis can be conveniently studied in gliding bacteria such as C . johnsonae.

Appl Environ Microbiol, 1996 May, 62(5), 1759 - 63
Mechanism by which gamma irradiation increases the sensitivity of Salmonella typhimurium ATCC 14028 to heat; Kim AY et al.; Effects of irradiation and heating on survival of Salmonella typhimurium ATCC 14028 were examined by measuring DNA damage and the integrity of the cytoplasmic membrane . S . typhimurium cells fell into two distinct groups following heating: (i) heat-sensitive cells, which were rapidly inactivated at 65 degrees C and (ii) heat-resistant cells, which were only slowly inactivated at 65 degrees C . Radiation sensitivity of S . typhimurium was greater in the presence of air than in the presence of N2 gas (radiation doses required to inactivate 90% of the cells, 0.394 +/- 0.029 in air and 0.561 +/- 0.035 in N2) . Recovery of the covalently closed circular form of plasmid pBR322 from S . typhimurium transformants (Ampr Tetr) was decreased by irradiation but not by heating . Heating prior to irradiation significantly decreased the recovery of plasmid DNA without affecting survival of S . typhimurium . Transformability of the recovered plasmid pBR322 was affected by neither irradiation nor heating, and mutation of antibiotic resistance genes was not detected in S . typhimurium . Heating, but not irradiation, caused destabilization of the cytoplasmic membrane, allowing penetration of hydrophobic dye . These results suggest that lethality of heating followed by irradiation for S . typhimurium was additive, reflecting irradiation-induced DNA damage and heat-induced membrane destabilization . When irradiation preceded heating in the absence of air, more cells were inactivated than was expected, because of heat-inactivating radiation-damaged DNA.

J Bacteriol, 1996 May, 178(10), 2989 - 90
Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli; Prival MJ; Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli . The S . typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E . coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.

J Bacteriol, 1996 May, 178(10), 2960 - 70
Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium; Williams AW et al.; Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype) . Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype) . The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK . Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region . By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined . Small deletions resulted in almost normal hook length control and almost wild-type swarming . Larger deletions resulted in loss of control of hook length and poor swarming . The largest deletions severely affected filament assembly as well as hook length control . Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T . Hirano, S . Yamaguchi, K . Oosawa, and S.-I . Aizawa, J . Bacteriol . 176:5439-5449, 1994; K . Kutsukake, T . Minamino, and T . Yokoseki, J . Bacteriol . 176:7625-7629, 1994) . They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus . The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data . We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.

J Bacteriol, 1996 May, 178(10), 2954 - 9
Characterization of the flagellar hook length control protein fliK of Salmonella typhimurium and Escherichia coli; Kawagishi I et al.; During flagellar morphogenesis in Salmonella typhimurium and Escherichia coli, the fliK gene product is responsible for hook length control . A previous study (M . Homma, T . Iino, and R . M . Macnab, J . Bacteriol . 170:2221-2228, 1988) had suggested that the fliK gene may generate two products; we have confirmed that both proteins are products of the fliK gene and have eliminated several possible explanations for the two forms . We have determined the DNA sequence of the fliK gene in both bacterial species . The deduced amino acid sequences of the wild-type FliK proteins of S . typhimurium and E . coli correspond to molecular masses of 41,748 and 39,246 Da, respectively, and are fairly hydrophilic . Alignment of the sequences gives an identity level of 50%, which is low for homologous flagellar proteins from S . typhimurium and E . coli; the C-terminal sequence is the most highly conserved part (71% identity in the last 154 amino acids) . The central and C-terminal regions are rich in proline and glutamine residues, respectively . Linker insertion mutagenesis of the conserved C-terminal region completely abolished motility, whereas disruption of the less conserved N-terminal and central regions had little or no effect . We suggest that the N-terminal (or N-terminal and central) and C-terminal regions may constitute domains . For several reasons, we consider it unlikely that FliK is functioning as a molecular ruler for determining hook length and conclude that it is probably employing a novel mechanism.

J Bacteriol, 1996 May, 178(10), 2911 - 5
The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin; Schmitt CK et al.; The flgM gene of Salmonella typhimurium encodes a negative regulator of flagellin synthesis that acts by inhibiting the flagellum-specific sigma factor FliA (sigma 28), but only when a mutation in a flagellar basal body, hook, or switch gene is present . We previously showed that FlgM is also necessary for the virulence of S . typhimurium in the mouse model of typhoid fever and proposed that FlgM is required to modulate the activity of the FliA sigma factor, which, in turn, regulates a gene involved in virulence . In this investigation, we observed that (i) the in vitro generation times of flgM mutant and wild-type strains of S . typhimurium were indistinguishable, as were the amounts of flagellin produced by the strains; (ii) the 50% lethal doses of fliA mutant and wild-type strains of S . typhimurium were similar in orally infected mice; and (iii) inactivation of the FliA-regulated flagellin gene fliC in an flgM S . typhimurium mutant resulted in a virulent phenotype . Therefore, we now conclude that expression of the FliC flagellin subunit in an flgM strain is responsible for the attenuated phenotype of an flgM mutant and that FliA does not appear to positively regulate virulence genes in S . typhimurium . Our results suggest that the normal regulation of flagellum synthesis appears to be necessary for virulence and that there may be an advantage conferred in vivo by expression of a particular flagellar phenotype of S . typhimurium.

J Bacteriol, 1996 May, 178(10), 2890 - 6
Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities; Clerch B et al.; Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli . recA mutants of S . typhimurium have already been isolated, but no mutations in lexA have been described yet . In this work, two different lexA mutants of S . typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized . The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively . SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant . Like lexA(Def) of E . coli, both lexA mutations only moderately increased the UV survival of S . typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E . coli UmuDC (UmuDCEc) . In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV . This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S . typhimurium . The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11 . These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.

J Bacteriol, 1996 May, 178(10), 2825 - 35
Surveying a supercoil domain by using the gamma delta resolution system in Salmonella typhimurium; Higgins NP et al.; A genetic system was developed to investigate the supercoil structure of bacterial chromosomes . New res-carrying transposons were derived from MudI1734 (MudJr1 and MudJr2) and Tn10 (Tn10dGn) . The MudJr1 and MudJr2 elements each have a res site in opposite orientation so that when paired with a Tn10dGn element in the same chromosome, one MudJr res site will be ordered as a direct repeat . Deletion formation was studied in a nonessential region (approximately 100 kb) that extends from the his operon through the cob operon . Strains with a MudJr insertion in the cobT gene at the 5' end of the cob operon plus a Tn10dGn insertion positioned either clockwise or counterclockwise from cobT were exposed to a burst of RES protein . Following a pulse of resolvase expression, deletion formation was monitored by scoring the loss of the Lac+ phenotype or by loss of tetracycline resistance . In exponentially growing populations, deletion products appeared quickly in some cells (in 10 min) but also occurred more than an hour after RES induction . The frequency of deletion (y) diminished with increasing distance (x) between res sites . Results from 15 deletion intervals fit the exponential equation y = 120 . 10(-0.02x) . We found that res sites can be plectonemically interwound over long distances ( > 100 kb) and that barriers to supercoil diffusion are placed stochastically within the 43- to 45-min region of the chromosome.

Invest Ophthalmol Vis Sci, 1996 May, 37(6), 1187 - 96
Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats; Jacquemin E et al.; PURPOSE . Inducible nitric oxide (NO) synthase (iNOS) has been implicated in the pathogenesis of endotoxin-induced uveitis (EIU) . This study was undertaken to localize the cells, in the eye, which express iNOS during EIU in the rat . METHODS . EIU was induced in Lewis rats by a single foot pad injection of 150 micrograms lipopolysaccharide (LPS) from Salmonella typhimurium . At different time intervals after LPS injection, the authors evaluated ocular inflammation (slit lamp observation), iNOS localization by in situ hybridization, and comparison of OX-42- and ED1-positive cell appearance and of glial response by specific immunohistochemistry . RESULTS . iNOS mRNA was not detected in the iris-ciliary body nor in the retina of control rats . It was detected strongly in the epithelial cells of the iris-ciliary body at 6 hours and also in stromal cells of the ciliary processes at 16 hours after LPS injection . In the neuroretina, iNOS mRNA was observed in the inner layers 16 hours after LPS injection . iNOS-positive cells were also present on the vitreous at this time . At 6 and approximately 16 hours after LPS injection, immunohistochemistry experiments revealed a large number of OX-42- and ED1-positive cells (microglia, macrophages, or polymorphonuclear leukocytes) colocalized in part with some iNOS-positive cells in the ciliary body and in the retina . Furthermore, expression of iNOS in Muller cells cannot be excluded . CONCLUSIONS . These observations confirm that subcutaneous injection of endotoxin dramatically induces NOS mRNA expression in the eye, and they demonstrate that epithelial cells of the iris-ciliary body and cells infiltrating the anterior segment of the eye and the retina are the major source of NO . These results support the hypothesis that both inflammatory and resident ocular cells are involved in iNOS expression during EIU.

J Bacteriol, 1996 May, 178(9), 2572 - 9
Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium; Bearson SM et al.; Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments . The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels . The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log- or stationary-phase growth . The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary-phase physiology . Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I . S . Lee, J . Lin, H . K . Hall, B . Bearson, and J . W . Foster, Mol . Microbiol . 17:155-167, 1995) . The acid shock induction of these proteins appears to be the result of an acid shock-induced increase in the level of sigmaS itself . We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+ . MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W . H . Benjamin, Jr., and P . D . Hall, abstr . B-67, p . 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993) . Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size . Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels . Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels . Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message . We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis . The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance . The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS-dependent gene expression during pathogenesis.

J Bacteriol, 1996 May, 178(9), 2533 - 8
Thiamine pyrophosphate (TPP) negatively regulates transcription of some thi genes of Salmonella typhimurium; Webb E et al.; In Salmonella typhimurium, thiamine is a required nutrient that is synthesized de novo . Labeling studies have demonstrated probable precursors for both the 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate moiety and the 4-methyl-5-(beta-hydroxyethyl) thiazole monophosphate moiety . The isolation of thiamine auxotrophs with mutations in at least five different genetic loci is reported . The majority (22 of 25) of the mutants required only the thiazole moiety of thiamine to satisfy their growth requirement . Most (14 of 25) of the mutants were affected in the thi cluster at min 90 on the S . typhimurium genetic map . Data provided herein indicate that this cluster encodes an operon whose transcription is regulated by thiamine and suggest that thiamine pyrophosphate, or a molecule derived form it, is the effector molecule . Mutants with altered regulation of this operon were isolated, and we propose that they are defective in thiamine phosphate kinase, the product of the thiL gene.

Clin Exp Immunol, 1996 May, 104(2), 228 - 35
Beneficial effect of Salmonella typhimurium infection and of immunoglobulins from S . typhimurium-infected mice on the autoimmune disease of (NZB x NZW) F1 mice; Matsiota-Bernard P et al.; Various infections can precede or aggravate autoimmune diseases . Yet a beneficial effect of infection has also been described an various mechanisms have been postulated to explain this effect . The aim of this study was to examine the hypothesis that infection can have an immunoregulatory effect on the autoimmune process via the increased production of natural polyreactive antibodies . The effect of Salmonella typhimurium infection on the lupus-like disease of (NZB x NZW)F1 (B/W) mice was therefore studied . The effect of IgM and IgG preparations isolated from the serum of S . typhimurium-infected C57B1/6 and CBA mice on the autoimmune disease of B/W mice was also tested . C57B1/6 and CBA mice were chosen because they are respectively genetically susceptible and resistant to S . typhimurium infection and they differ in their antibody response during the early phase of infection . CBA mice can mount a specific anti-bacterium antibody response, whereas C57B1/6 mice present increased production of polyreactive antibodies . The infection effect was evaluated on several disease parameters, i.e . survival, incidence of high grade proteinuria and serum IgM and IgG antibody activity directed against a panel of autoantigens . Our main findings were: (i) infection of B/W mice with an attenuated strain of S . typhimurium delayed the course of the autoimmune disease when performed before the appearance of autoimmune symptoms; and (ii) IgM and IgG preparations from S . typhimurium-infected C57B1/6 mice had a similar effect whereas the IgM and IgG preparations from infected CBA mice, as well as from normal C57B1/6 and CBA mice, were ineffective . These results suggest that S . typhimurium infection can beneficially influence the development of the autoimmune disease of B/W mice . The immunoregulatory effect of the infection seems to be related at least partially, to the increase of a particular population of antibodies, the polyreactive antibodies.

J Lab Clin Med, 1996 May, 127(5), 428 - 34
Safety evaluation of a polymerized hemoglobin solution in a murine infection model; Langermans JA et al.; Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals . This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial . Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication . Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection . At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXl) has been developed that has improved oxygen affinity and prolonged vascular retention . In the present study the potential risk of this solution on the promotion of infections has been evaluated . PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E . coli, or Candida albicans organisms . PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice . Also, the in vitro proliferation of L . monocytogenes, S . typhimurium, and E . coli was not increased by PolyHbXl . In conclusion, PolyHbXl does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections.

J Immunol, 1996 May 1, 156(9), 3321 - 6
Salmonella typhimurium aroA- infection in gene-targeted immunodeficient mice: major role of CD4+ TCR-alpha beta cells and IFN-gamma in bacterial clearance independent of intracellular location; Hess J et al.; Due to the dependency on aromatic precursors, the growth of Salmonella typhimurium aroA- is limited in immunocompetent mice . Here we show that H-21-A beta-/- mice (lacking MHC class II molecules and thus devoid of mature CD4+ TCR-alpha beta cells), TCR-beta-/- mice (devoid of TCR-alpha beta cells), and IFN-gamma R-/- mice (unresponsive to IFN-gamma) are highly susceptible to S . typhimurium aroA- infection compared with heterozygous controls . In contrast, beta 2m-deficient mice (lacking surface MHC class I and thus devoid of conventional CD8+ T cells) or TCR-delta-/- mice (devoid of TCR-gamma delta cells) were equally as resistant to S . typhimurium aroA- infection as their heterozygous littermates . These findings emphasize the vital role of CD4+ TCR-alpha beta cells and IFN-gamma in resistance against S . typhimurium aroA- . Sublethal inocula of S . typhimurium aroA- led to permanent infection in H-21-A beta-/- mice, suggesting that bacterial starvation is insufficient for sterile clearance in immunocompetent mice and that MHC class II-dependent immune mechanisms are required for pathogen eradication . The TCR-beta-/- mice suffered from salmonellosis more severely than the MHC class II-deficient mutants, suggesting an auxiliary function of CD8+ T cells . Recombinant S . typhimurium aroA-, secreting listeriolysin (Hly) of Listeria monocytogenes, are capable of escaping from the phagosome into the cytosol of the host cell . However, the course of infection of these recombinant S . typhimurium SL7207 Hlys and control strains did not differ in beta 2m-/- mutants . This finding argues against direct correlation of cytosolic location of S . typhimurium SL7207 Hlys with CD8+ T cell dependency of protection.

Cancer Res, 1996 May 1, 56(9), 2094 - 104
Pharmacological and toxicological aspects of new imidazoacridinone antitumor agents; Berger B et al.; Imidazoacridinones represent a new group of antitumor compounds developed by J . Konopa and coworkers in Gdansk, Poland (W.M . Cholody, J . Med . Chem., 33: 49-52, 1990) . The compounds exert activity against a broad spectrum of human tumors in the National Cancer Institute in vitro screening scheme . In this work, the in vitro cytotoxicity, cellular pharmacology, and genotoxic/transforming potential of five selected imidazoacridinones were studied . The compounds were highly cytotoxic (0.01-0.40 microM) to dividing cells, such as Friend erythroleukemia cells (line F4-6), V79 Chinese hamster cells, and exponentially growing C3H/M2 mouse fibroblasts . In contrast, nondividing primary rat hepatocytes and C3H/M2 cells in confluency were less sensitive to the toxicity of the imidazoacridinones . Multidrug-resistant-overexpressing F4-6 cells, 200-fold resistant to doxorubicin, showed only partial resistance (4-10 fold) to the imidazoacridinones . The cellular transport of the fluorescent imidazoacridinones occurred rapidly, and most of the drug fluorescence was localized in the nucleus . Cellular accumulation and retention of two selected imidazoacridinones (C-1310 and C-1311) in sensitive as well as in resistant F4-6 cells were determined with laser-excited flow cytometry . After an incubation with C-1311 and C-1310 for 60 min at 37 degrees C, the cellular accumulation of the less cytotoxic compound C-1310 was greater than that of C-1311, and for both compounds, the fluorescence in the resistant F4-6 cells was one-half of that in the sensitive F4-6 cells . Lowered temperature (4 degrees C) reduced the cellular accumulation for both compounds in the sensitive and in the resistant F4-6 cells and was comparable to the uptake in resistant F4-6 cells . The treatment of the resistant F4-6 cells with the multidrug-resistant modulator verapamil led to an enhanced accumulation of C-1310 and C-1311 by the cells . All five compounds produced a dose-dependent inhibition of {3H}uridine and {14C}thymidine incorporation and, except for C-1336, preferentially inhibited DNA synthesis . The affinity of the imidazoacridinones to DNA is also indicated by an increase of the DNA melting point by 9-11 degrees C . The mutagenic potential of the imidazoacridinones was investigated in the hypoxanthine guanine phosphoribosyl transferase test; the compounds C-1310 and C-1311 were additionally tested in the Salmonella typhimurium-microsome assay . Limited mutagenicity was detected in the hypoxanthine guanine phosphoribosyl transferase test, and in Salmonella typhimurium, mutagenicity was observed only in the strain TA1537 . Furthermore, no induction of DNA repair synthesis was observed after treatment of primary hepatocytes with the five imidazoacridinones . The compounds did not transform C3H/M2 fibroblasts . One derivative, C-1336, led to a significant induction of cell differentiation in Friend erythroleukemia cells . The results of this study show that the imidazoacridinones display a strong cytotoxic effect in rapidly dividing cells and only a partial resistance toward multidrug resistant cells; in addition, they showed a limited mutagenic potential in V79 fibroblasts and Salmonella typhimurium and no transforming potential in C3H/M2 cells . The imidazoacridinones are, therefore, an interesting group of new antitumor agents, and further in vivo studies are warranted to explore the usefulness of these compounds for the treatment of human cancer.

Infect Immun, 1996 May, 64(5), 1862 - 5
Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium; Baumler AJ et al.; The role of the Salmonella typhimurium fimbrial operons, lpf, fim, and pef, in adhesion to and invasion of epithelial cell lines was investigated . An S . typhimurium lpfC mutant was unable to adhere to or to invade HEp-2 cells, while an S . typhimurium fim deletion mutant did not attach to or enter HeLa cells . These results suggest that adhesion is a prerequisite for invasion and that distinct fimbrial adhesins select different target cells for invasion by S . typhimurium.

Infect Immun, 1996 May, 64(5), 1526 - 31
Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess; Zhang T et al.; Attenuated salmonellae represent attractive candidates for the delivery of foreign antigens by oral vaccination . In this report, we describe the high-level expression of a recombinant fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective antigen derived from virulent amebae, and a bacterially derived maltose-binding protein (MBP) in an attenuated strain of Salmonella typhimurium . Mice and gerbils immunized with S . typhimurium expressing SREHP-MBP produced mucosal immunoglobulin A antiamebic antibodies and serum immunoglobulin G antiamebic antibodies . Gerbils vaccinated with S typhimurium SREHP-MBP were protected against amebic liver abscess, the most common extraintestinal complication of amebiasis . Our findings indicate that the induction of mucosal and immune responses to the amebic SREHP antigen is dependent on the level of SREHP-MBP expression in S . typhimurium and establish that oral vaccination with SREHP can produce protective immunity to invasive amebiasis.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4197 - 201
Macrophage killing is an essential virulence mechanism of Salmonella typhimurium; Lindgren SW et al.; Phagocytic cells are a critical line of defense against infection . The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host . A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence . Salmonella is one of these pathogens . Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells . Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages . All four insertions were located in the ompR/envZ regulon . These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells.

Commun Dis Rep CDR Rev, 1996 Apr 26, 6(5), R76 - 8
MAFF statutory incident reports in surveillance, prevention, and control of human Salmonella typhimurium infection; Fone DL et al.; We surveyed consultants in communicable disease control (CCDCs) for their views on the current and potential value of Statutory Incident Reports--Salmonella in Animals, Birds and their Products received from the Ministry of Agriculture Fisheries and Food (MAFF), in the surveillance, prevention, and control of Salmonella typhimurium infections in humans . CCDCs from 103 (83%) of 124 district health authorities responded . Most CCDCs in rural areas used the reports either to cross reference information about animal and human isolates or to discuss with environmental health officers . Many believed that the reports' relevance to human infection could be improved if they were sent more quickly . Some CCDCs suggested that it would be useful to cross reference laboratory reports of animal and human infection at regional level and to have personal contact with local veterinary officers of MAFF . Close cooperation between public health doctors and MAFF and a coordinated approach is needed to prevent and control associations between animal and human zoonotic infections.

Science, 1996 Apr 19, 272(5260), 414 - 7
Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium; De Groote MA et al.; Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens . An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice . The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis . S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid . Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine . Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

Gene, 1996 Apr 17, 170(1), 69 - 72
Flagellar genes from Rhodobacter sphaeroides are homologous to genes of the fliF operon of Salmonella typhimurium and to the type-III secretion system; Ballado T et al.; A flagellar region of the genome of Rhodobacter sphaeroides was cloned and sequenced . Three ORFs were identified and arranged in the same order as fliH, fliI and fliJ of Salmonella typhimurium (St) . ORF2 is highly similar to FliI from St (49% similarity) showing Walker's A and B motifs . Similar scores were found with proteins of the type-III secretion system of virulence factors . ORF3 shows 16.4 and 11.1% similarity to FliJ from St and Bacillus subtilis, respectively . This work also shows that ORF3 is similar to HrpJ5 from Pseudomonas syringae (19.2% similarity) . It was found that ORF2 and ORF3 start immediately downstream from the adjacent coding region, suggesting a single transcriptional unit.

Biochemistry, 1996 Apr 16, 35(15), 4984 - 93
The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation; Wu J et al.; The principal locus for binding interactions between the aspartate and serine receptors of escherichia coli and the methyltransferase was found to be in the last five amino acids of the receptor . The thermodynamic parameters of transferase-receptor interactions were determined by isothermal titration calorimetry . the serine receptor and three C-terminal fragments (C-fragments) of the aspartate receptor consisting of ether the last 297, 88, or 38 amino acids gave comparable values for binding (n=1, deltaH approximately 13 kcal/mol, and Ka approximately 4 x 10(5)M-1) . Truncating either 16 or 36 amino acids form the C-terminus eliminated observable interactions . Finally the pentapeptide Asn-Trp-Glu-Thr-Phe, which corresponds to the last five amino acids of the receptor and is strictly conserved among E . coli serine amd aspartate receptors and the Salmonella typhimurium aspartate receptor, was found to have all the binding activity of the full-length receptor and the C-fragments . An in vitro methylation assay was used to obtain evidence for the physiological significance of this interaction in which excess peptide was able to completely block receptor methylation . The location of the binding site far from the methylation sites in the primary structure of the receptor suggests that the principle role of this interaction may be to hold the transferase in close proximity to all the methylation sites . Intersubunit methylation implication is proposed as plausible consequence of this "controlled proximity" mechanism since the ribose-galactose and dipeptide receptors lack the transferase binding sequence, and appear unable to bind transferase . Intersubunit methylation implies that transferase bound to eother the serine or aspartate receptor subunit may catalyze methylation of receptor subunits in a neighboring dimer, including those that have ligand specificity.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3188 - 92
Long-circulating bacteriophage as antibacterial agents; Merril CR et al.; The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages . The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system . In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them . Toxin levels were diminished by purifying phage preparations . To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time . By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22 . We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria . Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E . The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

Mutat Res, 1996 Apr 13, 351(2), 173 - 80
32P-Postlabeling analysis of a DNA adduct, an N2-acetyl derivative of guanine, formed in vitro by methylglyoxal and hydrogen peroxide in combination; Tada A et al.; Methylglyoxal is a direct-acting mutagen in Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide . In addition, a mixture of methylglyoxal and hydrogen peroxide reacts with 2'-deoxyguanosine to form N2-acetyl-2'-deoxyguanosine . We examined whether the guanine residues in DNA were acetylated by methylglyoxal in the presence of hydrogen peroxide using the 32P-postlabeling method . First, N2-acetyl-2'-deoxyguanosine 3'-monophosphate and N2-acetyl-2'-deoxyguanosine 3,5'-diphosphate were chemically synthesized as standard compounds for the analysis . Then calf thymus DNA (3.24 micromol) was treated with methylglyoxal (64.8 micromol) at pH 7.4 for 3 h at 37 degrees C, and subsequently with hydrogen peroxide (64.8 micromol) at 37 degrees C for 2 h . The adduct formation was analyzed using HPLC in combination with the 32P-postlabeling method under the standard conditions . N2-Acetyl-2'-deoxyguanosine was detected at levels of 2/10(6) nucleotides in double-stranded DNA and 1/10(5) nucleotides in single-stranded DNA . The estimated limit of detection by our method was 3 per 10(8) nucleotides.

J Biol Chem, 1996 Apr 12, 271(15), 8612 - 7
A thermally induced reversible conformational transition of the tryptophan synthase beta2 subunit probed by the spectroscopic properties of pyridoxal phosphate and by enzymatic activity; Ahmed SA et al.; A reversible thermally induced conformational transition of the beta2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5'-phosphate coenzyme as a spectroscopic probe . Increasing the temperature converts the major form of pyridoxal 5'-phosphate bound to the beta2 subunit from a ketoenamine species with lambdamax at 410 nm to a enolimine species with lambdamax at 336 nm (Tm = approximately 43 degrees C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm . The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5'-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5'-phosphate binding . The internal aldimine between pyridoxal 5'-phosphate and the epsilon-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60 degrees C results in covalent attachment of {4'-3H}pyridoxal 5'-phosphate . The thermal transition of the beta2 subunit below 60 degrees C produces reversible thermal inactivation (Ti = approximately 52 degrees C) and occurs at a much lower temperature than the major reversible unfolding at approximately 80 degrees C (Remeta, D . P., Miles, E . W., and Ginsburg, A . (1995) Pure Appl . Chem . 67, 1859-1866) . Our new results indicate that the 410 nm absorbing species of pyridoxal 5'-phosphate is the catalytically active form of the cofactor in the beta2 subunit and that the low temperature reversible conformational transition disturbs the active site and causes loss of catalytic activity.

Biochemistry, 1996 Apr 9, 35(14), 4568 - 77
Site-specific frame-shift mutagenesis by the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-y1)-1-aminopyrene located in the (CG)3 sequence: effects of SOS, proofreading, and mismatch repair; Malia SA et al.; 1-Nitropyrene (1-NP), the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagen and tumorigen . Nitroreduction is a major pathway by which 1-NP is metabolized . Reductively activated 1-NP forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), both in vitro and in vivo . In Salmonella typhimurium 1-NP induces a CpG deletion in a CGCGCGCG sequence . In Escherichia coli, however, mostly -1 and +1 frame-shifts are observed, which occur predominantly in 5'-CG, 5'-GC, and 5'-GG sequences . In order to determine the mechanism of mutagenesis by dGAP in a CpG repetitive sequence, we constructed a single-stranded M13 genome containing the adduct at the underscored deoxyguanosine of an inserted CGCGCG sequence . In E . coli strains with normal repair capability the adduct induced approximately 2% CpG deletions, which was 20-fold that of the control . With SOS, the frequency of frame-shift mutations increased to 2.6%, even though the frequency of CpG deletion accompanied 50% reduction . The enhancement in mutagenesis was due to a +1 frame-shift that occurred at a high frequency . In strains with a defect in methyl-directed mismatch repair, 50-70% increase in mutation frequency was observed . When these strains were SOS induced, frame-shift mutagenesis increased by approximately 100% . When transfections were carried out in dnaQ strains that are impaired in 3'-->5'exonuclease activity of DNA polymerase III, frame-shift mutagenesis increased 5-7-fold . dGAP-induced frame-shifts in the (CG)3 sequence, therefore, varied from 2% to 17% depending on the state of repair of the host cells . We conclude that dGAP induces both -2 and +1 frame-shifts in a CpG repetitive sequence and that these two mutagenic events are competing pathways . The CpG deletion does not require SOS functions, whereas the +1 frame-shifts are SOS-dependent . On the basis of the data in repair-deficient strains, it appears that both types of frame-shifts occurred as a result of misalignment, which are corrected primarily by the proofreading exonuclease of the DNA polymerase . Misaligned structures that escape the exonuclease are repaired by the methyl-directed mismatch repair, albeit with limited efficiency.

Mutat Res, 1996 Apr 6, 367(4), 177 - 85
Modulatory effects of polycyclic aromatic hydrocarbons on the mutagenicity of 1-nitropyrene: a structure-activity relationship study; Cherng SH et al.; Benzo{a}pyrene (B{a}P) is able to inhibit the mutagenicity of 1-nitropyrene (1-NP) through the reduction of nitroreductase activity and formation of adducts with DNA . The relationships between the chemical structure of 9 polycyclic aromatic hydrocarbons (PAHs) and antagonistic effects on the 1-NP-induced mutation were evaluated by the binary mixtures of 1-NP and PAHs with Salmonella typhimurium TA98 in the absence of S9 mix . Remarkably different antagonistic effects of 9 PAHs on the mutagenicity of 1-NP were observed . Among the tested PAHs, coronene demonstrates the most antagonistic potential followed by benzo{g,h,i}perylene (B{g,h,i}P), benzo{e}pyrene (B{e}P), dibenzo{a,h}pyrene (DB{a,h}P), benzo{a}pyrene (B{a}P) and pyrene . Naphthalene, anthracene, and chrysene had only minor inhibitory activity on the 1-NP mutagenicity . The modifying effects of PAHs on the nitroreductase activity of TA98 strains in the presence of 1-NP were further examined from the production of 1-AP . The statistical analytical data showed that the inhibitory effect of PAHs on the mutagenicity of 1-NP significantly correlated with their effects on the nitroreductase activity (r = -0.69, p < 0.05) . In addition, the formation of 1-NP-DNA adducts of the binary mixtures of 1-NP and PAH was determined by the 32P-postlabeling method . The results indicated that the modulatory effects of PAHs on the formation of 1-NP-DNA adducts were correlated well with their antagonistic activity (r = -0.91, P < 0.01) . From the above results, the relationships between the chemical structure of PAHs and the antagonistic effects on the 1-NP mutagenicity were revealed by the surface area and electronic parameters of PAHs . The planar molecular area of PAHs was more convincingly correlated with the antagonistic effect on the mutagenicity of 1-NP (r = -0.81, p < 0.01) than that with the difference in energy, delta E, between EHOMO and ELUMO (r = 0.69, p < 0.05) . According to the above, two possible mechanisms are involved in the interactive effect of the binary mixtures: (1) a higher binding affinity with nitroreductase for PAHs having a large planar surface area; and (2) a high energy of interaction between 1-NP and PAHs with a low delta E might decrease the nitroreductive capability.

J Toxicol Environ Health, 1996 Apr 5, 47(5), 443 - 51
Mutagenic potential of binary and complex mixtures using different enzyme induction systems; Markiewicz KV et al.; A series of experiments was conducted to investigate the mutagenic potential of binary and complex mixtures in the presence and absence of inducible liver enzyme systems prepared with several different chemical inducers . Liver homogenate (S9, or 9000 x g supernatant) fractions were obtained from Sprague-Dawley rats induced with either Aroclor 1254 (AR), phenobarbital (PB), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or corn oil (UI) . the mutagenic potential of test samples was measured with Salmonella typhimurium strain TA98 using each of the various S9 fractions . Test samples included benzo{a}pyrene (BaP), pentachlorophenol (PCP), a binary mixture of BaP and PCP, two five-component mixtures, a methylene chloride extract of wood preserving waste-amended soil, and a methanol extract of coal gasification waste . At a dose of 25 micrograms/plate, BaP produced 55, 83, 217, and 161 net revertants per plate with UI-, PB-, AR-, and TCDD-induced S9, respectively . The complex mixture extracted from the wood preserving waste-amended soil induced approximately equal responses with all four S9 mixes . At a dose of 250 micrograms/plate, the methanol extract of a coal gasification waste produced 56 net revertants using the uninduced S9; however, when Ar- and TCDD-induced S9 was used, 129 and 67 net revertants were observed, respectively . These data demonstrate the relative importance of the various induced cytochrome P-450 isozymes for the metabolism of mutagenic chemicals and complex mixtures.

Mutat Res, 1996 Apr 4, 359(3), 179 - 89
Palmitic acid is the major fatty acid responsible for significant anti-N-methyl-N'-nitro-N-nitroguanidine (MNNG) activity in yogurt; Nadathur SR et al.; We describe here the isolation and identification of palmitic acid as being responsible for significant anti-N-methyl-N'-nitro-N-nitroguanidine (MNNG) activity in yogurt . The Ames test (Salmonella typhimurium TA100) was used to direct fractionation of activity . Yogurt was freeze-dried and extracted with acetone to yield a crude extract . The crude extract was purified by normal phase silica gel, Sephadex LH-20, and reversed phase medium pressure liquid chromatographies . The major compound in the active medium pressure liquid chromatographic fractions was determined to be palmitic acid on GC and high pressure liquid chromatography (HPLC) systems, and by nuclear magnetic resonance (NMR) analysis . Other saturated straight chain and methyl branched fatty acids were detected by GC/MS and were later shown to possess anti-MNNG activity . Of the straight chain fatty acids, palmitic acid had the highest anti-MNNG activity . All omega - 1 methyl branched fatty acids tested were more active than their straight chain counterparts . A trace amount of isopalmitic acid (14-methyl pentadecanoic acid), a minor milk lipid, was detected in one of the active fractions, and was later shown to be five times more active than palmitic acid . Isopalmitic acid also inhibited mutagenesis induced 4-nitroquinoline-N-oxide (4NQO), and 7, 12-dimethyl benz{a}anthracene (DMBA), and was found to inhibit the metabolic activation of DMBA.

Mutat Res, 1996 Apr 4, 359(3), 159 - 63
Inhibition of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) mutagenicity by black and green tea extracts and polyphenols; Apostolides Z et al.; Solutions of lyophilized preparations of standard black and green tea extracts were made and tested over a range of six concentrations as inhibitors of the mutagenicity caused by the fool mutagen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) in the Salmonella typhimurium TA98 assay containing S9 fraction from rats induced with alpha-naphthoflavone and phenobarbital . Extracts of both black and green tea were equally good inhibitors of mutagenicity . Purified polyphenols were prepared from tea extracts by solvent extraction . The polyphenols of black tea were more potent inhibitors of mutagenicity than the polyphenols of green tea . These findings suggest that black tea may have similar health-promoting properties to those reported previously for green tea.

Biochemistry, 1996 Apr 2, 35(13), 4211 - 21
Exchange of K+ or Cs+ for Na+ induces local and long-range changes in the three-dimensional structure of the tryptophan synthase alpha2beta2 complex; Rhee S et al.; Monovalent cations activate the pyridoxal phosphate-dependent reactions of tryptophan synthase and affect intersubunit communication in the alpha2beta2 complex . We report refined crystal structures of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium in the presence of K+ at 2.0 angstrom and of Cs+ at 2.3 angstrom . Comparison of these structures with the recently refined structure in the presence of Na+ shows that each monovalent cation binds at approximately the same position about 8 angstrom from the phosphate of pyridoxal phosphate . Na+ and K+ are coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, and beta Gly-232 and to two or one water molecule, respectively . Cs+ is coordinated to the carbonyl oxygens of beta Phe-306, beta Ser-308, beta Gly-232, beta Val-231, beta Gly-268 and beta Leu-304 . A second binding site for Cs+ is located in the beta/beta interface on the 2-fold axis with four carbonyl oxygens in the coordination sphere . In addition to local changes in structure close to the cation binding site, a number of long-range changes are observed . The K+ and Cs+ structures differ from the Na+ structure with respect to the positions of beta Asp-305, beta Lys-167, and alpha Asp-56 . One unexpected result of this investigation is the movement of the side chains of beta Phe-280 and beta Tyr-279 from a position partially blocking the tunnel in the Na+ structure to a position lining the surface of the tunnel in the K+ and Cs+ structures . The results provide a structural basis for understanding the effects of cations on activity and intersubunit communication.

Biochemistry, 1996 Apr 2, 35(13), 3917 - 24
Energy coupling in Salmonella typhimurium nicotinic acid phosphoribosyltransferase: identification of His-219 as site of phosphorylation; Gross J et al.; Energy coupling between ATP hydrolysis and other enzyme reactions requires the phosphorylation of substrate-derived intermediates, or the existence of enzyme-derived intermediates capable of storage and transfer of energy . Salmonella typhimurium nicotinic acid phosphoribosyltransferase (NAPRTase, EC 2.4.2.11) couples net ATP hydrolysis to formation of NAMN and PPi from alpha-PRPP and nicotinic acid {Vinitsky, A., & Grubmeyer, C (1993) J . Biol . Chem . 268, 26004-26010} . In the current work, we have determined that the enzyme reacts with ATP to produce a covalently phosphorylated form of the enzyme (E-P), which is common to both the ATPase and NAMN synthesis functions of NAPRTase . We have isolated E-P and verified its catalytic competence . E-P showed acid lability and base stability, diagnostic of a phosphoramidate linkage . Pyridine and hydroxylamine-catalyzed hydrolysis of E-P gave second-order rate constants consistent with published values for phosphohistidine . Two-dimensional thin-layer chromatography of alkaline-hydrolyzed E-32P showed that the phosphorylated residue co-migrated with authentic 1-phosphohistidine . Chymotrypsin and trypsin proteolysis followed by HPLC and peptide sequencing localized the phosphopeptide to Ala-210 to Phe-222 of the 399-residue protein . This peptide contains a single histidine residue, His-219 . NAPRTase phosphorylated at His-219 is an intermediate in the energy transduction mechanism of NAPRTase.

Biochemistry, 1996 Apr 2, 35(13), 3909 - 16
Limited proteolysis of Salmonella typhimurium nicotinic acid phosphoribosyltransferase reveals ATP-linked conformational change; Rajavel M et al.; Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) . Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein . ATP and PRPP each provided protection against tryptic cleavage . Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates . Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes . The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 233 - 9
Identification of a new prp locus required for propionate catabolism in Salmonella typhimurium LT2; Hammelman TA et al.; A new propionate (prp) locus of S . typhimurium was defined by mutation, was located to minute 8 of the chromosome, and was shown to be transcribed in the clockwise direction . A plasmid carrying the wild-type prp+ locus was isolated by complementation and its initial physical characterization is presented . Transcriptional regulation of prp was studied using MudI1734(lacZ+) operon fusions . Propionate stimulated prp transcription in a merodiploid strain containing prp+ and a prp::MudI1734 fusion, but failed to stimulate transcription of the same fusion in a haploid genetic background . prp transcription was reduced by a factor of 2 in strains deficient in the synthesis of the global regulatory protein FruR; fruR mutants failed to grow on propionate . Propionate blocked growth of prp mutants on medium containing succinate as carbon/energy source.

Mol Microbiol, 1996 Apr, 20(1), 191 - 9
Induction of haemolytic activity in Escherichia coli by the slyA gene product; Oscarsson J et al.; The Salmonella typhimurium protein SlyA(ST), originally described as a cytolysin, shows sequence similarities to several known bacterial regulatory proteins . A homologue to the Slya(ST) gene has been localised to min 37 of the Escherichia coil K-12 chromosome and has been designated SlyA(EC) . When introduced in trans on a plasmid, the SlyA(EC) gene conferred a haemolytic phenotype on wild-type but not clyA-knockout strains of E . coli K-12 . The clyA gene encodes a novel haemolysin that is not expressed by wild-type E . coli under tested laboratory conditions . Western and Northern blot analyses, and DNA-band-shift assays support a model whereby the SlyA(EC) protein activates clyA expression by binding to the clyA promoter region, thereby supporting the sequence similarity data in suggesting that SlyA(ST) is a haemolysin activator rather than being a haemolysin per se.

Mol Microbiol, 1996 Apr, 20(1), 151 - 64
Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells; Stein MA et al.; Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (lgp)-containing vacuoles . Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable lgp-containing filamentous structures that connect with the Salmonella-containing vacuoles . Salmonella typhimurium SL1344::Tn l0dCm mutant strains unable to induce these structures were isolated . All contained insertions within a novel Salmonella induced filament gene A (sifA) . sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38 kDa and an apparent molecular mass of 35 kDa . sifA is flanked by 300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases . sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines . Fourteen-base-pair direct repeats mark the probable site of integration of sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52-54%) . Deletion mutant strains in sifA or in the downstream potC were constructed . Delta sifA does not produce Salmonella-induced filaments in epithelial cells, and is attenuated in mice . Delta potC produces Salmonella-induced filaments in epithelial cells, and was fully virulent . Collectively, these results suggest that sifA arose by horizontal gene transfer into Salmonella and its product is involved in a virulence-associated intracellular phenotype related to Salmonella-induced filament formation.

Avian Dis, 1996 Apr-Jun, 40(2), 391 - 7
Effect of a characterized continuous-flow culture of cecal bacteria on Salmonella typhimurium crop colonization in broiler chicks; Hume ME et al.; Broiler chicks were inoculated orally at 1 day of age with a continuous-flow (CF) culture of anaerobic cecal bacteria and challenged with 10(4) Salmonella typhimurium 48 hr (at 3 days old) after inoculation to determine the effect of the CF culture (CF3) on Salmonella crop colonization . Chicks were assigned to four groups: 1) untreated control chicks, 2) challenged at 3 days old with Salmonella, 3) inoculated at 1 day old (day-of-hatch) with CF3, and 4) inoculated at 1 day old with CF3 and challenged at 3 days old with Salmonella . Crop pH decreased significantly (P < 0.05) 24 hr after inoculation in chicks provided with CF3 . The pH of crops at 24 hr from control chicks (group 1) was 5.4 and the pH of crops from inoculated chicks (group 3) was 4.7 . Decreased pH was accompanied by a significant increase (P < 0.05) in corp lactic acid from approximately 0.1 mmol/ml in control chicks to about 0.2 mmol/ml in chicks given the culture . Salmonella crop colonization decreased (P < 0.05) 4 hr after challenge from a 2.6 log10 colony-forming units (cfu) in Salmonella-control (group 2) chicks to 0.6 log10 cfu in CF3-inoculated (group 4) chicks . Although at 4 and 8 hr after challenge, there were decreased (P < 0.05) numbers of crops testing culture-positive for Salmonella regardless of treatment, Salmonella colonization decreased (P < 0.05) in chicks inoculated with CF3 as compared with controls . The results indicated that CF3 can effectively reduce Salmonella crop colonization.

Vaccine, 1996 Apr, 14(6), 545 - 52
Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen; Londono LP et al.; Live vaccines based on BRD509, an attenuated S . typhimurium (aroA, aroD) strain, were constructed that directed the expression of hepatitis B core antigen particles (HBcAg) (BRD969) or HBcAg harbouring human papillomavirus type 16 E7 protein sequences (BRD974), under the control of the in vivo inducible nirB promoter . These strains were used to orally or intravenously immunise different inbred mouse strains and humoral, secretory and cellular anti-E7 and anti-HBcAg responses were monitored . Both BRD969 and BRD974 induced anti-HBcAg humoral IgG responses following oral or intravenous immunisation of B10 mice, although responses were higher in BRD969 immunised animals . IgG subclass analysis revealed a predominantly IgG2a response in these animals . BRD974, but not BRD969, induced anti-E7 humoral IgG responses . Anti-HBcAg (BRD969 and BRD974) and anti-E7 (BRD974) IgA responses were detected in the intestines of orally immunised mice . Anti-Salmonella but not anti-HBcAg or anti-E7 T helper cell responses were detected in mice immunised with BRD509, BRD969 and BRD974 . Thus Salmonella vaccine strains can be used to efficiently deliver HBcAg and E7 epitopes to the mucosal and systemic immune systems.

Environ Health Perspect, 1996 Apr, 104(4), 428 - 36
Seasonal and spatial variation of the bacterial mutagenicity of fine organic aerosol in southern california; Hannigan MP et al.; The bacterial mutagenicity of a set of 1993 urban particulate air pollution samples is examined using the Salmonella typhimurium TM677 forward mutation assay . Amibent fine particulate samples were collected for 24 hr every sixth day throughout 1993 at four urban sites, including Long Beach, central Los Angeles, Azusa, and Rubidoux, California, and at an upwind background site on San Nicolas Island . Long Beach and central Los Angeles are congested urban areas where air quality is dominated by fresh emissions from air pollution sources; Azuasa and Rubidoux are located farther downwind and receive transported air pollutants plus increased quantities of the products of atmospheric chemical reactions . Fine aerosol samples from Long Beach and Los Angeles show a pronounced seasonal variation in bacterial mutagenicity per cubic meter of- ambient air, with maximum in the winter and a minimum in the summer . The down-wind smog receptor site at Rubidoux shows peak mutagenicity (with postmitochondrial supernatant but no peak without postmitochondrial supernatant) during the September-October periods when direct transport from upwind sources can be expected . At most sites the mutagenicity per microgram of organic carbon from the aerosol is not obviously higher during the summer photochemical smog period than during the colder months . Significant spatial variation in bacterial mutagenicity is observed: mutagenicity per cubic meter of ambient air, on average, is more than an order of magnitude lower at San Nicolas Island than within the urban area . The highest mutagenicity values per microgram of organics supplied to the assay are found at the most congested urban sites at central Los Angeles and Long Beach . The highest annual average values of mutagenicity per cubic meter of air sampled occur at central Los Angeles . These findings stress the importance of proximity to sources of direct emissions of bacterial mutagens and imply that if important mutagen-forming atmospheric reactions occur, they likely occur in the winter and spring seasons as well as the photochemically more active summer and early fall periods.

Clin Infect Dis, 1996 Apr, 22(4), 638 - 41
Skull osteomyelitis due to Salmonella species: two case reports and review; Kamarulzaman A et al.; Osteomyelitis of the skull is a rare disease . We describe two cases due to Salmonella typhimurium and review 10 previously reported cases of salmonella osteomyelitis of the skull . This infection is frequently complicated by extradural abscess, which may be asymptomatic . Diagnostic imaging by means of computed tomographic scanning with contrast or gadolinium-enhanced magnetic resonance imaging should be performed to detect this complication . A good outcome can be expected with a combination of surgery and antibiotic therapy.

Ecotoxicol Environ Saf, 1996 Apr, 33(3), 236 - 45
PAH content and mutagenicity of marine sediments from the Venice lagoon; La Rocca C et al.; Sediments from the Venice lagoon, a polluted coastal environment in northeastern Italy, were assayed for mutagenicity and content of several toxic microcontaminants, which included selected polycyclic aromatic hydrocarbons (PAHs); the latter are specifically dealt with in this paper . Samples were collected at three lagoon sites with reasonably distinct environmental features--urban, industrial, or agricultural--and at two others considered to be under mixed pollution influences; a sixth sample was obtained from an open sea area to act as background control . The organic matter (EOM) associated with the mineral substrata was extracted; after cleanup, analyte determination was carried out by HRGC-LRMS(SIM) using isotopically labeled compounds as internal standards . Cumulative levels of the selected PAHs were found to be in the range of 0.065 to 0.46 micrograms/g of dry matrix at five sites; a much higher concentration (48 micrograms/g) was detected in the sample from the urban environment . The remarkable PAH level increase at this site was mostly accounted for by the concurrent, apparent increase of EOM contamination as PAH concentration was seen to reach 32 micrograms/mg of EOM from < 1 microgram/mg at the five remaining sites . Mutagenicity assays with Salmonella typhimurium strains TA98 and TA100 of marine sediment organic extracts also highlighted a distinct activity in the sample from the urban site . Further fractionation and analysis of this extract pointed to PAHs as the main mutagenic component present in the sediment matrix, possibly accounting for up to approximately 70-80% of the entire mutagenic potential detected.

APMIS, 1996 Apr, 104(4), 302 - 6
Antibiotic resistance mechanisms in Salmonella species causing bacteraemia in Malawi and Kenya; Leegaard TM et al.; In two studies on the causative agents of bacteraemia in Malawi and Kenya, 33 Salmonella strains were isolated . Fourteen strains of Salmonella typhimurium and Salmonella enteritidis were found to exhibit resistance to amoxicillin, amoxicillin/clavulanic acid and cotrimoxazole as well as decreased susceptibility to a range of aminoglycosides . The resistant strains were studied to establish their resistance mechanisms . Beta-lactamase co-focusing with TEM-1 was present in 12 strains . In two strains, both S . typhimurium from Kenya, an OXA-1 beta-lactamase was detected . The aminoglycoside-modifying enzyme ANT(2") was found in 10 strains . The presence of the encoding genes was confirmed by PCR . For comparison, susceptibility records of 73 Salmonella strains isolated during the past 14 years in our hospital were studied retrospectively . Only one of these strains was resistant to amoxicillin . This resistance was acquired during therapy.

J Bacteriol, 1996 Apr, 178(8), 2462 - 4
Stabilization of a HemA-LacZ hybrid protein against proteolysis during carbon starvation in atp mutants of Salmonella typhimurium; Archer CD et al.; Transposon insertions that stabilize the beta-galactosidase activity of a HemA-LacZ hybrid protein following carbon starvation were mapped to the atp operon of Salmonella typhimurium . This effect is similar to that seen with nuo mutants defective in the energy-conserving type I NADH dehydrogenase . Insertions in several other genes, including such highly pleiotropic mutants as rpoS, polA, and hfq, were isolated with the same phenotypic screen, but they do not affect the beta-galactosidase activity of HemA-LacZ . All of these mutants act indirectly to alter the colony color of many different fusion strains on indicator plates.

J Bacteriol, 1996 Apr, 178(8), 2272 - 8
Salmonella typhimurium LT2 possesses three distinct 23S rRNA intervening sequences; Mattatall NR et al.; The rrl genes for 23S rRNA of Salmonella typhimurium LT2 are known to carry intervening sequences (IVSs) at two sites, helix-25 and helix-45, which are excised by RNase III during rRNA maturation, resulting in rRNA which is fragmented but nevertheless functional . We isolated DNA fragments containing the seven rrl genes from BlnI, I-CeuI, and SpeI genomic digests following pulsed-field gel electrophoresis and used these DNA fragments as templates for PCRs utilizing primers upstream and downstream of helix-25 and helix-45 . Variance in amplicon length and cycle sequencing indicated that rrlG and rrlH have IVSs in helix-25 of approximately 110 bp which are only 56% identical . rrnA, rrnB, rrnC, rrnD, rrnE, and rrnH have IVSs of approximately 90 bp in helix-45, and all have the same nucleotide sequence . Twenty-one independent wild-type strains of S . typhimurium from Salmonella Reference Collection A were analyzed for IVSs by using PCRs with genomic DNAs and by denaturing agarose electrophoresis of RNAs . Many strains resemble LT2, but some have no IVSs in helix-25 and others have IVSs in helix-45 in all seven rrl genes . However, the IVSs in individual wild-type lines are relatively stable, for several LT2 isolates separated over many years by many single-colony isolations are indistinguishable from one another, with the exception of line LB5010, which differs by one helix-25 IVS . We postulate that IVSs have entered strain LT2 by three independent lateral-transfer events and that the IVS in helix-45 was dispersed to and maintained in the same sequence in six of the seven rrl genes by the mechanism of gene conversion.

J Bacteriol, 1996 Apr, 178(8), 2196 - 203
In vitro analysis of the interactions between the PocR regulatory protein and the promoter region of the cobalamin biosynthetic (cob) operon of Salmonella typhimurium LT2; Rondon MR et al.; The PocR protein of Salmonella typhimurium LT2 was overexpressed and used to demonstrate in vitro that it specifically binds to the cobalamin biosynthetic operon (cob) promoter region . Evidence is presented to show that PocR DNA-binding activity in vitro is regulated by the effector molecule 1,2-propanediol . Deletion analysis of the cob promoter (Pcob) suggested that two regions upstream of the promoter are needed for optimal activation of Pcob by PocR in vivo . DNase I footprinting experiments demonstrated that PocR binds to two sites within Pcob . The transcription initiation site of cob mRNA in response to 1,2-propanediol was identified and shown to be different from the one reported for transcription initiation under anoxic conditions in the absence of 1,2-propanediol.

Infect Immun, 1996 Apr, 64(4), 1321 - 7
Susceptibility of lipopolysaccharide-responsive and -hyporesponsive ItyS Mice to infection with rough mutants of Salmonella typhimurium; Mattsby-Baltzer I et al.; The R5 (chemotype Rb) but not the R10 (chemotype Rd) mutant of murine pathogen Salmonella typhimurium 395MS was extremely virulent in intraperitoneal infections of C57BL/10ScCr mice carrying the ityS and lpsD alleles . C57BL/6J (ityS lpsN) and C3H/HeJ (ityR lpsD) mice showed a much higher resistance to the R5 mutant . Further studies were performed with peritoneal macrophages in vitro in order to elucidate susceptibility in lipopolysaccharide (LPS)-hyporesponsive mice carrying ItyS . The intracellular killing capacity of the ItyS LpsD macrophages was lower than that of the ItyS LpsN macrophages for the R5 mutant and may partly explain the increased susceptibility of the ItyS LpsD mice . The deep rough mutant, R10, was rapidly killed intracellularly by the ItyS LpsD macrophages . Processing of the bacteria in macrophages that had phagocytosed R5 or R10 bacteria was followed for up to 18 days by endotoxin measurements (limulus assay) and immunostaining, with monoclonal antibodies to various parts of the LPS molecule being used . Only 0.1% or less of the macrophage-associated bacteria remained alive after 48 h of incubation, and none were alive on day 7 . Although immunostaining showed that LPS was present in both the LpsD and LpsN macrophages during the whole incubation period of 18 days, endotoxin activity in the LpsD macrophages on day 7 was lower than that in the LpsN macrophages, indicating that qualitative modifications of the chemical composition or physical state of the LPS molecule occurred . The interleukin-6 response in the ItyS LpsD macrophages was delayed and of shorter duration compared with that in the ItyS LpsN macrophages . The results suggest that the difference between the LPS-hyporesponsive and -responsive ItyS mice in susceptibility to infection with the R5 mutant was due to the lower activation state of the LpsD macrophages during infection, leading to a lower intracellular bactericidal systems of the macrophages . A rapid killing of the bacterium should restrict the infection and may partly compensate for a diminished inflammatory response . The persistence of LPS within the cells is discussed.

Infect Immun, 1996 Apr, 64(4), 1085 - 92
Role of the acid tolerance response in virulence of Salmonella typhimurium; Riesenberg-Wilmes MR et al.; During its life cycle, Salmonella typhimurium is exposed to a variety of acidic conditions . Survival in the acidic environments within the host may require the adaptive acid tolerance response (ATR), which is characterized by the induction of several Salmonella proteins upon exposure to mildly acidic conditions . These induced proteins protect the bacterium from death under severe acid challenge . The goal of this study was to examine the role of ATR in Salmonella pathogenesis . Initially, we observed that differences exist between the virulent S . typhimurium strains and the laboratory S . typhimurium strain LT2 with respect to their ATR . Mutations affecting the ATR of S . typhimurium LT2, including atrB, atrC (polA), atrD, atbR, and fur, were crossed into virulent Salmonella strains, and the resultant transductants were screened for virulence in mice and acid sensitivity . Surprisingly, with the exception of the fur mutation, none of the muatations had a major effect on acid resistance or virulence in the pathogenic strains . The fur mutants showed a 1-to 3-log increase in the 50% lethal dose; however, the magnitude of its effect was dependent on the strain background . Strains containing two or three different atr mutations were constructed, and these were also examined for acid sensitivity and virulence . The double and triple mutants that contained an atrC mutation no longer displayed an ATR . Those mutants which were more acid sensitive were also highly attenuated, suggesting a strong correlation between the ability to mount and ATR and virulence in S . typhimurium . Comparison of the ability of the various atr single, double, and triple mutants to survive within macrophages showed that strains containing an atrC mutation survived much less than the wild type in bone marrow-derived macrophages . No difference in survival within J774 macrophage like cells were detected.

Clin Exp Immunol, 1996 Apr, 104(1), 80 - 5
Comparative microbicidal activity of synovial fluid on arthritogenic organisms; Inman RD et al.; There is strong circumstantial evidence to support the concept that local microbial antigens play a key role in the synovitis of reactive arthritis (ReA) patients . It is not at all clear whether these antigens reflect the sequelae of previously viable organisms once resident in the joint . To address the microbicidal activity of synovial fluid (SF) we performed quantitative cultures of arthritogenic organisms (Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae) and controls (Escherichia coli, Staphylococcus aureus) in the presence of SF from patients with ReA . There was a dramatic inhibitory effect of SF on the Gram-negative organisms (mean 1.35x10(5) organisms at 3h; 0 organisms at 24 h) in contrast to Staph . aureus (1.61x10(5) at 3h; 5.70x10(5) at 24 h) . This SF bactericidal phenomenon was observed in 11/11 ReA patients, 5/8 rheumatoid arthritis (RA) patients and 1/8 osteoarthritis (OA) patients . Using a sandwich ELISA, we measured SF levels of bactericidal/permeability-increasing protein (BPI) . BPI was detectable in all ReA SF (range 4.6-333ng/ml)) and RA SF (range 343-2570ng/ml), but was absent in 5/6 OA SF tested . Anti-BPI antibodies, however, did not fully neutralize the bactericidal activity of inflammatory SF . In contrast to the SF effects observed on Gram-negative bacteria, Chlamydia trachomatis cultured within HeLa cells thrived in the presence of SF . Indeed extracellular Chlamydia could easily be passaged through cultured synovial fibroblasts in the presence of SF . These findings indicate that the potent microbicidal activity of SF may account for the failure to recover viable organisms from the joint in ReA . Chlamydia alone amongst these organisms demonstrates resistance to microbicidal effect of SF, which may relate to the pathogenesis of Chlamydia-induced arthritis.

Cancer Res, 1996 Apr 1, 56(7), 1533 - 8
Biomarker alterations produced in rat lung by intratracheal instillations of air particulate extracts and chemoprevention with oral N-acetylcysteine; Izzotti A et al.; Organic matter extracts were obtained from particulates recovered from 10,000-m3 air samples collected in Sicily (Italy) . The overall concentrations of acenaphthene, benzo(a)pyrene, phenanthrene, anthracene, fluoranthene, and pyrene were 526 ng/m3 air in a highly polluted urban area and 48 ng/m3 in a rural area affected by motor vehicle traffic pollution . After metabolic activation, both samples were mutagenic in Salmonella typhimurium his(-) strains of the TA and YG series, with potencies in TA100 of 140.7 and 11.8 revertants/m3 air, respectively . The samples, resuspended in tricaprylin, were instilled intratracheally in Sprague-Dawley rats for 5 consecutive days, accounting for a cumulative dose in each animal of the organic fractions extracted from 400 m3 air, which corresponds approximately to the volume of air inhaled by a man in 1 month . Treatment with the rural area sample and, at higher levels, with the urban area sample resulted in the formation of adducts to lung DNA, as assessed both by synchronous fluorescence spectrophotometry and by 32P postlabeling, which showed the appearance of up to six individual adducts emerging from diffuse diagonal radioactive zones . The adducts were more efficiently detected by extraction with butanol than by digestion with nuclease P1 . DNA binding of air particulate extracts was followed by alterations of early damage biomarkers only in the rats treated with the urban area sample . Repair of DNA damage in lung cells was inferred from a significant stimulation of the nuclear enzyme poly(ADP-ribose) polymerase compared with that in sham-exposed rats . Among the cells recovered by bronchoalveolar lavage, an increase in polymorphonucleate leukocytes and cells of the ciliated respiratory epithelium was accompanied by a relative decrease in pulmonary alveolar macrophages . The frequency of micronuclei was significantly enhanced both in epithelial cells and alveolar macrophages, and binucleated macrophages were also more frequent in treated rats . The thiol N-acetylcysteine, one of the most promising cancer chemopreventive agents, was administered with drinking water to a group of animals receiving the air particulate polycyclic aromatic hydrocarbon fraction from the urban area . N-acetylcysteine prevented or considerably attenuated the alterations of all monitored parameters . These findings provide evidence that, even under outstandingly high exposure conditions, it is possible to protect the respiratory tract from DNA-binding and DNA-damaging air particulate carcinogens.

Biochemistry, 1996 Mar 26, 35(12), 3803 - 9
A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase; Henriksen A et al.; Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides . Alpha-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5'-monophosphate (OMP) in the presence of divalent cations, preferably Mg2+ . OMP is thereafter converted to uridine 5'-monophosphate by OMP decarboxylase . We have determined the 2.4 angstrom structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data . In the structure of the E . coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis . The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion . Crystalline E . coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region . Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer . We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase.OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis . Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S . typhimurium OPRTase.OMP and E . coli OPRTase are found in the substrate binding regions: the 5'-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73).

Biochemistry, 1996 Mar 26, 35(12), 3746 - 53
Kinetics of the interaction between DNA and the type IC restriction enzyme EcoR124II; Ramsden JJ et al.; Optical waveguide mode spectroscopy was used to determine the binding constants characterizing the interaction of EcoR124II, a type IC restriction modification enzyme from Salmonella typhimurium, with DNA . The DNA is immobilized on the surface of an optical waveguide, and the enzyme is introduced in bulk solution flowing over the DNA under controlled hydrodynamic conditions . The binding kinetics of the protein to the DNA can be directly observed and the number of bound protein molecules per base pair determined to a high accuracy . Dissociation of the protein was measured by switching flowing protein to protein-free buffer . Binding to two different kinds of DNA, with and without the specific sequence recognized by EcoR124II, was investigated . Protein binding and dissociation ("nonspecific" binding), quantified by association and dissociation rate coefficients ka and kd, were the same for both types, but the DNA carrying the recognition site showed an additional process, "irreversible" association (i.e . dissociation was not observed on the time scale of the experiments) of the protein, quantified by a rate coefficient ks . Some inferences regarding the mechanism of base pair searching are made from the measured ka, kd, and ks values.

J Mol Biol, 1996 Mar 22, 257(1), 87 - 101
Structure, interactions and dynamics of PRD1 virus I . Coupling of subunit folding and capsid assembly; Tuma R et al.; Bacteriophage PRD1, which infects Escherichia coli and Salmonella typhimurium, consists of an icosahedral capsid enclosing a membrane-packaged double-stranded DNA genome . The viral shell has been investigated using time and temperature resolved Raman and ultraviolet-resonance Raman spectroscopy to reveal novel features of the capsid structure and its pathway of assembly from P3 subunits . Raman spectra show that the shell is thermostable to 50 degrees C, and disassembles between 50 and 70 C degrees with only a small change in P3 conformation . However, the products of thermal disassembly depend sensitively upon total protein concentration . Characterization by analytical ultracentrifugation indicates that below 8 mg/ml, the purified shell disassembles primarily into P3 trimers; at higher concentrations, larger multimers of P3 are formed . Guanidine hydrochloride (GuHCl) dissociation of the P3 shell yields similar results . Purified P3 trimers, isolated either by heat or GuHCl treatment, exhibit structure sensitivity between 30 and 50 degrees C . Thus, shell disassembly diminishes P3 thermostability . Both the lower temperature transition (30 degrees C to 50 degrees C) of the trimer and the higher temperature transition (50 degrees C to 70 degrees C) of the shell involve a conversion of approximately 5% of the P3 peptide backbone from alpha-helix to beta-strand . Deuterium exchange of the P3 peptide backbone reveals more rapid exchange in the shell than in the trimer, consistent with the observed non-specific polymerization of trimers at high concentration . Conversely, the exchange of indole 1NH groups shows that approximately 65% of tryptophan residues are protected against exchange in the assembled shell . The results suggest a mechanism for shell assembly in which the specific association of trimers into the correct shell architecture involves stabilization of a subunit alpha-helical domain and sequestering of selected side-chains from solvent access . We propose a capsid assembly model which couples P3 shell formation with the final step in folding of the P3 subunit.

Mol Gen Genet, 1996 Mar 20, 250(5), 570 - 80
recB recJ mutants of Salmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance; Garzon A et al.; recB recJ mutants of Salmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids . Plasmid instability is less severe in recD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type . Plasmids remain unstable in recA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability . recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair . In contrast to their severe defects in recombination, DNA repair and plasmid maintenance, recB recJ mutants of S . typhimurium behave similarly to the wild type in the segregation of chromosome duplications . The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2593 - 7
Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium; Shea JE et al.; Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7 . This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12 . Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system . To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2 . SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E . coli ydhE and pykF genes . Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E . coli . Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2545 - 50
Molecular mechanism of transmembrane signaling by the aspartate receptor: a model; Chervitz SA et al.; The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms . These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes . The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor . This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm . Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor . Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.

FEBS Lett, 1996 Mar 18, 382(3), 297 - 303
Mutants of EF-Tu defective in binding aminoacyl-tRNA; Abdulkarim F et al.; Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro . The substitutions are Leu120 Gln, Gln124 Arg and Tyr160 (Asp or Asn or Cys) . They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu.GTP configuration . The different EF-Tu variants exhibit a spectrum of phenotypes . First, k cat/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120 Gln, Gln124 Arg and Tyr160 Cys . Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation . Third, in four cases out of five the affinity of the complex between EF-Tu.GTP and aminoacyl-tRNA is significantly decreased . The most drastic reduction is observed for the Gln124 Arg change, where the association constant is 30-fold lower than in the wild-type case . Fourth, missense errors are increased as well as decreased by the different amino acid substitutions . Finally, the dissociation rate constant (kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160 Cys substitution, but remains unchanged in the four other cases . These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

J Biol Chem, 1996 Mar 15, 271(11), 6530 - 6
In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system; Cullen PJ et al.; Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter . In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters . To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB) . MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate . MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro . The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion . The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein . However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein . RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P . Therefore, the R . capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.

Vet Immunol Immunopathol, 1996 Mar, 50(1-2), 55 - 65
Intracellular survival of Brucella abortus, Mycobacterium bovis BCG, Salmonella dublin, and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus; Qureshi T et al.; Peripheral blood monocyte-derived macrophages were obtained from a herd of cows selected, bred, and confirmed as resistant or susceptible by in vivo challenge of Brucella abortus Strain 2308 . The ability to control in vitro intracellular bacterial replication of B . abortus Strain 2308, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Montreal Strain 9003, Salmonella dublin Strain 5631, and Salmonella typhimurium Strain 14028 was evaluated in a bactericidal assay . The macrophages from resistant cattle were significantly superior (P < 0.05) in controlling intracellular growth of B . abortus, M . bovis BCG, S . dublin but not of S, typhimurium than macrophages from susceptible animals . Controls of all four pathogens correlated strongly with each other in resistant or susceptible macrophages . Data from resistant cattle had a tighter grouping than that of susceptible cattle, while data from susceptible cattle overlapped considerably with data from resistant animals . Therefore, this assay was considered a phenotypic marker of the resistant trait . For each bacterial species a percent bacterial survival value was used as a cut-off point to designate animals as resistant or susceptible . These data were compared with the in vivo challenged resistant or susceptible classification by using the Chi-square analyses . A cut-off point of 70 percent bacterial survival for B . abortus designated 14 cattle as susceptible and seven as resistant and this correlated 100 percent with the number of animals designated as to the relevant category by in vivo challenge . A value of 65 percent bacterial survival for M . bovis BCG, and 100 percent bacterial survival for S . dublin correlated highly with actual numbers of animals designated as susceptible or resistant.

J Appl Toxicol, 1996 Mar-Apr, 16(2), 103 - 8
Mutagenic activity of acrolein in S . typhimurium and E . coli; Parent RA et al.; Acrolein was tested for mutagenic activity in seven strains of Salmonella typhimurium and one strain of Escherichia coli using a preincubation assay procedure . Cytotoxicity was evident at dosing levels above 33 and 67 micrograms acrolein per plate in the absence and presence of S-9 activation, respectively . Evidence of mutagenic activity was seen at non-toxic dosing levels in S . typhimurium strains TA98 and TA100 and E . coli strain WP2 uvrA . Responses in TA98 and E . coli were marginal at best, but a firm positive mutagenic activity was noted in TA100 at 20 micrograms per plate without S-9 activation and at 67 micrograms per plate with S-9 activation . The results of this study demonstrate the mutagenicity of acrolein under highly controlled conditions.

FEMS Microbiol Lett, 1996 Mar 1, 136(3), 263 - 8
Salmonella typhimurium InvA expression probed with a monoclonal antibody to the C-terminal peptide of InvA; Clark CG et al.; The Salmonella typhimurium InvA protein is a component of a sec-independent secretion apparatus necessary for full virulence of the bacteria . We generated a monoclonal antibody to the C-terminal portion of the InvA protein that recognized proteins in S . typhimurium and weakly in Y . enterocolitica, but not in several other species of bacteria, including S . flexneri . S . typhimurium grown without agitation produced relatively constant amounts of membrane InvA throughout the growth cycle, whereas bacteria grown with agitation had a sharp increase in the amount of membrane InvA at late exponential phase . Levels of InvA present in Salmonella membranes under some growth conditions do not appear to correlate with levels of invasion under the same conditions.

FEMS Immunol Med Microbiol, 1996 Mar, 13(3), 245 - 7
Role of two outer membrane antigens in the induction of protective immunity against Francisella tularensis strains of different virulence; Fulop M et al.; A crude outer membrane preparation from Francisella tularensis live vaccine strain was used to immunise mice . Immunised mice were completely protected from a F . tularensis challenge . We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA . We presented FopA to the immune system using an aromatic amino acid dependent Salmonella typhimurium as a vector . Although mice mounted an immune response to cloned FopA no significant protection was induced . However, lipopolysaccharide-immunised mice were completely protected from a F.tularensis live vaccine strain challenge . No increase in LD50 was observed using F . tularensis Schu4 as the challenge strain, although there was a significant increase in time to death . These data question the validity of the murine F . tularensis live vaccine strain model.

Anal Biochem, 1996 Mar 1, 235(1), 20 - 5
Use of computer-assisted motion analysis for quantitative measurements of swimming behavior in peritrichously flagellated bacteria; Amsler CD; An assay was developed which identifies individual bacterial tumbles and so allows rapid, quantitative measurements of tumble frequency in free-swimming bacteria . Tumble frequency is modulated by cells to enable chemotaxis . Mutations in the chemotaxis signal transduction pathway typically have phenotypes of altered tumble frequency . The purpose of this assay is to quantitatively measure steady-state tumble frequency to enable comparisons of mutant strain phenotypes . It was developed using Escherichia coli but should be applicable to other species with a peritrichous flagellation pattern, such as Salmonella typhimurium . Tumbles are defined by a combination of the parameters rate of change of direction and swimming speed, with a rapid change of direction defining the beginning of a tumble and increased swimming speed defining the end . These parameters have previously been shown to be correlated with tumbles in general but not used to identify discrete tumble events . The computer assay was validated by comparing its results with manual observations by eye . The assay was intended to be most sensitive to swimming patterns similar to wild type so as to resolve subtle changes which would result from partial-function mutations . It quantitatively detects extreme behavioral phenotypes as well and can be modified to increase resolution at either extreme if necessary.

Chem Res Toxicol, 1996 Mar, 9(2), 374 - 81
Mutational spectra of Salmonella typhimurium revertants induced by chlorohydroxyfuranones, byproducts of chlorine disinfection of drinking water; Knasmuller S et al.; The base substitution specificities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), and chloromalonaldehyde (CMA), a putative breakdown product of MCA, were examined in the hisG46 gene and in the hisG428 gene of Salmonella typhimurium using allele specific oligonucleotide hybridization . Although the compounds are structurally closely related, they induced substantially different mutation spectra: MCA and CMA caused primarily GC-->AT transitions in the hisG46 allele (target sequence CCC), in particular, at the second position of the codon in strain TA100 . In TA100 the mutation spectrum of MCA was similar to that of CMA . The mutational specificity of MCA can be explained as a consequence of misincorporation opposite to cyclic etheno adducts identical to those formed by the carcinogen vinyl chloride . The spectra induced by MX and CMCF in TA100 were almost identical but distinctively different from the spectra of MCA and CMA . Both compounds induced primarily GC-->TA transversions, in particular, at the second position of the codon, and to a lesser extent in the first position of the codon . An identical site bias is induced by carcinogens such as polycyclic aromatic hydrocarbons and heterocyclic amines as a consequence of formation of (noncyclic) guanosine adducts . In hisG428 (target sequence TAA) MX induced again primarily GC-->TA transversions in Tyr tRNA genes (supC/M) and, to a lesser extent, intragenic AT-->TA transversions (TAA-->AAA) . The possible involvement of guanosine and adenosine adducts in the mutational specificity of MX is addressed.

Poult Sci, 1996 Mar, 75(3), 342 - 5
Immune responses in chickens against lipopolysaccharide of Escherichia coli and Salmonella typhimurium; Sunwoo HH et al.; Immunization of chickens with whole bacteria results in the production of antibodies specific to lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria . However, there is relatively limited information available concerning immune response of purified LPS in this species . In the present study, immune responses were examined in serum and egg yolk from two groups of chickens injected with entire LPS from Escherichia coli and lipid A free LPS from Salmonella typhimurium . The results demonstrated that the increase of antibody activity occurs first in serum, and then in egg yolk with a lag in time of 1 to 3 wk in both groups of chickens . However, the time of elevated levels of antibodies activity was much shorter in chickens immunized with S . typhimurium LPS (< 1 wk) than in those immunized with E . coli LPS (4 wk) . A lack of lipid A is the S . typhimurium antigen may be a factor related to this difference.

Vaccine, 1996 Mar, 14(4), 251 - 9
Protection against oral challenge three months after i.v . immunization of BALB/c mice with live Aro Salmonella typhimurium and Salmonella enteritidis vaccines is serotype (species)-dependent and only partially determined by the main LPS O antigen; Hormaeche CE et al.; The role of the main LPS O antigen in the specificity of protection as mediated by systemic mechanisms following immunization with live attenuated Aro Salmonella vaccines was studied in mice . Innately Salmonella-susceptible (Itys) BALB/c mice were immunized intravenously with a single dose of either Salmonella typhimurium SL3261 aroA (LPS O4,5,12) or Salmonella enteritidis Se795aroA (LPS O1,9,12), and challenged orally 2-3 months later with either S . typhimurium C5 or S . enteritidis Thirsk . Nearly isogenic transductants of the two challenge strains expressing either their own LPS or that of the other serotype (S . typhimurium C5 O4 or O9, and S . enteritidis Thirsk O9 or O4) were also used . Both vaccines conferred similar high protection against the virulent strain of the homologous serotype expressing its own LPS . There was no protection against the heterologous serotype expressing its own LPS . However, when vaccinated mice were challenged with either the same serotype as the vaccine but expressing the heterologous LPS, or with the heterologous serotype expressing the LPS of the vaccine, protection was always lower than protection against the fully homologous serotype . Anti-smooth LPS antibodies showed higher titres against the homologous LPS, but with significant crossreactivity with the heterologous LPS . Antibodies to O-rough S . typhimurium and S . enteritidis LPS were present following immunization with either of the two vaccine strains . The LPS alone cannot fully account for the specificity of protection in this model; other (protein) antigens may be responsible . It remains to be seen whether there is a T-cell mediated component to the specificity of protection conferred by live Salmonella vaccines.

Ecotoxicol Environ Saf, 1996 Mar, 33(2), 193 - 200
A comparison of the Ames assay and Mutatox in assessing the mutagenic potential of contaminated dredged sediment; Jarvis AS et al.; The ability of the Ames assay and of Mutatox to identify the genotoxic potential of dredged sediments was compared . The Ames assay has been used extensively in the testing of environmental contaminants, whereas Mutatox, a new bacterial bioluminescence test, has only recently been used for this purpose . Ten sediments with varying degrees of contamination were soxhlet extracted . Each of the 10 extracts was split with half remaining in a crude form and half cleaned using silica gel chromatography, resulting in 20 extract samples . Both the Ames assay (using Salmonella typhimurium strains TA98 and TA100) and Mutatox were conducted with and without S9 metabolic activation . When metabolically activated, TA98 and TA100 indicated a positive mutagenic response in 80 and 50%, respectively, of the sediment extracts . Without S9 activation, TA98 indicated a positive mutagenic response with half the extracts, whereas only 10% did so with TA100 . Mutatox indicated a positive mutagenic response with S9 activation in 75% of the extracts and no mutagenic response in any of the sediment extracts without metabolic activation . In a side-by-side comparison of the Ames assay (TA98 with S9) and Mutatox, 80% of the sediment extracts had similar responses, both positive and negative . Fifty percent of the sediment extracts had similar responses when tested with TA100 and Mutatox in the presence of S9 . Mutatox compared reasonably well with the Ames assay but was insensitive to the presence of direct-acting mutagens in the sediments tested . Although Mutatox demonstrates promise as a screening tool to assess sediment genotoxicity, the authors consider it appropriate to use the Ames assay as a confirmation for definitive investigations.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Mar-Apr, (2), 85 - 7
{The morphometric characteristics of the immune system organs in dynamic experimental Salmonella infection}; Smoliagin AI et al.; Quantitative and morphological changes in the immune system (CBA x C57BL/6)F1 mice after their enteral (group 1) and intraperitoneal (group 2) inoculation with Salmonella typhimurium in a dose of 2 x (10)6 microbial cells per mouse were studied . The study revealed that on day 28 after inoculation the death rate of the animals was 12.5 +/- 6.75% in group 1 and 57.84 +/- 4.89% in group 2 . The same type of changes in the organs of the immune system was observed in both groups of mice, but in the animals of group 2 these changes were more pronounced . This was manifested by leukocytosis, exhaustion of thymic cells, hyperplasia, hypertrophy of the spleen and an increase in the proliferative activity in its B-zone, a decrease in the number of myelokaryocytes . Such changes are indicative of their importance in the resistance of the body to Salmonella infection.

Mutagenesis, 1996 Mar, 11(2), 177 - 81
Suppressive effects of methyl methacrylate on the mutagenicity and DNA adduct formation induced by 1-nitropyrene and benzo{a}pyrene; Chou LS et al.; Methyl methacrylate (MMA) is widely used as a cement in dentistry, orthopaedic surgery and ophthalmology . Studies based on short-term genotoxicity tests have produced conflicting results in the last two decades . In the present study, the effects of MMA on the mutagenicity of 1-nitropyrene (1-NP) and benzo{a}pyrene (B{a}P) were evaluated with the Salmonella typhimurium TA98 strain in the absence and presence of S9 mix . The direct-acting mutagenicity of 1-NP was markedly decreased by MMA in a dose-dependent manner . However, a low inhibitory effect of MMA on the metabolic-acting mutagenicity of B{a}P was observed . MMA did not show mutagenicity within the concentrations of 4.7-37.6 microM either with or without S9 mix . The inhibitory effect of MMA was not due to its cytotoxicity because very low and/or no cytotoxicity of MMA to S . typhimurium TA98 was observed . To confirm the antimutagenicity of MMA against 1-NP and B{a}P, a 32P-postlabelling method was used to determine whether MMA modified DNA adduct formation produced by both compounds in calf thymus DNA . MMA inhibits the formation of 1-NP- and B{a}P-DNA adducts in a dose-dependent manner . The DNA adduct of 1-NP reduced by MMA was greater than that of B{a}P . Thus, we suggested that MMA was possibly acting as an inhibitor of chemical carcinogenesis.

Mutagenesis, 1996 Mar, 11(2), 151 - 4
Mutagenic potential of Indian tobacco products; Niphadkar MP et al.; The mutagenic potential of aqueous extracts of masheri (ME), chewing tobacco alone (CTE) and a mixture of chewing tobacco plus lime (CTLE) was tested using the Ames assay . ME exhibited mutagenicity in Salmonella typhimurium TA98 upon metabolic activation with aroclor-1254-induced rat liver S9, while nitrosation rendered it mutagenic in TA100 and TA102 . CTE exhibited borderline mutagenicity in the absence or presence of S9 in TA98 and TA100 and after nitrosation in TA102, while nitrosation led to doubling of TA98 and TA100 revertants . In contrast, CTLE exhibited direct mutagenicity in TA98, TA100 and TA102, was mutagenic to TA98 upon S9 addition and induced mutagenic responses in all three tester strains after nitrosation . Experiments using scavengers of reactive oxygen species (ROS) suggested that CTLE-induced oxidative damage in TA102 was mediated by a variety of ROS . The high mutagenic potency of CTLE vis a vis that of CTE may be attributed to changes in the pH leading to differences in the amount and nature of compounds extracted from tobacco . Thus, exposure to a wide spectrum of tobacco-derived mutagens and promutagens may play a critical role in the development of oral cancer among users of tobacco plus lime.

Infect Immun, 1996 Mar, 64(3), 938 - 44
Effect of vaccination of hens with an avirulent strain of Salmonella typhimurium on immunity of progeny challenged with wild-Type Salmonella strains; Hassan JO et al.; The avirulent Salmonella typhimurium chi3985 was used to vaccinate white leghorn chickens at 16 and 18 weeks of age, and the effect of maternal antibody on Salmonella colonization of progeny of vaccinated hens was assessed with S . typhimurium F98 or chi3985 . Progeny of hens that had been vaccinated at 1 and 3 or 2 and 4 weeks of age with chi3985 were used to determine the effect of maternal immunity on vaccine efficacy . Vaccination of hens induced long-lasting Salmonella-specific antibodies which were transferred into eggs and were detected as immunoglobulin G (IgG) in the egg yolk . Maternal antibody was detected in the progeny of vaccinated birds as IgG and IgA in serum and intestinal fluid, respectively . The titer of maternally transmitted IgG or IgA was highest in the first week of life of the progeny and declined with age . Maternal antibodies prevented colonization of the chicks by S . typhimurium chi3985 and reduced colonization by S . typhimurium F98 . Overall, chicks from vaccinated hens had significantly higher antibody responses than did the progeny of nonvaccinated hens after oral infection with Salmonella strains . Maternal antibody reduced the efficacy of vaccination of progeny with chi3985 at 1 and 3 weeks of age . But vaccination at 2 and 4 weeks of age induced excellent protection against challenge with S . typhimurium F98 or S . enteritidis 27A PT 8 in birds from vaccinated hens and in specific-pathogen-free chickens . Vaccination of chickens at 2 and 4 weeks of age has been shown to protect the birds against challenge with homologous and heterologous Salmonella serotypes . A combination of vaccination of adult animals and use of the progeny of vaccinated birds will enhance effective control of Salmonella infections in the poultry industry . This will complement the present control of Salmonella-associated food poisoning caused by Salmonella enteritidis in eggs because the avirulent S . typhimurium vaccine strain chi3985 induced excellent protection against S . enteritidis in chickens.

Infect Immun, 1996 Mar, 64(3), 849 - 54
Gamma interferon and interleukin-10 gene expression in innately susceptible and resistant mice during the early phase of Salmonella typhimurium infection; Pie S et al.; Previous studies have shown that gamma interferon (IFN-gamma) plays a major role in natural resistance to Salmonella typhimurium during the early phase of infection . To assess whether the level of natural resistance in mice is related to the level of IFN-gamma gene expression, we compared IFN-gamma mRNA levels by means of reverse transcriptase-PCR in the spleens of genetically susceptible Itys (C57BL/6 and BALB/c) and resistant Ityr (CBA and DBA/2) mice during the first 5 days of infection . The mRNA expression of interleukin-10 (IL-10), a cytokine which antagonizes IFN-gamma effects, was also investigated . Mice were infected with 10(3) CFU of the virulent strain S . typhimurium C5, a dose which is lethal within a week for susceptible mice only . IFN-gamma mRNA increased to similar levels in both susceptible and resistant mice, suggesting that susceptibility to S . typhimurium infection is not related to defective IFN-gamma gene expression . In contrast, IL-10 mRNA reached much higher levels in susceptible than in resistant mice . Similar results were found in Ity congenic mice, confirming a link between the presence of the Itys allele and a high level of IL-10 gene expression during infection . High levels of IL-10 mRNA in susceptible mice correlated with high IL-10 serum levels (on day 5), whereas IL-10 was not detectable in the sera of resistant mice . However, administration of neutralizing anti-IL-10 monoclonal antibodies did not modify the course of infection . To evaluate the influence of bacterial multiplication on IL-10 mRNA expression, susceptible mice were infected with an attenuated strain of S . typhimurium . This strain induced a low level of IL-10 mRNA expression . When susceptible mice were immunized with an attenuated strain and challenged with the virulent strain, they inhibited the growth of the challenge bacteria and exhibited a low level of IL-10 mRNA . In contrast, when resistant mice were infected with a high (lethal) dose of the virulent strain, they exhibited a high level of IL-10 mRNA . Taken together, these results indicate that the level of IL-10 gene expression correlates with the level of bacterial multiplication in the organs and that the high level of IL-10 mRNA in Itys mice is a consequence rather than the cause of their susceptibility to S . typhimurium infection.

Infect Immun, 1996 Mar, 64(3), 1043 - 7
Neutrophils prevent extracellular colonization of the liver microvasculature by Salmonella typhimurium; Conlan JW; The early course of hepatic infection with the facultative intracellular bacterial pathogen Salmonella typhimurium was examined in control mice and in mice selectively depleted of neutrophils by treatment with a granulocyte-specific monoclonal antibody . The results show that >200-fold more salmonellae were recovered in livers of the latter group of mice than in livers of the former group by 24 h of parenterally initiated infection . Comparative histological examination of the livers from both groups of mice indicated that neutrophils participate in early anti-Salmonella defense in the liver in part by aborting infection in permissive hepatocytes and by inhibiting extracellular bacterial colonization of the hepatic microvasculature . It is shown in addition that systemic salmonellosis was also severely exacerbated in neutropenic mice infected intragastrically with the pathogen.

J Bacteriol, 1996 Mar, 178(5), 1476 - 9
Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium; Enos-Berlage JL et al.; purF mutants of Salmonella typhimurium are known to require a source of both purine and thiamine; however, exogenous pantothenate may be substituted for the thiamine requirement . We show here that the effect of pantothenate is prevented by blocks in the oxidative pentose phosphate pathway, gnd (encoding gluconate 6-phosphate {6-P} dehydrogenase) or zwf (encoding glucose 6-P dehydrogenase) . We further show that the defects caused by these mutations can be overcome by increasing ribose 5-P, suggesting that ribose 5-P may play a role in the ability of pantothenate to substitute for thiamine.

Carcinogenesis, 1996 Mar, 17(3), 555 - 8
Developmental changes in hepatic activation of 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline in rabbit; Rich KJ et al.; MeIQx, a potent bacterial mutagen formed when meat is cooked, requires metabolic activation to exert its genotoxicity, a reaction catalysed primarily by CYP1A2 in adult mammals . Little is known about mammalian developmental changes in the mutagneic activation of compounds such as MeIQx . In rabbits we have shown previously that expression of CYP1A2 increases with age and is inducible by 3-methylcholanthrene (MC) from as early as 4 days pre-parturition . We have therefore investigated the effect of age on rabbit liver activation of MeIQx (assessed in an Ames test using Salmonella typhimurium TA98) before and after treatment of the animals with MC . MeIQx activation could not be detected using hepatic microsomal fractions from rabbits of <17 days of age . Thereafter activation increased with age to peak in weanling animals . Following MC treatment MeIQx activation was increased, being detectable in samples from rabbits as young as 9-11 days . The inducibility of MeIQx activation increased with age, reaching a plateau between 17 and 35 days . These rates of activation were broadly parallel to the changes in CYP1A2 specific content . These results indicate that the ability of rabbit liver to activate MeIQx is dependent on CYP1A2 activity, the expression of which is developmentally regulated . Although it has been established that human activation of MeIQx is also CYP1A2 dependent, whether a similar situation exists in infant humans has yet to be determined, although there is evidence that CYP1A2-dependent activity reaches a peak in late childhood.

Carcinogenesis, 1996 Mar, 17(3), 413 - 9
Fibre-induced lipid peroxidation leads to DNA adduct formation in Salmonella typhimurium TA104 and rat lung fibroblasts; Howden PJ et al.; Certain end-products of lipid peroxidation bind to DNA forming a fluorescent chromophore . Incubation of both Salmonella typhimurium TA104 and a rat lung fibroblast cell line, RFL-6, with various types of mineral fibre resulted in a time- and dose-dependent increase in DNA fluorescence . The increase in DNA fluorescence was shown to be directly related to the amount of iron that could be mobilized from the fibre surface using in vitro studies in the absence of cells or bacteria . Crocidolite and man-made vitreous fibre-21 (MMVF-21) mobilized significant quantities of iron and were significantly more active than chrysotile and refactory ceramic fibre-1 (RCF-1) . Fibre-induced malondialdehyde-DNA adduct formation, the fluorescent product, was increased by incubating cells with buthionine sulfoximine and ameliorated by co-treatment with N-acetylcysteine, indicating a protective role for glutathione . Similarly, vitamin E was also shown to inhibit DNA adduct formation . These results suggest that mineral fibre-induced lipid peroxidation produced genotoxic products which can diffuse into nucleus and interact with cellular DNA . In conclusion, fibre-induced lipid peroxidation may be a possible mechanism in the genotoxic action of fibrous materials.

Eur J Immunol, 1996 Mar, 26(3), 563 - 70
Macrophage-T cell interaction in murine salmonellosis: selective down-regulation of ICAM-1 and B7 molecules in infected macrophages and its probable role in cell-mediated immunity; Gupta S et al.; Vaccine development and understanding of cellular immune modulatory mechanisms in salmonella infections have been impeded due to the paucity of data on antigens capable of eliciting effective immune responses . The present study was done to evaluate the efficacy of five major purified salmonella antigens (porins, pili, flagella, outer membrane proteins and heat shock proteins) in modulating T cell-macrophage interactions which play a central role in resistance to and recovery from infection with several intracellular pathogens, including salmonella . The results showed that the T cells recovered 10 days post-immunization (D10 T cells) from mice immunized with porins and outer membrane proteins showed maximum proliferation in the presence of macrophages incubated with dead bacteria; however, this response was decreased when T cells were co-cultured with live Salmonella typhimurium-infected macrophages . Delayed-type hypersensitivity responses, as measured by increased footpad thickness at 24 h, though induced effectively by porins, pili and flagella, were completely abrogated when D10 T cells were pre-incubated with macrophages infected with live bacteria . The phagocytic and bactericidal ability of normal macrophages, when grown in presence of T cell supernatants, was not influenced by the immunizing agents, but T cell supernatants obtained from mice immunized with porins and heat-shock protein triggered increased bactericidal activity . Further, the expression of the co-stimulatory molecules ICAM-1 and B7 increased with increasing bacteria (dead):macrophage ratio, but this expression was down-regulated upon incubation with live bacteria.

Mutat Res, 1996 Feb 29, 359(2), 85 - 93
Antimutagens reduce ofloxacin-induced bleaching in Euglena gracilis; Ebringer L et al.; The genotoxic effect of ofloxacin was significantly decreased by standard antimutagens (sodium selenite, ascorbic acid, butylated hydroxyanisole and butylated hydroxytoluene) in the unicellular flagellate Euglena gracilis . The antiofloxacin activity of sodium selenite was also documented by a bacterial test in which the repair-proficient strain Salmonella typhimurium TA102 was used.

Cancer Lett, 1996 Feb 27, 100(1-2), 235 - 40
Identification of antimutagenic substances in an extract of edible red alga, Porphyra tenera (Asakusa-nori)
Okai Y, Higashi-Okai K, Yano Y, Otani S.
Recently, a relatively strong antimutagenic activity has been detected in the extract of Porphyra tenera (Asakusa-nori in Japanese) which showed a suppressive effect on mutagen-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002 (Okai et al . (1994) Cancer Lett., 87, 25-32) . In the present paper, we analyzed the active principles for the antimutagenic activity in an extract of Porphyra tenera and detected three color spots on a silica gel TLC plate which indicated very similar Rf values and absorbance spectra of standard pigments such as beta-carotene, chlorophyll a and lutein . The seaweed pigments recovered from preparative silica gel TLC corresponding to beta-carotene, chlorophyll a and lutein exhibited significant suppressive activities against mutagen-induced umu C gene expression and combined treatment with these pigments showed an additive effect compared with single treatment with each pigment . Furthermore, the standard pigments prepared from other biological sources also exhibited similar anti-mutagenic activities . The significance of this finding is discussed from the protective role of seaweed pigments against mutagenesis probably associated with carcinogenesis.

Gene, 1996 Feb 22, 169(1), 75 - 80
The location of four fimbrin-encoding genes, agfA, fimA, sefA and sefD, on the Salmonella enteritidis and/or S . typhimurium XbaI-BlnI genomic restriction maps; Collinson SK et al.; Four fimbrin-encoding genes, fimA (type-1 or SEF21 fimbriae), agfA (thin aggregative or SEF17 fimbriae), sefA (SEF14 fimbriae and sefD (SEF18 fimbriae) from Salmonella enteritidis (Se) 27655-3b were located onto the XbaI-BlnI genomic restriction maps of Salmonella typhimurium (St) LT2 and Se strains SSU7998 and 27655-3b . The XbaI or BlnI genomic fragments carrying these genes were identified by hybridization with labeled oligodeoxyribonucleotides or fimbrin-encoding genes . The fimbrin-encoding genes were not encoded by the virulence plasmids, but were located on chromosomal DNA fragments . The position of each gene on a given XbaI fragment was determined by hybridization of a series of XbaI-digested genomic DNA samples from previously characterized Tn10 mutants of Se and St with its respective probe . The fimA gene mapped near 13 centisomes (Cs) between purE884::Tn10 at 12.6 Cs (11.8 min) and apeE2::Tn10 at 12.8 Cs (12.3 min) beside the first XbaI site at 13.0 Cs in St or between purE884::Tn10 at 12.6 Cs and the XbaI site at 13.6 Cs in Se . The agfA gene mapped near 26 Cs between putA::Tn10 and pyrC691::Tn10 in St, but near 40 Cs between pncX::Tn10 and the XbaI site at 43.3 Cs in Se . This difference in map position was due to the location of agfA near one end of the 815-kb chromosomal fragment inverted between Se and St . The sefA and sefD genes mapped precisely at 97.6 Cs in Se, but were absent from the genome of St LT2 . To verify the mapping procedures used herein, tctC was also mapped in both Salmonella serovars . As expected, tctC mapped near 60 Cs in both St and Se, thereby confirming previous studies.

Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1458 - 63
Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis; Hess J et al.; Vaccination provides the most potent measure against infectious disease, and recombinant (r) viable vaccines expressing defined pathogen-derived antigens represent powerful candidates for future vaccination strategies . In a new approach we constructed r-aroA- Salmonella typhimurium displaying p60 or listeriolysin (Hly) antigen of Listeria monocytogenes in secreted or somatic form in the host cell . Vaccination of mice with r-aroA- S . typhimurium induced protection against the intracellular pathogen L . monocytogenes only with secreted and not with somatic antigen . Secreted Hly was slightly more potent in inducing protective immunity than secreted p60 . Both r-aroA- S . typhimurium secreting p60 in the endosome and r-aroA- S . typhimurium secreting Hly in the cytosol induced protective CD4+ and CD8+ T-cells suggesting CD8+ T-cell stimulation independent from intracellular residence of r-aroA- S . typhimurium carriers . Hence, not only the type of antigen but also its display by the r-carrier within the host cell critically influences vaccine efficacy.

Mutat Res, 1996 Feb 19, 350(1), 93 - 102
Effects of phenethyl isothiocyanate on metabolism and on genotoxicity of dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo{4, 5-beta}pyridine (PhIP); Knasmuller S et al.; Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, inhibited the genotoxic effects of N-nitrosodimethylamine (DMN) and of 2-amino-1-methyl-6-phenylimidazo{4,5-beta}pyridine (PhIP) in differential DNA repair assays with E . coli K-12 strains in vitro and in animal mediated assays with mice . In Salmonella typhimurium, the mutagenic activities of DMN and PhIP measured after activation with S-9 homogenates from several organs of PEITC-treated mice were substantially lower than those obtained with homogenates of untreated animals as well . PEITC also reduced the formation of micronuclei by DMN in metabolically competent Hep-G-2 cells of human origin but was ineffective in combination with PhIP . Biochemical investigations showed that the prevention of genotoxic effects of DMN by PEITC results form an inhibition of its alpha-hydroxylation . The effect of oral administration of PEITC on the formation of DNA adducts of PhIP was examined in the colon and liver of mice . No inhibition of adduct formation was observed in these experiments . Biochemical experiments showed that PEITC reduces not only the metabolic activation of PhIP via 2-hydroxyamino PhIP but also inhibits a detoxification pathway (formation of 4-hydroxy PhIP) . The present results can be taken as an indication that the anticarcinogenic activities of isothiocyanates towards nitrosamines are paralleled by antimutagenic effects, and that probably no such protective effects occur in combination with heterocyclic amines . Furthermore, our findings show that the effects of chemopreventive agents demonstrated in bacteria in vitro cannot always be extrapolated to reactions occurring in intact mammalian cells.

Mutat Res, 1996 Feb 19, 350(1), 163 - 71
Plant activation and its role in environmental mutagenesis and antimutagenesis; Plewa MJ et al.; This paper reviews the use of in vitro and in vivo antimutagenicity studies that determined the role of plant peroxidases in the activation of arylamine promutagens . New information presented here suggests a model in which tobacco cell peroxidases exuded into the culture medium undergo a maturation process affecting their capacity to activate arylamine promutagens . Tobacco cell peroxidases are present in medium recovered from stationary phase cells and are associated with a fraction that sediments at 12000 x g . These peroxidases have a greater capacity to activate arylamines than do peroxidases present in the supernatant fluid . These data suggest that the plant activation of arylamines into products that are mutagenic in Salmonella typhimurium may be intimately involved in the process of lignification.

Biochemistry, 1996 Feb 13, 35(6), 1872 - 80
Allosteric regulation of tryptophan synthase: effects of pH, temperature, and alpha-subunit ligands on the equilibrium distribution of pyridoxal 5'-phosphate-L-serine intermediates; Peracchi A et al.; The equilibrium distribution of catalytic intermediates formed in the reaction of L-serine with the tryptophan synthase alpha 2 beta 2-complex from Salmonella typhimurium has been investigated by absorption and fluorescence spectroscopy as a function of pH, temperature, and alpha-subunit ligands . The novel result of this study is that the equilibrium between the two major catalytic species, the external aldimine and the alpha-aminoacrylate, is modulated by the ionization of two groups with apparent pK values of 7.8 +/- 0.3 and 10.3 +/- 0.2 . Protonation of these groups stabilizes the alpha-aminoacrylate Schiff base by an estimated 100-fold with respect to the external aldimine . Furthermore, the formation of the alpha-aminoacrylate from the external aldimine is an endothermic process . Temperature slightly affects the apparent pK values but remarkably influences the amplitude of the phase associated with the ionization of each group . At 20 degrees C, each phase accounts for nearly half of the titration . Since the isolated beta 2-dimer does not exhibit a pH-dependent distribution of intermediates, the alpha-beta-subunit interactions seem critical to the onset of this functional property of the beta-subunit . The modulation of intersubunit interactions by the alpha-subunit ligands DL-alpha-glycerol 3-phosphate and phosphate leads to significant changes in the pH-dependent distribution of intermediates . At saturating concentrations of either of these alpha-subunit ligands, the alpha-aminoacrylate Schiff base is the predominant species over a wide pH range while the apparent pK values of the groups that control the equilibrium are not significantly affected . The pH-dependent interconversion of catalytic intermediates here reported has not been previously detected because phosphate buffers have usually been employed in the studies of this enzyme . Our findings are discussed in the light of a model in which specific protein conformations are associated with the external aldimine and the alpha-aminoacrylate Schiff bases, the latter being stabilized by temperature, protons, and alpha-subunit ligands.

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