Microbiology, 1997 Jun, 143 ( Pt 6), 2057 - 63 The site-specific recombinase encoded by pinD in Shigella dysenteriae is due to the presence of a defective Mu prophage; Tominaga A; The DNA inversion systems are made up of an invertible DNA segment and a site-specific recombinase gene . Five systems are known in prokaryotes: the Salmonella typhimurium H segment and hin gene (H-hin), phage Mu G-gin, phage P1 C-cin, Escherichia coli e14 P-pin, and Shigella sonnei B-pinB systems . In this report a site-specific recombinase (pinD) gene of Shigella dysenteriae was cloned and sequenced . pinD mediated inversion of five known segments at the same extent in E . coli . Although one inv sequence was identified, no invertible region was detected in a cloned fragment . The predicted amino acid sequences of PinD and three ORFs showed high homology to those of Gin and its flanking gene products . An ORF homologous to Mom of Mu conserved a functional activity to modify intracellular plasmid DNA . Southern analysis showed that the cloned fragment contains two homologous regions corresponding to the left and right ends of the Mu genome . Together these results indicated that the pinD gene in S . dysenteriae is derived from a Mu-like prophage. J Bacteriol, 1997 Jun, 179(12), 4046 - 8 Identification of the ahp operon of Salmonella typhimurium as a macrophage-induced locus; Francis KP et al.; Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K . P . Francis and M . P . Gallagher, Infect . Immun . 61:640-649, 1993) . The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity . Plasmid-encoded ahp reverses sensitivity but reduces luminescence . This suggests that OxyR is titrated by the multicopy ahp promoter. J Bacteriol, 1997 Jun, 179(12), 3989 - 96 Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium; Enos-Berlage JL et al.; Mutants lacking the first enzyme in de novo purine synthesis (PurF) can synthesize thiamine if increased levels of pantothenate are present in the culture medium (J . L . Enos-Berlage and D . M . Downs, J . Bacteriol . 178:1476-1479, 1996) . Derivatives of purF mutants that no longer required pantothenate for thiamine-independent growth were isolated . Analysis of these mutants demonstrated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle . Results of phenotypic analyses suggested that a defect in Sdh decreased the thiamine requirement of Salmonella typhimurium . This reduced requirement correlated with levels of succinyl-coenzyme A (succinyl-CoA), which is synthesized in a thiamine pyrophosphate-dependent reaction . The effect of succinyl-CoA on thiamine metabolism was distinct from the role of pantothenate in thiamine synthesis. Microb Pathog, 1997 Jun, 22(6), 353 - 62 Bacterial phenotypes mediated by mviA and their relationship to the mouse virulence of Salmonella typhimurium; Swords WE et al.; The focus of this study was the phenotypic characterization of Salmonella typhimurium mutants lacking the function of the response regulator mviA . The inactivation of mviA+ (mviA::kan) is shown to induce a significant change in the growth of most virulent strains, as reflected in the size of the colonies formed on agar plates . The colony phenotype observed in these strains has been designated as the small colony morphology (Scm+) phenotype . Mutants exhibiting the Scm+ phenotype are shown to be significantly attenuated for virulence in susceptible (ItyB) mice . The Scm+ phenotype therefore provides an in vitro phenotypic marker for mviA+ activity . Further examination of Scm+ mutants has revealed that they lack expression of a 55 kDa periplasmic protein which is detected in isogenic mviA+ strains . This protein has been designated mviA+ related protein A (MrpA) and was expressed in direct correlation with virulence in all S . typhimurium strains examined. Glycobiology, 1997 Jun, 7(4), 559 - 63 Stereoselectivity of the Chinese hamster ovary cell sialidase: sialoside hydrolysis with overall retention of configuration; Kao YH et al.; The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, expressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spectroscopy . 4-Methyl umbelliferyl-N-acetyl neuraminic acid was employed as substrate, and the stereoselectivity of the enzyme catalysis was ascertained by monitoring the H3 axial and equatorial protons of the sialic acid product over the reaction course . At both high (3 U) and low concentrations (1 U) of the enzyme, the alpha anomer of the sialic acid was clearly observed as the initial reaction product . The corresponding beta anomer of sialic acid appeared much later in the reaction, arising from mutarotation of the alpha anomer . Similar studies were also carried out using the Salmonella typhimurium LT 2 sialidase, a protein of similar size and substrate specificity . Both enzymes apparently cleave the alpha linked sialoside substrate with retention of configuration . Based on the observations of a wide variety of other glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis with active site topology (Gebler et al., J . Biol . Chem . 267, 12559-12561, 1992), the results obtained here suggest that the microbial and mammalian sialidases have a homologous active site architecture even though the molecules do not share significant primary sequence similarities. Genetics, 1997 Jun, 146(2), 447 - 56 Transduction of low-copy number plasmids by bacteriophage P22; Mann BA et al.; The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers . Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22 . This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives . We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms . In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination . This chromosomal fragment is then packaged in the transducing particle . The second mechanism is a size-dependent mechanism involving a putative plasmid multimer . We propose that this multimer is formed by interplasmidic recombination . In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome . It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging . This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor . We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes. J Bacteriol, 1997 Jun, 179(11), 3797 - 800 Integration host factor is required for 1,2-propanediol-dependent transcription of the cob/pdu regulon in Salmonella typhimurium LT2; Rondon MR et al.; We show that integration host factor (IHF) is required for the activation of transcription of the cobalamin biosynthetic (cob) and 1,2-propanediol (1,2-PDL) utilization (pdu) operons in Salmonella typhimurium LT2 . A lack of IHF affected transcription of the cob/pdu regulon in at least two ways . First, the level of the regulatory protein PocR was decreased in ihfB (formerly himD) mutants, as judged by Western blot analysis with polyclonal antiserum raised against PocR . Second, even when PocR was available, in the absence of IHF, PocR was unable to activate transcription of cob/pdu in response to 1,2-PDL . This result suggested an additional role for IHF in PocR-dependent transcription activation . Consistent with these findings, ihfB mutants of this bacterium were unable to use 1,2-PDL as a carbon or energy source. J Bacteriol, 1997 Jun, 179(11), 3604 - 12 Specific detection of Salmonella typhimurium proteins synthesized intracellularly; Burns-Keliher LL et al.; Studies of the proteins Salmonella typhimurium synthesizes under conditions designed to more closely approximate the in vivo environment, i.e., in cell and tissue culture, are not easily interpreted because they have involved chemical inhibition of host cell protein synthesis during infection . The method which we have developed allows specific labeling of bacterial proteins without interfering with host cell metabolic activities by using a labeled lysine precursor which mammalian cells cannot utilize . We have resolved the labeled proteins using two-dimensional electrophoresis and autofluorography . We were able to detect 57 proteins synthesized by S . typhimurium during growth within a human intestinal epithelial cell line . Of the 57 proteins detected, 34 appear to be unique to the intracellular environment, i.e., they are not seen during growth of the bacteria in tissue culture medium alone . Current (and future) efforts are directed at organizing the 34 proteins into known stress response groups, determining the cellular locations of the proteins (outer or inner membrane, etc.), and comparing the pattern of proteins synthesized within an intestinal epithelial cell to the pattern synthesized during growth within other tissues. Infect Immun, 1997 Jun, 65(6), 2502 - 7 Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopneumoniae antigen (NrdF); Fagan PK et al.; Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia, a commercially expensive respiratory disease of swine . Salmonella typhimurium SL3261 was used as a live carrier of plasmid pKF1, which encodes a 15-kDa recombinant M . hyopneumoniae protein . This expressed recombinant protein consists of the carboxy-terminal 11 kDa of a 42-kDa M . hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein . Rabbit anti-15-kDa serum was able to inhibit the growth of viable M . hyopneumoniae J in vitro . When used as a live oral vaccine, S . typhimurium SL3261(pKF1) induced a significant secretory immunoglobulin A immune response in the lungs of mice orally immunized against the M . hyopneumoniae antigen . Utilization of live oral vaccines expressing potentially protective M . hyopneumoniae proteins, such as the NrdF antigen, which can stimulate a lung mucosal response against surface-accessible proteins may provide a cost-effective alternative to the present control strategies used for porcine enzootic pneumonia. Infect Immun, 1997 Jun, 65(6), 2451 - 3 Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene; Swords WE et al.; In order to identify the genetic basis for the attenuation of Salmonella typhimurium LT2 strains, experiments were performed to identify a gene(s) which restores virulence to an avirulent LT2 strain . These and further experiments confirmed that an rpoS mutation is the sole determinant of the attenuation of S . typhimurium LT2. Infect Immun, 1997 Jun, 65(6), 2402 - 12 Expression and immunogenicity of an Echinococcus granulosus fatty acid-binding protein in live attenuated Salmonella vaccine strains; Chabalgoity JA et al.; Fatty acid-binding proteins (FABPs) are candidate molecules for vaccines against several parasitic platyhelminths . A FABP from the cestode Echinococcus granulosus (EgDf1) was expressed in Salmonella vaccine strains as a C-terminal fusion to fragment C of tetanus toxin (TetC) by using expression vector pTECH . The fusion protein was equally expressed in several attenuated vaccine strains derived from bacteria with different genetic backgrounds and different attenuating mutations . Single-dose immunization experiments with the aroA Salmonella typhimurium strain SL3261 carrying the pTECH-EgDf1 construct were conducted with mice, using both the intravenous and the oral routes . Surprisingly, the antibody response to EgDf1 and the antigen-specific cytokine production in spleen cells were stronger in mice immunized orally . Furthermore, immune mouse sera strongly reacted with fixed sections of the worm's larval stage . Analysis of the isotype distribution of the specific anti-EgDf1 antibodies showed similar production of immunoglobulin G1 (IgG1) and IgG2a together with specific IgA antibodies . In addition, stimulation of spleen cells from mice immunized with the different constructs with either Salmonella lysate, TetC, or EgDf1 showed that, together with Th1-related cytokines (gamma interferon and interleukin 2 {IL-2}), significant levels of a Th2 cytokine (IL-5) were produced specifically, indicating a Th2 component to the response to the Salmonella carrier and to the recombinant antigens . Salmonellae expressing the TetC-rEgDfl fusion are currently under evaluation as potential vaccines against E . granulosus. Infect Immun, 1997 Jun, 65(6), 2396 - 401 Broad-spectrum antimicrobial activity of human intestinal defensin 5; Porter EM et al.; Defensins are antibiotic peptides expressed in human and animal myeloid and epithelial cells . Due to the limited availability of natural peptides, the properties of human epithelial defensins have not been studied . We assayed the microbicidal activity of recombinant human intestinal defensin 5 (rHD-5) in the presence of salt (O to 150 mM NaCl) with varied pH (pH 5.5 to pH 8.5) and trypsin (25 and 250 microg/ml) . rHD-5 exhibits microbicidal activity against Listeria monocytogenes, Escherichia coli, and Candida albicans . In contrast to cryptdins, the mouse intestinal defensins, rHD-5 is active against both mouse-virulent wild-type Salmonella typhimurium and its isogenic, mouse-avirulent phoP mutant . In the presence of salt, rHD-5 activity was reduced, and at 100 mM NaCl, activity against S . typhimurium was abolished . However, at all salt concentrations tested, rHD-5 remained bactericidal to L . monocytogenes . Activity against L . monocytogenes was not pH dependent but was diminished at pH 5.5 against wild-type S . typhimurium . This acid-induced resistance may have been mediated by the virulence gene regulator phoP, since the phoP mutant was equally sensitive at pH 5.5 and pH 7.4 . In the presence of trypsin, rHD-5 was partially cleaved, but even then, rHD-5 at 100 microg/ml decreased the number of CFU of wild-type S . typhimurium by more than 99% . The persistence of microbicidal activity of rHD-5 under these conditions supports the notion that naturally occurring human intestinal defensin is an effective arm of mucosal host defense. Infect Immun, 1997 Jun, 65(6), 2254 - 9 Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid; Baumler AJ et al.; Penetration of the intestinal mucosa at areas of Peyer's patches is an important first step for Salmonella typhimurium to produce lethal systemic disease in mice . However, mutations in genes that are important for intestinal invasion result in only moderately decreased virulence of S . typhimurium for mice . Here we report that combining mutations in invA and lpfC, two genes necessary for entry into Peyer's patches, results in a much stronger attenuation of S . typhimurium than inactivation of either of these genes alone . An S . typhimurium invA lpfC mutant was 150-fold attenuated by the oral route of infection but was fully virulent when the intestine was bypassed by intraperitoneal challenge of mice . During mixed-infection experiments, the S . typhimurium invA lpfC mutant showed a strong defect in colonizing Peyer's patches and mesenteric lymph nodes . These data suggest that mutations in invA and lpfC deactivate distinct pathways for intestinal penetration and colonization of Peyer's patches . While the inv-mediated pathway is widely distributed, the lpf operon is absent from many phylogenetic groups within the genus Salmonella . To investigate how acquisition of the lpf-mediated pathway for mucosal penetration contributed to evolution of virulence, we studied the relationship between the presence of the lpf operon and the pathogenicity for mice of 18 isolates representing 14 Salmonella serotypes . Only strains possessing the lpf operon were able to cause lethal infection in mice . These data show that both the invA- and lpfC-mediated pathways of intestinal perforation are conserved in mouse virulent Salmonella serotypes. Infect Immun, 1997 Jun, 65(6), 2041 - 51 Genetic and transcriptional analysis of flgB flagellar operon constituents in the oral spirochete Treponema denticola and their heterologous expression in enteric bacteria; Heinzerling HF et al.; Oral spirochetes possess many potential virulence factors, including the capacity for tissue invasion and persistence despite a vigorous host immune response . In an attempt to identify treponemal immunoreactive components, sera derived from individuals with advanced periodontal disease were used as a reagent to isolate recombinant bacteriophage lambda clones expressing antigens of the oral spirochete Treponema denticola ATCC 35405 . Nucleotide sequence analysis of a clone expressing three immunoreactive products has revealed seven T . denticola genes which appear to encode homologs of flagellar basal body constituents, FlgB, FlgC, FliE, and FliF, a flagellar switch component, FliG, and the putative flagellar export proteins, FliH and FliI, initially characterized in Salmonella typhimurium . Also identified was a gene resembling fliJ . Primer extension analysis identified a transcriptional start site 5' to the treponemal flgB gene . Appropriately spaced with respect to this start site was a sigma28 binding motif . The absence of additional identifiable sigma factor binding motifs within the treponemal sequence and the proximity of adjacent genes suggested operonic arrangement, and reverse transcriptase PCR provided evidence of cotranscription . Supporting the identification of these genes as flagellar components, heterologous expression in enteric bacteria of the putative switch basal body genes from T . denticola interfered with motility . Specifically, the presence of a plasmid expressing treponemal fliG reduced swarming motility in S . typhimurium, while in Escherichia coli, this plasmid conferred a nonmotile phenotype and a reduction in flagellar number . Thus, while spirochetal flagella are subject to unique synthetic and functional constraints, the organization of flagellar genes and the presence of sigma28-like elements are reminiscent of the flagellar systems of other bacteria, and there appears to be sufficient conservation of constituent proteins to allow interaction between T . denticola switch-basal body proteins and the flagellar machinery of gram-negative bacteria. Int J Food Microbiol, 1997 May 20, 36(2-3), 187 - 98 Water availability and the survival of Salmonella typhimurium in porous systems; Hills BP et al.; The survival of Salmonella Typhimurium LT2 in randomly packed beds of glass beads, microporous silica particles and Sephadex microspheres is examined . It is shown that the decrease in the percentage cell recovery in these porous materials at reduced water content is not correlated with the global water activity as determined by conventional vapour pressure measurements but rather with the osmotic shock induced by the sudden redistribution of water and air among the microscopic pores in the matrix surrounding the cells . For this reason the bacterial survival and growth data correlates best with physical measurements, such as NMR and electrical conductivity, which are sensitive to the microscopic air-water distribution . The implications of this observation in food safety and preservation are discussed. Biochemistry, 1997 May 13, 36(19), 5921 - 8 Flavin mononucleotide-binding domain of the flavoprotein component of the sulfite reductase from Escherichia coli; Coves J et al.; The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain . We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP . The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer . It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain . Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs . During photoreduction, the flavin radical accumulated first, followed by the fully reduced state . The redox potentials, determined at room temperature {E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV}, were very close to those previously reported for Salmonella typhimurium SiR-FP {Ostrowski, J., Barber, M . J., Rueger, D . C., Miller, B . E., Siegel, L . M., & Kredich, N . M . (1989) J . Biol . Chem . 264, 15796-15808} . Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively . Altogether, the results presented herein demonstrate that the N-terminal end of E . coli SiR-FP forms the FMN-binding domain . It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP . Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain. Mutat Res, 1997 May 12, 376(1-2), 43 - 51 Metabolism of isomeric nitrobenzo{a}pyrenes leading to DNA adducts and mutagenesis; Fu PP et al.; We have been interested in determining the structural and electronic features that may be useful in predicting the mutagenic activity of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) . We have previously found that a correlation between structural and electronic features and direct-acting mutagenicity in Salmonella typhimurium cannot be made using nitro-PAHs with different molecular size . In this study, a series of structurally related nitro-PAHs, the environmental contaminants 1-, 3-, and 6-nitrobenzo{alpha}pyrene (NBaP) and their derivatives, was used to determine structure-activity relationships . It was found that isomeric NBaPs are activated to DNA damaging and mutagenic derivatives by nitroreduction, ring-oxidation, or by a combination of these two pathways . A general finding was that NBaPs and derivatives with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in S . typhimurium strains TA98 and TA100 . In this paper, we also discuss the effect of the location of the nitro group on the metabolism and the mutagenicity of NBaPs and the effect of oxygen-containing functional groups on the mutagenicity of NBaP derivatives . These findings provide a useful molecular basis for interpreting and predicting the direct-acting mutagenicity of nitro-PAHs. J Mol Biol, 1997 May 9, 268(3), 655 - 65 Bacteriophage P22 scaffolding protein forms oligomers in solution; Parker MH et al.; The scaffolding protein of Salmonella typhimurium bacteriophage P22 is a 33.6 kDa protein required both in vivo and in vitro for the polymerization of the viral coat protein into closed T = 7 icosahedral procapsids . In vitro assembly reaction kinetics have previously been found to vary between second and third order with respect to scaffolding protein concentration, suggesting that dimers and/or higher-order oligomers may be the active species in assembly . Analytical ultracentrifugation experiments suggest that scaffolding protein undergoes a rapidly-reversible monomer/dimer/tetramer equilibrium, with higher association constants at 4 degrees C than at 20 degrees C . Under conditions in which in vitro assembly reactions are carried out (30 to 1000 microg/ml scaffolding protein, 20 degrees C), monomers are the predominant species, but the concentration of dimers is significant . A mutant scaffolding protein, R74C/L177I, which forms disulfide-linked dimers, catalyzed procapsid assembly at a higher rate than did the wild-type scaffolding protein; preincubation in dithiothreitol had little effect on the wild-type protein, but greatly reduced the activity of the mutant . These findings suggest that dimers and/or higher-order oligomers of scaffolding protein are active species in the assembly of P22. J Biol Chem, 1997 May 2, 272(18), 12083 - 90 DNA binding is not sufficient for H-NS-mediated repression of proU expression; Jordi BJ et al.; H-NS is a major component of bacterial chromatin and influences the expression of many genes . H-NS has been shown to exhibit a binding preference for certain AT-rich curved DNA elements in vitro . In this study we have addressed the factors that determine the specificity of H-NS action in vitro and in vivo . In bandshift studies, H-NS showed a slight binding preference for all curved sequences tested whether GC-based or AT-based; the specific architecture of the curve also influenced H-NS binding . In filter retention assays little difference in affinity could be detected for any sequence tested, including the downstream regulatory element (DRE) a downstream curved DNA element required for H-NS to repress transcription of the Salmonella typhimurium proU operon in vivo . A Kd of 1-2 microM was estimated for binding of H-NS to each of these sequences . In vivo, the distance between the proU promoter and the DRE, their relative orientations on the face of the DNA helix, and translation of the DRE had no major effect on proU regulation . None of the synthetic curved sequences tested could functionally replace the DRE in vivo . These data show that differential binding to curved DNA cannot account for the specificity of H-NS action in vivo . Furthermore, binding of H-NS to DNA per se is insufficient to repress the proU promoter . Thus, the DRE does not simply act as an H-NS binding site but must have a more specific role in mediating H-NS regulation of proU transcription. Berl Munch Tierarztl Wochenschr, 1997 May, 110(5), 171 - 5 {Vaccination of pigeons against Salmonella infections}; Grund S et al.; The efficiency of the live vaccine Zoosal T with a double marker mutant of Salmonella Typhimurium was tested on conditioned pigeons . For challenge infection we used a pigeon specific variation copenhagen strain in a defined state of virulence . The reduction of mortality and the persistence of Salmonella in organs were evaluated . An oral booster enhances the protection due to vaccination. Vet Microbiol, 1997 May, 56(1-2), 79 - 86 Comparison of an LPS-specific competitive ELISA with a motility enrichment culture method (MSRV) for detection of Salmonella typhimurium and S . enteritidis in chickens; Tan S et al.; We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp . in chicken organs and faeces . The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae . Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb . Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal . Stable coating and minimal false positive were achieved by conjugating LPS to poly-L-lysine . The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10(2) CFU/g, and did not allow migration of non-salmonella species . The c-ELISA detected 10(6) CFU of enriched culture or 10(2)-10(3) CFU of Salmonella/g of faeces . Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%. Food Addit Contam, 1997 May-Jun, 14(4), 389 - 98 An overview of the safety evaluation of the Thermomyces lanuginosus xylanase enzyme (SP 628) and the Aspergillus aculeatus xylanase enzyme (SP 578); Bergman A et al.; Xylanases SP 628 and SP 578 were produced by submerged fermentation of Aspergillus oryzae, containing a gene code originating from Thermomyces lanuginosus and Aspergillus aculeatus, respectively . Both enzymes were subject to the same series of toxicological tests to document their safety in use . The enzymes are to be applied as processing aids in the baking industry and in wheat starch separation . Neither enzyme was found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did they cause chromosomal aberrations in cultured human peripheral lymphocytes . No evidence of inhalation toxicity or skin and eye irritation was found . The enzymes are not regarded as skin-sensitizers, although the Buehler test with guinea-pigs revealed a minor potential . Oral administration up to 10.0 ml/kg bw/day (equivalent to a Total Organic Solids amount of 13.3% for SP 628 and of 11.3% for SP 578) in 13-week rat studies did not show any adverse effect. J Ethnopharmacol, 1997 May, 56(3), 223 - 6 Chemical composition and antimicrobial activity of Croton urucurana Baillon (Euphorbiaceae); Peres MT et al.; In the methanolic extract of Croton urucurana Baillon (Euphorbiaceae) a number of known compounds, such as acetyl aleuritolic acid, stigmasterol, beta-sitosterol, campesterol, beta-sitosterol-O-glucoside, sonderianin, catechin and gallocatechin were isolated and identified by MS and NMR spectroscopy, HRGC and data from literature . The antibacterial activity of the aqueous-EtOH extract, some fractions of the methanolic extract and some of the isolated compounds, were tested against Staphylococcus aureus and Salmonella typhimurium . Acetyl aleuritolic acid exhibits the best minimum inhibitory concentration (MIC) against both Staphylococcus aureus and Salmonella typhimurium. APMIS, 1997 May, 105(5), 410 - 3 Transfer of primed CD4+OX40- T lymphocytes induces increased immunity to experimental Salmonella typhimurium infections in rats; Thygesen P et al.; The protective effect of primed CD4 T cells against a lethal dose of Salmonella typhimurium was studied in Lewis rats . Primed CD4 T cells were obtained by inoculating Lewis rats with a non-lethal dose of S . typhimurium . Four weeks after the infection, spleen non-adherent mononuclear cells were isolated . The cells were separated according to their expression of CD4 and the OX40 antigen by FACS . OX40+ and OX40- CD4+ T-cell subpopulations were together with unsorted CD4+ T cells transferred to untreated rats 24 h prior to infection with S . typhimurium . Transfer of either unsorted CD4+ T cells or CD4+ T cells sorted into OX40- or OX40- subpopulations significantly increased animal survival compared to controls . Animals receiving OX40+CD4+ T cells did not differ significantly in survival probability from those receiving unsorted CD+ T cells . However, animals receiving OX40-CD4+ T cells had a significantly better survival compared to animals given unsorted CD4+ T cells . It is concluded that OX40-CD4+ T cells can induce significant protection against S . typhimurium infections in rats . This is most likely due to the fact that the OX40-CD4+ T-cell population contains a significant number of antigen-specific memory T cells that have returned to a resting state. Mol Microbiol, 1997 May, 24(4), 747 - 56 The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell; Collazo CM et al.; The ability of Salmonella typhimurium to interact with host cells is largely dependent on the function of a type III protein-secretion system encoded at centisome 63 of its chromosome . We have shown here that two targets of this protein-secretion system, SipB and SipC, are translocated into cultured intestinal Henle-407 cells . Translocation required the function of the type III secretion apparatus, as an S . typhimurium strain carrying a mutation in invA, which encodes an essential component of this system, failed to translocate the Sip proteins . Null mutations in the genes encoding SipB, SipC or SipD, prevented protein translocation, indicating that these proteins are involved in the translocation process . In contrast, mutations in sipA and sptP, which also encode secreted proteins, did not interfere with the translocation of SipC, indicating that only a subset of targets of the type III secretion system act as translocases . Externally or internally localized bacteria could direct protein translocation into Henle-407 cells as this process occurred in the presence of cytochalasin D at a concentration that prevented bacterial entry, or in the presence of gentamicin added shortly after bacterial internalization at a concentration that killed extracellular Salmonella . These results indicate that protein translocation into host cells may be a universal function of all type III secretion systems. Mol Microbiol, 1997 May, 24(4), 697 - 709 Non-invasive Salmonella typhimurium mutants are avirulent because of an inability to enter and destroy M cells of ileal Peyer's patches; Penheiter KL et al.; Salmonella typhimurium initiates infection of a host by invading M cells of Peyer's patches within the small intestine . The ability of the bacteria to invade mammalian cells has been shown to be regulated by environmental conditions, including oxygen concentrations, osmolarity, and growth phase . We have previously created oxygen-regulated Tn5lacZY S . typhimurium mutants that are defective in invasion . We have now identified the invasion genes disrupted by eight of the transposon insertions . These genes encode transcriptional regulators (hilA and invF), type III secretory components (orgA, invG and spaR) and secreted proteins (invC and invD) . Examination of the protein-secretion profiles of the non-invasive mutants indicated that each of the mutants was defective in secretion of between one and six proteins . We have also demonstrated that the loss of tissue culture cell invasiveness corresponds to an inability to invade and destroy M cells of Peyer's patches in a murine ligated loop model . Virulence studies, performed in mice, demonstrated that these defects significantly reduced the ability of the mutants to cause murine typhoid fever by an oral route of infection . Virulence by an intraperitoneal route of infection was unaffected . The data indicate that in vitro invasiveness, invasion-protein secretion, and M-cell invasion are critical indicators of S . typhimurium virulence. Mol Microbiol, 1997 May, 24(3), 499 - 510 Transcriptional regulation of the Yersinia pseudotuberculosis pH6 antigen adhesin by two envelope-associated components; Yang Y et al.; The Yersinia pseudotuberculosis pH6 antigen mediates haemagglutination and adhesion to cultured mammalian cells . The synthesis of pH6 antigen requires the products of the psaEFABC genes in both Yersinia pseudotuberculosis and Escherichia coli . In-frame deletion mutations of psaE and psaF caused defective haemagglutination . In contrast, we showed that the psaABC genes were sufficient for haemagglutination if they were expressed by a heterologous promoter . Environmental regulation of pH6 antigen by temperature and pH occurs via regulation of the major pilus protein PsaA at the transcriptional level . Northern blot analyses indicate that the psaA transcript was absent in either psaE or psaF mutant strains . Primer extension analyses indicate that, in Y . pseudotuberculosis, the transcription of the psaE and psaF genes is constitutive . Alkaline phosphatase fusion studies confirm the topology prediction that PsaE and PsaF are both inner-membrane-associated proteins . PsaE consists of an N-terminal cytoplasmic domain, containing sequence similarity to transcriptional regulators found in two-component systems as well as to the Salmonella typhimurium HIIA protein, with a C-terminal domain that is periplasmically localized . PsaF is predicted to be oriented with most of the protein in the periplasm, the hydrophobic N-terminus being either integrated in the inner membrane or cleaved as a signal peptide. Mutagenesis, 1997 May, 12(3), 159 - 62 Characterization of the CYP isozyme profile induced by cyclohexanol; Espinosa-Aguirre JJ et al.; In a previous report we described the ability of cyclohexanol to induce CYP activity . In order to characterize this induction we tested the capacity of liver S9 from rats orally treated with cyclohexanol for 5 days, to activate several carcinogenic nitrosamines into mutagens in the Salmonella typhimurium TA100 test system . Additionally, Western blot analysis of hepatic microsomes from the same treated animals were analysed with specific antibodies against P450 protein families 1A1/A2, 2B1/B2 and 2E1 . Cyclohexanol-S9 mixture was more efficient in activating the following nitrosamines: N-nitrosodimethylamine (NDMA), N-nitrosodipropylamine (NDPA), N-nitrosomethylpropylamine (NMPA), N-nitrosodibutylamine (NDBA), and N-nitrosopyrrolidine (NPYR) into bacterial mutagens than S9 from non-treated animals . The mutagenicity of N-nitrosodiethylamine (NDEA) was not modified in the presence of S9 from cyclohexanol-treated animals . Since the main metabolic pathway leading to the production of mutagenic intermediates of NDMA and NPYR is catalysed by isozyme CYP2E1 and that of NDPA, NMPA and NDBA by CYP2B1/B2, mutagenicity experiments predicted that cyclohexanol induces these two P450 isozyme families . Western blot analysis confirmed the results of the mutagenicity assay, showing an increase in the intensity of CYP2E1 and CYP2B1/B2 protein bands in hepatic microsomes from cyclohexanol treated rats in comparison with non-treated controls . Bacterial mutagenicity tests with specific pro-mutagens were good predictors of the P450 induction properties of cyclohexanol. J Appl Microbiol, 1997 May, 82(5), 610 - 4 The effects of growth dynamics upon pH gradient formation within and around subsurface colonies of Salmonella typhimurium; Walker SL et al.; pH measurements made in and around submerged colonies of Salmonella typhimurium grown within a model gelatin gel system using pH-sensitive micro- and macroelectrodes indicated some pH heterogeneity occurring in and around the bacterial colony . Inoculation density, initial pH and glucose concentration were all found to influence colony diameter and metabolism of Salmonella colonies . Colony growth in the presence of glucose, at pH 7.0 with an inoculation density of 1 cell ml-1 led to a pH fall of 1-2 pH units after 2 d . At pH 5.0, with glucose, colony growth rates were much slower than at pH 7.0, and the pH change varied by less than one pH unit often becoming alkaline . In the absence of glucose, only small pH changes were observed within the medium, although growth rates were similar to those in glucose-containing media . At the higher inoculation density (ca 1000 cells ml-1), isolated pH changes were not observed . Morphological changes, such as the production of annular rings, were noted in stationary phase colonies as was alkali production in colonies . These results are discussed in relation to observations with surface colonies. Toxicol Appl Pharmacol, 1997 May, 144(1), 183 - 8 Glutathione S-transferase-mediated mutagenicity of trihalomethanes in Salmonella typhimurium: contrasting results with bromodichloromethane off chloroform; Pegram RA et al.; Trihalomethanes (THMs) are the most prevalent disinfection by-products identified in chlorinated drinking water . Among the THMs, chloroform (CHCl3) generally occurs at the highest concentration in finished water, but the concentrations of each of the brominated THMs (CHBrCl2, CHBr2Cl, and CHBr3) can exceed that of CHCl3 . Each of these four THMs was carcinogenic in rodents in chronic oral dosing studies . This study assessed THM mutagenicity in a strain of Salmonella typhimurium TA1535 that was transfected with rat theta-class glutathione S-transferase T1-1 (+GST) . The +GST strain and its nontransfected parent strain (-GST) were employed in a plate-incorporation assay and exposed for 24 hr to the vapor of individual THMs at concentrations up to 25,600 ppm in sealed Tedlar bags . Base-substitution revertants were produced in the +GST strain in a dose-dependent fashion by CHBrCl2 but not by CHCl3 . At 4800 ppm CHBrCl2, which produced a calculated agar concentration of 0.67 mM, there were 419 +/- 75 revertants per plate compared to a spontaneous level of 23 +/- 5 . CHCl3 produced a doubling of revertants only at the two highest concentrations tested (19,200 and 25,600 ppm) . These results indicate that bromination of THMs confers the capability for theta-class GST-mediated transformation to mutagenic intermediates at low substrate concentrations, suggesting the possibility of a similar activation route in humans . Further, the very low affinity of the GSH-dependent pathway for CHCl3 demonstrates that different THMs can induce adverse effects via different mechanisms, indicating that risk evaluations of THMs should not treat members of this class as if they shared a common mode of action. Microbiology, 1997 May, 143 ( Pt 5), 1681 - 90 The flgK motility operon of Borrelia burgdorferi is initiated by a sigma 70-like promoter; Ge Y et al.; A cluster of flagellar genes of Borrelia burgdorferi was identified and sequenced . This cluster comprises an operon, designated the flgK operon, which is initiated by a sigma 70-like promoter . The flgK operon consists of flbF (function unknown), flgK (encoding HAP1), flgL (encoding HAP3) and orfX (function unknown), and maps at 185 kb on the chromosome . In other bacteria, the hook-associated proteins HAP1 and HAP3 connect the flagellar filament to the hook and are required for the last stage of flagellar assembly . Reverse transcriptase-PCR analysis indicated that flbF through to orfX are transcribed as a single mRNA, and primer extension analysis revealed that transcription of the flgK operon is initiated by a sigma 70-like promoter upstream of flbF . Subcloning the flgK promoter element into a promoter probe cat vector revealed that the flgK promoter element had strong activity in both Escherichia coli and Salmonella typhimurium . In addition, when this construct was transformed into a fliA mutant of S . typhimurium which lacked a functional flagellar-specific sigma 28 factor, the flgK promoter was still functional . Based on these results, the promoter element of the flagellin gene (fla, hereafter referred to as flaB) was re-examined . flaB encodes the flagellar filament protein, and a sigma gp33-34-like promoter has been reported to be involved in the transcription of this gene . A transcriptional start point was found 1 bp downstream of the reported start site . The sequence around -10 and -35 are consistent with the presence of a sigma 70-like promoter in addition to the putative sigma gp33-34-like promoter for flaB . In contrast to the flgK promoter element, no activity was detected after subcloning a flaB promoter element into the promoter probe cat vector . Because a sigma 70-like promoter rather than a unique flagellar sigma factor is involved in the later stage of flagellar assembly, the regulation of B . burgdorferi flagellar genes is evidently different from that of other bacteria. Microbiology, 1997 May, 143 ( Pt 5), 1539 - 47 The flagellin N-methylase gene fliB and an adjacent serovar-specific IS200 element in Salmonella typhimurium; Burnens AP et al.; The cloning and molecular genetic analysis of a locus mapping within the flagellar gene (fli) complex of Salmonella typhimurium is reported . A copy of the insertion element IS200 was located in a noncoding stretch of DNA upstream of the fliA gene . Comparative nucleotide sequence analysis showed that this copy of IS200 was 711 bp long and that its flanking regions contained no features common to other characterized insertion sites of this element . The element was located 37 bp downstream of an ORF whose product was shown by interspecific transfer and amino acid analysis to carry out N-methylation of selected lysine residues in Salmonella flagellin . The sequence and phenotype of this ORF identified it as fliB, encoding the only prokaryotic N-methylase acting on amino groups to have been characterized to date . It was found to be conserved among all clinically significant serovars of Salmonella . The IS200 insertion site is of particular interest since it was conserved in all but two rare evolutionary lines of S . typhimurium, and was absent from 85 Salmonella strains belonging to 37 other serovars . It is thus a phylogenetically significant marker at the serovar level. Microbiology, 1997 May, 143 ( Pt 5), 1533 - 7 The outer membrane of lipid A-deficient Escherichia coli mutant LH530 has reduced levels of OmpF and leaks periplasmic enzymes; Nurminen M et al.; We have previously described a new Escherichia coli K-12 mutant, LH530, which has a defective outer membrane . LH530 is very sensitive to hydrophobic antibiotics, does not grow at 42 degrees C and synthesizes reduced amounts of lipid A . Phenotypically LH530 is very similar to the known lipid A biosynthesis mutants of E . coli and Salmonella typhimurium . Its genetic defect is not known, but the defect is suppressed by multiple copies of ORF195 . Here we show that at 37 degrees C LH530 contains a reduced amount of the OmpF porin and that it leaks periplasmic beta-lactamase at 37 degrees C and 42 degrees C . We further show that ORF195, when present at low copy number, restores the antibiotic resistance and lipid A biosynthesis of LH530 at 28 degrees C, but not at higher temperatures . In contrast, OmpF expression is restored at 37 degrees C. Chem Res Toxicol, 1997 May, 10(5), 582 - 8 Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo{4,5-f}quinoline bioactivation to mutagenic metabolites: significance of cytochrome P450 peroxygenase; Anari MR et al.; We show that the naturally occurring hydroperoxide hydrogen peroxide is highly effective in supporting the cytochrome P450 1A2 peroxygenase-catalyzed metabolic activation of the heterocyclic aromatic amine 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) to genotoxic metabolites . Mutagenicity was assessed by the Ames assay with Salmonella typhimurium strain YG1012 and an activation system consisting of hydroperoxides plus either 3-methylcholanthrene-induced rat liver microsomes (rP4501A) or human P450 1A2-containing microsomes (hP4501A2) . The mutagenic response was dependent on the concentration of microsomal protein, IQ, and hydroperoxides . The addition of hydrogen peroxide or tert-butyl hydroperoxide to rP4501A greatly enhanced the yield of histidine prototrophic (His+) revertants . This increase was inhibited, in a concentration-dependent manner, by alpha-naphthoflavone, a P450 1A inhibitor . Hydrogen peroxide was the most effective peroxygenase cofactor, particularly with hP4501A2 (K(m) = 0.1 mM) . The hydroperoxide-supported activation of IQ produced reactive intermediates which bound to 2'-deoxyguanosine; LC/MS analysis of the adducts revealed the same major (protonated) adduct at m/z = 464.4 as previously reported for the DNA adduct formed (in vivo or in vitro) by the mixed function-catalyzed bioactivation system . None of the peroxidase-catalyzed IQ metabolites (nitro-, azo-, or azoxy-IQ) were detected . In conclusion, hydrogen peroxide in the physiological/pathological concentration range may be able to support the metabolic activation of arylamines to genotoxic products through the cytochrome P450 peroxygenase pathway. Pediatr Infect Dis J, 1997 May, 16(5), 482 - 5 Extraintestinal salmonellosis in a children's hospital; Schutze GE et al.; BACKGROUND: An increase in the number of patients presenting with extraintestinal salmonellosis has occurred at our institution . The purpose of this study was to review the extraintestinal salmonellosis cases in our institution and to investigate the possible reasons for this increase . METHODS: A retrospective review of patients from 1985 through 1996 was carried out to identify patients with extraintestinal infections with Salmonella . Demographic data were gathered and statistical evaluations comparing differences among groups (1985 to 1989, 1989 to 1992, 1993 to 1996) was done using the G statistic, adjusted (maximum likelihood) chi square or the Fisher's exact test . RESULTS: Thirty-nine patients were identified with extraintestinal salmonellosis and although the number of cases had increased from 8 in 1985 to 1988 to 18 in 1989 to 1992 and 13 from 1993 to 1996, the percentage of cases that were extraintestinal were similar (3.3%, 6%, 4.9%; P > 0.1) . Seventy-two percent of patients had underlying risk factors with the most common being age < 3 months (44%), sickle cell anemia (13%) and gastrointestinal surgery (10%) . Fever and diarrhea were more common presenting symptoms in patients < 3 months of age than in older patients (P < 0.05) . Salmonella typhimurium and Salmonella heidelberg were the most common serotypes isolated and an increasing trend of ampicillin resistance was noted from 0% in 1985 to 1988 to 39% from 1989 to 1992 and 23% from 1993 to 1996 . CONCLUSIONS: The reasons for an increasing trend in extraintestinal cases of human salmonellosis at our institution were not identified . This illness continues to occur in infants and children with well-recognized risk factors. J Bacteriol, 1997 May, 179(10), 3196 - 201 Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium; Zhulin IB et al.; Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E . coli . The threshold of the attractant response in both species was 10(-6) M glycerol . Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential . Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished . Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E . coli or S . typhimurium but is involved in the negative chemotaxis of E . coli to high concentrations of glycerol . We propose that positive chemotaxis to glycerol in E . coli and S . typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level. Protein Sci, 1997 May, 6(5), 1084 - 91 Energy profile of maltooligosaccharide permeation through maltoporin as derived from the structure and from a statistical analysis of saccharide-protein interactions; Meyer JE et al.; The crystal structure of the maltodextrin-specific porin from Salmonella typhimurium ligated with a maltotrioside at the pore eyelet is known at 2.4 A resolution . The three glucose units assume a conformation close to the natural amylose helix . The pore eyelet fits exactly the cross-section of a maltooligosaccharide chain and thus functions as a constraining orifice . The oligomer permeates the membrane by screwing along the amylose helix through this orifice . Because each glucose glides along the given helix, its interactions can be sampled at any point along the pathway . The interactions are mostly hydrogen bonds, but also contacts to aromatic rings at one side of the pore . We have derived the energy profile of a gliding maltooligosaccharide by following formation and breakage of hydrogen bonds and by assessing the saccharide-aromatics interactions from a statistical analysis of saccharide binding sites in proteins . The resulting profile indicates smooth permeation despite extensive hydrogen bonding at the orifice. J Bacteriol, 1997 May, 179(9), 3061 - 3 Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium; Frymier JS et al.; The transcriptional control of the kdpFABC (K+ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion . The kdpFABC operon of this organism was induced by K+ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K+ . In the trkA (sapG) kdp+ mutant background, high concentrations of K+ inhibited growth, along with repressing the kdp operon . This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss. J Bacteriol, 1997 May, 179(9), 2994 - 3003 An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB; Togashi F et al.; Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM . Most of the suppressing mutations lay in flagellar region IIIb of the chromosome . One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II . SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium . Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state . The location of the mutation further underscores the conclusion from a previous study of spontaneous missense mutants (V . M . Irikura, M . Kihara, S . Yamaguchi, H . Sockett, and R . M . Macnab, J . Bacteriol . 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically important for switching . All of the second-site mutations in motA and motB caused some impairment of motility, both in the pseudorevertants and in a wild-type fliG background . The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed . The mutations in the MotA and MotB sequences were clustered to a considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB . The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed. J Bacteriol, 1997 May, 179(9), 2907 - 14 Regulation of heme biosynthesis in Salmonella typhimurium: activity of glutamyl-tRNA reductase (HemA) is greatly elevated during heme limitation by a mechanism which increases abundance of the protein; Wang LY et al.; In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in heme biosynthesis . We report that when heme limitation is imposed on cultures of S . typhimurium, glutamyl-tRNA reductase (HemA) enzyme activity is increased 10- to 25-fold . Heme limitation was achieved by a complete starvation for heme in hemB, hemE, and hemH mutants or during exponential growth of a hemL mutant in the absence of heme supplementation . Equivalent results were obtained by both methods . To determine the basis for this induction, we developed a panel of monoclonal antibodies reactive with HemA, which can detect the small amount of protein present in a wild-type strain . Western blot (immunoblot) analysis with these antibodies reveals that the increase in HemA enzyme activity during heme limitation is mediated by an increase in the abundance of the HemA protein . Increased HemA protein levels were also observed in heme-limited cells of a hemL mutant in two different E . coli backgrounds, suggesting that the observed regulation is conserved between E . coli and S . typhimurium . In S . typhimurium, the increase in HemA enzyme and protein levels was accompanied by a minimal (less than twofold) increase in the expression of hemA-lac operon fusions; thus HemA regulation is mediated either at a posttranscriptional step or through modulation of protein stability. J Immunol, 1997 May 1, 158(9), 4310 - 9 Identification of a natural T cell epitope presented by Salmonella-infected macrophages and recognized by T cells from orally immunized mice; Cookson BT et al.; Murine infection with Salmonella typhimurium provides models for typhoid fever and long-lasting protective immunity conferred by oral vaccination with viable attenuated bacteria . To further understand the role of T cells in these systems, we identified a bacterial Ag recognized by murine T cells responding to a Salmonella infection . From orally infected mice, we derived a CD4+ Ak-restricted T cell clone (7.4.8) the stimulatory Ag of which was provided by S . typhimurium or its flagella, but not by other salmonellae or S . typhimurium mutants unable to synthesize the flagellar filament protein FliC . We mapped antigenic activity to FliC hypervariable region VI using a generally applicable method of sequential C-terminal truncation of recombinant MalE-FliC fusion proteins . Residues 339-350 are the minimal FliC structure capable of stimulating 7.4.8 and represent the first reported Salmonella-specific epitope recognized by T cells from infected mice . T cells with this specificity are generated by oral immunization, reactivity can be recovered for at least 5 mo afterwards, and FliC is the dominant recall Ag for CD4+ T cells from protectively immunized C3H/HeJ mice . FliC 339-350 is presented by macrophages infected with viable S . typhimurium, and presentation, but not bacterial uptake, is greatly enhanced by pretreatment of macrophages with IFN-gamma . These data point to the importance of IFN-gamma-activated macrophages in the stimulation of T cells responding to facultative intracellular pathogens like S . typhimurium and provide a model system for studying Ag-specific T cell responses in murine salmonellosis. J Immunol, 1997 May 1, 158(9), 4229 - 36 Bone marrow-derived dendritic cells can process bacteria for MHC-I and MHC-II presentation to T cells; Svensson M et al.; Dendritic cells can engulf particulate Ags and induce T cell proliferative responses after pulsing with particulate Ag . However, their capacity to process viable Gram-negative bacteria for presentation by MHC-I and MHC-II has not been shown . We therefore characterized the ability of murine bone marrow-derived dendritic cells to process Escherichia coli and Salmonella typhimurium, expressing defined epitopes for presentation by MHC-I and MHC-II molecules . The I-Ak-restricted 46-61 epitope from hen egg white lysozyme (HEL(46-61)) or the Kb-restricted 257-264 epitope from chicken egg OVA (OVA(257-264)) was expressed as fusion proteins in the bacterial cytoplasm as the Crl-HEL and Crl-OVA fusion proteins, respectively . Bacteria expressing Crl-HEL or Crl-OVA, or beads coated with HEL or OVA, were coincubated with murine bone marrow-derived dendritic cells, and Ag processing and presentation were quantitated using T cell hybridomas . The data show that granulocyte-macrophage CSF-stimulated dendritic cells can process live intact Gram-negative bacteria for peptide presentation by MHC-I and MHC-II . Cytochalasin D inhibition studies revealed that processing for both MHC-I and MHC-II presentation required cytoskeletal rearrangements . Processing for MHC-I and MHC-II presentation was inhibited by ammonium chloride, suggesting that acidic compartments were required . Thus, granulocyte-macrophage CSF-stimulated murine bone marrow dendritic cells are capable of processing exogenous particulate Ags, including bacteria with no known mechanism for phagosomal escape, for peptide presentation by both MHC-I and MHC-II . These data suggest that dendritic cells may be important in priming both CD4+ and CD8+ T cells to bacterial Ags. Infect Immun, 1997 May, 65(5), 1926 - 30 Expression of the Yersinia pestis capsular antigen (F1 antigen) on the surface of an aroA mutant of Salmonella typhimurium induces high levels of protection against plague; Titball RW et al.; The caf operon from Yersinia pestis encoding the structural subunit (caf1), the molecular chaperone (caf1M), the outer membrane anchor (caf1A), and the regulatory protein (caf1R) was cloned into Salmonella typhimurium SL3261 aroA . The recombinant Salmonella organisms were encapsulated when cultured at 37 degrees C but not when cultured at 28 degrees C . Oral inoculation of mice with the recombinant Salmonella induced predominantly an immunoglobulin G2a response to F1 antigen, and isolated T cells showed a recall response to soluble or Salmonella-associated F1 antigen . Mice immunized with S . typhimurium SL3261 aroA expressing F1 antigen intracellularly developed lower antibody responses to F1 antigen and showed a T-cell recall response only to Salmonella-associated F1 antigen . Mice immunized orally with two doses of the recombinant Salmonella which expressed F1 antigen on the surface were protected against 10(7) 50% lethal doses (LD50) of virulent Y . pestis given by the subcutaneous route of challenge, whereas mice immunized with the recombinant Salmonella expressing F1 antigen intracellularly were only partially protected against 10(5) LD50 of Y . pestis. Infect Immun, 1997 May, 65(5), 1814 - 23 Role of sigma factor RpoS in initial stages of Salmonella typhimurium infection; Nickerson CA et al.; The sigma factor RpoS mediates the stationary-phase expression of a large group of genes, including those involved in resistance to a variety of environmental stresses, such as starvation, oxidation, and low pH . In addition, RpoS has been shown to regulate Salmonella virulence . In Salmonella typhimurium, RpoS controls the expression of the Salmonella plasmid virulence (spv) genes, which are required for systemic infection . However, the mechanism by which RpoS affects the pathogenicity of Salmonella remains incompletely defined . In this study, we focused on the ability of rpoS to affect the early stages of the infection process of S . typhimurium . An rpoS mutant of S . typhimurium exhibited wild-type abilities to attach to and invade Int-407 cells and J774 macrophage-like cells . In addition, rpoS did not affect the intracellular survival of S . typhimurium in either J774 macrophage-like cells or rat bone marrow-derived macrophages . However, the rpoS mutant demonstrated a decreased ability to colonize murine Peyer's patches after oral inoculation than its wild-type virulent parent strain showed . In addition, virulence plasmid-cured derivatives of the rpoS mutant were recovered in lower numbers from murine Peyer's patches than were plasmid-cured derivatives of the isogenic wild-type S . typhimurium . This indicates that RpoS regulation of chromosomally encoded genes is important for colonization of the gut-associated lymphoid tissue (GALT) by S . typhimurium . Microscopic analysis of histological sections taken from Peyer's patches after peroral infection of mice showed that, unlike its wild-type virulent parent strain, the isogenic rpoS mutant did not destroy the follicle-associated epithelium of the GALT . Furthermore, the rpoS mutant demonstrated a decreased ability to adhere to histological sections of murine Peyer's patches than its wild-type parent showed . Our data provide evidence for a role of RpoS in the interaction of Salmonella with cells of the GALT, specifically the Peyer's patches . This implicates the involvement of rpoS in the initial stages of systemic infection by Salmonella as opposed to infection leading to gastroenteritis. Mol Gen Genet, 1997 Apr 28, 254(4), 440 - 8 Autogenous and global control of the flagellar master operon, flhD, in Salmonella typhimurium; Kutsukake K; Expression of the flagellar master operon, flhD, is known to be affected by growth conditions and by mutations in a variety of genes . In the present work, the transcriptional control of the Salmonella typhimurium flhD operon was investigated in various genetic backgrounds . First, we examined the effect of mutations in the global regulators cAMP-CRP, H-NS, OmpR and RpoS . Mutations in the cya, crp or hns gene reduced but did not eliminate flhD expression . However, expression was completely inhibited in the cya hns and crp hns double mutants . These results indicate that cAMP-CRP and H-NS independently activate the flhD operon and that maximal expression is attained in the presence of both regulators . On the other hand, the ompR and rpoS mutations did not affect either the motility phenotype or flhD expression . We next examined the expression of a chromosomal flhD-lac fusion gene in the presence of a plasmid carrying the wild-type flhD operon . It was found that under this condition the chromosomal flhD operon was repressed or activated, depending on the intracellular activity of FliA, an alternative sigma factor specific for late flagellar operons . In the absence of FliA or in the presence of both FliA and its cognate anti-sigma factor FlgM, the flhD operon was autogenously repressed, whereas in the flgM mutant background it was autogenously activated in the presence of FliA . This autoregulation was still observed in the crp or hns mutant background, indicating that the autogenous control is achieved by a mechanism that is independent of the cAMP-CRP and H-NS regulatory pathways. Mutat Res, 1997 Apr 24, 390(1-2), 45 - 50 Formation of 8-hydroxy-2'-deoxyguanosine following treatment of 2'-deoxyguanosine or DNA by hydrogen peroxide or glutathione; Abu-Shakra A et al.; We have demonstrated that free radicals generated by hydrogen peroxide (H2O2), in the presence of divalent iron (Fe2+) and a chelator (EDTA), oxidize 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) . The 8-OHdG formed by this reaction was isolated and quantitated using reverse-phase HPLC with UV and electrochemical detection . A 1-h incubation of dG with H2O2 caused a 50% increase in 8-OHdG over background, which increased to 100% after 2 h . However, when an H2O2-generating system {glutathione (GSH), Fe2+, EDTA} was used, there was no increase in 8-OHdG yield after the 1-h incubation, but up to a 50% increase over background was observed with GSH after 2-h incubation . Attempts to detect increased levels of 8-OHdG after H2O2- or GSH-treatment of purified calf thymus or rat DNA, or purified Salmonella typhimurium DNA were not successful . This may have been because the treatment procedures used generated 8-OHdG in the control samples at sufficiently high levels to mask any H2O2-induced responses that may have been present . This artifactual production of 8-OHdG has presented a problem in all in vitro studies to date . In contrast, treatment of Salmonella cells (strain TA104) with increasing concentrations of H2O2, caused a doubling in the 8-OHdG yield . GSH-treatment of strain TA104 cells under the same conditions did not result in an increase of 8-OHdG . The study presented here shows that the ubiquitous molecule H2O2 can play a major role in DNA oxidation, mutation, and damage. Mutat Res, 1997 Apr 24, 390(1-2), 11 - 9 Genotoxicity of ethyl carbamate (urethane) in Salmonella, yeast and human lymphoblastoid cells; Hubner P et al.; Ethyl carbamate is a known carcinogen occurring in fermented food and beverages and is therefore of interest for food safety assurance . We studied the genotoxicity of ethyl carbamate in Salmonella typhimurium, in Saccharomyces cerevisiae and in human lymphoblastoid TK6 cells . In absence of cytochrome P450 enzymes, no ethyl carbamate-mediated genotoxicity was observed in any of the three test systems in the non-cytotoxic range . In the presence of an activating system, ethyl carbamate was found to be mutagenic in Salmonella typhimurium strain TA100 but not in strains TA98 and TA102, indicating base-pair substitutions at G-C base pairs . In contrast, no significant mutagenicity of ethyl carbamate could be detected in human lymphoblastoid TK6 cells . However, applied in cytotoxic concentrations, ethyl carbamate was genotoxic for Saccharomyces cerevisiae in the absence of P450-mediated metabolic activation . Inhibitors of P450IIE1 (DMSO, ethanol and dithiodiethylcarbamate) diminished ethyl carbamate-mediated mutagenicity in Salmonella typhimurium strain TA100 in a dose dependent manner, suggesting that P450IIE1 is the activating enzyme. Gene, 1997 Apr 21, 189(2), 195 - 201 Identification of a large motility operon in Borrelia burgdorferi by semi-random PCR chromosome walking; Ge Y et al.; Motility has been implicated in the invasive process of Borrelia burgdorferi (Bb), the etiologic agent of Lyme disease . To identify Bb motility related genes, we used a method termed 'semi-random PCR chromosome walking' (SRPCW) to walk through a large motility gene cluster . The major advantage of this approach over other PCR walking methods is that it employs a secondary PCR amplification of cloned fragments which can be readily sequenced and analyzed . Starting with a primer specific to flgE, we identified and sequenced 14 open reading frames (ORFs) spanning 11 kb downstream of the flgE gene . The genes identified include flbD, motA, motB, fliL, fliM, fliN, fliZ, fliP, fliQ, fliR, flhB, flhA, flhF and flbE . Twelve of the deduced proteins shared extensive homology with flagellar proteins from other bacteria . The gene products and order of genes within this cluster are most similar to those of Treponema pallidum (Tp) and Bacillus subtilis (Bs) . One of the unique genes identified, flbD, demonstrated homology to an ORF from the same operon of Tp . Another ORF, flbE, showed similarity to genes from both Tp and Bs . RT-PCR and primer extension analysis revealed that this gene cluster is transcribed as a single unit indicating that it is part of a large motility operon spanning more than 21 kb . Antisera to Escherichia coli and Salmonella typhimurium FliN, FliM, FlhB and FlhA reacted with proteins of the predicted molecular weights in cell lysates of Bb . The results suggest that the flagellar system is highly conserved in evolution and thus underscore the importance of motility in bacterial survival and pathogenesis. Biochim Biophys Acta, 1997 Apr 17, 1335(1-2), 120 - 6 Adiabatic compressibility of flagellin and flagellar filament of Salmonella typhimurium; Tamura Y et al.; The partial specific volume and adiabatic compressibility of flagellin, its F40 fragment deprived of the disordered terminal regions, from Ala-1 to Arg-65 and from Ser-451 to Arg-494, and the flagellar filament of Salmonella typhimurium were determined from the density and the sound velocity measurements at 15 degrees C . The partial specific volumes were 0.728 cm3/g, 0.745 cm3/g, and 0.734 cm3/g, and the partial specific adiabatic compressibilities were 4.0 x 10(-12) cm2/dyn, 6.7 x 10(-12) cm2/dyn, and 4.7 x 10(-12) cm2/dyn, for flagellin, F40, and the filament, respectively . The smaller values of flagellin than those of F40 are reasonably explained by the presence of disordered terminal regions, which are supposed to be highly hydrated by water molecules . The volume increase upon polymerization of flagellin into the filament is also confirmed by depolymerization under a high pressure . The smaller volume and compressibility of the filament compared with those of F40 suggest an extensive hydration of the filament on its complex surface structure, which surpasses the effect on the volume and compressibility by a possible increase in the cavity volume at intersubunit interfaces upon polymerization. Structure, 1997 Apr 15, 5(4), 545 - 58 Crystal structure of the chemotaxis receptor methyltransferase CheR suggests a conserved structural motif for binding S-adenosylmethionine; Djordjevic S et al.; BACKGROUND: Flagellated bacteria swim towards favorable chemicals and away from deleterious ones . The sensing of chemoeffector gradients involves chemotaxis receptors, transmembrane proteins that detect stimuli through their periplasmic domains and transduce signals via their cytoplasmic domains to the downstream signaling components . Signaling outputs from chemotaxis receptors are influenced both by the binding of the chemoeffector ligand to the periplasmic domain and by methylation of specific glutamate residues on the cytoplasmic domain of the receptor . Methylation is catalyzed by CheR, an S-adenosylmethionine-dependent methyltransferase . CheR forms a tight complex with the receptor by binding a region of the receptors that is distinct from the methylation site . CheR belongs to a broad class of enzymes involved in the methylation of a variety of substrates . Until now, no structure from the class of protein methyltransferases has been characterized . RESULTS: The structure of the Salmonella typhimurium chemotaxis receptor methyltransferase CheR bound to S-adenosylhomocysteine, a product and inhibitor of the methylation reaction, has been determined at 2.0 A resolution . The structure reveals CheR to be a two-domain protein, with a smaller N-terminal helical domain linked through a single polypeptide connection to a larger C-terminal alpha/beta domain . The C-terminal domain has the characteristics of a nucleotide-binding fold, with an insertion of a small antiparallel beta sheet subdomain . The S-adenosylhomocysteine-binding site is formed mainly by the large domain, with contributions from residues within the N-terminal domain and the linker region . CONCLUSIONS: The CheR structure shares some structural similarities with small molecule DNA and RNA methyltransferases, despite a lack of sequence similarity among them . In particular, there is significant structural preservation of the S-adenosylmethionine-binding clefts; the specific length and conformation of a loop in the alpha/beta domain seems to be required for S-adenosylmethionine binding within these enzymes . Unique structural features of CheR, such as the beta subdomain, are probably necessary for CheR's specific interaction with its substrates, the bacterial chemotaxis receptors. Int J Food Microbiol, 1997 Apr 15, 35(3), 251 - 8 Effects of stress treatments on the detection of Salmonella typhimurium by in situ hybridization; Tolker-Nielsen T et al.; In order to assess the usefulness of quantitative in situ rRNA hybridization as an indicator of the physiological state of bacteria, we have used this method to measure the cellular contents of 16 S and 23 S rRNA in Salmonella typhimurium subjected to a number of different stress treatments . The contents of rRNA in S . typhimurium decreased when the bacteria were subjected to carbon starvation, heat stress, and osmotic stress prior to the hybridization, whereas no decrease in the intracellular contents of rRNA was observed when the bacteria were subjected to cold stress, acetic acid or ethanol treatment prior to the hybridization . We must conclude, that the content of 16 S rRNA and 23 S rRNA cannot be used as the sole indicator of the physiological state or viability of food borne pathogens . Viable as well as non-viable food borne bacteria will be detected when methods based on detection of rRNA are used. Nucleic Acids Res, 1997 Apr 15, 25(8), 1548 - 52 The mechanism of mutation induction by a hydrogen bond ambivalent, bicyclic N4-oxy-2'-deoxycytidine in Escherichia coli; Negishi K et al.; The triphosphate of the nucleoside deoxyribosyl dihydropyrimido{4,5-c}{1,2}oxazin-7-one (dP) is known to be incorporated into DNA efficiently by Taq polymerase and is a useful tool for polymerase-mediated in vitro mutagenesis . It is shown here that dP is a potent mutagen in Escherichia coli and Salmonella typhimurium . In E.coli , this deoxycytidine analog induces both GC-->AT and AT-->GC transitions . No induced transversions are observed . It is highly mutagenic in wild-type E.coli, but this is much reduced in a strain lacking thymidine kinase . Mutagenesis induced by dP is efficiently inhibited by the addition of thymidine . Partially purified thymidine kinase from E.coli catalyzes phosphorylation of dP to its 5'-monophosphate . When E.coli was grown in the presence of dP, the nucleoside analog was incorporated into its DNA . The content of dP in DNA was dependent on the concentration of dP added to the medium . The incorporation characteristics of the 5'-triphosphate of dP (dPTP) were also studied using E.coli DNA polymerase I large fragment . The results confirm that this triphosphate can be incorporated opposite A and G in the template with similar efficiencies . This indicates that dP is metabolized as a thymidine analog and that the resulting triphosphate induces a high rate of mutagenesis through replicational errors. Mutat Res, 1997 Apr 14, 375(1), 9 - 17 Targeted disruption of the gene encoding the classical nitroreductase enzyme in Salmonella typhimurium Ames test strains TA1535 and TA1538; Yamada M et al.; The gene encoding the 'classical nitroreductase' (CNR) of Salmonella typhimurium was disrupted . In this manner, cnr null mutant derivatives of strains TA1535 and TA1538 were constructed, and named YG7131 and YG7127, respectively . In both strain backgrounds, cnr gene disruption reduced nitrofurazone-reductase activity . This reduction almost completely eliminated the nitroreductase activity of strain TA1538 . In contrast, the nitroreductase activity of strain TA1535 was much higher than that in TA1538 . In this background, cnr gene disruption resulted in a reduction in nitroreductase activity by a similar absolute amount as in TA1538, but representing only about one-quarter of the original activity of TA1535 . The results suggest that S . typhimurium has originally at least two distinct nitroreductases, one of which is already deficient in strain TA1538; the CNR is present in both TA1535 and TA1538 . Also, these two strains (including their derivatives, TA98 and TA100) are not isogenic with regard to nitroreductase activity . After the introduction of plasmid pKM101, the sensitivities of the strains YG7132 and YG7128, the cnr-null mutants of TA98 and TA100, respectively, against several nitro compounds were compared with those of the conventional cnr-deficient strains TA98NR and TA100NR and the wild-type strains TA98 and TA100 . The mutagenicities of 2-nitrofluorene and 1-nitropyrene in YG7132 or TA98NR were ten-fold lower than those of the compounds in TA98 . Similarly, the mutagenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide in strain YG7128 or TA100NR was substantially lower than that of the compound in TA100 . However, the mutagenicity of 2-nitronaphthalene in YG7128 was between those observed with TA100 and TA100NR, suggesting that a nitroreductase in S . typhimurium other than CNR is involved in the metabolic activation of this compound . The cnr gene of S . typhimurium positively hybridized with DNA at 13 min on the E . coli chromosome where the nfsB and nfnB genes of E . coli are mapped . These results suggest that the cnr gene of S . typhimurium is a counterpart of the nfsB and nfnB genes of E . coli, and that the newly constructed cnr-deletion strains are useful to assess the role of nitroreductases in the metabolic activation of mutagenic nitro compounds. Science, 1997 Apr 11, 276(5310), 250 - 3 Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ; Guo L et al.; Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription . For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides . Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate . Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes . Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues. Eur J Epidemiol, 1997 Apr, 13(3), 347 - 52 Separation of Salmonella typhimurium DT2 and DT135: molecular characterization of isolates of avian origin; Baggesen DL et al.; In Denmark, 0.4 and 3.4% of the human Salmonella Typhimurium cases registered between 1988 and 1993 were caused by DT2 and DT135, respectively . Separation of these two phage types was, however, problematic as only minor differences in lysis pattern and lysis strength occurred . Molecular characterization of 23 Danish isolates, 10 German isolates and the two type strains have subsequently been performed . With only minor exceptions, strains examined could be separated by combination of 0.5 agglutination, ribotyping, and PFGE typing into two major groups in conformity with their phage types . The differences between the two groups were, however, very small and it has not been completely clarified whether this grouping is the result of two independent types or of two related lines developing in different environments . It is concluded that the classification of related phage types DT2 and DT135 has to be supported by molecular methods. Vaccine, 1997 Apr-May, 15(6-7), 739 - 46 Oral delivery of purified lipoprotein OspA protects mice from systemic infection with Borrelia burgdorferi; Luke CJ et al.; The lipoprotein outer surface protein A (OspA) of the Lyme disease agent . Borrelia burgdorferi, has provided protection to mice and other animals against systemic infection when delivered orally as a recombinant protein in Escherichia coli, bacille Calmette . Guerin or Salmonella typhimurium . In the present study purified recombinant strain B31 OspA or outer surface protein D (OspD), another lipoprotein of B . burgdorferi, were administered either subcutaneously (s.c.) or orally without cell carrier or adjuvant to mice . In comparison to the OspD preparation, the OspA protein was 256-fold more resistant to trypsin . Whereas OspA in the suspension was in regular complexes of 17-25 nm in size, OspD formed amorphous globules of different sizes . Animals received a primary immunization and at least one booster . Mice immunized s.c . with either OspA or OspD had detectable antibodies to B . burgdorferi by enzyme-linked immunosorbent assay (ELISA), growth inhibition assay (GIA) and immunoblot . Delivered orally, OspA but not OspD elicited a specific antibody response, including IgA, as determined by these assays . The geometric mean titre of sera from mice who received 4 micrograms of OspA orally on days 1, 2, 4, 21 and 22 was 1470 by Ig ELISA, 320 by IgA ELISA and 128 by GIA . In infectious challenge experiments with B . burgdorferi strain Sh2-2-82 (OspA+ OspD- ) inoculated intradermally at 100 x the ID 50 all eight mice immunized with the 4 micrograms dose of OspA were protected, none of the mice immunized with the 4 micrograms dose of OspD were protected (P < 0.001 by Fisher exact test) . These studies indicate that the lipoprotein OspA provides protection against systemic B . burgdorferi infection when delivered orally as a purified protein. Vaccine, 1997 Apr-May, 15(6-7), 587 - 96 Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein; Tijhaar EJ et al.; Salmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens at levels toxic for bacteria . A SL3261 strain expressing the B subunit of cholera toxin by a similar system (SL3261-CtxB) served as a control in FIV-immunization experiments . Cats immunized once orally or intraperitoneally with SL3261-MFG or SL3261-CtxB all developed serum antibodies to SL3261 lipopolysaccharide and against maltose binding protein or the B subunit of cholera toxin, respectively . Two intraperitoneal immunizations with SL3261-MFG also resulted in the development of Gag specific serum antibodies . Two oral immunizations with SL3261-MFG primed for a Gag specific response, which was demonstrated upon FIV challenge . All challenged cats became infected and no significant differences in viral loads were found between SL3261-MFG and SL3261-CtxB immunized cats. Immunology, 1997 Apr, 90(4), 618 - 25 Correlates of protection induced by live Aro- Salmonella typhimurium vaccines in the murine typhoid model; Harrison JA et al.; Live attenuated salmonella vaccines generally confer better protection than killed vaccines . The immune responses in BALB/c mice elicited by immunization with a live attenuated Aro Salmonella typhimurium vaccine given orally, intravenously or subcutaneously were compared with those elicited by killed whole-cell vaccines (acetone or heat-treated) given subcutaneously . Live vaccines given by all routes elicited higher interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) responses in spleen cells against an alkali-treated whole-cell salmonella lysate than did killed vaccines . Live and killed vaccines elicited high total antibody levels to smooth lipopolysaccharide (LPS) (enzyme-linked immunosorbent assay), but all live vaccine regimes elicited higher IgG2a, suggesting a Th1 response . Oral and intravenous vaccination with live organisms elicited IgA against smooth LPS which subcutaneous vaccination with live or killed salmonellae failed to evoke . Western blots using rough whole-cell lysates showed that all vaccines elicited a varied anti-protein response; however, all groups immunized with live organisms recognized three unidentified bands of MW 52,000, 46,000 and 18,000 which were consistently absent in groups immunized with killed organisms . The results indicate that immunization with live aroA salmonellae elicited a Th1 type of response, including bystander T-cell help to LPS, and a response to proteins not seen in mice that received killed vaccines. Br J Rheumatol, 1997 Apr, 36(4), 491 - 3 Antibodies against bacterial lipopolysaccharides in Japanese patients with ankylosing spondylitis; Tani Y et al.; We investigated IgG, IgA and IgM class specific antibodies to five bacterial (Klebsiella pneumoniae, Escherichia coli, Salmonella enteritidis, Salmonella typhimurium and Shigella flexneri) lipopolysaccharides (LPS) by enzyme-linked immunosorbent assay in 144 Japanese patients with ankylosing spondylitis (AS) . AS patients had significantly elevated IgA antibodies to K . pneumoniae LPS, Salmonella enteritidis LPS and Salmonella typhimurium LPS; however, there was no correlation between antibody level to LPS and acute-phase reactants, erythrocyte sedimentation rate and serum C-reactive protein. Mol Microbiol, 1997 Apr, 24(2), 399 - 410 Purification and characterization of the flagellar hook-basal body complex of Bacillus subtilis; Kubori T et al.; The flagellar hook-basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins . The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook . There were no outer (P and L) rings that are found in Gram-negative bacteria . The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium . The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B . subtilis FIgG, which was previously reported as a rod protein . The sequence of the reported FIgG protein of B . subtilis is more closely related to that of FIgE (the hook protein) rather than FIgG (the rod protein) of S . typhimurium, in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa) . The hook-basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation . The N-terminus of each of these proteins was sequenced . Comparison with protein databases revealed the following polypeptide-gene correspondences: 82 kDa, fIiF; 59 kDa, fIgK; 35 kDa, orfF; 32 kDa, yqhF; 23 kDa, orf3 of the fIaA locus; 20 kDa, fIgB and fIgC; 13 kDa, not determined . The band at 20 kDa was a mixture of FIgB and FIgC, as revealed by two-dimensional gel analysis . Characteristic features of B . subtilis HBB are discussed in comparison with those of S . typhimiurium. Mol Microbiol, 1997 Apr, 24(2), 233 - 9 Regulation of the Caulobacter flagellar gene hierarchy; not just for motility; Wu J et al.; The Caulobacter crescentus flagellum serves not only as a motility apparatus, but also as a key landmark in the differentiation of this asymmetrically dividing bacterium . A distinctive aspect of flagellum biosynthesis is the periodic expression of the flagellar genes during the cell cycle in a sequence corresponding to the order of gene product assembly into the growing flagellum . This program of gene expression is achieved in part by the organization of flagellar genes into a four-tiered regulatory hierarchy that controls their expression at both the transcriptional and post-transcriptional levels . Because of the close interconnection of the developmental program to the asymmetric cell-division cycle in C . crescentus, studies of flagellar gene regulation and motility have also begun to reveal basic mechanisms responsible for control of the cell cycle itself . Here, we review recent work on regulation of the flagellar gene hierarchy in C . crescentus and consider regulatory mechanisms that are distinct from those described in Escherichia coli and Salmonella typhimurium. Am J Public Health, 1997 Apr, 87(4), 580 - 4 A community waterborne outbreak of salmonellosis and the effectiveness of a boil water order; Angulo FJ et al.; OBJECTIVES: A 1993 large water-borne outbreak of Salmonella typhimurium infections in Gideon, Mo, a city of 1100 with an unchlorinated community water supply, was investigated to determine the source of contamination and the effectiveness of an order to boil water . METHODS: A survey of household members in Gideon and the surrounding township produced information on diarrheal illness, water consumption, and compliance with the boil water order . RESULTS: More than 650 persons were ill; 15 were hospitalized, and 7 died . Persons consuming city water were more likely to be ill (relative risk {RR} = 9.1, 95% confidence interval {CI} = 2.9, 28.4), and the attack rate increased with increased water consumption . S . typhimurium was recovered from samples taken from a city fire hydrant and a water storage tower . Persons in 31% (30/ 98) of city households had drunk unboiled water after being informed about the boil water order, including 14 individuals who subsequently became ill . Reasons for noncompliance included "not remembering" (44%) and "disbelieving" (25%) the order . CONCLUSIONS: Communities with deteriorating water systems risk widespread illness unless water supplies are properly operated and maintained . Effective education to improve compliance during boil water orders is needed. Microbiology, 1997 Apr, 143 ( Pt 4), 1405 - 13 Homologous regions of the Salmonella enteritidis virulence plasmid and the chromosome of Salmonella typhi encode thiol: disulphide oxidoreductases belonging to the DsbA thioredoxin family; Rodriguez-Pena JM et al.; The nucleotide sequence relatedness between the chromosome of Salmonella typhi and the virulence plasmid of Salmonella enteritidis was investigated using short DNA probes of < 2 kb covering the whole virulence plasmid sequence . Only one homologous region was detected . This region was subsequently cloned and partially sequenced . Sequences closely related to the pefl gene and the ORFs orf7, orf8 and orf9, which are located downstream of the fimbrial pef operon of the Salmonella typhimurium virulence plasmid, were detected . Sequencing of the cloned S . typhi DNA fragment also revealed identity with genes of the fimbrial sef operon characterized in the chromosome of S . enteritidis . These nucleotide sequences mapped upstream of the S . typhi chromosomal region homologous to the S . enteritidis virulence plasmid . The general organization of the cloned S . typhi chromosomal fragment was similar to the fimbriae-encoding region of the S . typhimurium virulence plasmid . The deduced product of orf8 in the S . typhimurium virulence plasmid, as well as those of the corresponding ORFs in the homologous region of the S . typhi chromosome and in the S . enteritidis virulence plasmid (designated dlt and dlp, respectively), appeared to be related to the thioredoxin family of thiol: disulphide oxidoreductases . The dlp gene was able to complement the DTT-sensitive phenotype, the inability to metabolize glucose 1-phosphate and the low alkaline phosphatase activity of a dsbA mutant of Escherichia coli . The dlt gene partially complemented the lack of alkaline phosphatase activity, but not the other mutant phenotypes . The products of both genes could be detected using the T7 RNA polymerase promoter expression system . The estimated molecular masses of the products of the dlt and dlp genes by SDS-PAGE were 26 and 23 kDa, respectively, the first being in agreement with the deduced amino acid sequence and the latter, somewhat smaller . The processing of a possible leader peptide in the Dlp protein, but not in the Dlt protein, could be responsible for this difference . The Dlp protein appeared as a doublet band on SDS-PAGE, which is characteristic of the oxidized and reduced states of this kind of protein. Mol Microbiol, 1997 Apr, 24(1), 155 - 67 Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2; Hensel M et al.; We have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium . ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb . ssaJ lles upstream of ssaK and is the terminal gene of another operon . The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp . genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively . The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system . Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells . However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain . We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains . SipC is a product of the SPI1 type III secretion system of S . typhimurium, and is important for epithelial cell invasion . Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems. Infect Immun, 1997 Apr, 65(4), 1566 - 9 Salmonella typhimurium aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice; Sinha K et al.; Athymic (nu/nu) BALB/c mice and their euthymic (nu/+) littermates were inoculated intravenously with live attenuated vaccine strains of Salmonella typhimurium . All strains caused progressive infections in the athymic mice but not in their euthymic littermates . Athymic mice given strain SL3261, an aroA derivative of SL1344, in doses between log 4.7 and 5.7 CFU were all severely ill and were killed by weeks 4 to 5 . Athymic mice given log 4.7 CFU of a derivative of S . typhimurium C5 carrying a mutation in htrA, encoding a stress protein, were ill and were killed by week 7 in one experiment but survived to week 13 in another . Athymic mice given log 4.6 CFU of a C5 aroD htrA double mutant were ill and were killed at week 7 . Athymic mice given SL3261 had high bacterial counts in the reticuloendothelial system at 4 weeks . Athymic mice given SL3261 or C5 htrA made immunoglobulin G3 (IgG3) (and to a lesser extent IgM) antibody to lipopolysaccharide (LPS), whereas euthymic mice made IgM, IgG1, IgG2a, IgG2b, and IgG3 anti-LPS antibodies . The results indicate that both aroA and htrA strains will produce slow, progressively lethal infections in athymic mice, that the htrA strain is more attenuated than the aroA strain as measured by time to death in this model, and that IgG3 anti-LPS antibody alone cannot suppress the progress of infections by very attenuated strains in athymic mice. Infect Immun, 1997 Apr, 65(4), 1527 - 30 Identification, characterization, and developmental regulation of Chlamydia trachomatis 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase and CMP-KDO synthetase; Wylie JL et al.; The kdsA and kdsB genes from Chlamydia trachomatis encoding 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase and CMP-KDO synthetase were identified by functional complementation of temperature-sensitive Salmonella typhimurium mutants, homology to known KDO-8-phosphate synthetase and CMP-KDO synthetase proteins, and in vitro enzyme activity . The kdsA gene was transcribed as part of a polycistronic mRNA with two downstream open reading frames (ORFs) . One of these ORFs appeared to encode a membrane-anchored protein, while the second encoded a protein showing homology to the ATP-binding component of periplasmic binding protein-dependent ABC transporters . Transcription of kdsA and kdsB in C . trachomatis was evident within 4 h of initiation of the C . trachomatis infection process and continued throughout the chlamydial life cycle. Infect Immun, 1997 Apr, 65(4), 1475 - 85 The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry; Rathman M et al.; Although it has been known for some time that Salmonella typhimurium is able to survive and even replicate in the normally bactericidal environment of the macrophage phagosome, the mechanisms by which this organism accomplishes this feat remain obscure . In this study, a murine macrophage cell line and confocal immunofluorescence microscopy were used to more thoroughly define the specific nature of phagosomes containing latex beads or wild-type S . typhimurium (viable or heat-killed organisms) . Live S . typhimurium organisms were observed to reside in phagosomes that diverge from the degradative pathway of the macrophage . These compartments contain lysosomal glycoproteins and lysosomal acid phosphatase, endocytic markers delivered to vacuoles by mannose 6-phosphate receptor-independent mechanisms, but are devoid of the mannose 6-phosphate receptor and cathepsin L . In contrast, phagosomes containing latex beads or heat-killed organisms appeared to be processed along the degradative pathway of the host cell; these compartments colocalized not only with lysosomal glycoproteins and lysosomal acid phosphatases but also with mannose 6-phosphate receptors and cathepsin L . The uniqueness of the phagosome containing viable S . typhimurium was confirmed by the observation that these compartments, in comparison to phagosomes containing latex beads, do not readily interact with incoming endocytic traffic . Finally, we show that an isogenic, noninvasive mutant of S . typhimurium, BJ66, ends up in an intracellular compartment identical to the wild-type S . typhimurium-containing phagosome . Thus, modifications of the Salmonella-containing compartment occur independently of the mechanism of bacterial entry. Infect Immun, 1997 Apr, 65(4), 1445 - 54 Mucosal immunogenicity of a recombinant Salmonella typhimurium-cloned heterologous antigen in the absence or presence of coexpressed cholera toxin A2 and B subunits; Harokopakis E et al.; An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice . Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen . Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen . A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S . typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration . Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors . These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B . Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 {Th1}/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen . Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment besides its ability to target antigen uptake into the mucosal inductive sites . CTA2/B may thus be useful as an S . typhimurium-cloned adjuvant for coexpressed protein antigens. Infect Immun, 1997 Apr, 65(4), 1313 - 6 Bacterial porins stimulate bone resorption; Meghji S et al.; Porins are abundant outer membrane proteins of gram-negative bacteria involved in transport of low-molecular-mass molecules . During the past decade, porins from a number of bacteria have also been shown to have proinflammatory activities including inducing the synthesis of proinflammatory mediators (cytokines, platelet-activating factor, and nitric oxide) in cultured cells and inducing inflammation in vivo . With this range of actions, it was possible that porins could also interact with bone cells to cause aberrant bone remodeling and that this could contribute to the bone destruction seen in gram-negative bone infections . By using purified preparations of Salmonella typhimurium and Pseudomonas aeruginosa porins, in the presence of polymyxin B, it was possible to induce concentration-dependent loss of calcium from cultured murine calvaria at porin concentrations in the range of 1 to 10 nM . The mechanism of action of the porins was determined by the inclusion of inhibitors of cyclooxygenase or inflammatory cytokines in the culture media . The bone-resorbing activity of both porins was not inhibited by the cyclooxygenase inhibitor indomethacin or by neutralizing the activity of tumor necrosis factor . Indeed, relatively high concentrations of these agents produced an unexpected increase in the bone resorption induced by the porins . In contrast, porin-induced bone resorption could be inhibited by relatively high concentrations of the natural inhibitor of interleukin-1 (IL-1 receptor antagonist) . It appears that these porins stimulate bone resorption by a mechanism distinct from that of lipopolysaccharide, and the possibility therefore exists that porins play a role in bone destruction in gram-negative bacterial infections of bone. Infect Immun, 1997 Apr, 65(4), 1286 - 92 Protection against murine listeriosis by an attenuated recombinant Salmonella typhimurium vaccine strain that secretes the naturally somatic antigen superoxide dismutase; Hess J et al.; A recombinant (r)-Salmonella typhimurium aroA vaccine strain was constructed which secretes the naturally somatic protein of Listeria monocytogenes, superoxide dismutase (SOD), by the HlyB/HlyD/TolC export machinery . Vaccine efficacy of the SOD-bearing carrier strain was compared with that of the p60-secreting construct, S . typhimurium p60s (J . Hess, I . Gentschev, D . Miko, M . Welzel, C . Ladel, W . Goebel, and S . H . E . Kaufmann, Proc . Natl . Acad . Sci . USA 93:1458-1463, 1996) . Vaccination of mice with both constructs induced protection against a lethal challenge with the intracellular pathogen, L . monocytogenes . While the somatic listerial antigen, SOD, is immunologically uncharacterized, the naturally secreted protein of L . monocytogenes, p60, is known to be highly immunogenic . Our data emphasize the high vaccine potential of r-Salmonella constructs secreting antigens of somatic or secreted origin . Moreover, they suggest that the HlyB/HlyD/TolC-based antigen delivery system with attenuated Salmonella spp . as the carrier is capable of potentiating the immune response against foreign proteins independent from their immunogenicity in and display by the natural host. Chem Res Toxicol, 1997 Apr, 10(4), 432 - 8 Synthesis, characterization, and mutagenicity of nitrated 7H-dibenzo{c,g}carbazole and its phenolic derivatives; Xue W et al.; The nitrated N-heterocyclic aromatic hydrocarbons (NAHs) are found in a variety of environmental sources; many of them have been determined to be mutagenic in short-term assays and/or carcinogenic in animal tests . In this laboratory, we synthesized and characterized nitrated 7H-dibenzo{c,g}carbazole (DBC) and the nitrophenolic metabolites of DBC as potential mutagenic and carcinogenic xenobiotics . The nitro group was formed exclusively at the 5 and/or the symmetric 9 position of DBC, 2-hydroxy-DBC, 3-hydroxy-DBC, and 4-hydroxy-DBC . Ames plate incorporation mutagenicity assays were conducted using Salmonella typhimurium strains TA98 and TA100, with or without rat liver homogenates (S9) . Mutagenicities of the nitrated DBCs were higher than the parent DBC in strain TA98, 5,9-Dinitro-DBC had stronger mutagenic responses than 5-nitro-DBC in all assays, particularly in strain TA98 with S9 . 5,9-Dinitro-DBC had a higher reduction potential relative to 5-nitro-DBC (-1.09 V and -1.37 V, respectively) . Hydroxyl derivatives of 5-nitro-DBC at the 2, 3, 4, 10, or 12 position, synthesized through nitration of the corresponding hydroxy-DBC, possessed greater mutagenicity than the parent 5-nitro-DBC, especially in strain TA100 with or without S9 . Our data suggest that nitrated DBC undergoes both nitroreduction and ring oxidation as the primary pathways for the metabolic activation leading to mutagenesis . The relative mutagenicities of the nitrohydroxy-DBC isomers are generally consistent with the resonance stabilization of the positive charge at the arylnitrenium ion, formed from the nitro functional group, as the proposed active electrophile responsible for genotoxic effects. Photochem Photobiol, 1997 Apr, 65(4), 714 - 22 Preclinical assessment of hypocrellin B and hypocrellin B derivatives as sensitizers for photodynamic therapy of cancer: progress update; Miller GG et al.; Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield . They are available in pure monomeric form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity . In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 {2-(N,N-dimethylamino)-propylamine-HB} . Extinction coefficients at 630 nm (epsilon 630) are 6230, 6190 and 4800, respectively; and 1O2 quantum yields, phi, 0.60, 0.32 and 0.42 . Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1) . Greatest uptake is associated with lysosomes and Golgi . The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions . Transcutaneous phototherapy with HBEA-R1 permanently ablates EMT6/Ed tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects . The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhimurium . Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted. J Lab Clin Med, 1997 Apr, 129(4), 470 - 81 Dimethylthiourea protects rats against gram-negative sepsis and decreases tumor necrosis factor and nuclear factor kappaB activity; Sprong RC et al.; The thiol-containing compound dimethylthiourea (DMTU) is a known protectant in various models of oxidant-mediated tissue damage . Protective effects of DMTU have also been reported in studies on endotoxin-induced (LPS-induced) tissue injury . DMTU may exert this protective effect by reducing oxidative stress . In this study we investigated the effect of DMTU on survival, oxidative stress, and tumor necrosis factor (TNF) activity in two rat models of gram-negative bacterial sepsis . Intraperitoneal injection of 500 mg DMTU/kg protected against the lethal effects of intraperitoneally injected LPS (5 mg/kg) and live Salmonella typhimurium (3.3 x 10(10) CFU/kg) . LPS injection resulted in oxidative stress, as indicated by an elevated concentration of hydrogen peroxide (H(2)O(2)) in normal and carbon monoxide-treated deproteinized blood . We also observed increased H(2)O(2) levels in animals injected with live Salmonella typhimurium . Although DMTU improved survival in both models, H(2)O(2) concentrations were not affected by it . This is consistent with our in vitro observation that DMTU is a weak H(2)O(2) scavenger . Serum TNF activity, however, was substantially decreased by DMTU, and this was associated with a reduced activation of nuclear factor kappaB in the peritoneal cells of LPS-treated rats . In addition, LPS-induced TNF production in vitro by rat peritoneal macrophages was inhibited by DMTU (p < 0.05) . These results suggest that the protective effect of DMTU in gram-negative bacterial sepsis may be the result of a reduction in TNF activity . DMTU does not exert this effect by H(2)O(2) scavenging but may inactivate toxic H(2)O(2) metabolites. J Leukoc Biol, 1997 Apr, 61(4), 469 - 80 Characterization of clonally derived, spontaneously transformed bone marrow macrophage cell lines from lipopolysaccharide hyporesponsive LPS(d) and normal LPS(n) mice; Monner DA et al.; Six macrophage cell lines, each derived from a bone marrow macrophage colony grown in soft agar, were established by expansion of the macrophage clones in liquid culture until spontaneous transformation occurred . Four lines originated from the LPS(d) nonresponder mouse strain C3H/HeJ and two from the LPS(n) responder strain CBA/J . The cell lines adhered to plastic and glass surfaces and displayed typical macrophage functions such as phagocytosis and nonspecific esterase activity . Flow c