Salmonella typhimurium

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 and curing departments, respectively, 430 and 279 rev/m(3) (YG1041), the Swedish company showed almost no mutagenic activity, respectively, 18 and 54 rev/m(3) (YG1041) . Further identification of the mutagenic profile showed that mutagenic activity was exclusively observed in S . typhimurium strains with elevated levels of O-acetyltransferase activity (YG1041 and YG1024) in the presence of a metabolic active liver S9 fraction, possibly indicating the presence of indirect mutagenic aromatic amines . These results show that although production processes and lay-out within rubber tire companies are comparable, differences in rubber chemicals used and overall level of control measures (e.g., good housekeeping, cleanliness) are likely to result in substantial differences in mutagenic exposure levels between companies.

Cancer Lett, 2000 Aug 11, 156(2), 199 - 205
Inhibitory effect of melatonin on 7, 12-dimethylbenz{a}anthracene-induced carcinogenesis of the uterine cervix and vagina in mice and mutagenesis in vitro; Anisimov VN et al.; Forty female CBA mice aged 3-4 months were exposed twice a week during 2 months to intravaginal applications of polyurethane sponges impregnated with 0.1% solution of 7,12-dimethylbenz{a}anthracene (DMBA) in triethyleneglycol . Three hours after each application the sponges were taken out . Starting from the day of the 1st DMBA application a part of mice was exposed five times a week during 4 months with melatonin in tap water (20 mg/l) given at night time (from 18:00 to 09:00 h) . Additional 20 female CBA mice were intact and served as a control . All mice were sacrificed in 6 months after start of the experiment . Seven of 20 mice exposed to DMBA alone developed malignancies in the vagina and cervix uteri and two mice developed benign cervical tumors . No malignancies in vagina and uterine cervix and three vaginal papillomas were observed in mice exposed to DMBA+melatonin . There were no any tumors in intact controls . Two in vitro tests were used for mutagenicity studies: the Ames test (strains TA 97 and TA 98 of Salmonella typhimurium) and the single cell gel electrophoresis assay (SCGE assay or COMET assay) performed on CHOK1 cells . In tested strains melatonin significantly reduced the mutagenicity of DMBA . In the SCGE assay preincubation with melatonin led to a strong inhibition of clastogenic activities of DMBA . Thus, our data indicate that pineal indole hormone melatonin inhibits cervical and vaginal carcinogenesis induced by DMBA in mice and possess antimutagenic and anticlastogenic effect in vitro.

Arch Med Res, 2000 Mar-Apr, 31(2), 156 - 61
Antimutagenesis of beta-carotene to mutations induced by quinolone on Salmonella typhimurium; Arriaga-Alba M et al.; BACKGROUND: Quinolone-induced mutagenesis in the Salmonella typhimurium hisG48 strains suggests that these antibiotics are oxygen free radical generators . The use of beta-carotene as antioxidant was evaluated as an alternative to reduce oxidative cell damage in patients who need therapy with nalidixic acid, norfloxacin, or pipemidic acid . The studied beta-carotene (30%), used by pharmaceutical laboratories as dietary complements, was not toxic or mutagenic for the S . typhimurium TA102 strain at a dose equivalent to 1,500 I.U . At the studied concentrations, the evaluated antimutagen did not modify the minimum inhibitory concentration of nalidixic acid, norfloxacin, or pipemidic acid against uropathogenic Escherichia coli strains . METHODS: The mutagenic effect of nalidixic acid and norfloxacin against hisG48 strains was inhibited with 1500 I.U . of beta-carotene . The antimutagenic effect of beta-carotene against mutations induced by pipemidic acid was observed even with 150 I.U . of beta-carotene . The antimutagenic effect against mutations induced on S . typhimurium TA102 or TA104 strains was observed only when the aroclor 1254 rat-induced liver S9 mixture was used . RESULTS: This mutagenic effect was detected only when the strains were exposed to quinolones and the beta-carotene simultaneously with the S9 mixture, suggesting that quinolones induce oxygen free radicals that may be scavenged by beta-carotene . CONCLUSIONS: The antimutagenic effect of this vitamin A precursor is probably due to the active molecule of vitamin A, a desmutagen with the ability of radical capture . A diet rich in beta-carotene or vitamin A could be a good alternative to reduce genotoxic risk to patients being treated with quinolone.

J Biol Chem, 2000 Oct 6, 275(40), 30878 - 85
Novel subtype of type IIs restriction enzymes . BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium; Sapranauskas R et al.; The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence . The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed . The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme . Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence . The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium . Biochemical analyses demonstrated that BfiI, like the nuclease of S . typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate . However, unlike the nonspecific S . typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically . We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein . The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S . typhimurium; therefore, BfiI did not require metal ions for catalysis . We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

EMBO J, 2000 Jul 3, 19(13), 3235 - 49
Salmonella maintains the integrity of its intracellular vacuole through the action of SifA; Beuzon CR et al.; A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice . Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA {required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs} are all involved in the same virulence function . sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA . A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains . Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol . We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Chemosphere, 2000 Oct, 41(7), 989 - 99
Chemical and biological profiles of sediments as indicators of sources of contamination in Hamilton Harbour . Part II: bioassay-directed fractionation using the Ames Salmonella/microsome assay; Marvin CH et al.; Bottom sediment and suspended sediment samples from Hamilton Harbour (western Lake Ontario) and from a major tributary were profiled using a bioassay-directed fractionation approach . Sample extracts were fractionated using an alumina/Sephadex gel clean-up procedure to afford non-polar aromatic fractions which were characterized using chemical analyses and the Ames/microsome bacterial assay in Salmonella typhimurium strains YG1025 with the addition of oxidative metabolism (S9), and YG1024 without S9 . Non-polar aromatic fractions of selected samples were separated by normal phase HPLC into 1-min fractions which were subjected to bioassay analyses . The bioassays using strain YG1025+S9, a TA100-type strain, were performed to assess genotoxicity arising from the presence of polycyclic aromatic hydrocarbons (PAH) . Fractions which exhibited mutagenic activity contained PAH with molecular masses of 252, 276 and 278 amu; these fractions contained over 80% of the genotoxicity attributable to PAH . Individual compounds identified using Gas Chromatography-Mass Spectrometry analyses in these active fractions included benzo{a}pyrene, indeno{cd}pyrene and dibenz{a,h}anthracene . The YG1025+S9 mutagenic activity profiles were similar for all samples . Mutagenic activity profiles generated using strain YG1024-S9, a TA98-type strain sensitive to compounds characteristic of mobile source emissions, were very different . The mutagenic activities in strain YG1024-S9 were greatest for harbour-suspended sediment samples collected from sites impacted by a major tributary . Suspended sediments collected near areas known to contain high levels of coal tar-contamination in the bottom sediments contained higher levels of genotoxic PAH than suspended sediments collected from other areas of the harbour.

Rev Esp Quimioter, 1999 Sep, 12(3), 250 - 4
{Antimicrobial susceptibility of a selection of Salmonella enterica strains of various origins isolated in Spain}; Cruchaga S et al.; The widespread use of antimicrobials in human and veterinary practice is increasingly causing the emergence of different multidrug-resistant human pathogens . This situation makes treating infections caused by these microorganisms difficult . Salmonella enterica is an ubiquitous organism and may be a good indicator of the influence of the use and abuse of antimicrobials on the appearance of multiresistant strains . One hundred and ninety S . enterica strains of different origins isolated in Spain in 1996 were randomly selected . The minimal inhibitory concentration (MIC) was studied using the agar dilution method according to NCCLS criteria in the following antimicrobials: ampicillin, ticarcillin, amoxicillin-clavulanic acid, cefazolin, cefuroxime, cefotaxime, imipenem, gentamicin, apramycin, ciprofloxacin, streptomycin, chloramphenicol, tetracycline, sulfamethoxazole and co-trimoxazole . Sixty-three percent of the S . enterica tested were resistant and 24% were multiresistant . The percentage of resistant and multiresistant strains of S . enterica of human origin was slightly higher than those of nonhuman origin . Statistically, ampicillin, ticarcillin and amoxicillin-clavulanic acid were significantly more resistant in strains of human origin . Ninety-one percent of the strains of Typhimurium serotype and phagotype 104 were multiresistant . The Salmonella Typhimurium serotype and phagotype 104 ACSTSu-resistant clone, which is widespread in various Western countries, was also isolated in this study . The use of different antimicrobials in human and veterinary practice needs to be rationalized.

Microbiology, 2000 Jul, 146 ( Pt 7), 1639 - 49
Interaction of Salmonella serotypes with porcine macrophages in vitro does not correlate with virulence; Watson PR et al.; The interaction between SALMONELLA: serotypes and macrophages is potentially instrumental in determining the outcome of infection . The nature of this interaction was characterized with respect to virulence and serotype-host specificity using pigs as the infection model . Experimental infection with SALMONELLA: typhimurium, SALMONELLA: choleraesuis or SALMONELLA: dublin resulted in enteric, systemic or asymptomatic infection, respectively, which correlates well with the association of S . choleraesuis with systemic disease in pigs in epidemiological studies . Persistence within porcine alveolar macrophages in vitro did not directly correlate with virulence since S . typhimurium persisted in the highest numbers, and S . choleraesuis in the lowest . Comparison to other studies revealed that the relatively high persistence of S . typhimurium in macrophages correlates with its virulence in a broad range of animals: this could be a virulence mechanism for broad-host-range serotypes . There were little or no significant differences in the induction of pro-inflammatory cytokines by macrophages infected with the three serotypes . S . typhimurium and S . dublin, but not S . choleraesuis, damaged porcine macrophages, and the mechanism of damage did not resemble apoptosis . In conclusion, the virulence of SALMONELLA: serotypes in pigs did not directly correlate with their interaction with porcine macrophages in vitro . The interaction of SALMONELLA: and macrophages in vitro may not accurately model their interaction in vivo, and this will form the basis of further study.

Res Vet Sci, 2000 Jun, 68(3), 261 - 4
The pharmacodynamic effect of amoxicillin and danofloxacin against Salmonella typhimurium in an in-vitro pharmacodynamic model; Lindecrona RH et al.; The pharmacodynamic effect of amoxicillin and danofloxacin against two strains of Salmonella typhimurium was examined in an in-vitro pharmacodynamic model . For amoxicillin, peak concentrations of 1, 2 and 4 microg ml(-1)and half-lives (t12) of 3 and 15 hours were evaluated . For danofloxacin peak concentrations of 0.25, 0.50 and 1 . 50 microg ml(-1)and half-lives of 7 and 15 hours were examined.For amoxicillin both the peak concentration and the half-life influenced the pharmacodynamic effect (P < 0.001) . Maximal pharmacodynamic effect was observed when the antibiotic concentration was greater than minimum inhibitory concentration for 79 per cent or more of the dosing interval . The MICS of the isolates increased when the amoxicillin concentrations were close to the MIC during the first hours of exposure.For danofloxacin the pharmacodynamic effect was dependent on the peak concentration only (P < 0.001) . Increases in MIC were found in two cases with the less susceptible strain, where peak concentration/ MIC ratios were equal to or less than 4 .

Res Vet Sci, 2000 Jun, 68(3), 211 - 6
Pharmacokinetics and penetration of danofloxacin into the gastrointestinal tract in healthy and in Salmonella typhimurium infected pigs; Lindecrona RH et al.; The pharmacokinetics and penetration of danofloxacin into the gastrointestinal tract in healthy pigs and in pigs experimentally infected with Salmonella typhimurium were studied.In the infected pigs, a decrease in body clearance and an increase in mean elimination half-life was observed (P < 0.01) . Moreover a significant reduction in the volume of the peripheral compartment was found . Danofloxacin distributed well to the gastrointestinal tract achieving high AUC / AUC(plasma)ratios in both groups of pigs . However, compared to the healthy pigs AUC / AUC(plasma)ratios decreased in the infected pigs . Salmonella infection led to an increase in mean residence time (MRT) in the small intestines and lymph nodes and a decrease in MRT in caecum and colon .

Nat Struct Biol, 2000 Jul, 7(7), 560 - 4
Structure of arylamine N-acetyltransferase reveals a catalytic triad; Sinclair JC et al.; Enzymes of the arylamine N-acetyltransferase (NAT) family are found in species ranging from Escherichia coli to humans . In humans they are known to be responsible for the acetylation of a number of arylamine and hydrazine drugs, and they are strongly linked to the carcinogenic potentiation of certain foreign substances . In prokaryotes their substrate specificities may vary and members of the gene family have been linked to pathways including amide synthesis during rifamycin production . Here we report the crystal structure at 2.8 A resolution of a representative member of this family from Salmonella typhimurium in the presence and absence of a covalently bound product analog . The structure reveals surprising mechanistic information including the presence of a Cys-His-Asp catalytic triad . The fold can be described in terms of three domains of roughly equal length with the second and third domains linked by an interdomain helix . The first two domains, a helical bundle and a beta-barrel, make up the catalytic triad using a structural motif identical to that of the cysteine protease superfamily.

Zh Mikrobiol Epidemiol Immunobiol, 1999 Nov-Dec, (6), 3 - 8
{The mechanism of action and the nature of the factors inducing the formation of uncultivatable forms of Salmonella typhimurium}; Gintsburg AL et al.; Approaches to the study of gene expression in the process of transition and prolonged stay of bacterial cells in the uncultivated state have been worked out with the use of the methods of reverse transcription and polymerase chain reaction . The differential expression of genes pqi and stn has been shown to occur in the uncultivated forms of S . typhimurium . The factor inducing the process of the transition of Salmonella cells into the uncultivated state has been found out.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 633 - 42
In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials; Heithoff DM et al.; Microbial pathogens possess a repertoire of virulence determinants that each make unique contributions to fitness during infection . Analysis of these in vivo-expressed functions reveals the biology of the infection process, encompassing the bacterial infection strategies and the host ecological and environmental retaliatory strategies designed to combat them (e.g . thermal, osmotic, oxygen, nutrient and acid stress) . Many of the bacterial virulence functions that contribute to a successful infection are normally only expressed during infection . A genetic approach was used to isolate mutants that ectopically expressed many of these functions in a laboratory setting . Lack of DNA adenine methylase (Dam) in Salmonella typhimurium abolishes the preferential expression of many bacterial virulence genes in host tissues . Dam- Salmonella were proficient in colonization of mucosal sites but were defective in colonization of deeper tissue sites . Additionally, Dam- mutants were totally avirulent and effective as live vaccines against murine typhoid fever . Since dam is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality worldwide, Dams are potentially excellent targets for both vaccines and antimicrobials.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 623 - 31
Salmonella interactions with host cells: in vitro to in vivo; Finlay BB et al.; Salmonellosis (diseases caused by Salmonella species) have several clinical manifestations, ranging from gastroenteritis (food poisoning) to typhoid (enteric) fever and bacteraemia . Salmonella species (especially Salmonella typhimurium) also represent organisms that can be readily used to investigate the complex interplay that occurs between a pathogen and its host, both in vitro and in vivo . The ease with which S . typhimurium can be cultivated and genetically manipulated, in combination with the availability of tissue culture models and animal models, has made S . typhimurium a desirable organism for such studies . In this review, we focus on Salmonella interactions with its host cells, both in tissue culture (in vitro) and in relevant animal models (in vivo), and compare results obtained using these different models . The recent advent of sophisticated imaging and molecular genetic tools has facilitated studying the events that occur in disease, thereby confirming tissue culture results, yet identifying new questions that need to be addressed in relevant disease settings.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 613 - 22
Identification and analysis of bacterial virulence genes in vivo; Unsworth KE et al.; Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens . Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches . A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes . We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium . This virulence determinant is required for intracellular survival . Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole . Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 565 - 74
DNA topology and adaptation of Salmonella typhimurium to an intracellular environment; Marshall DG et al.; The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells . A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth . Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS . The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced . Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S . typhimurium cells by the macrophage . These data indicate that DNA topology in S . typhimurium undergoes significant changes during adaptation to the intracellular environment . A model describing how this process may operate is discussed.

J Environ Sci Health B, 2000 Jul, 35(4), 517 - 25
Recovery of a marker strain of Salmonella typhimurium in litter and aerosols from isolation rooms containing infected chickens; Kwon YM et al.; Screening of poultry flocks for foodborne pathogen Salmonella contamination is critical for Salmonella control in preharvest stages of poultry production . In this study, two sampling methods (litter and air filter) were compared for detection of S . typhimurium from experimentally infected chicks some of which had received either a probiotic competitive exclusion culture or transfer of cecal contents from salmonellae-free adult birds . At 4, 9, and 11 days after inoculation, S . typhimurium samples were enumerated by selective plating . For both types of sampling, the control birds yielded the greatest levels of environmental contamination followed by the samples from the probiotic inoculated birds with the birds receiving the cecal transfer culture having the lowest levels of contamination . Although the two sampling methods responded in a similar fashion, detection sensitivity needs to be increased for air filter sampling.

J Environ Sci Health B, 2000 Jul, 35(4), 503 - 16
Response of selected poultry cecal probiotic bacteria and a primary poultry Salmonella typhimurium isolate grown with or without glucose in liquid batch culture; Durant JA et al.; The objective of this study was to determine whether fermentation by a cecal probiotic co-culture of an Enterococcus sp . and Veillonella sp . would inhibit the in vitro growth of a S . typhimurium poultry isolate . The growth rates of S . typhimurium and Enterococcus were significantly reduced at pH 5 . At the two pH levels, there was a significant (p < 0.001) increase at 24 h in colony forming units for each of the bacteria enumerated from the mixed culture compared to the respective pure culture enumerations . S . typhimurium was not inhibited in mixed cultures . The mixed cultures produced more acetate than any of the pure cultures and lactate produced by Enterococcus appeared to be utilized by Veillonella.

Structure Fold Des, 2000 Jun 15, 8(6), 655 - 67
The first crystal structure of a phospholipase D; Leiros I et al.; BACKGROUND: The phospholipase D (PLD) superfamily includes enzymes that are involved in phospholipid metabolism, nucleases, toxins and virus envelope proteins of unknown function . PLD hydrolyzes the terminal phosphodiester bond of phospholipids to phosphatidic acid and a hydrophilic constituent . Phosphatidic acid is a compound that is heavily involved in signal transduction . PLD also catalyses a transphosphatidylation reaction in the presence of phosphatidylcholine and a short-chained primary or secondary alcohol . RESULTS: The first crystal structure of a 54 kDa PLD has been determined to 1.9 A resolution using the multiwavelength anomalous dispersion (MAD) method on a single WO(4) ion and refined to 1.4 A resolution . PLD from the bacterial source Streptomyces sp . strain PMF consists of a single polypeptide chain that is folded into two domains . An active site is located at the interface between these domains . The presented structure supports the proposed superfamily relationship with the published structure of the 16 kDa endonuclease from Salmonella typhimurium . CONCLUSIONS: The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function . The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.

Virology, 2000 Jun 20, 272(1), 218 - 24
Characterization of phi8, a bacteriophage containing three double-stranded RNA genomic segments and distantly related to Phi6; Hoogstraten D et al.; The three double-stranded RNA genomic segments of bacteriophage Phi8 were copied as cDNA, and their nucleotide sequences were determined . Although the organization of the genome is similar to that of Phi6, there is no similarity in either the nucleotide sequences or the amino acid sequences, with the exception of the motifs characteristic of viral RNA polymerases that are found in the presumptive polymerase sequence . Several features of the viral proteins differ markedly from those of Phi6 . Although both phages are covered by a lipid-containing membrane, the protein compositions are very different . The most striking difference is that protein P8, which constitutes a shell around the procapsid in Phi6, is part of the membrane in Phi8 . The host attachment protein consists of two peptides rather than one and the phage attaches directly to the lipopolysaccharide of the host rather than to a type IV pilus . The host range of Phi8 includes rough strains of Salmonella typhimurium and of pseudomonads

Plasmid, 2000 Jul, 44(1), 24 - 33
The virulence plasmid of Salmonella typhimurium contains an autoregulated gene, rlgA, that codes for a resolvase-like DNA binding protein; Massey RC et al.; The virulence plasmid of Salmonella typhimurium contains a gene, rlgA, that shows strong homology to several reported resolvase-like proteins . This gene maps 5 kb upstream of spv locus, the major virulence determinant on the plasmid . Regulation of rlgA was studied using a lacZ transcriptional reporter fusion . The rlgA gene was found to be repressed at the level of transcription by its own product and to be expressed maximally in the late exponential phase of growth . The transcription start site of the rlgA gene was determined and the RlgA binding site was mapped and found to overlap with the transcription initiation signals . A derivative of the virulence plasmid was constructed with a knockout mutation in rlgA . This mutation did not alter the stability of the virulence plasmid nor did it affect the ability of S . typhimurium to cause systemic disease in mice .

Microb Pathog, 2000 Jul, 29(1), 53 - 9
Use of confocal microscopy to detect Salmonella typhimurium within host cells associated with Spv-mediated intracellular proliferation; Matsui H et al.; The major limitation in histological examination of orally inoculated mice of Salmonella typhimurium has been the difficulty of attaining high enough levels for immunochemical detection . This problem has been solved by the use of confocal laser scanning microscopy (CLSM) analysis, which allows detection of bacteria in the immunostained sections of mouse spleens at a minimum rate of approximately 1000 colony-forming units (cfu)/spleen . Here, we demonstrate that over 80% of salmonellae of the wild type of S . typhimurium were detected intracellularly within Mac-1 positive cells by the CLSM analysis of immunostained sections from spleens in orally or subcutaneously inoculated mice . Only 40% of salmonellae of the spv -deleted strain were detected inside Mac-1 positive cells . These data suggest that the spv genes play a key role in intracellular proliferation within phagocytes in the mouse spleen .

Eur J Oral Sci, 2000 Jun, 108(3), 202 - 6
Treatment of inflamed ferret dental pulps with recombinant bone morphogenetic protein-7; Rutherford RB et al.; Recombinant human BMP-7 (bone morphogenetic protein-7, osteogenic protein-1) is osteogenic, dentinogenic and cementogenic when implanted into the appropriate tissue in vivo . However, most studies characterizing the induction of these tissues have implanted BMP-7 into freshly surgerized, clinically healthy tissues . To determine if BMP-7 is dentinogenic in inflamed dental pulps, we applied BMP-7 to inflamed ferret pulps . A single application of 5 microg of a commercial preparation of lipopolysaccharide (LPS) from Salmonella typhimurium directly to the coronal pulp induced a reversible mixed inflammatory exudate of moderate intensity within 3 d . Treatment with a single application of 2.5, 7.5 or 25 microg recombinant human BMP-7/mg collagen (2 mg total mass/tooth) induced reparative dentinogenesis in controls but not LPS treated dental pulps . These data reveal that a single application of up to 50 microg/tooth of exogenous recombinant BMP-7 is insufficient to induce reparative dentinogenesis in ferret teeth with reversible pulpitis . Given that pulp cells in the inflamed tissues likely retain the capacity to respond to exogenous BMP-7, it is possible that insufficient active recombinant protein is available to induce tissue formation in experimentally inflamed dental pulps.

Virus Genes, 2000, 20(2), 149 - 57
Cloning, sequencing, expression and promoter analysis of a structural protein of bacteriophage MB78; Kolla V et al.; Bacteriophage MB78, a virulent phage of Salmonella typhimurium isolated in our laboratory . It is different from the well-known temperate phage P22 and 9NA . A detailed physical map has been constructed . To understand more about the physiology and genetics of this interesting phage it has become necessary to fragment the phage genome, clone the fragments and analyze in depth . A number of promoters of bacteriophage MB78 have been cloned and characterized recently . As a part of this program, in this investigation, we report cloning, sequencing and expression and promoter analysis of the ClaI G fragment . We identified the expressed protein as phage structural . Phage structural proteins play a vital role in forming the core head of the phage particle.

EMBO J, 1983, 2(8), 1345 - 50
The nucleotide sequence of the first externally suppressible--1 frameshift mutant, and of some nearby leaky frameshift mutants; Atkins JF et al.; Nine mutants within a 23 nucleotide sequence of the trpE gene of Salmonella typhimurium have been characterized . trpE91, a mutant which is externally suppressible has a single base deletion . Eight (or nine) nucleotides upstream of this deletion, two independently isolated mutations have the same transversion . In combination with trpE91 these mutations lead to partial restoration of synthesis of anthranilate synthetase in the absence of external suppressors . In the transversion the sequence A CA is changed to A AA and this new sequence may be the site where frameshifting occurs to allow leakiness . Leakiness is displayed by two further mutants of the same sign as trpE91, and one of the opposite sign, in the absence of any base substitution or external suppressors . Specific sequences, e.g., UUUC, may be especially prone to frameshifting and this sequence is created at the site of the +1 frameshift mutant which displays leakiness . In the new reading frame generated by the two -1 frame leaky mutants, a tryptophan codon is encountered . Leakiness is necessarily detected in the absence of tryptophan and under these conditions there will be a shortage of charged tryptophan tRNA . The possibility of such functional imbalance leading to frameshifting in these mutants is discussed.

J Biol Chem, 2000 Sep 8, 275(36), 27576 - 86
Analysis of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2 . Identification of residue His-46 as the site of guanylylation; Thomas MG et al.; CobU is a bifunctional enzyme involved in adenosylcobalamin (coenzyme B(12)) biosynthesis in Salmonella typhimurium LT2 . In this bacterium, CobU is the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase needed to convert cobinamide to adenosylcobinamide-GDP during the late steps of adenosylcobalamin biosynthesis . The guanylyltransferase reaction has been proposed to proceed via a covalently modified CobU-GMP intermediate . Here we show that CobU requires a nucleoside upper ligand on cobinamide for substrate recognition, with the nucleoside base, but not the 2'-OH group of the ribose, being important for this recognition . During the kinase reaction, both the nucleotide base and the 2'-OH group of the ribose are important for gamma-phosphate donor recognition, and GTP is the only nucleotide competent for the complete nucleotidyltransferase reaction . Analysis of the ATP:adenosylcobinamide kinase reaction shows CobU becomes less active during this reaction due to the formation of a covalent CobU-AMP complex that holds CobU in an altered conformation . Characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase reaction shows the covalent CobU-GMP intermediate is on the reaction pathway for the generation of adenosylcobinamide-GDP . Identification of a modified histidine and analysis of cobU mutants indicate that histidine 46 is the site of guanylylation.

J Nat Prod, 2000 Jun, 63(6), 773 - 6
Insecticidal and mutagenic evaluation of two annonaceous acetogenins; Guadano A et al.; Annonaceous acetogenins represent a new class of bioactive compounds whose primary mode of action is the inhibition of NADH-ubiquinone oxidoreductase . Given the potential pesticidal use of such a class of compounds, we have further evaluated the antifeedant and insecticidal effects of squamocin and annonacin, two annonaceous acetogenins, on Spodoptera littoralis, Leptinotarsa decemlineata, and Myzus persicae . Additionally, to partially assess their environmental risk, we have also tested their mutagenicity in Salmonella typhimurium strains TA98, TA100, and TA102 in the presence and absence of a metabolic activation system . Among the test compounds, annonacin showed antifeedant effects on L . decemlineata, while squamocin was toxic to L . decemlineata and M . persicae . Neither acetogenin was mutagenic, although both were toxic in the absence of a metabolic activation system . We compared these results with those obtained with rotenone, a well-known respiratory inhibitor that was highly toxic to L . decemlineata and M . persicae and showed no mutagenicity/toxicity in the S . typhimurium strains tested up to a concentration of 1000 microg per plate.

Vet Microbiol, 2000 Jul 3, 75(1), 73 - 82
Characterisation of streptomycin resistance determinants in Danish isolates of Salmonella Typhimurium; Madsen L et al.; Fifty six Danish streptomycin (Sm) resistant isolates of Salmonella enterica serotype Typhimurium from pigs (n=34), calves (n=3) and humans (n=19) were characterised with respect to co-resistances (14 drugs), transferability of Sm-resistance by conjugation, genetic determinants encoding Sm-resistance and diversity with respect to localisation of genes in the genome and DNA-sequences . Forty-six strains carried resistance(s) other than Sm-resistance . Nineteen different co-resistance patterns were observed and tetracycline was the most commonly observed resistance in these patterns . In 22 of the strains, Sm-resistance was transferred by conjugation . Eleven strains contained the gene aadA only, six strains contained aadA+strA+strB, and 35 strains contained strA+strB . Partial sequences of aadA were obtained from four strains . Three strains showed identical sequences to a published aadA sequence from the transposon Tn7, and in one strain the sequence showed one synonymous substitution compared to this sequence . Partial sequences were obtained of strA and strB in seven strains . The sequence of strB was identical to the published sequence of the plasmid RSF1010 in all strains . All seven sequences of strA were identical and differed from the sequence of strA in RSF1010 by two non-synonymous substitutions.

J Pharm Pharmacol, 2000 May, 52(5), 593 - 8
Anti-leukaemic and anti-mutagenic effects of di(2-ethylhexyl)phthalate isolated from Aloe vera Linne; Lee KH et al.; Extracts of Aloe vera Linne have been found to exhibit cytotoxicity against human tumour cell lines . This study examines the anti-tumour effects of di(2-ethylhexyl)phthalate (DEHP) isolated from Aloe vera Linne, in human and animal cell lines . Its anti-mutagenic effects were examined using Salmonella typhimurium TA98 and TA100 strains . Growth inhibition was specifically exerted by DEHP against three leukaemic cell lines at concentrations below 100 microg mL(-1) . At 100 microg mL(-1) DEHP, K562, HL60 and U937 leukaemic cell lines showed growth inhibition of 95, 97 and 95%, respectively . DEHP exhibited an inhibitory activity of 74, 83 and 81%, respectively, in K562, HL60 and U937 cell lines at a concentration of 10 microg mL(-1) . At a concentration of 1 microg mL(-1), DEHP exerted an inhibitory activity of 50, 51 and 52%, respectively, in K562, HL60 and U937 . In a normal cell line, MDBK, DEHP exerted 30% growth inhibition at a concentration of 100 microg mL(-1), and showed no inhibitory activity at concentrations below 50 microg mL(-1) . It was found that DEHP exerted anti-mutagenic activity in the Salmonella mutation assay . The number of mutant colonies of Salmonella typhimurium strain TA98 upon exposure to AF-2 (0.2 microg/plate) decreased in a concentration-dependent manner in the presence of different DEHP concentrations (decreasing to 90.4, 83.9, 75.4, 69.6 and 46.9%, respectively, for DEHP concentrations of 100, 50, 10, 5 and 1 microg/plate) . In the case of Salmonella typhimurium strain TA100, DEHP reduced AF-2-induced mutagenicity at 1, 5, 10, 50 and 100 microg/plate to 57.4, 77.5, 80.0, 89.0 and 91.5%, respectively . The isolated compound from Aloe vera Linne, DEHP, was considered to be the active principle responsible for anti-leukaemic and anti-mutagenic effects in-vitro.

Environ Mol Mutagen, 2000, 35(4), 312 - 8
Cell transformation and genotoxicity induced by bis(2, 3-dichloro-1-propyl) ether; Neurath G et al.; Bis(dichloropropyl) ether isomers have been identified in a petrochemical plant effluent through a toxicity identification evaluation study in the United States . They have also been observed in the microgram per liter range along one of the largest rivers in Europe, the Elbe River . In the present investigation, the genotoxic and transforming activity of a bis(dichloropropyl) ether isomer, bis(2,3-dichloro-1-propyl) ether, was assayed in vitro . The results demonstrate that bis(2,3-dichloro-1-propyl) ether is a potent mutagen in Salmonella typhimurium strains TA 100, TA 1535, and to a lesser extent in strain TA 98, but only when tested in the presence of a metabolic activation system (S9 mix) . We have also investigated the induction of micronuclei by bis(2,3-dichloro-1-propyl) ether in the metabolically competent cell line, MCL-5 . A linear, dose-dependent increase in micronuclei was observed following exposure to bis(2,3-dichloro-1-propyl) ether . The DNA strand-breaking capacity of this chemical was assessed in the alkaline single-cell gel electrophoresis ("comet") assay with MCL-5 cells . Bis(2,3-dichloro-1-propyl) ether clearly induced DNA strand breaks in the 4.5-45.5 microg/ml dose range . The ether also induced malignant transformation in C3H/M2 mouse fibroblasts after metabolic activation (S9 mix) . Thus, it must be suspected that bis(2, 3-dichloro-1-propyl) ether may possess a carcinogenic potential . Since the compound along with its isomers is present in considerable concentrations in surface water, their elimination is a matter of significant public concern .

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7539 - 44
A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium; Miao EA et al.; Type III secretion systems (TTSS) are important virulence factors that Gram-negative bacteria use to translocate proteins into the cytoplasm of eukaryotic host cells . Salmonellae encode two virulence-associated TTSS . The Salmonella pathogenicity island 1 (SPI1)-encoded TTSS is active on contact with host cells, whereas the Salmonella pathogenicity island 2 (SPI2)-encoded TTSS is expressed after phagocytosis of bacteria by host cells . Previously, no consensus signal sequence for translocation has been identified among TTSS effector proteins . In this work, seven proteins, termed Salmonella-translocated effectors (STE), are described that contain conserved amino acid sequences that direct translocation by TTSS in Salmonella typhimurium . STE that are coordinately regulated with SPI2 gene expression contain translocation signals that are recognized by the SPI2 but not by the SPI1 TTSS . STE that are constitutively expressed contain signals that direct translocation through both SPI1 and SPI2 TTSS . Of the seven STE examined, SspH1 and SspH2 have been previously shown to be translocated and involved in virulence; SlrP and SifA were identified as virulence factors, but were not previously known to be associated with TTSS; and SseI, SseJ, and SifB were previously unidentified . Three STE genes (sspH1, sspH2, and sseI) are located within temperate bacteriophages, suggesting a common mechanism for the dissemination of more recently evolved STE.

J Mol Biol, 2000 May 26, 299(1), 27 - 51
Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages; Juhala RJ et al.; We report the complete genome DNA sequences of HK97 (39,732 bp) and HK022 (40,751 bp), double-stranded DNA bacteriophages of Escherichia coli and members of the lambdoid or lambda-like group of phages . We provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of E . coli phage lambda and Salmonella typhimurium phage P22 . The comparisons confirm that these phages are genetic mosaics, with mosaic segments separated by sharp transitions in the sequence . The mosaicism provides clear evidence that horizontal exchange of genetic material is a major component of evolution for these viruses . The data suggest a model for evolution in which diversity is generated by a combination of illegitimate and homologous recombination and mutational drift, and selection for function produces a population in which most of the surviving mosaic boundaries are located at gene boundaries or, in some cases, at protein domain boundaries within genes . Comparisons of these genomes highlight a number of differences that allow plausible inferences of specific evolutionary scenarios for some parts of the genome . The comparative analysis also allows some inferences about function of genes or other genetic elements . We give examples for the generalized recombination genes of HK97, HK022 and P22, and for a putative headtail adaptor protein of HK97 and HK022 . We also use the comparative approach to identify a new class of genetic elements, the morons, which consist of a protein-coding region flanked by a putative delta 70 promoter and a putative factor-independent transcription terminator, all located between two genes that may be adjacent in a different phage . We argue that morons are autonomous genetic modules that are expressed from the repressed prophage . Sequence composition of the morons implies that they have entered the phages' genomes by horizontal transfer in relatively recent evolutionary time.

Int J Food Microbiol, 2000 Jun 1, 56(2-3), 167 - 77
Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes; Li X et al.; Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study . After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR . The slot blot assay was optimized and used to detect PCR products . The lowest detection level of this method was 10(3) cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction . Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples . The whole detection procedure was completed within 7 h . After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10(3) cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen . The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L . monocytogenes in food samples such as milk.

Comp Med, 2000 Apr, 50(2), 124 - 32
Histologic, cytologic, and bacteriologic examinations of experimentally induced Salmonella typhimurium infection in Lewis rats; Thygesen P et al.; BACKGROUND AND PURPOSE: Histopathologic changes, cellular composition, and bacterial spreading were studied in rat spleen after experimentally induced infection with Salmonella typhimurium . METHODS: Lewis rats were inoculated intraperitoneally with 10(6) bacteria . Spleen weight, cell numbers, and cell surface markers were studied together with histopathologic changes, and expression of inducible nitric oxide synthase (iNOS) . The spread of bacteria to blood, spleen, liver, mesenteric lymph nodes, lung, and kidney was studied at 12 hours, and 1, 3, 7, 14, and 28 days after inoculation . RESULTS: Experimentally induced infection caused an increase in spleen weight and leukocyte numbers, and a decrease in CD49d, on postinoculation days (PID) 3 through 7 . Numerous granulomas were disseminated throughout the splenic red pulp also on PID 3 through 7 . From PID 14 on, clearance of cellular exudate and regeneration of tissue structure were observed . Massive expression of iNOS was seen on PID 3 . Bacterial growth was observed in liver and spleen from 12 hours to 14 days after inoculation . Bacteria were detected in blood on PID 3 and mesenteric lymph nodes were infected from PID 3 through 14 . CONCLUSIONS: Salmonella typhimurium was rapidly taken up by the reticuloendothelial system . The infection induced weight increase and reversible changes in the spleen, peaking on PID 3 with granuloma formation and infiltration with macrophages . On PID 3, extensive production of iNOS within the granulomas was observed, suggesting initial killing of phagocytosed bacteria, followed by bacterial clearance and tissue regeneration . Cell surface marker expression on CD4+ T cells indicated no change in their numbers; however, there was a time-dependent change in expression of CD49d.

FEMS Microbiol Lett, 2000 Jun 15, 187(2), 145 - 50
Virulence and mutation rates of Salmonella typhimurium strains with increased mutagenic strength in a mouse model; Campoy S et al.; Two strains of Salmonella typhimurium presenting increased mutation rates, either spontaneous or mediated by DNA damage, have been constructed . One of the strains carries a null mutS mutation, while the other harbors plasmid pRW30, which contains the Escherichia coli umuDC operon . The virulence of these strains has been determined by inoculating BALB/c or Swiss mice . The 50% lethal dose of both strains is identical to that obtained for the wild-type . Likewise, the two strains and the wild-type contribute equally to animal death in mixed infections . The frequency of Nal(R) mutants recovered from animals inoculated with either wild-type or MutS(-) cells was not affected by the presence of pRW30 . These results indicate that the DNA damage which S . typhimurium cells can suffer during the infectious process by host cell metabolites does not cause induction of the SOS response at levels able to trigger the error-prone DNA repair pathway.

J Food Prot, 2000 Jun, 63(6), 747 - 52
Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation; Tsai GJ et al.; The antibacterial activity of a chitooligosaccharide mixture prepared by digestion of shrimp chitosan with cellulase at 50 degrees C for 14 h was evaluated . Sugars with 1 to 8 degrees of polymer (DP) were found in this chitooligosaccharide mixture, and the weight percentage of sugars with DP > or = 6 was 44.3% . Minimal lethal concentrations of this mixture against Aeromonas hydrophila, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholerae, and Vibrio parahaemolyticus in nutrient broth were 5 to 29 ppm, which were much lower than those of the chitosan reactant (50 to 1,000 ppm) . The antibacterial activity of this mixture in the sterilized milk against E . coli O157, L . monocytogenes, Salmonella Typhimurium, and S . aureus was much stronger at 4 degrees C than at 37 degrees C . When raw milk was supplemented with either 0.24% or 0.48% (wt/vol) of this oligosaccharide mixture and stored at 4 degrees C for 12 days, its mesophilic and psychrotrophic counts were reduced by at least 3 log cycles, and there was very little change in pH . In addition, this mixture retarded the growth of Salmonella species and caused quicker reduction of Staphylococcus species in raw milk . Accordingly, the shelf life of raw milk at 4 degrees C was extended by at least 4 days.

Cancer Biother Radiopharm, 1996 Apr, 11(2), 145 - 53
Attenuated Salmonella typhimurium containing interleukin-2 decreases MC-38 hepatic metastases: a novel anti-tumor agent; Saltzman DA et al.; Currently, there is no long-term effective treatment for unresectable hepatic malignancies . Salmonella sp . are known to naturally track to the liver during active infection . To develop a biological vector for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes, the avirulent and highly immunogenic chi 4550 strain of Salmonella typhimurium was used as a vector for IL-2 . The gene for human IL-2 was cloned into plasmid pYA292 (renamed pIL-2) and inserted into the attenuated Salmonella typhimurium and renamed {chi 4550 (pIL-2)} . This transformant was found to produced biologically active IL-2 demonstrated by NK cell activation in a 4 hour chromium release cytotoxicity assay . To determine anti-tumor potential, MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases and metastases were subsequently enumerated after 12 days . Statistical significance was determined by ANOVA with Fisher's test for significance . Hepatic metastases enumerated by blinded observers revealed that the mean number of metastases was 106.4 in control mice, 103.7 in mice gavage fed attenuated salmonella without IL-2 {chi 4550(pYA292)}, and 44.3 in mice fed the chi 4550(pIL2); (ANOVA: p < 0.01) . Culture of livers and spleens in mice administered a single gavage dose of salmonella demonstrated persistent colonization for up to 4 weeks . No observable toxicity was seen to either IL-2 or salmonella . These studies demonstrate that the chi 4550(pIL2) is a novel form of in vivo biotherapy which produces biologically active IL-2 and employs the oral route of administration to stimulate an immune response against malignancy in the liver.

Cancer Biother Radiopharm, 1997 Feb, 12(1), 37 - 45
Patterns of hepatic and splenic colonization by an attenuated strain of Salmonella typhimurium containing the gene for human interleukin-2: a novel anti-tumor agent; Saltzman DA et al.; Currently, there is no effective treatment for unresectable hepatic malignancies . Salmonella sp . are known to naturally track to the liver during active infection . A live biological vector was developed for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes . The avirulent and highly immunogenic c4550 strain of Salmonella typhimurium was used to express the IL-2 protein {renamed c4550(pIL-2)} . We have previously demonstrated that the c4550(pIL-2) produces biologically active IL-2 (up to 46.2 IU/ml) and that a single gavage feeding of 10(7) colony forming units (cfu) of c4550(pIL-2) significantly reduced the number of hepatic metastases when compared to animals fed salmonella lacking the IL-2 gene or non-treated controls . The goal of the current studies was to determine the pattern of splenic and hepatic colonization of Salmonella-IL2 . Hepatic and splenic colonization was determined following administration of 10(7) cfu of c4550(pIL-2) and c4550(pYA292) via a single gavage feeding to C57BL/6 mice . Five experiments of antibiotic regimen administration were conducted where splenic and hepatic homogenates were cultured after 14 days of parenteral and/or oral antibiotics . The natural history of hepatic and splenic colonization was also determined for animals without antibiotic treatment . Despite administration of various antibiotic regimens using different routes, eradication of salmonella with and without IL-2 was not achieved . Salmonella, however, was not cultured from hepatic and splenic tissue at 4 months after a single gavage feeding of salmonella with no specific treatment . In conclusion, oral administration of c4550(pIL-2) may represent a novel form of in vivo biotherapy for unresectable hepatic malignancies . Antibiotics do not accelerate eradication of this bacteria and it appears that c4550(pIL-2) follows the natural pathophysiological of salmonella infection in which eradication from the splenic and hepatic tissue occurs over a period of 2-4 months.

Invest Ophthalmol Vis Sci, 2000 Jun, 41(7), 1823 - 6
Recurrent intraocular inflammation in endotoxin-induced uveitis; Kozhich AT et al.; PURPOSE: Endotoxin-induced uveitis (EIU) in rats and mice peaks 24 hours after endotoxin injection and is commonly assumed to be a monophasic disease . This study examined intraocular inflammation at later time points to determine whether endotoxin injection can induce recurrent intraocular inflammation in strains of mice with high or moderate levels of susceptibility to EIU . METHODS: EIU was elicited in two mouse strains with high (C3H/HeN) and moderate (FVB/N) susceptibility, by means of intraperitoneal injections of Salmonella typhimurium endotoxin . Inflammatory cells in the anterior and posterior segments of the eye were counted by a masked observer on histologic sections of eyes from 1 to 17 days after endotoxin injection . RESULTS: A bimodal distribution of inflammatory cell infiltration was noted in eyes from C3H/HeN mice . As previously reported, inflammation peaked at 24 hours after endotoxin injection . However, a second, more pronounced peak of intraocular inflammation occurred approximately 5 days after endotoxin injection . FVB/N mice had a single peak of intraocular inflammation 4 days after injection . CONCLUSIONS: Endotoxin injection in C3H/HeN elicits recurrent intraocular inflammation . The previously unrecognized second peak of inflammation is more severe than the initial inflammatory disease . Studies on this second inflammatory peak may be useful in determining the pathogenesis of recurrent uveitis in humans.

Lett Appl Microbiol, 2000 Jun, 30(6), 461 - 7
Heat inactivation of Salmonella typhimurium DT104 in beef as affected by fat content; Juneja VK et al.; The heat resistance of an eight-strain cocktail of Salmonella typhimurium DT104 was determined at 58-65 degrees C in beef containing 7, 12, 18 or 24% fat . Inoculated beef was packaged in bags completely immersed in a circulating water bath and held at 58, 60, 62.5 and 65 degrees C for a predetermined length of time . The surviving cell population was enumerated by spiral plating heat-treated samples onto tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate . Preliminary studies on thermal inactivation of the Salmonellae isolates in chicken broth indicated no correlation between heat resistance and origin of the isolates . While linear survival curves were observed in chicken broth, inactivation kinetics in beef showed deviations from the first order kinetics, represented by an initial lag period or shoulder before any death occurred with time . Overall, increased fat levels in beef resulted in longer lag periods and lower D-values, suggesting that the lag periods must be taken into account and added to the D-values for calculating the time required at a specific temperature for achieving a specific lethality for Salm . typhimurium DT104 in beef . Thermal death times from this study will assist the retail food industry to design cooking regimes that ensure safety of beef contaminated with Salm . typhimurium DT104.

J Toxicol Sci, 2000 May, 25(2), 63 - 6
Mutagenicity of bovine lactoferrin in reverse mutation test; Yamauchi K et al.; The mutagenicity of bovine lactoferrin, which is an iron-binding glycoprotein in milk, was evaluated by the Ames mutagenicity test . A total of 5 test strains including 3 base-pair substitution-type strains, Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2uvrA, and 2 frameshift-type strains, TA98 and TA1537, were used in the test . The test was performed by both the direct method and the metabolic activation method with preincubation applied in each instance . The concentration range of the test solution was 0.16 to 5.00 mg/100 microliters (plate) . Results of the test revealed that the number of revertant colonies at each concentration of the test solutions was less than 1.4 times that of the control group . In the test system used, bovine lactoferrin did not exhibit mutagenicity.

Mol Microbiol, 2000 Jun, 36(5), 1024 - 33
Unravelling the mysteries of virulence gene regulation in Salmonella typhimurium; Lucas RL et al.; Salmonella typhimurium, which causes gastroenteritis in calves and humans as well as a typhoid-like disease in mice, uses numerous virulence factors to infect its hosts . Genes encoding these factors are regulated by many environmental conditions and regulatory pathways in vitro . Many virulence genes are specifically induced at particular sites during infection or in cultured host cells . The complex regulation of virulence genes observed in vitro may be necessary to restrict their expression to specific locations within the host . In vitro and in vivo studies provide clues about how virulence genes might be regulated in vivo . Future studies must assess the actual environmental signals and regulators that modulate each virulence gene in vivo and determine how multiple regulatory pathways are integrated to co-ordinate the appropriate expression of virulence factors at specific sites in vivo.

Mol Microbiol, 2000 May, 36(4), 962 - 72
Oligomerization of the chromatin-structuring protein H-NS; Smyth CP et al.; H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression . The mechanism by which this is achieved remains unclear . Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties . We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium . The N-terminal 89 amino acids are responsible for oligomerization . The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation . Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers . Similarly, in the full-length protein no single, defined oligomeric state is adopted . The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain . These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein . This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Immunol Lett, 2000 May 1, 72(2), 93 - 9
Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on activity of murine thymocytes and peritoneal macrophages; Navolotskaya EV et al.; The purpose of the present study was to investigate properties and mechanism of action of the synthetic adrenocorticotropin (ACTH)-like peptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain . The ACTH-like peptide was shown to act as an immunosuppressive agent in vitro: it inhibits the blast transformation of mouse thymocytes and reduces the spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against Salmonella typhimurium 415 virulent strain bacteria . High affinity receptors for the ACTH-like peptide were found on thymocytes and macrophages and shown to be at the same time the receptors for ACTH . The kinetic characteristics of the ACTH-like peptide and 125I-labeled ACTH (13-24) (ACTH 'address segment') specific binding to the receptors were determined . It was found that the ACTH-like peptide binding to the receptors on target cells is accompanied by an increase in both adenylate cyclase activity and intracellular cAMP content.

Microb Pathog, 2000 Jun, 28(6), 373 - 8
Evaluation of invasion-conferring genotypes and antibiotic-induced hyperinvasive phenotypes in multiple antibiotic resistant Salmonella typhimurium DT104; Carlson SA et al.; Antibiotic resistance in pathogenic bacteria is a problem in both industrialized and developing countries . This is especially evident in Salmonella typhimurium, a foodborne pathogen that causes gastrointestinal and systemic disease throughout the world . S . typhimurium DT104 further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes and its putative hypervirulent phenotype . Recently, we demonstrated that multiresistant S . typhimurium do not appear to be more invasive than non-resistant cohorts . In the present study, we evaluated the presence of Salmonella pathogenicity island 1 (SPI1) flanking and internal sequences in over 400 isolates of multiresistant S . typhimurium . With these same isolates, we also used a tissue culture invasion assay to evaluate a potential relationship between antibiotic exposure and a hyperinvasive phenotype . Our studies revealed that SPI1 flanking sequences are similar in multiresistant and non-resistant S . typhimurium . Furthermore, we failed to identify any isolates that were hyperinvasive in the presence of any of the 14 antibiotics evaluated . These results further indicate that the putative hypervirulence of multiresistant S . typhimurium is not likely to occur at the level of invasion .

Carcinogenesis, 2000 Jun, 21(6), 1227 - 32
Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase; Kushida H et al.; A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established . The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of SALMONELLA: Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR . Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined . CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN . CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK . The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine . In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN . We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s).

Gastroenterology, 2000 Jun, 118(6), 1061 - 71
Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells; Si-Tahar M et al.; BACKGROUND & AIMS: Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators . In the intestine, little is known about the potential endogenous anti-inflammatory molecules . Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells . METHODS: We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens . The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland . RESULTS: As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value) . SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules . SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans . Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner . Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium . In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport . CONCLUSIONS: These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.

J Biol Chem, 2000 Aug 11, 275(32), 24993 - 9
A maize homologue of the bacterial CMP-3-deoxy-D-manno-2-octulosonate (KDO) synthetases . Similar pathways operate in plants and bacteria for the activation of KDO prior to its incorporation into outer cellular envelopes; Royo J et al.; The eight-carbon acid sugar 3-deoxy-d-manno-2-octulosonate (KDO) is an essential component of Gram-negative bacterial cell walls and capsular polysaccharides . KDO is incorporated into these polymers as CMP-KDO, which is produced in an unusual activation step catalyzed by the enzyme CMP-KDO synthetase . CMP-KDO synthetase activity has traditionally been considered exclusive to Gram-negative bacteria . CMP-KDO synthetase inhibitors attract great interest owing to their potential as selective bactericides . The sugar KDO is also a component of the rhamnogalacturonan II pectin fraction of the primary cell walls of most higher plants and of the cell wall polysaccharides of some green algae . However, the metabolic pathway leading to its incorporation into the plant cell wall is unknown . This paper describes the isolation and characterization of a maize gene, which codes for a protein very similar in sequence and activity to prokaryotic CMP-KDO synthetases . Remarkably, the maize gene can complement a CMP-KDO synthetase (kdsB) Salmonella typhimurium mutant defective in cell wall synthesis . ZmCKS activity is novel in eukaryotes . The evolutionary origin of ZmCKS is discussed in relation to the high degree of conservation between the plant and bacterial genes and its atypical codon usage in maize.

Biochemistry, 2000 Jun 6, 39(22), 6602 - 15
AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC; Poole LB et al.; AhpF, the flavin-containing component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the NADH-dependent reduction of an active-site disulfide bond in the other component, AhpC, which in turn reduces hydroperoxide substrates . The amino acid sequence of the C-terminus of AhpF is 35% identical to that of thioredoxin reductase (TrR) from Escherichia coli . AhpF contains an additional 200-residue N-terminal domain possessing a second redox-active disulfide center also required for AhpC reduction . Our studies indicate that this N-terminus contains a tandem repeat of two thioredoxin (Tr)-like folds, the second of which contains the disulfide redox center . Structural and catalytic properties of independently expressed fragments of AhpF corresponding to the TrR-like C-terminus (F{208-521}) and the 2Tr-like N-terminal domain (F{1-202}) have been addressed . Enzymatic assays, reductive titrations, and circular dichroism studies of the fragments indicate that each folds properly and retains many functional properties . Electron transfer between F{208-521} and F{1-202} is, however, relatively slow (4 x 10(4) M(-)(1) s(-)(1) at 25 degrees C) and nonsaturable up to 100 microM F{1-202} . TrR is nearly as efficient at F{1-202} reduction as is F{208-521}, although neither the latter fragment, nor intact AhpF, can reduce Tr . An engineered mutant AhpC substrate with a fluorophore attached via a disulfide bond has been used to demonstrate that only F{1-202}, and not F{208-521}, is capable of electron transfer to AhpC, thereby establishing the direct role this N-terminal domain plays in mediating electron transfer between the TrR-like part of AhpF and AhpC.

Food Chem Toxicol, 2000 Jun, 38(6), 513 - 22
Bioactivation of the food mutagen 2-amino-3-methyl-imidazo{4, 5-f}quinoline (IQ) by prostaglandin-H synthase and by monooxygenases: DNA adduct analysis; Wolz E et al.; 2-Amino-3-methylimidazo{4,5-f}quinoline (IQ) is a known multisite carcinogen in rodents and a potent mutagen in acetyltransferase-proficient Salmonella typhimurium strains on activation by either monooxygenases (MFO) or by prostaglandin H synthase (PHS) . The primary metabolites formed by MFO- or PHS-mediated IQ-oxidation are different ({Wolz}), but secondary metabolism could ultimately result in the same DNA-binding intermediates . For further investigations, the DNA adduct pattern was now studied by means of (32)P-postlabelling analysis in vitro on PHS-activation and compared to that formed on MFO-mediated activation of IQ in hepatocytes . The C8-dG-IQ-adduct N-(deoxyguanosin-8-yl)-IQ was the major adduct in all samples, that is, in DNA isolated from S . typhimurium YG1024 treated with PHS-oxidized IQ or its nitro-derivative, from ovine seminal vesicle cells, and from hepatocytes exposed to IQ or nitro-IQ . This speaks for the formation of a common DNA-reactive species, presumably an arylnitrenium ion, generated by different pathways in these cellular model systems . The similarity of critical biochemical DNA lesions suggests that PHS can contribute to the bioactivation of IQ in vivo: this is of particular interest in extrahepatic tissues since expression of cytochrome P450 isoenzymes known to be involved in the N-oxidation of IQ is largely confined to the liver.

Biochim Biophys Acta, 2000 Jul 3, 1475(2), 169 - 74
Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells; Bertoloni G et al.; The photosensitizing action of hematoporphyrin (Hp) on two Staphylococcus aureus strains was investigated to determine if the photoprocess induces in vivo damage in DNA in addition to that occurring at the level of the cytoplasmic membrane . The results obtained demonstrate that the photokilling is dependent on the Hp dose even though the two strains, having a similar Hp-binding capacity, show different levels of photosensitivity . The electrophoretic analysis of cytoplasmic membrane proteins and DNA (chromosomal and plasmidial) suggests that the membrane represents the primary target of the photoprocess, while the DNA, that is damaged both in vivo and in vitro only at relatively long irradiation time, might be a secondary target . Moreover, the photoprocess results in mutagenesis for Salmonella typhimurium tester strains . This information is particularly important in view of the potential use of photodynamic therapy for the treatment of microbial infections.

Vet Microbiol, 2000 Jun 12, 74(4), 331 - 43
Adhesion and invasion of Escherichia coli from single and recurrent clinical cases of bovine mastitis in vitro; Dopfer D et al.; Seven strains of Escherichia coli, originating from clinical cases of bovine mastitis, and one Salmonella typhimurium control strain were tested for their ability to adhere to, and invade, bovine mammary epithelial cells (MAC-T cells) in vitro . Four of the seven strains were isolated from cows with chronic intramammary infections with recurrent episodes of clinical mastitis and three strains were isolated from single cases of clinical mastitis . Both adhesion and invasion of all strains were dose and time dependent . The four E . coli strains isolated from recurrent cases of clinical mastitis invaded twice as frequently as and three times faster than the strains isolated from single cases of clinical mastitis . By contrast, there was no difference in the amount or speed of adhesion between the two types of strains of E . coli . Adhesion and invasion curves of E . coli resembled a two-step chain reaction, where invasion was the rate-limiting step . Although adhesion and invasion of E . coli has not been demonstrated in vivo yet, the results of the present study may contribute to an understanding of the pathogenesis of chronic intramammary infections caused by E . coli.

J Food Prot, 2000 May, 63(5), 608 - 12
Fate of Listeria monocytogenes, Salmonella typhimurium DT104, and Escherichia coli O157:H7 in Labneh as a pre- and postfermentation contaminant; Issa MS et al.; Commercially pasteurized milk (approximately 2% milkfat) was heated at 85 to 87 degrees C/30 min, inoculated to contain 2,000 to 6,000 CFU/ml of Listeria monocytogenes, Salmonella typhimurium DT104, or Escherichia coli O157:H7, cultured at 43 degrees C for 4 h with a 2.0% (wt/wt) commercial yogurt starter culture, stored 12 to 14 h at 6 degrees C, and centrifuged to obtain a Labneh-like product . Alternatively, traditional salted and unsalted Labneh was prepared using a 3.0% (wt/wt) starter culture inoculum, similarly inoculated after manufacture with the aforementioned pathogens, and stored at 6 degrees C and 20 degrees C . Throughout fermentation, Listeria populations remained unchanged, whereas numbers of Salmonella increased 0.33 to 0.47 logs during the first 2 h of fermentation and decreased thereafter . E . coli populations increased 0.46 to 1.19 logs during fermentation and remained that these levels during overnight cold storage . When unsalted and salted Labneh were inoculated after manufacture, Salmonella populations decreased >2 logs in all samples after 2 days, regardless of storage temperature, with the pathogen no longer detected in 4-day-old samples . Numbers of L . monocytogenes decreased from 2.48 to 3.70 to < 1.00 to 1.95 logs after 2 days with the pathogen persisting up to 15 days in one lot of salted/unsalted Labneh stored at 6 degrees C . E . coli O157:H7 populations decreased from 3.39 to 3.7 to < 1.00 to 2.08 logs during the first 2 days, with the pathogen no longer detected in any 4-day-old samples . Inactivation rates for all three pathogens in Labneh were unrelated to storage temperature or salt content . Unlike L . monocytogenes that persisted up to 15 days in Labneh, rapid inactivation of Salmonella typhimurium DT104 and E . coli O157:H7 suggests that these emerging foodborne pathogens are of less public health concern in traditional Labneh.

J Food Prot, 2000 May, 63(5), 601 - 7
Antimicrobial effect of herb extracts against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium associated with beef; Cutter CN; The effects of plant extracts against pathogenic bacteria in vitro are well known, yet few studies have addressed the effects of these compounds against pathogens associated with muscle foods . A series of experiments was conducted to determine the effectiveness of a commercially available, generally recognized as safe, herb extract dispersed in sodium citrate (Protecta One) or sodium chloride (Protecta Two) against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes associated with beef . In the first experiment, E . coli O157:H7, Salmonella typhimurium, and L . monocytogenes inoculated onto beef and subjected to surface spray treatments with 2.5% solutions of Protecta One or Protecta Two were not affected by immediate application (day 0) of the herbal extracts . However, after 7 days of storage at 4 degrees C, E . coli O157:H7 was reduced by >1.3 log10 CFU/cm2 by Protecta Two; L . monocytogenes was reduced by 1.8 and 1.9 log10 CFU/cm2 by Protecta One and Protecta Two, respectively; Salmonella typhimurium was not reduced >0.3 log10 CFU/cm2 by either extract by day 7 . In the second experiment, 2.5% Protecta Two (wt/vol or wt/wt) added to inoculated lean and adipose beef trim, processed, and packaged as ground beef chubs (80% lean, 20% adipose), did not reduce pathogen populations >0.5 log10 CFU/g up to 14 days at 4 degrees C . In the third experiment, surface spray treatments of beef with 2.5% lactic acid or 2.5% solutions of Protecta One or Protecta Two, vacuum packaged, and stored up to 35 days at 4 degrees C did reduce E . coli O157:H7, L . monocytogenes, and Salmonella Typhimurium slightly . These studies suggest that the use of herb extracts may afford some reductions of pathogens on beef surfaces; however, the antimicrobial activity may be diminished in ground beef by adipose components.

J Food Prot, 2000 May, 63(5), 593 - 600
Antimicrobial activity of cetylpyridinium chloride washes against pathogenic bacteria on beef surfaces; Cutter CN et al.; Cetylpyridinium chloride (CPC), a water-soluble, neutral pH, colorless compound, is widely used in oral hygiene products to inhibit bacteria responsible for plaque . Previously, researchers have demonstrated that CPC not only reduces Salmonella typhimurium on poultry but also prevents cross-contamination . To determine the effectiveness of CPC against pathogens associated with lean and adipose beef surfaces, several spray-washing experiments (862 kPa, 15 s, 35 degrees C) with 1% (wt/vol) CPC were conducted . On lean beef surfaces, CPC immediately reduced 5 to 6 log10 CFU/cm2 of Escherichia coli O157:H7 and Salmonella typhimurium to virtually undetectable levels (0 log10 CFU/cm2), as well as after 35 days of refrigerated (4 degrees C), vacuum-packaged storage . On adipose beef surfaces, 5 log10 CFU/cm2 Salmonella typhimurium and E . coli O157:H7 were reduced immediately (>2.5 log10 CFU/cm2) with 1% CPC; by day 35 the reduction was <1.3 log10 CFU/cm2 . Further plate overlay analyses indicated that the effectiveness of CPC against pathogens on adipose surfaces was not hampered by the presence of meat components or fatty acids . Additional chemical and microbiological analyses of 1% CPC-treated beef surfaces subjected to a secondary water wash (following contact times of 0, 5, 10, 15, or 30 min) or grinding did reduce pathogenic bacteria and CPC levels . However, residual CPC levels following any of the treatments were considered excessive for human consumption . Despite the residual levels, this study is the first to demonstrate the effect of CPC on pathogenic bacteria associated with beef surfaces immediately after treatment and also after long-term, refrigerated, vacuum-packaged storage.

J Food Prot, 2000 May, 63(5), 579 - 92
Incidence of Salmonella in fish and seafood; Heinitz ML et al.; Field laboratories of the U.S . Food and Drug Administration collected and tested 11,312 import and 768 domestic seafood samples over a 9-year period (1990 to 1998) for the presence of Salmonella . The overall incidence of Salmonella was 7.2% for import and 1.3% for domestic seafood . Nearly 10% of import and 2.8% of domestic raw seafood were positive for Salmonella . The overall incidence of Salmonella in ready-to-eat seafood and shellfish eaten raw was 0.47% for domestic--one shucked oyster and one shark cartilage powder . The incidence in the 2,734 ready-to-eat import seafood was 2.6%--cooked shrimp, shellfish or fish paste, smoked fish, salted/dried fish, and caviar . The incidence in import shellfish consumed raw was 1% in oyster, 3.4% in clams, and 0% in mussels . The incidence in raw, import fish was 12.2% . Distribution of Salmonella in seafood on a regional basis indicated the incidence to be highest in central Pacific and Africa and lowest in Europe/Russia and North America (12% versus 1.6%) . Data on a country basis indicated Vietnam to have the highest (30%) and Republic of Korea the lowest (0.7%) . While the most frequent serotypes in import seafood were Salmonella Weltevreden (1st), Salmonella Senftenberg (2nd), Salmonella Lexington, and Salmonella Paratyphi-B (3rd, equal numbers for each serotype), the top 20 list included Salmonella enteritidis (5th), Salmonella Newport (6th), Salmonella Thompson (7th), Salmonella typhimurium (12th), and Salmonella anatum (13th), commonly involved in foodborne illness in the United States . Because the incidence in the present study is based on only a small fraction of the seafood imported into the United States, efforts should be directed toward implementation of hazard analysis and critical control points to reduce the incidence of Salmonella in seafood without relying on testing for Salmonella.

J Food Prot, 2000 May, 63(5), 573 - 8
Short-chain volatile fatty acids modulate the expression of the hilA and invF genes of Salmonella typhimurium; Durant JA et al.; The ability of Salmonella typhimurium to invade the intestinal mucosal cells is an important step in pathogenesis . This invasion process requires genes encoded on the Salmonella pathogenicity island 1 (SPI1) . Two transcriptional activators, HilA and InvF, encoded in SPII regulate the expression of invasion genes in response to environmental stimuli such as osmolarity, oxygen tension, and pH . During its pathogenic life cycle, Salmonella typhimurium is also exposed to short-chain fatty acids (SCFA), especially acetate, propionate, and butyrate, in the intestinal lumen, as well as the SCFA used as food preservatives . The effects of SCFA on the expression of hilA and invF-lacZY transcriptional fusions were examined to determine the potential role of SCFA in the pathogenesis of Salmonella typhimurium . Growth rates were reduced by increasing SCFA concentrations at pH 6 but not at pH 7 . At pH 7, hilA and invF expression was induced by acetate but not by propionate or butyrate, while at pH 6, all SCFA induced hilA and invF expression at 1 h . In general, hilA and invF expression levels when compared to respective control responses were higher at 1 h than at 4 and 8 h in the presence of most SCFA concentrations at pH 6 . However, expression levels at 4 and 8 h were either similar or higher than the 1-h responses for the hilA-lacZY fusion strain in the presence of acetate while exposure to 20 mM propionate yielded similar levels of expression at 1, 4, and 8 h . The pH-dependent manner of induction suggests that entry of SCFA into the cell was necessary for induction . We speculate that SCFA may serve as an environmental signal that triggers the expression of invasion genes in the gastrointestinal tract.

Vaccine, 2000 Jul 1, 18(26), 2991 - 8
Intranasal immunization with protein-linked phosphorylcholine protects mice against a lethal intranasal challenge with streptococcus pneumoniae; Trolle S et al.; Immunization against phosphorylcholine (PC) linked to a protein protects mice against Streptococcus pneumoniae when used parenterally, and against Salmonella typhimurium when used orally after entrapment in D,L-Lactide-co-Glycolide microspheres . Here, we immunized BALB/c mice intranasally with a serotype 3 S . pneumoniae strain . Immunization was followed by a rise in anti-PC IgA and IgG titers in serum and in pulmonary secretions, but not by any rise in anti ds-DNA antibody nor any glomerular Ig deposition . The survival rates were 91 and 76% in the two groups of mice, respectively . These rates were significantly higher than those in control mice immunized intranasally either with Thyr loaded in microspheres (0%), blank microspheres (22%), free Thyr (17%), and saline (18%) . This demonstrates that the mucosal route is effective for vaccination against S . pneumoniae pneumonia with PC linked to a protein carrier . It constitutes another important step forward in the development of the concept that PC can be used as a mucosal immunogen for protection against the different diseases caused by PC-bearing bacteria.

Life Sci, 2000 Apr 7, 66(20), 1955 - 67
Establishment of Salmonella strain expressing catalytically active human UDP-glucuronosyltransferase 1A1 (UGT1A1); Fujita K et al.; Human uridinediphosphate-glucuronosyltransferase 1A1 (UGT1A1) was expressed in Salmonella typhimurium TA1535 cells by transfection of the cells with plasmids carrying the UGT1A1 cDNA . UGT1A1 cDNA was isolated by a polymerase chain reaction from human liver total RNA and was inserted into the pSE420 plasmid, linked to the trc promoter and terminator . The plasmid thus constructed was introduced into Salmonella TA1535 cells . The expression of human UGT1A1 protein was confirmed by Western blot analysis . The maximal expression was observed at 24 h after the addition of isopropyl-beta-D-thiogalactopyranoside, an inducer . However, the bilirubin conjugation activity of the membrane fraction from the Salmonella cells was not detectable . When a beta-glucuronidase inhibitor such as saccharic acid 1,4-lactone, glycyrrhizin or 1-naphtyl-beta-D-glucuronide was added to the reaction mixture, the bilirubin conjugation activity of the human UGT1A1 was detected . When geniposide was added to the reaction mixture, the bilirubin conjugation activity of UGT1A1 was not seen . Taking these results into account, the established Salmonella strain possesses the beta-glucuronidase activity . Since the beta-glucuronidase activity of the Salmonella was lower than that of E . coli, it was concluded that Salmonella seemed to be a good host to express UGT protein . This is the first study to demonstrate the establishment of a bacterial strain expressing native human UGT protein showing catalytic activity.

J Immunol, 2000 Jun 1, 164(11), 5894 - 904
Salmonella typhimurium infection and lipopolysaccharide stimulation induce similar changes in macrophage gene expression; Rosenberger CM et al.; Changes in macrophage phenotype induced during infection result from the recognition of bacterial products as well as the action of bacterial virulence factors . We used the unprecedented opportunity provided by gene arrays to simultaneously study the expression of hundreds of genes during Salmonella typhimurium infection of macrophages and to assess the contribution of the bacterial virulence factor, LPS, in initiating the host responses to Salmonella . We found that S . typhimurium infection caused significant changes in the expression of numerous genes encoding chemokines, cell surface receptors, signaling molecules, and transcriptional activators at 4 h postinfection of the RAW 264.7 murine macrophage cell line . Our results revealed changes in the expression of several genes that had not been previously implicated in the host responses to S . typhimurium infection, as well as changes in the expression of several genes previously shown to be regulated by S . typhimurium infection . An overlapping spectrum of genes was expressed in response to virulent S . typhimurium and purified S . typhimurium LPS, reinforcing the major role of this surface molecule in stimulating the early response of macrophages to bacterial infection . The macrophage gene expression profile was further altered by activation with IFN-gamma, indicating that host cell responses depend on the activation state of the cell.

Chemosphere, 2000 Jul, 41(1-2), 129 - 32
Genotoxicity of surface water samples from Meiliang Bay, Taihu Lake, Eastern China; Shen L et al.; Taihu Lake is the third largest freshwater lake in China . Taihu Basin is one of the most densely populated and urbanized areas in this country . This area provides 15% of the GDP . Meiliang Bay is located in the north part of the Lake . It provides the municipal water source for Wuxi City . Some parts of the lake have been found to be highly polluted due to eutrophication for over a decade . Surface water (0-0.5 m) samples were collected from the Meiliang Bay by the aid of Global Position System (GPS) for positioning . Water samples were concentrated 5000 times with XAD-2 resin columns . A reverse mutation test using histidine-dependent Salmonella typhimurium strains was employed to assay the genotoxicity of the samples . The results showed that the sample from position 6 had the highest genotoxicity either in the case of activating with eucaryotic S9 system or without S9 . The genotoxic effect included, at least, two different molecular mechanisms: nucleotide point substitution on DNA molecules and reading frame shifting caused by nucleotide insertion or deletion . The genotoxicity of the water body in Meiliang Bay, Taihu Lake should be kept in close monitoring.

Biochem Soc Trans, 2000 Feb, 28(2), 1 - 6
An overview of bioactivation of chemical carcinogens; Glatt HR; Most environmental carcinogens require metabolic activation to reactive intermediates and are mutagenic in appropriate test systems . During the last decade, the cDNAs of numerous xenobiotic-metabolizing enzymes have been cloned . The individually expressed enzymes were used to study their substrate specificities and their inhibition by other compounds . Various enzymes were expressed directly in target cells of in vitro mutagenicity tests . This is illustrated in the present study for rat and human sulphotransferases (SULTs) expressed in Salmonella typhimurium TA1538 . Numerous compounds were mutagenic in the new test system . Some of these promutagens were activated by several different SULT forms, whereas many other promutagens were activated with high selectivity by a specific enzyme form, but not by genetically closely related forms from the same species (e.g . allelic variants) or orthologous enzymes from other species . Similar findings have been made using recombinant test systems for specific forms of other classes of enzymes (e.g . cytochromes P450) . This high selectivity in activation (and inactivation) may explain some organotropisms as well as species and inter-individual differences in the action of carcinogens . Many carcinogen-metabolizing enzymes are induced or inhibited by other xenobiotics . Such interactions can be exploited for chemo-prevention, which however may be carcinogen- and tissue-dependent.

Epidemiol Infect, 2000 Apr, 124(2), 193 - 200
Multiresistant Salmonella Typhimurium DT104 infections of humans and domestic animals in the Pacific Northwest of the United States; Besser TE et al.; Salmonella Typhimurium definitive type 104 with chromosomally encoded resistance to five or more antimicrobial drugs (R-type ACSSuT+) has been reported increasingly frequently as the cause of human and animal salmonellosis since 1990 . Among animal isolates from the northwestern United States (NWUS), R-type ACSSuT+ Typhimurium isolates increased through the early 1990s to comprise 73% of Typhimurium isolates by 1995, but subsequently decreased to comprise only 30% of isolates during 1998 . NWUS S . Typhimurium R-type ACSSuT+ were consistently (99%) phage typed as DT104 or the closely related DTu302 . S . Typhimurium isolates from cattle with primary salmonellosis, randomly selected from a national repository, from NWUS were more likely to exhibit R-type ACSSuT+ (19/24, 79%) compared to isolates from other quadrants (17/71, 24%; P < 0.01) . Human patients infected with R-type ACSSuT+ resided in postal zip code polygons of above average cattle farm density (P < 0.05), while patients infected with other R-types showed no similar tendency . Furthermore, humans infected with R-type ACSSuT+ Typhimurium were more likely to report direct contact with livestock (P < 0.01) than humans infected with other R-types.

Int J Mol Med, 2000 Jun, 5(6), 615 - 8
Salmonella typhimurium-induced reactivation of latent HIV-1 in promonocytic U1 cells is inhibited by trovafloxacin; Gollapudi S et al.; We have previously reported that virulent Salmonella typhimurium induces replication of latent HIV-1 in U1 cells, via activation of tumor necrosis factor-alpha (TNF-alpha) production . In the present study, we show that Trovafloxacin, a new quinolone antibiotic, inhibits S . typhimurium-induced TNF-alpha production and HIV-1 replication . In addition, Trovafloxacin inhibits TNF-alpha-induced reactivation of latent HIV-1 in U1 cells . The concentrations of Trovafloxacin that inhibited HIV-1 replication are comparable to the plasma and tissue levels achieved by therapeutic dosage used in the treatment of bacterial infections . Therefore, Trovafloxacin is a potential candidate for adjunct therapy in HIV-1 infection.

Bioorg Khim, 2000 Jan, 26(1), 31 - 8
{Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1}; Navolotskaia EV et al.; The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied . At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415 . By using a 125I-labeled "addressing" fragment of ACTH inverted question mark{125I}ACTH-(13-24) inverted question mark, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM) . Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of {125I}ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively . Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized . The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.

Ecotoxicol Environ Saf, 2000 May, 46(1), 73 - 80
Assessment of the water-extractable genotoxic potential of soil samples from contaminated sites; Ehrlichmann H et al.; A screening method for the evaluation of the water-extractable genotoxic potential of soil is proposed . Due to the low sensitivity of genotoxicity test systems, PAD-1 resin was used as solid phase to concentrate less hydrophilic compounds from aqueous soil extracts . Concentrated and nonconcentrated aqueous soil extracts from 19 soil samples were evaluated using three genotoxicity assays: the umu test according to the German standard method with Salmonella typhimurium TA1535/pSK1002, the NM2009 test with S . typhimurium NM2009, and the SOS Chromotest with Escherichia coli PQ37 . All tests were performed according to a uniform protocol using microplates . Results indicate that the nonconcentrated water elutriates should be tested because the genotoxic potential of some soil samples is extremely high . Additionally, water elutriates should be concentrated by solid phase extraction to avoid false-negative results . The results of this study show that the genotoxic risk of sites contaminated with PAHs or mineral oils may be underestimated if only nonconcentrated samples are tested . The solid-phase extraction method with a concentration factor of 30 for these low soluble compounds is suitable for distinguishing between background genotoxicity of noncontaminated sites and anthropogenic contaminations .

Sci Total Environ, 2000 Mar 20, 247(2-3), 213 - 25
New methods to use fish cytochrome P4501A to assess marine organic pollutants; Cousinou M et al.; A new methodology has been developed to assess cytochrome P4501A expression in two South Atlantic Spanish fish, guilthead seabream (Sparus aurata) and grey mullet (Liza aurata), used as pollution bioindicators . Degenerate oligos were used to amplify by reverse transcription and PCR (RT-PCR) specific cyp1A cDNA sequences, used subsequently to design specific primers to get the full cDNA by rapid amplification of cDNA ends . A new assay has been developed to quantitate cyp1A expression by RT-PCR in an automated DNA sequencer . The effect of beta-naphthoflavone inducing biotransformation has been used to compare three distinct pollution biomarkers: EROD activity, ELISA determination of CYP1A, and 2-aminoanthracene (2-AA) activation . Immunodetection by ELISA or Western blot was inconsistent in S . aurata and L . aurata . EROD activity yielded satisfactory results; the higher induction was observed by bioactivation of 2-AA to mutagens detected with strain BA149 of Salmonella typhimurium, in agreement with the high sensitivity previously described for this biomarker . The present paper summarizes the current status of our research.

Epidemiol Mikrobiol Imunol, 2000 Feb, 49(1), 34 - 8
{Salmonelloses in the Czech Republic 1989-1998}; Sramova H et al.; In the Czech Republic in 1989 a triple increase of the incidence of salmonelloses was recorded: 34,435 cases . Since that year the morbidity trend varies between 400 and 500 cases per 100,000 population . The dominating agent (95% and more) is Salmonella Enteritidis PT8 . The epidemic incidence was recorded on the whole territory of the Czech Republic mainly in Moravia and in the East Bohemian region . The specific morbidity is highest in 0 and 1-4 year-old children . The seasonal incidence has two peaks with the exception of 1997 . During the period between 1989 and 1996 there was a significant increase of epidemics of salmonellosis in conjunction with food production incl . private confectionery shops, restaurants and the sale of foods in the streets . The most important vehicle are eggs and egg products, in particular confectionery . Salmonella Typhimurium DT104 is found only rarely in the Czech Republic . The first epidemic (15 cases) developed in 1998.

FEMS Microbiol Lett, 2000 May 15, 186(2), 281 - 6
Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis; Williams K et al.; The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens . For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines . A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs . An isogenic Y . pestis htrA mutant was constructed using a reverse genetics approach . In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model . However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.

J Biol Chem, 2000 Jul 7, 275(27), 20302 - 7
Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge . Evidence from arrhenius plots, absorption spectra, and primary kinetic isotope effects; Fan YX et al.; This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium . Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between alpha subunit Asp(56) and beta subunit Lys(167) play allosteric roles . Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an alpha subunit ligand, alpha-glycerol 3-phosphate . Arrhenius plots of the activity data under these conditions were nonlinear . The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects . We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, "open" conformation to a high activity, "closed" conformation under certain conditions . The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms . The results suggest that alpha subunit Asp(56) and beta subunit Lys(167) are not essential for catalysis and for allosteric communication between the alpha and beta subunits but that their mutual interaction is important in stabilization of the active, closed form of the alpha(2)beta(2) complex.

Am J Physiol Regul Integr Comp Physiol, 2000 May, 278(5), R1232 - 9
Differential inducible nitric oxide synthase expression in systemic and pulmonary vessels after endotoxin; Pulido EJ et al.; Inducible nitric oxide synthase (iNOS) is associated with vascular hypocontractility in systemic vessels after endotoxin lipopolysaccharide (LPS) administration . Although lung iNOS is increased after LPS, its role in the pulmonary circulation is unclear . We hypothesized that whereas iNOS upregulation is responsible for LPS-induced vascular dysfunction in systemic vessels, iNOS does not play a significant role in the pulmonary artery (PA) . Using isolated aorta (AO) and PA rings, we examined the effect of nonselective NOS inhibition {N(G)-monomethyl-L-arginine (L-NMMA); 100 micromol/l} and selective iNOS inhibition (aminoguanidine, AG; 100 micromol/l) on alpha(1)-adrenergic-mediated vasoconstriction (phenylephrine; 10(-9) to 10(-3) M) after LPS (Salmonella typhimurium, 20 mg/kg ip) . We also determined the presence of iNOS using Western blot and immunohistochemistry . LPS markedly impaired AO contractility (maximal control tension 1,076 +/- 33 mg vs . LPS 412 +/- 39 mg, P < 0.05), but PA contractility was unchanged (control 466 +/- 29 mg vs . LPS 455 +/- 27 mg, P > 0.05) . Selective iNOS inhibition restored the AO's response to vasoconstriction (LPS + AG 1,135 +/- 54 mg, P > 0.05 vs . control and P < 0.05 vs . LPS), but had no effect on the PA (LPS + AG 422 +/- 38 mg, P > 0.05 vs . control and LPS) . Western blot and immunohistochemistry revealed increased iNOS expression in the AO after LPS, but iNOS was not detected in the PA . Our results suggest that differential iNOS expression after LPS in systemic and pulmonary vessels contributes to the phenomenon of sepsis/endotoxemia-induced systemic hypotension and pulmonary hypertension.

Aust N Z J Med, 2000 Feb, 30(1), 28 - 32
HLA-B27 expression and reactive arthritis susceptibility in two patient cohorts infected with Salmonella Typhimurium; McColl GJ et al.; BACKGROUND: Reactive arthritis (ReA) is an inflammatory arthritis triggered by certain gastrointestinal and genitourinary infections . Single source outbreaks of triggering infections provide an opportunity to elucidate host susceptibility factors in this disease . AIM: To determine the role of Major Histocompatibility Complex (MHC) Class I alleles in ReA susceptibility after two large single source outbreaks of Salmonella Typhimurium gastroenteritis . METHODS: A questionnaire screening for features of ReA and a request for HLA class I typing were sent to all patients affected by two single source outbreaks of S . Typhimurium gastroenteritis . Individuals with arthritis of recent onset were interviewed, examined and diagnostic criteria for ReA applied . RESULTS: Nineteen cases of reactive arthritis, 11 female, were diagnosed in the 424 respondents with S . Typhimurium gastroenteritis from both outbreaks . Clinical features of the arthritis were similar to those described after other large single source outbreaks of Salmonella infection . HLA-B27 was expressed by only two of the 19 ReA patients and therefore did not predict susceptibility to this form of arthritis . Caucasians were, however, more likely to develop reactive arthritis than Asians . CONCLUSIONS: In this study, susceptibility to ReA was not increased in HLA-B27 positive individuals or males but was greater in those of Caucasian descent.

Int J Antimicrob Agents, 2000 May, 14(4), 321 - 5
Ecological impact of antibiotic use in animals on different complex microflora: environment; Witte W; Different means of interaction between microecological systems in different animal hosts (including humans) and the environment may occur during the transfer of resistant bacteria and their resistance genes . Spread of resistance takes place in different ways with respect to clonal spread of resistance strains by the spread of wide host range plasmids and translocatable elements . Commensals in ecosystems have a special significance and a pronounced capacity for acquisition and transfer of resistance genes as with Enterococcus faecium and Escherichia coli in the gut flora or Pseudomonas spp . in aquatic environments . The route of transmission from animals to humans by meat products is well established . Other routes via water and food plants (vegetables) have been investigated less, although resistance genes transfer in aquatic environments as evidenced from sequence comparison of such genes (e.g . tetR, floR in Salmonella typhimurium DT104) . Whether this is due to rare but important transfer events or whether there is a more frequent exchange in aquatic or terrestrial environments needs further elucidation.

Lett Appl Microbiol, 2000 Apr, 30(4), 320 - 4
Comparison of methods for the recovery and detection of low levels of injured Salmonella in ice cream and milk powder; Baylis CL et al.; This study compared the ability of four rapid methods and a standard cultural method to detect low levels of heat-injured cells of Salmonella typhimurium in ice cream and skimmed milk powder . The detection of Salmonella in samples contaminated with low levels (< 10 cfu 25 g-1) was significantly greater with the novel broth method than with the other methods (P </= 0.01) . At contamination levels > 10 cfu 25 g-1, there was no significant difference between the methods except for the novel broth method and a dipstick-based immunoassay (P </= 0.05) . The novel broth method, S.P.R.I.N.T . Salmonella, which incorporates a specifically formulated peptone and Oxyrase(R) combination followed by the timed release of selective agents into the recovery medium, was shown to improve the rate of detection of low numbers of injured cells of Salmonella after 24 h enrichment.

J Appl Microbiol, 2000 Apr, 88(4), 720 - 7
Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo; Naughton PJ et al.; The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm . enteritidis 857 . Con A significantly increased numbers of Salm . typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone . Con A had much less effect on rats infected with Salm . enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study . GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm . typhimurium S986 and significantly improved rat growth . GNA had little effect on infection by Salm . enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.

Immunology, 2000 Apr, 99(4), 607 - 14
Bacterial lipopolysaccharide acts as an adjuvant to induce autoimmune arthritis in mice; Yoshino S et al.; We investigated the ability of lipopolysaccharide (LPS) as an adjuvant to induce autoimmune arthritis . LPS from Escherichia coli was intraperitoneally injected into DBA/1J mice together with the joint cartilage component type II collagen (CII) on day 0 . Thereafter, the injection of CII and LPS was continued every 2 weeks up to day 56 . The results showed that mice injected with CII plus LPS had signs of arthritis on day 55 and the joint inflammation reached a peak on day 75 . Injection of CII or LPS alone induced no arthritis . Histologically, marked oedema of synovium and intense infiltration of inflammatory cells, including neutrophils, were observed 3 days after the onset of joint inflammation . Twenty-one days later, there were marked proliferation of synovial tissues with many mononuclear cells and destruction of cartilage . Anti-CII immunoglobulin G (IgG) and IgG2a antibodies were markedly produced in mice injected with CII plus LPS . Pronounced secretion of cytokines, including interleukins-12 and -1beta, interferon-gamma and tumour necrosis factor-alpha, was also observed in these animals . Arthritis was passively transferred into naive syngeneic mice with sera but not with lymphoid cells from mice given CII with LPS . Other types of LPS from Salmonella enteritidis, Salmonella typhimurium and Klebsiella pneumoniae as well as lipid A from E . coli, induced inflammation in joints when administered with CII . Polymixin B sulphate mixed with LPS or lipid A blocked the induction of joint inflammation . These results indicate that LPS appears to play an important role as an adjuvant in the induction of arthritis in which autoimmunity to CII is involved.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 133 - 6
Mechanism of action of pulsed high electric field (PHEF) on the membranes of food-poisoning bacteria is an 'all-or-nothing' effect; Russell NJ et al.; Salmonella typhimurium (CRA 1005) was more sensitive than Listeria monocytogenes (NCTC 11994) to pulsed high electric field (PHEF) treatment in distilled water (10, 15 and 20 kV/cm), 10 mM tris-maleate buffer pH 7.4 (15 kV/cm) and model beef broth (0.75% w/v: 15 kV/cm) . Sublethal injury could not be detected using a selective medium plating technique, indicating that bacterial inactivation by PHEF may be an 'all-or-nothing' event . PHEF-induced membrane permeabilization resulted in increased UV-leakage and a decreased ability of L . monocytogenes to maintain a pH gradient.

Int J Food Microbiol, 2000 Apr 10, 55(1-3), 115 - 9
Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium; Ritz M et al.; Salmonella typhimurium is a leading cause of foodborne diseases . Today high hydrostatic pressure treatments are considered as alternative methods of preservation . To select optimal conditions of treatment, we have to characterize the cell targets of pressure . In this study the action of pressure on the bacterial membrane proteins is analysed . The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation . Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain . Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes . Outer membrane protein content is drastically modified after treatments . In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear . LamB is more resistant to hyperbaric exposure when the pH of the media is acidic . This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH . This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments . Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.

Clin Rheumatol, 2000, 19(2), 167 - 8
Psoriatic arthritis exacerbated by Salmonella infection; Punzi L et al.; Psoriatic arthritis (PsA) is an inflammatory joint disease in which environmental factors, particularly trauma and infections, are thought to play an important role . The authors describe the case of a patient with a mild and long-untreated form of PsA which was severely exacerbated by Salmonella typhimurium infection . This case confirms the importance of infectious agents in the occurrence and course of PsA.

J Biol Chem, 2000 May 5, 275(18), 13542 - 51
High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents . Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium; Zhou Z et al.; When Escherichia coli are grown on LB broth containing 25 mm NH(4)VO(3), complex modifications to the lipid A anchor of lipopolysaccharide are induced . Six modified lipid As (EV1-EV6) have been purified . Many of these variants possess 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (pEtN) substituents . Here we use NMR spectroscopy to investigate the attachment sites of the l-Ara4N and pEtN moieties on underivatized, intact EV3 and EV6 and on precursors II(A) and III(A) from kdsA mutants of Salmonella . CDCl(3)/CD(3)OD/D(2)O (2:3:1, v/v) is shown to be a superior solvent for homo- and heteronuclear one- and two-dimensional NMR experiments . The latter were not feasible previously because available solvents caused sample decomposition . Selective inverse decoupling difference spectroscopy is used to determine the attachment sites of substituents on EV3, EV6, II(A), and III(A) . l-Ara4N is attached via a phosphodiester linkage to the 4'-phosphates of EV3 and EV6 and has the beta anomeric configuration . pEtN is attached by a pyrophosphate linkage to the 1-phosphate of EV6 . The l-Ara4N and pEtN substituents of lipids II(A) and III(A) are attached in the opposite manner, with l-Ara4N on the 1-phosphate of II(A) and pEtN on the 4'-phosphate of III(A) . Determination of the proper attachment sites of these substituents is necessary for elucidating the enzymology of lipid A biosynthesis and for characterizing polymyxin-resistant mutants, in which l-Ara4N and pEtN substituents are greatly increased.

J Mol Biol, 2000 May 12, 298(4), 577 - 83
Overproduced Salmonella typhimurium flagellar motor switch complexes; Lux R et al.; Three Salmonella typhimurium flagellar motor proteins, FliG, FliM and FliN, are required for the switching of rotation sense . The proteins have been localized to the cytoplasmic module of the flagellar base . Structures, which were morphologically indistinguishable from the native transmembrane MS-ring and cytoplasmic C-ring basal body modules, formed in Escherichia coli upon plasmid-encoded synthesis of these proteins together with FliF . The structures localized to the cell membrane and contained all three motor proteins, as determined by immuno-electron microscopy . This result supports the deduction, based on earlier biochemical analysis, that the C-ring is composed entirely of these proteins and, therefore, functions as a dedicated motor component . In addition, it demonstrates that the morphologically correct assembly of the C-ring onto the MS-ring proceeds independently of other structural components of these modules .

Acta Vet Scand Suppl, 1999, 92, 15 - 22
Antibiotic resistance: genetic mechanisms and mobility; Olsen JE; Based on the current knowledge, resistance genes seems mainly to originate in the organisms which produce antibiotics (Davies 1994) . We lack considerably in the understanding of how these genes were transferred to pathogenic bacteria, and due to the enormous diversity of e.g . the soil flora, it is doubtful that we will ever obtain more that a faint picture of this . In Gram negative bacteria, more and more resistance genes are demonstrated to be located in integrons (e.g . beta-lactamase and streptomycin resistance genes in Salmonella Typhimurium DT104 (Sandvang et al . in press)) . Integrons seem primarily to act as insertion sites for resistance genes . The origin of integrons as well as the resistance gene cassettes that are the other essential element of this system, is largely unknown (Hall & Collis 1995) . Integrons can be located in the chromosome, in transposons, which have the ability to copy them themselves to other DNA molecules, or on plasmids . The emergence of resistant bacteria normally happens because of selection for a resistant clone of bacteria . Several mechanisms, however, exists by which the resistance genes can be transferred from one bacteria to another . Conjugation, mediated by plasmids or conjugative transposons, is currently the most well established of these mechanisms . Still, however, the selection pressure created by the use of antibiotics determines whether bacteria that have newly acquired a resistance gene expand to dominate in the population or remains a blink in history.

Vaccine, 2000 Jun 1, 18(24), 2668 - 76
Antibody responses to Yersinia pestis F1-antigen expressed in Salmonella typhimurium aroA from in vivo-inducible promoters; Bullifent HL et al.; Attenuated mutants of Salmonella typhimurium are being evaluated as delivery systems for a variety of heterologous vaccine antigens . Gene promoters which are induced in vivo can direct the stable expression of genes encoding these antigens . We have investigated the utility of the phoP, ompC, pagC and lacZ gene promoters for expression of the Y . pestis F1-antigen in S . typhimurium SL3261 (aroA) . After i.g . (intragastric) dosing the highest level of spleen colonisation was found with recombinant Salmonella expressing F1-antigen from the phoP gene promoter, and this recombinant was most effective in inducing serum and mucosal antibody responses . The use of the pagC gene promoter to direct expression of F1-antigen resulted in the induction of serum and mucosal antibody responses even though the recombinant Salmonella were unable to colonise spleen tissues suggesting that colonisation of these tissues is not essential for the induction of antibody responses.

Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5492 - 7
An autologous oral DNA vaccine protects against murine melanoma; Xiang R et al.; We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp100(25-33) and TRP-2(181-188) . These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2D(b) and H-2K(b), respectively . The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier . Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8(+) T cells that secreted T(H)1 cytokine IFN-gamma and induced tumor rejection and growth suppression after a lethal challenge with B16G3 . 26 murine melanoma cells . Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp100(25-33) peptide, which differs in its three NH(2)-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity . The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen . This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Jpn J Infect Dis, 2000 Feb, 53(1), 15 - 6
Salmonella typhimurium DT104 from livestock in Japan; Sameshima T et al.; We examined the distribution of multidrug-resistant Salmonella enterica serotype Typhimurium definitive phage type 104 (DT104) among Japanese livestock from 1973 to 1998 . The 144 S . Typhimurium field isolates were tested for susceptibility to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, kanamycin, trimethoprim, nalidixic acid, and norfloxacin . Thirty-six of 68 strains which exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 . Results of plasmid profiling showed that all DT104 strains retain a 90-kb virulence plasmid, while 20 of 36 strains possessed a few additional small plasmids ranging from 2 to 4 kb . These results showed that DT104 strains have existed in Japanese livestock since 1990, and that this phage type may be an important pathogen for cattle in Japan.

Int J Antimicrob Agents, 2000 Apr, 14(3), 249 - 51
The anti-bacterial action of diclofenac shown by inhibition of DNA synthesis; Dastidar SG et al.; Most strains of Gram-positive and Gram-negative bacteria were inhibited by 50-100 mg/l of the anti-inflammatory agent, diclofenac sodium (Dc) . In vivo test using 30 or 50 microg Dc per 20 g mouse (Swiss Albino variety) significantly (P <0.001) protected the animals when challenged with 50 MLD of a virulent Salmonella typhimurium . The anti-bacterial action of Dc was found to be due to inhibition of DNA synthesis which was demonstrated using 2 micro Ci (3H) deoxythymidine uptake.

Int J Antimicrob Agents, 2000 Apr, 14(3), 225 - 9
The effects of chlorpromazine on the outer cell wall of Salmonella typhimurium in ensuring resistance to the drug; Amaral L et al.; Chlorpromazine (CPZ), a compound employed for the management of psychosis, has a wide ranging antibacterial activity . The growth of Salmonella typhimurium100 mg/l), was initially inhibited during the first 8-16 h of exposure to concentrations of CPZ below the MIC . During this period of transient susceptibility, the distribution of ribosomes was markedly altered in a concentration dependent manner; the rough cell wall was transformed into a smooth form . The protein composition of the outer cell wall of 55 kDa was markedly decreased, whilst there was an increased number of high molecular weight proteins . After 16 h of exposure to sub-MIC levels of CPZ, the inhibitory effect of the drug was no longer apparent whereas the effects noted on the cell wall were retained . These Salmonella were, as the control, agglutinated by O antigen specific antibody . Whereas agglutination of the control Salmonella was blocked by the presence of CPZ at concentrations that induced the cell-wall effects, agglutination of CPZ exposed-Salmonella for periods in excess of 16 h was not blocked by any concentration of CPZ . These results suggested that eventual resistance to CPZ was dependent upon changes induced by CPZ at the cell wall level . The results also suggested that the CPZ binds to the 55 kDa protein and that such binding interfered with the recognition of the O antigen by antibody.

J Food Prot, 2000 Apr, 63(4), 451 - 6
Inactivation of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg by ultrasonic waves under pressure; Manas P et al.; The resistance of Salmonella Enteritidis (ATCC 13076), Salmonella Typhimurium (ATCC 13311), and Salmonella Senftenberg 775W (ATCC 43845) to ultrasonic waves under pressure treatments, at sublethal (manosonication) and lethal temperatures (manothermosonication) in citrate-phosphate buffer and in liquid whole egg was investigated . The influence of treatment parameters on the inactivation rate of manosonication was also studied . Decimal reduction times (Dt) of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg 775W corresponding to a heat treatment at 60 degrees C in pH 7 buffer and in liquid whole egg were 0.068, 0.12, and 1.0 min for buffer, and 0.12, 0.20, and 5.5 min for liquid whole egg . Those corresponding to a manosonication treatment (117 microns, 200 kPa, 40 degrees C) in both media were 0.73, 0.78, and 0.84 min, and 0.76, 0.84, and 1.4 min, respectively . When the amplitude of ultrasonic waves was increased linearly, the inactivation rate of manosonication increased exponentially . The inactivation rate also increased when pressure was raised . However, the magnitude of this increase was progressively smaller at higher pressures . The magnitude of the influence of the amplitude of ultrasonic waves and static pressure on the inactivation rate of manosonication was the same in the three serotypes investigated . Whereas a heat treatment at 60 degrees C only attained a 1/2-log cycle reduction in the number of Salmonella Senftenberg 775W survivors, a manothermosonication treatment (117 microns and 200 kPa) at this temperature attained a 3-log cycle reduction.

Mutat Res, 2000 Apr 13, 467(1), 69 - 82
From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of nitroaromatics in Salmonella typhimurium . Part II . Substituents far away from the nitro group; Klein M et al.; Derivatives of 4-nitrobiphenyl, 4-nitrosobiphenyl, 2-phenyl-5-nitropyridine (2-aza-4-nitrobiphenyl) and 2-nitrofluorene, bearing various alkyl substituents far away from the nitro group (4'-position in nitrobiphenyls, 7-position in 2-nitrofluorenes) were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium . In the absence of S9 in both strains, mutagenicity of all4'-Ad (Ad=adamantyl) . Changes of the molecular shape from 'planar' to non-planar caused by the bulk of the introduced substituents (without influencing the twisting of the nitro substituent or the phenyl rings in the biphenyl compounds) may be responsible for this effect by interfering with an efficient intercalation into DNA.A comparison between experimental and theoretical values as calculated from recently developed equations (QSAR) confirmed our previous results (see the preceding paper) that mutagenicity of alkyl-substituted nitroaromatics cannot be predicted by hydrophobicity and LUMO-energies alone without including steric parameters.

Mutat Res, 2000 Apr 13, 467(1), 55 - 68
From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium . Part I . Substituents ortho to the nitro group and in 2'-position; Klein M et al.; Eleven alkyl substituted derivatives of 4-nitrobiphenyl (4NBp) and two corresponding nitroso compounds were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium . The mutagenicity of compounds substituted ortho to the nitro group (3-methyl-, 3-ethyl-, 3-isopropyl-, 3-tertbutyl-, 3, 5-diethyl-, 3,5-diisopropyl-, and 3,5-ditertbutyl-4NBp) decreased with growing steric demand of the alkyl substituents in both tester strains . The most sterically hindered compounds were non-mutagenic even at highest concentrations . This reduction of mutagenicity is correlated with deviations from the coplanar orientation of the nitro group relative to the aromatic plane . Since a comparable decrease of mutagenicity for the nitroso compounds (4NOBp and 3-tertbutyl-4NOBp) was not observed, the first reduction step of non-planar nitro groups must be inhibited.Alkyl groups in the 2'-position of 4NBp (2'-methyl-, 2'-ethyl-, 2'-isopropyl-, and 2',4', 6'-trimethyl-4NBp) also reduced mutagenic activity with increasing size and removed it completely for the most sterically hindered species (2'-isopropyl-, 2',4',6'-trimethyl-4NBp) . In this case, the co-planarity of the nitro group is not affected but the twisting of the two aromatic rings, which is associated with a less effective charge delocalisation of the nitrenium ion.The experimental mutagenicities of all nitro compounds were compared to predicted values, that are based on recently developed empirical equations . While there was reasonable correspondence for the parent compound (4NBp), its ortho methylated derivative (3-methyl-4NBp) and two highly hydrophobic dialkylated species (3,5-diisopropyl- and 3, 5-ditertbutyl-4NBp), predictions for all other alkyl substituted compounds were too high . Thus, steric parameters should be included to improve the general validity of predictions by means of quantitative structure-activity relationships (QSAR).

J Leukoc Biol, 2000 Apr, 67(4), 457 - 63
Immune response to infection with Salmonella typhimurium in mice; Mittrucker HW et al.; Infection of mice with Salmonella typhimurium results in systemic infection and a disease similar to that seen in humans after infection with S . typhi . The innate immune system can restrict replication of S . typhimurium to a certain degree, but for effective control and eradication of bacteria, acquired immunity is essential . Salmonella infection induces the generation of specific CD4+ and CD8+ T cells, and both T cell populations are important for protection during primary and secondary responses, although the mechanisms underlying T cell-mediated protection are not yet completely understood . Infection with S . typhimurium also results in a strong antibody response to Salmonella antigens and, in contrast to most other intracellular bacteria, this antibody response participates in protection . In summary, the response to S . typhimurium involves both T and B cell-mediated immunity, and mechanisms mediated by both lymphocyte populations are important for control of primary infection and protection against secondary infection.

FEMS Immunol Med Microbiol, 2000 May, 28(1), 25 - 35
Identification and characterization of mutants with increased expression of hilA, the invasion gene transcriptional activator of Salmonella typhimurium; Fahlen TF et al.; Induction of invasion gene transcription and expression of the invasive phenotype of Salmonella strains are regulated by environmental conditions . Experimental evidence indicates that oxygen, pH, and osmotic conditions need to closely resemble those of the host intestinal lumen for invasion gene activation . The hilA gene, encoded on Salmonella pathogenicity island 1 (SPI-1), is a transcriptional activator which is required for invasion and whose expression is modulated by oxygen, pH, and osmolarity . Additionally, hilA is regulated by genetic elements encoded on SPI-1 (hilC/sirC/sprA and hilD), as well as by elements which reside outside of SPI-1 (phoP/phoQ and sirA), although how environmental signals modulate hilA is unknown . In an effort to further characterize the Salmonella invasion gene regulon, we have created and preliminarily characterized 18 Tn5 insertions which result in upregulation of a hilA::lacZY fusion . We have classified the mutations based on location and phenotype into three classes . Six class 1 and six class 2 mutants have insertions in SPI-1 near the invasion gene orgA or the invasion gene regulator hilD, respectively . Six class 3 mutants reside outside of SPI-1 in four different loci . The class 2 and 3 mutations induce overexpression of an episomal hilA::lacZY fusion and significantly increase S . typhimurium invasion of HEp-2 cells in a standard invasion assay . These data implicate new regions of SPI-1 as being involved in the regulation of invasion by S . typhimurium and identify new invasion gene regulators located outside of SPI-1.

J Biol Chem, 2000 Apr 21, 275(16), 12123 - 8
The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance; Jordi BJ et al.; The intracellular concentration of K(+)-glutamate, chromatin-associated proteins, and a downstream regulatory element (DRE) overlapping with the coding sequence, have been implicated in the regulation of the proU operon of Salmonella typhimurium . The basal expression of the proU operon is low, but it is rapidly induced when the bacteria are grown in media of high osmolarity (e.g . 0.3 M NaCl) . It has previously been suggested that increased intracellular concentrations of K(+)-glutamate activate the proU promoter in response to increased extracellular osmolarity . We show here that the activation of the proU promoter by K(+)-glutamate in vitro is nonspecific, and the in vivo regulation cannot simply be mimicked in vitro . In vivo specificity requires both the chromatin-associated protein H-NS and the DRE; they are both needed to maintain repression of proU expression at low osmolarity . How H-NS and the DRE repress the proU promoter in vivo has so far been unclear . We show that, in vivo, the DRE acts at a distance to inhibit open complex formation at the proU promoter.

J Biol Chem, 2000 Jun 23, 275(25), 18801 - 9
The AhpC and AhpD antioxidant defense system of Mycobacterium tuberculosis; Hillas PJ et al.; The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized . It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues . Mutagenesis studies show that all three cysteine residues are important for catalytic activity . Analysis of the M . tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter . This protein has also been cloned, expressed, purified, and characterized . AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase . The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF from Salmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol . AhpC and AhpD reduce alkylhydroperoxides more effectively than H(2)O(2) but do not appear to interact with each other . These two proteins appear to be critical elements of the antioxidant defense system of M . tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies.

Arch Ophthalmol, 2000 Apr, 118(4), 521 - 7
Biphasic ocular inflammatory response to endotoxin-induced uveitis in the mouse; Shen DF et al.; OBJECTIVE: To examine the kinetics and mechanisms of endotoxin-induced uveitis in the mouse . METHODS: C3H/HeN mice were injected subcutaneously with 0.3 mg of Salmonella typhimurium lipopolysaccharide (LPS) in 0.1 mL of phosphate-buffered saline solution or phosphate-buffered saline solution alone in 3 separate experiments; mice were killed after 1, 3, 5, and 7 days . In 2 other separate experiments, mice were killed 1, 3, 6, and 24 hours after LPS injection . All eyes were collected for histological examination, immunohistochemical analyses, aqueous protein level determination, and reverse transcriptase-polymerase chain reaction for ocular interleukin (IL)1alpha, IL-6, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor messenger RNA (mRNA) . Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor alpha and IL-6 levels in aqueous and serum samples . RESULTS: Results were consistent for all experiments . Numbers of ocular inflammatory cells and levels of aqueous protein peaked 1 and 5 days after LPS injection . Control mice did not develop inflammation . Serum and aqueous IL-6 and ocular IL-6 mRNA levels peaked at 1 day and subsided at 3 days . However, ocular IL-1alpha, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor mRNA appeared, peaked, and subsided at 3, 5, and 7 days, respectively . Predominant infiltrating cells were neutrophils at 1 day and macrophages at 5 days . Although no ocular inflammatory cells were detected before 24 hours after LPS injection, tumor necrosis factor alpha mRNA was noticed at 1 hour, peaked at 3 hours, and disappeared at 6 hours and granulocyte-macrophage colony-stimulating factor mRNA was spotted only at 3 hours after LPS injection . CONCLUSIONS: The ocular inflammatory response to C3H/ HeN mouse endotoxin-induced uveitis is biphasic for 7 days . The first wave appears at day 1 and subsides by day 3 . A second, higher peak appears at day 5 . The 2 inflammatory waves are related to the kinetics of the different cytokines released in the eye . This is in contrast to the rat monophasic endotoxin-induced uveitis model, which has only one peak of intense inflammation associated with cytokine release . CLINICAL RELEVANCE: A biphasic inflammatory response associated with cytokine release lasting several days is observed in C3H/HeN mice with endotoxin-induced uveitis . Because human anterior uveitis has a tendency to be recurrent in nature, this might be a better experimental model.

Vopr Virusol, 2000 Mar-Apr, 45(2), 10 - 4
{Isolation and study of recombinant strains of Salmonella typhimurium SL 7207, producing HBcAG and HBcAG-HBs}; Karpenko LI et al.; Recombinant strains producing hepatitis B virus (HBcAg) core protein and chimeric core protein exposing on its surface the major immunogenic epitope of HBsAg (HBcAg-HBs) were constructed on the base of attenuated S . typhimurium SL 7202 strain . The resultant Salmonella strains produced proteins which were capable of self-assembly into virus-like particles and showed antigenic properties of both core and surface hepatitis B proteins . A single rectal immunization with recombinant S . typhimurium induced humoral and cellular immune response to HBcAg and HBsAg . Specific anti-HBcAg were detected in animal sera and intestinal tissues, which indicated the formation of specific mucosal immunity.

Food Chem Toxicol, 2000 May, 38(5), 429 - 42
Toxicity and mutagenicity studies of DN-50000((R)) and RP-1((R)) enzymes; Burdock GA et al.; Improved yields of 5'-nucleotides from yeast extract for food flavouring purposes is possible through use of microbial nucleotidases, which will be available to food processors as the flavour enhancer Aromild . The safety of these enzymes, 5'-phosphodiesterase (RP-1) and the 5'-adenylic deaminase (DN-50000) was investigated in male and female rats . Feeding rats a diet admixed with 500, 2000 and 8000 mg/kg body weight of DN-50000 for 35 days resulted in no significant dose-related changes in body weight, water consumption, urinalysis, haematological profiles, blood chemistry or histopathological profiles of either male or female rats from consumption of the enzyme preparation . There was an increase in the absolute and/or relative organ weights of the submaxillary (salivary) glands in both sexes at 8000 mg/kg . The no-observed-effect level (NOEL) for DN-50000 was clearly evident at 2000 mg/kg . Consumption of RP-1 enzyme for 35 days at dietary levels of 500, 2000 and 8000 mg/kg body weight resulted in no significant changes in the above mentioned parameters, which could be directly attributed to a dose-related effect, with the exception of an increase in the absolute and relative weights of submaxillary glands of both male and female rats in the 2000 and 8000 mg/kg groups . The increase in weight of the submaxillary glands was associated with histological evidence of acinar cell hypertrophy . The NOEL for dietary consumption of RP-1 was clearly evident at 500 mg/kg . In a follow-up study in which rats were gavaged with 2000 mg/kg RP-1, submaxillary gland hypertrophy did not occur . These studies suggest that DN-50000 and RP-1 exert an action on submaxillary glands similar to that which has been previously reported for the enzyme pancreatin . Neither DN-50000 nor RP-1 were mutagenic in the Ames assay using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 or Escherichia coli strain WP2uvrA, in the presence or absence of S9 mix.

J Infect Dis, 2000 Apr, 181(4), 1350 - 8 Epub 2000 Apr 13.
Morphine increases susceptibility to oral Salmonella typhimurium infection; MacFarlane AS et al.; This study examined the effect of morphine on oral infection with virulent Salmonella typhimurium . Animals were treated with a 75-mg slow-release morphine pellet followed by inoculation with salmonellae . Morphine markedly sensitized mice to oral infection, as assessed by survival, mean survival time, and colony culture . By 24 h after Salmonella inoculation, morphine-treated mice had a 105-fold difference in number of organisms in the Peyer's patches, compared with controls . The opioid antagonist naltrexone significantly blocked Salmonella colonization in Peyer's patches and reduced Salmonella burden in other organs, indicating that morphine acts at least in part via an opioid receptor-mediated pathway . The data show that morphine markedly potentiates Salmonella infection at the gastrointestinal portal of entry and enhances subsequent dissemination of Salmonella organisms . The results have implications for potentiating gastrointestinal opportunistic infections in intravenous drug abusers and in opioid-medicated postsurgical patients.

Mol Microbiol, 2000 Apr, 36(1), 10 - 23
AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways; Romling U et al.; The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter . AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf) . To detect additional components contributing to the multicellular morphotype in S . typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins . The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level . In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion . Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour . Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli . Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae . AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins . AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion . An agfBA adrA double mutant behaved like the agfD mutant . Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S . typhimurium.

Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 945 - 7
Recognition of nomenclatural standing of Salmonella typhi (Approved Lists 1980), Salmonella enteritidis (Approved Lists 1980) and Salmonella typhimurium (Approved Lists 1980), and conservation of the specific epithets enteritidis and typhimurium . Request for an opinion; Ezaki T et al.; In 1994, the Judicial Commission of the ICSB announced that Le Minor and Popoff's Request for an Opinion in 1987 to designate Salmonella enterica sp . nov., nom . rev . as the type and only species of the genus Salmonella was denied . Thus, the current species of the genus Salmonella are Salmonella typhimurium, Salmonella enteritidis, Salmonella typhi, Salmonella choleraesuis (including six subspecies) and Salmonella bongori, with the type species, S . choleraesuis (Smith 1894) Weldin 1927 (Approved Lists 1980) . Because the decision of the Judicial Commission about the request by Le Minor in 1987 was suspended for 7 years, the non-validated name 'S . enterica' has been used among microbiologists and has caused confusion in the nomenclature of Salmonella . In order to overcome such confusion, and because of their importance as human pathogens, we herein propose to recognize the nomenclatural status of S . typhi, S . enteritidis and S . typhimurium as species and request to issue an Opinion to conserve the specific epithets enteritidis and typhimurium in the species names Salmonella enteritidis and Salmonella typhimurium.

Arzneimittelforschung, 2000 Mar, 50(3), 316 - 21
Mutagenicity of recombinant antihemophilic factor (GC-gamma AHF); Shin M et al.; This study was carried out to evaluate the mutagenic potential of recombinant antihemophilic factor VIII (GC-gamma AHF) . Salmonella typhimurium (S . typhimurium) reversion assay with/without histidine moiety, chromosomal aberration assay on Chinese hamster lung (CHL) fibroblast cells and in vivo micronucleus assay using mouse bone marrow cells and supravital micronucleus assay using peripheral blood were performed . GC-gamma AHF containing histidine did show inconsistent and irregular mutagenic effects on S . typhimurium TA98, TA100, TA1535 and TA1537 both in the absence and presence of the metabolic activation system, however, GC-gamma AHF without histidine showed no mutagenic effects regardless of the metabolic activation system, thus suggesting that the histidine moiety in GC-gamma AHF might cause inconsistent mutagenic effect . Also GC-gamma AHF did not increase the number of cells having structural or numerical chromosome aberration in the cytogenetic test . In classical and supravital micronucleus assay, no significant increases were observed in the occurrence of micronucleated polychromatic erythrocytes and micronucleated peripheral lymphocytes in male ICR mice . These results strongly indicate that GC-gamma AHF has no genetic toxicity under these experimental conditions.

Genetics, 2000 Mar, 154(3), 985 - 97
Compensatory mutations, antibiotic resistance and the population genetics of adaptive evolution in bacteria; Levin BR et al.; In the absence of the selecting drugs, chromosomal mutations for resistance to antibiotics and other chemotheraputic agents commonly engender a cost in the fitness of microorganisms . Recent in vivo and in vitro experimental studies of the adaptation to these "costs of resistance" in Escherichia coli, HIV, and Salmonella typhimurium found that evolution in the absence of these drugs commonly results in the ascent of mutations that ameliorate these costs, rather than higher-fitness, drug-sensitive revertants . To ascertain the conditions under which this compensatory evolution, rather than reversion, will occur, we did computer simulations, in vitro experiments, and DNA sequencing studies with low-fitness rpsL (streptomycin-resistant) mutants of E . coli with and without mutations that compensate for the fitness costs of these ribosomal protein mutations . The results of our investigation support the hypothesis that in these experiments, the ascent of intermediate-fitness compensatory mutants, rather than high-fitness revertants, can be attributed to higher rates of compensatory mutations relative to that of reversion and to the numerical bottlenecks associated with serial passage . We argue that these bottlenecks are intrinsic to the population dynamics of parasitic and commensal microbes and discuss the implications of these results to the problem of drug resistance and adaptive evolution in parasitic and commmensal microorganisms in general.

Genomics, 2000 Mar 15, 64(3), 230 - 40
Cloning and characterization of the murine toll-like receptor 5 (Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptible MOLF/Ei mice; Sebastiani G et al.; Toll-like receptors (TLRs) are a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens . In this paper, we describe the cloning and characterization of the mouse homologue of human TLR5 . Mouse Tlr5 encodes a 859-amino-acid protein that contains an N-terminal signal sequence, a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll/interleukin-1R signaling domain characteristic of all TLR proteins . The mouse Tlr5 protein shows 81% homology to human TLR5 and approximately 40% similarity to other TLR family members . Northern blot analysis reveals that Tlr5 is expressed predominantly in liver and lung with low-level expression in most other tissues examined . We have mapped Tlr5 to distal chromosome 1 using the (C57BL/6J x Mus spretus) x C57BL/6J Jackson BSB panel as well as a (C57BL/6J x MOLF/Ei)F(2) panel with the following position: D1Mit112-8.0 cM-Tlr5-9.6 cM-D1Mit17 . The presence of a quantitative trait locus for susceptibility to Salmonella typhimurium on distal chromosome 1 prompted the examination of Tlr5 in susceptible MOLF/Ei mice . Polymorphic sequence variants in Tlr5 allowed us to identify a unique 4-allele haplotype in MOLF/Ei . Furthermore, using both Northern blot analysis and reverse transcription-polymerase chain reaction, we have shown a reduced expression of Tlr5 during infection of MOLF/Ei mice with Salmonella . The assignment of Tlr5 to a chromosomal region known to harbor a Salmonella-susceptibility locus together with decreased expression of Tlr5 mRNA in liver of susceptible MOLF/Ei mice suggests the possibility that, as with other members of this family, Tlr5 may play a role in host response to bacterial gram-negative infections .

Vet Hum Toxicol, 2000 Apr, 42(2), 77 - 84
Acute toxicity, primary irritancy, and genetic toxicity studies with 3-(methylthio)propionaldehyde; Ballantyne B et al.; Basic acute toxicity, primary irritancy, and genetic toxicity studies were conducted with 3-(methylthio)propionaldehyde (3-MTP) . The acute rat peroral LD50 (with 95% confidence limits) for 3-MTP as a 25% (v/v) dilution in corn oil was 1.00 (0.59-1.70) ml/kg (males) and 1.68 (0.95-2.99) ml/kg (females); most deaths occurred 1.5 to 4 h postdosing . By 24-h occluded contact with undiluted 3-MTP, the rabbit acute percutaneous LD50 was 0.71 (0.43-1.15) ml/kg (males) and 0.79 (0.49-1.30) ml/kg (females): times to death ranged from 2 h to 2 d after the start of dosing . Exposure of rats to a statically generated saturated atmosphere killed all 5 males with a 40 min exposure and all 5 females with a 24 min exposure . In contrast, a 4-h exposure of rats to a dynamically generated saturated vapor atmosphere of 3-MTP did not produce any mortalities or signs of toxicity . A 4-hr occluded contact with 0.5 ml undiluted 3-MTP caused moderate to severe erythema and severe edema resolving by 7 to 17 d . Five/6 animals had necrosis apparent on removal of the occlusive dressing and persisting 10 to 17 d . On the rabbit eye, 0.1 ml undiluted 3-MTP produced moderate to severe corneal injury with iritis and moderate conjunctival inflammation which persisted 21 d in 3/6 animals; 0.01 ml caused moderate diffuse corneal injury and moderate conjunctival inflammation with healing by 7 d . 3-MTP did not produce mutagenic activity either in the absence or presence of metabolic activation with a Salmonella typhimurium reverse mutation assay using strains TA98, TA100, TA1535, TA1537 and TA1538 . In a mouse lymphoma cell (L5178Y/tk +/-) assay, 3-MTP produced concentration-related increases in mutant colonies, both in the absence and presence of metabolic activation . Increases were mainly in the sigma (chromosomal damaging) colonies . In a mouse bone marrow micronucleus study, with vapor exposures to 37.4, 88.5 and 155.6 ppm for 1 h/d for 2 consecutive d, there were exposure concentration-related increases in micronucleated erythrocytes which were statically significant for male mice.

J Biol Chem, 2000 Jun 2, 275(22), 16767 - 73
Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system; Kucharski LM et al.; Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+) . We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium . Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx . Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA . The compounds inhibited uptake by the bacterial S . typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain . Cation hexaammines also inhibited growth of S . typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system . In contrast, hexacyano-cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake . The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase . These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation.

Int J Food Microbiol, 2000 Mar 10, 54(1-2), 127 - 32
Application of thin agar layer method for recovery of injured Salmonella typhimurium; Kang DH et al.; Xylose lysine decarboxylase (XLD) medium, a selective plating medium, can inhibit heat-injured Salmonella typhimurium from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not . To facilitate recovery of heat-injured S . typhimurium cells while providing selectivity of isolation of S . typhimurium from other bacteria in the sample, a thin agar layer (TAL) procedure was developed by overlaying 14 ml of nonselective medium (TSA) onto prepoured and solidified XLD medium in a 8.5 cm diameter Petri dish . During the first few hours of incubating the plate, the injured S . typhimurium repaired and started to grow in the TSA . During the resuscitation of injured cells, the selective agents from XLD were diffused to the TSA top layer part . Once the selective agents diffused to the top part of the TAL, the resuscitated S . typhimurium started to produce a typical reaction (black color) and other microorganisms were inhibited by the selective agents . The recovery rate for heat-injured (55 degrees C for 15 min) S . typhimurium with the TAL method was compared with TSA, XLD, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar 3-4 h after incubation) . No significant difference occurred among TSA, OV, and TAL (P > 0.05) for enumeration of heat-injured S . typhimurium, but they recovered significantly higher numbers than from XLD agar (P < 0.05).

Eur J Intern Med, 2000 Apr, 11(2), 96 - 97
Salmonella endocarditis presenting as cerebral hemorrhage; Gomez-Moreno J et al.; The case of a 35-year-old male with cerebral hemorrhage as a presenting complication of infective endocarditis of the aortic valve, caused by Salmonella typhimurium, is described . We emphasize the infrequent etiology and review the mechanisms of intracranial hemorrhage in infective endocarditis.

Microbes Infect, 2000 Feb, 2(2), 145 - 56
Salmonella pathogenicity islands: big virulence in small packages; Marcus SL et al.; Reflecting a complex set of interactions with its host, Salmonella spp . require multiple genes for full virulence . Many of these genes are found in 'pathogenicity islands' in the chromosome . Salmonella typhimurium possesses at least five such pathogenicity islands (SPI), which confer specific virulence traits and may have been acquired by horizontal transfer from other organisms . We highlight recent progress in characterizing these SPIs and the function of some of their genes . The role of virulence genes found on a highly conserved plasmid is also discussed . Collectively, these packages of virulence cassettes are essential for Salmonella pathogenesis.

J Biochem (Tokyo), 2000 Apr, 127(4), 577 - 83
Characterization of the binding of spike H protein of bacteriophage phiX174 with receptor lipopolysaccharides; Inagaki M et al.; The spike H protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged fusion (HisH) . On enzyme-linked plate assaying, HisH was found to bind specifically to the lipopolysaccharides (LPSs) of phiX174-sensitive strains, Escherichia coli C and Salmonella typhimurium Ra chemotype, having the complete oligosaccharide sequence of the R-core on the LPSs . In sharp contrast, HisH bound weakly to the LPSs of phiX174-insensitive strains, i.e . E . coli F583 (Rd(2)) lacking some terminal saccharides and E . coli O111: B4 (smooth strain) having additional O-repeats on the R-core . The fluorescence spectra of HisH changed dose-dependently in the case of the LPS of E . coli C, the intensity increasing and the emission peak shifting to the shorter wavelength side, which was attributable to the hydrophobic interaction of HisH with the LPS . The binding equilibrium was analyzed by fluorometric titration to determine the dissociation constant K(d), 7.02 +/- 0.37 microM, and the Gibbs free energy change DeltaG(0), -29.1 kJ mol(-1) (at 22 degrees C, pH 7.4) . Based on the temperature dependence of (K)d in a van't Hoff plot, the standard enthalpy change DeltaH(0) and the entropy change DeltaS(0) were calculated to be +23.7 kJ mol(-1) and 179 J mol(-1) K(-1) at 22 degrees C, respectively, and this binding was thereby concluded to be an entropy-driven reaction.

Biosci Biotechnol Biochem, 2000 Feb, 64(2), 414 - 6
Antimutagenicity of the purple pigment, hordeumin, from uncooked barley bran-fermented broth; Deguchi T et al.; The novel purple pigment hordeumin, an anthocyanin-tannin pigment, was produced from barley bran-fermented broth . The mutagenicity or antimutagenicity of hordeumin was investigated according to the Ames method, an indication of the safety of food, using Salmonella typhimurium TA98 . Despite the presence of S-9 mix, hordeumin was not mutagenic . On the other hand, hordeumin effectively decreased a reverse mutation from Trp-P-1, Trp-P-2, IQ, and B{a}P . Furthermore, hordeumin also decreased the reverse mutation from dimethyl sulfoxide extracts of grilled beef.

Commun Dis Intell, 1999 Apr 15, 23(4), 101 - 3
An outbreak of Salmonella typhimurium RDNC A045 at a wedding feast in South Australia; Brennan P et al.; In April 1998 an outbreak of salmonellosis amongst guests at a wedding feast was investigated . Of the 58 attendees interviewed 38 (66%) subsequently developed gastrointestinal symptoms . Stool cultures from 7 cases grew Salmonella Typhimurium RDNC A045 . Food samples were culture-negative for Salmonella spp . A cohort study implicated spatchcock (RR 2.5, 95% CI 1.09-5.77) and scampi (RR 2.0, 95% CI 1.05-3.89) . Temperature abuse and cross-contamination within the kitchen during preparation and cooking are likely to have been the main contributing factors to this outbreak . Control measures included staff education in safe food handling and improvements in poultry processing methods to minimise carcass contamination.

Biochemistry, 2000 Apr 4, 39(13), 3624 - 35
Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system . In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization; Brokx SJ et al.; Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17) . The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain . Complementation results in the formation of unstable heterodimers . None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I . Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer . The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM . It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type . In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay . Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site . Gly356Ser mutation impairs enzyme I dimerization . The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site . A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.

J Appl Microbiol, 2000 Feb, 88(2), 213 - 9
Fluorometric assessment of gram-negative bacterial permeabilization; Helander IM et al.; Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria . An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence . With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria . Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps . aeruginosa, Salm . typhimurium) or almost totally (E . coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation . Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2 . In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.

J Appl Microbiol, 2000 Feb, 88(2), 202 - 12
Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium; Tholozan JL et al.; The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions . Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers . Increasing pressure treatments resulted in an exponential decrease of cell counts . Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments . Progressive morphological changes were evident with the pressure increase . Cell lysis only appeared with the highest pressure treatments . Cell volume was not affected by pressure treatment . A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase . A parallel lowering of membrane potentials was measured.

Vet Microbiol, 2000 Apr 4, 73(1), 25 - 35
Augmentation of antibiotic resistance in Salmonella typhimurium DT104 following exposure to penicillin derivatives; Carlson SA et al.; Antibiotic resistance in pathogenic bacteria has been a problem in both developed and developing countries . This problem is especially evident in Salmonella typhimurium, one of the most prevalent foodborne pathogens . While performing in vitro gentamicin protection-based invasion assays, we found that certain isolates of multiresistant S . typhimurium can be 'induced' to exhibit new resistance profiles . That is, bacteria become resistant to a wider range of antibiotics and they also exhibit quantitative increases in MIC values for antibiotics that were part of their pre-induction antibiograms . This 'induction' process involves growing the bacteria to stationary phase in the presence of antibiotics such as ampicillin, amoxicillin or ticarcillin . Since the isolates studied exhibited resistance to ampicillin, amoxicillin and ticarcillin prior to exposing the bacteria to these antibiotics, the observed phenomenon suggests that resistant Salmonella not only have a selective advantage over non-resistant Salmonella but their resistance phenotypes can be accentuated when an inappropriate antibiotic is used therapeutically.

J Vet Diagn Invest, 2000 Mar, 12(2), 130 - 5
Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide-derived antigens covalently attached to polystyrene microwells; Wiuff C et al.; Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation . Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate . The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs . This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low day-to-day variations and low interplate variations . Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA) . The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs.

Mutat Res, 2000 Mar 23, 466(2), 173 - 8
Genotoxicity of drinking water from three Korean cities; Park JH et al.; Organic content of drinking tap water from Seoul, Taejon, and Suwon was extracted with an XAD-2 resin column and organic solvents . Four doses of the extract equivalent to 4, 2, 1, and 0.5 l water were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix . The organic extracts of the water from all three cities were mutagenic in TA 98 without S9 mix and in TA 100 with and without S9 mix . The highest number of revertants per plate was found in the absence of S9 mix . Three doses of the extract (equivalent to 22, 11, and 3.7 l water) were also tested in the bone marrow micronucleus test using BDF1 mice . At the highest dose, a significant increase of the micronucleus frequency was observed . The time required to be on the effect, however, varied with the source of the water . Our results indicate that the drinking tap waters from the three cities were genotoxic clearly in the bacterial test and also in the in vivo assay with mice . As we found no genotoxicity of the source water as seen in a previous study, it is likely that the chlorination process leads to the genotoxicity of the tap water.

Mutat Res, 2000 Mar 23, 466(2), 143 - 59
Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons; Kappers WA et al.; Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene . Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix.Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite . In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells . However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX) . The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline . No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested.In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways.

FEMS Immunol Med Microbiol, 2000 Apr, 27(4), 341 - 9
Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier; Darji A et al.; The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines . Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids . Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice . Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration . Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen . A single dose was already partially protective . The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host . Evidence for such an event could be obtained in vivo and in vitro . Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes . Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c . Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.

FEMS Immunol Med Microbiol, 2000 Apr, 27(4), 333 - 40
Molecular analysis of hemolysin-mediated secretion of a human interleukin-6 fusion protein in Salmonella typhimurium; Li Y et al.; Previously, we reported a plasmid-bearing Salmonella typhimurium strain capable of secreting human interleukin-6 (hIL-6) when genetically fused to the Escherichia coli hemolysin transport signal (HlyA(S)) . Stationary phase culture supernatants of this strain revealed three major forms of hIL-6-HlyA(S) fusion protein (apparent molecular masses 32.4, 30.3, 27.0 kDa), at which the largest protein presumably represented full-length hIL-6-HlyA(S) . The biological activity of the hIL-6-HlyA(S) protein mixture was similar to that of mature hIL-6 . Accumulation of hIL-6-HlyA(S) in the culture supernatant occurred only during the initial growth phase, whereas in stationary phase and under in vitro conditions successive cleavage into the two truncated forms was observed . On the other hand, in whole cell lysates only full-length hIL-6-HlyA(S) could be detected, accounting for more than 50% of the totally synthesized protein . Upon cell fractionation, cellular hIL-6-HlyA(S) was exclusively found in the membrane fraction . These results suggest, that in S . typhimurium production and secretion of hIL-6-HlyA(S) is restricted to growing cells . A specific processing by a Salmonella-derived protease did not affect the biological activity of the fusion protein.

FEMS Immunol Med Microbiol, 2000 Apr, 27(4), 313 - 20
Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response; Yrlid U et al.; Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells . However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria . Thus, DC may also be important APC for initiating an immune response to bacterial infections . Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e . Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo . Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S . typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II . These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules . In contrast, DC process E . coli and S . typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules . We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S . typhimurium infection of BMDC and BMMPhi . These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86 . Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12 . Finally, injecting mice with BMDC that have been loaded in vitro with S . typhimurium primes naive CD4(+) and CD8(+) T cells to Salmonella-encoded antigens . Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.

FEMS Immunol Med Microbiol, 2000 Apr, 27(4), 283 - 9
Protection against murine tuberculosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the 30-kDa antigen of Mycobacterium bovis BCG; Hess J et al.; A recombinant (r-) Salmonella typhimurium aroA vaccine that secretes the naturally secreted protein of Mycobacterium bovis strain BCG, Ag85B, by means of the HlyB/HlyD/TolC export machinery (termed p30 in the following) was constructed . In contrast to r-S . typhimurium control, oral vaccination of mice with the r-S . typhimurium p30 construct induced partial protection against an intravenous challenge with the intracellular pathogen Mycobacterium tuberculosis, resulting in similar vaccine efficacy comparable to that of the systemically administered attenuated M . bovis BCG strain . The immune response induced by r-S . typhimurium p30 was accompanied by augmented interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) levels produced by restimulated splenocytes . These data suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated r-S . typhimurium as carrier is capable of inducing an immune response against mycobacterial antigens.

Pathobiology, 1999, 67(5-6), 227 - 9
Fighting infection: the role of lipopolysaccharide binding proteins CD14 and LBP; Schutt C; An invading pathogen must be held in check by the innate immune system until a specific immune response is mounted . Nonclonal pattern recognition receptors like CD14 or lipopolysaccharide (LPS) binding protein (LBP) recognize ubiquitous pathogen-associated molecular patterns, e.g . LPS . LBP mediates the binding of minute amounts of LPS to membrane-bound CD14 (mCD14) triggering a proinflammatory response of macrophages, which is crucial for keeping an infection under control . Moreover, in vitro mCD14 and LBP are involved in recognition and phagocytosis of heat-killed bacteria . Living Salmonella typhimurium or Escherichia coli depend on the presence of LBP to induce the generation of reactive oxygen species in human or murine macrophages . Using LBP-deficient mice it could be demonstrated that LBP is essential to control low dose (100 CFU S . typhimurium) infection . Therefore, LPS binding proteins play a pivotal role in physiology as well as pathophysiology of Gram-negative infection .

J Agric Food Chem, 2000 Mar, 48(3), 642 - 7
Antimutagenic activity of flavonoids from Pogostemon cablin; Miyazawa M et al.; A methanol extract from Pogostemon cablin showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) . The methanol extract was re-extracted with hexane, dichloromethane, butanol, and water . A dichloromethane fraction showed a suppressive effect . Suppressive compounds against furylfuramide in the dichloromethane fraction were isolated by SiO(2) column chromatography and identified as 7,4'-di-O-methyleriodictyol (1), 7, 3',4'-tri-O-methyleriodictyol (2), and 3,7,4'-tri-O-methylkaempferol (3) . In addition, three flavonoids, ombuine (4), pachypodol (5), and kumatakenin (6), were isolated and identified from the dichrolomethane fraction . Compounds 1 and 3 suppressed >50% of the SOS-inducing activity at <0.6 micromol/mL, and the ID(50) values of both compounds were 0.25 micromol/mL . Compound 2 showed a weakly suppressive effect (17%) at a concentration of 0.6 micromol/mL, and compounds 4-6 did not . These compounds were also assayed with 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which requires liver metabolizing enzymes . Compounds 3-6 suppressed >80% of the SOS-inducing activity of Trp-P-1 at <0.06 micromol/mL, and compounds 1 and 2 suppressed 87 and 63% at a concentration of 0.3 micromol/mL . In addition, these compounds were assayed with activated Trp-P-1, and the suppressed effects of these compounds were further decreased when compared to Trp-P-1 . The antimutagenic activities of these compounds against furylfuramide, Trp-P-1, and activated Trp-P-1 were assayed by the Ames test using S . typhimurium TA100.

Chem Res Toxicol, 2000 Mar, 13(3), 165 - 9
Isolation and chemical--structural identification of a novel aromatic amine mutagen in an ozonized solution of m-phenylenediamine; Kami H et al.; The mutagenicity of a m-phenylenediamine (m-PD) solution was markedly enhanced by oxidation with ozone . The ethyl acetate extracts from a m-PD solution ozonized at pH 10.7 were fractionated by normal-phase and reversed-phase column chromatography to isolate mutagens by monitoring mutagenic activities on Salmonella typhimurium TA98 in the presence of a mammalian metabolic activation system (S9 mix) . From fraction 5-3-2, which exhibited the strongest mutagenicity (308000 revertants/mg), a major mutagenic compound was isolated . On the basis of the high-resolution EI-mass, (1)H NMR and (13)C NMR spectral, and X-ray crystallography data, the structure of this compound was determined to be 2-amino-5-{(3-aminophenyl)amino}-4-{(3-aminophenyl)imino}-2, 5-cyclohexadien-1-one (PDT-1), which is a novel compound . PDT-1 is a newly identified frame-shift type mutagen, inducing 65400 revertants and 295000 revertants of S . typhimurium TA98 and YG1024 per micromole, respectively, in the presence of S9 mix . When a m-PD solution was oxidized with 1 or 2 mol of ozone at pH 4.0, 7.0, and 10.7, the contribution of PDT-1 to the mutagenicity of ethyl acetate extracts from the ozonized m-PD solution was 5-23%.

Toxicol Lett, 2000 Mar 15, 112-113, 341 - 8
Sulfotransferases: genetics and role in toxicology; Glatt H et al.; The mammalian xenobiotic-metabolizing sulfotransferases are cytosolic enzymes, which form a gene superfamily (SULT) . Ten distinct human SULT forms are known . Two SULT forms represent splice variants, the other forms are encoded by separate genes . Common functional polymorphisms of the transcribed region are known for two of the forms . We have expressed 16 separate rat and human SULTs as well as some of their allelic variants, in Salmonella typhimurium TA1538 and/or V79 cells, which are target cells of commonly used mutagenicity assays . The expressed SULTs activated numerous compounds to mutagens in both assay systems . However, some promutagens were activated by only one or several of the human SULTs . Pronounced differences in promutagen activation were also detected between orthologous rat and human SULTs, and between allelic variants of human SULTs.

Mol Cell Biochem, 2000 Jan, 204(1-2), 107 - 17
Intestinal mucins: the binding sites for Salmonella typhimurium; Vimal DB et al.; Mucus-bacterial interactions in the gastrointestinal tract and their impact on subsequent enteric infections are poorly delineated . In the present study, we have examined the binding of Salmonella typhimurium to rat intestinal mucus and characterized a mucus protein (Mucus-Rs) which specifically binds to S . typhimurium . Both virulent (1402/84), and avirulent (SF 1835) S . typhimurium were observed to bind to crude mucus, however, the virulent strain showed 6 fold more binding as compared to avirulent strain . Fractionation of crude mucus on sepharose CL-6B resolved it into three major peaks . Maximal bacterial binding was observed with a high mol . wt . glycoprotein corresponding to neutral mucin . SDS-PAGE of purified protein (termed Mucus-Rs) under non reducing conditions showed it to be a homogenous glycoprotein (mol . wt . 250 kDa), while under reducing conditions, three bands corresponding to mol . wt . of 118,75 and 60 kDa were observed . Pretreatment of Mucus-Rs with pronase, trypsin and sodium metaperiodate markedly inhibited bacterial binding . GLC analysis of Mucus-Rs showed it to contain Mannose, Glucose, Galactose, Glucosamine, Galactosamine and Sialic acid as main sugars . Competitive binding in the presence of various sugars and lectins indicated the involvement of mannose in the mucus-bacterial interactions . The Mucus-Rs binding was highly specific for S . typhimurium; no significant binding was seen with E . coli and V . cholerae . Thus, we conclude that S . typhimurium specifically binds to a 250 kDa neutral mucin of intestinal tract . This binding appears to occur via specific adhesin-receptor interactions involving bacterial pili and mannose of neutral mucin.

J Mol Biol, 2000 Mar 24, 297(2), 355 - 64
Co-evolution of the tuf genes links gene conversion with the generation of chromosomal inversions; Hughes D; The tufA and tufB genes in Salmonella typhimurium co-evolve by recombination and exchange of genetic material . A model is presented which predicts that co-evolution is achieved by gene conversions and chromosomal inversions . Analysis of recombinants reveals that conversion and inversion each occur with similar rates and each depends on RecBCD activity . The model predicts sequence structures for different classes of post-recombination tuf genes . Sequence analysis reveals the presence of each of these structures and classes, with a predicted bias in the absence of mismatch repair . An implication of these data is that co-evolution of gene families can be linked with the generation of chromosomal rearrangements .

Mol Microbiol, 2000 Mar, 35(5), 1146 - 55
The putative iron transport system SitABCD encoded on SPI1 is required for full virulence of Salmonella typhimurium; Janakiraman A et al.; Salmonella typhimurium is an invasive pathogen that causes diseases ranging from mild gastroenteritis to enteric fever . During the infection process, S . typhimurium induces a number of virulence genes required to circumvent host defences and/or acquire nutrients in the host . We have used the in vivo expression technology (IVET) system to select for S . typhimurium genes that are induced after invasion of a murine cultured cell line . We have characterized a putative iron transporter in Salmonella pathogenicity island 1, termed sitABCD . The sitABCD operon is induced under iron-deficient conditions in vitro and is repressed by Fur . This locus is induced in the animal specifically after invasion of the intestinal epithelium . We show that a sit null mutant is significantly attenuated in BALB/c mice, suggesting that SitABCD plays an important role in iron acquisition in the animal.

Mutat Res, 2000 Feb 16, 465(1-2), 165 - 71
Comparison of the mutagenic specificity induced by four nitro-group-containing aromatic amines in Salmonella typhimurium his genes; Chung K et al.; Four nitrated aromatic amines (2-nitro-p-phenylenediamine {2NPD}, 3-nitro-o-phenylenediamine {3NPD}, 4-nitro-o-phenylenediamine {4NPD} and 4,4'-dinitro-2-biphenylamine {DNBA}) are direct-acting mutagens in Salmonella typhimurium strain TA100 . These compounds were tested further using the Xenometrix strains of S . typhimurium: TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006, with and without S9 mix in the plate incorporation assay . The direct-acting mutagenicity of 2NPD, 4NPD, and DNBA was detected with TA7002, TA7004 and TA7005 . 2NPD and DNBA showed some activity in TA7006; DNBA also showed some activity in TA7003 . Mutagenicity was generally decreased in these strains when S9 was added . 3NPD was mutagenic in TA7004 without S9 and in TA7005 with and without S9 . These data suggest that 2NPD, 4NPD and DNBA induced TA-->AT and CG-->AT transversions as well as GC-->AT transitions in the his gene . 3NPD induced CG-->AT transversions and GC-->AT transitions . 2NPD and DNBA also induced a small portion of CG-->GC transversions.

Mutat Res, 2000 Feb 16, 465(1-2), 85 - 90
Bioactivation of the carcinogen 11-methoxy-16, 17-dihydro-15H-cyclopenta{a}phenanthrene; Catterall FS et al.; The title compound is a more potent carcinogen than would be anticipated from its simple phenanthrene structure lacking further D-ring conjugation . In vitro it undergoes microsomal metabolism to yield as major metabolites its 15- and 17-alcohols and its 16, 17-diol; other minor metabolites are also derived from attack at the 5-membered ring, but no evidence of aromatic oxidation is apparent . The title compound is a weak mutagen in the Ames' test with Salmonella typhimurium TA100, but only with microsomal bio-activation . The 17-ol and 16,17-diol are inactive, with or without biological activation . By contrast the 15-alcohol, a rather reactive compound, is a strong mutagen both in the presence and absence of the bio-activation system . This, therefore, may be the proximate carcinogen, and its structural analogy to the naturally occurring hepato-carcinogen safrole is noted.

Biochim Biophys Acta, 2000 Mar 7, 1477(1-2), 90 - 7
S-nitrosylated human alpha(1)-protease inhibitor; Miyamoto Y et al.; Alpha(1)-protease inhibitor (alpha(1)PI) is an acute phase plasma protein, and possesses a single cysteine residue at position 232 . A single cysteinyl sulfhydryl of human alpha(1)PI is found to be readily S-nitrosylated by nitric oxide (NO) in vitro without affecting the inhibitory capacity against bovine trypsin or elastase, a major target protease of alpha(1)PI in vivo . S-nitroso-alpha(1)PI (S-NO-alpha(1)PI) was also formed by the reaction of alpha(1)PI with NO produced excessively by a murine macrophage cell line (RAW264 cells) upon infection with Salmonella typhimurium and in an ex vivo perfusion system of the liver obtained from lipopolysaccharide-treated rats . S-NO-alpha(1)PI (10(-9)-10(-6) M) induces a dose-dependent relaxation of the ring preparation of rabbit aorta . Also, S-NO-alpha(1)PI but not alpha(1)PI shows a potent inhibitory effect on platelet aggregation . Unprecedented observation is that S-NO-alpha(1)PI showed a potent bacteriostatic effect against a wide range of bacteria at the concentration of 1-10 microM, which was 10-1000-fold stronger than that of NO and other S-nitrosylated compounds including S-nitrosylated albumin and S-nitrosylated glutathione . These results suggest that S-NO-alpha(1)PI is produced as an NO sink under inflammatory conditions, where production of both alpha(1)PI and NO is highly up-regulated, and it may function as a soluble factor which consists of an innate defense system through not only the protease inhibitory activity but also its antibacterial activity and facilitating the peripheral blood flow . Therefore, S-nitrosylation of alpha(1)PI occurring under physiological conditions in vivo should diversify the biological functions contributing to cytoprotective effects of alpha(1)PI.

Tsitol Genet, 1999 Nov-Dec, 33(6), 19 - 25
{The antimutagenic activity of biomass extracts from the cultured cells of medicinal plants in the Ames test}; Duhan OM et al.; Antimutagenic activity of 20 and 40% ethanol extracts from the biomass of Rhodiola rosea, Polyscias filicifolia, Panax ginseng and Ungernia victoris cultured cells have been studied . DDDTDP, ethidium bromide, benz(a)pyrene, benzidine served as model mutagens for Salmonella typhimurium TA 98 strain (the latter two were tested in presence of metabolic activation system); for S . typhimurium TA 100 strain these were tio-tefa, bichromate potassium and sodium azide and heavy metal compounds (chlorides of manganese, zinc, cadmium, lead acetate) for both strains . Higher capacity of the extracts from the biomass of R . rosea and P . filicifolia to counteract gene mutations induced by various mutagens was demonstrated (ca . 90% inhibition in isolated cases) . In the experiment with the metabolic activation most effective proved to be the extracts from the P . ginseng biomass (up to 34% and 47% mutagenicity inhibition).

Planta Med, 2000 Feb, 66(1), 70 - 1
Baicalein: an in vitro antigenotoxic compound from Scutellaria baicalensis; Lee BH et al.; Bioassay-guided fractionation of an H2O extract of the root of Scutellaria baicalensis has furnished an in vitro antigenotoxic flavonoid, baicalein (1) and 2',5,5',7-tetrahydroxy-6',8-dimethoxyflavone (2) . Compound 1 exhibited a dose-dependent inhibition of aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidine mutagenicity in the Salmonella typhimurium bacterial mutation assay . In the chromosome aberration assay, compound 1, at a concentration of 5 microM, reduced the frequency of chromosome aberration induced by AFB1 but increased the clastogenic effect of AFB1 at a concentration of 50 microM.

Gen Physiol Biophys, 1999 Oct, 18 Spec No, 92 - 8
Genetic risk assessment of acid waste water containing heavy metals; Miadokova E et al.; The mutagenic/cancerogenic potential of acid-mine water from the Slovak mining area Rudnany containing a high load of toxic metals was evaluated after its application to three model test organisms (bacteria Salmonella typhimurium, yeast Saccharomyces cerevisiae and plant Vicia sativa L.) . The results obtained from the modified preincubation Ames assay proved that 1000-fold diluted waste water exhibited mutagenic effect in three (TA97, TA98, TA102) of four bacterial strains . In the test on yeast the toxicity and genotoxicity increased as a function of the concentration . At the highest concentration used (0.06%) the frequency of revertants increased 6 times and convertants increased 4.5 times above the control level . In the simultaneous phytotoxicity and clastogenicity assay, concentration dependent toxicity and statistically significant clastogenicity was proved . We can conclude that heavy metals might be responsible for the genotoxic/cancerogenic potential of the test water . However, we do not entirely exclude the possibility that its genotoxicity might be promoted by its high acidity.

Microb Pathog, 2000 Mar, 28(3), 157 - 67
Immune recognition of porin and lipopolysaccharide epitopes of Salmonella typhimurium in mice; Singh SP et al.; We investigated the antigenic specificity of the humoral immune response to infection by Salmonella typhimurium, by competitive inhibition enzyme-linked immunosorbent assay and Western immunoblots . A panel of eight murine monoclonal antibodies, raised to OmpC and OmpD porins and lipopolysaccharide (LPS)-O antigens, was used to define the specificity of the polyclonal immune response in mice . The monoclonal antibody panel recognized five distinct epitopes; these were localized to surface-exposed loops of OmpC and OmpD porin, to the "eye-let" forming loop L3 of OmpC/OmpD, and to LPS-O4 and O5 factors . The immune mouse serum raised to infections with S . typhimurium LT-2 strain WB600 (wild-type) competitively inhibited the binding of biotin-labelled monoclonal antibodies to the epitopes that they recognize, indicating that all five epitopes were targets of the host immune response to natural infection . However, only two epitopes, one within a surface-exposed loop of OmpC porin, and the other in the LPS-O4 factor, were immunodominant . Furthermore, the bacterial LPS core and O-antigen structure influenced the immune response to the porins . Surface epitopes of porins were dominant in the rough strain SH5014 (rfa), whereas the immune recognition of LPS epitopes was predominant in mice infected with the smooth, wild-type strain (WB600) . Finally, the immune response to LPS epitopes O4 and O5 was more pronounced in mice immunized with heat-killed cells than those infected with live S . typhimurium .

Free Radic Biol Med, 2000 Feb 1, 28(3), 330 - 6
Cyto- and genotoxic effects of novel aromatic nitroxide radicals in vitro; Damiani E et al.; Because of the increasing interest in the use of nitroxide radicals as antioxidants and probes for various applications in biological systems, the question of their toxicity is of paramount importance . Cytotoxicity and mutagenicity studies have been extensively performed with the commercially available aliphatic nitroxides, and the general outcome is that these compounds are nonmutagenic and relatively noncytotoxic . In this study, the cytotoxicity and genotoxicity of a new class of aromatic nitroxides that we have synthesized (i.e., indolinonic and quinolinic nitroxides), whose antioxidant activity has been established in both chemical and biological systems, were evaluated and compared with those of two commercial nitroxides and with that of butylated hydroxytoluene (BHT) . The mutagenicity assay was performed using Salmonella typhimurium tester strains TA98, TA100, and TA102, chosen on the basis of their ability to detect various types of mutations and their sensitivity to oxidative damage . None of the compounds tested were found to be mutagenic . The colony-forming assay (CFA) using Chinese hamster ovary (CHO) AS52 cells was employed for determining the cytotoxicity of the test compounds . On comparing the effective dose that inhibits the CFA by 50% (IC(50)), most of the compounds tested on an equal molar concentration basis were less toxic than BHT . Therefore, the overall results obtained correlate well with the data reported in the literature on the toxicity of aliphatic nitroxides and lend support to the possible use of these compounds as therapeutic antioxidants.

Science, 2000 Mar 3, 287(5458), 1655 - 8
Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase; Vazquez-Torres A et al.; A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages . Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages . The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst . Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.

APMIS, 2000 Jan, 108(1), 45 - 50
Identification of a Salmonella typhimurium genomic region involved in invasion of HeLa and Henle-407 epithelial cells; Park JU et al.; To identify the invasion determinant, a cosmid library was constructed by cloning a genomic library of Salmonella typhimurium 82/6915 into a cosmid vector, pLA2917 . A genomic region involved in invasion of cultured HeLa and Henle-407 cells was subcloned into plasmid pGEM-7Z . E . coli strain DH1 carrying pSV6235 consisting of a S . typhimurium 4.6 kb genomic region in pGEM-7Z showed invasion of cultured HeLa and Henle-407 cells . Nested sequential deletions were introduced into the 4.6 kb genomic region of pSV6235 . The E . coli recombinants which contained less than 1.5 kb deletions from the 5' end (SmaI site) of the genomic region invaded the cells as effectively as DH1 (pSV6235) . The invasion of the recombinants carrying over 2.0 kb deletions from the end of pSV6235 was significantly inactivated compared to DH1 (pSV6235) . Restriction enzyme analysis showed that the 3.1 kb fragment from the 3' end of the 4.6 kb genomic region was distinguished from the Salmonella pathogenicity I genes of S . typhimurium such as the inv, spa, and hil regions showing invasion of the cultured eukaryotic cells.

Antonie Van Leeuwenhoek, 2000 Jan, 77(1), 13 - 20
Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2; Horton AJ et al.; Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans . S . typhimurium LT2 cultures, when transferred from 37 degrees C to 5 degrees C or 10 degrees C, showed an initial lag period in growth with an approximate generation time of 10-25 h . Western blot assay using E . coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S . typhimurium cultures after exposure to 10 degrees C or 5 degrees C showed elevated expression of a major cold shock protein, CS7.4 . Identification of a decreased level of CS7.4 at 37 degrees C suggests that the expression of this protein may require a large temperature downshift . Putative regulatory protein binding segment on the 5'-untranslated region referred as 'Fragment 7' in S . typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E . coli and S . enteritidis, respectively . The differences in the nucleotide sequence within the Fragment 7 between S . typhimurium and S . enteritidis may explain the differential expression of CspA at 37 degrees C . The nucleotide sequence of the open reading frame of S . typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine . In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S . typhimurium were exposed to 10 degrees C or 5 degrees C . Differential expression of the CspA and other proteins in S . typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.

Toxicol Sci, 2000 Feb, 53(2), 253 - 63
Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs; Zhou HR et al.; Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity . LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins . Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure . The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis . Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone . Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice . Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone . When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone . The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

J Biol Chem, 2000 Mar 10, 275(10), 6783 - 9
The study of guanosine 5'-diphosphate 3'-diphosphate-mediated transcription regulation in vitro using a coupled transcription-translation system; Choy HE; The effects of the "alarmone" guanosine 5'-diphosphate 3'-diphosphate (ppGpp) on regulation of the Salmonella typhimurium histindine operon and the Escherichia coli tRNA(leu) operon were analyzed in vitro using a DNA-dependent transcription-translation system, S-30 . The expression of the hisG promoter is positively regulated by ppGpp, whereas that of the leuV promoter (of tRNA(1eu)) is negatively regulated by ppGpp . In an attempt to understand the global regulatory mechanism of ppGpp control, interrelationship between ppGpp-dependent activation and repression of gene expression was examined using these promoters as models . It has been traditionally supposed that the ppGpp-dependent regulation, at least for the activation, is by a passive mode of control: the activation of gene expression by ppGpp is a consequence of the repression of stable RNA gene expression in the condition of RNA polymerase limiting . To test this model, the ppGpp-dependent regulations of both an activable promoter (hisGp) and a repressible promoter (leuVp) were determined in vitro simultaneously using a mixed template setup . The rationale for this exercise was to see whether the ppGpp-dependent activation and repression are inversely correlated in the in vitro condition in which RNA polymerase is limiting . No correlation was observed . It was concluded that the ppGpp-dependent activation is independent of the repression . Moreover, it was proposed that ppGpp-dependent activation and repression are mediated by titratable factors, each of which operate independently.

J Food Prot, 2000 Feb, 63(2), 155 - 61
Multidrug-resistant Salmonella Typhimurium DT104 in poultry; Rajashekara G et al.; Salmonella Typhimurium isolates from feed ingredients or poultry sources isolated during 1995 to 1997 from different geographical locations within Minnesota were examined for the presence of Salmonella Typhimurium definitive type 104 (DT104) . Antibiotic susceptibility studies indicated that 15 of 50 isolates of Salmonella Typhimurium had an antibiotic resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) that is usually observed with multidrug-resistant Salmonella Typhimurium DT104 . Of the 15 isolates showing the antibiotic resistance pattern, 8 isolates were phage type 104, 3 isolates were typed as phage type 104 complex, and the remaining 4 isolates belonged to phage types 193, 81, and 126 . DT104 was recovered from both feed ingredients and poultry samples . Of the seven feed ingredient-associated Salmonella Typhimurium isolates, four were DT104, whereas only 7 of 43 poultry-associated Salmonella Typhimurium isolates were DT104 . A repetitive sequence-based polymerase chain reaction (rep-PCR) of 50 isolates of Salmonella Typhimurium representing 13 phage types identified seven distinct fingerprint profiles . No correlation between phage type and rep-PCR type was noticed . Eleven Salmonella Typhimurium isolates belonging to DT104 and its complex were grouped into two closely related rep-PCR types.

J Immunol, 2000 Mar 1, 164(5), 2644 - 9
Neutralization of IL-18 reduces neutrophil tissue accumulation and protects mice against lethal Escherichia coli and Salmonella typhimurium endotoxemia; Netea MG et al.; In addition to stimulating IFN-gamma synthesis, IL-18 also possesses inflammatory effects by inducing synthesis of the proinflammatory cytokines TNF and IL-1beta and the chemokines IL-8 and macrophage inflammatory protein-1alpha . We hypothesized that neutralization of IL-18 would have a beneficial effect in lethal endotoxemia in mice . IL-1beta converting enzyme (ICE)-deficient mice, lacking the ability to process mature IL-18 and IL-1beta, were completely resistant to lethal endotoxemia induced by LPS derived from either Escherichia coli or Salmonella typhimurium . In contrast, both wild-type and IL-1beta-/- mice were equally susceptible to the lethal effects of LPS, implicating that absence of mature IL-18 or IFN-gamma but not IL-1beta in ICE-/- mice is responsible for this resistance . However, IFN-gamma-deficient mice were not resistant to S . typhimurium LPS, suggesting an IFN-gamma-independent role for IL-18 . Anti-IL-18 Abs protected mice against a lethal injection of either LPS . Anti-IL-18 treatment also reduced neutrophil accumulation in liver and lungs . The increased survival was accompanied by decreased levels of IFN-gamma and macrophage inflammatory protein-2 in anti-IL-18-treated animals challenged with E . coli LPS, whereas IFN-gamma and TNF concentrations were decreased in treated mice challenged with S . typhimurium . In conclusion, neutralization of IL-18 during lethal endotoxemia protects mice against lethal effects of LPS . This protection is partly mediated through inhibition of IFN-gamma production, but mechanisms involving decreased neutrophil-mediated tissue damage due to the reduction of either chemokines (E . coli LPS) or TNF (S . typhimurium LPS) synthesis by anti-IL-18 treatment may also be involved.

J Immunol, 2000 Mar 1, 164(5), 2281 - 4
Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease; Matsushita M et al.; Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different . A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL . On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19) . We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP . MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3 . The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement . These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.

Environ Mol Mutagen, 2000, 35(1), 39 - 47
Nitric oxide-induced mutations in the HPRT gene of human lymphoblastoid TK6 cells and in Salmonella typhimurium; Zhuang JC et al.; Characterization of mutations induced by NO in different experimental systems will facilitate elucidation of mechanisms underlying its genotoxicity . The mutagenic specificity of NO in human cells is of particular interest in view of its potential role in inflammation-associated carcinogenesis . We compared mutagenesis in human lymphoblastoid TK6 cells and in Salmonella typhimurium induced by exposure to NO delivered into the medium at rates approximating its production by activated macrophages . Exposure of TK6 cells continuously for 60 min decreased viability by 88%, and survivors exhibited a sixfold increase in mutant fraction in the hprt gene . Independent mutants were isolated and mutations characterized by RT-PCR and DNA sequencing . Among a total of 68 mutants analyzed, RT-PCR products were obtained in 41 (60%), and cDNA sequencing revealed that 26 (63%) of them contained mutations located in the hprt coding region . Base substitutions were present in 18 mutants, 12 occurring at A:T base pairs . Seven mutants contained deletions of 1-27 bp and one a 13-bp insertion; the 15 remaining RT-PCR products contained whole-exon deletions, 14 involving single exons . Six tester strains of S . typhimurium, each containing one of the six possible point mutations in the target codon of a gene in the histidine biosynthetic pathway, were similarly treated with NO and induction of mutation was detected by reversion to histidine auxotrophy . Significant increases were observed in frequencies of each of the six possible base mutations, with the highest occurring in G:C --> A:T transitions . The pattern of NO-induced hprt mutations in TK6 cells was similar to a recently published spectrum in spontaneous mutants, suggesting that reactive species derived from NO may contribute to spontaneous mutagenesis of the endogenous hprt gene in human cells .

Mol Microbiol, 2000 Feb, 35(4), 949 - 60
The putative invasion protein chaperone SicA acts together with InvF to activate the expression of Salmonella typhimurium virulence genes; Darwin KH et al.; SigD and SigE (Salmonella invasion gene) are proteins needed for optimal invasion of Salmonella typhimurium into eukaryotic cells in vitro . SigD is a secreted protein and SigE is a putative chaperone required for SigD stability and/or secretion . SigD is secreted by a type III secretion apparatus encoded within a pathogenicity island on the Salmonella chromosome known as Salmonella pathogenicity island 1 (SPI1) . The expression of sigDE, which is not linked to SPI1, is co-ordinately regulated with the SPI1 genes and is dependent on the transcriptional regulators SirA, HilA and InvF . These three proteins alone are unable to activate transcription from the sigD promoter in Escherichia coli, therefore it is likely that other factors are needed for expression . A screen for genes required for the expression of a sigD-lacZYA reporter fusion found a mutant with a transposon insertion in spaS, an SPI1 gene which encodes a putative inner-membrane component of the type III secretion system . The expression of a SPI1 operon encoding a putative chaperone (SicA) and several secreted proteins (Sips B, C, D and A) was also reduced in this mutant . The regulation defect of the spaS mutant was complemented by sicA and not by spaS . Because sicA is encoded immediately downstream of spaS, the mutation in spaS was likely to be polar on the expression of sicA . In addition, a sicA disruption mutant was as defective as an invF deletion mutant for the expression of sigD, sicA and sipC reporter fusions . The introduction of plasmids encoding invF and sicA into a non-pathogenic E . coli K-12 strain stimulated the transcription of both a sicA- and a sigD-lacZYA promoter fusion . This result suggests that InvF and SicA are sufficient for the expression of these genes . This is the first demonstration of a positive regulatory role for a putative type III secretion system chaperone in the expression of virulence genes.

Mol Microbiol, 2000 Feb, 35(4), 777 - 90
In vivo and in vitro studies of transmembrane beta-strand deletion, insertion or substitution mutants of the Escherichia coli K-12 maltoporin; Charbit A et al.; LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane . Each monomer is composed of an 18-stranded antiparallel beta-barrel . In the present work, on the basis of the known X-ray structure of LamB, the effects of modifications of the beta-barrel domain of maltoporin were studied in vivo and in vitro . We show that: (i) the substitution of the pair of strands beta13-beta14 of the E . coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of beta-strands (deletion beta13-beta14) from the E . coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E . coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro . We also inserted into deletion beta13-beta14 the portion of the E . coli LamB protein comprising strands beta13 to beta16 . This resulted in a protein expected to have 20 beta-strands and which completely lost all LamB-specific activities in vivo and in vitro.

Mol Microbiol, 2000 Feb, 35(4), 728 - 42
DNA methylation-dependent regulation of pef expression in Salmonella typhimurium; Nicholson B et al.; Plasmid-encoded fimbriae (Pef) expressed by Salmonella typhimurium mediate adhesion to mouse intestinal epithelium . The pef operon shares features with the Escherichia coli pyelonephritis-associated pilus (pap) operon, which is under methylation-dependent transcriptional regulation . These features include conserved DNA GATC box sites in the upstream regulatory region as well as homologues of the PapI and PapB regulatory proteins . Unlike Pap fimbriae, which are expressed in a variety of laboratory media, Pef fimbriae were expressed only in acidic, rich broth under standing culture conditions . Analysis of S . typhimurium grown under these conditions indicated that Pef production was regulated by a phase variation mechanism, in which the bacterial population was skewed between fimbrial expression (phase ON) and non-expression (phase OFF) states . Leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam) were required for pef transcription . In contrast, the histone-like protein (H-NS) and the stationary-phase sigma factor (RpoS) repressed pef transcription . Methylation of the pef GATC II site appeared to be required for pef fimbrial expression based on analysis of a GCTC II mutant that did not express Pef fimbriae . Analysis of the DNA methylation states of pef GATC sites indicated that, under acidic growth conditions, which induced Pef production, most GATC I sites were non-methylated, whereas GATC II and GATC X were predominantly methylated . The methylation protection at GATC I and GATC II was dependent upon Lrp and was modulated by PefI . Together, these results indicate that Pef production is regulated by DNA methylation, which is the first example of methylation-dependent gene regulation outside of E . coli.

Mol Microbiol, 2000 Feb, 35(4), 718 - 27
Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa; Walsh AG et al.; Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance . We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P . aeruginosa lipopolysaccharide . The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P . aeruginosa . Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful . We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene . These results are consistent with the fact that P . aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P . aeruginosa viability . A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability . Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant . The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively . The S . typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs . The inner core phosphates of P . aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.

Biochemistry, 2000 Feb 22, 39(7), 1643 - 54
Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6; Tseng TY et al.; The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity . The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides . GTP and CTP are both required for the initiation of oligoribonucleotide synthesis . In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position . On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3' . This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites . SP6 primase shares some properties with the well-characterized E . colibacteriophage T7 primase . The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis . However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E . coli- or the T7-encoded ssDNA binding protein . An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.

Poult Sci, 2000 Jan, 79(1), 33 - 40
Effects of Salmonella typhimurium lipopolysaccharide on broiler chickens; Xie H et al.; The effects of Salmonella typhimurium lipopolysaccharide (LPS) on the physiology of 3-wk-old broiler chickens were studied at 12, 24, and 48 h after a single intravenous injection of saline or LPS . Lipopolysaccharide elevated cloacal temperature by 3 h after injection, induced a diuretic response, and decreased BW gain . An increase in the relative liver weight was evident in LPS-treated birds at all time intervals, whereas a decrease in the relative weight of bursa of Fabricius was observed only at the 48-h time point . The plasma interleukin (IL)-6 and the blood heterophil concentrations were elevated at 12 and 24 h following LPS administration . These changes were not observed in control chickens or in LPS-treated chickens at 48 h . A decrease in the blood glucose concentration in LPS-treated birds at 12 h was accompanied by an elevation in the blood phosphate level . An increase in total plasma protein concentration was observed only at 24 and 48 h after LPS treatment . Comparative SDS-PAGE analysis of plasma proteins from these birds under nonreducing conditions showed some quantitative differences in four bands of proteins between saline and LPS-treated chickens . A protein corresponding to an approximate molecular weight (MW) of 65 kDa increased in LPS-treated chickens, and three other proteins with MW of approximately 39, 49, and 56 kDa showed reductions in concentration compared with saline-treated controls . These results show that LPS induces a number of physiological changes that may be responsible for the regulation of the acute phase response in chickens.

J Periodontal Res, 1999 Oct, 34(7), 393 - 9
Porphyromonas gingivalis virulence factors and invasion of cells of the cardiovascular system; Progulske-Fox A et al.; Our laboratory is interested in the genes and gene products involved in the interactions between Porphyromonas gingivalis (Pg) and the host . These interactions may occur in either the periodontal tissues or other non-oral host tissues such as those of the cardiovascular system . We have previously reported the cloning of several genes encoding hemagglutinins, surface proteins that interact with the host tissues, and are investigating their roles in the disease process . Primary among these is HagA, a very large protein with multiple functional groups that have significant sequence homology to protease genes of this species . Preliminary evidence indicates that an avirulent Salmonella typhimurium strain containing hagA is virulent in mice . These data indicate that HagA may be a key virulence factor of Pg . Additionally, we are investigating the invasion of primary human coronary artery endothelial cells (HCAEC) by Pg because of the recent epidemiological studies indicating a correlation between periodontal disease (PD) and coronary heart disease (CHD) . We found that some, but not all, strains of Pg are able to invade these cells . Scanning electron microsopy of the infected HCAEC demonstrated that the invading organisms initially attached to the host cell surface as aggregates and by a "pedestal"-like structure . By transmission electronmicroscopy it could be seen that internalized bacteria were present within multimembranous compartments localized with rough endoplasmic reticulum . In addition, invasion of the HCAEC by Pg resulted in an increase in the degradation of long-lived cellular proteins . These data indicate that Pg are present within autophagosomes and may use components of the autophagic pathway as a means to survive intracellularly . However, Pg presence within autophagosomes in KB cells could not be observed or detected . It is therefore likely that Pg uses different invasive mechanisms for different host cells . This and the role of HagA in invasion is currently being investigated further.

Food Chem Toxicol, 2000 Jan, 38(1), 79 - 98
Review of the toxicologic properties of medium-chain triglycerides; Traul KA et al.; Medium chain triglycerides (MCTs) are a family of triglycerides, containing predominantly, caprylic (C(8)) and capric (C(10)) fatty acids with lesser amounts of caproic (C(6)) and lauric (C(12)) fatty acids . MCTs are widely used for parenteral nutrition in individuals requiring supplemental nutrition and are being more widely used in foods, drugs and cosmetics . MCTs are essentially non-toxic in acute toxicity tests conducted in several species of animals . In ocular and dermal irritation testing MCTs exhibit virtually no potential as ocular or dermal irritants, even with prolonged eye or skin exposure . MCTs exhibit no capacity for induction of hypersensitivity . Ninety-day toxicity tests did not result in notable toxicity, whether the product was administered in the diet up to 9375mg/kg body weight/day or by intramuscular (im) injection (up to 0 . 5ml/kg/day, rabbits) . There was no evidence that intravenous (iv) or dietary administration of MCTs adversely affected the reproductive performance of rats or resulted in maternal toxicity, foetal toxicity or teratogenic effects at doses up to 4.28g/kg body weight/day (iv) or 12,500mg/kg body weight/day (dietary) . There was no evidence that dietary administration of MCTs adversely affected the reproductive performance of pigs or resulted in maternal toxicity, foetal toxicity or teratogenic effects at doses up to 4000mg/kg body weight/day in the diet . In rabbits, following iv administration, the maternal and foetal no-observed-adverse-effect levels (NOAELs) were between 1.0 and 4.28g/kg body weight/ day . A 2-year study in rats, conducted with a closely related compound (tricaprylin, a triglyceride with C(8) fatty acids), provided no evidence of a carcinogenic effect when the material was administered by oral gavage at levels up to 10ml/kg (9.54g/kg) per day . Although tricaprylin was found to be positive in one of five strains of Salmonella typhimurium in the presence of metabolic activation in an Ames mutagenicity assay, the results of the carcinogenicity test with tricaprylin and mutagenicity tests with caprylic acid indicate that MCTs do not have the potential to be carcinogenic or mutagenic . The safety of human dietary consumption of MCTs, up to levels of 1g/kg, has been confirmed in several clinical trials.

J Exp Med, 2000 Feb 21, 191(4), 613 - 24
Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells; Yrlid U et al.; Salmonella typhimurium is a gram-negative bacterium that survives and replicates inside vacuolar compartments of macrophages . Infection of macrophages with S . typhimurium grown under conditions allowing expression of the type III secretion system results in apoptotic death of the infected cells . Here, we show that infection of bone marrow-derived macrophages (MPhi) with wild-type S . typhimurium 14028 results in presentation of epitopes derived from a bacteria-encoded antigen on major histocompatibility complex (MHC) class I and MHC class II molecules after internalization of apoptotic MPhi by bystander dendritic cells (DCs) . In contrast, infection of MPhi with the phoP constitutive mutant strain CS022, which does not induce apoptosis in infected MPhi, does not result in presentation of a bacteria-derived antigen by bystander DCs unless the infected MPhi are induced to undergo apoptosis by treatment with lipopolysaccharide and ATP . DCs appear to be unique in their ability to present antigens derived from MPhi induced to undergo apoptosis by Salmonella, as bystander MPhi are not capable of presenting the bacteria-derived antigen despite the fact that they efficiently internalize the apoptotic cells . These data suggest that apoptosis induction by bacterial infection of MPhi may not be a quiescent death that allows the bacteria to escape recognition by the immune system, but rather may contribute to an antimicrobial immune response upon engulfment by bystander DCs.

J Wildl Dis, 2000 Jan, 36(1), 161 - 4
Antibodies to selected disease agents in translocated wild turkeys in California; Charlton KG; Wild turkeys (Meleagris gallopavo) trapped within California (n = 715) or imported into California from other states (n = 381) from 1986 to 1996 were tested for exposure to certain disease agents . Prevalence of antibody to Mycoplasma gallisepticum, Mycoplasma meleagridis, Salmonella pullorum, Salmonella typhimurium, Newcastle disease virus, and avian influenza virus was low (0-4%) for wild turkeys trapped within California . With the exception of antibody prevalence to M . meleagridis of 33%, the same was true for wild turkeys imported into California from other states . Antibody prevalence to Mycoplasma synoviae was 8-10% for both groups.

Curr Opin Microbiol, 2000 Feb, 3(1), 35 - 42
Reactive nitrogen intermediates and the pathogenesis of Salmonella and mycobacteria; Shiloh MU et al.; Over the past decade, reactive nitrogen intermediates joined reactive oxygen intermediates as a biochemically parallel and functionally non-redundant pathway for mammalian host resistance to many microbial pathogens . The past year has brought a new appreciation that these two pathways are partially redundant, such that each can compensate in part for the absence of the other . In combination, their importance to defense of the murine host is greater than previously appreciated . In addition to direct microbicidal actions, reactive nitrogen intermediates have immunoregulatory effects relevant to the control of infection . Genes have been characterized in Mycobacterium tuberculosis and Salmonella typhimurium that may regulate the ability of pathogens to resist reactive nitrogen and oxygen intermediates produced by activated macrophages.

Curr Opin Microbiol, 2000 Feb, 3(1), 97 - 101
Novel approaches to monitor bacterial gene expression in infected tissue and host; Lee SH et al.; Elucidating the complex and dynamic host-microbe interactions during infection requires, among other things, detailed knowledge of microbial gene expression in vivo . Recently, advances in fluorescence and bioluminescence detection techniques, as well as recombinase-based in vivo expression technology, have rendered monitoring virulence gene expression in vivo a feasible task . These techniques have been adapted by several laboratories to study the spatial and temporal patterns of virulence gene expression by organisms such as Salmonella typhimurium, Listeria monocytogenes, Yersinia entercolitica and Vibrio cholerae during infection of tissue culture or animal models of infection.

Biochem Biophys Res Commun, 2000 Jan 27, 267(3), 918 - 23
Novel functions of human alpha(1)-protease inhibitor after S-nitrosylation: inhibition of cysteine protease and antibacterial activity; Miyamoto Y et al.; alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)) . We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase . S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity . Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione . NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap . Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase . As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated .

Mol Microbiol, 2000 Feb, 35(3), 677 - 85
A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence; Revell PA et al.; The primary invasion factor of Yersinia enterocolitica, invasin, is encoded by inv . inv expression is regulated in response to pH, growth phase and temperature . In vitro, inv is maximally expressed at 26 degrees C, pH 8.0, or 37 degrees C, pH 5.5, in early stationary phase . At 37 degrees C, pH 8.0, inv is weakly expressed . To identify which gene(s) are required for inv regulation, we screened for transposon insertions that decreased expression of an inv-'phoA chromosomal reporter at 26 degrees C . Of 30 000 mutants screened, two were identified that had negligible inv expression in all conditions tested . Both of these independent mutants had an insertion into the same gene, designated rovA (regulator of virulence) . RovA has 77% amino acid identity to the Salmonella typhimurium transcriptional regulator SlyA . Complementation with the wild-type rovA allele restores wild-type inv expression as monitored by Western blot analysis, tissue culture invasion assay and alkaline phosphatase assay . There is also a significant decrease in invasin levels in bacteria recovered from mice infected with the rovA mutant; therefore, RovA regulates inv expression in vivo as well as in vitro . In the mouse infection model, an inv mutant has a wild-type LD50, even though the kinetics of infection is changed . In contrast, the rovA mutant has altered kinetics, as well as a 70-fold increase in the LD50 compared with wild type . Furthermore, because the rovA mutant is attenuated in the mouse model, this suggests that RovA regulates other virulence factors in addition to inv . Analysis of other proposed virulence factors such as Ail, YadA and the Yop proteins shows no regulatory role for RovA . The more severe animal phenotype combined with the lack of impact on known virulence genes aside from inv suggests RovA regulates potentially novel virulence genes of Y . enterocolitica during infection.

Mol Microbiol, 2000 Feb, 35(3), 635 - 46
Characterization of two novel regulatory genes affecting Salmonella invasion gene expression; Altier C et al.; A Salmonella typhimurium chromosomal deletion removing approximately 19 kb of DNA at centisome 65 reduces invasion of cultured epithelial cells as well as the expression of lacZY operon fusions to several genes required for the invasive phenotype . As the deleted region contains no genes previously known to affect Salmonella invasion, we investigated the roles of individual genes in the deleted region using a combination of cloning, complementation and directed mutation . We find that the deletion includes two unrelated regulatory genes . One is the Salmonella homologue of Escherichia coli barA (airS ), which encodes a member of the multistep phosphorelay subgroup of two-component sensor kinases . The action of BarA is coupled to that of SirA, a member of the phosphorylated response regulator family of proteins, and includes both HilA-dependent and HilA-independent components . The other regulatory gene removed by the deletion is the Salmonella homologue of E . coli csrB, which specifies a regulatory RNA implicated in controlling specific message turnover in E . coli . These results identify a protein that is likely to play a key role in the environmental control of Salmonella invasion gene expression, and they also suggest that transcriptional control of invasion genes could be subject to refinement at the level of message turnover.

Shock, 2000 Feb, 13(2), 160 - 5
Suppression of lethal toxicity of endotoxin by biscoclaurine alkaloid cepharanthin; Maruyama H et al.; Suppressive effects of Cepharanthin (CE) on lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNFalpha) production followed by liver injury were investigated . Pretreatment with CE reduced limulus activity of LPS . Intraperitoneal treatment with CE 10 min before an i.v . challenge of LPS resulted in protection from LPS lethality in D-galactosamine (GalN)-sensitized BALB/c but not in C57BL/6 and C57BL/10ScSn mice . Treatment with CE before the LPS challenge significantly reduced serum TNF levels in a dose-dependent manner . The suppression was most effective when CE was administered 10 min before the LPS challenge . Increased levels of enzymes released from hepatocytes into the circulation, as a result of LPS-induced liver injury, were reduced by CE administration . Histological evaluation demonstrated that massive cell infiltration after severe injury developed in liver of mice injected with LPS plus D-GalN unless they were pretreated with CE . Apoptotic cells decreased by treatment with CE . Treatment with CE retarded lethal shock induced by an infection with 10(8) CFU Salmonella typhimurium deltaaroA mutant . These results suggest that action of CE is initiated through suppression of LPS-induced TNF production.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 287 - 99
Novel allosteric effectors of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling; Marabotti A et al.; Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the formation of L-tryptophan . The functional properties of one subunit are allosterically regulated by ligands of the other subunit . Molecules tailored for binding to the alpha-active site were designed using as a starting model the three-dimensional structure of the complex between the enzyme from Salmonella typhimurium and the substrate analog indole-3-propanol phosphate . On the basis of molecular dynamics simulations, indole-3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few other structurally related compounds were found to be good candidates for ligands of the alpha-subunit . The binding of the designed compounds to the alpha-active site was evaluated by measuring the inhibition of the alpha-reaction of the enzyme from Salmonella typhimurium . The inhibition constants were found to vary between 0.3 and 1.7 mM . These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the rate of the beta-reaction in the isolated beta(2) dimer . A small inhibitory effect on the activity of the alpha(2)beta(2) complex was caused by indole-3-acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory effect was caused by indole-3-acetamide . Furthermore, indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base . These results indicate that (i) indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind to the alpha-subunit and act as allosteric effectors whereas indole-3-acetyl-valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii) the terminal phosphate present in the already known allosteric effectors of tryptophan synthase is not strictly required for the transmission of regulatory signals.

J Mol Biol, 2000 Feb 18, 296(2), 385 - 401
Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination; Murphy KC; Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination . Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo . UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity . Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme . In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not . In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand . In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi . However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity . These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway . This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently .

Biotechnol Appl Biochem, 2000 Feb, 31 ( Pt 1), 1 - 4
Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase; Roh HJ et al.; D-Tagatose is a potential bulking agent in food as a non-calorific sweetener . To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose . The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E . coli, B . subtilis and S . typhimurium respectively . In the cultures of recombinant E . coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively . The enzyme extract of E . coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

J Infect Dis, 2000 Feb, 181(2), 602 - 12
Expression of and cytokine activation by Escherichia coli curli fibers in human sepsis; Bian Z et al.; Curli organelles are expressed by commensal Escherichia coli K12 and by Salmonella typhimurium at temperatures <37 degrees C, which bind serum proteins and activate the contact-phase system in vitro . This study demonstrates, by means of an anti-CsgA (curli major subunit) antibody, that a significant fraction of E . coli isolates (24 of 46) from human blood cultures produce curli at 37 degrees C in vitro . Serum samples from 12 convalescent patients with sepsis, but not serum from healthy controls, contained antibodies against CsgA (n=12) . This study further demonstrates that a curli-expressing E . coli strain and a noncurliated mutant secreting soluble CsgA induce significantly (P<.05) higher levels of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin {IL}-6, and IL-8) in human macrophages differentiated from THP-1 cells . These data, therefore, provide direct evidence that curli are expressed in vivo in human sepsis and suggest a possible role for curli and CsgA in the induction of proinflammatory cytokines during E . coli sepsis.

Toxicon, 2000 Mar, 38(3), 337 - 46
Occurrence of the toxin dehydroabietic acid in Salmonella typhimurium; Beier RC et al.; Many strains of Salmonella typhimurium studied in our lab demonstrated marked differences in the pathogenicity for guinea pig, chicken and Hela cells . As a result, a pathogenic strain of S . typhimurium, strain 9SR2, was evaluated for lipophilic components that may be associated with virulence using gas chromatography/mass spectrometry . The hydroxylated fatty acids 2-hydroxytetradecanoic acid (2-OH-14:0) and 3-hydroxytetradecanoic acid (3-OH-14:0) often present in lipid A, a potent endotoxin, were observed as their methyl esters . The cyclic fatty acids methylene-hexadecanoic acid (C17delta) and methyleneoctadecanoic acid (C19delta) also were detected . The nephrotoxic and neurotoxic diterpenoid resin acid, dehydroabietic acid, was observed for the first time from S . typhimurium in both the total lipid and diglyceride fractions and determined as its methyl ester at m/z 314.2246 . Due to its previously established toxicity, dehydroabietic acid may be a factor associated with virulence of S . typhimurium.

Blood, 2000 Feb 15, 95(4), 1258 - 63
An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium; Urashima M et al.; CD40 ligand (CD40L) has a great potential as a novel treatment for B-cell lymphoma (BCL) . It has previously been demonstrated that a nonvirulent strain of Salmonella typhimurium mutant (ST) can be used not only as a vehicle in oral genetic immunization via the intestinal mucosa, but also as an enhancer of interferon gamma- and tumor necrosis factor alpha-mediated immunity . After confirming that human CD40L can up-regulate expression of Fas, B7-1, and B7-2 molecules on murine BCL cells in vitro, we transfected the human CD40L gene into S typhimurium mutant (ST40L), which was administrated orally to determine whether it was able to prevent the growth of BCL in mice . Expression of human CD40L was confirmed immunohistochemically with protein being detected in the Peyer's patches of mice immunized with ST40L . Moreover, human soluble CD40L had been detectable until 7 to 8 weeks after oral administration of ST40L . Although ST alone exhibited some protective effects, ST40L demonstrated a significantly greater protection against the development of CD40 positive BCL compared with the control . In the surviving mice that had been treated with ST40L, a small and hard nodule was formed at the injection site, which was found to be composed of infiltrating lymphocytes expressing Fas ligand . These results have the potential to be a simple, effective, and above all, safe immune-gene therapy against BCL . (Blood . 2000;95:1258-1263)

Lett Appl Microbiol, 1999 Nov, 29(5), 303 - 7
Selection of Salmonella typhimurium as an indicator for pathogen regrowth potential in composted biosolids; Sidhu J et al.; In order to select a suitable indicator for monitoring the pathogen regrowth potential of composted biosolids, the growth kinetics of selected bacteria were investigated . Growth parameters of six serovars of Salmonella and three strains of Escherichia coli in sterilized compost were compared . Seeded Salmonella and E . coli grew rapidly, reaching population densities of more than 10(8) g-1 after 30 h of incubation . The specific growth rates of Salmonella serovars and E . coli strains were similar and varied from 0.49 to 0.55 h-1 . The specific growth rate of the Salm . Typhimurium isolates was significantly higher than the other bacterial strains . It was concluded that an antibiotic-resistant strain of Salm . Typhimurium can be used as an indicator for a pathogen regrowth potential test.

J Biol Chem, 2000 Feb 11, 275(6), 3943 - 9
Engineered salt-insensitive alpha-defensins with end-to-end circularized structures; Yu Q et al.; We designed a retro-isomer and seven circularized "beta-tile" peptide analogs of a typical rabbit alpha-defensin, NP-1 . The analogs retained defensin-like architecture after the characteristic end-to-end, Cys(3,31) (C I:C VI), alpha-defensin disulfide bond was replaced by a backbone peptide bond . The retro-isomer of NP-1 was as active as the parent compound, suggesting that overall topology and amphipathicity governed its antimicrobial activity . A beta-tile design with or without a single cross-bracing disulfide bond sufficed for antimicrobial activity, and some of the analogs retained activity against Escherichia coli and Salmonella typhimurium in NaCl concentrations that rendered NP-1 inactive . The new molecules had clustered positive charges resembling those in protegrins and tachyplesins, but were less cytotoxic . Such simplified alpha-defensin analogs minimize problems encountered during the oxidative folding of three-disulfide defensins . In addition, they are readily accessible to a novel thia zip cyclization procedure applicable to large unprotected peptide precursors of 31 amino acids in aqueous solutions . Collectively, these findings provide new and improved methodology to create salt-insensitive defensin-like peptides for application against bacterial diseases.

Cancer Lett, 1999 Dec 1, 147(1-2), 1 - 4
Assessment of aniline derivatives-induced DNA damage in the liver cells of B6C3F1 mice using the alkaline single cell gel electrophoresis ('comet') assay; Przybojewska B; The alkaline single cell gel electrophoresis (SCGE) or 'comet' assay under alkaline conditions was used to measure DNA damage in the liver cells of B6C3F1 male mice exposed to 2,4-dimethylaniline and 2,4,6-trimethylaniline . Cells embedded in agarose were lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope . The effect of 2,4-dimethylaniline and 2,4,6-trimethylaniline was studied after single intraperitoneal injections at doses equal to 100, 200 mg/kg and 150, 300 mg/kg body wt., respectively . It was found that 2,4-dimethylaniline and 2,4,6-trimethylaniline were able to damage DNA in the liver cells of the mice . As has been published elsewhere, the DNA damaging effect of the studied compounds was also observed in bone marrow cells of the mice . In conclusion, taking into account the results mentioned above and the results obtained by the other researchers who reported mutagenic activity of 2,4-dimethylaniline and 2,4,6-trimethylaniline in a Salmonella typhimurium assay and DNA repair test using Chinese hamster hepatocytes, it can be stated that both aromatic amines are genotoxic.

Adv Exp Med Biol, 1999, 473, 281 - 9
Phase variable switching of in vivo and environmental phenotypes of Salmonella typhimurium; Isaacson RE et al.; Previously it was shown that S . typhimurium strain 798, which is known to cause persistent asymptomatic infections in pigs, exists in two phenotypes . One phenotype, which is called adhesive, was shown to produce pili, is adhesive to porcine enterocytes, is readily phagocytized, and then survives intracellularly in phagocytes . The other phenotype, termed non-adhesive, does not produce pili, does not attach to enterocytes, is phagocytized less efficiently, and does not survive within the phagocyte . Cells in each phenotype can freely switch to the other phenotype at a fairly high frequency and thus the shift between each phenotype is phase variation . Further analysis of these phenotypes identified 4 additional characteristics that were co-regulated by phase variation . The first is the enterocyte-specific adhesin, which was shown to be type 1 fimbriae . Mutations in fimA, the major pilin molecule, led to a decreased ability to colonize the gut of pigs and mice . The second characteristic is O-antigen production . Adhesive cells produce a long O-antigen (up to 18 subunits) while non-adhesive cells do not (only 1-2 subunits) . The long O-antigen produced by the adhesive cells leads to resistance to serum and appears to be the result of phase variable expression of rfaL . A third locus, ebu, has been identified based on differential color production of colonies growing on Evans blue-Uranine plates . The relationship of this trait to in vivo survival or virulence is not known but ebu is genetically related to a family of transcriptional activators . The fourth locus, prv is located on the virulence plasmid and a mutation in prv results in delayed time to death in mice . It is hypothesized that the adhesive phenotype is the in vivo, virulent form, while the non-adhesive phenotype is the environmental, avirulent form . By modulating the fraction of cells in each phase, persistent asymptomatic infections can be promoted.

Adv Exp Med Biol, 1999, 473, 261 - 74
Of mice, calves, and men . Comparison of the mouse typhoid model with other Salmonella infections; Tsolis RM et al.; Numerous Salmonella typhimurium virulence factors have been identified and characterized using experimental infection of mice . While the murine typhoid model has been used successfully for Salmonella typhi vaccine development and to infer virulence mechanisms important during typhoid fever, information derived from infection of mice has been of limited value in elucidating the mechanisms by which S . typhimurium causes enteritis in humans . Progress in our understanding of virulence mechanisms contributing to diarrheal disease comes from recent studies of bovine enteritis, a S . typhimurium infection, which manifests as acute gastroenteritis . This review compares virulence genes and mechanisms required during murine typhoid, typhoid fever, and bovine enteritis . Comparison of illnesses caused in different animal hosts identifies virulence mechanisms involved in species specific disease manifestations . The determination of the relative importance of virulence factors for disease manifestations in different host species provides an important link between the in vitro characterization of genes and their role during host pathogen interaction.

Chem Biol Interact, 2000 Jan 3, 124(1), 29 - 51
Chemical features of flavonols affecting their genotoxicity . Potential implications in their use as therapeutical agents; Silva ID et al.; Flavonls are natural compounds present in edible plants and possess several biological activities that can be useful in drug design . Conversely some of these compounds have been shown to be genotoxic to prokaryotic and eukaryotic cells . In this study we tried to establish the chemical features responsible for the genotoxicity of flavonols and to study the conditions that can modulate their genotoxicity namely pH, the presence of antioxidants and metabolism . We assessed the induction of revertants in Salmonella typhimurium TA98 and the induction of Chromosomal aberrations in V79 cells by eight different flavonols and one catechin in the presence and in the absence of metabolizing systems . We have also studied the generation of hydroxyl radical by these flavonoids using the deoxyribose degradation assay . The results obtained in this study suggest that flavonols having a free hydroxyl group at position 3 of the C ring, a free hydroxyl group at position 7 of the A ring and a B ring with a catechol or pyrogallol structure, or a structure that after metabolic activation is transformed into a catechol or a pyrogallol, are flavonols whose genotoxicity in eukaryotic cells depends on their autooxidation . These flavonols can autooxidize when the pH value is slightly alkaline, such as in the intestine, and therefore can induce genotoxicity in humans . Given the above mentioned considerations it is necessary to clarify the mechanisms and the conditions that mediate the biological effects of flavonols before considering them as therapeutical agents.

J Immunol, 2000 Feb 15, 164(4), 1648 - 52
Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection; Mittrucker HW et al.; Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S . typhi . Since S . typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S . typhimurium is a longstanding matter of debate . By infecting mice on a susceptible background and deficient in B cells (Igmu-/- mice) with different strains of S . typhimurium, we could for the first time formally clarify the role of B cells in the response against S . typhimurium . Compared with Igmu+/+ mice, LD50 values in Igmu-/- mice were reduced during primary, and particularly secondary, oral infection with virulent S . typhimurium . After systemic infection, Igmu-/- mice cleared attenuated aroA- S . typhimurium, but vaccine-induced protection against systemic infection with virulent S . typhimurium involved both B cell-dependent and -independent effector mechanisms . Thus, B cell-mediated immunity plays a distinct role in control of S . typhimurium in susceptible mice.

Free Radic Biol Med, 2000 Jan 1, 28(1), 108 - 20
Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein; Poole LB et al.; Nox-1 from Streptococcus mutans, the bacteria which cause dental caries, was previously identified as an H2O2-forming reduced nicotinamide adenine dinucleotide (NADH) oxidase . Nox-1 is homologous with the flavoprotein component, AhpF, of Salmonella typhimurium alkyl hydroperoxide reductase . A partial open reading frame upstream of nox1, homologous with the other (peroxidase) component, ahpC, from the S . typhimurium system, was also identified . We report here the complete sequence of S . mutans ahpC . Analyses of purified AhpC together with Nox-1 have verified that these proteins act as a cysteine-based peroxidase system in S . mutans, catalyzing the NADH-dependent reduction of organic hydroperoxides or H2O2 to their respective alcohols and/or H2O . These proteins also catalyze the four-electron reduction of O2 to H2O2, clarifying the role of Nox-1 as a protective protein against oxygen toxicity . Major differences between Nox-1 and AhpF include: (i) the absolute specificity of Nox-1 for NADH; (ii) lower amounts of flavin semiquinone and a more prominent FADH2 to NAD+ charge transfer absorbance band stabilized by Nox-1; and (iii) even higher redox potentials of disulfide centers relative to flavin for Nox-1 . Although Nox-1 and AhpC from S . mutans were shown to play a protective role against oxidative stress in vitro and in vivo in Escherichia coli, the lack of a significant effect on deletion of these genes from S . mutans suggests the presence of additional antioxidant proteins in these bacteria.

Emerg Infect Dis, 2000 Jan-Feb, 6(1), 70 - 3
Salmonellosis in the Republic of Georgia: using molecular typing to identify the outbreak-causing strain; Sulakvelidze A et al.; In May 1998, three large outbreaks of salmonellosis, affecting 91 persons, were identified in the Republic of Georgia . Eighteen Salmonella Typhimurium strains were characterized by arbitrary primed polymerase chain reaction and pulsed-field gel electrophoresis; the results suggested that all cases were part of a single outbreak caused by a distinct clonal strain.

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