J Biotechnol, 2000 Sep 29, 83(1-2), 19 - 26 Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes; Gentschev I et al.; Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria . The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA(s)) at the C-terminus, across both membranes of gram-negative bacteria . This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane . This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope . Suitable HlyA(s)-fused antigens are secreted with high efficiency by E . coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica . The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA . This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants . After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules . This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models. Biotechniques, 2000 Sep, 29(3), 514 - 6, 518-20, 522 Infection by bacterial pathogens expressing type III secretion decreases luciferase activity: ramifications for reporter gene studies; Savkovic SD et al.; Pathogenic microbes influence gene regulation in eukaryotic hosts . Reporter gene studies can define the roles of promoter regulatory sequences . The effect of pathogenic bacteria on reporter genes has not been examined . The aim of this study was to identify which reporter genes are reliable in studies concerning host gene regulation by bacterial pathogens expressing type III secretory systems . Human intestinal epithelial cells, T84, Caco-2 and HT-29, were transfected with plasmids containing luciferase (luc), chloramphenicol acetyltransferase (CAT) or beta-galactosidase (beta-gal) as reporter genes driven by the inducible interleukin-8 (IL-8) or constitutively active simian virus 40 (SV40) promoter . Cells were infected with enteropathogenic E . coli or Salmonella typhimurium, and the reporter activity was assessed . Luc activity significantly decreased following infection, regardless of the promoter . The activity of recombinant luc was nearly ablated by incubation with either EPEC or Salmonella in a cell-free system . Activity was partially preserved by protease inhibitors, and immunoblot analysis showed a decreased amount and molecular weight of recombinant luc, suggesting protein degradation . Neither beta-gal nor CAT activity was altered by infection . Disruption of type III secretion prevented the loss of luc activity . We conclude that CAT or beta-gal, but not luc, can be used as reliable reporter genes to assess the impact of pathogenic microbes, especially those expressing type III secretion on host cell gene regulation. Folia Microbiol (Praha), 1999, 44(5), 513 - 8 Antimutagenicity of milk fermented by Enterococcus faecium; Belicova A et al.; The diethyl ether extracts isolated from unfermented milk and milk fermented by Enterococcus faecium exhibited dose-dependent inhibition of mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), nitrovin (NIT), 5-nitro-2-furylacrylic acid (NFA) and UV-irradiation on the Ames bacterial test (Salmonella typhimurium strains TA97 and TA100) and the unicellular flagellate Euglena gracilis . Overall, the fermented milk extract was the most active against UV-irradiation, less active against NIT and MNNG, and the least active against NFA on bacteria . The highest antibleaching effects were observed against MNNG . The differences between antimutagenic effects from fermented and unfermented milk extracts were determined to be statistically significant at the 0.95 CI level. J Biol Chem, 2000 Dec 22, 275(51), 40244 - 51 Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase; Bettati S et al.; Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types . The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions . The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail . The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group . Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques . Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization . Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form . The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type. J Agric Food Chem, 2000 Sep, 48(9), 4377 - 80 Suppression of furylfuramide-induced SOS response by acetophenones using Salmonella typhimurium TA1535/pSK1002 umu test; Miyazawa M et al.; The recently isolated paeonol (2-hydroxy-4-methoxyacetophenone), as one of the antimutagenic compounds from Discorea japonica, was used as a lead compound for detailed structure-activity relationship studies . Nine acetophenones (2-hydroxy-4-methoxy, 2-hydroxy-5-methoxy, 2-hydroxy-6-methoxy, 4-hydroxy-3-methoxy, o-methoxy, m-methoxy, p-methoxy, and 2,5-dimethoxyacetophenone and acetophenone) were investigated for their ability of suppression of furylfuramide-induced SOS response using Salmonella typhimurium TA1535/pSK1002 in the umu test, against the mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) . The results showed that 2-hydroxy-6-methoxyacetophenone displayed the strongest activity (EC(50) = 0.6 micromol/mL), and a hydroxyl group at C-2 is necessary feature for acetophenone derivatives to show the suppressive effects of furylfuramide-induced SOS response. Teratog Carcinog Mutagen, 2000, 20(5), 301 - 11 Assessment of the in vitro and in vivo genotoxicity of Thalomid (thalidomide); Teo S et al.; Thalomid is the FDA-approved commercial formulation of thalidomide currently used in the US to treat erythema nodosum leprosum, a complication of leprosy . The genotoxicity of Thalomid thalidomide was assessed in the Ames reverse mutation, AS52/XPRT mammalian cell forward gene mutation, and mouse bone marrow micronucleus assays . The Ames and AS52 assays were performed with and without S9 . In the Ames, Salmonella typhimurium strains TA1535, 1537, 98, 100, and 102 and Escherichia coli strain WP2 uvrA were used . Assays were performed by using plate incorporation and liquid pre-incubation systems at thalidomide doses of 50-10,000 microg/plate . In the AS52 assay, Chinese hamster ovary cells were plated with fortified Ham's F12 medium and incubated overnight . The medium was then incubated with 1-1000 microg/ml thalidomide . After a series of aspirations, washings, reconstitutions, and incubations, mutant AS52 cells were fixed and stained . Colonies were then counted and the relative survival frequencies compared to negative controls . In the mouse micronucleus assay, Crl:CD-1 albino mice were dosed with 500, 2,500, and 5,000 mg/kg thalidomide and sacrificed over 72 h . Femurs were flushed with fetal bovine serum and the suspensions centrifuged . The supernatant was aspirated and the cell pellet resuspended and stained . Polychromatic erythrocytes were scored for micronucleated polychromatic and normochromatic erythrocytes . Thalidomide did not increase revertant frequencies in all bacterial strains . It also did not produce any significant increase in the average mutant frequencies of AS52 cells and mouse micronucleated polychromatic erythrocytes . We conclude that Celgene's Thalomid thalidomide is non-genotoxic. Clin Infect Dis, 2000 Aug, 31(2), 488 - 92 Epub 2000 Sep 05. Risk factors for the occurrence of sporadic Salmonella enterica serotype typhimurium infections in children in France: a national case-control study; Delarocque-Astagneau E et al.; To determine risk factors for the occurrence of sporadic Salmonella typhimurium infections among children in France, we conducted a matched case-control study . Cases were identified between 15 June and 30 September 1996 . We interviewed 101 pairs of case patients and control subjects, matched for age and place of residence . The risk of illness was greater for children who ate undercooked ground beef than for those who did not (odds ratio {OR}, 5.0; 95% confidence interval {CI}, 1.9-13.1) . Case patients were more likely than control subjects to have taken antibiotics during the month before onset of disease (OR, 2.2; 95% CI, 1.0-4.9) . Case patients <5 years of age were more likely to have been in contact with a household member with diarrhea 3-10 days before onset (P=.05) . Consumption of undercooked ground beef is a risk factor for the sporadic occurrence of S . typhimurium infection among children, and antibiotics may facilitate the occurrence of illness . The possibility of person-to-person transmission among young children needs to be considered. Mutat Res, 2000 Oct 10, 470(1), 71 - 6 Sesamol exhibits antimutagenic activity against oxygen species mediated mutagenicity; Kaur IP et al.; The effects of sesamol, a phenolic compound responsible for the high resistance of sesame oil to oxidative deterioration as compared with other vegetable oils, have been investigated after mutagen treatment in various strains of Salmonella typhimurium . Sesamol was shown to exhibit strong antimutagenic effects in the Ames tester strains TA100 and TA102 . The TA102 strain has been shown to be highly sensitive to reactive oxygen species . Mutagenicity was induced by the generation of oxygen radicals by tert-butylhydroperoxide (t-BOOH) or hydrogen peroxide (H(2)O(2)); therefore, the antimutagenic property of sesamol was attributed to its antioxidant properties . The superoxide and hydroxyl radical scavenging capabilities have further been elucidated using in vitro test systems . It was further shown to have a desmutagenic effect on t-BOOH-induced mutagenesis in TA102 strain . Sesamol also inhibited the mutagenicity of sodium azide (Na-azide) in TA100 tester strain while it had no effect on nitroquinoline-N-oxide (NQNO)-induced mutagenesis in TA98 strain of Salmonella typhimurium . Since active oxygen species are involved in multiple stage processes of carcinogenicity, this compound may also exhibit anticarcinogenic properties. J Immunol Methods, 2000 Aug 28, 242(1-2), 133 - 43 New technology for regiospecific covalent coupling of polysaccharide antigens in ELISA for serological detection; Jauho ES et al.; In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA) . Lipopolysaccharides were cleaved by mild acid hydrolysis into the lipid A part and the polysaccharide part . The polysaccharide was conjugated regiospecifically to a photochemically active compound, anthraquinone, resulting in a polysaccharide-anthraquinone conjugate . Anthraquinones forms active radicals when exposed to soft UV irradiation (350 nm) permitting the formation of stable covalent bonds to polymers e.g . microtiter plates . By this technique the polysaccharides are bound through the anthraquinone part of the polysaccharide-anthraquinone conjugates to the microtiter plates . This minimizes denaturation of O-antigen epitopes during binding to the microtiter plates and avoids cross-reactivity due to conserved domains in the lipid A . Furthermore, the covalent binding of the polysaccharide antigens are compatible with harsh assay conditions, such as extensive washing procedures and buffers with high salt concentrations with no risk of antigen leakage . Here we describe the use of this technique for the immobilization of lipopolysaccharide derived polysaccharides from Salmonella Typhimurium and Salmonella Choleraesuis lipopolysaccharides, representing the O-antigens 1, 4, 5, 6, 7, and 12 . The functional polysaccharide surface gave similar ELISA results to plates coated passively with the corresponding unmodified lipopolysaccharide antigens . The plates were highly reproducible, showed very low inter- and intra-plate variation and were stable at room temperature for more than 8 months. Proc Natl Acad Sci U S A, 2000 Sep 26, 97(20), 11008 - 13 Contribution of Salmonella typhimurium type III secretion components to needle complex formation; Kimbrough TG et al.; The prgHIJK operon encodes components of the Salmonella typhimurium pathogenicity island 1 type III secretion system (TTSS) . Previously, prgH and prgK were shown to be required for formation of the supramolecular type III secretion needle complex (NC) {Kubori, T., et al . (1998) Science 280, 602-605} . This work indicates that all prg operon genes are required for NC formation . PrgH multimerizes into a distinct tetrameric-shaped structure that may be an early intermediate of NC assembly and may provide the structural foundation required for PrgK oligomerization . PrgH and PrgK, in the absence of other TTSS components, oligomerize into ring-shaped structures identical in appearance and size to the base of the NC, indicating that they are likely the major inner membrane structural components required for secretion . PrgI and PrgJ cofractionate with the NC and are secreted into the culture supernatant . NC from prgI and prgJ mutants have an identical morphology to the envelope-spanning (basal body) NC components, but are missing the external needle, indicating that PrgI and PrgJ are required for full NC assembly and are likely components of the external needle . Therefore, PrgI and PrgJ are secreted through the NC basal body, composed in part of PrgH/K and InvG/H rings, to participate in assembly of the more distal components of the NC. J Food Prot, 2000 Sep, 63(9), 1268 - 72 Inhibition of Salmonella on poultry skin using protein- and polysaccharide-based films containing a nisin formulation; Natrajan N et al.; The objective of this study was to examine the use of protein- arid polysaccharide-based films containing bacteriocin formulations for inhibiting salmonellae on fresh broiler skin . The lethality of the films containing a nisin-based formulation was determined against Salmonella Typhimurium-contaminated broiler drumstick skin samples coated with the film . In the first study, varying concentrations of nisin (0, 100, 300, and 500 microg/ml) plus 3% citric acid, 5.0 mM EDTA, and 0.5% Tween 80 were incorporated into 0.5% calcium alginate films at a 20% level (wt/wt) and then applied to Salmonella TyphimuriumNAr-contaminated skin samples (log10 5.0) at a 1:2 weight ratio (film:skin) . Salmonella TyphimuriumNAr skin population reductions ranged from 1.98 to 3.01 log cycles after a 72-h exposure at 4 degrees C . In comparison to the 0- and 100-microg/ml nisin concentrations, significantly greater population reductions were achieved at nisin concentrations of 300 and 500 microg/ml . In related studies, the 500-degreesg/ml nisin formulation was incorporated into 0.75 and 1.25% agar gels and applied to contaminated broiler drumstick skin samples (log10 7.0) . Salmonella TyphimuriumNAr skin population reductions following a 96-h exposure at 4 degrees C were 1.8-(1.25% agar gel) and 4.6-log cycles (0.75% agar gel) . These results demonstrated that the inclusion of nisin-based treatments into either calcium alginate or agar gels that were subsequently applied to contaminated broiler drumstick skin yielded significant Salmonella TyphimuriumNAr population reductions ranging between 1.8 to 4.6 log cycles after 72 to 96 h of exposure at 4 degrees C . The level of kill was affected by film type and gel concentration (i.e., gel network formation), exposure time, and nisin concentration. FEMS Microbiol Lett, 2000 Sep 1, 190(1), 109 - 14 Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene; So JS et al.; A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced . One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da . Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis . A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria . The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide . The cloned B . japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789 . Transformation of this mutant with the B . japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin . An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene. Med Parazitol (Mosk), 2000 Jul-Sep, (3), 32 - 5 {The reciprocal influence of Escherichia coli and Salmonella bacteria in a mixed infection of Ornithodoros papillipes Birula ticks.}; Podboronov VM et al.; The paper presents and analyzes the results of experiments on the Escherichia coli-Salmonella relationships in combined infection of Ornithodoros papillipes ticks . The findings have led to the following conclusions . The body of hungry adult O . papillipes ticks can retain the pathogen of Salmonella infection for a month . This model object is also favourable for avirulent E . coli strain persistent in the body's cavity both alone and in combination with Salmonella typhimurium strain in a 1:1 ratio . Binary carriage in hungry Ornithodoros papillipes ticks revealed that the ticks associated with Salmonella typhimurium and E . coli 083 mcl + lyz No 225, microcine and lysozyme producers, are freed of pathogenic bacteria during 24 hours . The initial bacteria E . coli 083 mcl also suppress salmonellae; however, their elimination occurs much later--in 5 days . Calculating the correlation between the pairs (S . typhi and E . coli) has revealed a linear functional relationship of the rate of E . coli growth to S . typhimurium suppression. Yi Chuan Xue Bao, 2000, 27(5), 462 - 7 {Regulation of purine biosynthetic genes expression in Salmonella typhimurium}; Long HX et al.; To study binding funtion of 4 consensus bases in 16 bp PUR box with purR protein, the directed site mutation for each was carried out, which mutate from C to G, A to G, A to G, T to C, respectively . Gel retardation showed that the PUR box carrying a reserved mutation could not bind with purR protein . It suggested that all these consensus base pairs are necessary to hold the normal binding function of PUR box with purR protein. J Biol Chem, 2000 Dec 1, 275(48), 37718 - 24 Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD; Steele-Mortimer O et al.; The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival . Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308) . We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells . A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation . In HeLa cells, wild type S . typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity . In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles . Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype . This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB . Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form . SigD is also the first bacterial effector to be identified as an activator of Akt. Arch Pharm Res, 2000 Aug, 23(4), 407 - 12 Identification and characterization of nitric oxide synthase in Salmonella typhimurium; Choi DW et al.; The presence of the nitric oxide synthase (NOS) enzyme from Salmonella typhimurium (S . typhimurium) was identified by measuring radiolabeled L-{3H}citrulline and NO, and Western blot analysis . NOS was partially purified by both Mono Q ion exchange and Superose 12HR size exclusion column chromatography, sequentially . The molecular weight of NOS was estimated to be 93.3 kDa by Western blot analysis . The enzyme showed a significant dependency on the typical NOS cofactors; an apparent Km for L-arginine of 34.7 mM and maximum activity between 37 degrees C and 43 degrees C . The activity was inhibited by NOS inhibitors such as aminoguanidine and N(G),N(G)-dimethyl-L-arginine . Taken together, partially purified NOS in S . typhimurium is assumed to be a different isoform of mammalian NOSs. Mol Gen Mikrobiol Virusol, 2000, (3), 21 - 6 {Ultrastructural organization of Salmonella typhimurium cells during long-term starvation and transfer to an unculturable state}; Didenko LV et al.; Electron microscopic and immunocytochemical studies of Salmonella typhimurium culture were carried out under conditions of cell transfer into an unculturable state induced by carbon, phosphorus, and nitrogen starvation . Morphological variants of bacterial cells were detected in the course of cell culturing under conditions of starvation . Electron microscopy showed that O-antigen was retained in salmonella after long starvation and transfer into an unculturable state. Trans R Soc Trop Med Hyg, 2000 May-Jun, 94(3), 310 - 4 Clinical presentation of non-typhoidal Salmonella bacteraemia in Malawian children; Graham SM et al.; We report the clinical presentation and outcome of 299 Malawian children with non-typhoidal Salmonella (NTS) bacteraemia and no evidence of focal sepsis, admitted to Queen Elizabeth Central Hospital (QECH), Blantyre, over a 26-month period (February 1996-April 1998) . A peak incidence during the rainy season was noted . Salmonella typhimurium (79%) and S . enteritidis (13%) were the commonest isolates . For children aged > 6 months, NTS bacteraemia was significantly associated with malarial parasitaemia (RR 1.5 {1.2, 2.2}, P < 0.01) and with severe anaemia (RR 7.2 {3.4, 15.3}, P < 0.0001), when compared to other common pathogens causing childhood bacteraemia . Clinical overlap with malaria and anaemia, and the presence of malarial parasitaemia on admission, may delay diagnosis . NTS bacteraemia was commonly diagnosed following blood transfusion . Resistance in vitro to ampicillin (79%), co-trimoxazole (72%) and gentamicin (55%) was very common, and was rare to chloramphenicol (0.3%) which is the antibiotic of choice for NTS sepsis at QECH . Overall mortality was high (23%) . Young age and clinical HIV infection were risk factors for mortality . Recurrences of NTS bacteraemia following antibiotic therapy were common among children with clinical HIV infection. Gene, 2000 Aug 22, 254(1-2), 209 - 17 Synthesis of a bacteriophage MB78 late protein by novel ribosomal frameshifting; Kolla V et al.; MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression . A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level . Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting . In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa . A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame . Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein . Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium. Nat Med, 2000 Sep, 6(9), 1048 - 51 Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens; Matkowskyj KA et al.; Galanin is widely distributed in enteric nerve terminals lining the gastrointestinal tract . We previously showed that pathogenic Escherichia coli, but not normal commensal organisms, increase galanin-1 receptor expression by epithelial cells lining the colon (i.e., colonocytes) . When present, galanin-1 receptor activation by ligand causes colonocyte Cl- secretion . We herein demonstrate that disparate pathogens including Salmonella typhimurium and Shigella flexerii also increase colonocyte galanin-1 receptor expression, whose activation is responsible for a principal component of the increased colonic fluid secretion observed . Although eliminating the GAL1R gene by homologous recombination does not alter basal colonic fluid secretion, removal of one or both alleles completely attenuates the increase in fluid secretion due to infection with enteric pathogens . Galanin-1 receptor up-regulation therefore represents a novel mechanism accounting for the increased colonic fluid secretion observed in infectious diarrhea due to several different pathogens. Mol Microbiol, 2000 Sep, 37(5), 1220 - 31 Completion of the hook-basal body complex of the Salmonella typhimurium flagellum is coupled to FlgM secretion and fliC transcription; Karlinsey JE et al.; The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon . The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon . Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present . No flagella were visible under the electron microscope when cells were grown without inducer . The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated . After addition of tetracycline, the class 1 flhDC operon was transcribed immediately . Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction . At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated . Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium . This also coincides with the completion of hook-basal body structures . Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time . At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour . Multiple flagella assemble and grow on individual cells after induction of the master operon . These results confirm that the flagellar regulatory hierarchy of S . typhimurium is temporally regulated after induction . Both FlgM secretion and class 3 gene expression occur upon completion of the hook-basal body structure. Mol Microbiol, 2000 Sep, 37(5), 1133 - 45 The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection; Scherer CA et al.; Salmonella species translocate effector proteins into the host cell cytoplasm using a type III secretion system (TTSS) . The translocation machinery probably contacts the eukaryotic cell plasma membrane to effect protein transfer . Data presented here demonstrate that both SspB and SspC, components of the translocation apparatus, are inserted into the epithelial cell plasma membrane 15 min after Salmonella typhimurium infection . In addition, a yeast two-hybrid interaction between SspC and an eukaryotic intermediate filament protein was identified . Three individual carboxyl-terminal point mutations within SspC that disrupt the yeast two-hybrid interaction were isolated . Strains expressing the mutant SspC alleles were defective for invasion, translocation of effector molecules and membrane localization of SspC . These data indicate that insertion of SspC into the plasma membrane of target cells is required for invasion and effector molecule translocation and that the carboxyl terminus of SspC is essential for these functions. Mutagenesis, 2000 Sep, 15(5), 391 - 7 Mutagenicity of diesel exhaust particles from two fossil and two plant oil fuels; Bunger J et al.; Particulate matter of diesel engine exhaust from four different fuels was studied for content of polynuclear aromatic compounds and mutagenic effects . Two so-called biodiesel fuels, rapeseed oil methylesters (RME) and soybean oil methylesters (SME), were compared directly with two fossil diesel fuels with the normal (DF) and a low sulfur content (LS-DF) . Diesel exhaust particles were sampled on filters from the diluted and cooled exhaust of a test engine at five different speeds and loads . Filters were weighed for total particulate matter, Soxhlet extracted with dichloromethane and the content of insoluble material determined . The soluble organic fraction was analysed for polynuclear aromatic compounds . Mutagenicity was determined using the Salmonella typhimurium/mammalian microsome assay with strains TA98 and TA100 . Compared with DF, the exhaust particles of LS-DF, RME and SME contained less insoluble material, which consisted mainly of the carbon cores of diesel exhaust particles . The concentrations of individual polynuclear aromatic compounds varied widely among the different exhaust extracts, but total concentrations of the compounds were approximately double for DF and SME compared with LS-DF and RME . In TA98 significant increases in mutation rates were obtained for the soluble organic fractions of all fuels for engines running at full speed (load modes A and D), but for DF revertants were 2- to 10-fold more frequent as compared with LS-DF, RME and SME . Revertant frequencies for DF and partly for LS-DF were also elevated in TA100, while RME and SME gave no significant increase in mutations . The results indicate that diesel exhaust particles from RME, SME and LS-DF contain less black carbon and total polynuclear aromatic compounds and are significantly less mutagenic in comparison with DF . A high sulfur content of the fuel and high engine speeds (rated power) and loads are associated with an increase in mutagenicity of diesel exhaust particles. J Theor Biol, 2000 Sep 21, 206(2), 307 - 11 A comparative study of mutations in Escherichia coli and Salmonella typhimurium shows that codon conservation is strongly correlated with codon usage; Alff-Steinberger C; Escherichia coli and Salmonella typhimurium are closely related species of enteric bacteria, having diverged from 120 to 160 million years ago, according to the estimate of Ochman & Wilson (1987 . J . Mol . Evol.26, 74-86) . In order to study base substitution mutations in the genomes of these bacteria, we have compared pairs of genes for the same product in the two species, and have selected a sample in which the protein length is the same in both E . coli and S . typhimurium . From the alignment of these gene pairs, we observe that frequently used codons are more conserved than infrequently used codons, i.e., the apparent mutation rate is higher for rare codons than for popular codons . Proc Soc Exp Biol Med, 2000 Sep, 224(4), 231 - 9 Host immune response to intracellular bacteria: A role for MHC-linked class-Ib antigen-presenting molecules; Soloski MJ et al.; MHC-linked class-Ib molecules are a subfamily of class-I molecules that display limited genetic polymorphism . At one time these molecules were considered to have an enigmatic function . However, recent studies have shown that MHC-linked class-Ib molecules can function as antigen presentation structures that bind bacteria-derived epitopes for recognition by CD8+ effector T cells . This role for class-Ib molecules has been demonstrated across broad classes of intracellular bacteria including Listeria moncytogenes, Salmonella typhimurium, and Mycobacterium tuberculosis . Additionally, evidence is emerging that MHC-linked class-Ib molecules also serve an integral role as recognition elements for NK cells as well as several TCR alpha/beta and TCR gamma/delta T-cell subsets . Thus, MHC-linked class-Ib molecules contribute to the host immune response by serving as antigen presentation molecules and recognition ligands in both the innate and adaptive immune response to infection . In this review, we will attempt to summarize the work that supports a role for MHC-linked class-Ib molecules in the host response to infection with intracellular bacteria. Cell Mol Life Sci, 2000 Jul, 57(7), 1033 - 49 The invasion-associated type III secretion system of Salmonella typhimurium: common and unique features; Sukhan A; Several bacterial pathogens make use of a specialized protein secretion system to inject effector proteins into host cells . This system, commonly referred to as type III secretion, is always associated with phenotypes related to intimate interactions between the pathogen and its respective host cells . The enteric pathogen Salmonella typhimurium utilizes a type III secretion system to invade nonphagocytic intestinal epithelial cells . Whereas the invasion-associated type III system of S . typhimurium has evolved to perform a specific function, many of the components of this system are conserved among the type III systems of other bacterial pathogens . This review will discuss the common and unique features of the S . typhimurium system in relation to the type III systems of other human pathogens . Topics discussed include the phenotypes associated with various type III systems, the genetic loci encoding these systems, the components of the type III secretion apparatus, the effector proteins and the mechanisms by which they enter host cells as well as the mechanisms used to regulate the expression of type III systems. Drug Chem Toxicol, 2000 Aug, 23(3), 477 - 84 In vitro inhibition of carcinogen-induced mutagenicity by Cassia occidentalis and Emblica officinalis; Sharma N et al.; Aqueous extracts of Cassia occidentalis Linn . (Leguminoceae) and Emblica officinalis Gaertn . (Euphorbiaceae) were screened for effectiveness in inhibiting mutagenicity of aflatoxin B1 (AFB1) and benzo{a}pyrene (B{a}P) in the Ames test . Antimutagenicity was evaluated using Salmonella typhimurium strains TA 98 and TA 100 . In the assay, metabolic activation of AFB1 (0.5 microg/plate) and B{a}P (1 microg/plate) was mediated by rat liver S9 preparation . Although both plants inhibited mutagenicity, E . officinalis had more inhibitory effect than C . occidentalis . Their action is possibly mediated through interactions with microsomal activating enzymes . Their inhibitory action on chromosomal aberrations together with present results suggest that these plants have potent antimutagenic and anticarcinogenic activities against mutagens requiring metabolic activation. J Microbiol Methods, 2000 Aug, 41(3), 195 - 9 Efficient amplification of multiple transposon-flanking sequences; Kwon YM et al.; Transposon mutagenesis is a very useful tool for gene identification in bacteria . Once the transposon mutants of interest are isolated, it is often necessary to identify the sequences that flank the transposon insertions . We devised an efficient method for specific amplification of transposon-flanking sequences that requires the sequence information of only transposon-specific sequences . The basic steps for this method consists of (1) digestion with a restriction enzyme, (2) ligation with a Y-shaped linker and (3) polymerase chain reaction amplification using a transposon-specific primer and a primer specific to the Y-shaped linker . The feasibility of this method was demonstrated with mini-Tn5 mutants of Salmonella typhimurium . We also found that this method can be used for simultaneous amplification of multiple transposon-flanking sequences. J Agric Food Chem, 2000 Aug, 48(8), 3256 - 66 Antimutagens in gaiyou (Artemisia argyi levl . et vant.); Nakasugi T et al.; Antimutagens from gaiyou (Artemisia argyi Levl . et Vant., Compositae) were examined . The methanol extract prepared from aerial parts of this plant strongly reduced the mutagenicity of 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), when Salmonella typhimurium TA98 was used in the presence of the rat liver microsomal fraction . The antimutagens were purified chromatographically while monitoring the antimutagenic activity against Trp-P-2 with a modified Ames test employing a plate method . This purification resulted in the isolation of four strong antimutagens, 5,7-dihydroxy-6,3',4'-trimethoxyflavone (eupatilin), 5, 7,4'-trihydroxy-6,3'-dimethoxyflavone (jaceosidin), 5,7, 4'-trihydroxyflavone (apigenin) and 5,7, 4'-trihydroxy-3'-methoxyflavone (chrysoeriol) from the methanol extract . These antimutagenic flavones exhibited strong antimutagenic activity against not only Trp-P-2 but also against other heterocyclic amines, such as 3-amino-1,4-dimethyl-5H-pyrido{4, 3-b}indole (Trp-P-1), 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), 2-amino-3, 8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) and 2-amino-3-methyl-9H-pyrido{2,3-b}indole (MeA(alpha)C) in S . typhimurium TA98 . In contrast, they did not exhibit antimutagenic activity against benzo{a}pyrene (B{a}P), 4-nitroquinoline-1-oxide (4-NQO), 2-aminofluorene (2-AF), 2-nitrofluorene (2-NF) or furylfuramide (AF-2) in S . typhimurium TA98, or B{a}P, 4-NQO, 2-NF, AF-2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium azide (SA) in Salmonella typhimurium TA100, whereas they decreased the mutagenicity caused by aflatoxin B(1) (AFB(1)) and 2-aminoanthracene (2-AA) in both of these tester strains . Regarding the structure-activity relationship, the tested flavones had distinct differences in the intensities of their antimutagenic activities according to the differences of their substitution patterns . Namely, the intensity of antimutagenic activities against Trp-P-2 decreased in the order of: 5,7,3',4'-tetrasubstituted flavones (IC(50): <0.1 mmol/plate), 5,7,4'-trisubstituted flavones (IC(50): 0.120-0.260 mmol/plate), 5,6,7,3',4'-pentasubstituted flavones (IC(50): 0.440-0 . 772 mmol/plate) . The four isolated flavones were also studied regarding their antimutagenic mechanisms with preincubation methods of the modified Ames test and emission spectroscopic analysis . The results suggested that all isolated flavones were desmutagens which directly inactivated Trp-P-2 or inhibited its metabolic activation. Am J Vet Res, 2000 Aug, 61(8), 992 - 6 Pharmacokinetics and tissue distribution of amoxicillin in healthy and Salmonella Typhimurium-inoculated pigs; Agerso H et al.; OBJECTIVE: To determine pharmacokinetics and tissue distribution of amoxicillin in healthy and Salmonella Typhimurium-inoculated pigs . ANIMALS: 12 healthy pigs and 12 S Typhimurium-inoculated pigs . PROCEDURE: Concentration of amoxicillin in tissue was measured by use of high-performance liquid chromatography 4, 8, 12, and 24 hours after IM administration . Pharmacokinetic values of amoxicillin in plasma were assessed by use of a 1-compartment model with first-order absorption . RESULTS: Inoculation caused diarrhea and increased rectal temperature and WBC count . Absorption half-life was shorter in inoculated pigs (0.26 hours) than in healthy pigs (0.71 hours), and inoculated pigs had longer elimination half-life . Distribution ratios in healthy pigs ranged from 0.31 to 0.56 and in inoculated pigs ranged from 0.14 to 0.48 . Ratios for distribution to intestinal mucosa ranged from 0.34 to 1.16 in healthy pigs and from 0.22 to 0.36 in inoculated pigs . CONCLUSIONS AND CLINICAL RELEVANCE: Salmonella Typhimurium inoculation altered absorption of amoxicillin from the injection site and prolonged elimination half-life . However, distribution of amoxicillin to intestinal tract tissue was only affected to a minor degree. Mutat Res, 2000 Aug 21, 469(1), 71 - 82 Genotoxicity of urban air pollutants in the Czech Republic . Part I . Bacterial mutagenic potencies of organic compounds adsorbed on PM10 particulates; Cerna M et al.; As part of a long-term program to investigate the impact of air pollution on the health of a population in a polluted region in Northern Bohemia, mutagenicity of extractable organic matter (EOM) from air particles PM10 was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay . The air samples were collected in both the polluted and the control districts during the summers and winters of 1993-1994 . In the polluted district, the collection was repeated during the winter of 1996-1997 . The crude extracts from filters pooled according to the locality and the season were fractionated by acid-base partitioning into acid, base, and neutral fractions . The neutral fractions were further fractionated by silica gel column chromatography into five subfractions . The induction of revertants with the crude extracts was higher in winter samples than in summer samples . Both indirect-acting and direct-acting mutagenicity were observed . The indirect mutagenic potency of aromatic subfractions containing polycyclic aromatic hydrocarbons (PAHs) was generally low . The mutagenic potency detected with TA98 was more distinct only in the winter sample 1993-1994 from the polluted area, where the aromatic subfraction accounted for 23% of total mutagenicity . In both strains, the highest direct-acting mutagenicity was found in slightly polar fractions containing nitro-PAHs . The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98 . No substantial locational- or time-related variances in the mutagenic potencies of EOM, or in the spectrum of chemical components identified in individual fractions were found . The polluted district, in comparison to the control district, was found to have higher amounts of EOM, carcinogenic PAHs and mutagenicity of air particles (rev/m(3)) . The fractionating process, combined with the bacterial mutagenicity test, confirmed that nitro-derivatives are the most important contributors to the bacterial mutagenicity of air particles . However, this study did not fulfill the expectancy to bring substantially new, clear-cut information on the composition and the biological activity of air pollution in both districts. J Appl Microbiol, 2000 Jul, 89(1), 90 - 9 Changes in viability and macromolecular content of long-term batch cultures of Salmonella typhimurium measured by flow cytometry; Turner K et al.; Exposure of many Gram-negative bacteria to prolonged starvation induces alternative programmes of gene expression, along with a transition into a dormant condition sometimes referred to as a viable non-culturable (VBNC) state . Knowledge of how pathogenic species respond to nutrient limitation is therefore important for their detection and dissemination . This study used flow cytometry, coupled with fluorescent dyes for viability and macromolecular content, to study the responses of the pathogen Salmonella typhimurium to prolonged batch culture . Statistical analysis of the flow cytometric data, together with total and culturable cell counts, failed to demonstrate a VBNC state in this pathogen, contrary to reports from other workers . Analysis of rRNA and protein content identified a small proportion of cells in 110 day-old cultures that represented an active sub-population . This observation may provide an explanation for the long-term survival properties of this organism during prolonged exposure to nutrient limitation, as well as the high degree of heterogeneity observed in labelled cells. J Appl Microbiol, 2000 Jul, 89(1), 63 - 9 Expression of the hilA Salmonella typhimurium gene in a poultry Salm . enteritidis isolate in response to lactate and nutrients; Durant JA et al.; Pathogens express virulence genes in response to the combination of environmental conditions present in the host environment . The crop is the first gastrointestinal environment encountered in birds . However, feed withdrawal alters the crop environment resulting in an increased pH, and decreased concentrations of lactate, glucose and amino acids compared with unmoulted birds . Salmonella enteritidis infections increase significantly in hens that have been force moulted by feed withdrawal . The present study examined the effects of pH, carbohydrate sources, amino acids and lactate on expression of Salm . enteritidis virulence by measuring expression of hilA . The hilA gene encodes a transcriptional activator that regulates expression of Salmonella virulence genes in response to environmental stimuli . HilA expression was determined using a poultry isolate of Salm . enteritidis carrying a hilA-lacZY transcriptional fusion from Salm . typhimurium . The media used were Luria Bertani (LB) broth and LB broth diluted 1:5 (DLB) . The expression of hilA was 2.9-fold higher in DLB broth compared with LB broth which suggested that there is a nutritional component to the regulation of hilA . Addition of 0.2% glucose, fructose or mannose to LB and DLB reduced hilA expression 1.5 to twofold . Addition of 0.2% Casaminoacids, arabinose, fucose, or lactose had little effect on hilA expression . Lactate (25 and 50 mmmol 1-1) reduced hilA expression at pH 6, 5 and 4, with the lowest expression occurring at pH 4 . Based on these results it appears that the composition of the crop lumen could potentially influence Salm . enteritidis virulence expression. J Food Prot, 2000 Aug, 63(8), 1038 - 42 Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella typhimurium DT104 in rumen contents in vitro; Anderson RC et al.; Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter . Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion . Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E . coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes . In support of this hypothesis, we found that concentrations of E . coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate . In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml) . Thus, chlorate was bactericidal to E . coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria . The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6). Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10225 - 30 Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system; Kubori T et al.; Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells . These proteins stimulate or interfere with host cellular functions for the pathogen's benefit . The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells . Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex . This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface . We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base . We show that the length of the needle segment is determined by the type III secretion associated protein InvJ . We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex . PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants . We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells. Cent Eur J Public Health, 2000 Jul, 8 Suppl, 66 - 7 Results of the general toxicity and genetic studies of an insecticide intermediate; Beres E et al.; Methyl-chrysanthemate is one of the intermediates of pyrethroid type insecticides . The acute toxicity of the test item was investigated in rats after single oral, dermal and inhalation applications . The irritation effect was determined by Draize method . Buehler method was applied to evaluate the sensitization potential of the test item . The mutagenic effect was assessed on Salmonella typhimurium strains . Furthermore metaphase chromosome aberration assay was performed on CHO cell line to check the structural chromosome aberrations. J Biol Chem, 2000 Oct 27, 275(43), 33969 - 73 Characterization of luminal paneth cell alpha-defensins in mouse small intestine . Attenuated antimicrobial activities of peptides with truncated amino termini; Ouellette AJ et al.; Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins . Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules . Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography . Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms . The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues . In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen . In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active . The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E . coli, and S . typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity . Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion. Microbiol Immunol, 2000, 44(6), 447 - 54 Constitutively expressed phoP inhibits mouse-virulence of Salmonella typhimurium in an Spv-dependent manner; Matsui H et al.; In Salmonella typhimurium, the transcription of several virulence genes including spvB is regulated by the PhoP/PhoQ regulatory system . To further examine the relationship between the PhoP/PhoQ and Spv systems for virulence in mice, we examined a non-polar phoP mutation combined with different virulence plasmid genotypes for effects on virulence of S . typhimurium in the mouse model . PhoP-/Spv+ and PhoP-/Spv- mutants were not detectably recovered from the spleens of subcutaneously or orally inoculated mice . The phoP gene constitutively expressed from the lacZ promoter of a low copy number vector (phoP(C)) only partially complemented the non-polar phoP mutation for mouse-virulence in both the Spv+ and Spv- backgrounds; both PhoP(C) strains exhibited virulence equal only to a PhoP+/Spv- strain . Interestingly, in a PhoP+ background, the phoP(C) gene reduced splenic infection of the Spv+ but not Spv- salmonellae after subcutaneous or oral inoculation compared with the PhoP+ parents . Additionally, the phoP(C) gene in an Spv+ background reduced the net growth of salmonellae in macrophages in vitro; phoP(C) in an Spv- background was without effect . These data suggest that the constitutive expression of the phoP gene attenuates the virulence of S . typhimurium in mice in an Spv-dependent manner. J Mol Microbiol Biotechnol, 2000 Apr, 2(2), 245 - 54 Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX; Moreno M et al.; The alternate sigma factor sigmaS plays an important role in the survival of Salmonella typhimurium following sudden encounters with a variety of stress conditions . The level of sigmaS is very low in rapidly growing cells but dramatically increases as those cells encounter environmental stress or enter into stationary phase . This increase is due in large measure to the stabilization of sigmaS protein against degradation by the ClpXP protease . The MviA protein, also known as RssB or SprE in Escherichia coli, is a putative member of a two component signal transduction system that plays a central role in facilitating sigmaS degradation by ClpXP . In contrast to most two-component systems, MviA does not appear to regulate gene expression but is believed to interact directly with sigmaS and somehow facilitate degradation . We now provide evidence that MviA(RssB) directly interacts both with sigmaS and ClpX in vivo, presumably enabling presentation of sigmaS to the ClpP protease . Interactions were demonstrated using a bacterial two-hybrid system in which sigmaS, MviA, and ClpX were fused to separate moieties of Bordetella pertussis CyaA (adenylate cyclase) . Paired hybrid plasmids containing Cya'-MviA/RpoS-'Cya or Cya'-MviA/ClpX-'Cya successfully reconstituted adenylate cyclase activity in both S . typhimurium and E . coli . However, no direct interactions were detected between ClpX and RpoS . A second series of experiments has indicated that the interaction between MviA and sigmaS requires the N-terminus but not the C-terminus of MviA . Cellular levels of MviA appear to be very low in the cell based on lacZ fusion, Western blot and Northern blot analyses suggesting a catalytic role for MviA in sigmaS degradation . Mutagenesis of MviA residue D58, a canonical residue subject to phosphorylation in many two-component systems, decreased the ability of MviA to facilitate sigmaS turnover in vivo confirming that phosphorylation of MviA increases MviA activity. Mol Microbiol, 2000 Aug, 37(3), 583 - 94 Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome; El Hanafi D et al.; The notion that transcription can generate supercoils in the DNA template largely stems from work with small circular plasmids . In the present work, we tested this model in the bacterial chromosome using a supercoiling-sensitive promoter as a functional sensor of superhelicity changes . The leu-500 promoter of Salmonella typhimurium is a mutant and inactive variant of the leucine operon promoter that regains activity if negative DNA supercoiling rises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants) . Activation of the leu-500 promoter was analysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon . Some insertions inhibited leu-500 promoter activation in the absence of inducer . This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the mechanism responsible for activation . Superimposed on these effects, transcription of the inserted gene stimulated or inhibited leu-500 promoter activity depending on whether this gene was oriented divergently from the leu operon or in the same direction respectively . Interestingly, transcription-mediated inhibition of leu-500 promoter was observed with inserts as far as 5 kb from the leu operon, and it could be relieved by the introduction of a strong gyrase site between the inserted element and the leu-500 promoter . These results are consistent with the idea that transcriptionally generated positive and negative supercoils can diffuse along chromosomal DNA and, depending on their topological sign, elicit opposite responses from the leu-500 promoter. Mol Microbiol, 2000 Aug, 37(3), 515 - 27 The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence; Black DS et al.; A variety of pathogenic bacteria use type III secretion pathways to translocate virulence proteins into host eukaryotic cells . YopE is an important virulence factor that is translocated into mammalian cells via a plasmid-encoded type III system in Yersinia spp . YopE action in mammalian cells promotes the disruption of actin filaments, cell rounding and blockage of phagocytosis . It was reported recently that two proteins with sequence similarity to YopE, SptP of Salmonella typhimurium and ExoS of Pseudomonas aeruginosa, function as GTPase-activating proteins (GAPs) for Rho GTPases . YopE contains an 'arginine finger' motif that is present in SptP, ExoS and other Rho GAPs and is essential for catalysis by this class of proteins . We show here that a GST-YopE fusion protein stimulated in vitro GTP hydrolysis by the Rho family members Cdc42, RhoA and Rac1, but not by Ras . Conversion of the essential arginine in the arginine finger motif to alanine (R144A) eliminated the in vitro GAP activity of GST-YopE . Infection assays carried out with a Yersinia pseudotuberculosis strain producing YopER144A demonstrated that GAP function was essential for the disruption of actin filaments, cell rounding and inhibition of phagocytosis by YopE in HeLa cells . Furthermore, the GAP function of YopE was important for Y . pseudotuberculosis pathogenesis in a mouse infection assay . Transfection of HeLa cells with a vector that produces a constitutively active form of RhoA (RhoA-V14) prevented the disruption of actin filaments and cell rounding by YopE . Production of an activated form of Rac1 (Rac1-V12), but not RhoA-V14, in HeLa cells interfered with YopE antiphagocytic activity . These results demonstrate that YopE functions as a RhoGAP to downregulate multiple Rho GTPases, leading to the disruption of actin filaments and inhibition of bacterial uptake into host cells. Mol Microbiol, 2000 Jul, 37(1), 125 - 35 Pathways for the utilization of N-acetyl-galactosamine and galactosamine in Escherichia coli; Brinkkotter A et al.; Among enteric bacteria, the ability to grow on N-acetyl-galactosamine (GalNAc or Aga) and on D-galactosamine (GalN or Gam) differs . Thus, strains B, C and EC3132 of Escherichia coli are Aga+ Gam+ whereas E . coli K-12 is Aga- Gam-, similarly to Klebsiella pneumoniae KAY2026, Klebsiella oxytoca M5a1 and Salmonella typhimurium LT2 . The former strains carry a complete aga/kba gene cluster at 70.5 min of their gene map . These genes encode an Aga-specific phosphotransferase system (PTS) or IIAga (agaVWE) and a GalN-specific PTS or IIGam (agaBCD) . Both PTSs belong to the mannose-sorbose family, i.e . the IIB, IIC and IID domains are encoded by different genes, and they share a IIA domain (agaF) . Furthermore, the genes encode an Aga6P-deacetylase (agaA), a GalN6P deaminase (agaI), a tagatose-bisphosphate aldolase comprising two different peptides (kbaYZ) and a putative isomerase (agaS), i.e . complete pathways for the transport and degradation of both amino sugars . The genes are organized in two adjacent operons (kbaZagaVWEFA and agaS kbaYagaBCDI) and controlled by a repressor AgaR . Its gene agaR is located upstream of kbaZ, and AgaR responds to GalNAc and GalN in the medium . All Aga- Gam- strains, however, carry a deletion covering genes agaW' EF 'A; consequently they lack active IIAga and IIGam PTSs, thus explaining their inability to grow on the two amino sugars . Remnants of a putative recombination site flank the deleted DNA in the various Aga- Gam- enteric bacteria . Derivatives with an Aga+ Gam- phenotype can be isolated from E . coli K-12 . These retain the DeltaagaW' EF 'A deletion and carry suppressor mutations in the gat and nag genes for galactitol and N-acetyl-glucosamine metabolism, respectively, that allow growth on Aga but not on GalN. Mol Microbiol, 2000 Jul, 37(1), 98 - 107 Fusidic acid-resistant EF-G perturbs the accumulation of ppGpp; MacVanin M et al.; Reductions in growth rate caused by fusidic acid-resistant EF-G mutants in Salmonella typhimurium correlate strongly with increased mean cell size . This is unusual because growth rate and cell size normally correlate positively . The global transcription regulator molecule ppGpp has a role in co-ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division . We show that fusidic acid-resistant EF-G mutants have perturbed ppGpp basal levels during steady-state growth and perturbed induced levels during starvation . One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one-third of that found in the wild-type strain . Other fusA mutants with intermediate or wild-type growth rates and cell sizes have either normal or increased basal levels of ppGpp . There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF-G is inhibited by ppGpp in vitro . This enhanced interaction between mutant EF-G and ppGpp correlates with an increased KM for GTP . Our results suggest that mutant EF-G modulates the production of ppGpp by the RelA (PSI) pathway . In conclusion, fusidic acid-resistant EF-G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division . It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns. Mol Microbiol, 2000 Jun, 36(6), 1206 - 21 Identification of SopE2 from Salmonella typhimurium, a conserved guanine nucleotide exchange factor for Cdc42 of the host cell; Stender S et al.; Salmonella typhimurium translocates effector proteins into host cells via the SPI1 type III secretion system to induce responses such as membrane ruffling and internalization by non-phagocytic cells . Activation of the host cellular RhoGTPase Cdc42 is thought to be a key event during internalization . The translocated Salmonella protein SopE is an activator for Cdc42 . Because SopE is absent from most S . typhimurium strains it remains unclear whether all S . typhimurium strains rely on activation of Cdc42 to invade host cells . We have identified SopE2, a translocated effector protein common to all S . typhimurium strains . SopE2 is a guanine nucleotide exchange factor for Cdc42 and shows 69% sequence similarity to SopE . Analysis of S . typhimurium mutants demonstrated that SopE2 plays a role in recruitment of the actin-nucleating Arp2/3 complex to the membrane ruffles and in efficient host cell invasion . Transfection experiments showed that SopE2 is sufficient to activate host cellular Cdc42, to recruit the actin-nucleating Arp2/3 complex and to induce actin cytoskeletal rearrangements and internalization . In conclusion, as a result of SopE2 all S . typhimurium strains tested have the capacity to activate Cdc42 signalling inside host cells which is important to ensure efficient entry. Acta Crystallogr D Biol Crystallogr, 2000 Jul, 56 ( Pt 7), 924 - 6 Crystallization of peptidase T from Salmonella typhimurium; Hakansson K et al.; Aminotripeptidase (peptidase T) from Salmonella typhimurium and a derivative carrying a C-terminal His tag have been crystallized . In both cases, the space group was found to be C2, with a single molecule in the asymmetric unit . Crystals of the native peptidase T diffract to 2.9 A, but a selenomethionine derivative of this protein did not yield good crystals . Crystals of the His-tag peptidase T diffracted to 2.6 A, however, and could be used for the production of good-quality selenomethionine crystals . All 15 methionines, a native metal ion and two mercury reactive sites could be located and crystals suitable for MAD data collection have been produced. Food Chem Toxicol, 2000 Sep, 38(9), 801 - 9 5-Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione; Janzowski C et al.; 5-(hydroxymethyl)-2-furfural (HMF), a common product of the Maillard reaction, occurs in many foods in high concentrations, sometimes exceeding 1 g/kg (in certain dried fruits and caramel products) . The toxicological relevance of this exposure has not yet been clarified . Induction of aberrant colonic crypt foci had been reported for HMF, in vitro studies on genotoxicity/mutagenicity have given controversial results . To elucidate the toxic potential of HMF, cytotoxicity (trypan blue exclusion), growth inhibition (SRB assay), mutagenicity (HPRT assay), DNA damage (single-cell gel electrophoresis) and depletion of cellular glutathione were investigated in mammalian cells . Genotoxicity (SOS repair) was monitored in Salmonella typhimurium (umu assay) . HMF induced moderate cytotoxicity in V79 cells (LC(50): 115 mM, 1 hr incubation) and in Caco-2 cells (LC(50): 118 mM, 1 hr incubation) . Growth inhibition was monitored following 24 hr of incubation (V79, IC(50): 6.4 mM) . DNA damage was detectable neither in these cell lines nor in primary rat hepatocytes up to the cytotoxic threshold concentration (75% absolute viability) . Likewise, in primary human colon cells, obtained from biopsy material, DNA damage was not measurable . At 120 mM, already exhibiting some reduction in cell viability, HMF was weakly mutagenic at the hprt-locus in V79 cells (mutants/10(6) cells: HMF 120 mM: 16 vs control: 3) . Intracelluar glutathione was depleted by HMF (>/=50 mM) in V79 cells, in the human colon adenocarcinoma cell line Caco-2 and in primary rat hepatocytes down to approximately 30% of control (120 mM) . Genotoxicity was observed with HMF in the umu assay without external activation (16 mM: 185 rel . umu units, %, P<0.001) . The genotoxic potential was not altered by addition of rat liver microsomes . By comparison, the natural flavour constituent (E)-2-hexenal (HEX) was already cytotoxic, mutagenic and depleted glutathione at about 1000-fold lower concentrations . It induced DNA damage in mammalian cells (200-400 microM) . These results suggest that HMF does not pose a serious health risk, even though the highest concentrations in specific foods approach the biologically effective concentration range in cell systems. Food Chem Toxicol, 2000 Sep, 38(9), 783 - 92 Application of a dynamic in vitro gastrointestinal tract model to study the availability of food mutagens, using heterocyclic aromatic amines as model compounds; Krul C et al.; The TNO gastro-Intestinal tract Model (TIM) is a dynamic computer-controlled in vitro system that mimics the human physiological conditions in the stomach and small intestine . In the current TIM physiological parameters such as pH, temperature, peristaltic movements, secretion of digestion enzymes, bile and pancreatic juices, and absorption of digested products-by removal through dialysis-was simulated . Heterocyclic aromatic amines (HAA; viz . IQ, MeIQ, MeIQx and PhIP) were used as model compounds for food mutagens, and the passage through TIM was investigated for each of these compounds separately . Subsequently, the influence of a matrix and different rates of passage on the availability for absorption and distribution were studied in experiments with prepared meat, supplemented with MeIQx . Samples taken at various time points from the jejunal and ileal dialysates and from the lumen at the end of the small intestine (ileal delivery) were tested for the presence of mutagenic activity in the Ames test with Salmonella typhimurium strain TA98 as indicator, in the presence of mammalian metabolic activation (rat S9 mix) . The results show that, comparable with the human in vivo situation, all four HAA are quickly removed (approx . 50% in 2 hr; approx . 95% in 6 hr) and mainly recovered from the lumen into the jejunal and ileal dialysates (94% of recovery) . Only 5+/-1.5% is recovered in the chyme at the end of the small intestine . When MeIQx was added to meat, its availability for absorption was slower, although the influence of the gastrointestinal passage time on the availability of MeIQx was more pronounced than this matrix effect . More MeIQx was found in the jejunal dialysate (23%; P<0.01) and less in the ileal delivery (8%; P<0.01) when simulating the gastrointestinal passage of solid meals was compared to simulating that of liquid meals . The present experiments demonstrate that TIM can be applied to study in vitro the availability of heterocyclic aromatic amines in the gastrointestinal tract . More generally, these studies indicate that TIM shows promise as a useful tool for various research purposes dealing with the availability for absorption of mutagenic as well as antimutagenic components in food. Indian J Exp Biol, 2000 Mar, 38(3), 285 - 6 Effect of norepinephrine on growth of Salmonella and its enterotoxin production; Rahman H et al.; Salmonella typhimurium was cultured in presence or absence of norepinephrine in conditioned media . Two conditioned media containing bovine and pig serum were prepared . Supplementation of fresh cultures with norepinephrine (5 x 10(-5) M per mL of medium) resulted in ten-fold increase in growth as compared to controls . No significant difference in growth of organisms in media containing bovine and pig serum was observed . Growth was more in culture incubated under shaking condition than in non-shaking condition . Enterotoxin production increased by two to eight-folds in the medium supplemented with norepinephrine. Biochemistry, 2000 Aug 8, 39(31), 9486 - 93 Attractant regulation of the aspartate receptor-kinase complex: limited cooperative interactions between receptors and effects of the receptor modification state; Bornhorst JA et al.; The manner by which the bacterial chemotaxis system responds to a wide range of attractant concentrations remains incompletely understood . In principle, positive cooperativity between chemotaxis receptors could explain the ability of bacteria to respond to extremely low attractant concentrations . By utilizing an in vitro receptor-coupled kinase assay, the attractant-dependent response curve has been measured for the Salmonella typhimurium aspartate chemoreceptor . The attractant chosen, alpha-methyl aspartate, was originally used to quantitate high receptor sensitivity at low attractant concentrations by Segall, Block, and Berg {(1986) Proc . Natl . Acad . Sci . U.S.A . 83, 8987-8991} . The attractant response curve exhibits limited positive cooperativity, yielding a Hill coefficient of 1.7-2.4, and this Hill coefficient is relatively independent of both the receptor modification state and the mole ratio of CheA to receptor . These results disfavor models in which there are strong cooperative interactions between large numbers of receptor dimers in an extensive receptor array . Instead, the results are consistent with cooperative interactions between a small number of coupled receptor dimers . Because the in vitro receptor-coupled kinase assay utilizes higher than native receptor densities arising from overexpression, the observed positive cooperativity may overestimate that present in native receptor populations . Such positive cooperativity between dimers is fully compatible with the negative cooperativity previously observed between the two symmetric ligand binding sites within a single dimer . The attractant affinity of the aspartate receptor is found to depend on the modification state of its covalent adaptation sites . Increasing the the level of modification decreases the apparent attractant affinity at least 10-fold in the in vitro receptor-coupled kinase assay . This observation helps explain the ability of the chemotaxis pathway to respond to a broad range of attractant concentrations in vivo. Environ Mol Mutagen, 2000, 36(1), 52 - 8 Genotoxic activity of five haloacetonitriles: comparative investigations in the single cell gel electrophoresis (comet) assay and the ames-fluctuation test; Muller-Pillet V et al.; Halogenated acetonitriles (HANs) are known to be water disinfectant by-products . Their mutagenicity and carcinogenicity have been shown in different test systems in vivo and in vitro . They also have clastogenic properties . In this study, the ability of HAN to induce single-strand breaks on the DNA of HeLa S3 cells was investigated using the single-cell gel electrophoresis (SCGE) assay, which could be a good tool with which to evaluate the genotoxicity of chlorinated water . The results were compared to those obtained in the Ames fluctuation test using the Salmonella typhimurium TA 100 strain without activation . With the Ames fluctuation test, a mutagenic effect was observed for chloroacetonitrile (MCAN), dichloroacetonitrile (DCAN), and trichloroacetonitrile (TCAN) . No mutagenic effect was found with bromoacetonitrile (MBAN) or dibromoacetonitrile (DBAN) . In the SCGE assay, all five HANs induced DNA damage in HeLa S3 cells, increasing the mean tail moment significantly . For each compound, a dose-effect relation was observed . This study shows that the SCGE assay has greater sensitivity for assessing the genotoxicity of HAN than does the Ames-fluctuation test . Brominated acetonitriles were more genotoxic than chlorinated acetonitriles in the SCGE assay, and the genotoxicity increased with the number of halogenated atoms of the compound . This behavior had already been found with other genotoxicity tests . Environ Mol Mutagen, 2000, 36(1), 13 - 21 Genetic toxicity evaluation of octamethylcyclotetrasiloxane; Vergnes JS et al.; Octamethylcyclotetrasiloxane (OMCTS; CAS No . 556-67-2) was evaluated in a genetic toxicity battery . In preincubation tests with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, no mutagenicity was detected (maximum dose = 5 mg/plate) with or without S9 in two independent trials . Treatment of cultured Chinese hamster ovary (CHO) cells was limited by cytotoxicity at OMCTS concentrations greater than 0.003 mg/mL without S9 and 0.03 mg/mL with S9 . CHO cells treated with up to 0.003 mg/mL without S9 and 0.03 mg/mL with S9 showed no significant dose-related increases in chromosomal aberration frequencies . No significant dose-related increases in sister chromatid exchanges (SCEs) occurred in OMCTS-treated CHO cells (maximum OMCTS concentration = 0.003 mg/mL without S9; 0.03 mg/mL with S9) . Therefore, OMCTS was concluded to be negative in the SCE assay . In a screen for in vivo clastogenic potential, Sprague-Dawley rats received 700 ppm OMCTS by whole-body vapor inhalation 6 hr daily for 5 days . A negative control group received filtered air on the same schedule . A positive control group was exposed to filtered air on the same schedule and received cyclophosphamide 24 hr before termination . The OMCTS-treated animals were terminated 6 and 24 hr after the final exposure . Positive and negative control animals were terminated 24 hr after the last exposure . No significant, treatment-related increases in chromosomal aberrations were detected . The results of these studies indicate that OMCTS does not possess significant in vitro genotoxic potential . No adverse genetic findings were seen in the in vivo screen for chromosome aberrations . Curr Microbiol, 2000 Sep, 41(3), 172 - 6 Expression of the rfa, LPS biosynthesis promoter in Salmonella typhimurium during invasion of intestinal epithelial cells; Maurer JJ et al.; Salmonella regulates transcription of many of its genes in response to environmental conditions encountered inside or outside the eukaryotic cells it infects . In this paper, we examined Salmonella typhimurium gene expression within epithelial cells, by using bacterial luciferase as a reporter . We focused on gene expression controlled by Salmonella rfa promoter, using lac promoter as a control . We observed down regulation for both promoters during the initial 2 h of invasion . The decreased levels of luciferase activity appeared to be due to metabolic changes, since we observed similar results with tissue culture medium alone . Gene expression stabilized to a new steady state for the Salmonella rfa promoter, while a lac promoter activity steadily decreased . Bacterial luciferase activity was a good indicator of intracellular numbers and allowed us to detect as few as 1000 bacterial cells/infected monolayer . Both promoters were not dependent on host protein synthesis for expression. J Infect Dis, 2000 Aug, 182(2), 482 - 9 Epub 2000 Jul 28. Alcohol consumption by C57BL/6 mice is associated with depletion of lymphoid cells from the gut-associated lymphoid tissues and altered resistance to oral infections with Salmonella typhimurium; Sibley D et al.; Studies were done to test whether ethanol (ETOH) consumption alters resistance to mucosal and systemic infections by Salmonella typhimurium . S . typhimurium-immune and -nonimmune mice were fed 1 of 3 diets (an ETOH-containing liquid diet, an isocaloric liquid diet equal in volume to that of the ETOH-treated group, or laboratory chow) in a pair-feeding design and were infected orally or intravenously with S . typhimurium . The number of bacteria in spleen and liver and the effect of ETOH feeding and infection on the number of lymphoid cells in the gut-associated lymphoid tissues (GALT) were determined . ETOH feeding resulted in profound loss of GALT lymphoid cells and an increased number of Salmonella organisms in the intestines, liver, and spleen of infected nonimmune, but not of immune, mice . These data show that ETOH consumption in this model impairs host defense mechanisms that control mucosal infections and inhibits the mechanisms that control levels of bacteria in the central organs. J Food Prot, 2000 Jul, 63(7), 945 - 52 Genotoxicity testing of cooked cured meat pigment (CCMP) and meat emulsion coagulates prepared with CCMP; Stevanovic M et al.; The preformed cooked cured meat pigment (CCMP) synthesized directly from bovine red blood cells or through a hemin intermediate was found to be a viable colorant for application to comminuted pork as a nitrite substitute . However the genotoxicity of CCMP and meat emulsion coagulates prepared with CCMP has not been evaluated . Therefore the objectives of this work were to investigate genotoxicity of CCMP and the influence of CCMP addition on genotoxicity and the content of residual nitrite in model meat emulsion coagulates . Meat emulsions were prepared from white (musculus longissimus dorsi) and red (musculus quadriceps femoris) pork muscles with two different amounts of synthesized pigment CCMP . Comparatively, emulsions with fixed addition of nitrite salt and emulsions without any addition for color development were made . Genotoxicity of CCMP and meat emulsion coagulates was tested with the SOS/umu test and the Ames test . Neither CCMP nor meat emulsion coagulates prepared with CCMP or nitrite salt were genotoxic in the SOS/umu test . In the Ames test using Salmonella Typhimurium strains TA98 and TA100 samples of coagulates prepared with CCMP and with nitrite showed weak mutagenic activity in Salmonella Typhimurium strain TA100 but only in the absence of the metabolic activation, while CCMP was not mutagenic . Coagulates prepared with CCMP contained significantly less residual nitrite than coagulates prepared with nitrite salt . These results indicate that from the human health standpoint the substitution of nitrite salt with CCMP would be highly recommendable. Biochemistry, 2000 Aug 1, 39(30), 8859 - 69 Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity; Reynolds CM et al.; AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli . Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate . Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction . We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF . In addition, Nt-TrR retains essentially full TrR activity . Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR . The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis . These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties . These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction. J Biol Chem, 2000 Oct 13, 275(41), 32141 - 6 A single point mutation in 3-deoxy-D-manno-octulosonate-8-phosphate synthase is responsible for temperature sensitivity in a mutant strain of Salmonella typhimurium; Taylor WP et al.; Salmonella typhimurium mutants conditionally deficient in 3-deoxy-d-manno-octulosonate-8-phosphate (KDO8P) synthase activity play a central role in our understanding of lipopolysaccharide function in enteric bacteria . The detailed characterization of KDO8P synthase from such a mutant, however, has not been previously reported . To address this issue KDO8P synthase from S . typhimurium AG701 and from a related temperature-sensitive strain (S . typhimurium AG701i50) have been overexpressed in Escherichia coli and purified to homogeneity . The enzyme from the temperature-sensitive strain has a single proline to serine substitution at position 145, leading to an increase in K(m) for both substrates, d-arabinose 5-phosphate and phosphoenolpyruvate . Analytical gel filtration and native polyacrylamide gel electrophoresis indicate that this enzyme also has an altered oligomeric state . These observations are rationalized through an examination of the structure of E . coli KDO8P synthase, which has 93% sequence identity to the enzyme from S . typhimurium. Blood, 2000 Aug 1, 96(3), 1125 - 9 Wild-type HFE protein normalizes transferrin iron accumulation in macrophages from subjects with hereditary hemochromatosis; Montosi G et al.; Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases . A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload . Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein . The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood . To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier . The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency . The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE . Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface . The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells . These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells. J Anim Sci, 2000 Jul, 78(7), 1885 - 91 Acute phase responses of pigs challenged orally with Salmonella typhimurium; Balaji R et al.; This study evaluated responses of the systemic endocrine stress (cortisol) and growth (IGF-I, GH) axes, as well as those of inflammatory mediators (prostaglandin E2 {PGE2} and tumor necrosis factor alpha {TNFalpha}), to active infection with Salmonella typhimurium . Eighteen crossbred barrows were penned individually with ad libitum access to feed and water . After an acclimation period, jugular catheters were placed in all animals . Control pigs received sterile broth orally (CON, n = 7), whereas the treated pigs (S.TYP, n = 11) received 3 x 10(9) cfu of S . typhimurium orally . Plasma was collected at 6-h intervals from -48 to 120 h . Body weights, feed intake, and rectal temperatures also were monitored . Rectal temperatures were elevated in S.TYP pigs (P < .01) relative to CON pigs by 12 h, peaked at 42 h (P < .001), and remained elevated throughout the remainder of the study . Feed intake was reduced maximally in S.TYP pigs at 48 h (P < .001) and remained reduced through 120 h after the challenge . Daily body weight gain also was reduced during the 2 wk following infection (P < .001) . Plasma cortisol concentrations increased (P < .05) at 18 h after the challenge in S.TYP pigs and remained elevated generally until 60 h after infection . A marked suppression of plasma IGF-I occurred in S.TYP pigs beginning at 30 h after infection (P < .001), and it remained lower through 108 h . Plasma GH was not affected consistently by treatment, nor did infection alter plasma TNFalpha and PGE2 . Taken together, the results reveal that infectious processes produce profound alterations in the endocrine stress and the somatotropic axis, and this may occur in the absence of significant changes in systemic proinflammatory mediators. Mol Gen Genet, 2000 Jun, 263(5), 867 - 76 The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system; Eckert SE et al.; The tryptophan synthase-encoding gene, trpB, of Aspergillus nidulans was cloned and characterized . It was mapped to chromosome I, between the gene medA, which is required for sexual and asexual development, and an ORF encoding a protein with significant similarity to subunit B of vacuolar ATP synthases . The 5' untranslated region was found to be at least 142 nucleotides (nt) long, the poly(A) addition site was localized at position + 216 relative to the stop codon by sequencing of several independent cDNA clones . The trpB gene contains two exons separated by an intron of 105 nt, which is located close to the 5' end of the ORF . Directly upstream of the transcriptional start site, one well conserved potential binding site for the cross-pathway control transcriptional activator CPCA was found . The level of trpB transcript was shown to be regulated by cross-pathway control . A knockout mutant for trpB displays tryptophan auxotrophy, no trpB transcript is detectable, and development is perturbed to an extent that is dependent on the amount of tryptophan added to the medium . The trpB gene encodes a protein of 723 amino acids, with a calculated molecular weight of 77.6 kDa . The deduced amino acid sequence shows 72.6% similarity to the tryptophan synthase of Neurospora crassa . Most amino acid residues essential for catalytic activity in the tryptophan synthase of Salmonella typhimurium are conserved . The linker region joining the two domains of the enzyme is 13 residues longer than the longest connector found so far in tryptophan synthases from fungi. J Biol Chem, 2000 Oct 20, 275(42), 32940 - 9 Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moiety in Salmonella typhimurium lipid A . Function of LpxO, A new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue; Gibbons HS et al.; Lipid A molecules of certain Gram-negative bacteria, including Salmonella typhimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacyl chains . S . typhimurium has recently been shown to synthesize its S-2-hydroxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis . We postulated that 2-hydroxylation might be catalyzed by a novel dioxygenase . Lipid A was extracted from a PhoP-constitutive mutant of S . typhimurium grown in the presence or absence of O(2) . Under anaerobic conditions, no 2-hydroxymyristate-containing lipid A was formed . Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipid A from cells grown in the presence of (18)O(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxyacyl-modified lipid A . Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading frame(s) with similarity to mammalian aspartyl/asparaginyl beta-hydroxylases in bacteria known to make 2-hydroxyacylated lipid A molecules . The S . typhimurium aspartyl/asparaginyl beta-hydroxylase homologue (designated lpxO) was cloned into pBluescriptSK and expressed in Escherichia coli K-12, which does not contain lpxO . Analysis of the resulting construct revealed that lpxO expression is sufficient to induce O(2)-dependent formation of 2-hydroxymyristate-modified lipid A in E . coli . LpxO very likely is a novel Fe(2+)/alpha-ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of lipid A (or of a key precursor) . The S . typhimurium lpxO gene encodes a polypeptide of 302 amino acids with predicted membrane-anchoring sequences at both ends . We hypothesize that 2-hydroxymyristate chains released from lipopolysaccharide inside infected macrophages might be converted to 2-hydroxymyristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myristoyl transferase. J Exp Med, 2000 Jul 17, 192(2), 249 - 58 Salmonella exploits caspase-1 to colonize Peyer's patches in a murine typhoid model; Monack DM et al.; Salmonella typhimurium invades host macrophages and induces apoptosis and the release of mature proinflammatory cytokines . SipB, a protein translocated by Salmonella into the cytoplasm of macrophages, is required for activation of Caspase-1 (Casp-1, an interleukin {IL}-1beta-converting enzyme), which is a member of a family of cysteine proteases that induce apoptosis in mammalian cells . Casp-1 is unique among caspases because it also directly cleaves the proinflammatory cytokines IL-1beta and IL-18 to produce bioactive cytokines . We show here that mice lacking Casp-1 (casp-1(-/)- mice) had an oral S . typhimurium 50% lethal dose (LD(50)) that was 1,000-fold higher than that of wild-type mice . Salmonella breached the M cell barrier of casp-1(-/)- mice efficiently; however, there was a decrease in the number of apoptotic cells, intracellular bacteria, and the recruitment of polymorphonuclear lymphocytes in the Peyer's patches (PP) as compared with wild-type mice . Furthermore, Salmonella did not disseminate systemically in the majority of casp-1(-/)- mice, as demonstrated by significantly less colonization in the PP, mesenteric lymph nodes, and spleens of casp-1(-/)- mice after an oral dose of S . typhimurium that was 100-fold higher than the LD(50) . The increased resistance in casp-1(-/)- animals appears specific for Salmonella infection since these mice were susceptible to colonization by another enteric pathogen, Yersinia pseudotuberculosis, which normally invades the PP . These results show that Casp-1, which is both proapoptotic and proinflammatory, is essential for S . typhimurium to efficiently colonize the cecum and PP and subsequently cause systemic typhoid-like disease in mice. J Exp Med, 2000 Jul 17, 192(2), 237 - 48 Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis . II . Effects on microbial proliferation and host survival in vivo; Mastroeni P et al.; The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox(-/)-, iNOS(-/)-, and congenic wild-type mice . Although both gp91phox(-/)- and iNOS(-/)- mice demonstrated increased susceptibility to infection with S . typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox(-/)- and iNOS(-/)- mouse strains . Greater bacterial numbers were present in the spleens and livers of gp91phox(-/)- mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox(-/)- mice succumbed to infection within 5 d . In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS(-/)- mice only beyond the first week of infection . Influx of inflammatory CD11b(+) cells, granuloma formation, and serum interferon gamma levels were unimpaired in iNOS(-/)- mice, but the iNOS-deficient granulomas were unable to limit bacterial replication . The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection. J Exp Med, 2000 Jul 17, 192(2), 227 - 36 Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis . I . Effects on microbial killing by activated peritoneal macrophages in vitro; Vazquez-Torres A et al.; The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91phox(-/)-, iNOS(-/)-, and doubly immunodeficient phox(-/)-iNOS(-/)- mice . The respiratory burst and NO radical (NO.) made distinct contributions to the anti-Salmonella activity of macrophages . NADPH oxidase-dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity . NO-derived species initially synergize with oxyradicals to kill S . typhimurium, and subsequently exert prolonged oxidase-independent bacteriostatic effects . Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O(2).(-)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) production . However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S . typhimurium . Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth . Interferon gamma appears to augment antibacterial activity predominantly by enhancing NO . production, although a small iNOS-independent effect was also observed . These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species. J Exp Med, 2000 Jul 17, 192(2), 183 - 92 A macrophage invasion mechanism for mycobacteria implicating the extracellular domain of CD43; Fratazzi C et al.; We studied the role of CD43 (leukosialin/sialophorin), the negatively charged sialoglycoprotein of leukocytes, in the binding of mycobacteria to host cells . CD43-transfected HeLa cells bound Mycobacterium avium, but not Salmonella typhimurium or Shigella flexneri . Quantitative bacteriology showed that macrophages (M(phi)) from wild-type mice (CD43(+/+)) bound M . avium, Mycobacterium bovis (bacillus Calmette-Guerin), and Mycobacterium tuberculosis (strain H37Rv), whereas M(phi) from CD43 knockout mice (CD43(-/)-) did not . Fluorescence microscopy demonstrated that the associated M . avium had been ingested by the CD43(+/+) M(phi) . The inability of CD43(-/)- M(phi) to bind M . avium could be restored by addition of galactoglycoprotein (Galgp), the extracellular mucin portion of CD43 . The effect of Galgp is not due to opsonization of the bacteria, but required its interaction with the M(phi) other mucins had no effect . CD43 expression by the M(phi) was also required for optimal induction by M . avium of tumor necrosis factor (TNF)-alpha production, which likewise could be reconstituted by Galgp . In contrast, interleukin (IL)-10 production by M . avium-infected M(phi) was CD43 independent, demonstrating discordant regulation of TNF-alpha and IL-10 . These findings describe a novel role of CD43 in promoting stable interaction of mycobacteria with receptors on the M(phi) enabling the cells to respond specifically with TNF-alpha production. Chem Res Toxicol, 2000 Jul, 13(7), 535 - 40 Isolation and identification of a new 2-phenylbenzotriazole-type mutagen (PBTA-3) in the Nikko river in Aichi, Japan; Shiozawa T et al.; We have previously determined the chemical structures of two 2-phenylbenzotriazole mutagens (PBTA-1 and PBTA-2) in blue cotton-adsorbed material from the Nishitakase River in Kyoto, Japan . In the present study, further analysis of mutagenic substances in the Nikko River, which flows through Aichi Prefecture in Japan, allowed the isolation of a new mutagen . Material (2.2 g) adsorbed on blue cotton (3 kg) at a site below the sewage plant on the Nikko River was purified by various column chromatographies, and a mutagen (120 microg) accounting for 11% of the total mutagenicity was isolated . On the basis of data from UV, mass, and (1)H NMR spectra of the mutagen, the compound was deduced to be a PBTA-1 analogue . As with PBTA-1, the mutagen was able to be synthesized from the azo dye 2-{(2-bromo-4, 6-dinitrophenyl)azo}-4-methoxy-5-{(2-hydroxyethyl)amino}acetanilide by reduction and chlorination . Since all spectra of the mutagen isolated from the river water were the same as those of the synthesized form, the structure was concluded to be 2-{2-(acetylamino)-4-{(2-hydroxyethyl)amino}-5-methoxyphenyl}-5-amino -7-bromo-4-chloro-2H-benzotriazole (PBTA-3) . PBTA-3 is a potent mutagen, inducing 81 000 and 3 000 000 revertants per microgram of Salmonella typhimurium TA 98 and YG1024 respectively, in the presence of an S9 mix . In addition to its detection in the water of the Nikko River, PBTA-3 was detected in water samples from three other rivers flowing through regions where dyeing industries have been developed . Like PBTA-1 and PBTA-2, PBTA-3 might have also been produced from azo dyes during industrial processes in dyeing factories and/or through treatment at sewage plants. Chem Res Toxicol, 2000 Jul, 13(7), 531 - 4 A reactive metabolite of furan, cis-2-butene-1,4-dial, is mutagenic in the Ames assay; Peterson LA et al.; Furan is classified as a nongenotoxic hepatocarcinogen . It is thought to be activated to a toxic metabolite, cis-2-butene-1,4-dial, which is acutely toxic to liver cells . The resulting cytotoxicity is followed by compensatory cell proliferation, increasing the likelihood of tumor production . We examined the genotoxic activity of cis-2-butene-1,4-dial in several strains of Salmonella typhimurium commonly used in the Ames assay . This reactive compound tested positive in TA104, a strain that is sensitive to aldehydes . Mutagenic activity was concentration-dependent (1000 +/- 180 revertants/micromol) . Incubation of cis-2-butene-1,4-dial with glutathione prior to addition of bacteria inhibited both the acute toxic and genotoxic activity of this compound . No evidence of mutagenic activity was seen at nontoxic concentrations in TA97, TA98, TA100, and TA102 . Our findings are consistent with the hypothesis that cis-2-butene-1,4-dial reacts with DNA to form mutagenic adducts . Our data suggest that cis-2-butene-1,4-dial may be an important genotoxic as well as toxic intermediate in furan-induced tumorigenesis. Int J Food Microbiol, 2000 Jun 30, 58(1-2), 49 - 58 Probability of detection of Salmonella using different analytical procedures, with emphasis on subspecies diarizonae serovar 61:k:1,5,(7) {S . IIIb 61:k:1,5,(7)}; Alvseike O et al.; A parameter for assessing qualitative methods is suggested: 'The probability of detection' has been used in this study to compare the efficiency of commonly used media for detecting Salmonella in mutton and faeces . Mutton and ovine faeces spiked with strains of Salmonella IIIb 61:k: 1,5,(7) and Salmonella Typhimurium were analysed and the performance of each combination of media was estimated . Extensive variation between serovars and strains of identical serovars were recorded . In meat, an inoculum of up to 10(6)/g was needed to detect salmonellae with 90% probability, while in faeces even 10(7)/g was not enough for some strains of S . IIIb 61:k:1,5,(7) . All method combinations thus had a rather low efficiency . Based on our experiments, we recommend using a combination of the XLD medium and any of the three selective broths for detection of S . IIIb 61:k:1,5,(7) from mutton, while we recommend using the SC broth with any of the three plating media for detection from ovine faeces. Reprod Fertil Dev, 1999, 11(4-5), 219 - 28 Antigen-specific systemic and reproductive tract antibodies in foxes immunized with Salmonella typhimurium expressing bacterial and sperm proteins; de Jersey J et al.; Attenuated Salmonella typhimurium strains are potential 'safe' delivery vectors of an oral immunocontraceptive vaccine for the European red fox (Vulpes vulpes) . In the present study, model bacterial (Escherichia coli heat-labile enterotoxin B subunit, LTB) and fox sperm (fSP10) antigens were expressed in S . typhimurium SL3261 (delta aroA) under the control of the trc promoter . Adult female foxes were given three oral immunizations with SL3261 containing either LTB (SL3261/pLTB), fSP10 (SL3261/pFSP10) or a control plasmid (pKK233-2 or pTrc99A) . All foxes raised serum (IgG) and vaginal (IgG and IgA) antibodies against S . typhimurium lipopolysaccharide (LPS) . Each fox that received SL3261/pLTB raised high titre LTB-specific serum and vaginal IgG antibodies . However, only one of four foxes immunized with SL3261/pFSP10 raised an anti-fSP10 immune response, in the form of low titre serum and vaginal IgG antibodies . No vaginal IgA antibodies were raised against either LTB or fSP10 in these experiments . The immune responses against recombinant LTB and fSP10 resulted chiefly from the initial dose of antigen in the inocula and were minimally influenced by continued in vivo antigen expression . This study demonstrates for the first time in the female red fox that oral Salmonella can elicit specific systemic and reproductive tract antibodies against heterologous, recombinant proteins. Mutat Res, 2000 Jul 20, 452(1), 139 - 44 Enhancing effect of saccharides on the mutagenicity of 2-chloro-4-methylthiobutanoic acid; Kimura S et al.; The mutagenicity of 2-chloro-4-methylthiobutanoic acid (CMBA), a nitrite-treated Sanma fish mutagen, in Salmonella typhimurium TA100 was enhanced by addition of D-glucose during the CMBA-treatment . Several other monosaccharides also enhanced the mutagenicity of CMBA, and the order of the enhancing potency was found to be D-mannose, D-glucose>D-fructose, D-ribose, D-galactose . A disaccharide, maltose, showed only little enhancement . No enhancement was found with L-glucose . We investigated whether saccharides affect uptake of {methyl-14C}CMBA into S . typhimurium TA100 . Saccharides which enhanced CMBA-induced mutagenesis increased the uptake . L-Glucose did not enhance the uptake . There was good correlation between the enhanced mutagenesis and increased radioactivity in Salmonella, suggesting that the enhancing effect of monosaccharide on the CMBA-induced mutagenesis results from the enhanced uptake of the mutagen into bacteria. J Agric Food Chem, 2000 Jun, 48(6), 2271 - 5 Mutagenicity of heated sugar-casein systems: effect of the Maillard reaction; Brands CM et al.; The formation of mutagens after the heating of sugar-casein model systems at 120 degrees C was examined by the Ames test, using Salmonella typhimurium strain TA100 . Several sugars (glucose, fructose, galactose, tagatose, lactose, and lactulose) were compared in their mutagenicities . Mutagenicity could be fully ascribed to Maillard reaction products and strongly varied with the kind of sugar . The differences in mutagenicity among the sugar-casein systems were caused by a difference in reaction rate and a difference in reaction mechanism . Sugars with a comparable reaction mechanism (glucose and galactose) showed a higher mutagenic activity corresponding with a higher Maillard reactivity . Disaccharides showed no mutagenic activity (lactose) or a lower mutagenic activity (lactulose) than their corresponding monosaccharides . Ketose sugars (fructose and tagatose) showed a remarkably higher mutagenicity compared with their aldose isomers (glucose and galactose), which was due to a difference in reaction mechanism. Yi Chuan Xue Bao, 2000, 27(2), 170 - 5 {Studies of gene regulation of de novo biosynthetic pathway of purine in Salmonella typhimurium . X . Isolation of purR(am) mutants and preliminary studies of amino acid substitution}; Zhang HS et al.; Starting from a super-repressed mutant of purR, 3-18, 439 independent candidates of purR- mutants were isolated by using NCE selecting plate with lactose as sole carbon source . Among these mutants . 11 amber mutants were detected by introducing a tRNA suppressor gene . Cotransduction analysis proved that the amber mutation sites of 11 amber mutants all located on purR . Amino acid substitution experiments were performed with three tRNA suppressors, supD, supE and supF, for each purR(am) . The results showed that the same amino acid substitution occurred in different site of PurR protein could result in varied effects on purR function; different amino acid substitution occurred at the same position of PurR protein also could produced varied effects on purR function. Mutagenesis, 2000 Jul, 15(4), 325 - 8 A new approach to evaluate mechanistic relationships among genotoxic phenomena: validation; Rosenkranz HS et al.; In order to determine its applicability for the study of genotoxicity, a recently developed method to probe for possible mechanistic relationships among toxicological phenomena was applied to the induction of mutations in Salmonella typhimurium . Since the basis of this phenomenon is understood, this would provide a test of the applicability of the new method to DNA-based mechanisms . The results presented indicate that significant relationships are indeed found among phenomena involving damage to or modification of DNA but not between them and non-genotoxic phenomena . The present results suggest that the newly developed approach could be applied to test mechanistic hypotheses involving genotoxic phenomena. Mutagenesis, 2000 Jul, 15(4), 317 - 23 A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay; Ohta T et al.; Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene . Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion . We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds . We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems . Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet . Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) . Escherichia coli WP3105P was also more sensitive than S . TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine . The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens. Mutat Res, 2000 Jul 10, 468(2), 165 - 71 Mutagenic profile of rubber dust and fume exposure in two rubber tire companies; Vermeulen R et al.; The aim of this study was to evaluate current mutagenic activity of ambient rubber dust and fume exposure in the mixing and curing departments of two rubber tire companies situated in The Netherlands and Sweden . Salmonella typhimurium strains YG1021, YG1024 and YG1041 were used to study the possible presence of mutagenic nitroarenes and aromatic amines . A large difference in mutagenic activity was found between the two companies . While the rubber tire company situated in The Netherlands revealed overall high mutagenic activity of rubber dust and fumes in the mixing