Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16619 - 24 Epub 2002 Dec 10. Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron; D'Autreaux B et al.; Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences . The function of Fur is not limited to iron homeostasis . A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control . Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds . Fur recently was found to regulate hmp in Salmonella typhimurium, and in Escherichia coli, the iron-chelating agent 2,2'-dipyridyl induces hmp expression . We now establish direct inhibition of E . coli Fur activity by NO . By using chromosomal Fur-regulated lacZ reporter fusion in E . coli, Fur activity is switched off by NO at micromolar concentration . In vitro Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO . NO reacts with Fe(II) in purified FeFur protein to form a S = 12 low-spin FeFur-NO complex with a g = 2.03 EPR signal . Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition . The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air . This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein . These data establish a link between control of iron metabolism and the response to NO effects. Microbes Infect, 2002 Nov, 4(14), 1409 - 15 Co-localization of quantitative trait loci regulating resistance to Salmonella typhimurium infection and specific antibody production phenotypes; Trezena AG et al.; Salmonella enterica serotype typhimurium is a facultative intracellular bacteria that induces systemic infection in mice . Resistance to this pathogen is under polygenic control in which Nramp1 is the major gene involved . Lines of mice obtained by selective breeding for high (HIII) or low (LIII) antibody response to flagellar antigens of salmonellae showed significant susceptibility differences, although both the lines display Nramp1(R) alleles . The HIII line was extremely susceptible to infection, while the LIII line was resistant . In order to examine the cellular and genetic mechanisms involved in this distinct pattern of resistance, HIII and LIII mice were analyzed for IFNgamma and IL4 production and screened for quantitative trait loci involved in S . typhimurium infection, using several polymorphic microsatellites . In the present work, HIII mice showed an IFNgamma downregulation in the early phase of infection when compared with LIII animals . No interline differences in IL4 production were verified . The loci screening was performed on immunized F2 intercrosses obtained from HIII and LIII mice . Three antibody-controlling chromosomal regions were coincident, and another was mapped near one of the four loci known to affect susceptibility to S . typhimurium . These results indicate a major role of IFNgamma in our model, and suggest the co-localization of quantitative trait loci modulating both infection and antibody production phenotypes. Microbes Infect, 2002 Nov, 4(14), 1379 - 87 Polymorphonuclear leukocyte migration across model intestinal epithelia enhances Salmonella typhimurium killing via the epithelial derived cytokine, IL-6; Nadeau WJ et al.; The host response to Salmonella typhimurium involves movement of polymorphonuclear leukocytes (PMN) across the epithelium and into the intestinal lumen . Following their arrival in the lumen, the PMN attempt to combat bacterial infection by activating antimicrobial defenses such as granule release, oxidative burst, phagocytosis, and cell signaling . We sought to examine PMN-S . typhimurium interaction following PMN arrival in the lumenal compartment . Here, for the first time, we demonstrate that PMN that have transmigrated across model intestinal epithelia have an enhanced ability to kill S . typhimurium . Our data provide evidence to indicate that the extracellular release of the primary and secondary granules of PMN, myeloperoxidase and lactoferrin, respectively, is correlated with enhanced bacterial killing . Furthermore, epithelial cells, during PMN transmigration, release the cytokine IL-6 . IL-6 is known to increase intracellular stores of Ca(2+), and we have determined that this epithelial released cytokine is not only responsible for priming the PMN to release their granules, but also stimulating the PMN to kill S . typhimurium . These results substantiate the pathway in which PMN transmigration activates the epithelial release of IL-6, which in turn increases intracellular Ca(2+) storage . Our results, herein, extend this pathway to include an enhanced PMN granule release and an enhanced killing of S . typhimurium. Pharmacogenetics, 2002 Dec, 12(9), 677 - 89 Differential activation of promutagens by alloenzymes of human sulfotransferase 1A2 expressed in Salmonella typhimurium; Meinl W et al.; Various enzymatically formed sulfuric acid esters are chemically reactive and mutagenic . This metabolic activation pathway is not detected in standard in-vitro mutagenicity test systems . We describe the construction of Salmonella typhimurium TA1538-derived strains expressing alloenzymes *1, *2, *3, *5, *6 of human sulfotransferase 1A2 (SULT1A2) . The reference compounds, 1-hydroxymethylpyrene (1-HMP), N-hydroxy-2-acetylaminofluorene (OH-AAF) and 2-hydroxylamino-5-phenylpyridine (OH-APP), were activated to mutagens in these strains . Their activity differed 7- to 16-fold between strains expressing various alloenzymes . It was strongest and weakest in the strains expressing the common alloenzymes, *1 and *2, respectively . The SULT1A2 protein expression levels, and the V(max) and K(m) values with the reference substrate 4-nitrophenol, varied 2.5-, 4-, and 110-fold, respectively, in cytosolic preparations from strains TA1538-SULT1A2*1 and *2 . Strains with varying protein levels were constructed via insertion of silent mutations in the 5'-part of the cDNA . TA1538-SULT1A2*1Z and TA1538-SULT1A2*2Y showed equal expression levels of alloenzymes *1 and *2, respectively, which were 3 times above those of TA1538-SULT1A2*1 . The mutagenicity of OH-AAF and OH-APP was unchanged in strain TA1538-SULT1A2*1Z versus *1, and moderately increased in TA1538-SULT1A2*2Y versus *2 . The influence of the protein level was stronger with 1-HMP . Nevertheless, mutagenic activity of 1-HMP was still 11 times higher in TA1538-SULT1A2*1Z than in TA1538-SULT1A2*2Y . Thus, differences in the properties between alloenzymes can lead to differences in the activation of promutagens . The model compounds were also tested in strains expressing the other ten human SULTs identified . Whereas OH-AAF and OH-APP showed the highest mutagenic activities in strains expressing SULT1A2, 1-HMP was more potent in strains expressing other SULT forms . With the limitation that little is known about the tissue distribution and regulation of SULT1A2, the findings suggest that its polymorphism may affect the individual susceptibility towards procarcinogens, in particular certain aromatic amines and amides . Arch Inst Pasteur Madagascar, 1996, 63(1-2), 67 - 75 {Street-vendor foods: quality of ice creams, sherbets and sorbets sold in the urban agglomeration of Antananarivo}; Ravaonindrina N et al.; A survey of selling conditions and bacteriological examinations of ice-cream was carried-out in Antananarivo from June 1996 to May 1997 . The way of investigation by vendors and of bacteriological examinations were widely described . Sellers had classic features of a street-vended food vendor: uneducated, no having professional training and mishandling foodstuffs . 202 samples of ice-cream were collected . The contamination prevalence rate was of 95% +/- 3.7% . Salmonella typhimurium was isolated from one sample . Immediate and rigourous measures ought to be put into effect by authorities to right this alarming situation. Clin Ther, 2002 Oct, 24(10), 1585 - 94 Results of a 5-year prospective surveillance study of antibiotic resistance among Salmonella enterica isolates and ceftriaxone therapy among children hospitalized for acute diarrhea; Chiappini E et al.; BACKGROUND: The spread of resistant Salmonella strains continues to increase worldwide . It is necessary to establish epidemiologic information to determine an appropriate empiric antibiotic regimen (when indicated) in infants and children with suspected Salmonella infections for whom the results of susceptibility tests are not yet available . OBJECTIVES: The aim of the present study was to investigate resistance rates and their modifications among Salmonella enterica strains isolated from Italian children hospitalized for acute diarrhea over 5 years . In addition, when antibiotic treatment was indicated, we assessed the in vivo success of parenteral ceftriaxone therapy . METHODS: This study included children admitted consecutively for acute diarrhea to the Division of Pediatrics and Infectious Diseases, Department of Pediatrics, University of Florence, Italy, from January 1, 1997, to December 31, 2001 . S enterica strains were isolated from stool cultures, biochemically identified, and serotyped . These isolates were tested by disk-diffusion assay, using the Kirby-Bauer method, for susceptibilities to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, neomycin, tetracycline, and trimethoprim/sulfamethoxazole . The limits used for definition of resistance were those established by the guidelines of the National Committee for Clinical Laboratory Standards . RESULTS: A total of 2003 children (1051 boys, 952 girls; median age, 10.3 years; age range, 1 month-16.8 years) with acute diarrhea were admitted to the study . S enterica strains were isolated using stool cultures from 218 (10.9%) children (108 boys, 110 girls; median age, 3.3 years; age range, 2 months-15.8 years) . A total of 148 (67.9%) isolates were resistant to at least 1 antibiotic and 57 (26.1%) were multiresistant . The highest rates of resistance were those to tetracycline (132/218 {60.6%}), ampicillin (102/218 {46.8%}), and chloramphenicol (47/218 {21.6%}) . The lowest rate of resistance was to ceftriaxone (4/218 {1.8%}) . Overall, the rate of resistance to ciprofloxacin (19/218 {8.7%}) was significantly higher than that for ceftriaxone (P = 0.003) . Salmonella typhimurium (119/218 {54.6%}) and Salmonella enteritidis (62/218 {28.4%}) were the most frequently identified serotypes . Ceftriaxone was effective in vivo in all 56 children who required antibiotic therapy . CONCLUSIONS: There was a high prevalence of resistant S enterica strains . Ceftriaxone was used effectively in the treatment of S enterica infection in the population studied. Front Biosci, 2003 Jan 01, 8, d430 - 9 Transcription-driven DNA supercoiling and gene expression control; Chen CC et al.; DNA supercoiling plays important roles in gene expression regulation, although, the underlying mechanisms whereby DNA supercoiling modulates gene expression remain elusive . The fact that the transcription process itself generates DNA supercoiling has further complicated the issue . Transcription-driven DNA supercoiling is local and transient . Such a DNA supercoiling effect is likely to play important roles in controlling complex gene expression regulation . Using the suppression of the leu-500 mutation in Salmonella typhimurium topA mutants as a model system, we put forward our view of the effects of transcription-driven DNA supercoiling on gene expression control. Poult Sci, 2002 Nov, 81(11), 1653 - 60 Evaluation of aviguard, a commercial competitive exclusion product for efficacy and after-effect on the antibody response of chicks to Salmonella; Nakamura A et al.; The competitive exclusion (CE) action of Aviguard (AG) and its effects on the antibody response of chicks were evaluated in this study . We observed that AG protected the chicks from overwhelming colonization . Fourteen days after infection, fewer AG-pretreated than nonpretreated chicks shed salmonellae from their coloaca in both infected groups, although much less from SE-infected chicks . Antibody titers of sera produced to Salmonella typhimurium (ST) and SE in pretreated and non-pretreated chicks were not significantly different . Immunoblotting showed that these antibodies reacted with SDS-PAGE-separated 71.4, 67.7, 44.0, and 30.3 kDa proteins detectable in the test strains . Few weak bands of doubtful significance were observed in the cross-reaction between the sera of ST- and SE-infected chicks with ST and SE antigens, respectively . Our study showed that AG protected chicks from overwhelming colonization by salmonellae, and neither altered the antigenic proteins of infecting salmonellae nor their recognition by specific antibodies produced in response to the infection. Curr Drug Targets Infect Disord, 2001 Nov, 1(3), 287 - 302 Bacterial carriers and virus-like-particles as antigen delivery devices: role of dendritic cells in antigen presentation; Beyer T et al.; Replicating attenuated strains of intracellular bacteria like Salmonella typhimurium, Listeria monocytogenes or Mycobacterium bovis Bacille Calmette Guerin (BCG), and non-replicating virus-like-particles (VLP) consisting, for instance, of the VP1-surface component of polyoma virus offer great potential as heterologous carriers delivering foreign protein antigens for immune recognition . Moreover, attenuated S . typhimurium and L . monocytogenes strains hold also great promise as delivery vehicles for DNA vaccines . Polyoma virus-specific VLP consisting of VP1-pentamers are also of interest as carrier devices for eukaryotic expression plasmids . At first sight these different replicating and non-replicating types of vehicles have little in common, but from an immunological point of view viable bacteria and non-viable VLP are both well suited for evoking protective immune responses via several routes of vaccine administration . As these antigen carriers generate humoral and cell-mediated immunity, the heterologous antigens are not only targeted to appropriate pathways of major histocompatibility (MHC) class I and class II antigen processing and presentation, but also generate an adequate cytokine milieu for promoting antigen-specific responses . The most prominent advantage of these carrier devices is presented by their capacity to directly target antigenic proteins or DNA vaccines to immature dendritic cells (DC) along their maturation pathway . Mature DC are the key antigen presenting cell population which efficiently mediates antigen transport to organised lymphoid tissues for the initiation of T cell responses . In general, uptake of these diverse antigen delivery systems by antigen presenting cells (APC) finally lead to efficacious immune responses in the control of pathogenic microorganisms and tumours. Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2159 - 61 Epub 2002 Nov 23. Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium; Simanshu DK et al.; In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle . The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30) . Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique . Crystals belong to the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 63.600, b = 100.670, c = 204.745 A . A complete data set to 2.5 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator. Food Chem Toxicol, 2003 Jan, 41(1), 107 - 18 Effects of cis-nonachlor, trans-nonachlor and chlordane on the immune system of Sprague-Dawley rats following a 28-day oral (gavage) treatment; Tryphonas H et al.; The immunotoxicity of cis- and trans-nonachlor and chlordane were investigated in adult male and female Sprague-Dawley rats following a 28-day oral (gavage) treatment . Rats were randomly assigned to six experimental groups: cis-nonachlor, females; trans-nonachlor, females; technical chlordane females; cis-nonachlor, males; trans-nonachlor, males; technical chlordane, males . The immunologic endpoints included: quantification of the total serum immunoglobulin (Ig) levels and subclasses and flow cytometric analysis of peripheral blood leukocytes and T-lymphocyte subsets, evaluation of the lymphoproliferative activity of splenocytes in response to concanavalin A (Con A) and Salmonella typhimurium (STM) mitogens, and natural killer (NK) cell activity of splenocytes . Satellite experiments to examine the delayed-type hypersensitivity (DTH) response to oxazolone, and resistance to Listeria monocytogenes were set up for female rats treated with cis- or trans-nonachlor . Statistically significant (P<0.05) effects included: increased serum immunoglobulin M (IgM) levels in the chlordane-treated females at the 25 mg/kg dose (pairwise comparison); increased serum IgG(1) and IgG(2c) in the cis-nonachlor-treated males at the 2.5 and 25 mg/kg doses and increased serum IgG(2a) levels at all doses; increased serum IgG(2b) at the 25 mg/kg dose and decreased (dose-related) serum IgM levels in the cis-nonachlor-treated male rats; increased (linear trend) IgG(1) and IgG(2a) in the cis-nonachlor-treated females with effects on IgG(2a) significant at the 25 mg/kg dose compared with control; increased serum IgG(2a) in the trans-nonachlor-treated male and female rats at the 2.5 mg/kg dose; increased absolute numbers (linear trend) of peripheral white blood cells, B lymphocytes, natural killer (NK) cells, T-suppressor/cytotoxic lymphocytes, and the double positive (T-helper/inducer, T-suppressor/cytotoxic) cells in the trans-nonachlor-treated females; increased (non-linear trend) lymphoproliferative activity in the Con A-stimulated splenocytes and decreased (linear trend) activity in the S . typhimurium mitogen-stimulated splenocytes of the cis-nonachlor-treated females; reduced resistance to L . monocytogenes in the cis-nonachlor (day 3, P=0.034)- and trans-nonachlor (day 2, P=0.0001)-treated females, and reduced (linear trend) NK cell activity in the cis-nonachlor-treated males . The present data indicated that the chlordane compounds tested in this study had significant effects on a number of immunologic endpoints . In comparison to technical chlordane, cis- and trans-nonachlors were more immunotoxic . Therefore, an evaluation of the risk these chlorinated compounds may pose to human health should consider the potential effects different chlordane compounds may have on the immune system. Nucl Med Biol, 2002 Nov, 29(8), 849 - 53 Fluoro-A-85380 demonstrated no mutagenic properties in in vivo rat micronucleus and Ames tests; Valette H et al.; The potential mutagenic properties (micronucleus and the Ames tests) of fluoro-A-85380 (2-fluoro-3-{2(S)-2-azetidinylmethoxy}pyridine) were evaluated as a mandatory pre-clinical step . No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was found in animals treated at any dose tested . No biologically significant increase in the mean number of revertants was noted in all the Salmonella typhimurium strains tested with fluoro-A-85380 . Therefore, fluoro-A-85380 demonstrated no mutagenic properties using these two tests. Mol Microbiol, 2002 Dec, 46(5), 1451 - 64 Intestinal short-chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA; Lawhon SD et al.; Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI-1) . BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI-1 invasion genes . We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia . This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA . We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant . Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl-phosphate . These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl-phosphate in the bacterial cytoplasm, a pathway that bypasses barA . We also found that a Delta(ackA-pta) mutation alone had no effect on virulence but, in combination with Delta(barA), it increased the oral LD50 24-fold . Thus, the combined loss of the BarA- and acetate-dependent pathways is required to reduce virulence . Two other short-chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild-type strain . Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild-type strain . These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella, whereas those of the large intestine inhibit invasion. Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 481 - 6 Defined competitive exclusion cultures in the prevention of enteropathogen colonisation in poultry and swine; Nisbet D; A competitive exclusion culture (CE) containing a mixture of 29 different bacterial isolates obtained from the cecae of broiler chickens was developed utilizing continuous-flow culture techniques . This culture (CF3) has been efficacious in controlling gut colonization by enteropathogens in both experimentally infected broilers and under commercial field conditions . In day-old broiler chicks provided CF3, and challenged with 10,000 CFU Salmonella typhimurium greater than a 99% reduction in Salmonella cecal colonization levels was observed compared to control chicks . Similarly, CF3 has also been shown to protect experimentally infected broiler chicks from cecal colonization by S . enteritidis (Phage types 4 and 13), S . gallinarum, Listeria Monocytogenes, and Escherichia coli O157:H7 . A commercial product was developed from CF3 and is sold under the tradename PREEMPT . In a Food and Drug Administration approved, double blinded, pivotal field trial, chicks treated with PREEMPTT' had significantly fewer salmonellae than untreated chicks at end-of-growout . This product is the first of its kind available to the U.S . poultry industry . Using similar technology a product has also been developed that decreases shedding of salmonellae in neonate and weaned pigs, and also has been shown to reduce mortality associated with enteropathogens in young pigs both in the laboratory and in a commercial swine herd. Biomed Mater Eng, 2002, 12(3), 225 - 37 A new porous titanium-nickel alloy: Part 1 . Cytotoxicity and genotoxicity evaluation; Assad M et al.; Porous titanium-nickel (PTN) alloys represent new biomaterials for long-term implantation . Their porosity properties might confer them the capacity to trigger fluid capillarity, tissue ingrowth, as well as good tissue-implant apposition and fixation . Before PTN materials are used as long-term implants, their biocompatibility level must be assessed . In this study, porous titanium-nickel was therefore extracted in a saline semi-physiological solution and materials were evaluated for potential cytotoxicity and genotoxicity reactions . The cytocompatibility elution test was performed in order to determine PTN toxic potential at the in vitro cellular level: no reactivity was detected in cell layers exposed to PTN extracts or the negative controls . In parallel, the genocompatibility of porous titanium-nickel was evaluated using three different assays in order to assess potential damage at the DNA level: the test for chemical induction of chromosome aberrations, the Salmonella typhimurium and Escherichia coli reverse mutation assay, and the mouse micronucleus test . No significant increase in the number of chromosomal aberrations, bacterian revertant colonies, or micronuclei was observed in presence of PTN extracts when compared to negative control exposition . Based on the above results, porous titanium-nickel can be considered completely cytocompatible and genocompatible, and therefore represents a good candidate for long-term implantation. Biosens Bioelectron, 2003 Jan, 18(1), 91 - 9 Impedance characterization of a piezoelectric immunosensor part II: Salmonella typhimurium detection using magnetic enhancement; Kim GH et al.; This study investigated the usefulness and characteristics of a 5-MHz quartz crystal resonator as a sensor of biological pathogens such as Salmonella typhimurium . An impedance analyzer measured the impedance behavior of the oscillating quartz crystal exposed to various concentrations of Salmonella (10(2)-10(8) cells per ml) . The Salmonella cells were captured by antibody-coated paramagnetic microspheres, and then these complexes were moved magnetically to the sensing quartz and were captured by antibodies immobilized on the crystal surface . The response of the crystal was expressed in terms of equivalent circuit parameters . The motional inductance and the motional resistance increased as a function of the concentration of Salmonella . The viscous damping was the main contributor to the resistance and the inductance in a liquid environment . The load resistance was the most effective and sensitive circuit parameter . A magnetic force was a useful method to collect the complexes of Salmonella-microspheres on the crystal surface and enhance the response of the sensor . In this system, the detection limit, based on resistance monitoring, was about 10(3) cells per ml. J Vet Sci, 2001 Dec, 2(3), 181 - 8 Multidrug-resistant Salmonella typhimurium and Salmonella enteritidis identified by multiplex PCR from animals; Yang SJ et al.; Antibiotic resistance in Salmonella enteritidis and S . typhimurium, one of most frequent etiologic pathogens of food-borne bacterial gastroenteritidis in humans, is a serious health problem worldwide . Fifteen and 22 each of S . enteritidis and S . typhimurium were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns and phage types . S . enteritides isolates were highly resistant to sulfonamides (86.7%) and four of them (26.6%) showed multiple antibiotic resistance . The most frequent phage type (PT) of S . enteritids was PT1 (33.3%) even though none of them had multiple antibiotic resistance . S . typhimurium isolates were highly resistant to streptomycin, sulfonamides, and tetracycline, 100%, 95.5%, and 86.4% respectively . The incidence of multiple antibiotic resistance of S . typhimurium isolates was extremely high (100%) comparing to S . enteritidis isolates (26.7%) . Two of the five ACSSuT type S . typhimurium isolates, resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline, were phage type DT104 . All S . typhimurium isolates were sensitive to florfenicol . For the rapid detection of multiple antibiotic resistant S . enteritidis and S . typhimurium isolates, particularly ACSSuT type S . typhimurium DT104, antibiotic resistance genes, cmlA/tetR, PSE-1, and TEM, and Salmonella spp . Specific gene, SipB/C, were amplified using four pairs of primers in hot-started multiplex polymerase chain reaction . Two Korean isolates of S . typhimurium DT104 showed TEM amplicons instead of PSE-1 for the ampicillin resistance . The multiplex PCR used in this study was useful in rapid detection of ACSSuT type S . typhimurium and identification of b-lactamase gene distribution among Salmonella isolates. Proc Natl Acad Sci U S A, 2002 Nov 26, 99(24), 15705 - 10 Epub 2002 Nov 19. Host-pathogen interactions: Host resistance factor Nramp1 up-regulates the expression of Salmonella pathogenicity island-2 virulence genes; Zaharik ML et al.; Nramp1 (Natural resistance-associated macrophage protein-1; also known as Slc11a1) is a host resistance gene that provides protection against several intracellular pathogens, including Salmonella enterica serovar Typhimurium . Little is known about the dynamic interplay that occurs between mammalian host resistance determinants such as Nramp1 and pathogens during infection . To explore these interactions, we examined the effect of Nramp1 on expression of Salmonella typhimurium (STM) virulence factors . We demonstrate that Salmonella pathogenicity island 2 (SPI2) is essential for replication of STM in spleens of infected Nramp1(+/+) mice . Furthermore, the presence of Nramp1 in transfected cell lines and congenic knockout mice resulted in the up-regulation of STM SPI2-associated virulence genes critical for intramacrophage survival . This Nramp1-dependent up-regulation of SPI2 was mimicked in vitro by chelation of iron, demonstrating the iron-responsive nature of expression of STM SPI2-associated virulence genes . We propose that acquisition of SPI2 by S . enterica not only enabled this bacterium to become an effective intracellular pathogen but also allowed the bacterium to withstand the effects of macrophage defense mechanisms such as Nramp1 early in the evolution of its pathogenic character . These dynamic Nramp1-pathogen interactions may be essential for regulating the course of an infection . This study demonstrates the presence of a previously undescribed direct influence of a mammalian innate host resistance locus on a pathogen at the genetic level. Mutat Res, 2002 Nov 26, 521(1-2), 19 - 27 Mutagenic activity of structurally related oxiranes and siloranes in Salmonella typhimurium; Schweikl H et al.; Ring-opening molecules like oxiranes (epoxides) maybe suitable for the development of non-shrinking dental composite materials . Since oxiranes are reactive molecules, they can cause adverse biological effects in living organisms . The introduction of siloranes, a merger of silane and oxirane, may solve this problem . Here, new oxiranes and siloranes were analyzed for the induction of mutations in Salmonella typhimurium (TA97a, TA98, TA100, and TA102), and a reactive oxirane molecule served as a reference . This chemical, epoxy cyclohexyl methyl-epoxy cyclohexane carboxylate (Est-Ep) tested positive in S . typhimurium TA100 . The numbers of mutants were about 3-10-fold higher than controls in the presence of a metabolically active S9 fraction isolated from rat liver . Only a weak mutagenic effect was observed after direct testing (without S9) . Di(cyclohexene-epoxidemethyl)ether (Eth-Ep) also caused a slight increase of mutant numbers in TA100 both in the presence and absence of S9 . In contrast, no effects were detected with the large oxirane molecules, 2,2-bis(4,1-phenylenoxy-3,1-propanediyl-3-oxatricyclo {3.2.1.0(2,4)}octylcarboxy) propylidene (Nor-BP-Ep) and 2,2-bis(4,1-phenylenoxy-3,1-propanediyl-3,4-epoxycyclo-hexylcarboxylic-acid) propylidene (Est-BP-Ep) . As to the siloranes, 1,4-bis(2,3-epoxypropyloxypropyl-dimethylsilyl)-benzene (Phen-Glyc) was a direct mutagen in S . typhimurium TA100 and TA102 . This weak but dose-related increase of revertants was even enhanced by S9 . Other siloranes, like di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil), methyl-bis{2-(7-oxabicyclo{4.1.0}hept-3-yl)phenyl silane (Ph-Sil), and 1,3,5,7-tetrakis(ethyl cyclohexane epoxy)-1,3,5,7-tetramethyl-cyclotetrasiloxane (TET-Sil) tested negative in all S . typhimurium strains . All compounds will be further analyzed for the formation of chromosomal aberrations in mammalian cell cultures. Biomaterials, 2003 Feb, 24(4), 611 - 7 Effect of sample preparation on the in vitro genotoxicity of a light curable glass ionomer cement; Muller BP et al.; The glass ionomer cement Vitrebond showed a clear genotoxic effect in the in vitro Mammalian Cell Gene Mutation Test (HPRT Test) with CHO cells as well as in the bacterial umu-test with Salmonella typhimurium TA1535/pSK1002 . Both DMSO and Ham's F12 cell culture medium extracts according to ISO 10993-12 (Biological evaluation of medical devices-Part 12: sample preparation and reference materials, Geneva, Switzerland) exhibit a clear genotoxic effect in the umu-test . The effect is independent of the extraction volume in a range from 0.5 to 4 ml Ham's F12 cell culture medium . Subsequent extractions of Vitrebond showed no significant difference in the genotoxic response although weight loss and content of 2-hydroxyethyl-methacrylate dropped significantly . In vivo conditions of Vitrebond were simulated by extractions with artificial and collected human saliva . These extracts showed a clear genotoxic effect in the umu-test, even if only a few seconds of extraction time were applied . In conclusion, sample preparations for genotoxicity testing according to ISO 10993-12 reflect the in vivo conditions of Vitrebond applications . This seems to be mostly due to the hydrophilic nature of the genotoxic ingredients. Structure (Camb), 2002 Nov, 10(11), 1569 - 80 Structural studies of Salmonella typhimurium ArnB (PmrH) aminotransferase: a 4-amino-4-deoxy-L-arabinose lipopolysaccharide-modifying enzyme; Noland BW et al.; Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as Salmonella, resistance to cationic antimicrobial peptides, including those derived from the innate immune system . ArnB catalysis of amino group transfer from glutamic acid to the 4"-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway . Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses . High-resolution crystal structures were solved for two native forms and one covalently inhibited form of S . typhimurium ArnB . These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl cis peptide bond in the active site. Avian Pathol, 2002 Feb, 31(1), 41 - 7 Quantitative comparison of intestinal invasion of zoonotic serotypes of Salmonella enterica in poultry; Aabo S et al.; The aim of the present study was to compare the invasion of selected zoonotic Salmonella serotypes of poultry in an in vivo chicken intestinal loop model and also in vitro in epithelial cell cultures . Invasion was measured relative to a reference strain, Salmonella Typhimurium 4/74 invH201::TnphoA . Two serotypes demonstrated intracellular log(10) counts that differed significantly from all other serotypes tested: Salmonella Enteritidis PT4 being 1.5 log(10) colony forming units (CFU) (31-fold) higher, and Salmonella Tennessee being 0.7 log(10) CFU (fivefold) lower than the reference strain (P < or = 0.0001) . A group of serotypes, which can be vertically transmitted, showed significantly higher intracellular counts (fourfold to eightfold) than the reference strain . The group included S . Typhimurium 4/74, S . Typhimurium DT104 (poultry and porcine isolates), S . Enteritidis PT1, S . Enteritidis PT6, S . Enteritidis PT8, and Salmonella Berta . The serotypes Salmonella Hadar, Salmonella Virchow, S . 4,12:b:-, S . Typhimurium DT41, and Salmonella Infantis, most of which are considered horizontally transmitted, did not show significantly different intracellular counts from the reference strain . Results from the cell culture invasion studies agreed with the in vivo data, with the exception of S . Berta and the poultry isolate of S . Typhimurium DT104. J Chemother, 2002 Aug, 14(4), 346 - 50 Antimicrobial-resistant Salmonella enterica serovars isolated from chickens in Spain; Hernandez T et al.; In order to analyze the antibiotic resistance of Salmonella enterica serovars, a total of 112 Salmonella strains were tested (54 S . enteritidis, 32 S . typhimurium, 11 S . heidelberg, 7 S . infantis, 4 S . virchow and 4 S . hadar) . The bacteria were isolated from 691 samples of frozen and fresh chicken meat . Identification of microorganisms and antimicrobial sensitivity testing were undertaken by means of the automated MicroScan AutoScan 4 method (Baxter in Spain) . 45.5% of 112 strains tested were susceptible to all antibiotics . The highest percentage of resistance was found to: chloramphenicol (44.6%), ampicillin (34.8%) and tetracycline (33.9%) . Multiple resistance was observed in 49 strains (43.7%), whereas single resistance was seen in 12 isolates (10.7%) . We found 12 different patterns of resistance in Salmonella enterica serovar enteriditis . Resistance to chloramphenicol was the most common single resistance . The most frequent patterns of multiresistant strains were ampicillin + amoxicillin/clavulanate + cefazolin + imipenem and chloramphenicol + impipenem . In this serotype, 49 isolates belonged to phagetype 4 . Salmonella typhimurium showed the highest percentages of resistance to the tested drugs, with six different resistance patterns found . 25 strains out of 32 S . typhimurium isolates belonged to phagotype 120 and 13 of these showed the same resistance pattern: chloramphenicol + tetracycline + ampicillin . The high incidence of antibiotic resistant salmonellae found in chickens in our study suggests the need for public health interventions to decrease selective pressure on bacterial strains by antimicrobial agents. Carcinogenesis, 2002 Nov, 23(11), 1937 - 45 Metabolic activation of the environmental contaminant 3-nitrobenzanthrone by human acetyltransferases and sulfotransferase; Arlt VM et al.; 3-Nitrobenzanthrone (3-NBA) an extremely potent mutagen and suspected human carcinogen identified in diesel exhaust and in airborne particulate matter was shown to form multiple DNA adducts in vitro and in vivo in rats . In order to investigate whether human N,O-acetyltransferases (NATs) and sulfotransferases (SULTs) contribute to the metabolic activation of 3-NBA we used a panel of newly constructed Chinese hamster lung fibroblast V79MZ derived cell lines expressing human NAT1, human NAT2 or human SULT1A1, as well as TA1538-derived Salmonella typhimurium strains expressing human NAT1 (DJ400) or human NAT2 (DJ460) and determined DNA binding and mutagenicity . The formation of 3-NBA-derived DNA adducts was analysed by (32)P-postlabelling after exposing V79 cells to 0.01 micro M 3-NBA or 0.1 micro M N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA), a potential metabolite of 3-NBA . Similarly up to four major and two minor adducts were detectable for both compounds, the major ones being identical to those detected previously in DNA from rats treated with 3-NBA . Comparison of DNA binding between different V79MZ derived cells revealed that human NAT2 and, to a lesser extent, human NAT1 and human SULT1A1, contribute to the genotoxic potential of 3-NBA and N-Ac-N-OH-ABA to form DNA adducts . However, the extent of DNA binding by 3-NBA was higher in almost all V79 cells at a 10-fold lower concentration than by N-Ac-N-OH-ABA, suggesting that N-Ac-N-OH-ABA is not a major intermediate in the formation of 3-NBA-derived adducts . 3-NBA showed a 3.8-fold and 16.8-fold higher mutagenic activity in Salmonella strains expressing human NAT1 and human NAT2, respectively, than in the acetyltransferase-deficient strain, whereas N-Ac-N-OH-ABA was only clearly (but weakly) mutagenic in Salmonella DJ460 expressing human NAT2 . This finding suggests that N-Ac-N-OH-ABA is not a major reactive metabolite responsible for the high mutagenic potency of 3-NBA in Salmonella . Collectively our results indicate that O-acetylation and O-sulfonation by human NATs and SULTs may contribute significantly to the high mutagenic and genotoxic potential of 3-NBA . Moreover, the yet-unidentified four major 3-NBA-derived adducts may be DNA adducts without an N-acetyl group. J Biotechnol, 2003 Jan 9, 100(1), 1 - 12 Antimicrobial properties of the Escherichia coli R1 plasmid host killing peptide; Pecota DC et al.; The 52 amino acid host killing peptide (Hok) from the hok/sok post-segregational killer system of the Escherichia coli plasmid R1 was synthesized using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, and its molecular weight was confirmed by mass spectroscopy . Hok kills cells by depolarizing the cytoplasmic membrane when it is made in the cytosol . Six microorganisms, E . coli, Bacillus subtilis, Pseudomonas aeruginosa, P . putida, Salmonella typhimurium, and Staphylococcus aureus were exposed to the purified peptide but showed no significant killing . However, electroporation of Hok (200 microgml(-1)) into E . coli cells showed a dramatic reduction (100000-fold) in the number of cells transformed with plasmid DNA which indicates that the synthetic Hok peptide killed cells . Electroporation of Hok into P . putida was also very effective with a 500-fold reduction in electrocompetent cells (100 microgml(-1)) . Heat shock in the presence of Hok (380 microgml(-1)) resulted in a 5-fold reduction in E . coli cells but had no effect on B . subtilis . In addition, three Hok fragments (Hok(1-28), Hok(31-52) and Hok(16-52)) killed cells when electroporated into E . coli at 200 microgml(-1) (over 1000-fold killing for Hok(1-28), 50-fold killing for Hok(16-52) and over 1000-fold killing for Hok(31-52)) . E . coli cells electroporated with Hok and visualized using transmission electron microscopy showed the same morphological changes as control cells to which Hok was induced using a plasmid inside the cell. Poult Sci, 2002 Oct, 81(10), 1598 - 605 Comparison of electrolyzed oxidizing water with various antimicrobial interventions to reduce Salmonella species on poultry; Fabrizio KA et al.; Foodborne pathogens in cell suspensions or attached to surfaces can be reduced by electrolyzed oxidizing (EO) water; however, the use of EO water against pathogens associated with poultry has not been explored . In this study, acidic EO water {EO-A; pH 2.6, chlorine (CL) 20 to 50 ppm, and oxidation-reduction potential (ORP) of 1,150 mV}, basic EO water (EO-B; pH 11.6, ORP of -795 mV), CL, ozonated water (OZ), acetic acid (AA), or trisodium phosphate (TSP) was applied to broiler carcasses inoculated with Salmonella Typhimurium (ST) and submerged (4 C, 45 min), spray-washed (85 psi, 25 C, 15 s), or subjected to multiple interventions (EO-B spray, immersed in EO-A; AA or TSP spray, immersed in CL) . Remaining bacterial populations were determined and compared at Day 0 and 7 of aerobic, refrigerated storage . At Day 0, submersion in TSP and AA reduced ST 1.41 log10, whereas EO-A water reduced ST approximately 0.86 log10 . After 7 d of storage, EO-A water, OZ, TSP, and AA reduced ST, with detection only after selective enrichment . Spray-washing treatments with any of the compounds did not reduce ST at Day 0 . After 7 d of storage, TSP, AA, and EO-A water reduced ST 2.17, 2.31, and 1.06 log10, respectively . ST was reduced 2.11 log10 immediately following the multiple interventions, 3.81 log10 after 7 d of storage . Although effective against ST, TSP and AA are costly and adversely affect the environment . This study demonstrates that EO water can reduce ST on poultry surfaces following extended refrigerated storage. Mol Microbiol, 2002 Oct, 46(2), 355 - 66 Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium; Maisnier-Patin S et al.; Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria . This biological cost can often be reduced by compensatory mutations . In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin . The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased . Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs . From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation . The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness . Only four of the evolved lineages contained streptomycin-sensitive revertants . The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations . Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively . Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations. Epidemiol Infect, 2002 Oct, 129(2), 287 - 93 The use of sequential studies in a salmonellosis outbreak linked to continental custard cakes; Ward B et al.; We investigated an outbreak of 54 cases of Salmonella Typhimurium phage type 9 (STM9) with a specific antibiotic resistance pattern . We used sequential analytic studies: two retrospective cohort studies, a case-control study, and a modified case-control study . An outbreak of salmonellosis due to Salmonella Typhimurium PT9 SSu (resistant to streptomycin and sulphafurazole) was identified . Fifty-four cases had illness onset from November 1998 to March 1999 . Notifications commenced following a restaurant birthday party in December 1998 . An initial cohort and case control study found no association with consumption of custard cake . However, case follow-up identified another cohort of people who had attended a birthday party in February at which 8/27 people who consumed a continental custard cake were ill compared to 0/10 who did not (P = 0.07) . A revised case control study found illness was strongly associated with consumption of a particular continental custard cake (Mantel-Haenszel matched OR infinity, P = 0.00004) . This report highlights the epidemiological value of using sequential study types, and persisting with the investigation of apparently sporadic food-borne outbreaks. Curr Microbiol, 2002 Dec, 45(6), 456 - 8 Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph; Xu K et al.; To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene . A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain . The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22 . Faster-growing colonies indicated complementation of the hemE mutation . Each clone was tested by backcross transduction of the mutant . Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella . HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone . This clone showed an active hemE gene within a 4-kb insert. Blood, 2003 Jan 15, 101(2), 649 - 54 Epub 2002 Sep 12. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma; Pertl U et al.; The successful induction of a T-cell-mediated tumor-protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy . We achieved this by immunization with a tyrosine hydroxylase (mTH)-based DNA vaccine, enhanced with the posttranscriptional regulatory acting RNA element (WPRE), derived from woodchuck hepatitis virus in combination with an antibody-cytokine fusion protein (ch14.18-IL-2) that targets interleukin-2 (IL-2) to the tumor microenvironment . This DNA vaccine mTH-WPRE was carried by attenuated Salmonella typhimurium and applied by oral gavage in a mouse model of neuroblastoma . Mice immunized with the mTH-WPRE vaccine, and which additionally received a boost with suboptimal doses of ch14.18-IL-2, were completely protected against hepatic neuroblastoma metastases . In contrast, all controls presented with disseminated metastases . Both T-cell and natural killer (NK) cell-dependent mechanisms were involved in the induction of a systemic tumor-protective immunity . Thus, up-regulation of interferon-gamma (IFN-gamma) expression in CD8(+) T cells occurred only in those animals that received the mTH-WPRE vaccine plus the ch14.18-IL-2 boost . Up-regulation of this proinflammatory cytokine was not observed in mice immunized with mTH-WPRE vaccine alone . A role for NK cells was indicated by the complete abrogation of systemic tumor-protective immunity in all animals that were depleted of NK cells in vivo . Taken together, these data demonstrate that immunization with a posttranscriptionally enhanced DNA vaccine encoding the WPRE sequence, combined with a boost of the ch14.18-IL-2 fusion protein, completely protects against hepatic metastases in a murine model of neuroblastoma and therefore may lead to a new strategy for immunotherapy and prevention of metastatic neuroblastoma. Chem Res Toxicol, 2002 Oct, 15(10), 1288 - 94 Structure of DNA adduct formed with aminophenylnorharman, being responsible for the comutagenic action of norharman with aniline; Totsuka Y et al.; A mutagenic heterocyclic amine (HCA), 9-(4'-aminophenyl)-9H-pyrido{3,4-b}indole (aminophenylnorharman, APNH), is produced in the presence of S9 mix by the reaction of norharman and aniline, both of which are nonmutagenic and abundantly present in our environment . It has been previously reported that APNH-DNA adducts were detected in DNA of Salmonella typhimurium strain incubated with APNH and S9 mix . In the present study, we examined the structures of APNH-DNA adducts using the (32)P-postlabeling method and various spectrometry techniques . When the reaction mixture of N-acetoxy-APNH and 2'-deoxyguanosine 3'-monophosphate (3'-dGp) was analyzed, three adduct spots (two major and one minor) were observed by (32)P-postlabeling under modified-standard conditions . No adduct formation was observed for reaction mixtures of N-acetoxy-APNH with 3'-dAp, 3'-dTp, or 3'-dCp . The two major adduct spots (spots 1 and 2) detected by TLC were extracted and subjected to HPLC along with the standards 3',5'-pdGp-C8-APNH and 5'-pdG-C8-APNH, which were independently chemically synthesized . On the basis of the results of co-chromatography, spots 1 and 2 were identified to be 5'-monophosphate and 3',5'-diphosphate forms of dG-C8-APNH . When the extract of spot 2 (3',5'-pdGp-C8-APNH) was further digested with nuclease P1 and phosphodiesterase I, a spot corresponding to spot 1 (5'-pdG-C8-APNH) was newly observed on TLC . From these observations, both of the two major spots were concluded to be dG-C8-APNH . A similar DNA adduct pattern to that apparent in vitro was observed in various organs of F344 rats fed 40 ppm of APNH for 4 weeks . The levels of APNH-DNA adducts were highest in the liver and colon, with RAL values of 1.31 +/- 0.26 and 1.32 +/- 0.11 adducts/10(7)nucleotides, respectively . Thus, APNH was demonstrated to form DNA adducts primarily at the C-8 position of guanine residues in vitro and in vivo, like other mutagenic and carcinogenic HCAs. Food Chem Toxicol, 2002 Oct, 40(10), 1475 - 82 Antimutagenic activities of acetone and methanol fractions of Terminalia arjuna; Kaur K et al.; The antimutagenic effect of benzene, chloroform, acetone and methanol fractions from Terminalia arjuna, a well-known medicinal plant, was determined against Acid Black dye, 2-aminofluorene (2AF) and 4-nitro-o-phenylenediamine (NPD) in TA98 Frameshift mutagen tester strain of Salmonella typhimurium . Among the different fractions, the antimutagenic effect of acetone and methanol fractions was more than that observed with other fractions . Co-incubation and pre-incubation modes of experimentation did not show much difference in the antimutagenic activity of the extracts . Moreover, these fractions inhibited the S9-dependent mutagens, 2AF and Acid Black dye more effectively than the direct-acting mutagens . Studies are under way to isolate and elucidate the nature of the antimutagenic factor in acetone and methanol fractions. Nippon Eiseigaku Zasshi, 2002 Sep, 57(3), 547 - 55 {Low dose exposure to cadmium and its health effects (1) . Genotoxicity and carcinogenicity}; Koyama H et al.; We reviewed studies on genotoxicity and carcinogenicity of cadmium (Cd) . Salmonella typhimurium and Escherichia coli exposed to Cd did not show mutagenicity, whereas cultured mammalian cells exposed to Cd showed mutation, DNA strand breaks, and chromosomal aberrations . Carcinogenicity tests showed that exposure to Cd increased the occurrence of tumors in testis, lung, prostate, hematopoietic tissues, and injection sites . On the other hand, recent epidemiologic studies are not supportive of earlier observations on the association between Cd and prostate cancer . The US NIOSH data on a possible association between Cd and lung cancer may need reevaluation . No studies which show a positive relationship between oral Cd exposure and carcinogenesis have been reported . All available data suggest that Cd should be reassigned to IARC Group 2A (probably carcinogenic to humans) from the current Group 1. J Surg Res, 2002 Sep, 107(1), 101 - 7 Liver and circulating NK1.1(+)CD3(-) cells are increased in infection with attenuated Salmonella typhimurium and are associated with reduced tumor in murine liver cancer; Feltis BA et al.; An attenuated (DeltacyA, Deltacrp) strain of Salmonella typhimurium (chi4550) containing a gene for human IL-2 (chi4550pIL2) reduces hepatic tumor burden when orally inoculated into mice with liver cancer; however, wild-type S . typhimurium is also associated with cancer regression . Therefore, experiments were designed to clarify the invasiveness and the anti-tumor properties of three strains of S . typhimurium . S . typhimurium chi4550pIL2, chi4550, or wild type (WT) was incubated with mature Caco-2 and HT-29 enterocytes, and S . typhimurium internalization was assessed . For infectivity experiments, mice were orally inoculated with saline or 10(9)S . typhimurium chi4550pIL2, chi4550, or WT; 48 h later mice were sacrificed for analysis of cecal bacteria and S . typhimurium translocation to mesenteric lymph nodes . For experiments involving tumor implantation, four groups were studied: saline control, tumor alone, chi4550pIL2+tumor, and chi4550+tumor . Mice were orally inoculated with saline or S . typhimurium and underwent laparotomy 24 h later with 5 x 10(4) MCA38 murine adenocarcinoma cells injected into the spleen . On day 14, liver tumors were counted and peripheral blood and hepatic lymphocyte populations were analyzed by FACScan . Attenuated S . typhimurium exhibited decreased internalization by cultured enterocytes and decreased infectivity after oral inoculation . Mice treated with chi4550pIL2 or chi4550 had fewer liver tumors and increased populations of hepatic and circulating NK1.1(+)CD3(-) lymphocytes compared to mice treated with saline (P < 0.01) . These data suggest that attenuated S . typhimurium may have an application as an anti-tumor agent. Regul Toxicol Pharmacol, 2002 Aug, 36(1), 69 - 79 The toxicity of behenyl alcohol . I . Genotoxicity and subchronic toxicity in rats and dogs; Iglesias G et al.; The genotoxic potential of behenyl alcohol, a saturated long-chain (C22:0) fatty alcohol, was examined in the Ames Salmonella typhimurium reverse mutation assay, the gene mutation, and chromosome aberrations assays in Chinese hamster V79 cells, and the micronucleus assay in NMRI mice . Behenyl alcohol did not increase the number of revertants per plate compared to controls in the S . typhimurium assay, with or without metabolic activation . No significant increases in the number of mutant colonies or in structural chromosome aberrations were observed in Chinese hamster V79 cells . In addition, behenyl alcohol did not increase the frequency of bone marrow polychromatic erythrocyte (PCE) micronuclei in mice in vivo . In two subchronic toxicity studies, CD rats and beagle dogs were administered behenyl alcohol by oral gavage for at least 26 weeks at doses of 0, 10, 100, or 1000 mg behenyl alcohol/kg body weight/day for rats and 0, 20, 200, or 2000 mg behenyl alcohol/kg body weight/day for dogs . Adverse effects were not observed following gross and histopathological evaluations of dosed rats . Compound-related effects in dogs were limited to observations of pale feces, indicative of unabsorbed behenyl alcohol, at doses of 2000 mg/kg body weight/day . There were no histopathological changes observed in dogs dosed with behenyl alcohol . The no-observed-adverse-effect-level (NOAEL) for behenyl alcohol was 1000 mg/kg body weight/day for rats, and 2000 mg/kg body weight/day for dogs, the highest doses used in these studies. J Biol Chem, 2002 Dec 27, 277(52), 51025 - 32 Epub 2002 Oct 14. Cdc42 and Rac1 regulate late events in Salmonella typhimurium-induced interleukin-8 secretion from polarized epithelial cells; Hobert ME et al.; Salmonella typhimurium colonization of the intestinal epithelium initiates biochemical cross-talk between pathogen and host that results in the secretion of chemokines, such as interleukin (IL)-8, that direct neutrophil migration to the site of infection . In nonpolarized cells, Rac1 and Cdc42 have been shown to regulate both bacterial invasion and signaling events leading to nuclear responses and IL-8 secretion . However, because the underlying actin cytoskeleton and the associated signaling machinery are distributed much differently in polarized epithelial cells, we used polarized Madin-Darby canine kidney monolayers to investigate the role of Rac1 and Cdc42 in S . typhimurium-induced pro-inflammatory responses in the more physiologically relevant polarized state . In Madin-Darby canine kidney monolayers expressing dominant-negative Rac1 or Cdc42, both Salmonella- and tumor necrosis factor alpha-induced activation of NFkappaB and mitogen-activated protein kinase signaling cascades proceeded normally, but IL-8 secretion was inhibited . We found that Rac1 and Cdc42 were not involved in early pro-inflammatory signaling events, as in nonpolarized cells, but rather regulated the basolateral exocytosis and secretion of IL-8 . In contrast, dominant-negative Rac1 inhibited apical actin pedestal formation, indicating that pedestal formation and nuclear signaling for pro-inflammatory activation are not linked . These findings indicate that there are significant differences in the requirements of pathogen-induced host cell signaling pathways in polarized and nonpolarized cells. Int J Food Microbiol, 2003 Jan 25, 80(2), 153 - 9 Salmonella DNA adenine methylase mutants prevent colonization of newly hatched chickens by homologous and heterologous serovars; Dueger EL et al.; Salmonella mutants lacking DNA adenine methylase (Dam) are highly attenuated for virulence and confer protection against oral challenge with homologous and heterologous Salmonella serovars in mice and chicken broilers . To determine whether vaccines based on Dam are efficacious in preventing early colonization of newly hatched chickens, a Salmonella typhimurium Dam(-) vaccine was evaluated for the protection of chicks against oral challenge with homologous and heterologous Salmonella serovars . Vaccination of chicks elicited protection 2 and 6 days post-challenge as evidenced by a significant reduction in colonization of the gastrointestinal tract (ileum, cecum and feces) and visceral organs (spleen and bursa) when challenged with homologous S . typhimurium . Moderate protection was observed following challenge with heterologous S . enteritidis and Salmonella O6, 14, 24:e, h-monophasic) serovars . These data suggest that Salmonella Dam mutant strains conferred cross-protection, presumably via competitive exclusion mechanisms that prevent superinfection of chicks by other Salmonella strains . Such protection may reduce pre-harvest Salmonella contamination in poultry, decreasing the potential for food-borne transmission of this pathogen to humans. Mutat Res, 2002 Oct 31, 508(1-2), 147 - 56 Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds, purpurin and alizarin; Takahashi E et al.; Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test . To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics . The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid . Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5 . Carminic acid did not affect the activities of any CYPs tested . CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study . From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive . The values of slopes obtained from Lineweaver-Burk plots are proportional to the square of purpurin concentration . This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1 . The K(m) value of CYP1B1 was 11 microM, and the K(i) value of purpurin and alizarin against CYP1B1 was 0.7 microM(2) and 0.5 microM, respectively . We also examined the effects of these pigments on the mutagenicities of MeIQx and B{a}P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR) . The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid . Purpurin also reduced the mutagenicity of B{a}P in TA1538 1A1/OR or 1B1/OR . These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by inhibition of CYP activities responsible for activating the mutagens. J Leukoc Biol, 2002 Oct, 72(4), 743 - 51 Salmonella virulence factor SipB induces activation and release of IL-18 in human dendritic cells; Dreher D et al.; Interleukin-18 (IL-18) plays an important role in innate and acquired immunity, in particular against intracellular pathogens . However, little is known about the microbial factors that trigger IL-18 secretion by dendritic cells (DCs) . To determine the influence of bacterial virulence factors on the activation and release of IL-18, we infected human monocyte-derived DCs with virulence mutants of the facultative intracellular pathogen Salmonella typhimurium . Our results show that infection by S . typhimurium causes caspase-1-dependent activation of IL-18 and triggers the release of IL-18 in human DCs . The secretion of IL-18 by the DCs was closely correlated with the ability of the S . typhimurium strains to induce apoptosis . We demonstrate that activation and release of IL-18 are blocked by mutations in the Salmonella sipB gene, which encodes a virulence factor that activates caspase-1 to induce apoptosis . These findings indicate that the activation and release of IL-18 induced by bacterial virulence factors may represent one component of innate immunity against the intracellular bacteria. J Rheumatol, 2002 Oct, 29(10), 2154 - 8 Reactive arthritis and other sequelae following sporadic Salmonella typhimurium infection in British Columbia, Canada: a case control study; Buxton JA et al.; OBJECTIVE: To describe sequelae occurring in the 3 months after sporadic Salmonella typhimurium (ST) infection in British Columbia (BC), Canada . METHODS: We compared the incidence of sequelae to similar symptoms in controls; identified risk factors for developing sequelae; identified the incidence of reactive arthritis (ReA) as diagnosed by a rheumatologist, and assessed primary care physician diagnosis of ReA . A questionnaire was administered by telephone to cases of ST occurring in BC between December 1, 1999, and November 30, 2000; and to controls obtained from the BC provincial client registry . Cases reporting symptoms were followed up by a rheumatologist . RESULTS: Thirty-five of 66 (53%) cases reported any symptom, 17 (26%) reported joint symptoms . The Mantel-Haenszel odds ratio (weighted by sex and pediatric/adult) of a salmonella case reporting "any symptom" compared to controls was 5.42; 95% confidence interval (CI) 2.18-16.27; and reporting joint symptoms was 4.40; 95% CI: 1.25-19.53 . The sex distribution of cases reporting joint symptoms was not significantly different . No medication taken during the salmonella infection was significantly different between the cases who had joint symptoms and those who did not . Four cases (2 adults, 2 children) were considered by the rheumatologist to have symptoms consistent with ReA, 2 of these had been told by a physician that their symptoms were related to their ST infection . CONCLUSION: Cases were more than 4 times more likely to report joint symptoms than controls; and despite the loss of many cases to followup, 6% of all cases were considered to have ReA. J Environ Qual, 2002 Sep-Oct, 31(5), 1702 - 9 Salmonella typhimurium survival and viability is unaltered by suspended particles in freshwater; Maki RP et al.; Rolling microcosm experiments were conducted to determine whether suspended particles affect the survival and viability of a model pathogen, Salmonella choleraesuis, serotype typhimurium (American Type Culture Collection no . 23567), in a freshwater microbial community . Water from the Duluth, MN harbor of Lake Superior (including native microorganisms) was inoculated with clay, silt, or flocculent organic particles in a range of concentrations and a streptomycin-resistant strain of S . typhimurium . Microcosms (incubated at 20 degrees C) were rolled horizontally (3 rpm) and sampled periodically for total bacteria and total, viable, and culturable S . typhimurium . Total S . typhimurium abundance decreased rapidly in all experiments (8.5-73.1% d-1) . Total bacteria did not decrease as rapidly as the S . typhimurium population in any experiment, suggesting that a microcosm effect was not responsible for the decline in S . typhimurium populations . Loss rates of attached and free cells were similar, indicating that attachment to particles did not enhance the persistence of Salmonella cells beyond our minimum detectable differences . After eight days, only 0.1 to 11.9% of the initial S . typhimurium inocula were detected by direct counts . Suspended particles had a minimal effect on the survival and viability of S . typhimurium; the losses of total, viable, or culturable Salmonella were generally the same across particle treatments and concentrations . Silt and flocculent particles affected loss rates of total and viable S . typhimurium similarly to inorganic particles (clay) . It appears unlikely that suspended particles would provide a means for S . typhimurium to persist at hazardous levels in freshwater. J Immunol, 2002 Oct 15, 169(8), 4475 - 80 Neutrophil influx in response to a peritoneal infection with Salmonella is delayed in lipopolysaccharide-binding protein or CD14-deficient mice; Yang KK et al.; The induction of an adaptive immune response to a previously unencountered pathogen is a time-consuming process and initially the infection must be held in check by the innate immune system . In the case of an i.p . infection with Salmonella typhimurium, survival requires both CD14 and LPS-binding protein (LBP) which, together with Toll-like receptor 4 and myeloid differentiation protein 2, provide a sensitive means to detect bacterial LPS . In this study, we show that in the first hours after i.p . infection with Salmonella a local inflammatory response is evident and that concomitantly neutrophils flood into the peritoneum . This rapid neutrophil influx is dependent on TNF since it is 1) abolished in TNF KO mice and 2) can be induced by i.p . injection of TNF in uninfected animals . Neutrophil influx is not strictly dependent on the presence of either LBP or CD14 . However, in their absence, no local inflammatory response is evident, neutrophil migration is delayed, and the mice succumb to the infection . Using confocal microscopy, we show that the neutrophils which accumulate in CD14 and LBP null mice, albeit with delayed kinetics, are nevertheless fully capable of ingesting the bacteria . We suggest that the short delay in neutrophil influx gives the pathogen a decisive advantage in this infection model. J Immunol, 2002 Oct 15, 169(8), 4262 - 72 Pentoxifylline functions as an adjuvant in vivo to enhance T cell immune responses by inhibiting activation-induced death; Suresh R et al.; Modalities for inducing long-lasting immune responses are essential components of vaccine design . Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability . We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization . PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers . Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo . Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants. Br Poult Sci, 2002 Sep, 43(4), 501 - 7 Genetic resistance to Salmonella typhimurium in two lines of chickens selected as resistant and sensitive on the basis of heterophil/lymphocyte ratio; Al-Murrani WK et al.; 1 . A study was conducted to test for the validity of the heterophil/lymphocyte (H/L) ratio as a criterion for selection for resistance to Salmonella typhimurium in chickens . 2 . An infective dose of S . typhimurium was given, directly in the crop, to two groups of chicken selected as Resistant (R) and Sensitive (S) on the bases of H/L ratio . The 99% lower confidence limit was used as a borderline; individuals below the limit were considered R and those above S . Many aspects of immune response were compared, namely: H/L ratio, antibody titre, cellular immunity, phagocytic activity, cortisol concentration, bursa and body weight . 3 . The R group exceeded the S in all the studied variables of the immune response, indicating the possibility of using the H/L ratio and its confidence limit to select for general resistance . 4 . Due to the within-strain variability in resistance, it seems that immunological and genetic studies should take into consideration separation of individuals into R and S before grouping . Failing to do so might lead to erroneous conclusions as a difference may simply be due to the different numbers of R and S included in each group. Nat Cell Biol, 2002 Oct, 4(10), 766 - 73 Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella; Terebiznik MR et al.; Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling . Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium . Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions . Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB) . Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles . Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission . Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis. J Med Microbiol, 2002 Sep, 51(9), 771 - 6 Increased in-vitro and in-vivo biological activity of lipopolysaccharide extracted from clinical low virulence vacA genotype Helicobacter pylori strains; Salgado F et al.; Helicobacter pylori infection in man is associated with chronic gastritis and peptic ulcer disease . The virulence factors of the species are still under investigation . Among these, the lipopolysaccharide (LPS) is a potential pathogenic factor of the micro-organism, whose biological activity can be estimated by immunological parameters . The aim of this study was to determine the ability of pure LPS extracted from clinical isolates of H . pylori to induce mitogenicity, secretion of tumour necrosis factor-alpha (TNF-alpha), and spleen growth in a murine model . Rough and smooth LPS from Salmonella typhimurium were used as controls . The results showed that, like the control LPS, all extracts of LPS induced mitogenic activity, stimulated synthesis of TNF-alpha and induced spleen growth, although the effects produced by the majority of the H . pylori LPS samples analysed were less intensive than those produced by the S . typhimurium LPS . The immunological parameters analysed allowed the detection of two types of H . pylori LPS: one of low biological activity and one of high biological activity . The most active LPS was extracted from strains isolated from patients with increased mucous damage associated with epithelial regeneration . Surprisingly, these strains were cagA negative and belonged to a low virulence genotype according to vacA gene (slbm2 and s2m2) . The results suggest the need to re-evaluate the role of the LPSas a virulence factor for some strains of H . pylori. Eur J Immunol, 2002 Oct, 32(10), 2800 - 6 Toll-like receptor 4 is not required for the full maturation of dendritic cells or for the degradation of Gram-negative bacteria; Rescigno M et al.; Toll-like receptor 4 (TLR4) has been recently associated with cellular responses to lipopolysaccharide (LPS), and mice mutated in tlr4, such as C57BL/10ScCr or C3H/HeJ mice, become hyporesponsive to LPS . In this study, we have analyzed the capacity of bone marrow-derived dendritic cells (BMDC) from C57BL/10ScCr (ScCr-BMDC) or C3H/HeJ (HeJ-BMDC) mice to respond to LPS or to Gram-negative bacteria . We show that ScCr- or HeJ-BMDC are insensitive to LPS, but can mature in response to live and killed Gram-negative bacteria . Interestingly, only ScCr-BMDC but not HeJ-BMDC, stimulated with bacteria, have reduced capacity to produce pro- and anti-inflammatory cytokines as compared to BMDC from control mice, probably due to genetic defects unrelated to the tlr4 mutation . Nevertheless, ScCr-BMDC and ScCr BM-macrophages (BM-Mphi) phagocytose Salmonella typhimurium similarly to control cells, indicating that TLR4 is not compulsory for bacterial uptake . Moreover, BM-Mphi, but not BM-DC from B10ScCr or C3H/HeJ mice, are impaired in their capacity to kill intracellular bacteria and to produce NO as compared to wild type controls . However, the bacteria killing property of BM-Mphi is completely restored by pretreating the cells with IFN-gamma . Hence, TLR4 plays different roles in DC versus Mphi. J Infect Dis, 2002 Oct 15, 186(8), 1122 - 30 Epub 2002 Sep 20. Inhibition of Salmonella typhimurium enteropathogenicity by piperidine, a metabolite of the polyamine cadaverine; Kohler H et al.; Piperidine is a 1-ring heterocyclic compound formed from the polyamine cadaverine in the human intestine . Because heterocyclic compounds are routinely used in the promotion of antimicrobial treatment strategies, it was considered whether piperidine could be used against infection with enteric pathogens . This study demonstrates that piperidine treatment prevented the invasion of Salmonella typhimurium into model intestinal epithelium by nearly 95% . In vivo studies also revealed that it increased mouse survival and reduced S . typhimurium translocation into and colonization of various organs and tissues . Initial evaluations demonstrated that piperidine reduced the S . typhimurium-induced polymorphonuclear leukocyte transepithelial migration response in vitro by inhibiting activation of protein kinase C . Piperidine did not affect the ability of S . typhimurium to elicit interleukin-8 secretion by epithelial cells or to activate extracellular-regulated kinase signal transduction pathways . These results show that piperidine does not exhibit paninhibitory activity and suggest that piperidine may be useful in down-regulating active inflammation at mucosal surfaces. Mutat Res, 2002 Sep 30, 506-507, 41 - 8 The effect of deuterium and fluorine substitution upon the mutagenicity of N-hydroxy-2,6-dimethylaniline; Matilde Marques M et al.; 2,6-Dimethylaniline (2,6-DMA) is an intermediate in the manufacture of several products, including pesticides, dyestuffs, and synthetic resins . It is also present in nanogram amounts in tobacco smoke, and is a major metabolite of the potent anesthetic and antiarrhythmic drug lidocaine, as well as a nasal carcinogen in rats . As with other aromatic amines, 2,6-DMA can undergo metabolic activation through cytochrome p450-mediated N-hydroxylation, followed by O-esterification to a reactive derivative capable of forming DNA adducts . We have recently characterized four DNA adducts resulting from this metabolic pathway . Three of the adducts arose from reaction of the exocyclic heteroatoms of deoxyadenosine and deoxyguanosine with the carbon para to the arylamine nitrogen . The fourth adduct resulted from reaction of the 2,6-DMA nitrogen with the C8 atom of deoxyguanosine . In order to investigate the relative contribution of the exocyclic heteroatom adducts as compared to the C8-deoxyguanosine adduct to the toxicities elicited by 2,6-DMA, we synthesized and compared the mutagenicity of N-hydroxy-2,6-DMA, N-hydroxy-4-deutero-2,6-DMA, 2,6-dimethylnitrosobenzene, 4-deutero-2,6-dimethylnitrosobenzene, and N-hydroxy-4-fluoro-2,6-DMA . In Salmonella typhimurium TA100, the two deuterated compounds and their non-deuterated analogues gave similar mutagenic responses ( approximately 25 revertants/nmol) . Likewise in S . typhimurium TA98, a similar mutant frequency ( approximately 0.7 revertants/nmol) was obtained with the four compounds . With N-hydroxy-4-fluoro-2,6-DMA, the mutant frequency was reduced by approximately 90% in S . typhimurium TA100 and approximately 50% in S . typhimurium TA98 . The results suggest that multiple adducts contribute to base substitution mutations detected by S . typhimurium TA100 while the C8-deoxyguanosine adduct is primarily responsible for the frameshift mutations detected by S . typhimurium TA98. Cent Eur J Public Health, 2002 Sep, 10(3), 104 - 6 Antibacterial efficacy of disinfectants against some gramnegative bacteria; Majtan V et al.; The antibacterial effect of 11 new commercially manufactured disinfectants on clinical isolates of Salmonella typhimurium DT104, Serratia marcescens and Pseudomonas aeruginosa was studied . The substances tested represented six pure quaternary ammonium substances (QAS) and five QAS combined with other ingredients . The antibacterial efficacy was characterized by influencing the growth of bacterial cells expressed by MIC and ED50 values . The disinfectants are divided into three groups according to their efficacy . The antibacterial efficacy of disinfectants on S . typhimurium DT104 in the study is the highest in comparison with S . marcescens and P . aeruginosa strains . The highest inhibition of growth was caused by Diesen forte on S . typhimurium DT 104 and by Benzalkonium chloride on both S . marcescens and P . aeruginosa strains. Mutat Res, 2002 Sep 26, 520(1-2), 119 - 24 Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay; Catterall F et al.; The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay . CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98 . Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response . Under the same conditions DCNB failed to display mutagenic activity . The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain . It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen. Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13681 - 6 Epub 2002 Sep 23. Macrophage migration inhibitory factor (MIF) plays a pivotal role in immunity against Salmonella typhimurium; Koebernick H et al.; The cytokine macrophage migration inhibitory factor (MIF) exerts a multitude of biological functions . Notably, it induces inflammation at the interface between the immune system and the hypothalamus-pituitary-adrenal stress axis . The role of MIF in infectious diseases is not understood completely . Here, we show that MIF-deficient (MIF(-/-)) knockout mice fail to control an infection with wild-type Salmonella typhimurium . Increased susceptibility was accompanied by a reduced Th1 response, demonstrated by decreased levels of IL-12, IFNgamma, and tumor necrosis factor alpha . In Salmonella-infected MIF(-/-) mice, levels of IL-1beta were markedly increased . Additionally, infected MIF(-/-) mice showed elevated serum levels of nitric oxide and corticosterone as compared with control mice . Our results point to MIF as a key mediator in the host response to S . typhimurium . MIF not only promotes development of a protective Th1 response but ameliorates disease by altering levels of reactive nitrogen intermediates and corticosteroid hormones, which both exert immunosuppressive functions. J Immunol, 2002 Oct 1, 169(7), 3900 - 7 Divergent role for TNF-alpha in IFN-gamma-induced killing of Toxoplasma gondii and Salmonella typhimurium contributes to selective susceptibility of patients with partial IFN-gamma receptor 1 deficiency; Janssen R et al.; Patients with defects in IFN-gamma- or IL-12-mediated immunity are susceptible to infections with Salmonella and non-tuberculous mycobacteria, but rarely suffer from infections with other intracellular pathogens such as Toxoplasma gondii . Here we describe macrophage and T cell function in eight individuals with partial IFN-gamma receptor 1 (IFN-gammaR1) deficiency due to a mutation that results in elevated cell surface expression of a truncated IFN-gammaR1 receptor that lacks the intracellular domain . We show that various effector mechanisms dependent on IFN-gammaR signaling are affected to different extents . Whereas TNF-alpha production was normally up-regulated in response to IFN-gamma, IL-12 production and CD64 up-regulation were strongly reduced, and IFN-gamma-mediated killing of the intracellular pathogens Salmonella typhimurium and T . gondii was completely abrogated in patient's macrophages . Since these patients suffer selectively from infections with non-tuberculous mycobacteria and Salmonella, but not T . gondii, despite sero-immunity in six of eight patients, which indicates previous contact with this pathogen, we next studied the role of TNF-alpha as a possible immune compensatory mechanism . IFN-gamma-induced killing of T . gondii appeared to be partially mediated by TNF-alpha, and addition of TNF-alpha could compensate for the abrogated killing of T . gondii in the patient's macrophages . In contrast, IFN-gamma-mediated killing of S . typhimurium appeared to be independent of TNF-alpha . We propose that the divergent role of TNF-alpha in IFN-gamma-induced killing of T . gondii and S . typhimurium may at least partially explain the highly selective susceptibility of patients. Vet Microbiol, 2002 Oct 22, 89(2-3), 167 - 79 A laboratory study of an inactivated bivalent iron restricted Salmonella enterica serovars Enteritidis and Typhimurium dual vaccine against Typhimurium challenge in chickens; Clifton-Hadley FA et al.; A commercial inactivated iron restricted Salmonella Typhimurium and Salmonella Enteritidis vaccine was used to vaccinate chicks at 1 day and again at 4 weeks of age, with challenge by a high and a low dose of S . Typhimurium given either orally or by contact with seeder birds inoculated orally with a high dose of S . Typhimurium . In all three challenge regimes, the shedding of challenge strain was reduced significantly (p < 0.05) in vaccinated birds compared with unvaccinated controls . Vaccination reduced colonisation of internal organs after challenge by contact seeder birds . However, no effect of vaccination upon colonisation of internal organs after either high or low oral challenge was apparent . In conclusion, the data indicate that the vaccine should be a useful tool in the control of S . Typhimurium infection in chickens. Mol Gen Mikrobiol Virusol, 2002, (3), 34 - 40 {NRAMP1 gene: structure, function, and human infectious diseases}; Puzyrev VP et al.; The human infectious disease become of the great importance for Health Welfare . The infectious diseases mortality rate reaches one third of total mortality among 51 million patients died annually . The genetic factors seem to be most responsible for potency of human body to withstand to infections, caused by a variety of causative agents . The detection of the coincident factors and understanding the mechanisms of formation of susceptibility and resistance to infectious agents appeared to be important aspects for development of the new methods of prevention and treatment the infectious diseases . In inbred mice the natural resistance to infections, caused by Mycobacterium bovis, Mycobacterium avium, Mycobacterium lepraemurium, Leishmania donovani and Salmonella typhimurium is controlled by gene Nrampl (natural resistance associated macrophage protein gene) . The gene codes for integral membrane protein, expressed by phagocytes . Protein is localized in the endosomal/lysosomal compartment of the silent macrophage, being recruited to the membrane of the phagosome containing particles under phagocytosis . This function is to transfer bivalent cations of metals from phagosome inward macrophage, that appears to affect negatively on destiny of microbes consumed . The human homologue of the Nrampl gene, denoted as NRAMP1, is situated on human chromosome 2q35 . The gene Nrampl consists of 15 exones of different spread disparted by intrones of various sizes . Several polymorphous variants of the gene are described . The experimental presuppositions to more extensive investigation of the role of the gene NRAMPl in development of human pathology are pointed out. J Biol Chem, 2002 Nov 22, 277(47), 45068 - 74 Epub 2002 Sep 17. Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence . A slipped frameshift intermediate model for dinucleotide deletion; Hilario P et al.; In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP) . Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role . It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 ()-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP) . In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence . Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli . A remarkable difference in the induced CpG deletion frequency between these adducts was noted . In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively . With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively . This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence . In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III . Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability . This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself. J Food Prot, 2002 Sep, 65(9), 1475 - 9 Salmonella spp . on chicken carcasses in processing plants in Poland; Mikolajczyk A et al.; Chickens at selected points in the slaughter process and after slaughter on the dressing line in poultry plants were sampled and analyzed for Salmonella . These chickens came from the northeast part of Poland . The examinations were carried out in quarters I, II, III, and IV of 1999 . All the birds were determined to be healthy by a veterinary inspection . Swab samples were taken from the cloaca after stunning and from the skin surface and body cavity of the whole bird after evisceration, after rinsing at the final rinse station but before chilling in the spin-chiller, and after cooling in the continuous cooling plant at the end of the production day . In 1999, 400 whole chickens were examined . The percentage of these 400 chickens from which Salmonella spp . were isolated was relatively high (23.75%; Salmonella-positive results were observed in 95 cases) . Salmonella spp . were found after stunning in 6% of the chickens (6 of 100 samples), after evisceration in 24% (24 of 100), before cooling in 52% (52 of 100), and after cooling in 13% (13 of 100) . These results show that Salmonella spp . were found more often at some processing points than at others . The lowest Salmonella spp . contamination rate (6%) for slaughter birds was found after stunning, and the highest contamination rate was found before chilling (52%) . The serological types of Salmonella spp . isolated from whole chickens were Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Saintpaul, Salmonella Agona, and Salmonella Infantis . The results of these investigations indicate that Salmonella Enteritidis is the dominant serological type in infections of slaughter chickens, as it is in many countries. J Food Prot, 2002 Sep, 65(9), 1463 - 9 Evaluation of antioxidative and mutagenic properties of 50% ethanolic extract from red beans fermented by Aspergillus oryzae; Chou ST et al.; Various bean products fermented by microorganisms are commonly consumed in Asian diets; however, the safety or functional properties of fermented beans can vary with different microbial species and with different processes being applied to different beans . The objectives of this study were to evaluate the antioxidative and mutagenic properties of 50% ethanolic extracts from red beans fermented by Aspergillus oryzae . The extracts' antioxidative activities, including alpha,alpha;-diphenyl-beta-picryl-hydrazyl (DPPH) radical-scavenging effects, Fe(2+)-chelating ability, and reducing power, were studied in vitro . The antioxidative effects provided by the extracts depended strongly on their concentrations . In general, antioxidative activity increased with extract concentration to a certain point and then leveled off as the concentration further increased . The fermented red bean extracts showed less of a scavenging effect on the DPPH radical and less reducing power than the commercial antioxidants alpha-tocopherol and butylated hydroxytoluene, but better Fe(2+)-chelating ability . No mutagenicity or toxicity effect on any of the tested strains (Salmonella Typhimurium TA97, TA98, TA100, TA102, and TA1535) was found for the 50% ethanolic extracts of fermented red beans with the Ames mutagenicity assay . These results suggest that the 50% ethanolic extracts were not mutagenic. Plant Physiol, 1993 Mar, 101(3), 1073 - 1080 Pyrophosphorylases in Solanum tuberosum (IV . Purification, Tissue Localization, and Physicochemical Properties of UDP-Glucose Pyrophosphorylase); Sowokinos JR et al.; The enzyme UDP-glucose pyrophosphorylase (UGPase) from potato (Solanum tuberosum L . cv Norchip) tubers was purified 177-fold to near homogeneity and to a specific activity of 1099 international units/mg of protein . The molecular mass of the purified enzyme was 53 kD as determined by SDS-PAGE and gel filtration . Immunological and activity assays detected UGPase at similar levels in potato stems, stolons, and tubers . Leaves and roots contained lower levels of UGPase activity and protein . Lineweaver-Burk plots for substrates inorganic pyrophosphate and UDP-glucose were linear in the pyrophosphorolytic direction, yielding Km values of 0.13 and 0.14 mM, respectively . However, Lineweaver-Burk plots for the substrates glucose-1-P and UTP were biphasic in nature when UGPase was assayed in the direction of UDP-glucose synthesis . At physiological substrate concentrations (i.e . from 0.05-0.20 mM), Km values of 0.08 mM (glucose-1-P) and 0.12mM (UTP) were obtained . When substrate concentrations increased above 0.20 mM, Km values increased to 0.68 mM (glucose-1-P) and 0.53 mM (UTP) . These kinetic patterns of potato UGPase suggest a "negative cooperative effect" (A . Conway, D.E . Koshland, Jr . {1968} Biochemistry 7: 4011-4022) with respect to the substrates glucose-1-P and UTP . The biphasic substrate saturation curves were similar to the kinetics of the dimeric form of UGPase purified from Salmonella typhimurium (T . Nakae {1971} J Biol Chem 246: 4404-4411) . The in vivo significance of the enzyme's "negative cooperativity" in the direction of UDP-glucose synthesis and potato sweetening is discussed. J Endotoxin Res, 2002, 8(4), 263 - 71 Lipopolysaccharide binds to and activates A(1) adenosine receptors on human pulmonary artery endothelial cells; Wilson CN et al.; Previously, it was reported that A(1) adenosine receptor antagonists prevent endotoxin-induced acute lung injury and pulmonary arterial endothelial cell damage . In competition radioligand binding experiments in membranes prepared from human pulmonary artery endothelial cells (PAECs), lipopolysaccharides (LPSs) of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa displaced the binding of a selective A(1) adenosine receptor antagonist {(125)I}-BWA844U (IC(50) values: 195 ng/ml, 290 ng/ml, 602 ng/ml, and 693 ng/ml, respectively) in a dose-dependent, competitive manner . There was no displacement of this radioligand by enterotoxin (< or = 10 microg/ml), diphosphoryl lipid A (< or = 10 microg/ml), and glycolipids, monosialoganglioside (< or = 1 microg/ml), lactocerebroside (< or = 100 microg/ml), or NBD galactocerebroside (< or = 100 microg/ml) . Based on calculated IC(50) values, LPS (E . coli, IC(50) 111 ng/ml) displaced the selective A(1) adenosine receptor agonist, {(3)H}-2-chloro, N(6)-cyclopentyladenosine (CCPA) in human PAECs with a potency profile, CCPA > LPS > 2-phenylaminoadenosine (CV 1808), a selective A(2) adenosine receptor agonist . The potency profile for displacement of the selective A(2a) adenosine receptor agonist {(3)H}-CGS 21680 was CV 1808 > CCPA . LPS (E . coli 0.1 pg/ml-10 microg/ml) did not displace {(3)H}-CGS 21680 binding . In human PAECs, IL-6 and TXA(2) release induced by LPS (0-1 microg/ml) or CCPA (0-1 microM) at high doses was significantly reduced by the selective A(1) adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 microM) . These data suggest that LPS binds to and activates A(1) adenosine receptors on human PAECs to induce the release of IL-6 and TXA(2) . Activation of A(1) adenosine receptors on human PAECs by LPS, may contribute to the pathophysiology of acute lung injury associated with Gram-negative septicemia and endotoxemia. Int J Food Microbiol, 2002 Oct 25, 78(3), 227 - 34 PCR amplification of the Salmonella typhimurium fimY gene sequence to detect the Salmonella species; Yeh KS et al.; This study evaluated the suitability of fimY gene amplification by PCR as an effective means of detecting Salmonella species . Although fimY gene of Salmonella typhimurium is involved in regulating type 1 fimbrial expression, the amino acid sequence of FimY shares very little homology with other known prokaryotic proteins in the GenBank database . Therefore, fimY is a promising target gene to detect the presence of Salmonella species . Herein, two primers internal to the fimY gene of S . typhimurium are used to investigate the distribution of the fimY homologous sequence among 45 Salmonella serovars and 20 non-Salmonella species by using PCR . Experimental results indicated that only Salmonella species possessed the fimY homologous sequence, subsequently generating the specific 526-bp DNA fragments . The sensitivity of the fmY-specific primer set was demonstrated on a Salmonella-free swab sample from a pork carcass surface, which was then artificially contaminated with different concentrations of S . typhimurium . A combining of pre-enrichment step in buffered peptone water and PCR amplification of fimY allowed the detection of S . typhimurium at the concentration of 3.4 x 10(0) CFU/ml from the swab sample . With an additional enrichment step in Rappaport-Vassiliadis (RV) broth, this procedure can also detect pork carcass surface naturally contaminated with Salmonella species in a slaughterhouse . Results in this study demonstrate that fimY is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms. Biosci Biotechnol Biochem, 2002 Jul, 66(7), 1450 - 4 2-{3-(2-Thioxopyrrolidin-3-ylidene)methyll-tryptophan, a novel yellow pigment in salted radish roots; Matsuoka H et al.; The structure of the yellow pigment found in salted radish roots was studied . It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment . The yellow pigment was isolated and identified as 2-{3-(2-thioxopyrrolidin-3-ylidene)methyl}-tryptophan (TPMT) by IR, MS, 1H-, and 13C-NMR spectroscopy . In addition, we proved that this compound was the main yellow pigment in salted radish roots . This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation. Biol Chem, 2002 Jun, 383(6), 977 - 82 N-hydroxyarylamine O-acetyltransferase-deficient Escherichia coli strains are resistant to the mutagenicity of nitro compounds; Josephy PD et al.; In Salmonella typhimurium, a single enzyme catalyzes both the acetyl CoA-dependent O-acetylation of hydroxylamines (a key step in the activation of mutagenic nitroaromatic compounds and related aromatic and heterocyclic amines) and the N-acetylation of aromatic amines . S . typhimurium Ames test mutants lacking this activity are highly resistant to the genotoxic effects of nitro compounds . However, such mutants have not yet been obtained in Escherichia coli . We used a PCR-based method to engineer a null mutation (deletion) of the nhoA gene encoding the enzyme in E . coli and we transduced this mutation into a lacZ strain background suitable for use in mutation assays . In E . coli, as in S . typhimurium, nhoA mutants show marked resistance to nitro compound mutagenicity . The new strains provide a clean background for expression of recombinant N-acetyltransferases. East Afr Med J, 2001 Nov, 78(11), 576 - 80 Salmonella, Shigella and growth potential of other food-borne pathogens in Ethiopian street vended foods; Muleta D et al.; OBJECTIVE: To evaluate the bacteriological safety of food items sold by street vendors with regard to Salmonella and Shigella and to assess the growth potential of some foodborne pathogens in some street foods . DESIGN: Collection of street-vended foods and laboratory based microbiological analysis . Setting: Microbiology Laboratory, Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia . RESULTS: Most of the street food samples had aerobic mesophilic counts >10(7) cfu/g . Nine "kitfo" and one "egg sandwich" samples yielded Salmonella . Shigella was isolated from three "macaroni" samples . The Salmonella isolates were sensitive to all ten drugs tested but the Shigella isolates had multiple resistance against five drugs . In a challenge study, Salmonella typhimurium, Shigella flexneri and Staphylococcus aureus grew in street-vended food samples to hazardous levels within eight to twelve hours . CONCLUSION: Street foods are heavily contaminated with micro-organisms and are potential sources of food borne infections . Health hazards from street foods may be significantly minimised by consumption within four hours of preparation. Eur J Immunol, 2002 Sep, 32(9), 2664 - 71 Processing of viable Salmonella typhimurium for presentation of a CD4 T cell epitope from the Salmonella invasion protein C (SipC); Musson JA et al.; We have identified Salmonella invasion protein C (SipC) as a target antigen for CD4 T cell recognition in mice infected with Salmonella typhimurium . SipC is a product of the type III secretion system encoded by S . typhimurium pathogenicity island 1 . A SipC-specific T cell response was induced by infection with either the C5 wild type or attenuated SL3261 vaccine strain of S . typhimurium . We localized the response of T cell lines from infected mice to an epitope near the carboxyl terminus of SipC (SipC(381-394)) and studied the way it was processed from viable S . typhimurium . We demonstrated that CD4 T cell recognition of this epitope required actin-dependent uptake of S . typhimurium . Presentation also occurred when transport of newly synthesized MHC class II from the endoplasmic reticulum was disrupted and when the pH of intracellular compartments was raised, suggesting presentation by mature MHC class II recycled from the macrophage surface into neutral intracellular compartments . Salmonellae are known to colonize macrophages by localizing to compartments that do not make contact with the bactericidal environment of late endosomes or lysosomes, and thus might avoid lysosomal antigen processing . However, we demonstrate that a CD4 T cell response to S . typhimurium-secreted proteins may be induced by an alternative pathway capable of antigen presentation in conditions similar to those in the compartments where Salmonella localize. J Immunol, 2002 Sep 15, 169(6), 3275 - 83 Induction of CD8+ T lymphocytes by Salmonella typhimurium is independent of Salmonella pathogenicity island 1-mediated host cell death; Wijburg OL et al.; Salmonella are intracellular bacterial pathogens that reside and replicate inside macrophages, and attenuated strains of Salmonella typhimurium can be used to deliver heterologous Ags for MHC class I and/or MHC class II-restricted presentation . Recently, it was shown that invasion of macrophages by S . typhimurium may result in the death of host macrophages via a mechanism harboring features of apoptotic and necrotic cell death . However, it is unknown whether this bacterial-induced host cell death affects immunity . In addition, it has been hypothesized that macrophage death following infection with S . typhimurium and subsequent uptake of apoptotic cells by APC are fundamental to the induction of CTL responses . In this study we investigated the in vivo induction of Ag-specific CD8+ T lymphocyte responses and compared CD8+ T lymphocyte responses elicited with S . typhimurium strains carrying a mutation in their invA gene, and therefore an inability to induce Salmonella pathogenicity island 1 (SPI-1)-mediated macrophage death, with responses elicited by an attenuated deltaaroAD strain . Ag-specific CD8+ T lymphocyte responses were analyzed using IFN-gamma ELISPOT, tetramer binding, and in vivo and in vitro CTL assays . Our results showed that deltaaroAD and deltaaroADdeltainvA induced comparable levels of Ag-specific CD8+ T lymphocyte responses as well as protective, Ag-specific B and CD4+ T lymphocyte immunity . Furthermore, experiments in macrophage-depleted mice showed that CD8+ T lymphocyte responses were effectively induced in the absence of macrophages . Together, our results imply that in this infection model, SPI-1-mediated cell death does not affect the immunological defense response and is not important for the induction of CD8+ T lymphocyte responses. J Immunol, 2002 Sep 15, 169(6), 2846 - 50 Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway; Collier-Hyams LS et al.; Secreted prokaryotic effector proteins have evolved to modulate the cellular functions of specific eukaryotic hosts . Generally, these proteins are considered virulence factors that facilitate parasitism . However, in certain plant and insect eukaryotic/prokaryotic relationships, effector proteins are involved in the establishment of commensal or symbiotic interactions . In this study, we report that the AvrA protein from Salmonella typhimurium, a common enteropathogen of humans, is an effector molecule that inhibits activation of the key proinflammatory NF-kappaB transcription factor and augments apoptosis in human epithelial cells . This activity is similar but mechanistically distinct from that described for YopJ, an AvrA homolog expressed by the bacterial pathogen YERSINIA: We suggest that AvrA may limit virulence in vertebrates in a manner analogous to avirulence factors in plants, and as such, is the first bacterial effector from a mammalian pathogen that has been ascribed such a function. BMC Evol Biol . 2002 Sep 08;2(1):14. Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells; Tracy BS et al.; BACKGROUND: A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes . Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy . RESULTS: Extensive and variable losses and a few gains of catabolic functions were observed by this standardized method . Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates . CONCLUSION: While the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment . Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog system. Drug Metab Rev, 2002 Aug, 34(3), 667 - 76 Role of human cytochrome P450 (CYP) in the metabolic activation of nitrosamine derivatives: application of genetically engineered Salmonella expressing human CYP; Kamataki T et al.; The role of human cytochrome P450 (CYP) in the metabolic activation of tobacco-related N-nitrosamines was examined by Salmonella mutation test using a series of genetically engineered Salmonella typhimurium YG7108 strains each co-expressing a form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) together with human NADPH-cytochrome P450 reductase . Seven tobacco-related N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosonornicotine, N-nitrosoanabasine, and N-nitrosoanatabine were used . The CYP2A6 was found to be responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined . On the basis of the evidence, genetic polymorphism of the CYP2A6 gene appeared to be one of the factors determining cancer susceptibility caused by smoking . Previously, we found the whole deletion of the CYP2A6 gene (CYP2A6*4C) as a type of genetic polymorphism in Japanese . We hypothesized that individuals possessing the gene homozygous for CYP2A6*4C were incapable of activating tobacco-related N-nitrosamines and showed lower susceptibility to lung cancer induced by tobacco smoke . Thus, the relationship between the CYP2A6*4C and the susceptibility to the lung cancer was evaluated . The frequency of the CYP2A6*4C was significantly lower in the lung cancer patients than healthy volunteers, suggesting that the subjects carrying the CYP2A6*4C alleles are resistant to carcinogenesis caused by N-nitrosamines because of the poor metabolic activation capacity . Taking these results into account, CYP2A6 is an enzyme enhancing lung cancer risk. Poult Sci, 2002 Aug, 81(8), 1224 - 30 Pulmonary hypertensive response to endotoxin in cellulose-primed and unprimed broiler chickens; Wang W et al.; Previous studies indicate that individual broilers vary widely in their pulmonary vascular responsiveness to i.v . injections of endotoxin . This individual variability may reflect differences acquired during previous respiratory challenges or genetic variability that may be associated with susceptibility to pulmonary hypertension syndrome (ascites) . In the present study, we compared the endotoxin responses of 4- to 5- wk-old control broilers (unprimed) and broilers in which the pulmonary vasculature had been immunologically challenged 48 h previously by an i.v . injection of cellulose micro-particles (primed) . The injected cellulose micro-particles are carried in the venous blood to the lungs, where they become trapped in the pulmonary vasculature and initiate acute focal inflammatory responses within the surrounding lung parenchyma . Physiological variables (respiratory rate, heart rate, pulmonary and systemic arterial pressures) were evaluated prior to and following the i.v . administration of 1 mg of Salmonella typhimurium endotoxin . Prior to endotoxin injection, the respiratory rate was higher in primed than in unprimed broilers; however, the heart rate, pulmonary arterial pressure, and systemic arterial pressure did not differ between groups . Broilers in both groups exhibited similar ranges of individual variability in their endotoxin responses . The overall time of onset, magnitude, and duration of the pulmonary hypertensive responses were similar for both groups . Accordingly, the initiation of a preexisting inflammatory response within the lung parenchyma did not alter the timing, amplitude, or variability of the subsequent pulmonary hypertensive response to endotoxin in broilers. Environ Mol Mutagen, 2002, 40(1), 63 - 70 Genotoxicity of 5-aminolevulinic and 4,5-dioxovaleric acids in the salmonella/microsuspension mutagenicity assay and SOS chromotest; Onuki J et al.; 5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in some porphyric disorders and in lead poisoning which can undergo metal-catalyzed oxidation producing reactive oxygen species and the keto-aldehyde, 4,5-dioxovaleric acid (DOVA) . Evidence in vitro of ALA-induced DNA lesions suggests that ALA and DOVA have mutagenic potential that could possibly contribute to an increased frequency of hepatocellular carcinoma (HCC) in patients with acute intermittent porphyria (AIP) . In this study, we evaluated the genotoxic potential of ALA and DOVA . In the absence of exogenous metabolic activation, ALA and DOVA were mutagenic in Salmonella typhimurium tester strain TA104 . ALA was also mutagenic in S . typhimurium TA102, but not in TA98, TA100, or TA1535, indicating an oxidative mechanism . Removal of H(2)O(2) with catalase gave only partial protection, suggesting generation of other mutagenic species . Both ALA and DOVA damaged the DNA of Escherichia coli PQ37, inducing the SOS response detected by an increase in beta-galactosidase activity . These results verified the potential mutagenic activity of ALA and DOVA and reinforce the hypothesis that DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP . Toxic Rep Ser, 1995 Apr, 30, 1 - G5 NTP technical report on the toxicity studies of Dibutyl Phthalate (CAS No . 84-74-2) Administered in Feed to F344/N Rats and B6C3F1 Mice; Marsman D; Dibutyl phthalate is a phthalate ester with extensive use in industry in such products as plastic (PVC) piping, various varnishes and lacquers, safety glass, nail polishes, paper coatings, dental materials, pharmaceuticals, and plastic food wrap . Concomitant with this extensive worldwide use is the high potential for human exposure to dibutyl phthalate in the workplace and the home environment through direct sources as well as indirectly, through contamination of water, air, and foodstuffs . Because existing toxicity information was considered inadequate, the effects of exposure to dibutyl phthalate were examined in male and female F344/N rats and B6C3F1 mice in 13-week feed studies . Furthermore, due to concern over the potential for pervasive exposure of humans to dibutyl phthalate, additional perinatal studies examined rats and mice exposed as pups in utero, for the 4 weeks of lactation, and for an additional 4 weeks postweaning . Additional studies examined the effects on rats of combining perinatal and adult subchronic exposure . Due to the recognized biologic activity of this and other phthalates, hepatic peroxisome proliferation during the in utero and lactational phases and testicular toxicity during the perinatal period were also examined . Finally, reproductive assessment by continuous breeding (including crossover mating trials and offspring assessment) and genetic toxicity studies were also conducted . In the maximum perinatal exposure (MPE) determination study in rats, dibutyl phthalate was administered in the diet to dams during gestation and lactation, and to the pups postweaning for four additional weeks, at concentrations of 0, 1,250, 2,500, 5,000, 7,500, 10,000, and 20,000 ppm . Decreased weight gains were noted in dams exposed to 20,000 ppm during gestation and to dams exposed to 10,000 ppm during lactation . The gestation index (number of live pups per breeding female) was significantly lower in the 20,000 ppm group than in the controls, and pup mortality in this group was marked (100% by Day 1 of lactation); however, survival was 89% or greater in all other treatment groups . The mean body weight of pups in the 10,000 ppm group at Day 28 of lactation was approximately 90% of the mean weight of control pups . Pups were weaned onto diets containing dibutyl phthalate at the same concentrations fed to dams . After an additional 4 weeks of dietary administration, final mean body weights of pups in the 10,000 ppm groups were 92% of the control value for males and 95% of the control value for females . Hepatomegaly (increased relative liver weight) was observed in males in all exposed groups and in females receiving 2,500 ppm or greater . No gross lesions were observed at necropsy . Moderate hypospermia of the epididymis was diagnosed in all male rats in the 7,500 and 10,000 ppm groups; mild hypospermia of the epididymis was diagnosed in 2 of 10 males in the 5,000 ppm group . No degeneration of the germinal epithelium was detected in the testis of these rats . Thus, although toxicologically important, the epididymal hypospermia was not considered to be life threatening, and 10,000 ppm was recommended as the MPE concentration for male and female rats . In the subsequent subchronic toxicity study of dibutyl phthalate with perinatal exposure, dams were administered diets containing 0 or the MPE concentration (10,000 ppm) during gestation and lactation, and weaned pups were administered the same diets as their dams received for an additional 4 weeks, until the beginning of the 13-week exposure phase . Male and female rats then received diets containing dibutyl phthalate at concentrations of 0, 2,500, 5,000, 10,000, 20,000, and 40,000 ppm for 13 weeks . No mortality or toxicity was observed in dams during the perinatal phase of the study; however, before pups were culled at 4 days postpartum, the percentage of live pups per litter was 86% to 93% that of the controls . Through weaning, litter weights of exposed pups ranged from 89% to 92% of the control values . Ten control and ten exposed pups per sex were examined at the time of trol and ten exposed pups per sex were examined at the time of weaning; hepatomegaly and markedly increased peroxisomal enzyme activities (approximately 9-fold greater than the control values) were observed in exposed pups . Body weights of the perinatally exposed pups remained lower than those of the controls throughout the 4-week period before the 13-week adult exposures began . During the 13-week adult exposure phase, the final mean body weight of males in the MPE: 0 ppm control group (MPE rats, returned to the base diet for 13 weeks), was 95&percnt; that of the controls . The body weight gain of females in the MPE:0 ppm group was greater than that of the unexposed controls, and the final body weights of these two groups were similar . Body weight gains of rats treated with dibutyl phthalate as adults decreased with increasing exposure concentration; for rats that received the MPE concentration followed by 40,000 ppm for 13 weeks, final body weights were 51&percnt; of the control value for males and 74&percnt; of the control value for females . Hepatomegaly apparently regressed in rats in the MPE:0 ppm groups but was observed in male rats receiving 5,000 ppm or greater and in females receiving 2,500 ppm or greater . In males that received 20,000 ppm as adults, testis and epididymal weights were less than in the controls; males in the 40,000 ppm group also had a lower testis weight than the controls . Results of hematologic analyses conducted at the end of the 13-week exposure period suggested a mild anemia in male rats administered 10,000 ppm or greater as adults and female rats administered 40,000 ppm as adults . Hypocholesterolemia and hypotriglyceridemia were observed in male and female rats at the higher exposure concentrations . Hypotriglyceridemia was detected in females receiving 20,000 or 40,000 ppm and in males receiving 10,000 ppm or greater . Elevations in alkaline phosphatase activities and bile acid concentrations in male and female rats receiving 20,000 or 40,000 ppm as adults were indicative of cholestasis . Microscopic examination revealed hepatocellular cytoplasmic alteration, consistent with glycogen depletion, in male and female rats receiving a concentration of 10,000 ppm or greater . In the liver of rats receiving 40,000 ppm, small, fine, eosinophilic granules were also observed in the cytoplasm of hepatocytes . Ultrastructural examination suggested the presence of increased numbers of peroxisomes . Lipofuscin accumulation was detected in rats that received 10,000 ppm or greater . Consistent with the regression of the hepatomegaly in rats in the MPE:0 and MPE:2,500 ppm groups, peroxisomal enzyme activity was not elevated in these groups . Marked elevations of peroxisomal enzyme activity were detected, however, in males receiving 5,000 ppm or greater and in females receiving 10,000 ppm or greater; at the 40,000 ppm concentration, the highest concentration tested, enzyme activities were approximately 20 fold greater than the control values . Histopathologic examination of the testes revealed degeneration of the germinal epithelium, a mild to moderate focal lesion in rats in the 10,000 and 20,000 ppm groups and a marked, diffuse lesion in all males receiving 40,000 ppm; at 40,000 ppm, an almost complete loss of the germinal epithelium resulted . Testicular zinc concentrations were lower in the 40,000 ppm group than in the controls, a finding consistent with the marked loss of germinal epithelium at this exposure concentration . Spermatogenesis was evaluated in rats in the 0, 2,500, 10,000, and 20,000 ppm groups; rats administered 20,000 ppm had fewer spermatid heads per testis than the unexposed controls, and epididymal spermatozoal concentration was less than that in the MPE:0 ppm group . For comparison with the perinatal subchronic study, a standard 13-week evaluation of the toxicity of dibutyl phthalate in male and female rats was also conducted . In this study, rats received dibutyl phthalate at the same dietary concentrations used in the 13-week exposure phase of the study with perinatal exposure: 0, 2,500, 5,000, 10,000, 20,000, and 40,000 ppm . No deaths occurred in the standard study . Markedly reduced final mean body weights were observed in males and females in the 40,000 ppm groups (45&percnt; and 73&percnt; of control body weights, respectively); final mean body weights of males receiving 10,000 ppm or greater and females receiving 20,000 ppm or greater were lower than those of the controls . Hepatomegaly was observed in males that received 5,000 ppm or greater and in females that received 10,000 ppm or greater . Testis and epididymal weights of males in the 20,000 and 40,000 ppm groups were lower than those of the controls . A minimal anemia was detected in male rats receiving 5,000 ppm or greater . Hypocholesterolemia was observed in male and female rats receiving 20,000 or 40,000 ppm, and hypotriglyceridemia was detected in males in all exposed groups and in females receiving 10,000 ppm or greater . Elevations in alkaline phosphatase activity and bile acid concentration in male and female rats were considered indicative of cholestasis . Morphologic evaluation again confirmed the toxicity of dibutyl phthalate to the liver and testes of rats . Microscopic examination of the liver revealed hepatocellular cytoplasmic alterations, consistent with glycogen depletion, in male and female rats receiving 10,000 ppm or greater . In the liver of rats in the 40,000 ppm groups, small, fine, eosinophilic granules were also observed in the cytoplasm of hepatocytes . Ultrastructural examination suggested the presence of increased numbers of peroxisomes, and peroxisomal enzyme activity was elevated in the livers of male and female rats administered 5,000 ppm or greater; the enzyme activities in the 40,000 ppm groups were approximately 13-fold greater than the control value for males and 32-fold greater than the control value for females . Lipofuscin accumulation was detected in rats receiving 10,000 ppm or greater . Histopathologic examination of the testes revealed degeneration of the germinal epithelium, a mild to marked focal lesion in the 10,000 and 20,000 ppm groups and a marked, diffuse lesion in all males in the 40,000 ppm group; at 40,000 ppm, an almost complete loss of the germinal epithelium resulted . Testicular zinc concentrations were lower in the 20,000 and 40,000 ppm groups than in the controls . Serum testosterone values were also lower at these concentrations than in the controls . Spermatogenesis was evaluated in males in the 0, 2,500, 10,000, and 20,000 ppm groups; at 20,000 ppm, spermatid heads per testis and per gram testis, epididymal spermatozoal motility, and the number of epididymal spermatozoa per gram epididymis were lower than in the controls . All of these findings are consistent with the marked loss of germinal epithelium at these exposure concentrations . In the continuous breeding study, Sprague-Dawley rats received 0, 1,000, 5,000, or 10,000 ppm dibutyl phthalate in feed . Mean body weights of exposed dams at delivery and during lactation generally decreased with increasing exposure concentration . The mean pup weight at birth in the 10,000 ppm group was significantly lower than the control pup weight . The average number of live pups per litter in all exposed groups was lower than in the controls . Crossover mating trials in the F(0) generation revealed no effects on the fertility of male or female rats receiving 10,000 ppm . In contrast to the F(0) rats, mating, pregnancy, and fertility indices of F(1) rats were lower in the 10,000 ppm group than in the controls . Germinal epithelial degeneration of the testes and absence or under development of the epididymides were noted in F(1) males in the 10,000 ppm group . Interstitial cell hyperplasia was noted in 7 of 10 males in the 10,000 ppm group . These effects document the male and female reproductive toxicity of dibutyl phthalate in F(1) rats receiving 10,000 ppm and do not exclude the possibility of developmental toxicity to F2 offspring . In the MPE determination study in mice, dams received 0, 1,250, 2,500, 5,000, 7,500, 10,000, or 20,000 ppm dibutyl phthalate in feed during gestation and lactation; pups were weaned onto the same diets as the dams received and were exposed for an additional 4 weeks . The gestation period was longer in dams that received 2,500 ppm or greater than in the controls, and gestational body weight gain depressions were noted in dams receiving 7,500 ppm or greater . Only 5 of 20 females in the 10,000 ppm group delivered live pups, and none of the 20 females receiving 20,000 ppm delivered live pups . Only one pup in the 10,000 ppm group survived past Lactation Day 1; the number of live pups per litter in the 7,500 ppm group also remained low throughout lactation . No deaths of either male or female pups occurred after weaning . Initial (postweaning) and final body weights of male pups receiving 2,500 ppm or greater were significantly less than those of the control group . The mean body weights of exposed female pups were similar to the control body weight at weaning and remained similar throughout the 4 weeks postweaning . Hepatomegaly was present in male mice in all exposed groups, and the absolute liver weight of males administered 7,500 ppm was greater than that of the controls; although a similar change was apparent in females, no statistical differences between the liver weights of exposed and control females were detected . No treatment-related gross lesions were identified at necropsy, and no histopathologic lesions definitively associated with treatment were observed in male or female mice in the 7,500 ppm groups . The one surviving male pup in the 10,000 ppm group had cytoplasmic alteration in the liver, consistent with peroxisome proliferation . Developmental toxicity and fetal and pup mortality were suggested at concentrations as low as 7,500 ppm . No subchronic toxicity study with prior MPE exposure was conducted with mice, although an MPE concentration of 5,000 ppm was suggested by the data . In a standard 13-week toxicity study, mice received 0, 1,250, 2,500, 5,000, 10,000, or 20,000 ppm dibutyl phthalate in feed . No deaths occurred during this study . Mean body weights and weight gains of male and female mice decreased with increasing exposure concentration, and the decreases were significant for males and females that received 5,000 ppm or greater . Relative liver weights were greater in males and females receiving 5,000 ppm or greater than in the controls . A minimal anemia was suggested in female mice in the 20,000 ppm group . Although no gross lesions were observed at necropsy, microscopic examination revealed hepatocellular cytoplasmic alterations, consistent with glycogen depletion, in male mice receiving 10,000 or 20,000 ppm and female mice receiving 20,000 ppm . Small, fine, eosinophilic granules, consistent with peroxisome proliferation, were also observed in the cytoplasm of hepatocytes in males and females in the 20,000 ppm groups . Lipofuscin accumulation in the liver was detected in mice receiving 10,000 ppm or greater . In a continuous breeding study using Swiss (CD-1&reg;) mice, animals received 0, 300, 3,000, or 10,000 ppm dibutyl phthalate in feed . The fertility index, average number of litters per breeding pair, and average number of live pups per litter in the 10,000 ppm group were lower than in the controls . Crossover mating trials of mice receiving 10,000 ppm revealed effects on dams in the F(0) generation, with a lower fertility index, number of live pups per litter, and pup weight than in the controls . Liver weights were greater in males and females, and the uterine weight was less in exposed dams than in the controls . No other changes were observed at necropsy or on histopathologic examination . These data document the female reproductive toxicity of dibutyl phthalate in F(0) mice . Dibutyl phthalate was not mutagenic in Salmonella typhimurium strain TA98, TA100, TA1535, or TA1537 with or without exogenous metabolic activation but did induce mutations in L5178Y mouse lymphoma cells treated without metabolic activation . In peripheral blood samples obtained from male and female mice at the end of the 13-week study, frequencies of micronucleated normochromatic erythrocytes were similar between exposed and control mice . Together, the studies in rodents suggest that young rodents (in utero and perinatal) respond in a manner qualitatively similar to that of adult rats and mice . Dibutyl phthalate induced toxic effects in rodents as pups in utero and during the lactational phases of development and also affected young adults, as evidenced by fetotoxicity and lethality, body weight gain decrements, increased liver weights, hepatic peroxisome proliferation, testicular toxicity, and female reproductive toxicity . Dibutyl phthalate was lethal to rat fetuses and rat and mouse neonates at dietary concentrations that were not toxic to dams . Otherwise, there was no teratogenic or morphologic evidence that rodent young were uniquely sensitive to the effects of short-term dibutyl phthalate treatment . Synonyms: 1,2-Benzenedicarboxylic acid dibutyl ester; benzene-o-dicarboxylic acid di-n-butyl ester; o-benzenedicarboxylic acid dibutyl ester; butyl phthalate; n-butyl phthalate; DBP; dibutyl 1,2-benzene dicarboxylate; dibutylphthalate; di-n-butylphthalate; di(n-butyl) phthalate; dibutyl-o-phthalate; phthalic acid dibutyl ester . Trade Names: Celluflex DBP; Elaol; Ergoplast FDB; Ersoplast FDA; Genoplast B; Hexaplas M/B; Palatinol C; Polycizer DBP; PX 104; RC Plasticizer DBP; Staflex DBP; Uniflex DBP; Unimoll DB; Witcizer 300; Witicizer 300 . (NOTE: These studies were supported in part by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund (Superfund) by an interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S . Public Health Service.) Toxic Rep Ser, 1995 Jan, 31, 1 - G5 NTP technical report on the toxicity studies of Isoprene (CAS No . 78-79-5) Administered by Inhalation to F344/N Rats and B6C3F1 Mice; Melnick R; Isoprene, the 2-methyl analogue of 1,3-butadiene, has a high production volume and is used largely in the manufacture of synthetic rubber . Isoprene is also the major endogenous hydrocarbon exhaled in human breath . Two-week and 13-week inhalation toxicology studies were conducted in male and female F344/N rats and B6C3F1 mice to characterize potential adverse effects of isoprene . Male rats and male mice were also exposed to isoprene vapors for 6 months followed by a 6-month recovery period (stop- exposure protocol) to determine if isoprene produces a carcinogenic response similar to that of 1,3-butadiene after intermediate exposure durations . In addition to histopathology, evaluations included clinical pathology, tissue glutathione analyses, forelimb and hindlimb grip strength analyses, and sperm motility and vaginal cytology . Data from inhalation teratology studies of isoprene in rats and mice are also reported . In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells . In conjunction with the inhalation studies in mice, evaluations were also made of sister chromatid exchanges and chromosomal aberrations in bone marrow cells and micronuclei in peripheral blood of male mice exposed to isoprene for 12 days or 13 weeks . Target concentrations of isoprene in the inhalation chambers were 0, 438, 875, 1,750, 3,500, and 7,000 ppm in the 2-week studies; 0, 70, 220, 700, 2,200, and 7,000 ppm in the 13-week and stop-exposure studies; and 0, 280, 1,400, and 7,000 ppm in the teratology studies . In the 2-week studies, no changes related to chemical administration were observed in survival, body weight gain, clinical signs, hematologic or clinical chemistry parameters, or the incidence of gross or microscopic lesions in rats . In mice, there were no effects on survival; the mean body weight of males in the 7,000 ppm group was less than that of the controls . In mice, exposure to isoprene caused decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts, atrophy of the testis and thymus, cytoplasmic vacuolization of the liver, olfactory epithelial degeneration in the nasal cavity, and epithelial hyperplasia in the forestomach . Exposure to isoprene for 13 weeks produced no discernible toxicologic effects in rats . In the stop-exposure study, interstitial cell hyperplasia of the testis was observed in all male rats in the 7,000 ppm group after 6 months of exposure . Following the 6-month recovery period, male rats exposed to 700, 2,200, or 7,000 ppm isoprene had slightly greater incidences of interstitial cell adenomas of the testes than the controls . Exposure to isoprene for 13 weeks or 6 months produced no clear exposure-related effects on body weight gain in male or female mice; however, survival was decreased for male mice exposed to 7,000 ppm isoprene for 6 months . More notably, toxic and carcinogenic effects were induced at multiple organ sites in mice exposed to isoprene . After 6 months of exposure and 6 months of recovery, male mice exposed to 700 ppm or higher concentrations of isoprene had greater incidences of neoplasms of the liver (0 ppm, 7/30; 70 ppm, 3/30; 220 ppm, 7/29; 700 ppm, 15/30; 2,200 ppm, 18/30; 7,000 ppm, 17/28), lungs (2/30, 2/30, 1/29, 5/30, 10/30, 9/28), forestomach (0/30, 0/30, 0/30, 1/30, 4/30, 6/30), and harderian gland (2/30, 6/30, 4/30, 14/30, 13/30, 12/30) than the controls . In addition to the higher neoplasm incidences in male mice exposed to 700 ppm or greater, incidences of multiple neoplasms and/or neoplasms of greater malignancy were also higher than in the controls . Hematologic effects similar to those occurring in exposed mice in the 2-week study, plus greater mean cell volume values than in the controls, were observed after 24 days and after 13 weeks of exposure to isoprene . These hematologic effects, which were not accompanied by greater reticulocyte counts or a higher frequency of polychromatic erythrocytes than controls, were indicative of a nonresponsive, macrocytic anemia . In male mice in the stop-exposure study, partial hindlimb paralysis in the 7,000 ppm group and a dose-related decrease in grip strength were observed near the end of the 6-month exposure period . Other nonneoplastic effects in mice exposed to isoprene included spinal cord and sciatic nerve degeneration, skeletal muscle atrophy, degeneration of the olfactory epithelium, epithelial hyperplasia of the forestomach, increased estrous cycle length, testicular atrophy, and decreased epididymal weight, sperm head count, sperm concentration, and sperm motility . The inhalation teratology studies did not show maternal or developmental toxicity in Sprague-Dawley rats at exposures of up to 7,000 ppm isoprene; in CD-1&reg; Swiss mice, exposure to isoprene resulted in lower fetal weights and a higher percentage of fetuses per litter with supernumerary ribs . Isoprene was not mutagenic in Salmonella typhimurium and did not induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells with or without exogenous metabolic activation; however, in mice, isoprene induced increases in the frequency of sister chromatid exchanges in bone marrow cells and in the frequency of micronucleated erythrocytes in peripheral blood . These inhalation studies showed that isoprene caused toxic effects in the testis of rats and at multiple organ sites in mice . In F344/N rats, exposure to 7,000 ppm isoprene for 6 months caused an increase in the incidence of testicular interstitial cell hyperplasia, and after 6 months of recovery there was a marginally increased incidence of benign testicular adenomas that may have been related to isoprene administration . No-observable-adverse-effect levels (NOAELs) for isoprene-induced toxic lesions in mice were: 70 ppm for nonresponsive, macrocytic anemia, decreased hindlimb grip strength, olfactory epithelial degeneration, and decreases in epididymal weights, spermatid head counts, sperm concentration, and sperm motility; 220 ppm for forestomach epithelial hyperplasia; 700 ppm for increased estrous cycle length; and 2,200 ppm for testicular atrophy, sciatic nerve degeneration, and muscle atrophy . A NOAEL was not achieved for spinal cord degeneration (less than 70 ppm) or developmental toxicity (less than 280 ppm, based on lower body weights of female fetuses) . In addition, the 6-month inhalation exposure plus 6-month recovery (stop-exposure) study provided clear evidence of carcinogenicity of isoprene in the liver, lung, forestomach, and harderian gland of mice . Because these studies involved exposures of male rats and male mice to isoprene for only 6 months, they do not necessarily reveal the full carcinogenic potential of isoprene in these species . Most of the toxic and carcinogenic effects seen with isoprene were also caused by inhalation exposure to 1,3-butadiene . Synonyms: isopentadiene; 2-methyl-1,3-butadiene; beta-methylbivinyl. Toxic Rep Ser, 1993 Dec, 32, 1 - E7 NTP technical report on the toxicity studies of Methylene Bis(thiocyanate) (CAS No . 6317-18-6) Administered by Gavage to F344/N Rats and B6C3F1 Mice; Burka LT; Methylene bis(thiocyanate) is used as a biocide in a number of applications . Its major use is in water cooling systems and paper mills as an inhibitor of algae, fungi, and bacteria . Methylene bis(thiocyanate) was selected for study because of the potential for human exposure to the compound and because of the interest in organothiocyanates as a chemical class . Toxicity studies of methylene bis(thiocyanate) (approximately 98% pure) were conducted with male and female F344/N rats and B6C3F1 mice; the compound was administered to the animals by gavage in an aqueous methyl cellulose vehicle for 2 weeks or 13 weeks . In addition to these studies, the genetic toxicity of methylene bis(thiocyanate) was evaluated by determining mutagenicity in Salmonella typhimurium with and without S9 activation and frequency of micronucleated normochromatic erythrocytes in the peripheral blood of mice . In the 2-week studies, groups of five rats and five mice per sex were administered methylene bis(thiocyanate) at concentrations of 0, 10, 20, 40, 80, and 160 mg/kg body weight . All animals in the two highest dose groups (80 and 160 mg/kg) died by Day 2 of the studies . Except for one female rat, all animals receiving 40 mg/kg methylene bis(thiocyanate) also died before the end of the studies . Few significant gross lesions were observed in the 80 and 160 mg/kg groups . Clinical observations were similar to those reported for cyanide toxicity and included dyspnea, tremors . and ataxia . The stomach, which was identified as the target organ in rats and mice surviving for at least 24 hours, had necrotic inflammatory lesions of the mucosal surface of both the glandular and nonglandular portions . In the 13-week studies, groups of 10 rats and 10 mice per sex were administered methylene bis(thiocyanate) at concentrations of 0, 1, 2, 4, 8, and 16 mg/kg body weight . In the rat study, deaths occurred in the 2, 4, 8, and 16 mg/kg groups, while in the mouse study, deaths occurred only in the 8 and 16 mg/kg groups . As in the 2-week studies, the stomach was identified as the primary target organ . However, the lower doses administered in the 13-week studies resulted in gastric effects that were limited to the forestomach and consisted primarily of squamous mucosal hyperplasia and hyperkeratosis . Rats receiving the higher doses of methylene bis(thiocyanate) developed a mild anemia, and sperm motility was decreased in male rats receiving 4 or 8 mg/kg . Methylene bis(thiocyanate) was not mutagenic in S . typhimurium, with or without S9 activation . The frequencies of micronucleated normochromatic erythrocytes in the peripheral blood of dosed and control mice were similar . Chemical disposition studies of {14C}-labeled methylene bis(thiocyanate) were conducted in male F344 rats . In these studies, more than 90% of the administered radioactivity was eliminated in 48 hours . However, as the dose was increased from 0.2 to 1 to 10 mg/kg, greater percentages of the administered radioactivity remained in the tissues . Blood cyanide concentrations were increased shortly after the administration of 10 mg/kg {14C}-methylene bis(thiocyanate) but were similar to control values 2 hours after dosing . Overall, the toxic effects of methylene bis(thiocyanate) were consistent with those of an irritant chemical administered by gavage . There was also some indication that the release of cyanide may result in acute toxicity at the higher dose levels used in these studies . The no-observed-adverse-effect level for forestomach lesions in the 13-week studies was 4 mg/kg for male rats and 2 mg/kg for female rats and male and female mice . Synonyms: MBT; methylene-bis-thiocyanate; methylene bisthiocyanate; methylene dithiocyanate. Toxic Rep Ser, 1993 Aug, 35, 1 - I12 NTP technical report on the toxicity studies of a Chemical Mixture of 25 Groundwater Contaminants Administered in Drinking Water to F344/N Rats and B6C3F(1) Mice; Yang R; Toxicity studies were performed with a chemically defined mixture of 25 groundwater contaminants, using dose levels considered to have environmental relevance . The mixture contained 19 organic compounds and six metals (shown below); the selection of these compounds was based primarily on the frequency of their occurrence in United States Environmental Protection Agency surveys of groundwater contamination in the vicinity of hazardous waste disposal sites . This report focuses primarily on 26-week drinking water toxicity studies with male and female F344/N rats and B6C3F(1) mice . The endpoints evaluated included histopathology, clinical pathology, neurobehavioral studies, and reproductive toxicity . Additional studies using this same chemical mixture are briefly reviewed in this report and include an evaluation of spermatogenesis in B6C3F(1) mice exposed to the chemical mixture for 13 weeks, a continuous breeding study with Sprague-Dawley rats and CD-1(R) Swiss mice, studies of myelotoxicity in B6C3F(1) mice exposed to the chemical mixture for up to 31.5 weeks, studies of immunosuppression in B6C3F(1) mice exposed for up to 13 weeks, in vitro mutagenicity assays in Salmonella typhimurium and Escherichia coli, and measures of genetic damage in bone marrow and peripheral blood of F344/N rats and B6C3F(1) mice in 2-week drinking water studies . In a 26-week drinking water study in which rats were administered the chemical mixture at composite contaminant concentrations of 0, 11, 38, 113, or 378 ppm, no deaths occurred and the body weight gain of high-dose males was slightly less than that of the controls . Water consumption decreased with dose and was 24% to 28% less than that of the controls at the highest concentration . Changes in organ weights occurred primarily in high-dose rats and included increased absolute and relative liver and kidney weights in females, increased relative kidney weight in males, and decreased absolute and relative thymus weights in males and females . Hematologic assessments indicated that rats receiving 378 ppm developed a microcytic anemia consistent with that accompanying iron depletion . Multiple foci of inflammation occurred in the liver of exposed rats . In high-dose females, these liver lesions were especially prominent and included bile duct and oval cell hyperplasia . Inflammation also occurred in the mesenteric lymph nodes, the adrenal gland, and the spleen . The amount of hemosiderin in the spleens of rats receiving the higher concentrations of the chemical mixture was less than normal . Components of a chemical mixture of 25 groundwater contaminants include acetone, aroclor 1260, arsenic, benzene, cadmium, carbon tetrachloride, chlorobenzene, chloroform, chromium, 1,1-dichloroethane, 1,2-dichloroethane, 1,1-dichloroethylene, 1,2-trans-dichloroethylene, di(2-ethylhexyl) phthalate, ethylbenzene, lead, mercury, methylene chloride, nickel, phenol, tetrachloroethylene, toluene, 1,1,1-trichloroethane, trichloroethylene, xylenes . In a 26-week study in which mice were exposed to the chemical mixture at concentrations of 0, 11, 38, 113, and 378 ppm in drinking water, there were no clear adverse effects noted in survival, weight gain, clinical pathology parameters, or histopathologic evaluations . Water consumption decreased with increasing dose, and water consumption by high-dose mice was approximately 40% less than that by the controls . In neurobehavioral assessments, no clear treatment-related effects were observed in measures of forelimb and hindlimb grip strength, hindlimb footsplay, motor activity, response to a thermal stimulus, or startle response in rats or mice evaluated at 6-week intervals throughout the 26- week drinking water studies . There were no effects on sperm morphology or motility or on estrous cycle length in rats or mice receiving the chemical mixture during the 26-week studies . Sperm concentration was decreased in F(1) CD-1(R) Swiss mice during continuous breeding studies, although there were no clear adverse effects on the fertility of Sprague-Dawley rats or CD-1(R) Swiss mice in th CD-1&reg; Swiss mice in these studies . Pup weight, the number of live males, and the number of male pups per litter were slightly decreased in dosed rats in the continuous breeding study in rats; the number of live female mouse pups in litters born of the F(0) and F(1) generations was decreased in the 378 ppm group . The significance of these observations, if any, is not known . F(1) mice receiving 378 ppm had increased incidences of hepatic inflammation compared to the controls . In female B6C3F(1) mice that received the chemical mixture in drinking water at concentrations as high as 756 ppm for 2 weeks or 378 ppm for 13 weeks, assessments of immune function showed suppression of hematopoietic stem cells and antigen-induced antibody-forming cells . This was manifested by impaired resistance to challenge with a nonlethal strain of mouse malaria, Plasmodium yoelii . Additional evidence of an adverse effect on hematopoietic stem cells was demonstrated by decreases in the in vitro colony-forming ability of granulocyte-macrophage progenitor cells and erythroid precursor cells isolated from female mice that had received the chemical mixture at a concentration of 378 or 756 ppm in 31.5 week studies . Potential genotoxic effects of the chemical mixture to the bone marrow of F344/N rats and B6C3F(1) mice were assessed in 2-week drinking water studies with concentrations as high as 756 ppm . Small increases in sister chromatid exchanges and micronucleated polychromatic erythrocytes occurred in the bone marrow of dosed male mice, and micronucleated polychromatic erythrocytes were also increased in dosed female mice . The chemical mixture did not induce mutations in Salmonella typhimurium strains TA98 and TA100 and did not induce DNA damage in Escherichia coli with or without metabolic activation . In summary, rats receiving drinking water containing a mixture of 25 common groundwater contaminants at levels of potential environmental relevance developed inflammatory lesions in the liver, spleen, lymph nodes, and adrenal gland, as well as evidence of an iron deficiency anemia . The inflammatory lesions could not be predicted based on the known toxic effects of the individual components of the chemical mixture . Mice exposed to similar concentrations of the chemical mixture did not show adverse effects in a standard toxicity study but developed deficits in bone marrow function, evidence of genetic damage, hepatic inflammation, and immunosuppression in other studies that generally included exposures to higher concentrations or exposures of longer duration . A no-observed-adverse-effect level for histologic injury (granulomatous inflammation of the liver) was 11 ppm in rats; however, no clear evidence for histologic injury was seen in mice exposed to concentrations of the chemical mixture as high as 378 ppm in a standard 26-week study . NOTE: These studies were supported in part by funds from the Comprehensive Environmental Response, Compensation, and Liability Act trust fund (Superfund) by an interagency agreement with the Agency for Toxic Substances and Disease Registry, U.S . Public Health Service. Toxic Rep Ser, 1992 Oct, 20, 1 - D10 NTP technical report on the toxicity studies of Diethanolamine (CAS No . 111-42-2) Administered Topically and in Drinking Water to F344/N Rats and B6C3F1 Mice; Melnick R; Diethanolamine is a high-production chemical used in cosmetics, in cutting fluids, as a dispersing agent for agricultural chemicals, and as an absorbent for acidic gases . Toxicology studies of diethanolamine were conducted in F344/N rats and B6C3F1 mice of both sexes for 2 weeks (5/sex/species/dose) and 13 weeks (10/sex/species/dose) to characterize and compare the effects of oral and dermal exposure . In addition to histopathology, evaluations included clinical pathology, urinalyses, and sperm morphology or vaginal cytology . In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and mouse lymphoma L5178Y cells, analysis of chromosomal aberrations and sister chromatid exchange in Chinese hamster ovary cells, and determination of micronuclei formed in mice during the 13-week dermal exposure study . Groups of rats and mice received drinking water containing diethanolamine at concentrations of up to 10000 ppm during studies of 2 or 13 weeks duration . In the 2-week studies, rats and mice of both sexes received in the were 0, 630, 1250, 5000, and 10000 ppm diethanolamine in the drinking water . In the 13-week studies, rats received 0, 320, 630, 1250, 2500, and 5000 ppm (males) or 0, 160, 320, 630, 1250, and 2500 ppm (females) in drinking water; male and female mice received 0, 630, 1250, 2500, 5000, and 10000 ppm . All female rats in the 2 highest dose groups and 2 males in the 10000 ppm group in the 2-week study died before the end of the study . In the 13-week study, deaths of mice occurred in the 3 highest dose groups; 2 male rats in the top dose group also died . Surviving animals in the higher concentration groups in both studies exhibited depressed weight gains . Rats receiving diethanolamine developed a poorly regenerative, microcytic anemia in both studies . In the 2-week study, dosed male and female rats had increased kidney weights, renal tubular cell necrosis, and decreased renal function; rats in the 13-week study also showed increased incidences or severity of nephropathy, tubular necrosis, and mineralization . Degeneration of the seminiferous tubules of the testis was noted in dosed males in both the 2- and 13-week studies, and sperm motility and count were decreased in the 13-week study . Demyelination in the brain (medulla oblongata) and spinal cord was observed in male and female rats in the 13-week study . In mice, dose-dependent increases in liver weight were observed in males and females in the 2-week study; cytologic alteration and necrosis of individual hepatocytes were observed in the highest dose group . In the 13-week drinking water study in mice, nephropathy and tubular necrosis were observed in males, and degeneration of cardiac myocytes, and hepatocellular necrosis were seen in males and females . Cytologic alteration in the submandibular salivary gland was noted in male and female mice . Hepatocyte cytologic alteration also was noted in all dosed groups of mice . In the 2-week dermal studies, groups of rats and mice were administered daily doses of diethanolamine in 95% ethanol, ranging from 160 to 2500 mg/kg for mice, and from 125 to 2000 mg/kg for rats, 5 days per week . In 13-week studies, dermal doses ranged from 32 to 500 mg/kg for rats, and from 80 to 1250 mg/kg for mice . In the 2-week study, early deaths of male rats and male and female mice occurred in the highest dose groups and in female rats in the 2 highest dose groups (1000 and 2000 mg/kg) . Body weight gains were reduced in rats and mice in the higher dose groups . Early deaths in the 13-week study were observed in the highest dose groups of rats (500 mg/kg) and mice (1250 mg/kg) . Body weight gains were reduced in rats and mice given the higher doses . Rats in the dermal studies exhibited dose-dependent hematologic and renal function changes similar to those observed in rats in the drinking water study . In addition, in the 2-week study, rats exhibited ulcerative skin lesions at the site of application, accompanied by inflammatory cell infiltration, hyperkeratosis, and acanthosis (hyperplasia) of the epidermis.dermis . Hyperkeratosis, without ulceration, was observed in some animals . Ulceration at the site of application was observed in male and female mice . Acanthosis, without ulceration or inflammatory cell infiltration, was observed in mice in all lower dose groups . In the 13-week study, skin lesions at the site of application included ulceration and inflammation, hyperkeratosis, and acanthosis . Liver weights were increased in male and female rats, but there were no associated histopathological changes . Other treatment-related effects observed in rats included demyelination in the brain and spinal cord, and nephropathy, renal tubular necrosis, and/or tubular mineralization; mice exhibited cytological alterations in the liver and/or hepatocellular necrosis, renal tubular epithelial necrosis, and cardiac myocyte degeneration . In in vitro genetic toxicity studies, diethanolamine was not mutagenic in Salmonella typhimurium or mouse L5178Y TK&plusmn; cells . Diethanolamine did not induce sister-chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells, nor did it induce micronuclei in peripheral blood erythrocytes in mice exposed by topical application for 13 weeks . All in vitro studies were conducted with and without S9 activation . Target organs of diethanolamine toxicity identified in these studies included bone marrow, kidney, brain, spinal cord, testis, and skin in rats, and liver, kidney, heart, salivary gland, and skin in mice . A no-observed-adverse-effect-level (NOAEL) was not achieved for hematological changes or nephropathy in rats (&lt;160 ppm), or for cytologic alteration of the liver in mice (&lt;630 ppm) in the drinking water studies . In the dermal studies, a NOAEL was not achieved for hematological changes, nephropathy, or hyperkeratosis of the skin in rats (&lt;32 mg/kg), or for cytologic alteration of the liver or acanthosis of the skin in mice (&lt;80 mg/kg) . Synonyms: 2,2&vprime;-iminodiethanol; 2,2&vprime;-iminobisethanol; diethylolamine; bis(hydroxy-ethyl)amine; 2,2'dihydroxydiethylamine; 2,2&vprime;-aminodiethanol. Toxic Rep Ser, 1992 Oct, 21, 1 - E14 NTP technical report on the toxicity studies of 2-Hydroxy-4-methoxybenzophenone (CAS No . 131-57-7) Adminstered Topically and in Dosed Feed to F344/N Rats and B6C3F1 Mice; French JE; 2-Hydroxy-4-methoxybenzophenone (HMB) occurs naturally in flower pigments and is synthesized for use in sunscreens, as a UV stabilizer in various cosmetic products, and in plastic surface coatings and polymers . Toxicity studies of HMB were performed in F344/N rats and B6C3F1 mice, by administering HMB in feed and by topical application, in studies of 2 weeks' (5 animals/sex, dose and species) and 13 weeks' (10 animals/sex, dose and species) duration . Assessments included hematology, clinical chemistry, urinalysis, reproductive toxicity, and histopathologic evaluations . In both 2- and 13-week dosed feed studies, rats received diets containing 0, 3125, 6250, 12500, 25000, or 50000 ppm HMB . One high-dose female rat died during the 2-week study . Body weight gains of high-dose male and female rats were reduced in the 13-week study . Liver and kidney weights were increased in dosed rats in both studies . In the 2-week studies, enlarged livers were associated with a marked hepatocyte cytoplasmic vacuolization in rats receiving diets containing concentrations of 6250 ppm HMB or higher; renal lesions, consisting of dilated tubules and regeneration of tubular epithelial cells, were found primarily in high-dose rats . In the 13-week studies, kidney lesions progressed to include papillary degeneration, or necrosis, and inflammation, while the liver lesion appeared to regress; liver enzymes in serum remained elevated . Rats receiving a diet with 50000 ppm HMB showed markedly lower epididymal sperm density and an increase in the length of the estrous cycle at the end of the 13-week studies . In 2-week dermal studies, rats received topical applications of 1.25 to 20 mg of HMB in an acetone or lotion vehicle . The only effects noted were small and variable increases in liver and kidney weights, reaching statistical significance primarily in the higher dose groups . In 13-week studies, rats received topical doses from 12.5 to 200 mg/kg HMB in acetone . Kidney weights were elevated in dosed groups of female rats . No other findings were attributed to HMB treatment . In 2- and 13-week dosed feed studies, mice received feed containing 0, 3125, 6250, 12500, 25000, or 50000 ppm HMB . A dose- related increase in liver weight associated with hepatocyte cytoplasmic vacuolization was the only finding in mice in the 2- week studies . Decreased body weight gains were dose-related in mice in the 13-week studies; mild increases in liver weights were seen in dosed mice of both sexes . Kidney weights were increased variably in dosed females . Microscopic lesions were noted only in the kidneys of males receiving 50000 ppm HMB; these included eosinophilic protein casts in dilated renal tubules and a mild inflammation associated with the dilated tubules . Mice in the highest dose group exhibited a decrease in epididymal sperm density and an increase in length of the estrous cycle . In 2-week dermal studies, mice received topical applications from 0.5 to 8 mg HMB in an acetone or lotion vehicle . The only effects noted were minimal, variable increases in liver and kidney weights, primarily in the higher dose groups . In 13-week studies, mice received topical doses of 22.75 to 364 mg/kg in acetone . Kidney weights were increased variably in dosed male mice . Epididymal sperm density was decreased at all 3 dose levels evaluated (22.75, 91, and 200 mg/kg) . The genetic toxicity of HMB also was evaluated in mutagenicity studies with Salmonella typhimurium, in cytogenetic studies with Chinese hamster ovary (CHO) cells, and by evaluation of micronucleated erythrocytes in peripheral blood smears from mice in the 13-week studies . HMB was weakly mutagenic in Salmonella with metabolic activation, and induced sister-chromatid exchanges and chromosomal aberrations in CHO cells in the presence of a metabolic activation system . There was no increase in the frequency of micronucleated erythrocytes in the blood of mice receiving HMB . In summary, HMB produced generally similar effects following topical and oral administration to rats and mice . Consistent findings included decreases in epididymal sperm density, lengthened estrous cycle, and increased liver and kidney weights . Mice in the dosed feed studies exhibited microscopic changes in the kidneys, comprising tubular dilatation with eosinophilic protein casts . Dilatation, tubular regeneration, papillary degeneration, and inflammation were noted in the kidneys of rats; and liver lesions consisting of an apparently reversible hepatocyte cytoplasmic vacuolization occurred in both rats and mice . A no-observed-adverse-effect level (NOAEL) for microscopic lesions was 6250 ppm HMB in the diet for rats and mice . A NOAEL was not reached for decreased epididymal sperm density in the 13- week dermal study in mice (&lt;23 mg/kg/day) . Synonyms: Oxybenzone; 4-Methoxy-2-hydroxy-benzophenone; Cyasorb UV; Uvinul M 40; (2-hydroxy-4-methoxyphenyl)phenyl-methanone; NSC-7778; Spectra-sorb UV; Syntase 62; UF 3; USAF CY-9; NCI-C60957. Toxic Rep Ser, 1992 Nov, 22, 1 - D20 NTP technical report on the toxicity studies of N,N-Dimethylformamide (CAS No . 68-12-2) Administered by Inhalation to F344/N Rats and B6C3F1 Mice; Lynch D; N,N-Dimethylformamide (DMF), a colorless liquid with a high boiling point, is a solvent used in a large number of industrial processes . Male and female F344/N rats (30/sex/group) and B6C3F1 mice (10/sex/group) were exposed to DMF vapors at concentrations of 0, 50, 100, 200, 400, or 800 ppm, 6 hours/day, 5 days/week, for 13 weeks in whole body exposure inhalation studies . In addition to histopathology, sperm morphology, and vaginal cytology, which were evaluated in both species, the studies examined clinical pathology, cardiovascular, and renal function in rats only . In genetic toxicity studies, DMF was not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, or TA98, with or without S9 activation, nor did it induce germ cell mutations in male Drosophila melanogaster treated by feeding or injection . No induction of sister chromatid exchanges or chromosomal aberrations was noted in cultured Chinese hamster ovary cells treated in vitro with DMF, with or without an S9 metabolic activation system . In one laboratory, a marginal increase in mutant colonies was observed after treatment of mouse lymphoma L5178Y/TK+/- cells with DMF in the absence of S9; results from studies in 2 other laboratories were negative . In the 13-week studies, all rats survived exposures to DMF . Body weight gains were reduced by 50-65% in rats exposed at 800 ppm and to a lesser extent in the 400 ppm group . Evidence of hepatocellular injury was noted as early as day 4, based on increases in activities of liver-specific enzymes in serum in rats of both sexes exposed at 200-800 ppm . Serum cholesterol levels were increased at all exposure concentrations . Relative liver weights were increased in male rats exposed at 100 ppm and higher concentrations, and in female rats at all concentrations . Minimal to moderate centrilobular hepatocellular necrosis was seen in rats of both sexes exposed at 400 and 800 ppm; the lesion was more severe in females . There were no clear, adverse effects seen in urinalyses, in electrocardiographic studies, or in male reproductive system evaluations that could be related to DMF exposure . Hematologic studies showed mild hemoconcentration in males and females . Prolonged diestrus was observed in females exposed at 800 ppm . Among mice exposed to DMF for 13 weeks, there was no chemically related mortality . Body weight gains were approximately 30% less than controls in females exposed at 800 ppm . Relative liver weights were increased in males and females at all exposure concentrations . Centrilobular hepatocellular hypertrophy (minimal to mild) was found in all groups of male mice exposed to DMF, and in female mice exposed at 100 ppm and higher concentrations . The length of the estrous cycle in mice increased with increasing DMF exposure . In summary, DMF-related effects were seen in the liver of both rats and mice, with rats being more severely affected . For rats of both sexes, the no-observed-adverse-effect level (NOAEL) was 200 ppm, based on the absence of liver histopathology, although liver function assays and liver weights showed changes at all exposure levels (as low as 50 ppm) . For mice, hepatocellular hypertrophy or increased liver weights occurred at all exposure concentrations . Synonyms: DMF, DMFA. Toxic Rep Ser, 1993 Mar, 23, 1 - E4 NTP technical report on the toxicity studies of ortho-, meta-, and para- Nitrotoluenes (CAS Nos . 88-72-2, 99-08-1, 99-99-0) Administered in Dosed Feed to F344/N Rats And B6C3F1 Mice; Dunnick J; Nitrotoluenes are high production volume chemicals used in the synthesis of agricultural and rubber chemicals and in various dyes . Because of differences in the metabolism of the 3 isomers and their capability to bind to DNA, comparative toxicity studies of o-, m-, or p-nitrotoluene were conducted in F344 rats and B6C3F1 mice . Animals were evaluated for histopathology, clinical pathology, and toxicity to the reproductive system . The nitrotoluenes were also studied in several in vitro and in vivo assays for genetic toxicity . In 14-day studies, o-nitrotoluene, m-nitrotoluene, or p-nitrotoluene was administered in the feed to male and female rats and mice at concentrations ranging from 388 to 20000 ppm (5 animals/chemical/species/sex/dose) . There were no effects on survival or clinical signs of toxicity in these studies, although animals at the higher doses showed decreases in body weight gains relative to controls . In the 13-week studies, o-, m-, or p-nitrotoluene was given to male and female rats and mice (10 animals/chemical/species/ sex/dose) in the feed at concentrations between 625 and 10000 ppm . The estimated daily doses based on measures of feed consumption were 40 to 900 mg nitrotoluene/kg body weight/day for rats and 100 to 2000 mg/kg/day for mice and were similar for each of the 3 isomers when compared for each dietary level/sex/species . There were no effects on survival in any of the studies, and clinical signs of toxicity were limited to decreases in feed consumption . Decreased body weight gains occurred in dosed rats and mice in all studies at the higher dose levels and were most pronounced in rats receiving o-nitrotoluene . In rats, histopathologic analyses after 13 weeks of dosing showed toxicity to kidney, spleen, and testis in animals receiving any of the 3 isomers, and toxicity to the liver and mesothelium in male rats given o-nitrotoluene . Kidney toxicity observed in male rats was characterized by the presence of hyaline droplets in tubular epithelial cells, attributed to an increase in the level of alpha-2&mgr;-globulin . Pigment, possibly lipofuscin, and karyomegaly in the p-nitrotoluene study were present in the renal tubular epithelium of dosed male and female rats . In the spleen of treated male and female rats, there was a mild increase in hematopoiesis, hemosiderin deposition, and/or congestion; this effect was most severe with the para-isomer, followed by the ortho- and then the meta-isomer . Administration of o-, m-, or p-nitrotoluene impaired testicular function of the rat, shown by degeneration of the testis and reduction in sperm concentration, motility, and spermatid number . All 3 isomers increased the length of the estrous cycle in rats . Hepatic toxicity was characterized by cytoplasmic vacuolization and oval cell hyperplasia and by an increase in the level of serum bile acids, SDH, and ALT activities in male rats given o-nitrotoluene . There was no histopathologic evidence for liver toxicity in male or female rats with the m- or p-isomers, or in female rats with the o-isomer; but evidence of liver injury was observed in these groups, indicated by increases in relative liver weights and elevations in bile acids and liver enzymes in serum . Mesotheliomas of the tunica vaginalis were observed in 3/10 male rats receiving o-nitrotoluene at 5000 ppm, and mesothelial cell hyperplasia was observed in 2/10 male rats receiving o-nitrotoluene at 10000 ppm . The only histopathologic evidence for toxicity in mice in the 13- week studies occurred in the olfactory epithelium in mice receiving o-nitrotoluene, where the chemical caused degeneration and metaplasia . No liver lesions were noted in mice, but the 3 isomers caused increases in relative liver weights . There was no toxicity to the reproductive system in male or female mice treated with any of the nitrotoluene isomers . The 3 nitrotoluene isomers were not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 . Only p-nitrotoluene induced chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells, and this required metabolic activation . Sister-chromatid exchanges were increased in CHO cells following exposure to each isomer; the requirement for metabolic activation varied . Only p-nitrotoluene was studied in the mouse lymphoma L5178Y test; it caused mutations with metabolic activation . Unscheduled DNA synthesis (UDS) was increased in in vitro incubations of hepatocytes isolated from both sexes of rats and mice after receiving a single in vivo oral dose of o-nitrotoluene . UDS was not increased in a similar study with male rats given m- or p-nitrotoluene . o-Nitrotoluene also induced s-phase DNA synthesis in hepatocytes of rats but not in those of mice . In summary, the 3 nitrotoluene isomers were toxic to the kidney, spleen and/or reproductive system in rats; o-nitrotoluene also caused lesions in the liver of male rats . No treatment-related lesions were noted in mice except with o-nitrotoluene where olfactory epithelium degeneration occurred . The increase in relative liver weights and the increase in UDS in liver indicate that all 3 isomers affected the liver of female rats and of male and female mice, even though histopathologic lesions were not observed . In general, the extent of the toxicity was most severe with the o-isomer in both rats and mice . o-Nitrotoluene was carcinogenic in male rats in 13-week studies, based on the occurrence of mesothelioma and mesothelial cell hyperplasia in dosed groups . Synonyms: o-NT, 2NT, 2-nitrotoluene, 2-methylnitrobenzene, 2-nitrotoluol; m-NT, 3NT, 3-nitrotoluene, 3-methylnitrobenzene, 3-nitrotoluol; p-NT, 4NT, 4-nitrotoluene, 4-methylnitrobenzene, 4-nitrotoluol. Toxic Rep Ser, 1993 Mar, 24, 1 - D8 NTP technical report on the toxicity studies of 1,6-Hexanediamine Dihydrochloride (CAS No . 6055-52-3) Administered by Drinking Water and Inhalation to F344/N Rats and B6C3F1 Mice; Hebert C; 1,6-Hexanediamine (HDA) is an aliphatic amine that is produced in large volumes in the United States . HDA is widely used as a corrosion inhibitor in lubricants and as an intermediate in the industrial synthesis of paints, resins, inks, and textiles . Toxicity studies of the dihydrochloride salt of HDA (HDDC) were conducted in male and female Fischer 344/N rats and B6C3F1 mice by the drinking water (2-week studies only) and whole-body inhalation routes (2-week and 13-week studies) . Animals were evaluated for histopathology, clinical chemistry, hematology, and reproductive toxicity . In addition, the genetic toxicity of HDA was assessed in Salmonella typhimurium and in Chinese hamster ovary cells in vitro; HDDC was evaluated in the mouse micronucleus assay in vivo . In the 2-week drinking water studies, groups of 5 rats of each sex received HDDC at doses of 0.75 to 6.7 mg/mL, and groups of 5 mice of each sex received doses of 0.2 to 3.0 mg/mL for 14 or 15 days . All animals survived to the end of the studies . No gross or microscopic pathologic changes and no clinical abnormalities related to HDDC consumption were seen in any dose group . The only statistically significant change was a slight decrease in absolute and/or relative liver weights of female rats in the 1.7, 5.0, and 6.7-mg/mL treatment groups, in male rats in the 3.0 mg/mL treatment group, and in female mice in the 0.8 mg/mL treatment group . Because there was no significant toxicity in these studies, 13-week drinking water studies were not conducted . In the 2-week inhalation studies, 5 rats and 5 mice of each sex were exposed to 0, 10, 30, 89, 267, or 800 mg HDDC/m(3) for 6-hours per day for 12 days . In the highest exposure group (800 mg/m(3)), all male and female rats, all female mice, and 2 male mice died before the end of the studies . In the remaining groups, there was a dose-dependent depression in body weight gain in male and female mice, but not in rats . Clinical signs were primarily related to upper respiratory tract irritation and included dyspnea and nasal discharge in rats and mice . Absolute and relative liver weights were reduced in some male mice, but this did not occur in a dose- dependent manner . In rats, histopathologic lesions that were considered related to chemical exposure included inflammation and necrosis of laryngeal epithelium as well as focal inflammation and ulceration of the respiratory and olfactory nasal mucosa . In mice, focal areas of inflammation and necrosis were present in the respiratory mucosa of the larynx and trachea in the 2 highest exposure groups . Nasal lesions, including focal inflammation and ulceration, and degeneration and necrosis of the olfactory and respiratory epithelium were also seen in mice . In addition, mild testicular degeneration was present in 2 mice from the highest exposure group (800 mg/m(3)) . In the 13-week inhalation studies, 10 rats and 10 mice of each sex were exposed to 0, 1.6, 5, 16, 50, or 160 mg HDDC/m(3) for 6 hours per day, 5 days per week for 13 weeks . In addition special groups of 20 male and 40 female rats and mice (mating trial animals) at each exposure level were included to assess the effect of HDDC on reproduction . All rats and mice in the base-study groups survived to the end of the studies, and there were no exposure-related changes in body weight . In the mating trials, 3 female mice exposed to 16 mg/m(3) and 1 female and 1 male mouse exposed to 50 mg/m(3) died before scheduled termination . These deaths, however, were not considered to be chemical related . In male mice in the base study, liver weights were increased relative to controls in the 2-highest exposure groups . No exposure-related changes in absolute or relative organ weights and no exposure-related clinical signs or gross lesions were seen in either species . In female rats, a dose-related decrease in white blood cell count was observed . Chemical-related microscopic lesions in male and female rats and mice were limited to the upper respiratory tract (larynx and nasal passages) in the 2 highest exposure groups and were similar in both species . These lesions included minimal to mild focal erosion/ulceration, inflammation, and hyperplasia of the laryngeal epithelium as well as degeneration of the olfactory and respiratory nasal epithelium . HDDC caused no significant changes in sperm morphology or in the length of the estrous cycle of rats or mice . In mating trials, HDDC demonstrated no adverse effects on reproduction of rats . The only statistically significant changes in reproductive parameters of mice were a slight increase in gestation length in the 50 mg/m(3) and 160 mg/m(3) exposure groups and a decrease in mean pup weight on Day 21 in the highest exposure group . These changes were not considered to be biologically significant . 1,6-Hexanediamine was not mutagenic in 4 strains of Salmonella typhimurium, and it did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells . These in vitro tests were conducted with and without exogenous metabolic activation (S9) . Negative results were also obtained in an in vivo test that measured the frequency of micronucleated erythrocytes in peripheral blood of male and female mice . In summary, the toxicity of HDDC to rats and mice resulted from irritant properties of the chemical and was consistent with the effects of other irritant chemicals administered by inhalation . This toxicity was limited to the nose and airways . In the 2-week inhalation studies, deaths occurred in both rats and mice at the highest exposure level (800 mg/m(3)) . In the 13-week studies, the no-observed-adverse-effect-level (NOAEL) for respiratory damage was 5 mg/m(3) for rats and mice . HDDC had no adverse effect on reproduction of either species and was not genotoxic . Synonyms: Hexamethylenediamine dihydrochloride; 1,6-diaminohexane dihydrochloride; 1,6-hexamethylenediamine dihydrochloride; 1,6- hexylenediamine dihydrochloride; 1,6-diamino-n-hexane dihydrochloride; HMDA; HDA; HDDC. Toxic Rep Ser, 1993 Mar, 25, 1 - E10 NTP technical report on the toxicity studies of Glutaraldehyde (CAS No . 111-30-8) Adminstered by Inhalation to F344/N Rats and B6C3F1 Mice; Kari F; Glutaraldehyde is a potent sensory irritant with the capability to cross-link, or fix, proteins . It is used industrially as an antimicrobial agent and as a cold sterilant in hospitals, and it has a variety of other industrial uses . The toxicity of glutaraldehyde was evaluated in 2-week and 13-week inhalation exposure studies in F344/N rats and B6C3F1 mice . In addition to histopathology, evaluations included clinical pathology and assessments of sperm morphology and estrous cycle length . In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and in mouse lymphoma L5178Y cells and analysis of chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells . The ability of glutaraldehyde to induce sex-linked recessive lethal mutations was also studied in vivo in Drosophila melanogaster . In 2-week inhalation studies, groups of five rats and five mice of each sex were exposed to glutaraldehyde by whole-body inhalation at concentrations of 0, 0.16, 0.5, 1.6, 5, and 16 ppm for 6 hours per day, 5 days per week . All rats and mice exposed to 5 or 16 ppm glutaraldehyde died before the end of the studies; all mice exposed to 1.6 ppm also died . Rats exposed to 1.6 ppm did not gain weight . Deaths were attributed to severe respiratory distress . Mice appeared to be more sensitive than rats because the small airways of the nasal passage of mice were more easily blocked by cell debris and keratin . Lesions noted in the nasal passage and larynx of rats and mice included necrosis, inflammation, and squamous metaplasia . At higher exposure concentrations, similar lesions were present in the trachea of rats and mice and in the lung and on the tongue of rats . In 13-week studies, groups of 10 rats and 10 mice of each sex were exposed to glutaraldehyde by whole-body inhalation at concentrations of 0, 62.5, 125, 250, 500, and 1000 ppb for 6 hours per day, 5 days per week . There were no exposure-related deaths in rats, but all mice exposed to 1000 ppb and two female mice exposed to 500 ppb died before the end of the study . Body weight gains were reduced in male rats exposed to 1000 ppb and in female rats exposed to 500 or 1000 ppb . Body weight gains of male mice exposed to 125, 250, or 500 ppb and female mice exposed to 250 or 500 ppb were reduced in a concentration-related manner . There was no clear evidence of systemic toxicity in rats or mice by histopathologic or clinical pathology assessments; however, exposure-related lesions in the respiratory tract were observed, and resembled those noted in the 2-week studies . In rats, the most severe lesions occurred in the anterior portions of the nasal passages and involved both the respiratory and olfactory epithelium . Hyperplasia and squamous metaplasia were most commonly noted on the lateral wall of the nasal cavity and on the tips of the nasoturbinates . Lesions were most extensive in rats exposed to 1000 ppb, but were also noted in the 250 and 500 ppb groups and in one male exposed to 125 ppb . In mice, histopathologic lesions in the respiratory tract were most severe in animals in the 1000 ppb group and consisted of minimal to mild squamous metaplasia of the laryngeal epithelium, suppurative inflammation in the anterior parts of the nasal cavity, and minimal squamous metaplasia on the tips of the nasoturbinates . Necrosis and inflammation were noted at lower concentrations, primarily in the anterior portion of the nasal passage . In genetic toxicity studies, glutaraldehyde was mutagenic with and without S9 metabolic activation in Salmonella typhimurium strains TA100, TA102, and TA104 . Glutaraldehyde was mutagenic in mouse L5178Y lymphoma cells in the absence of S9 and induced sister chromatid exchanges in Chinese hamster ovary cells with and without S9 . In one laboratory, chromosomal aberrations were induced in Chinese hamster ovary cells by glutaraldehyde in the absence of S9 only; no increase in chromosomal aberrations was observed with or without S9 in a second laboratory . Glutaraldehyde did not induce sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster treated as adults by feeding or injection or treated as larvae by feeding . In summary, exposure of rats and mice to glutaraldehyde by inhalation for up to 13 weeks resulted in a spectrum of necrotic, inflammatory, and regenerative lesions confined to the upper respiratory tract . Mice were somewhat more sensitive than rats because the small airways of the nasal passage in mice were more prone to blockage with cellular debris, bacteria, and keratin . The no-observed-adverse-effect level (NOAEL) was 125 ppb for respiratory lesions in rats . An NOAEL was not reached for mice, as inflammation was found in the anterior nasal passage at concentrations as low as 62.5 ppb . Synonyms: 1,5-Pentanedial; glutaral; glutaric dialdehyde; 1,3-diformylpropane. Toxic Rep Ser, 1993 Jul, 26, 1 - G15 NTP technical report on the toxicity studies of Ethylene Glycol Ethers: 2-Methoxyethanol, 2-Ethoxyethanol, 2-Butoxyethanol (CAS Nos . 109-86-4, 110-80-5, 111-76-2) Administered in Drinking Water to F344/N Rats and B6C3F1 Mice; Dieter M; Glycol alkyl ethers represent a class of high-production-volume chemicals with widespread industrial applications as solvents and chemical intermediates . Comparative toxicity studies with three glycol ethers, 2-methoxyethanol, 2-ethoxyethanol, and 2-butoxyethanol, were conducted in F344/N rats and B6C3F1 mice in both 2-week and 13-week drinking water studies . Toxicologic endpoints evaluated in animals included histopathology, hematology, clinical chemistry, urinalysis, and reproductive system parameters . Genetic toxicity was also evaluated for each glycol ether in several in vitro and in vivo assays . In the 2-week studies, groups of five male and five female rats and mice received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water . Estimates of compound consumption based on water consumption by male and female rats ranged from 100 to 400 mg/kg for 2-methoxyethanol, 200 to 1600 mg/kg for 2-ethoxyethanol, and 70 to 300 mg/kg for 2-butoxyethanol . For mice, consumption values ranged from 200 to 1300 mg/kg for 2-methoxyethanol, 400 to 2800 mg/kg for 2-ethoxyethanol, and 90 to 1400 mg/kg for 2-butoxyethanol . There were no chemical-related effects on survival for rats or mice in the 2-week studies . Decreased body weight gains were noted for both male and female rats treated with 2-methoxyethanol or 2-ethoxyethanol for 2 weeks, and there were dose-related decreases in water consumption for rats of each sex treated with the ethylene glycol ethers . Most of the changes in organ weights for rats and mice treated with the glycol ethers were sporadic (mice) or related to low final mean body weights (rats), except for thymic atrophy in male and female rats and testicular atrophy in males of both species receiving 2-methoxyethanol or 2-ethoxyethanol . In the 13-week studies in rats, groups of 10 males and 10 females received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water at concentrations ranging from 750 to 6000 ppm, 1250 to 20,000 ppm, or 750 to 6000 ppm, respectively . In the 13-week studies in mice, groups of 10 males and 10 females received 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol in the drinking water at concentrations ranging from 2000 to 10,000 ppm, 2500 to 40,000 ppm, or 750 to 6000 ppm, respectively . Estimates of compound consumption based on water consumption by male and female rats ranged from 70 to 800 mg/kg for 2-methoxyethanol, 100 to 2200- mg/kg for 2-ethoxyethanol, and 70 to 500 mg/kg for 2-butoxyethanol . For-mice, consumption values ranged from 300 to 1800 mg/kg for 2-methoxyethanol, 600 to 11,000 mg/kg for 2-ethoxyethanol, and 100 to 1300 mg/kg for 2-butoxyethanol . Chemical-related mortality occurred in male and female rats administered 4500 or 6000 ppm 2-methoxyethanol and in male and female rats administered 20,000 ppm 2-ethoxyethanol . No deaths occurred in rats administered 2-butoxyethanol or in mice administered 2-methoxyethanol, 2-ethoxyethanol, or 2-butoxyethanol . Decreased body weight gains occurred in dosed rats and mice in all three studies; the greatest reductions in body weight gain were seen with 2-methoxyethanol . In rats administered 2-methoxyethanol or 2-ethoxyethanol, treatment-related histopathologic changes were observed in the testes, thymus, and hematopoietic tissues (spleen, bone marrow, and liver) . A dose-related degeneration of the germinal epithelium in the seminiferous tubules of the testes was more severe in 2-methoxyethanol-treated rats than in rats treated with 2-ethoxyethanol . In special stop-exposure studies in male rats in which administration of the glycol ethers was stopped after 60 days, marked degeneration of the seminiferous tubules was present in rats treated with 3000 ppm 2-methoxyethanol, and mild to moderate degeneration was observed in rats treated with 1500 ppm . Moderate to marked testicular degeneration was present in rats treated with 10,000 or 20,000 ppm 2-ethoxyethanol but not in rats treated with 5000 ppm . After 30 and 56 days of recovery from treatment with these chemicals, only partial recovery from testicular degeneration was observed . There was no testicular degeneration after 60 days of treatment with 1500 to 6000 ppm 2-butoxyethanol . 2-Methoxyethanol treatment for 13 weeks resulted in a progressive anemia associated with a cellular depletion of bone marrow and fibrosis of the splenic capsule . Anemia was also seen with 2-ethoxyethanol, but evidence of an adaptive response was indicated by increased hematopoiesis in the bone marrow, spleen, and liver . Toxicity with 2-butoxyethanol was limited to the liver and hematopoietic system . Cytoplasmic alteration and a minimal hepatocellular degeneration were present in the liver of male and female rats . A minimal anemia was present, and a hematopoietic response was evident in the bone marrow and spleen . In mice, 2-methoxyethanol and 2-ethoxyethanol had similar effects on the testes, spleen, and adrenal gland (females only) . A dose-related degeneration of the germinal epithelium in seminiferous tubules of the testes was more severe with 2-methoxyethanol than with 2-ethoxyethanol . A dose-related increase in splenic hematopoiesis was also more prominent with 2-methoxyethanol . Both 2-methoxyethanol and 2-ethoxyethanol caused a prominent lipid vacuolization of the X-zone of the adrenal gland in female mice . There were no chemical-related lesions attributed to 2-butoxyethanol administration in mice . All three of the glycol ethers were negative in Salmonella typhimurium mutation tests conducted with and without induced hamster and rat liver S9 . In the mouse lymphoma L5178Y cell mutation assay, 2-ethoxyethanol was negative without S9 but was weakly positive in the presence of induced rat liver S9; 2-methoxyethanol and 2-butoxyethanol were not tested in this assay . At high concentrations, 2-ethoxyethanol induced sister chromatid exchanges (SCEs) in Chinese hamster ovary cells with and without S9 . Chromosomal aberrations (Abs) were also induced by 2-ethoxyethanol, but only in the absence of S9 and without a delay in cell cycle . In contrast, 2-butoxyethanol induced cell cycle delay but did not induce SCEs or Abs with or without S9 . 2-Ethoxyethanol was the only glycol ether tested for induction of sex-linked recessive lethal mutations in germ cells of Drosophila melanogaster; both feeding and injection trials were negative . In summary, based on survival, decreased body weight gains, and histopathologic effects, the rank order of toxicity for the three glycol alkyl ethers was 2-methoxyethanol>2-ethoxyethanol>2-butoxyethanol; the toxic effects were more severe in rats than in mice . In the 13-week study of 2-methoxyethanol in rats, a no-observed-adverse-effect level (NOAEL) was not reached, since testicular degeneration in males and decreased thymus weights in males and females occurred at the lowest concentration administered (750 ppm) . In the 13-week study of 2-ethoxyethanol in rats, the NOAEL for decreased thymus weights in males was 1250 ppm; for female rats treated with 2-ethoxyethanol for 13 weeks, the NOAEL for all histopathologic and hematologic effects was 5000 ppm . In rats treated with 2-butoxyethanol for 13 weeks, the NOAEL for liver degeneration was 1500 ppm in males and females . For male mice treated with 2-methoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 2000 ppm . A NOAEL was not reached for female mice treated with 2-methoxyethanol, since adrenal gland hypertrophy and increased hematopoiesis in the spleen occurred at the lowest concentration administered (2000 ppm) . For male mice treated with 2-ethoxyethanol for 13 weeks, the NOAEL for testicular degeneration and increased hematopoiesis in the spleen was 20,000 ppm . For female mice in the 13-week study of 2-ethoxyethanol, the NOAEL for adrenal gland hypertrophy and increased hematopoiesis in the spleen was 5000 ppm . No clear chemical-related effects were seen in male or female mice administered 2-butoxyethanol for 13 weeks at concentrations as high as 6000 ppm . Synonyms: 2-Methoxyethanol: Ethylene glycol monomethyl ether; methyl cellosolve; 2-Ethoxyethanol: Ethylene glycol monoethyl ether; cellosolve; 2-Butoxyethanol: Ethylene glycol monobutyl ether; butyl cellosolve. Toxic Rep Ser, 1993 Dec, 27, 1 - D9 NTP technical report on the toxicity studies of Riddelliine (CAS No . 23246-96-0) Administered by Gavage to F344 Rats and B6C3F1 Mice; Chan P; Riddelliine is a naturally occurring pyrrolizidine alkaloid, a class of compounds occurring in rangeland plants of the genera Crotalaria, Amsinckia, and Senecio . Two-week and 13-week rodent toxicity studies of riddelliine were conducted because riddelliine can be a contaminant of foodstuffs, such as meat, grains, seeds, milk, herbal tea, and honey . In addition to histopathology, evaluations included clinical pathology and reproductive toxicity . In vitro genetic toxicity studies included assessments of mutagenicity in Salmonella typhimurium and of the induction of chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells . Riddelliine was also evaluated in vivo for the induction of micronuclei in mouse bone marrow and in peripheral blood and for the induction of S-phase synthesis and unscheduled DNA synthesis in the liver of rats and mice . In the 2-week studies, groups of five male and five female F344/N rats and B6C3F1 mice were administered riddelliine in 0.1 M phosphate buffer by gavage at dose levels of 0, 0 . 33, 1.0, 3.3, 10, or 25 mg/kg body weight five times per week, for a total of 12 doses . Four of five male rats in the 25 mg/kg group died or were killed moribund before the end of the study . Mean body weight gains of male rats in the 10 and 25 mg/kg groups were depressed . No deaths or body weight effects were observed in female rats . Male rats had dose-related hemorrhagic centrilobular hepatic necrosis, hepatocytic karyomegaly and cytologic alterations, pulmonary hemorrhage and/or edema, splenic extramedullary hematopoiesis, and pancreatic edema . Female rats exhibited fewer and less severe lesions than identically treated male rats . Heart weights of treated male and female rats were lower than those of the controls . No deaths or effects on body weight were observed in treated mice . Dose-related increases in absolute and relative liver weights and increased incidences of hepatic cytomegaly were the only treatment-related findings in male and female mice administered riddelliine . In the 13-week studies, groups of 20 male and 20 female F344/N rats and B6C3FI mice were administered riddelliine in 0.1 M phosphate buffer by gavage five times per week for 13 weeks . Rats received 0, 0.1, 0.33, 1.0, 3.3, or 10 mg/kg and mice received 0, 0.33, 1.0, 3.3, 10, or 25 mg/kg . Ten animals from each dose group were killed after 13 weeks of treatment . The remaining 10 animals in each dose group were observed without further treatment for up to 14 weeks; five animals from each dose group were killed after 7 weeks of recovery, and the remaining five animals per dose group were killed at the end of the 14-week recovery period . During the 13-week treatment period, 19 of 20 male rats in the high-dose group died; all others survived . Body weight gains were decreased with increasing dose at Week 13 . During the 14-week recovery period, all male rats survived, but five high-dose females died . Mean body weight gains of dosed and control male rats were similar throughout the 1 4-week recovery period; the final mean body weights of the treated males approached the final mean body weight of the controls . Similarly, mean body weight gains among the treated female rats were similar to the control value at the end of the 14- week recovery period . However, the final mean body weight of female rats given 1.0 or 3.3 mg/kg remained lower than that of controls at the end of the 14-week recovery period . In the 13-week study, the most significant treatment-related histopathologic lesions in rats occurred in the liver and included hepatocyte cytomegaly and karyomegaly, cytoplasmic vacuolization, centrilobular necrosis, mixed inflammatory cell infiltration, and bile duct hyperplasia . Vascular lesions in the kidneys and lungs were observed in most high- dose rats after 13 weeks of riddelliine administration . Additional lesions were found in the heart, spleen, kidneys, and pancreas at 13 weeks . At the end of the 14-week recovery period, hepatocyte karyomegaly, cytomegaly, and cytoplasmic vacuolization persisted . In addition, the incidence of bile duct hyperplasia was markedly increased in dosed female rats, and foci of cytologic alteration or hyperplastic hepatocytes were observed in dosed rats that were allowed to recover for up to 14 weeks . Adenomas of the liver occurred in 2 of 10 females in the 10 mg/kg group at 13 weeks and in one of five females in this group after the 14-week recovery period; no adenomas were found in the livers of control females . Serum activities of alkaline phosphatase in male rats and sorbitol dehydrogenase in female rats increased with increasing dose . Reticulocyte counts consistently increased and platelet counts consistently decreased with increasing dose in treated male and female rats . The clinical pathology findings were indicative of liver damage and erythrocyte and platelet sequestration . In mice in the 13-week study, no deaths related to riddelliine treatment occurred . Body weight gains were depressed at the two highest dose levels (10 and 25 mg/kg); the depression in body weight persisted throughout the 14- week recovery period . Dose-related increases in erythrocyte counts in male mice and in reticulocyte counts in female mice were observed . Dose-related decreases in platelet counts were also observed in both males and females . Centrilobular cytomegaly in the liver was noted at 13 weeks in males and females administered 25 mg/kg riddelliine; this lesion persisted through the recovery period in females . At the end of the 14-week recovery period, bile duct hyperplasia was seen in the liver in high-dose female mice . Epithelial hyperplasia of the forestomach was noted in male and female mice in the 10 and 25 mg/kg groups after 13 weeks of treatment, but this lesion became less severe during the recovery period . In male rats administered up to 3.3 mg/kg and in male mice administered up to 25 mg/kg for 13 weeks, riddelliine did not adversely affect any of the reproductive end points evaluated . In female rats given 10 mg/kg and in female mice given 25 mg/kg, the length of the estrous cycle was increased . However, no unequivocal adverse effects were noted on fertility, pup growth and survival, or weight gain of dams during pregnancy during the mating trial in rats, although mean body weights of dams given 0.1 or 1.0 mg/kg were significantly lower than the mean body weight of the controls throughout gestation and lactation . In contrast, riddelliine administered at a dose of 25 mg/kg was toxic to the dams in the mouse mating trial, resulting in lower body weights at the beginning of gestation and throughout lactation . Administration of 25 mg/kg riddelliine to mouse dams also affected fetal growth and survival; the average live litter size was significantly reduced, the number of pups born dead was increased, and the average pup weight was reduced throughout the 21-day postpartum period . Riddelliine was mutagenic in Salmonella typhimurium strain TA100 with, but not without, S9 activation; results of mutagenicity testing were negative in strains TA97, TA98, and TA1535 . Riddelliine induced sister chromatid exchanges in Chinese hamster ovary (CHO) cells with and without S9 . Chromosomal aberrations were induced in CHO cells only in the presence of S9 . The frequency of micronucleated erythrocytes in mouse peripheral blood samples was not elevated after 4 or 13 weeks of daily gavage treatments; however, a weakly positive response was noted in the peripheral blood and bone marrow of male mice administered a single, high dose of riddelliine by gavage . Unscheduled DNA synthesis was detected in cultured hepatocytes from male and female rats and mice following 5 or 30 days of riddelliine treatment by gavage . In addition, an increase in S-phase DNA synthesis was observed in cultured hepatocytes of male and female rats treated for either time period . In summary, the administration of riddelliine to rodents by gavage for up to 13 weeks resulted in a spectrum of neoplastic and nonneoplastic effects similar to those previously described for other pyrrolizidine alkaloids . Rats were found to be somewhat more sensitive than mice, and males more sensitive than females, to the toxic effects of riddelliine . The no-observed-adverse-effect level (NOAEL) for histopathologic changes in the 13-week studies was 3.3 mg/kg body weight for mice and 0.1 mg/kg body weight for rats . The liver was the primary target of riddelliine-induced injury that resulted in lesions characterized by cytomegaly and cytologic alteration in rats and mice and also by marked necrotic and proliferative changes in rats . Riddelliine is carcinogenic to female F344/N rats, based on the occurrence of hepatocellular adenomas . Synonyms: 13,19-didehydro-12,18-dihydroxy senecionan-11,16- dione; trans-15-ethylidine-12b-hydroxy-12a-hydroxymethyl-13-methylenesenec-1-enine; 3-ethylidine-3,4,5,6,9,11,13,14,14a,14b-decahydro-6-hydroxy-6-(hydroxymethyl)-5-methylene (1,6)di-oxacyclododecino(2,3,4-gh)-pyrrolizidine-2,7-dione. Toxic Rep Ser, 1993 Jan, 28, 1 - D10 NTP technical report on the toxicity studies of Tetrachlorophthalic Anhydride (CAS No . 117-08-8) Administered by Gavage to F344/N Rats and B6C3F1 Mice; Mahler J; Tetrachlorophthalic anhydride (TCPA) is primarily used as a flame retardant in plastics . Toxicology studies were conducted by administering TCPA by oral gavage to F344/N rats and B6C3F1 mice for 13 weeks . Evaluations included histopathology, clinical pathology, and analyses of reproductive system parameters . The genetic toxicity of TCPA was assessed with in vitro tests of mutagenicity in Salmonella typhimurium and induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells; sister chromatid exchanges and chromosomal aberrations were also determined in mouse bone marrow cells following in vivo exposure . The ability of TCPA to induce sex-linked recessive lethal mutations was also studied in vivo in Drosophila melanogaster . Groups of 10 rats and 10 mice of each sex received TCPA in corn oil vehicle by oral gavage (5 days/week) at doses of 0, 94, 187, 375, 750, and 1500 mg/kg . The deaths of 5 male rats and 1 female rat in the 1500 mg/kg dose group and 1 female rat in the 750 mg/kg dose group were considered due to chemical toxicity . Mean final body weights and body weight gains were depressed in male rats in the 375, 750, and 1500 mg/kg groups and in all groups of female rats receiving TCPA . Relative liver weights were slightly increased in males and females at doses of 187 mg/kg and higher, although a dose relationship was not apparent . Heart weights of surviving male rats in the high-dose group were also increased . Male and female rats exhibited dose-dependent increases in kidney weights and in the incidence and severity of renal tubule necrosis and/or dilation . No clinical pathology changes were clearly associated with chemical exposure . There were no chemical-related effects on survival, body weights, or organ weights in dosed mice . No chemical-related lesions were identified in organs examined microscopically . Decreases in red blood cell parameters consistent with a mild, poorly regenerative anemia were the only evidence of possible compound toxicity in dosed mice . Sperm morphology and vaginal cytology evaluations in rats and mice revealed no adverse changes related to TCPA exposure . In genetic toxicology studies, TCPA, tested with and without exogenous metabolic activation (S9), was not mutagenic in Salmonella typhimurium and did not induce sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells . In the Drosophila melanogaster sex-linked recessive lethal test, TCPA gave equivocal results when administered by feeding and negative results when administered by injection . No induction of chromosomal aberrations was observed in bone marrow cells of mice 17 hours after intraperitoneal injection of TCPA, although an increase in sister chromatid exchanges was detected in these cells 23 hours after injection . In summary, clear evidence of organ toxicity following administration of TCPA in corn oil by gavage for 13 weeks was limited to the kidney of rats . The no-observed-adverse-effect level for histopathologic lesions in this tissue was not achieved with doses as low as 94 mg/kg per day . No significant adverse effects were seen in mice given doses as high as 1500 mg/kg per day for 13 weeks . Synonyms: 4,5,6,7-Tetrachloro-1,3-isobenzofurandione; 1,3-dioxy- 4,5,6,7-tetrachloroisobenzofuran; 3,4,5,6-tetrachloro-1,2-benzene- dicarboxylic anhydride; niagathal; tetrathal. Toxic Rep Ser, 1982 Mar, 12, 1 - B5 NTP technical report on the toxicity studies of Castor Oil (CAS No . 8001-79-4) In F344/N Rats And B6C3F1 Mice (Dosed Feed Studies); Irwin R; Castor oil is a natural oil derived from the seeds of the castor bean, Ricinus communis . It is comprised largely of triglycerides with a high ricinolin content . Toxicity studies with castor oil were performed by incorporating the material at concentrations as high as 10% in diets given to F344/N rats and B6C3F1 mice of both sexes for 13 weeks . Genetic toxicity studies also were performed and were negative for mutation induction in Salmonella typhimurium, for induction of sister chromatid exchanges or chromosomal aberrations in Chinese hamster ovary cells, and for induction of micronuclei in the peripheral blood erythrocytes of mice evaluated at the end of the 13-week studies . Exposure to castor oil at dietary concentrations as high as 10% in 13-week studies did not affect survival or body weight gains of rats or mice (10 per sex and dose) . There were no biologically significant effects noted in hematologic analyses in rats . Mild increases in total bile acids and in serum alkaline phosphatase were noted at various times during the studies in rats receiving the higher dietary concentrations of castor oil . Liver weights were increased in male rats receiving the 10% dietary concentration and in male and female mice receiving diets containing 5% or 10% castor oil . However, there were no histopathologic lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ in rats or mice . No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of estrous cycles of rats or mice given diets containing castor oil . Thus, no significant adverse effects of castor oil administration were noted in these studies . Synonyms: Ricinus Oil, oil of Palma Christi, tangantangan oil, phorboyl, Neoloid. Toxic Rep Ser, 1992 Jul, 14, 1 - C2 NTP technical report on the toxicity studies of para-Chloro-alpha,alpha,alpha Trifluorotoluene (CAS NO: 98-56-6) Administered in Corn Oil and alpha-Cyclodextrin to F344/N Rats and B6C3F1 Mice in 14-Day Comparative Gavage Studies; Jameson CW et al.; p-Chloro-alpha,alpha,alpha trifluorotoluene (CTFT) is a volatile, aromatic liquid used as a chemical intermediate in the manufacture of dinitroaniline herbicides . To evaluate the toxicity of CTFT, groups of F344/N rats and B6C3F1 mice of each sex were administered CTFT by gavage once a day for 14 consecutive days in either corn oil or in an experimental molecular complex vehicle, a-cyclodextrin (alpha-CD) . Dose levels selected for CTFT with the alpha-CD vehicle were 10, 50, and 400 mg/kg; dose levels used with the corn oil vehicle were 10, 50, 400, and 1000 mg/kg . The toxicokinetics of CTFT also were compared by gavage with the different vehicles and by i.v . administration . In genetic toxicity studies, CTFT was not mutagenic in Salmonella typhimurium . The elimination of an intravenous dose of CTFT from blood is best described by a triexponential equation . The data best fit a 3-compartment kinetic model with a very rapid distribution phase . A biexponential equation was found to best fit the elimination of CTFT from blood following a gavage dose in either corn oil or an aqueous molecular complex suspension, alpha-CD . However, the biological half-life (t 1/2) was the same in both routes, approximately 20 hours . Absorption of CTFT from the alpha-CD vehicle was found to be much faster than from corn oil . The average t 1/2 of the absorption phase for a 10 mg/kg dose of CTFT in the alpha-CD and corn oil vehicles was 7 and 150 minutes, respectively . Despite the differences in absorption, no statistical difference was observed in the calculated area under blood concentration versus time curves (AUC) obtained from rats dosed with CTFT in either vehicle . Blood concentrations of CTFT were proportional to dose, at levels as high as 400 mg/kg in both vehicles . The bioavailability of CTFT was shown to be complete in both vehicles, through comparing the AUC following oral and i.v . dosing . In 14-day toxicity studies, 1 of 10 female rats given the top dose of 1000 mg/kg CTFT in corn oil died on day 8; no deaths of male rats or of mice of either sex were attributable to the administration of CTFT . Body weight gains in all groups of rats and mice were similar with the exception of the top dose (1000 mg/kg) groups of male and female rats, which lost weight during the first week and resumed weight gain during the second . CTFT was found to accumulate in the kidneys of male rats, and there was a linear relationship between the kidney CTFT concentrations and the kidney levels of a2u-globulin, as determined by an ELISA assay . Microscopic changes in male rats included a dose-related toxic nephropathy consistent with that previously described as "hyaline droplet nephropathy." Dosed male and female rats also had hepatocyte hypertrophy and cytoplasmic vacuolization of the adrenal cortex . Clinical pathology findings suggested a mild anemia and cholestasis in rats . In contrast to rats, mice did not show appreciable CTFT concentrations in any tissue evaluated, suggesting a more rapid elimination of the chemical . However, hepatocellular hypertrophy, and clinical pathology findings consistent with cholestasis and mild liver injury, were noted in mice in the 400 and 1000 mg/kg dose groups . These studies demonstrated that oral doses of CTFT of 400 mg/kg or higher caused liver hypertrophy in rats and mice and adrenal changes in rats . Doses of 50 mg/kg or higher caused "hyaline droplet nephropathy" in male rats . The results were similar with CTFT administered either in corn oil or in alpha-CD (although absorption of CTFT was somewhat more rapid with alpha-CD), suggesting that alpha-CD may be an appropriate vehicle for toxicity studies with other chemicals . Synonyms: CTFT; p-Chloro-4-(trifluoromethyl) benzene; (p-chlorophenyl) trifluoromethane; 4-chlorobenzotrifluoride; Benzene, 1-chloro-4-(trifluoromethyl)-; p-(trifluoromethyl) chloro-benzene; p-chlorobenzotrifluoride; p-chlorotri-fluoromethylbenzene; p-trifluoromethylphenyl chloride; parachloro-alpha,alpha,alpha trifluorotoluene; parachlorobenzotrifluoride; parachlorotrifluoro-methylbenzene. Toxic Rep Ser, 1992 Jul, 17, 1 - E2 NTP technical report on the toxicity studies of Black Newsprint Inks Administered Topically to F344/N Rats and C3H Mice; Mahler J; Toxicity studies were conducted by applying black newsprint inks or mineral oils to clipped skin of the dorsal interscapular area of C3H mice and F344/N rats of both sexes, to determine systemic and local effects . Four lots of both letterpress and offset types of newsprint ink were studied, either as composite mixtures or as individual lots . An industrial grade mineral oil, used as an extender for newsprint ink formulation, and USP medicinal grade mineral oil also were studied . Analyses for the presence of polycyclic aromatic hydrocarbons (PAHs) were conducted on composite ink mixtures and mineral oils; letterpress and offset ink mixtures were found to have cumulative concentrations of 206 and 105 ppm, respectively; the concentration of PAHs in the printing ink mineral oil sample was 208 ppm, while none were detected in the USP grade mineral oil . In genetic toxicity studies, letterpress and offset newsprint ink composite mixtures were each mutagenic in Salmonella typhimurium strains TA98 and TA100 when tested in a preincubation protocol with added hamster liver S9 . With rat liver S9, results for both inks were positive in strain TA98 and negative in strain TA100 . Neither type of ink was mutagenic in the absence of S9 activation . In 30-day studies, 5 rats and mice per sex were given single, daily dermal applications of letterpress or offset newsprint inks, 5 days per week, for a total of 21 - 22 applications . Dose groups for each type of ink received either the neat (undiluted) composite ink mixture, or the 3:1, 1:1, or 1:3 dilutions (ink:USP mineral oil), with a total dose volume of 100 (mice) or 250 (rats) &mgr;l . All animals survived until the end of the studies . Toxicity attributed to ink administration was limited to decreased body weight gains in female rats treated with neat and the 3:1 dilution of letterpress ink, and to scaliness at the site of application in 1 or more mice in each letterpress ink treatment group . As a result of grooming activity and the large amount of test chemical applied, chemicals were spread over the body, and there was evidence that some oral ingestion had occurred . In 13-week studies, various ink and mineral oil formulations were administered dermally to 10 rats and mice per sex . To prevent accumulation of inks and distribution over the body as seen in the 30-day studies, the frequency of application was reduced to twice weekly and the total dose volume was decreased to 20 microliters for mice and 50 microliters for rats . Treatment groups for rats consisted of letterpress ink mixture, offset ink mixture, printing ink mineral oil, USP mineral oil, and clipped, untreated controls . Groups of mice were administered each of the 4 individual lots of both letterpress and offset inks, the composite mixtures of each, and printing ink and USP mineral oils; clipped, untreated groups served as controls . All rats, all male mice, and all female mice except one administered offset ink-lot E survived to the end of the studies . Effects attributable to compound administration in rats were limited to decreased body weight gains in females treated with printing ink mineral oil and letterpress ink mixture, and increased liver and kidney weights in both males and females exposed to USP mineral oil; there were no local toxic effects at the site of application . In mice, there were no body weight effects, but liver weights were increased in most ink and mineral oil treatment groups of both sexes . Dermal toxicity was evidenced in mice by scaliness and irritation at the site of application of both sexes treated with USP mineral oil and letterpress ink-lot C . Microscopically, local toxicity at the site of application was observed in mice of all treatment groups and was characterized by acanthosis and inflammation . In summary, results of these studies indicate that topical administration of black newsprint inks and mineral oils produces local toxicity at the site of application in mice; toxic effects on the skin in this species are consistent with those of a primary cutaneous irritant . In rats, possible evidence for toxicity was limited to decreased body weight gains in females treated with letterpress ink formulations. Toxic Rep Ser, 1993 Feb, 18, 1 - C10 NTP technical report on the toxicity studies of Methyl Ethyl Ketone Peroxide (CAS No . 1338-23-4) in Dimethyl Phthalate (CAS No . 131-11-3) (45:55) Administered Topically in F344/N Rats and B6C3F1 Mice; Zeiger E; Methyl ethyl ketone peroxide (MEKP) is an unstable organic peroxide used in the manufacture of acrylic resins, as a hardening agent for fiberglass-reinforced plastics, and as a curing agent for unsaturated polyester resins . It is commercially available as a 40% to 60% solution in dimethyl phthalate (DMP) . Because exposure to MEKP is typically through dermal contact, 2-week and 13-week toxicity studies were conducted by topical application of MEKP in DMP (45:55 w/w) to the clipped dorsal region of male and female Fischer 344/N rats and mice . Animals were evaluated for histopathology and for reproductive endpoints . In vitro genetic toxicity studies of MEKP included assessments of mutagenicity in Salmonella typhimurium and in mouse lymphoma L5178Y cells and analysis of chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells . In addition, the peripheral blood of mice from the 13-week study was evaluated in the micronucleus assay . In the 2-week studies, groups of 5 animals of each species and sex were administered MEKP in DMP for 5 days per week at doses of 50.6, 101.3, 202.5, 405, and 810 mg/kg body weight per day for rats and 112.5, 225, 450, 900, and 1800 mg/kg body weight per day for mice . Control groups received DMP or no treatment . No rats died during the studies, but at least 1 mouse in each group receiving MEKP died . Body weight gains of rats decreased with increasing doses of MEKP; body weight gains of mice were not affected by treatment . The primary effects of topical administration of MEKP in both rats and mice were an extensive coagulative necrosis of the epidermis and dermis, variable degrees of inflammation of the adnexa, and epidermal regeneration and hyperplasia at the application site . Lesions considered secondary to the dermal lesions included increased hematopoiesis in the spleen in rats and mice and increased myeloid hyperplasia of the bone marrow in mice, primarily at the higher doses . Mice showed a marked, dose-related increase in liver weight . In the 13-week dermal studies, groups of 10 rats and 10 mice of each sex were administered MEKP in DMP for 5 days per week at doses of 1.07, 3.57, 10.7, 35.7, and 107 mg/rat and 0.357, 1.19, 3.57, 11.9, and 35.7 mg/mouse . All high-dose mice, 3 high-dose female rats, and 1 female mouse in the 11.9 mg/animal group died or were sacrificed during the first week of the studies . Skin lesions similar to those seen in the 2-week studies were judged of sufficient severity to warrant early termination of surviving rats and mice in the 2 highest dose groups . All rats and mice in the remaining dose groups survived to the end of the studies, and weight gains were generally lower with increasing doses of MEKP . Skin lesions at the application site for the remaining animals (rats and mice) in the 10.7 mg/rat and 3.57 mg/mouse dose groups involved a spectrum of necrosis, inflammation, and acanthosis (epidermal hyperplasia) . Lesions in the lower dose groups were limited to acanthosis and hyperkeratosis in rats (1.07 and 3.57 mg/rat) and acanthosis in mice (0.357 and 1.19 mg/mouse) . While splenic and bone marrow lesions similar to those described in the 2-week studies were seen in animals that died early in the 13-week studies and in the rats and mice that showed ulcerative or necrotic injury, no other systemic changes were noted in animals that did not show ulcerative skin lesions . In genetic toxicity studies, MEKP in DMP (45:55 w/w) was not mutagenic in Salmonella typhimurium strains TA100, TA1535, TA1537, or TA98, with or without S9activation . A positive response was obtained in the mouse lymphoma assay for induction of trifluorothymidine resistance in L5178Y cells without S9 . In cytogenetic tests with Chinese hamster ovary cells, MEKP induced sister chromatid exchanges and chromosomal aberrations, with and without S9 . No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples obtained from male and female mice at the termination of the 13-week toxicity study . In summary, topical administration of MEKP in DM of MEKP in DMP resulted in a spectrum of necrotic, inflammatory, and regenerative skin lesions limited to the application site . Histopathologic changes in the spleen and bone marrow were also seen in rats and mice with ulcerative skin lesions, and were considered a secondary response . A no-observed-adverse-effect level (NOAEL) for histopathologic skin lesions could not be determined from these studies, as lesions were observed with administration of daily doses as low as 1.07 mg for rats and 0.357 mg for mice . Methyl Ethyl Ketone Peroxide Synonyms: 2-Butanone Peroxide . Dimethyl Phthalate Synonyms: 1,2-Benzenedicarboxylic acid dimethyl ester; phthalic acid dimethyl ester; methyl phthalate; dimethyl 1,2-benzene- dicarboxylate; DMP Environ Mol Mutagen, 2002, 40(2), 134 - 42 Mammalian cell cytotoxicity and genotoxicity analysis of drinking water disinfection by-products; Plewa MJ et al.; Cytotoxicity and genotoxicity assays were used to analyze drinking water disinfection by-products (DBPs) in Chinese hamster ovary (CHO) AS52 cells . The DBPs were chosen because they are common in drinking water, resulting from conventional disinfection using chlorination and chloramination . Data were also available to compare these results with cytotoxicity and mutagenicity studies in Salmonella typhimurium . The rank order in decreasing chronic cytotoxicity measured in a microplate-based assay was bromoacetic acid (BA) >> 3-chloro-4-(dichloromethyl)-5-hydroxy-2{5H}-furanone (MX) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > KBrO(3) > tribromoacetic acid (TBA) > EMS (ethylmethanesulfonate, positive control) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA) . The induction of DNA strand breaks by these agents was measured by alkaline single-cell gel electrophoresis (SCGE, comet assay) and the rank order in decreasing genotoxicity was BA >> MX > CA > DBA > TBA > EMS > KBrO(3), while DCA and TCA were refractory . BA was more cytotoxic (31x) and genotoxic (14x) than MX in CHO cells . BA was over 400x more genotoxic than potassium bromate . The brominated haloacetic acids (HAAs) were more cytotoxic and genotoxic than their chlorinated analogs . The HAAs expressed a statistically significant inverse relationship in CHO cell cytotoxicity and genotoxicity as a function of increased numbers of halogen atoms per molecule . A quantitative comparison was conducted with results from a previous study with cytotoxicity and mutagenicity in S . typhimurium . There was no correlation between chronic CHO cell and bacterial cell cytotoxicity . DBP-induced CHO cell cytotoxicity was not related to mutagenic potency in S . typhimurium . Cytotoxicity in CHO cells was statistically significant and highly correlated to CHO cell genotoxicity . Finally, we determined that the DBP genotoxic potency in CHO cells and the mutagenic potency in S . typhimurium were not related . This suggests that toxicity data in S . typhimurium did not quantitatively predict the toxic effects of DBPs in mammalian cell systems . The microplate CHO cell cytotoxicity and genotoxicity assays were well suited for the analysis of DBPs, especially when the quantity of test material is limited . Mutagenesis, 2002 Sep, 17(5), 439 - 44 Comparative potencies of induction of point mutations and genetic duplications by the methylating agents methylazoxymethanol and dimethyl sulfate in bacteria; Hoffmann GR et al.; Methylazoxymethanol (MAM) and dimethyl sulfate (DMS) are mutagens whose genetic effects can be ascribed to the methylation of DNA . While both methylate the N7 position of guanine heavily, only MAM strongly methylates the O(6) position of guanine . We evaluated the relative effectiveness and specificity of MAM and DMS in bacterial assays for the induction of point mutations and the formation of chromosomal duplications by genetic recombination . Salmonella typhimurium strain TS1121 was used to measure the formation of genetic duplications on the basis of the aroC321 allele and mutations by reversion of the hisG46 allele . Specific base pair substitutions and frameshift mutations were measured in a reversion assay based on lacZ alleles of Escherichia coli . The results show MAM to be the more potent mutagen and DMS the stronger recombinagen in the Salmonella assay . In the lacZ assay DMS induced several classes of base pair substitutions (GC-->AT transitions, GC-->TA transversions and AT-->TA transversions), as well as lower frequencies of +1, -1 and -2 frameshift mutations . The activity of MAM as a base pair substitution mutagen was more specific than that of DMS, inducing only GC-->AT transitions . It also induced +G, -G, -A and -CG frameshift mutations, though more weakly than it induced GC-->AT transitions . Long known as a base pair substitution mutagen, the induction of frameshifts by MAM was unexpected . The results show that both DMS and MAM are effective inducers of base pair substitutions and modest inducers of frameshifts and that DMS exhibits a broader spectrum of mutagenic activity than does MAM. Indian J Med Res, 2002 Mar, 115, 108 - 12 Some aspects of molecular epidemiology & characterisation of Salmonella Typhimurium isolated from man & animals; Rahman H; BACKGROUND & OBJECTIVES: Salmonellae are widely distributed in nature and cause a wide spectrum of diseases in man and animals . Among the different serovars of Salmonella enterica, S . Typhimurium is most commonly associated with enteric infection in man and animals . The present paper reports on the phage typing, plasmid profile, antimicrobial resistance and genetic diversity of Indian strains of S . Typhimurium isolated from man and animals . METHODS: A total of 17 strains of S . Typhimurium were subjected to phage typing and tested for plasmid and antimicrobial resistance . Genetic variability of the strains was studied by cleaving the whole cell DNA with restriction endonuclease, XbaI and detecting the restriction fragments by pulse-field gel electrophoresis (PFGE) . RESULTS: All the 17 strains were typeable and belonged to 4 phage types (DT003, DT004, DT096 and DT193) . Three plasmid profiles were observed . Multiresistance was common . Strains belonged to DT193 were highly resistant to most of the antimicrobial agents tested and harboured 3-4 plasmids . Strains belonged to DT003, DT004 and DT096 were found to be less resistant to antibiotics and harboured only 1 plasmid . A genetic variability was observed among the strains irrespective of the source of isolation . Twelve restriction patterns observed among the 17 strains as analyzed by PFGE indicated a considerable genetic heterogeneity among human and animal isolates . INTERPRETATION & CONCLUSION: A relationship was observed between resistance patterns, plasmid profiles and phage typing . Restriction analysis obtained by PFGE was found to be more discriminating in the differentiation of strains than the other methods used, and thus provides a valuable alternative tool for molecular characterisation of S . Typhimurium isolated from man and animals. Aquat Toxicol, 2002 Oct 30, 60(3-4), 223 - 31 Effects of enteric bacterial and cyanobacterial lipopolysaccharides, and of microcystin-LR, on glutathione S-transferase activities in zebra fish (Danio rerio); Best JH et al.; Cyanobacteria (blue-green algae) can produce a variety of toxins including hepatotoxins e.g . microcystins, and endotoxins such as lipopolysaccharides (LPS) . The combined effects of such toxins on fish are little known . This study examines the activities of microsomal (m) and soluble (s) glutathione S-transferases (GST) from embryos of the zebra fish, Danio rerio at the prim six embryo stage, which had been exposed since fertilisation to LPS from different sources . A further aim was to see how activity was affected by co-exposure to LPS and microcystin-LR (MC-LR) . LPS were obtained from Salmonella typhimurium, Escherichia coli, a laboratory culture of Microcystis CYA 43 and natural cyanobacterial blooms of Microcystis and Gloeotrichia . Following in vivo exposure of embryos to each of the LPS preparations, mGST activity was significantly reduced (from 0.50 to between 0.06 and 0.32 nanokatals per milligram (nkat mg(-1)) protein) . sGST activity in vivo was significantly reduced (from 1.05 to between 0.19 and 0.22 nkat mg(-1) protein) after exposure of embryos to each of the cyanobacterial LPS preparations, but not in response to S . typhimurium or E . coli LPS . Activities of both m- and sGSTs were reduced after co-exposure to MC-LR and cyanobacterial LPS, but only mGST activity was reduced in the S . typhimurium and E . coli LPS-treated embryos . In vitro preparations of GST from adult and prim six embryo D . rerio showed no significant changes in enzyme activity in response to the LPS preparations with the exception of Gloeotrichia bloom LPS, where mGST was reduced in adult and embryo preparations . The present study represents the first investigations into the effects of cyanobacterial LPS on the phase-II microcystin detoxication mechanism . LPS preparations, whether from axenic cyanobacteria or cyanobacterial blooms, are potentially capable of significantly reducing activity of both the s- and mGSTs, so reducing the capacity of D . rerio to detoxicate microcystins . The results presented here have wide ranging implications for both animal and human health. Dis Colon Rectum, 2002 Aug, 45(8), 1023 - 8 Cyclooxygenase-2 inhibition augments the hepatic antitumor effect of oral Salmonella typhimurium in a model of mouse metastatic colon cancer; Feltis BA et al.; INTRODUCTION: Oral inoculation with a nontoxic, attenuated strain of Salmonella typhimurium reduces tumor burden and improves survival in a mouse model of metastatic colon cancer . These effects are likely mediated by S . typhimurium-induced increases in hepatic natural killer leukocytes . Cyclooxygenase-2 inhibitors may mediate antitumor effects through antiangiogenic, immune, or proapoptotic pathways . We hypothesized that cyclooxygenase-2 inhibitors would act synergistically with S . typhimurium, resulting in additional antitumor effects . METHODS: Four groups of mice were studied: control, S . typhimurium alone, cyclooxygenase-2 inhibitor alone, and S . typhimurium plus cyclooxygenase-2 inhibitor . Mice were given normal drinking water (control, S . typhimurium alone) or water with 1,600 parts per million cyclooxygenase-2 inhibitor (cyclooxygenase-2 inhibitor alone, and S . typhimurium plus cyclooxygenase-2 inhibitor) and orally inoculated with saline (control, cyclooxygenase-2 inhibitor alone) or 10(9) S . typhimurium (S . typhimurium alone, S . typhimurium plus cyclooxygenase-2 inhibitor) . Twenty-four hours later, all mice underwent laparotomy, and 5 x 10(4) MCA38 murine adenocarcinoma cells were injected into the spleen . On Day 14, hepatic tumor number and tumor volume was quantitated and hepatic leukocytes were analyzed by flow cytometry . RESULTS: Compared with control mice orally inoculated with saline, S . typhimurium-treated mice had fewer and smaller tumors; mice treated with cyclooxygenase-2 inhibitor alone had tumor burden similar to control mice, and mice treated with S . typhimurium plus cyclooxygenase-2 inhibitor had fewer and smaller tumors compared with all other groups . Increased liver natural killer cells and decreased CD4+ and CD8+ T cells were observed in both S . typhimurium-treated groups . No alterations in hepatic leukocyte phenotype were observed in mice receiving cyclooxygenase-2 inhibitor alone . CONCLUSION: Oral cyclooxygenase-2 inhibitor appeared to act synergistically with S . typhimurium to reduce tumor burden . This combination therapy may have clinical application in the treatment or prevention of hepatic metastases associated with colorectal cancer. J Immunol, 2002 Sep 1, 169(5), 2545 - 52 Role of IL-12-independent and IL-12-dependent pathways in regulating generation of the IFN-gamma component of T cell responses to Salmonella typhimurium; John B et al.; Clearance of facultative intracellular pathogens such as Salmonella requires IFN-gamma from CD4 T cells . Mechanisms linking intracellular pathogen recognition with induction of IFN-gamma-producing T cells are still poorly understood . We show in this study that IL-12 is not required for commitment to the IFN-gamma-producing T cell response in infection with Salmonella typhimurium, but is needed for its maintenance . The IL-12-independent signals required for commitment depend on events during the first hour of infection and are related to Ag presentation . Even transient attenuation of Ag presentation early during infection specifically abrogates the IFN-gamma component of the resulting CD4 T cell response . The IL-12 needed for maintenance is also better induced by live rather than dead bacteria in vivo, and this difference is due to specific suppression of IL-12 induction by dead bacteria . Presence of exogenous IL-4 down-modulates IL-12 production by macrophages activated in vitro . Furthermore, macrophages from IL-4-null mice secrete high levels of both IL-12 and IL-18 in response to stimulation in vivo even with dead bacteria, but this does not lead to induction of IFN-gamma-secreting T cells in response to immunization with dead S . typhimurium . Early IL-4 is contributed by triggering of CD4 NK T cells by dead, but not live, bacteria . Thus, Ag presentation-related IL-12-independent events and IL-4-sensitive IL-12-dependent events play crucial complementary roles in the generation of the IFN-gamma-committed CD4 T cell component of the immune response in Salmonella infection. Mikrobiol Z, 2002 May-Jun, 64(3), 75 - 80 {Immunosuppressive components of extracellular lipopolysaccharide highly virulent strain Salmonella typhimurium 1468}; Molozhavaia OS et al.; Immunosuppressive activity of culture liquid substrate (CFS) of highly virulent strain Salmonella typhimurium has been studied . A model of delayed hypersensitivity (DHS) to nonbacterial antigen in mice, a method of gel-filtration through the sephadex column G-200, immunosorbents were used . Three components with immunosuppressive activity: thermolabile component and thermostable one with direct immunosuppressive action and the third thermolabile component which manifested inductive immunosuppressive activity only after redox treatment have been revealed in the strain CFS . O-specific and lipid parts were found in the composition of all the components . This allowed them to be related to lipopolysaccharide. Carcinogenesis, 2002 Sep, 23(9), 1537 - 40 Mutation, DNA strand cleavage and nitric oxide formation caused by N-nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity; Arimoto-Kobayashi S et al.; N-Nitrosoproline (NPRO) is endogenously formed from proline and nitrite . NPRO has been reported to be nonmutagenic and noncarcinogenic . In this study, we have detected the direct mutagenicity of NPRO plus natural sunlight towards Salmonella typhimurium . Furthermore, formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a mutagenic lesion, was observed in calf thymus DNA treated with NPRO plus simulated sunlight . The treatment with NPRO and sunlight induced single strand breaks in the superhelical replicative form of phage M13mp2 DNA . Single-strand DNA breaks also occurred in the human fibroblast cells on treatment with NPRO plus UVA, as detected by the comet assay . An analysis using scavengers suggested that both reactive oxygen species and NO radical mediate the strand breaks . The formation of nitric oxide was observed in NPRO solution irradiated with UVA . We analyzed the photodynamic spectrum of mutation induction and DNA breakage using monochromatic radiation at a series of wavelengths between 300 and 400 nm . Both mutation frequencies and DNA breakage were highest at the absorption maximum of NPRO, 340 nm . The co-mutagenic and co-toxic actions of NPRO and sunlight merit attention as possible mechanisms increasing the carcinogenic risk from UVA irradiation. Protein Expr Purif, 2002 Aug, 25(3), 437 - 47 Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli; Li Y et al.; Previously, we constructed human interleukin-6 (hIL-6)-secreting Escherichia coli and Salmonella typhimurium strains by fusion of the hIL-6 cDNA to the HlyA(s) secretional signal, utilizing the hemolysin export apparatus for extracellular delivery of a bioactive hIL-6-hemolysin (hIL-6-HlyA(s)) fusion protein . Molecular analysis of the secretion process revealed that low secretion levels were due to inefficient gene expression . To adapt the codon usage in hIL-6 cDNA to the E . coli codon bias, a synthetic hIL-6Ec gene variant was constructed from 20 overlapping oligonucleotides, yielding a 561-bp fragment, which comprises the complete hIL-6 cDNA sequence . Genetic fusion of the hIL-6Ec gene with the hlyA(s) secretional signal as an integral part of the hemolysin operon resulted in 3-fold higher hIL-6-HlyA(s) secretion levels in E . coli, compared to a strain expressing the original hIL-6-hlyA(s) fusion gene . An increase in the electrophoretic mobility of secreted hIL-6-HlyA(s) in non-reducing SDS-PAGE, similar to that found for recombinant mature hIL-6, and the absence of such a mobility shift in the intracellular hIL-6-HlyA(s) protein fraction indicated that in hIL-6-HlyA(s) most probably correct intramolecular disulfide bond formation occurred during the secretion step . To confirm the disulfide bond formation, hIL-6-HlyA(s) was purified by a single-step immunoaffinity chromatography from culture supernatant in yields of 18 microg/L culture supernatant with purity in the range of 60% . These results demonstrate that codon usage has an impact on the hemolysin-mediated secretion of hIL-6 and, furthermore, provide evidence that the hemolysin system enables secretory delivery of disulfide-bridged proteins. Eur J Biochem, 2002 Aug, 269(16), 4074 - 85 Functional characterization of the maltose ATP-binding-cassette transporter of Salmonella typhimurium by means of monoclonal antibodies directed against the MalK subunit; Stein A et al.; The maltose ATP-binding cassette transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic receptor (MalE) . In addition to its role in transport, the complex acts as a repressor of maltose-regulated gene expression and is subject to inhibition in the process of inducer exclusion . These activities are thought to be mediated by interactions of the ATPase subunit, MalK, with the transcriptional activator, MalT, and nonphosphorylated enzyme IIA of the glucose phosphotransferase system, respectively . To gain further insight in protein regions that are critical for these functions, we have generated nine MalK-specific monoclonal antibodies . These bind to four nonoverlapping linear epitopes: 60-LFig-63 (5B5), 113-RVNQVAEVLQL-123 (represented by 4H12), 309-GHETQI-314 (2F9) and 352-LFREDGSACR-361 (represented by 4B3) . All mAbs recognize their epitopes in soluble MalK and in the MalFGK2 complex with Kd values ranging from 10-6 to 10-8 m . ATP reduced the affinity of the mAbs for soluble MalK, indicating a conformational change that renders the epitopes less accessible . 4H12 and 5B5 inhibit the ATPase activity of MalK and the MalE/maltose-stimulated ATPase activity of proteoliposomes, while their Fab fragments displayed no significant effect . The results suggest a similar solvent-exposed position of helix 3 in the MalK dimer and in the intact complex and might argue against a direct role in the catalytic process . 4B3 and 2F9 exhibit reduced binding to the MalFGK2 complex in the presence of MalT and enzyme IIAGlc, respectively, thereby providing the first direct evidence for the C-terminal domain of MalK being the site of interaction with the regulatory proteins. Int J Pharm, 2002 Aug 21, 242(1-2), 15 - 24 Biodegradable microparticles for the mucosal delivery of antibacterial and dietary antigens; Fattal E et al.; Mucosal administration of antigen is known to be appropriate for vaccine purposes as well as tolerance induction . Biodegradable poly(DL-lactide-co-glycolide) (PLGA) microparticles were used to deliver both antibacterial phosphorylcholine (PC) and dietary antigen beta lactoglobulin (BLG) by mucosal route . In a first study, the protective immunity elicited by intragastric vaccination with PC encapsulated in microparticles was evaluated in a mouse model against intestinal infection by Salmonella typhimurium and pulmonary infection by Streptococcus pneumoniae . A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after oral immunization with PC-loaded microparticles compared with the titers of mice immunized with free PC-thyr or blank microparticles . This antibody response correlated with a highly significant resistance to oral challenge by S . typhimurium . IgA in pulmonary secretion were not able to protect against S . pneumoniae infection . BALB/c mice were, therefore, immunized intranasally (i.n.) . Immunization was followed by a rise in anti-PC IgA and IgG titers in serum and in pulmonary secretions by both free and encapsulated PC-Thyr . The survival rates were 91 and 76% in the two groups of mice, respectively . In a second study and in order to prevent allergy against milk by inducing oral tolerance, one of the major allergenic milk protein, BLG was entrapped into microparticles . Oral administration of microparticles containing BLG reduced significantly (by 10000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response . These studies demonstrate the ability of microparticles to induce both mucosal immunity and oral tolerance. Food Chem Toxicol, 2002 Nov, 40(11), 1589 - 94 Mutagenicity and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt; Kitano K et al.; Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution . In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay . Effective lethality was observed in the rec-assay . No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix) . In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix . The mutagenic activity became stronger increasing with the reaction period . Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity . PS, ascorbic acid and Fe-salts were inactive when they were used separately . Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive . These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity. Cell Microbiol, 2002 Aug, 4(8), 531 - 40 SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins; Yu XJ et al.; Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV) . We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies . To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria . We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly . The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors . SpiC was investigated further in view of its proposed role as an effector . Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria . However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria . Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro . An antibody against SpiC detected the protein on immunoblots from total cell lysates of S . typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins . Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria . Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein. Int J Toxicol, 2002 Jul-Aug, 21(4), 277 - 85 Genotoxicity and carcinogenicity studies of soy isoflavones; Misra RR et al.; The potential cancer preventive efficacy of soy isoflavones is being investigated in preclinical and phase 1 clinical studies sponsored by the U.S . National Cancer Institute . Although 90-day oral toxicity studies with PTI G-2535 (an investigational soy isoflavone drug product) in rats and dogs, as well as teratology studies, indicated no signs of toxicity, there remains a mechanistic concern associated with the ability of isoflavones (i.e., genistein) to inhibit topoisomerase, possibly leading to DNA strand breaks . The present report describes results from two in vitro genotoxicity studies, one in vivo genotoxicity study, and a single carcinogenicity study conducted in p53 knockout mice . Bacterial mutagenesis experiments using six tester strains without metabolic activation revealed no evidence that PTI G-2535 was mutagenic . In similar experiments with exogenous metabolic activation there were statistically significant increases in revertants, but less than twofold, in a single (Salmonella typhimurium TA100) tester strain . Mouse lymphoma cell mutagenesis experiments conducted with and without metabolic activation revealed statistically significant increases in mutation frequency at PTI G-2535 concentrations > or = 0.8 and 12 microg/ml, respectively; such increases were dose related and increases in the frequency of both small and large colonies were observed . A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was also seen 24 hours after treatment in male, but not female, mice who received 500 and 1000 mg/kg body weight PTI G-2535; however,such increases were small, were not dose related, and were not observed 48 hours after treatment . In contrast, dietary genistein had no effect on survival, weight gain, or the incidence or types of tumors that developed in cancer-prone rodents lacking the p53 tumor suppressor gene, p53 knockout mice . The apparent risk/benefit of isoflavone ingestion may ultimately depend on the dose and developmental timing of exposure. Pathol Biol (Paris), 2002 Jul, 50(6), 361 - 8 Subtyping of Salmonella typhimurium by pulsed-field gel electrophoresis and comparisons with phage types and resistance types; Lailler R et al.; One-hundred and sixty-eight Salmonella enterica subsp . enterica serotype Typhimurium isolates have been analysed by phage typing, by pulsed-field gel electrophoresis and for their antimicrobial susceptibility . Those independent strains, isolated from food animal production including cattle, poultry and pig sectors have been collected by the French non human Salmonella network, during the first semester in 1999 . Isolates encompassed 14 phage types . The majority of S . Typhimurium isolates was found to be definitive phage type DT104, representing 39% of all isolates . Other phage types were mainly DT8, PT U302, DT120, DT193 and DT135 . Forty-six pulsotypes were obtained using Xbal restriction enzyme, and amongst them, ten were associated to the DT104 phage type . A major pulsotype (px1), was represented by 79% of DT104 isolates and was also found among DT120 . Forty-eight percent of isolates showed a classic DT104 resistance profile to ampicillin, streptomycin, chloramphenicol, tetracycline, sulfonamides (ASCTSu) . Among this resistance type, 84% were DT104 and 12% were DT120 . Some pulsotypes were found associated to this resistant type . The pulsed field gel electrophoresis showed to be a useful typing method for discrimination of S . Typhimurium strains and for tracing clone through different sectors of origin in order to control their spread. Cancer Lett, 2002 Nov 28, 185(2), 123 - 30 Effects of cacao liquor proanthocyanidins on PhIP-induced mutagenesis in vitro, and in vivo mammary and pancreatic tumorigenesis in female Sprague-Dawley rats; Yamagishi M et al.; The effects of cacao liquor proanthocyanidins (CLPr) on 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP)-induced mutagenesis in vitro and on in vivo carcinogenesis in female Sprague-Dawley (SD) rats were investigated . In the Ames assay using Salmonella typhimurium TA98, CLPr showed strong antimutagenic effects against PhIP when assayed in the presence of S-9 mixture . For determination of the influence on initiation and subsequent development of lesions, CLPr (0.025% or 0.25%) were fed during the period of PhIP application (100 mg/kg given to rats via gastric tubes eight times over 4 weeks), or thereafter until the termination at 48 weeks . CLPr treatments did not affect body or organ weights . The incidences, multiplicities and volumes of mammary tumors in the 0.25% CLPr (post-initiation) group showed a tendency to decrease as compared to PhIP alone group values, although without statistical significance . The incidences of preneoplastic eosinophilic foci in the exocrine pancreas were significantly (P<0.05) decreased in a dose-dependent manner when CLPr were given during the initiation period . These results indicate that CLPr inhibit in vitro mutagenicity of PhIP, as well as rat pancreatic carcinogenesis in the initiation stage, but not mammary carcinogenesis induced by PhIP. J Anim Sci, 2002 Jul, 80(7), 1947 - 53 Effects of Ascophyllum nodosum extract on growth performance and immune function of young pigs challenged with Salmonella typhimurium; Turner JL et al.; Ninety-five pigs (initially 7.1 kg and 24 d of age) were used in a 28-d experiment to determine the effects of Ascophyllum nodosum seaweed extract (ANOD) on young pig growth performance and immune function in response to enteric disease challenge with Salmonella typhimurium (ST) . Experimental treatments were arranged in a 2 x 4 factorial with main effects of disease challenge (control vs ST-challenge) and dietary addition of ANOD (0, 0.5, 1.0, and 2.0% of diet) . Pigs were fed ANOD diets for 14 d and then challenged orally with ST or sterile media . There were no main effects of ANOD on growth performance end points, although there were significant quadratic effects of ANOD on ADG (P < 0.04) and final weight (P < 0.003), both being greatest at 1.0% ANOD . There was a positive linear effect of ANOD inclusion on ADFI (P < 0.07) and a negative linear effect on the gain-to-feed ratio (G/F) (P < 0.05) . ST-challenge reduced ADG (P < 0.05), ADFI (P < 0.05), and G/F (P < 0.05) in the first week following challenge . Daily estimates revealed reductions in feed intake in ST-infected pigs on d 2 to 4 following infection (P < 0.05) . Rectal temperature was increased maximally 2 d following ST-infection (P < 0.05) . A disease challenge x time interaction (P < 0.001) was observed for serum haptoglobin and alpha1-acid glycoprotein . Serum immunoglobulin M (IgM) was not influenced by disease challenge, but IgM declined (P < 0.001) in all pigs over time . Serum immunoglobulin G (IgG) also was not influenced by disease challenge, but IgG tended (P < 0.08) to increase over time . In vitro culture of porcine alveolar macrophages with 10 mg/mL ANOD elevated (P < 0.05) prostaglandin E2 (PGE2) production over that of controls at 3 and 24 h of culture . There was no interleukin-10 response by porcine splenocytes cultured in vitro with 0.005, 0.05, 0.5, or 5 mg/mL ANOD . We conclude that this model of enteric disease elicits an acute phase response that is accompanied by increased rectal temperature and diminished feed intake . Furthermore, our results indicate some beneficial effects of dietary ANOD on growth performance and no influence of dietary ANOD on immune response in the presence or absence of ST-challenge . However, high ANOD concentrations are capable of activating porcine alveolar macrophages in vitro to secrete PGE2. J Anim Sci, 2002 Jul, 80(7), 1939 - 46 Effects of a Quillaja saponaria extract on growth performance and immune function of weanling pigs challenged with Salmonella typhimurium; Turner JL et al.; Ninety-six pigs (initially 8.9 kg and 24 d of age) were used in a 28-d experiment to determine the effects of Quillaja saponaria extract (QS) on weanling pig growth performance and immune function in response to enteric disease challenge with Salmonella typhimurium (ST) . Experimental treatments were arranged in a 2 x 4 factorial with main effects of disease challenge (control vs ST-challenge) and dietary addition of QS (0, 125, 250, or 500 mg/kg) . Pigs were fed QS diets for 14 d and then challenged orally with ST or sterile media . There were no differences in ADG or ADFI among dietary treatments, but gain/feed ratio (G/ F) was depressed (P < 0.06) in pigs fed 250 mg/kg QS . ST-challenge reduced ADG (P < 0.05), ADFI (P < 0.05), and G/F (P < 0.05) 1 wk after challenge . Daily estimates revealed reductions in feed intake in ST-infected pigs on d 2 to 5 following infection (P < 0.05), and rectal temperature was increased maximally 2 d following infection (P < 0.05) . There was a marked decline in serum IGF-I during the 6 d after ST-infection (P < 0.05) . ST-challenge produced a rise (P < 0.05) in serum haptoglobin on d 7 after challenge, and serum alpha1-acid glycoprotein (AGP) in ST-challenged pigs also was elevated (P < 0.05) above controls on d 7 and 14 after challenge . Serum immunoglobulin (Ig) M increased (P < 0.05) over time in both groups, and serum IgM of ST-challenged pigs was greater than controls on d 7 after challenge (P < 0.05) . Serum IgG was not affected by enteric disease challenge; however, on d 7 and 14 after disease challenge, serum IgG for both groups was greater (P < 0.05) than on d 0 . Dietary QS had no significant influence on any of the end points used to characterize the acute phase response to ST-challenge . Phagocytic cell function was depressed in pigs fed 250 (P < 0.05) and 500 (P < 0.05) mg/kg as compared to pigs fed 125 mg/kg QS . Yet, there was no difference in phagocytic function among pigs fed 0, 250, or 500 mg/kg QS . We conclude that this model of enteric disease invokes an acute phase response accompanied by decreases in feed intake and serum IGF-I . Furthermore, dietary QS, at the levels fed in this study, appears to offer little benefit to growth performance or immune function in the presence or absence of ST-challenge. Mutat Res, 2002 Aug 26, 519(1-2), 199 - 204 Genotoxic activation of benzophenone and its two metabolites by human cytochrome P450s in SOS/umu assay; Takemoto K et al.; The genotoxic potential of benzophenone and its metabolically related compounds, benzhydrol and p-benzoylphenol, was investigated using human cytochrome P450 (P450) enzymes . Benzophenone and its two metabolites (0.1-1mM) showed a suppression of bacterial growth without any P450 system, but no induction of umu gene expression was observed in Salmonella typhimurium TA1535/pSK1002 . Human liver microsomes induced the bacterial cytotoxicity of these compounds without any umu gene expression . On the other hand, with the addition of Escherichia coli membranes expressing recombinant human P450 2A6 and NADPH-cytochrome P450 reductase (NPR), benzophenone showed umu gene expression (64 umu units/min/nmol) P450 2A6) . Moderate activation of benzophenone by P450 1A1/NPR membranes, 1A2/NPR membranes, or 1B1/NPR membranes was also observed . Activation of benzhydrol and p-benzoylphenol by the P450/NPR system was similar to that of benzophenone . These results suggest that benzophenone and its metabolically related benzhydrol and p-benzoylphenol can be bioactivated by P450 2A6 and P450 family 1 enzymes . Until now, benzophenone has been investigated mainly in terms of estrogenic activity and cytotoxicity, however, the genotoxic activation of benzophenone by human cytochrome P450s should be examined in terms of the risks to humans. Mutat Res, 2002 Aug 26, 519(1-2), 187 - 97 Seasonal fluctuation of the mutagenicity of river water in Fukui, Japan, and the contribution of 2-phenylbenzotriazole-type mutagens; Watanabe T et al.; To clarify their mutagenic potential, samples of water from the Mawatari, Asuwa and Kitsune rivers, which flow through the central area of Fukui, Japan, were seasonally collected at six sites using blue rayon from July 1998 to August 2000 . Forty-five of 52 (87%) of the water samples exhibited mutagenicity toward Salmonella typhimurium YG1024 and YG1029 with and without S9 mix, and the highest potencies were observed in YG1024 with S9 mix . The samples collected in summer and autumn tended to be more mutagenic than those collected in winter and spring . Fractionation using high-performance liquid chromatography (HPLC) suggests that several compounds are responsible for the mutagenicity of river water samples, and some of the major mutagens seem to be common among the samples . Three 2-phenylbenzotriazole (PBTA)-type mutagens, 2-{2-(acetylamino)-4-{(2-hydroxyethyl)amino}-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-3), 2-{2-(acetylamino)-4-amino-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) and 2-{2-(acetylamino)-4-{bis(2-hydroxyethyl)amino}-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6), were quantified in samples collected between July 1998 and April 1999 . At least one of these PBTA-type mutagens was detected in 23/24 (96%) of the samples . The amounts of PBTA-3, -4 and -6 were <0.08-58.7, <0.1-15.0 and <0.07-467.9 ng/g of blue rayon, respectively, and high levels of PBTA congeners were detected in the samples collected from each river in July and November 1998 . The contributions of these PBTA congeners to the mutagenicity of water samples were also high in July and November 1998 . The highest total contribution was observed for samples from the Asuwa river (67.6%) . These findings suggest that these three rivers were continually and heavily contaminated with mutagens, and PBTA congeners were some of the major mutagens in these rivers. Mutat Res, 2002 Aug 26, 519(1-2), 133 - 40 Chemical models for cytochrome P450 as a biomimetic metabolic activation system in mutation assays; Inami K et al.; DNA damage is a critical factor in carcinogenesis . The Ames assay is a short-term test that screens for DNA-damaging agents . To be detected in the assay, most carcinogens require oxidation by cytochrome P450, a component of the liver homogenate preparation (S9 mix) that is traditionally used to metabolize promutagens to an active form in vitro . A combination of iron(III) porphyrin plus an oxidant activates many promutagens by mimicking cytochrome P450 metabolism . We previously reported that the mutagenicity of the N-nitrosodialkylamines was detected following reaction with tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride (Fe(F(5)P)Cl) plus tert-butyl hydroperoxide (t-BuOOH), which yielded the same alcohols and aldehydes as the enzymatic reaction . In the present study, to extend the scope of biomimetic models, we tested the mutagenicity of other carcinogens exposed to chemical oxidation systems.We investigated the optimal assay conditions for the models in Salmonella typhimurium TA1538, a strain sensitive to frame-shift mutagens . We activated 2-aminofluorene (AF), benzo{a}pyrene (B{a}P), a tryptophane pyrolysate 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), and 2-acetylaminofluorene (AAF) with Fe(F(5)P)Cl plus an oxidant-t-BuOOH, m-chloroperoxybenzoic acid (mCPBA), or magnesium monoperoxyphthalate (MPPT)-and we noted the effect of three solvents-acetonitrile (CH(3)CN),1,4-dioxane, and N,N-dimethylformamide (DMF)-on AF activation.All the promutagens became mutagenic in the presence of Fe(F(5)P)Cl plus an oxidant, with the effectiveness of the oxidant varying with the chemical . Aromatic amines, for example, showed the strongest mutagenicity with t-BuOOH whereas polycyclic hydrocarbons showed the strongest mutagenicity with mCPBA . All the promutagens were mutagenic in the presence of Fe(F(5)P)Cl plus MPPT . For AF activation, the order of effectiveness of the solvents was CH(3)CN>1,4-dioxane>DMF . The results suggested that these systems would serve as useful models for microsomal activating systems. Mutat Res, 2002 Aug 26, 519(1-2), 121 - 31 Particulate matter, PM 10 & PM 2.5 levels, and airborne mutagenicity in Chiang Mai, Thailand; Vinitketkumnuen U et al.; Daily levels of particulate matter (PM) in the ambient air (PM 2.5 and PM 10) were measured in a northern city of Thailand (Chiang Mai) from March 1998 to October 1999 . Twenty-four-hour air particulate matter samples were collected each day with Airmetric Minivol portable air samplers . Monthly averages of PM 2.5 from four stations in Chiang Mai varied from 15.39 to 138.31microg/m(3) and 27.29 to 173.40 microg/m(3) for PM 10 . The PM 2.5 annual average was 58.48 mg/m(3) and PM 10, 86.38 microg/m(3) . Daily PM 2.5 (24h values) during the winter months in Chiang Mai frequently exceeded 200-300 microg/m(3) . The maximum concentrations of PM 2.5 (24h average) in Chiang Mai air from December 1998 to April 1999 were 2.8-, 3.5-, 4.2-, 6.5- and 3.2-fold higher than the US Environmental Protection Agency (US EPA), PM 2.5, 24h standard of 65 microg/m(3) . From May to October, the mean 24h levels of PM 2.5 and PM 10 were at acceptable levels . The data shows that during the winter season (December to March), levels of PM 2.5 and PM 10 in the Chiang Mai atmosphere are very high, and there may be significant health implications associated with these high concentrations . During the summer season, the fine particles were generally within the acceptable levels . To our knowledge, these are the first measurements of PM 2.5 to be reported for the city of Chiang Mai and they indicate considerable ambient fine particle exposures to the Chiang Mai population . In addition, dichloromethane extracts of airborne particulate matter PM 2.5 or PM 10 collected in the months of winter in the city of Chiang Mai were mutagenic to Salmonella typhimurium strain TA100 without metabolic activation . The mutagenicity appeared to track particle concentrations and increased in the presence of S9 mix. Toxicol Sci, 2002 Aug, 68(2), 437 - 43 Morphological transformation and oxidative stress induced by cyanide in Syrian hamster embryo (SHE) cells; Kamendulis LM et al.; Cyanide is a well-established poison known for its rapid lethal action and toxicity . Although long-term mammalian studies examining the carcinogenic potential of cyanide have not been previously reported, cyanide was reported to be positive in Salmonella typhimurium mutagenesis assay and induced aneuploidy in DROSOPHILA: To further evaluate the carcinogenic potential of cyanide, the ability of cyanide to induce morphological transformation in Syrian hamster embryo (SHE) cells was studied . Cyanide induced a dose-dependent increase in morphological transformation in SHE cells following a 7-day continuous treatment . A significant increase in transformation was observed at potassium cyanide doses of 200 microM and greater . Transformation induced by cyanide was inhibited in a dose-related manner by vitamin E, suggesting a role of oxidative stress in the induction of morphological transformation by cyanide . Further, it was shown that 500 microM cyanide induced oxidative DNA damage in SHE cells, evidenced by the formation of 8-hydroxy-2'-deoxyguanosine (50-66% increase over control) . The induction of oxidative stress by cyanide involved an early and temporal inhibition of antioxidant enzymes (catalase and superoxide dismutase) as well as an increased production of reactive oxygen species (1.5- to 2.0-fold over control). Biochemistry, 2002 Aug 6, 41(31), 9863 - 72 Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation; Gill HS et al.; The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site . The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5% . Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2% . The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions . A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis . These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state . We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop. Water Res, 2002 Jun, 36(11), 2802 - 12 Rapid detection of six types of bacterial pathogens in marine waters by multiplex PCR; Kong RY et al.; A rapid multiplex PCR (m-PCR) method that allows the simultaneous detection, in a single tube, of six commonly encountered waterborne pathogens is developed . The target genes used were: the aerolysin (aero) gene of Aeromonas hydrophila, the invasion plasmid antigen H (ipaH) gene of Shigella flexneri, the attachment invasion locus (ail) gene of Yersinia enterocolitca, the invasion plasmid antigen B (ipaB) gene of Salmonella typhimurium, the enterotoxin extracellular secretion protein (epsM) gene of Vibrio cholerae and a species-specific region of the 16S-23S rDNA (Vpara) gene of Vibrio parahaemolyticus were used as the gene targets . Multiplex PCR using the six pairs of primers produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in seawater and from pure cultures . The m-PCR assay was specific and rapid, with a turnaround time of < 12 h . The detection limit of the assay for the bacterial targets was estimated at 10(0)-10(2) cfu . Multiplex PCR analysis was performed on 19 seawater samples collected around Hong Kong and the results indicated significant levels of four bacterial pathogens at several sites where primary sewage wastes are discharged, and the levels of which showed no correlation with E . coli counts . Overall, both laboratory and field validation results demonstrated that the m-PCR assay developed in this study could provide a cost-effective and informative supplement to conventional microbiological methods for routine monitoring and risk assessment of water quality. Mol Biol Evol, 2002 Aug, 19(8), 1350 - 8 Gene location and bacterial sequence divergence; Mira A et al.; Previous comparison of a relatively small set of homologous genes from Escherichia coli and Salmonella typhimurium revealed that genes nearer to the origin of replication had substitution rates lower than genes closer to the replication terminus . The recently completed sequences of numerous bacterial genomes have allowed us to test whether this effect of distance from the replication origin on substitution rates, as observed for the E . coli-S . typhimurium comparison, is a general feature of bacterial genomes . Extending the analysis to all 3,000 E . coli-S . typhimurium homologs confirmed the significant association between chromosomal position and synonymous site divergence . However, the effect, though still significant, is not as dramatic as originally thought . A similar association between relative chromosomal location and synonymous substitution rate was detected in the majority of other bacterial species comparisons within alpha- and gamma- Proteobacteria, and Firmicutes but was absent in Chlamydiales . The opposite trend, i.e., a decrease in synonymous divergence with distance from the replication origin, was detected in Mycobacteria . Analysis of the patterns of nucleotide substitutions revealed that the distance effect is not affected by gene orientation and is mainly caused by an increase in rates of transversions, suggesting that this effect may not be caused by recombinational repair or biased gene conversion, as originally suggested. Mol Microbiol, 2002 Aug, 45(3), 627 - 35 Mutations in Bacillus subtilis glutamine synthetase that block its interaction with transcription factor TnrA; Fisher SH et al.; In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein- protein interaction with glutamine synthetase . During growth with excess nitrogen, the feedback-inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA . Missense mutations in glutamine synthetase that constitutively express the TnrA-regulated amtB gene were characterized . Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA-binding activity of TnrA . Two of the mutant proteins exhibited enzymatic properties similar to those of wild-type glutamine synthetase . A model of B . subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme . Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site . These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity. Bioresour Technol, 2002 Sep, 84(2), 191 - 6 The effects of feeding broiler litter on microbial contamination of beef carcasses; Davis JR et al.; Two experiments were conducted to test the effects of feeding broiler litter, either directly in the diet or indirectly through pasture-fertilization, to beef cattle on the incidence of Salmonella typhimurium (S) and Escherichia coli O157:H7 (EC) contamination of carcasses and ground beef . In Experiment 1, beef cows (n = 32) were allotted either ad libitum access to grass hay or a formulated diet (80% deep-stacked broiler litter and 20% corn) . In Experiment 2, beef cows (n = 32) were assigned to graze on pastures fertilized with a commercial fertilizer or fresh broiler litter . Cows in Experiment 1 were harvested following a 56-d feeding period; whereas, cows in Experiment 2 were harvested after 5, 10, 20, and 40 d of grazing pastures . All samples of muscle, purge, and ground beef were culture-negative for S and EC, suggesting that beef cattle may consume properly handled deep-stacked broiler litter, or pastures fertilized with fresh litter, without increasing the likelihood of carcass/meat contamination with S and (or) EC. Vet Microbiol, 2002 Aug 25, 88(2), 175 - 88 Herd prevalence of Salmonella spp . in Danish pig herds after implementation of the Danish Salmonella Control Program with reference to a pre-implementation study; Christensen J et al.; The problems addressed are: (1) comparison of prevalences of Salmonella spp . in different herd types in the Danish pig population after implementation of the Danish Salmonella Control Program (DSCP), and (2) to make a reference to a study from 1993/1994 (pre-implementation) with a discussion of possible biases when diagnostic methods differ slightly . The objectives were to present the prevalences of Salmonella spp., Salmonella Typhimurium, and multiresistant S . Typhimurium DT104 in Danish pig herds in 1998 . Further, to discuss how herd prevalences may be compared to a previous study.A bacteriological study in 1998 comprised: (a) a random sample of slaughter pig producing herds (N=1962); (b) a random sample of farrow-to-grower (sow) herds (N=305); and (c) all breeding and multiplying (genetic) herds (N=366) . A previous bacteriological study on Salmonella presence in 1993/1994 served as a model for the present study . The results of the study were that multiresistant S . Typhimurium DT104 was detected in one herd producing slaughter pigs . The herd apparent prevalences (HAPs) of Salmonella spp . were 11.7, 16.7 and 11.4% in genetic, sow, and slaughter pig herds, respectively . The conclusion of the study was that prevalence of multiresistant S . Typhimurium DT104 was low in the examined slaughter pig herds . The herd true prevalence (HTP) of Salmonella spp . in pigs had declined from before the start of the DSCP in 1993/1994 to 4 years later (1998). Arch Pharm Res, 2002 Jun, 25(3), 293 - 9 Cytotoxic and antimutagenic stilbenes from seeds of Paeonia lactiflora; Kim HJ et al.; Cytotoxic and antimutagenic effects of a novel cis-epsilon-viniferin and five known stilbenes, transresveratrol, trans-epsilon-viniferin, gnetin H, suffruticosols A and B, isolated from the seeds of Paeonia lactiflora Pall . (Paeoniaceae) were determined against five different cancer cell lines, and mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Salmonella typhimurium TA100, respectively . Six stilbenes showed cytotoxic activity in a dose-dependent manner, and especially did potent cytotoxic activity against C6 (mouse glioma) cancer cell with IC50 values ranging from 8.2 to 20.5 microg/ml . trans-Resveratrol showed significant cytotoxic activity against HepG2 (liver hepatoma) and HT-29 (colon) human cancer cell lines with IC50 values of 11.8 and 25.2 g/ml, respectively . In contrast, trans-epsilon-viniferin and cis--viniferin, and gnetin H exhibited marked cytotoxic activity against Hela (cervicse) and MCF-7 (breast) human cancer cell lines with IC50 values of 20.4, 21.5, and 12.9 microg/ml, respectively . However, suffruticosol A and B had less cytotoxic effect against all cancer cells except C6 . Meanwhile, six stilbenes exerted antimutagenic activity in a dose-dependent fashion . Of them, trans-resveratrol exhibited the strongest antimutagenic effect against MNNG with IC50 value of 27.0 microg/plate, while other five resveratrol oligomers also did moderate antimutagenic activity with IC50 values ranging from 31.7 to 35.2 microg/plate. J Infect Dis, 2002 Aug 1, 186(3), 372 - 8 Epub 2002 Jul 11. Salmonella serotype Typhimurium infection of bovine Peyer's patches down-regulates plasma membrane calcium-transporting ATPase expression; Santos RL et al.; To identify host genes differentially expressed during Salmonella enterica serotype Typhimurium infection, an RNA differential display was made with total RNA extracted from ileal loops that were infected with Salmonella Typhimurium 2.5 h after infection . Down-regulated cDNA was identified in bovine Peyer's patches after infection that was highly homologous to a human plasma membrane calcium-transporting ATPase (PMCA) . Differential expression of PMCA, evaluated by Northern analysis, was found to have more than a 4.6-fold decrease in expression of mRNA (size, approximately 5.1 kb) . PMCA mRNA was detected by in situ hybridization exclusively within epithelial cells in the Peyer's patches . cDNA (4.4 kb) was amplified by rapid amplification of cDNA ends, cloned, and sequenced and showed a high homology to hPMCA . Bovine PMCA is down-regulated in epithelial cells of Peyer's patches after infection with Salmonella Typhimurium and, subsequently, may influence cellular calcium levels that contribute to the inflammatory processes in the pathogenesis of diarrhea. J Immunol Methods, 2002 Aug 1, 266(1-2), 33 - 44 Surface plasmon resonance (BIACORE) detection of serum antibodies against Salmonella enteritidis and Salmonella typhimurium; Jongerius-Gortemaker BG et al.; We have used a surface plasmon resonance biosensor (BIACORE 3000) to detect serum antibodies in chickens having current or recent infections . Three well-defined Salmonella flagellar recombinant DNA antigens reflecting Salmonella enteritidis (H:g,m flagellin) and Salmonella typhimurium (H:i and H:1,2 flagellins) expressed in Escherichia coli were each immobilized in a single flow cell of a biosensor chip . Glutathione-S-transferase was immobilized on the surface of another flow cell to monitor non-specific binding . Sera collected from chickens with no history of Salmonella infection, and from chickens infected with Salmonella serotypes infantis, pullorum, gallinarum were used to test the performance of the system . The sensitivity exhibited to a range up to 900 arbitrary response units (RU) for the most positive S . typhimurium serum at a dilution of 1/40 . Sera from Salmonella infantis, Salmonella pullorum and Salmonella gallinarum infected birds gave responses less than the cut-off point, which was determined as the averaged response of sera from specific pathogen-free chickens plus three times the standard deviation . A positive response was obtained when these sera and whole blood were fortified with S . enteritidis and S . typhimurium positive serum . The sensitivity, specificity, precision and reproducibility obtained suggested that this approach could be used for detecting past or present infection with a range of pathogens in animals. Hum Gene Ther, 2002 Jul 1, 13(10), 1225 - 33 Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent; King I et al.; The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium expressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU) . The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated . TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration . TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were detected in tumors in mice treated with both TAPET-CD and 5-FC . No 5-FU could be detected in normal tissues . Inhibition of tumor growth was observed in mice treated with either TAPET-CD alone or TAPET-CD in combination with 5-FC (TAPET-CD/5-FC), but not with 5-FC alone . TAPET-CD/5-FC inhibited tumor growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79% . These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given. AIDS Patient Care STDS, 2002 Jun, 16(6), 247 - 50 Nontyphoidal salmonella bacteremia and pneumonia as the initial manifestation of human immunodeficiency virus infection in a four-year-old child; Eaton EE et al.; This report describes the case of a 4-year-old human immunodeficiency virus (HIV)-infected girl with Salmonella typhimurium bacteremia and pneumonia . The girl presented with a history of fever for 1 month and mild pulmonary symptoms . Subsequent studies revealed previously undiagnosed HIV disease. Chem Pharm Bull (Tokyo), 2002 Jul, 50(7), 1007 - 10 Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677; Terai T et al.; Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been used as a non-caloric sugar substitute in Japan . Pezzuto et al . demonstrated that steviol shows a dose-dependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the presence of metabolic activation system (Aroclor induced rat liver S9 fraction) . Our studies were carried out to identify the genuine mutagenic active substance from among the eight steviol derivatives . Steviol indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al . 15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence of S9 mixture . Interestingly, specific mutagenicity of the lactone derivative under the presence of S9 mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture. FEMS Microbiol Lett, 2002 Jul 16, 213(1), 81 - 5 Salmonella typhimurium displays cyclical patterns of sensitivity to UV-C killing during prolonged incubation in the stationary phase of growth; Child M et al.; Stationary phase cells of Salmonella typhimurium were more resistant to killing by UV-C irradiation than those from the exponential phase . Analysis of the tolerance of cells taken at different stages of prolonged incubation as batch cultures to 60 or 100 J m(2) doses of UV-C revealed cycles of resistance and tolerance . The possible involvement of rpoS-controlled functions in mediating these cycles could be discounted because they were also detected in an rpoS minus mutant of S . typhimurium . The results are discussed in the context of heterogeneity in cells of stationary phase cultures of S . typhimurium. J Ethnopharmacol, 2002 Aug, 81(3), 381 - 6 Antimutagenic potential of homoisoflavonoids from Muscari racemosum; Miadokova E et al.; The potential antimutagenic effect of the plant extract of Muscari racemosum bulbs, rich on 3-benzylidene-4-chromanones, was evaluated on three genetic model organisms . The mixture of three homoisoflavonoids was applied together with diagnostic mutagens in the Ames assay on four bacterial strains Salmonella typhimurium TA97, TA98, TA100, TA102, in the toxicity and mutagenicity/antimutagenicity assay on the yeast strain Saccharomyces cerevisiae D7, and in the simultaneous phytotoxicity and clastogenicity/anticlastogenicity assay on Vicia sativa (L.) . The extract exerted antimutagenic and anticlastogenic effects due to the presence of homoisoflavonoids, which may be included in the group of natural antimutagens . This genotoxicological study suggests that homoisoflavonoids from M . racemosum (L.) owing to antimutagenic and anticlastogenic properties are of great pharmacological importance, and might be beneficial for prevention of cancer. Vaccine, 2002 Jul 26, 20(23-24), 2878 - 86 DNA immunization using a secreted cell wall antigen Mp1p is protective against Penicillium marneffei infection; Wong LP et al.; None of the vaccines used in dimorphic fungal infections utilized the mucosal route for immunization, whereas only one utilized a secreted protein as antigen, despite knowing that infections caused by dimorphic fungi are usually acquired through inhalation . In this study, we investigated the usefulness of Mp1p (a secreted cell wall antigen encoded by MP1)-based vaccines for generation of protective immune responses against Penicillium marneffei infection using a mouse model, and compared the relative effectiveness of intramuscular MP1 DNA vaccine, oral mucosal MP1 DNA vaccine delivered by live-attenuated Salmonella typhimurium, and intraperitoneal recombinant Mp1p protein vaccine . The serum IgM level of the Mp1p protein vaccine group at day 7 and the serum IgG levels of the Mp1p protein vaccine group at days 7 and 21 were significantly higher than those of the other groups (P<0.0001) . The serum IgG level of the MP1 DNA vaccine group was significantly higher than that of the corresponding control group and oral mucosal MP1 DNA vaccine group (one dose) at day 21 (P<0.0001 and <0.05, respectively) . The groups of mice immunized with intramuscular MP1 DNA vaccine, oral mucosal MP1 DNA vaccine, and intraperitoneal Mp1p protein vaccine showed significantly higher Mp1p-specific lymphocyte proliferation index (LPI) than the control groups . The interferon-gamma (IF-gamma) levels of supernatant of splenic cell cultures obtained from mice after intramuscular MP1 DNA vaccine, mucosal MP1 DNA vaccine (three doses), or intraperitoneal Mp1p protein vaccine administration were higher than that which occurred after mucosal MP1 DNA vaccine (one dose) administration or those of controls . Interleukin-4 (IL-4) was not detectable in the supernatant of splenic cell cultures obtained from all groups of mice . The percentage survival of the mice immunized with intramuscular MP1 DNA vaccine, oral mucosal MP1 DNA vaccine (three doses), oral mucosal MP1 DNA vaccine (one dose), intraperitoneal recombinant Mp1p protein, oral live-attenuated S . typhimurium control, and intramuscular pJW4303 DNA control at day 60 after wild type P . marneffei challenge were 100, 60, 40, 40, 40, and 0%, respectively . The survival of mice in the MP1 DNA vaccine group was significantly better than those of the oral mucosal MP1 DNA vaccine (three doses) group (P<0.05), oral mucosal MP1 DNA vaccine (one dose) group (P<0.005), recombinant Mp1p protein group (P<0.005), S . typhimurium aroA strain group (P<0.05), and pJW4303 group (P<0.00001) . Although, the mechanism by which intramuscular MP1 DNA vaccine offered the best protection against P . marneffei infection remains to be elucidated, the present observation prompted further clinical trials on the use of MP1 DNA immunization on asymptomatic human immunodeficiency virus carriers in P . marneffei endemic areas. J Biol Chem, 2002 Sep 27, 277(39), 36748 - 54 Epub 2002 Jul 15. Organization of the receptor-kinase signaling array that regulates Escherichia coli chemotaxis; Levit MN et al.; Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors . In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles . We found that these complexes have at least 6 receptors per CheA . CheW is not required for CheA binding to receptors, but is essential for kinase activation . The kinase activity per mole of bound CheA is proportional to the total bound CheW . Similar results were obtained with the E . coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar . In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is approximately 6 Tsr:4 CheW:1 CheA . Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules. Microb Drug Resist, 2002 Summer, 8(2), 107 - 22 Diversity in antimicrobial resistance and other characteristics among Salmonella typhimurium DT104 isolates; Poppe C et al.; Multiresistant Salmonella enterica subspecies enterica serovar Typhimurium definitive type 104 (S . Typhimurium DT104 or DT104) bacteria are important pathogens in animals and humans . DT104 isolates are often called pentaresistant strains that spread clonally . The purpose of this study was to determine phenotypic, genotypic, and epidemiologic characteristics of 175 S . Typhimurium DT104 strains isolated from food-producing animals in Canada . More than 90% of the isolates were resistant to ampicillin (Amp), chloramphenicol (Chl), florfenicol (Flo), sulfisoxazole (Sul), and tetracycline (Tet), 53% of the isolates were additionally resistant to spectinomycin (Spc) and streptomycin (Str), and 28% to kanamycin (Kan) and neomycin (Neo) . Sixty-one percent of the strains harbored a single 60-MDa plasmid, 21% contained 60- and 2.0-MDa plasmids, and 4% had 60, 4.6- and 2.0-MDa plasmids . Resistance to Kan and Neo was encoded by the aminoglycoside aphA-1 gene on 2.0-MDa plasmids, whereas resistance to trimethoprim (Tmp) and Sul was encoded by the dhfrIb gene on 4.6-MDa plasmids . Polymerase chain reactions (PCR) showed the presence of integrons with the ant (3")-Ia aminoglycoside adenyltransferase and the bla(PSE-1) beta-lactamase gene cassettes, and the presence of the flost gene in all but one strain resistant to Spc and Str, Amp, and Chl and Flo, respectively . DT104 isolates from cattle at six feedlots represented a separate clone; they were sensitive to Str and Spc and lacked the ant (3")-Ia gene . Pulsed-field gel electrophoresis (PFGE) using Bln I, Spe I, and Xba I resulted in 15, 12, and 8 PFGE patterns, respectively . In summary, we observed considerable diversity in phenotypic, genotypic, and epidemiological characteristics among the DT104 isolates. Toxic Rep Ser, 2002 Apr, (55), 1 - F12 NTP Technical Report on the toxicity studies of trans-1,2-dichloroethylene (CAS no . 156-60-5) administered in microcapsules in feed to F344/N rats and B6C3F(1) mice; Ress NB; 1,2-Dichloroethylene exists in two isomeric states: trans-1,2-dichloroethylene and cis-1,2-dichloroethylene . The trans isomer is used more widely in industry than the cis isomer . trans-1,2-Dichloroethylene is used as a solvent for waxes, resins, and acetylcellulose . It is also used in the extraction of rubber, as a refrigerant, and in the manufacture of pharmaceuticals and artificial pearls . F344/N rats and B6C3F1 mice were administered trans-1,2-dichloroethylene in microcapsules in feed for 14 weeks . Animals were evaluated for clinical pathology, reproductive system effects, and histopathology . Genetic toxicity studies were conducted in vitro in Salmonella typhimurium and Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow cells and peripheral blood erythrocytes . In the 14-week feed studies, groups of 10 male and 10 female rats and mice were fed diets containing microcapsules with a chemical load of 45% trans-1,2-dichloroethylene . Dietary concentrations of 3,125, 6,250, 12,500, 25,000, and 50,000 ppm microencapsulated trans-1,2-dichloroethylene resulted in average daily doses of 190, 380, 770, 1,540, and 3,210 mg/kg for male rats; 190, 395, 780, 1,580, and 3,245 mg/kg for female rats; 480, 920, 1,900, 3,850, and 8,065 mg/kg for male mice; and 450, 915, 1,830, 3,760, and 7,925 mg/kg for female mice . Additional groups of 10 male and 10 female rats and mice served as untreated and vehicle controls . There were no exposure-related deaths of rats or mice . Mean body weights of male rats and male and female mice in the 50,000 ppm groups were significantly less than those of the vehicle controls . The mean body weight gains of female mice in the 12,500 and 25,000 ppm groups were also significantly less than that of the vehicle controls . On day 21 and at week 14, there were mild decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts in groups of male and female rats in the 25,000 and 50,000 ppm groups . At week 14, these effects were seen in male rats exposed to 6,250 and 12,500 ppm . There were no exposure-related alterations in clinical chemistry parameters in rats or mice . The liver weights of female rats exposed to 6,250 ppm or greater were significantly greater than those of the vehicle controls . The absolute kidney weights of male rats exposed to 25,000 or 50,000 ppm were significantly decreased . No gross or microscopic lesions were observed in rats or mice that could be attributed to trans-1,2-dichloroethylene exposure . Neither cis-, trans-, nor cis,trans-1,2-dichloroethylene was mutagenic in S . typhimurium strain TA97 (cis isomer only), TA98, TA100, TA1535, or TA1537, with or without S9 metabolic activation enzymes . In CHO cells in vitro, cis- 1,2-dichloroethylene induced sister chromatid exchanges (SCEs) in the absence of S9; with S9, the single trial that was performed yielded equivocal results . The cis,trans isomer induced significant increases in SCEs in cultured CHO cells with and without S9 . In contrast to these positive results, trans-1,2-dichloroethylene gave negative results in the SCE test, with and without S9 . Neither cis-, trans-, nor cis,trans-1,2-dichloroethylene induced chromosomal aberrations (Abs) in cultured CHO cells, with or without S9 . In vivo, no induction of SCEs or Abs was noted in bone marrow cells of male mice administered cis- or trans-1,2-dichloroethylene by intraperitoneal injection once, with sampling performed 23 hours (for SCE analyses) or 17 hours (for Abs analyses) after injection . In addition, negative results were obtained in a peripheral blood micronucleus test in male and female mice administered trans- 1,2-dichloroethylene in microcapsules in feed for 14 weeks . Very little toxicity was associated with ingestion of microencapsulated trans-1-2-dichloroethylene . Histopathology and clinical chemistry data, combined with body and organ weight data, revealed that the maximum tolerated dose was not reached in these studies. J Food Prot, 2002 Jul, 65(7), 1142 - 5 Effects of UV irradiation on selected pathogens in peptone water and on stainless steel and chicken meat; Kim T et al.; Effects of intensity and processing time of 254 nm UV irradiation on Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Typhimurium were investigated . Intensities measured at 5.08, 10.1, 15.2, and 20.3 cm from the light source were 1.000, 500, 250, and 150 microW/cm2, respectively . Intensities of 250 or 500 microW/cm2 reduced all suspended pathogen cells in peptone water about 5 log cycles after 2 min and completely inactivated L . monocytogenes and E . coli O157:H7 after 3 min by reductions of 8.39 and 8.64 log cycles, respectively . Intensities of 250 or 500 microW/cm2 also reduced (P < or = 0.05) the tested pathogens inoculated on stainless steel (SS) chips, and E . coli O157:H7 was completely destroyed at 500 microW/cm2 for 3 min . After UV treatment for 3 min at 500 microW/cm2, all selected pathogens on chicken meat with or without skin showed reduction ranges from 0.36 to 1.28 log cycles . Results demonstrated that UV irradiation could effectively decrease pathogens in peptone water and on SS but that it was less effective on chicken meat. J Food Prot, 2002 Jul, 65(7), 1088 - 92 Inhibition of Salmonella Typhimurium and Listeria monocytogenes in mung bean sprouts by chemical treatment; Lee SY et al.; This study was undertaken to compare the efficacies of chlorous acid (268 ppm), sodium hypochlorite (200 ppm), and lactic acid (2%) in eliminating total mesophilic microorganisms, Salmonella Typhimurium, and Listeria monocytogenes on commercial mung bean sprouts immediately after treatment and during posttreatment refrigerated storage . Treatment with sodium hypochlorite for 10 min did not reduce the total aerobic count . However, treatment with lactic acid and chlorous acid for 10 min initially reduced the total aerobic count by 0.6 and 0.8 log CFU/g, respectively, and maintained the same level or a lower level of the total aerobic count during the storage time . Treatment with chlorous acid reduced Salmonella Typhimurium from 5.0 log to undetectable levels (<0.48 log CFU/g), and the pathogen remained undetectable over a 9-day storage period . Treatment with lactic acid resulted in an initial 3-log reduction and further reduced the number of Salmonella Typhimurium cells to undetectable levels after 3 days . For L . monocytogenes, treatment with chlorous acid resulted in an initial 5-log reduction, and treatment with lactic acid resulted in a 2-log reduction at the beginning and undetectable levels after 9 days . When chemically injured cells were investigated by the selective overlay method, no statistical difference was observed (P < 0.05) between the number of injured cells recovered following treatment with chlorous acid and the number of bacteria counted on selective media, whereas sodium hypochlorite generated more injured cells than the other treatments did . These data suggest that treatment with chlorous acid may be useful in reducing total mesophilic microorganisms, Salmonella Typhimurium, and L . monocytogenes in commercial mung bean sprouts. J Food Prot, 2002 Jul, 65(7), 1081 - 7 Inactivation of Salmonella Typhimurium in orange juice containing antimicrobial agents by pulsed electric field; Liang Z et al.; Combinations of different hurdles, including moderately high temperatures (<60 degrees C), antimicrobial compounds, and pulsed electric field (PEF) treatment, to reduce Salmonella in pasteurized and freshly squeezed orange juices (with and without pulp) were explored . Populations of Salmonella Typhimurium were found to decrease with an increase in pulse number and treatment temperature . At a field strength of 90 kV/cm, a pulse number of 20, and a temperature of 45 degrees C, PEF treatment did not have a notable effect on cell viability or injury . At and above 46 degrees C, however, cell death and injury were greatly increased . Salmonella numbers were reduced by 5.9 log cycles in freshly squeezed orange juice (without pulp) treated at 90 kV/cm, 50 pulses, and 55 degrees C . When PEF treatment was carried out in the presence of nisin (100 U/ml of orange juice), lysozyme (2,400 U/ml), or a mixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cell viability loss was increased by an additional 0.04 to 2.75 log cycles . The combination of nisin and lysozyme had a more pronounced bactericidal effect than did either nisin or lysozyme alone . An additional Salmonella count reduction of at least 1.37 log cycles was achieved when the two antimicrobial agents were used in combination . No significant difference (P > 0.05) in cell death was attained by lowering the pH value; only cell injury increased . Inactivation by PEF was significantly more extensive (P < 0.05) in pasteurized orange juice than in freshly squeezed orange juice under the same treatment conditions . This increase might be due to the effect of the chemical composition of the juices. Pharmazie, 2002 Jun, 57(6), 416 - 20 Screening of Basidiomycete mushroom extracts for antigenotoxic and bio-antimutagenic activity; Filipic M et al.; In this study we screened crude methanol:water extracts of 89 different mushroom species for their antigenotoxic and bio-antimutagenic activity . The screening was performed with the SOS/umu test and we monitored the ability of extracts to inhibit UV induced expression of umuC gene in Salmonella typhimurium TA1535/pSK1002 . Seventeen extracts inhibited umuC expression by more than 50% . These extracts were further evaluated for the ability to inhibit UV induced mutations in Escherichia coli WP2 . Five extracts (Cortinarius evernius, Rozites caperatus, Lactarius vellereus, Russula integra and Pleurotus cornucopiae) inhibited also UV induced mutations . The study showed that certain mushrooms contain substances with bio-antimutagenic potential . Particularly interesting for further investigations are Pleurotus cornucopiae (Lentinaceae), which was the most effective and species of Russulaceae and Cortinaceae families, which might contain common family specific bio-antimutagenic substance(s). Plant Physiol, 2002 Jul, 129(3), 1265 - 75 Glyphosate-resistant goosegrass . Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase; Baerson SR et al.; The spontaneous occurrence of resistance to the herbicide glyphosate in weed species has been an extremely infrequent event, despite over 20 years of extensive use . Recently, a glyphosate-resistant biotype of goosegrass (Eleusine indica) was identified in Malaysia exhibiting an LD(50) value approximately 2- to 4-fold greater than the sensitive biotype collected from the same region . A comparison of the inhibition of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity by glyphosate in extracts prepared from the resistant (R) and sensitive (S) biotypes revealed an approximately 5-fold higher IC(50)(glyphosate) for the (R) biotype . Sequence comparisons of the predicted EPSPS mature protein coding regions from both biotypes revealed four single-nucleotide differences, two of which result in amino acid changes . One of these changes, a proline to serine substitution at position 106 in the (R) biotype, corresponds to a substitution previously identified in a glyphosate-insensitive EPSPS enzyme from Salmonella typhimurium . Kinetic data generated for the recombinant enzymes suggests that the second substitution identified in the (R) EPSPS does not contribute significantly to its reduced glyphosate sensitivity . Escherichia coli aroA- (EPSPS deficient) strains expressing the mature EPSPS enzyme from the (R) biotype exhibited an approximately 3-fold increase in glyphosate tolerance relative to strains expressing the mature EPSPS from the (S) biotype . These results provide the first evidence for an altered EPSPS enzyme as an underlying component of evolved glyphosate resistance in any plant species. Mutat Res, 2002 Jul 25, 518(2), 181 - 94 Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound; Suter W et al.; AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity . AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR . The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100 . AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9 . A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9 . AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with {14C}-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration . Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo . In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue . These data were considered to provide sufficient safety to initiate clinical development of the compound. Epidemiol Infect, 2002 Jun, 128(3), 373 - 82 Molecular epidemiology of Salmonella typhimurium isolates from human sporadic and outbreak cases; Heir E et al.; The molecular epidemiology of a representative collection of sporadic foreign and domestically acquired Salmonella Typhimurium (S . Typhimurium) isolates from Norwegian patients in 1996-9 was studied by numerical analysis of pulsed-field gel electrophoresis (PFGE) profiles . Three subclusters (E5, F1 and G1) comprised 47% of the 102 sporadic isolates investigated and 45% of the domestically acquired isolates fell in subclusters E5 and F1 . Distinct seasonal and geographic variations were evident for these strains which have been responsible for both local outbreaks (E5) and a national epidemic (F1) where salmonella-infected hedgehogs and birds constituted the suggested primary source of infection . Subcluster G1 was dominated by imported multi-resistant definitive type (DT) 104 isolates . All multi-resistant isolates contained integron-associated gene cassette-structures . This study presents valuable information on the relative significance, geographic distribution and antibiotic resistance features of distinct S . Typhimurium clones causing human salmonellosis among Norwegians. Environ Toxicol, 2002, 17(3), 219 - 31 Assessment of mutagenicity and toxicity of different-size fractions of air particulates from La Plata, Argentina, and Leipzig, Germany; Massolo L et al.; Airborne particulates, especially fine particles and bound chemical compounds, are a potential mediator of adverse health effects . In this study an analysis was done of the concentration and size distribution of air particulate matter, the content of bound polycyclic aromatic hydrocarbons (PAHs), and the biological effects of organic extracts from different fractions of dust that had been influenced by urban and industrial emissions from the regions of La Plata, Argentina, and Leipzig, Germany, along with samples from control areas . Air particulate matter was sampled in summer and winter in each region using a high-volume sampler with a six-stage cascade impactor, classifying dust in six size fractions from 10-microm particles to those less than 0.49 microm in size . Organic extracts of dust were tested for mutagenicity (Ames test, Salmonella typhimurium TA98 strain, S9+) and cytotoxicity (Tetrahymena pyriformis test system, growth rate, cell respiration) . The content of PAHs was analyzed by high-performance liquid chromatography (HPLC) with diode array and fluorescence detection . Mutagenic and cytotoxic effects were found to be associated with very fine (<0.49 microm) and fine (<1.5 microm) particle-bound compounds, corresponding to a higher content of PAHs . The mutagenic potency (revertants/m(3)) associated with particles less than 0.49 microm from urban areas of La Plata was about 1 order of magnitude higher than in particles in the range of 3.0 to 0.49 microm . Fine fractions from sites with an industrial burden were also found to exhibit high mutagenic potency . A similar tendency was observed in cytotoxicity tests with T . pyriformis . This cell system proved to be very sensitive to toxicants in tested dust fractions . The observed biological effects were found to be correlated significantly with concentrations of total PAH, carcinogenic PAHs, and benzo{a}pyrene . Eur J Clin Microbiol Infect Dis, 2002 Jun, 21(6), 449 - 54 Epub 2002 Jun 14. Differences between reference laboratories of the European community in their ability to detect Salmonella species; Voogt N et al.; The ability of national reference laboratories for Salmonella of the European Union member states to detect Salmonella bacteria was tested in four collaborative studies during the period 1995 through 1999 . Three different methods were prescribed in the four studies . Capsules containing various numbers of Salmonella Typhimurium or Salmonella Enteritidis were tested . In studies II, III and IV, Salmonella bacteria were isolated in the presence of competitive microorganisms . Significant differences were found between the four studies due to varying levels of difficulty with regard to the level of contamination, the use of serotypes and the presence of competitive organisms . There were also significant differences between the laboratories in the results obtained . Possible reasons for these differences will be further investigated by the European Union Community Reference Laboratory for Salmonella. Mutagenesis, 2002 Jul, 17(4), 313 - 6 Characterization of Trp(+) reversions in Escherichia coli strain WP2uvrA; Ohta T et al.; The Escherichia coli strain WP2uvrA is widely used in general mutagenicity screening tests because of its high sensitivity to many kinds of mutagens and it serves as a supplement to the standard Salmonella typhimurium tester strains . In contrast to Salmonella His(+) revertants, E.coli Trp(+) revertants have not been characterized at the molecular level . In this study we found that in the trpE65 allele of WP2uvrA the triplet that codes for the fourth amino acid from the N-terminus of anthranilate synthetase was an ochre stop codon (TAA) instead of a glutamine codon (CAA) . In spontaneous Trp(+) revertants the ochre codon had been changed to glutamine (CAA), lysine (AAA), glutamic acid (GAA), leucine (TTA), serine (TCA) or tyrosine (TAC, TAT) . Since tryptophan prototrophy could also be restored by ochre suppressor mutations at the anticodon sites in the genes for tRNA(Glu) (glnU), tRNA(Lys) (lysT) and tRNA(Tyr) (tyrT, tyrU), the Trp(+) reversion system with E.coli WP2uvrA detected five types of base substitutions, A.T-->T.A, A.T-->C.G, A.T-->G.C, G.C-->A.T and G.C-->T.A . About 30-50% of Trp(+) revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, captan and angelicin plus UVA irradiation were attributable to reversion at the trpE65 ochre locus; the others were attributable to suppressor mutations . In contrast, almost all revertants induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and furylfuramide were caused by suppressor mutations . Thus, the high mutagen sensitivity of WP2uvrA is due to several target sites consisting of A.T base pairs (trpE65, lysT) and G.C base pairs (glnU, tyrT, tyrU). Mutagenesis, 2002 Jul, 17(4), 293 - 9 Mutagenicity of two 2-phenylbenzotriazole derivatives, 2-{2-(acetylamino)-4-(diethylamino)-5-methoxyphenyl}-5-amino- 7-bromo-4-chloro-2H-benzotriazole and 2-{2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole and their detection in river water in Japan; Watanabe T et al.; We recently detected five 2-phenylbenzotriazole (PBTA)-type mutagens (PBTA-1, PBTA-2, PBTA-3, PBTA-4 and PBTA-6) in concentrates from several rivers that flow in geographically different areas in Japan containing textile-related industries . On the basis of synthesis studies, these five PBTA derivatives were deduced to have originated from the corresponding dinitrophenylazo dyes, which are industrial chemicals used in textile dyeing, via reduction and chlorination . 2-{(2-Bromo-4,6-dinitrophenyl)azo}-5-(diethylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 291, CAS registry no . 56548-64-2) and 2-{(2-bromo-4,6-dinitrophenyl)azo}-5-(diallylamino)-4-methoxyacetanilide (Color Index name Disperse Blue 373, CAS registry no . 51868-46-3) are used in textile dyeing and have 2-{(2-bromo-4,6-dinitrophenyl)azo}-4-methoxyacetanilide moieties in their structures, which are thought to be essential for their conversion to mutagenic PBTA derivatives . In the present study we have synthesized 2-{2-(acetyl-amino)-4-(diethylamino)-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-7) and 2-{2-(acetylamino)-4-(diallylamino)-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-8) from Disperse Blue 291 and Disperse Blue 373, respectively, by reduction with iron powder and subsequent chlorination with sodium hypochlorite . Both PBTA-7 and PBTA-8 exerted strong mutagenicity in Salmonella typhimurium TA98 and YG1024 in the presence of S9 mix (43 000 and 1 430 000 revertants/nmol for PBTA-7 and 40 700 and 2 213 000 revertants/nmol for PBTA-8 in TA98 and YG1024) . To clarify whether PBTA-7 and PBTA-8 exist in the environment, water samples were collected at seven sites in six rivers flowing through two different regions where textile dyeing industries are located . All water samples were mutagenic in Salmonella typhimurium YG1024 with S9 mix and their potencies ranged from 108 000 to 1 990 000 revertants/g blue rayon . PBTA-7 and PBTA-8 were detected in water samples from both regions at levels of <0.1-101.4 ng/g blue rayon and <0.1-48.9 ng/g blue rayon, respectively . In some samples PBTA-7 and PBTA-8 could contribute up to 15% of the water mutagenicity. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Jul, 34(4), 488 - 93 Expression of the newcastle disease virus fusion glycoprotein in vero cells using attenuated Salmonella typhimurium as transgenic carrier; Fang WH et al.; The full-length cDNA of fusion protein (F) gene of newcastle disease virus (NDV)strain F48E9 was amplified by RT-PCR and inserted into pcDNA3 under the control of human cytomegalovirus (hCMV) immediate early enhancer and promoter . The resulting recombinant plasmid pcDNA3-F was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam(-) and phoP(-)), which was then used to transfect the Vero cells . The DNA and RNA dot blotting revealed that the F gene was transcribed into mRNA in the Vero cells . There was expression of the F protein as shown by indirect immunofluorescent assay . The expression began at 48 h post-infection and increased thereafter, as indicated by ELISA . A 55 kD band of the F protein was identified by SDS-PAGE and Western blotting . These results clearly show that the expressed fusion protein was immuno-reactive with chicken anti-NDV serum. Emerg Infect Dis, 2002 Jul, 8(7), 732 - 4 Role of electronic data exchange in an international outbreak caused by Salmonella enterica serotype Typhimurium DT204b; Lindsay EA et al.; From July through September 2000, patients in five European countries were infected with a multidrug-resistant strain of Salmonella Typhimurium DT204b . Epidemiologic investigations were facilitated by the transmission of electronic images (Tagged Image Files) of pulsed-field gel electrophoresis profiles . This investigation highlights the importance of standardized protocols for molecular typing in international outbreaks of foodborne disease. EMBO J, 2002 Jul 1, 21(13), 3286 - 95 Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE; Buchwald G et al.; The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol . These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements . One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors . To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42 . SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release . This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1 . However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids . Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins. Biochemistry, 2002 Jul 9, 41(27), 8580 - 8 Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B(12)-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol; Bandarian V et al.; The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor . Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co(2+) in cob(II)alamin (B(12r)) produces a dominant splitting of the EPR signals of both the radical and the Co(2+) . Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by approximately 11 A . Experiments with (13)C- and with (2)H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report {Babior, B . M., Moss, T . H., Orme-Johnson, W . H., and Beinert, H., (1974) J . Biol . Chem . 249, 4537-4544} . Experiments with perdeutero-S-2-aminopropanol and {2-(15)N}-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol . The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1 . Experiments with adenosylcobalamin enriched in (15)N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co(2+) in a functional steady-state reaction intermediate. J Food Prot, 2002 Jun, 65(6), 1041 - 4 Salmonella serotypes isolated from nonhuman sources in São Paulo, Brazil, from 1996 through 2000; Tavechio AT et al.; A total of 4,581 Salmonella strains isolated from nonhuman sources, including foodstuffs associated with foodborne Salmonella outbreaks, from January 1996 through December 2000 were serotyped at the Enteropathogens Laboratory, Instituto Adolfo Lutz, Sao Paulo, Brazil . Among the 123 different serotypes identified, Salmonella enterica subsp . enterica serotype Enteritidis (Salmonella Enteritidis) was the most prevalent (32.7%), ranking first for almost every kind of source . The next most common serotypes were Salmonella Senftenberg (10.3%), Salmonella Hadar (6.8%), Salmonella Agona (5.1%), and Salmonella Typhimurium (2.4%) . Rough strains belonging to the subspecies S . enterica subsp . enterica (4.8%), S . enterica subsp . arizonae (<1%), S . enterica subsp . diarizonae (<1%), and S . enterica subsp . houtenae (<1%) were also detected . Foodstuffs (including poultry meat for consumption) contained 38.1% of the studied Salmonella strains, poultry flocks (from several farms under salmonellosis control by the owners) contained 21.7%, the environment contained 10.6%, sewage contained 9.4%, water contained 6.6%, animal feed contained 4.4%, chill water from poultry-processing operations contained 2.2%, and other sources contained 7.0% . Foodstuffs extensively contaminated with Salmonella strains were poultry meat (40%), cow meat (11%), desserts (8%), mayonnaise (6%), sausage (5%), and unpasteurized shell eggs (4%), and there were several other food sources (26%) . Homemade mayonnaise was the most common vehicle for Salmonella foodborne outbreaks, and Salmonella Enteritidis was the serotype most isolated (95%) from that source . According to these data and previously published data concerning Salmonella strains isolated in Sao Paulo State, almost the same serotypes have predominated among nonhuman sources for the last decade. Nippon Rinsho, 2002 Jun, 60(6), 1083 - 8 {Development of vaccine for enterohemorrhagic Escherichia coli infection}; Yamasaki S; Efforts on the development of vaccines against enterohemorrhagic Escherichia coli (EHEC) infection has been described in this review . Two kinds of vaccines were developed and these have been targeted for in humans and cattle . One vaccine candidate is toxoid, which uses an inactive form of Shiga toxin(Stx) . A part of B subunit, each B or A subunit or one or two amino acid mutated holotoxin were developed as a toxoid vaccine candidate . The other candidate was bacterial surface antigen such as a live attenuated EHEC and hybrid between non-toxic LPS and toxoid . A live attenuated vaccine against EHEC O26: H11, O157: H7, O139: H1 were developed . Further a live attenuated vaccine candidate of Vibrio cholerae O1 expressing Stx1-B, Shigella flexneri expressing S . dysenteriae O-antigen and Stx1-B, or Salmonella Typhimurium expressing O111 antigen were developed . Hybrid type vaccine candidates were also developed with O111 LPS and tetanus toxoid, O157 LPS and exotoxin, and O157 LPS and Stx1-B. J Immunol, 2002 Jul 1, 169(1), 108 - 16 Antigen presentation capacity and cytokine production by murine splenic dendritic cell subsets upon Salmonella encounter; Yrlid U et al.; Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice . However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8alpha(+), CD8alpha(-)CD4(-), and CD8alpha(-)CD4(+) DC, from these organs in response to SALMONELLA: Infection of purified splenic DC with S . typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation . Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8alpha(+) and CD8alpha(-) splenic DC subsets . Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II . Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-alpha was produced by a large percentage of CD8alpha(-) DC, while only a minor proportion of CD8alpha(+) DC produced this cytokine following bacterial exposure . In contrast, the greatest number of IL-12p40-producing DC were among CD8alpha(+) DC . Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production . Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion. J Immunother, 2002 Mar-Apr, 25(2), 162 - 75 Unexpected induction of unresponsiveness by vaccination with transformed Salmonella typhimurium; Zoller M; Rats vaccinated with attenuated Salmonella typhimurium transformed with a vector containing the v2 exon of CD44 (SL-v2) were not protected and developed thymic metastases at a high rate . This was surprising because there was evidence for concomitant induction of a CD44v2-specific helper and cytotoxic T-cell response . The inefficacy of vaccination was partly caused by tumor escape and tumor-induced immunosuppression . More important were the facts that (i) BSpl2v2 cells migrated from the intraperitoneal implantation site to the thymus and (ii) after vaccination with transformed attenuated Salmonella typhimurium, a small number of dendritic cells, which had transcribed the cDNA insert, were detected in the thymus . In the thymic environment, these v2 presenting dendritic cells, as well as the BSp12v2 tumor cells, supported tolerance induction . Thus, vaccination with tumor-associated differentiation antigens, which in many instances have induced antitumor response, may deteriorate survival time and rate if vaccination is accompanied by presentation of the antigen during intrathymic T-cell selection. Eur J Clin Microbiol Infect Dis, 2002 Apr, 21(4), 290 - 3 Epub 2002 Apr 10. Analysis of risk factors for bacteremia in children with nontyphoidal Salmonella gastroenteritis; Yang YJ et al.; To identify the risk factors for Salmonella bacteremia in infants and children with Salmonella gastroenteritis, a retrospective study of a 10-year period was conducted to evaluate 456 infants and children with culture-proven nontyphoidal Salmonella infection . Salmonella typhimurium was the most common isolate found . Among the 257 patients with gastroenteritis who had a concomitant blood culture performed, 50 exhibited bacteremia . Statistically significant differences were noted between patients with gastroenteritis and bacteremia and those without bacteremia in duration of fever > or = 5 days ( P < 0.001; OR, 5.6; 95% CI, 2.6 - 12.1) and infection with group D1 Salmonella ( P < 0.001; OR, 6.5; 95% CI, 2.5 - 16.9) after adjustment for multivariate analysis . Of the 320 Salmonella strains that were serotyped, Salmonella panama was shown to be strongly associated with bacteremia ( P<0.001) in children with gastroenteritis . In summary, in children with nontyphoidal Salmonella gastroenteritis, prolonged fever lasting 5 days or more and infection with a specific Salmonella serotype were risk factors closely associated with development of bacteremia. Microb Pathog, 2002 May, 32(5), 207 - 18 Migration of Salmonella typhimurium --harboring bone marrow--derived dendritic cells towards the chemokines CCL19 and CCL21; Cheminay C et al.; Macrophages are considered as main cellular target encountered by the facultative intracellular bacterium Salmonella typhimurium . However, in orally infected mice these pathogens are first internalized by dendritic cells (DCs) that are located in the subepithelial dome of Peyer's patches . Moreover, DCs can penetrate the intestinal epithelium to sample bacteria . Here, we examined the interaction of Salmonella with bone marrow-derived DCs (BM-DCs) . In order to study the role of DCs as vehicles for the dissemination of Salmonella, an in vitro model was established . In this model, Salmonella -activated BM-DCs enhanced surface expression of MHC class II and co-stimulatory molecules . We found that, upon maturation, BM-DCs upregulated chemokine receptor 7 (CCR7) mRNA and surface molecule expression . Salmonella -exposed DCs as well as mature DCs, but not immature DCs, were recruited towards the CC chemokines CCL19 and CCL21, two ligands of CCR7 . The maturation process of DCs did neither require bacterial internalization nor viability . About one third of the migrated BM-DCs harbored intracellular bacteria, whereas the remaining two third did not contain bacteria . Salmonella, but not an apathogenic E . coli laboratory strain was capable to survive within BM-DCs . Taken together, our data implicate that DCs are first activated and subsequently utilized as carriers by Salmonella . FEMS Microbiol Rev, 2002 Jun, 26(2), 141 - 8 Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-borne infections; Threlfall EJ; Strains of Salmonella spp . with resistance to antimicrobial drugs are now widespread in both developed and developing countries . In developed countries it is now increasingly accepted that for the most part such strains are zoonotic in origin and acquire their resistance in the food-animal host before onward transmission to humans through the food chain . Of particular importance since the early 1990s has been a multiresistant strain of Salmonella typhimurium definitive phage type (DT) 104, displaying resistance to up to six commonly used antimicrobials, with about 15% of isolates also exhibiting decreased susceptibility to ciprofloxacin . Mutations in the gyrA gene in such isolates have been characterised by a PCR LightCycler-based gyrA mutation assay, and at least four different mutations have been identified . Multiple resistance (to four or more antimicrobials) is also common in the poultry-associated pathogens Salmonella virchow and Salmonella hadar, with an increasing number of strains of these serotypes exhibiting decreased susceptibility to ciprofloxacin . Multiple resistance is also being found in other serotypes in several other European countries, and has been associated with treatment failures . For Salmonella typhi, multiple drug resistance is now the norm in strains originating in the Indian subcontinent and south-east Asia . Such multiresistant strains have been responsible for several epidemics and some of these have been associated with contaminated water supplies . Furthermore, an increasing number of multiresistant strains of S . typhi are now exhibiting decreased susceptibility to ciprofloxacin, with concomitant treatment failures . In developed countries antimicrobial resistance in zoonotic salmonellas has been attributed to the injudicious use of antimicrobials in food-producing animals . It is hoped that the application of Codes of Practice for the use of such agents, which have been prepared by the pharmaceutical industry in response to widespread international concern about the development of drug resistance in bacterial pathogens, will now result in a widespread reduction in the incidence of drug-resistant salmonellas in food production animals and humans on an international scale. Prev Vet Med, 2002 Jun 25, 54(2), 169 - 78 Prevalence of and risk factors for Salmonella in water offered to weaned dairy calves in California, USA; Kirk J et al.; Water from troughs used by weaned dairy calves was sampled on California, USA dairies to determine the prevalence and associated risk factors for Salmonella contamination . Salmonella were found on 4 of 48 dairies (4/82 water samples) in fall 1998 and on 8 of the same 37 dairies (8/83 water samples) in summer, 1999 . Serotypes isolated from the water were Salmonella meleagridis and Salmonella typhimurium . Primary risk factors associated with the increased prevalence of Salmonella in water offered to weaned dairy calves were a continuous water tank-filling method compared to a valve (an "on-demand" procedure) and a water pH>8. Food Chem Toxicol, 2002 Aug, 40(8), 1063 - 8 Heterologous expression of human N-acetyltransferases 1 and 2 and sulfotransferase 1A1 in Salmonella typhimurium for mutagenicity testing of heterocyclic amines; Muckel E et al.; A variety of carcinogenic heterocylic amines (HAs) are found in cooked food . They can be metabolised to reactive intermediates via N-hydroxylation catalysed by cytochrome P450 1A2, followed by conjugation of the resulting N-hydroxyl group by N-acetyltransferase (NAT) or sulfotransferase (SULT) . In order to compare the role of O-acetylation and O-sulfonation by human enzymes in the activation of HAs, we have introduced the cDNAs for wild-type forms of human NAT1, NAT2 and SULT1A1 in the acetyltransferase-deficient Salmonella typhimurium strain TA1538/1,8-DNP . Functional expression of recombinant proteins was demonstrated using immunoblot analysis and determination of enzyme activity with characteristic substrates . The established strains were used to study the mutagenicity of the N-hydroxy derivatives of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . The results demonstrate that N-hydroxy-HAs are activated by different human enzymes . At the concentrations used in the mutagenicity assay, N-hydroxy-IQ was activated by human NAT2, but not by NAT1 or SULT1A1 . In contrast, N-hydroxy-PhIP was activated specifically by human SULT1A1, but not by NAT1 or NAT2 . Therefore, both O-acetylation and O-sulfonation by human enzymes have to be regarded as important determinants for HA genotoxicity in humans. J Appl Microbiol, 2002, 93(1), 46 - 51 Immunoassays to detect the serum antibody response of cattle to infection with Salmonella Typhimurium definitive type 104 and following vaccination with Bovivac S; Chart H et al.; AIMS: To use ELISA and immunoblotting assays to examine the serum antibody response of cattle infected with Salmonella Typhimurium DT104 and following vaccination with Bovivac S . METHODS AND RESULTS: Three hundred and twenty-nine cattle, including 16 shedding multiresistant Salmonella Typhimurium DT104, were screened for serum antibodies binding to O=1, 4, 5, 12 lipopolysaccharide (LPS) antigens before and after vaccination with Bovivac S . Sera with an ELISA reading of 0.9A405 or above were shown to contain antibodies, of the IgG-class only, to the LPS of Salmonella Typhimurium using immunoblotting . Prior to vaccination, only 11 cattle had serum IgG-class antibodies to the O=4, 5 LPS antigens, and of these one also had antibodies to outer membrane proteins and H=i flagellar antigens . Following vaccination, 87 out of 315 cattle developed serum antibodies to the LPS of Salmonella Typhimurium . CONCLUSIONS: Evidence of infection of cattle with Salmonella Typhimurium was readily obtained with an LPS-based ELISA in association with an immunoblotting procedure, supplementing existing bacteriological procedures . This enabled the detection of an increase in the number of cattle with serum antibodies to Salmonella Typhimurium LPS following vaccination with Bovivac S . SIGNIFICANCE AND IMPACT OF THE STUDY: The immunoassays described provided evidence of infection with Salmonella Typhimurium and served as a valuable adjunct to established bacteriology. Cell Microbiol, 2002 Jun, 4(6), 315 - 28 The Salmonella-containing vacuole is a major site of intracellular cholesterol accumulation and recruits the GPI-anchored protein CD55; Catron DM et al.; Intracellular, pathogenic Salmonella typhimurium avoids phago-lysosome fusion, and exists within a unique vacuolar niche that resembles a late endosome . This model has emerged from studying the trafficking of host proteins to the Salmonella-containing vacuole (SCV) . Very little is known about the role of major host lipids during infection . Here, we show using biochemical analyses as well as fluorescence microscopy, that intracellular infection perturbs the host sterol biosynthetic pathway and induces cholesterol accumulation in the SCV . Cholesterol accumulation is seen in both macrophages and epithelial cells: at the terminal stages of infection, as much as 30% of the total cellular cholesterol resides in the SCV . We find that accumulation of cholesterol in the SCV is linked to intracellular bacterial replication and may be dependent on Salmonella pathogenicity island 2 (SPI-2) . Furthermore, the construction of a three-dimensional space-filling model yields novel insights into the structure of the SCV: bacteria embedded in cholesterol-rich membranes . Finally, we show that the glycosylphosphatidylinositol (GPI)-anchored protein CD55 is recruited to the SCV . These data suggest that, in contrast to prevailing models, the SCV accumulates components of cholesterol-rich early endocytic pathways during intracellular bacterial replication. J Ethnopharmacol, 2002 Jul, 81(2), 257 - 64 Genotoxicity of Brosimum gaudichaudii measured by the Salmonella/microsome assay and chromosomal aberrations in CHO cells; Varanda EA et al.; The root bark of Brosimum gaudichaudii Trecul (Moraceae) is popularly used for treatment of vitiligo . In the present study the mutagenic activity of the aqueous and methanolic extract as well as of the n-butanolic fraction of this medicinal plant were evaluated using Salmonella typhimurium assays, TA100, TA98, TA102 and TA97a strains, while the clastogenic effect in Chinese hamster ovary (CHO) cells in the G(1)/S, S and G(2)/S phases of the cell cycle . The results showed mutagenic activity of the aqueous extract against TA102 in the presence of S9, and of methanolic extract, with and without metabolic activation . TA100 mutagenicity was only observed for the methanolic extract in the absence of S9 . The n-butanolic fraction did not present mutagenic activity . In CHO cells only the methanolic extract induced a significant increase of chromosomal aberrations in the G(1)/S and S phases, whereas a decrease in the mitotic index was observed in the G(1)/S and G(2)/S phases . No clastogenicity was observed for the aqueous extract . The furocoumarins (psoralen and bergapten) presented in the extracts might contribute to the mutagenicity . The lower activity of the aqueous extract was probably due to the presence of smaller amount of furocoumarins compared to the methanolic extract. Gene, 2002 May 15, 290(1-2), 153 - 61 Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in bacteria; Ellermeier CD et al.; A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described . Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions . Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed . These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the lambda Red recombination method . The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest . Transcriptional fusions to the Salmonella typhimurium genes sodCII and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur . Translational fusions were also constructed using this method . In this case, expression of beta-galactosidase was dependent on translation of the target protein . Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms. Indian J Pediatr, 2002 May, 69(5), 393 - 6 Rotavirus and enteric pathogens in infantile diarrhoea in Manipal, South India; Ballal M et al.; The etiology of Rotavirus in acute diarrhoeal illness in children 0-5 years of age, admitted to the pediatric wards of Kasturba Medical College Hospital, Manipal was studied over a period of 5 years . Rotavirus in the faeces detected by Latex agglutination test accounted for 19.56% of the diarrhoea with maximum incidence (65%) in the 7-12 months of age group . Bacterial aetiological agents continued to play a significant role (69.6%) in diarrhoeal diseases . Enteroaggregative E . coli was common in the age group between 25-36 months, Shigellosis in 37-60 months and Salmonella typhimurium enteritis in 7-12 months of age . The other pathogens isolated were vibrio cholerae (4.98%), species of aeromonas (15.92%), along with cryptosporidium (6.47%) and candida albicans (3.98%) . In a control group consisting of 100 children without history of diarrhoea, 2 were positive for rotavirus, 3 for cryptosporidium and 12 for Escherichia coli. Vet Res, 2002 May-Jun, 33(3), 291 - 7 Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium; Splichal I et al.; Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals . Germ-free animals, however, enable to study response against infection without background effect of other microorganisms . Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA . In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups . TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection . In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella . These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis. Nat Immunol, 2002 Jul, 3(7), 667 - 72 Epub 2002 Jun 10. Essential role of MD-2 in LPS responsiveness and TLR4 distribution; Nagai Y et al.; Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types . MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro . We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection . We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts . Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4. J Immunol, 2002 Jun 15, 168(12), 6396 - 403 The role of lipopolysaccharide binding protein in resistance to Salmonella infections in mice; Fierer J et al.; Polymorphonuclear leukocytes (PMN) and LPS-binding protein (LBP) are both components of the innate immune system . LBP is a plasma protein that binds to lipid A and enhances the biological activity of LPS 100- to 1000-fold . Recently it was reported that LBP-deficient mice are more susceptible to Salmonella typhimurium infection . Here we report that LBP KO mice are more susceptible to Salmonella peritonitis, but not to oral or i.v . infection . LBP knockout (KO) mice responded normally to i.p . injections of Staphylococcus aureus and casein, but not to i.p . injection of S . typhimurium or Salmonella LPS . Mice with a mutation in Toll-like receptor 4 (C3H/HeJ) have a similar defect in PMN chemotaxis . In normal mice S . typhimurium stimulated production of the CXC chemokines macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant, but levels of cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein-2 were greatly reduced in the LBP KO mice . LBP KO mice pretreated with casein to attract PMN in an LBP-independent manner were more resistant to Salmonella infection, but neutropenic mice were not protected by casein . Splenic TNF-alpha mRNA levels were also lower in LBP KO than in control mice infected with SALMONELLA: Since TNF-alpha can activate PMN, LBP KO mice may have both fewer and less active PMN in the first few hours after Salmonella are injected, making LBP KO mice more susceptible . This work confirms the importance of PMN in resistance to Salmonella infections and shows that this is facilitated by LBP. J Mol Biol, 2002 May 10, 318(4), 941 - 50 Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH; Auvray F et al.; The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly . We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation . Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body . Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP . Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain . The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity . The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids . The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion . They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly . (c) 2002 Elsevier Science Ltd. Biochem Biophys Res Commun, 2002 May 24, 293(5), 1497 - 501 The proteolytic release of genotoxins from cooked beef; Martin FL et al.; Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs . Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels . We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis . Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K) . Genotoxicity was measured using the alkaline single cell-gel electrophoresis ('Comet') assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019 . Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated. Int J Food Microbiol, 2002 Jun 25, 76(3), 177 - 90 Development and validation of a tertiary simulation model for predicting the potential growth of Salmonella typhimurium on cooked chicken; Oscar TP; The growth of Salmonella typhimurium (ATCC 14028) on the surface of autoclaved ground chicken breast and thigh burgers incubated at constant temperatures from 8 to 48 degrees C in 2 degrees C increments was investigated and modeled . Growth curves at each temperature were fit to a two-phase linear primary model to determine lag time (lambda) and specific growth rate (mu) . Growth of S . typhimurium on breast and thigh meat was not different . Consequently, secondary models that predicted lag time and specific growth rate as a function of temperature were developed with the combined data for breast and thigh meat . Five secondary models for lag time and three secondary models for specific growth rate were compared . A new version of the hyperbola model and a cardinal temperature model were selected as the best secondary models for lag time and specific growth rate, respectively . The secondary models were combined in a computer spreadsheet to create a tertiary simulation model that predicted the potential growth (log10) increase) of S . typhimurium on cooked chicken as a function of time and temperature . Probability distributions and simulation were used in the tertiary model to model the secondary model parameters and the times and temperatures of abuse . The outputs of the tertiary model were validated (prediction bias of -4% for lambda and 1% for mu and prediction accuracy of 10% for lambda and 8% for mu) and integrated with a previously developed risk assessment model for Salmonella. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2001, 33(2), 233 - 237 Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella; Li YH et al.; To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice . After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells . Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection . Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes . The inhibition of the tumor and the survival time of the mice were also investigated . Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue . And the GFP expression was much stronger in spleen and tumor than in other tissues . Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo . The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05) . Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy. Vopr Virusol, 2002 Mar-Apr, 47(2), 25 - 8 {Construction and study of antigenic characteristics of recombinant salmonella strain producing TBI protein}; Karpenko LI et al.; A recombinant strain producing TBI protein (artificial protein containing HIV-1 B and T cell epitopes) was constructed on the base of attenuated Salmonella typhimurium SL7207 strain and BALB/c mice were immunized with this strain in a dose of 10(9) live cells orally or rectally . A single immunization with the recombinant salmonella strain induced humoral and cellular immune response to HIV-1; hence, this strain is a promising candidate for vaccine against HIV. World J Gastroenterol, 2002 Jun, 8(3), 540 - 5 Evidences for vagus nerve in maintenance of immune balance and transmission of immune information from gut to brain in STM-infected rats; Wang X et al.; AIM: To determine whether Salmonella Typhimurium (STM)in gastrointestinal tract can induce the functional activation of brain, whether the vagus nerve involves in signaling immune information from gastrointestinal tract to brain and how it influences the immune function under natural infection condition . METHODS: Animal model of gastrointestinal tract infection in the rat was established by an intubation of Salmonella Typhimurium (STM) into stomach to mimic the condition of natural bacteria infection . Subdiagphragmatic vagotomy was performed in some of the animals 28 days before infection . The changes of Fos expression visualized with immunohistochemistry technique in hypothalamic paraventricular nucleus (PVN) and superaoptic nucleus (SON) were counted . Meanwhile, the percentage and the Mean Intensities of Fluorescent (MIFs) of CD4+ and CD8+ T cells in peripheral blood were measured by using flow cytometry (FCM), and the pathological changes in ileum and mesenteric lymph node were observed in HE stained sections . RESULTS: In bacteria-stimulated groups, inflammatory pathological changes were seen in ileum and mesenteric lymph node . The percentages of CD4+ T cells in peripheral blood were decreased from 42%+/-4.5% to 34%+/-4.9% (P<0.05) and MIFs of CD8+ T cells were also decreased from 2.9+/-0.39 to 2.1+/-0.36 (P<0.05) with STM stimulation . All of them proved that our STM-infection model was reliable . Fos immunoreactive (Fos-ir) cells in PVN and SON increased significantly with STM stimulation, from 189+/-41 to 467+/-62 (P<0.05) and from 64+/-21 to 282+/-47 (P<0.05) individually, which suggested that STM in gastrointestinal tract induced the functional activation of brain . Subdiagphragmatic vagotomy attenuated Fos expression in PVN and SON induced by STM, from 467+/-62 to 226+/-45 (P<0.05) and from 282+/-47 to 71+/-19 (P<0.05) individually, and restored the decreased percentages of CD4+ T cells induced by STM from 34%+/-4.9% to original level 44%+/-6.0% (P<0.05) . In addition, subdiagphragmatic vagotomy itself also decreased the percentages of CD8+ T cells (from 28%+/-3.0% to 21%+/-5.9%, P<0.05) and MIFs of CD4+ (from 6.6+/-0.6 to 4.9+/-1.0, P<0.05) and CD8+ T cells (from 2.9+/-0.39 to 1.4+/-0.34, P<0.05) . Both of them manifested the important role of vagus nerve in transmitting immune information from gut to brain and maintaining the immune balance of the organism . CONCLUSION: Vagus nerve does involve in transmitting abdominal immune information into the brain in STM infection condition and play an important role in maintenance of the immune balance of the organism. J Chemother, 1991 Jan, 3 Suppl 1, 69 - 72 Endotoxin release after antibiotic treatment and its potential pathogenetic role in coagulative disorders of patients with gram-negative sepsis; Miragliotta G; The liberation of endotoxin by gram-negative bacteria upon antibiotic treatment might be of clinical relevance in terms of adverse effects of antibiotic therapy in patients with gram-negative infections . In this paper we have taken into consideration the endotoxic material released by Salmonella typhimurium SH9178 after treatment with either ofloxacin and norfloxacin (which act as DNA-gyrase inhibitors) or imipenem (which affects the bacterial cell wall) in its capacity to induce in-vitro production of procoagulant activity, tissue factor-like, by human mononuclear cells . Endotoxin released from Salmonella typhimurium after treatment with the above antibiotics behaves as the typical endotoxin of gram-negative bacteria in its capacity to induce procoagulant production . Indeed human mononuclear cells after prolonged incubation with supernatants of antibiotic-treated Salmonella were able to shorten the recalcification time of normal but not Factor VII deficient plasma (tissue factor-like activity) . On this basis we hypothesize that this mechanism, leading to the activation of blood coagulation through the extrinsic pathway, might be closely involved in thrombohemorrhagic disorders sometimes complicating the antimicrobial therapy of gram-negative sepsis. Immunol Lett, 2002 Jul 3, 82(3), 197 - 204 Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV; Vecino WH et al.; An immunization strategy using attenuated bacteria to deliver DNA vaccine plasmids to mucosal sites may induce protective T cell responses against sexual HIV transmission . In a murine intranasal (i.n.) immunization model, we demonstrate that transiently persistent Deltaasd Shigella flexneri strain 15D harboring DNA vaccines induces HIV- and SIV-specific gamma interferon (IFN-gamma) producing CD8+ T cells among splenocytes more efficiently than either a longer persisting DeltaaroD Salmonella typhimurium strain SL7207 or transiently persistent S . typhi strain Ty21a harboring DNA vaccines . Also, the frequency of antigen-specific gamma interferon (IFN-gamma) producing cells induced by Shigella 15D harboring a DNA vaccine were comparable to that induced by intramuscular (i.m.) immunization with purified DNA vaccine . Moreover, the magnitude of mucosal and systemic antigen-specific IgA and IgG responses after immunization were dependent upon the route (i.m . vs . i.n.) of inoculation, with i.n . Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than i.m . purified DNA vaccine . Deltaasd S . flexneri is a promising vector for mucosal DNA vaccine immunization against HIV. Poult Sci, 2002 May, 81(5), 632 - 41 In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium; Lee EN et al.; Chicken egg yolk antibody (IgY) raised against Salmonella enteritidis or Salmonella typhimurium was found in highly specific activity levels by ELISA . S . enteritidis- and S . typhimurium-specific IgY powder, prepared by freeze-drying the egg yolk water-soluble fraction, contained 15.5 and 10.0% of specific IgY, respectively . Anti-S . enteritidis IgY cross-reacted 55.3% with S . typhimurium . The cross-reactivity of anti-S . typhimurium IgY with S . enteritidis was 42.4% . Salmonella-specific IgY was demonstrated to inhibit Salmonella growth in liquid medium . The growth rate of S . enteritidis incubated with S . enteritidis-specific IgY was fourfold less than that of the control group during a 4-to-6-h incubation . Cell counts of S . typhimurium incubated with S . typhimurium-specific IgY were reduced by 1.6 log cfu/mL in comparison to that of the control group after 6 h of incubation . The specific binding activity of IgY was further evaluated by using immunofluorescence and immunoelectron microscopy . It was found that Salmonella-specific IgY could bind to the antigens expressed on the Salmonella surface, resulting in structural alterations of the bacterial surface. Lett Appl Microbiol, 2002, 34(6), 428 - 32 Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types; Lawson AJ et al.; AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types . METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE) . The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%) . LPS profiling was able to discriminate between isolates of identical PFGE type . Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10 . All 10 outbreaks were identical by PFGE analysis . CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles . The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS . SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations. Mikrobiologiia, 2002 Mar-Apr, 71(2), 194 - 9 {The effect of anabiosis autoinducers on the bacterial genome}; Il'inskaia ON et al.; The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d1 factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to anabiotic state, was studied using the Ames test . In the range of concentrations studied (0.1 to 100 micrograms/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C1-AHB) and hexylresorcinol (C6-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects . C6-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol . C1-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect . Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity . The growth-inhibiting effect of elevated C6-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies . The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes (the phenotypic dissociation of microbial populations and the formation of resting microbial forms) is discussed . The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions. Genetika, 2002 Apr, 38(4), 453 - 7 {Study of mutagenic activity of fullerene and some of its derivatives using His+ reversions of Salmonella typhimurium as an example}; Babynin EV et al.; The influence of three new derivatives of fullerence C60 ({61}dimethoxyphosphoryl{61}carbethoxy-methano{60}fullerene, {61}-(dimethoxyphosphoryl-{61}-carbmethoxy-methanofullerene, and 1-methyl-2-(3,5-di-tertbutyl-4-hydroxy-phenyl)-3,4-fulleropyrrolidine) on the appearance of His+ reversions in the Salmonella typhimurium strain BA13 was studied . It was ascertained that the effect of fullerene derivatives on the occurrence of mutations depends on the type of the molecular group with which fullerene interacts . The biological effect is determined not only by the action of the group associated with fullerene . The dependence between the mutagenic effect and properties of the solvents was detected . Exposure to visible light of the culture treated with fullerene derivatives was found to have an antimutagenic effect in the case of {61}dimethoxyphosphoryl{61}carbethoxy-methanofullerene{60} . For two other derivatives, the differences between experimental and control variants were statistically nonsignificant. J Biol Chem, 2002 Jul 26, 277(30), 27094 - 102 Epub 2002 May 16. A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators; Rodriguez-Pachon JM et al.; Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells . Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells . Furthermore, it promotes the activation of the cell integrity MAPK Slt2 . Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs . These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway . Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway . Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S . cerevisiae, down-regulating MAPK-mediated signaling . Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways . Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes. Protein Sci, 2002 Jun, 11(6), 1565 - 74 Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin); Michels J et al.; The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels . However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density . The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin . In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation . The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization . Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46 . Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data . The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide . The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding. Zhonghua Zhong Liu Za Zhi, 2002 Mar, 24(2), 110 - 3 {Development of oral DNA vaccine based on MG(7)-Ag mimotope of gastric cancer}; Guo C et al.; OBJECTIVE: To develop an oral DNA vaccine based on MG(7)-Ag mimotope of gastric cancer using attenuated Salmonella typhimurium and evaluate its efficacy and protective effect . METHODS: The eukaryotic expression vector including the MG(7)-Ag mimotope and a Th epitope was constructed, and then transduced into an attenuated Salmonella typhimurium to get the oral DNA vaccine . C57BL/6 J mice were orally immunized with 1 x 10(8) cfu Salmonella transfectants, with Salmonella harboring empty plasmid, with phophate buffered saline (PBS) as control . At the 6th week, serum titer of MG(7) antibody was detected by ELISA . In the 8th week, a {(3)H}-thymidine incorporation assay was performed to test the proliferation of murine spleen cells to the stimulant of MG(7)-Ag mimicry peptide . At the same time, Ehrlich ascites carcinoma cells expressing MG(7)-Ag were used in tumor challenge assay to evaluate the protective effect of the immunization . RESULTS: The oral DNA vaccine induced MG(7) antibody in mice, while in vivo unprimed proliferation assay of the spleenocytes showed no difference among the three groups . Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while none in the control group was protected . CONCLUSION: Oral DNA vaccine based on the MG(7)-Ag momitope is immunogenic . It is able to induce specific immunity response against tumor in mice, and the vaccine is partially protective. J Appl Microbiol, 2002, 92(6), 1097 - 104 The effect of toluidine blue on the survival, dormancy and outer membrane porin proteins (OmpC and OmpF) of Salmonella typhimurium LT2 in seawater; Ozkanca R et al.; AIMS: To study the relationship between changes in the composition of the outer membrane proteins and the survival of Salmonella typhimurium LT2 in filtered autoclaved seawater containing Toluidine Blue (TB) dye as a photosensitizer . METHODS AND RESULTS: In samples exposed to TB and excited by artificial visible light, the total viable (TVC) and respiring cell counts (RCC) showed that, although the TVC declined to an undetectable level in 6.5 h, the RCC showed that some cells were still capable of respiration . The porin protein composition changed gradually with OmpC and OmpF becoming undetectable by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after 8 h of incubation . Hydrogen peroxide-pretreated cells survived longer compared with the control . CONCLUSIONS: Oxidative pretreatment of Salm . typhimurium protects cells from some of the effects of sunlight in the presence of photosensitizers . The changes in porin proteins may play a role in this protection . SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the survival of bacteria under conditions of stress is the result of a linked series of reactions. Traffic, 2002 Jun, 3(6), 407 - 15 SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules; Brumell JH et al.; A unique feature of Salmonella enterica serovar typhimurium (S . typhimurium) is its ability to enter into (invade) epithelial cells and elongate the vacuole it occupies into tubular structures called Salmonella-induced filaments (Sifs) . This phenotype is dependent on SifA, a Salmonella virulence factor that requires the SPI-2-encoded type III secretion system for delivery into host cells . Previous attempts to study SifA and other type III secreted proteins have been limited by a lack of suitable reagents . We examined SifA function by expressing SifA with two internal hemagglutinin epitope tags . By employing subcellular fractionation techniques, we determined that translocated SifA was membrane associated in infected HeLa cells . Confocal microscopy revealed that SifA associated with the Salmonella vacuole and with Sifs . Our analysis also revealed that microtubules serve as a scaffold for Sifs, and that SifA colocalizes with microtubules at sites of interaction between lysosomal glycoprotein-containing vesicles and Sifs . Treatment with the microtubule inhibitor nocodazole blocked Sif formation but did not prevent SifA translocation into the Salmonella vacuole . While polymerized actin has been observed on Sifs, this phenotype was transient and did not play a role in promoting or maintaining Sif formation . Our findings demonstrate the essential role of microtubule dynamics in the formation of Sifs and the utility of this epitope tagging strategy for the study of bacterial type III secreted proteins. Vet Pathol, 2002 Mar, 39(2), 200 - 15 Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves; Santos RL et al.; The host response to Salmonella plays a major role in the outcome of infection . The present study was undertaken to further characterize Salmonella typhimurium infection in neonatal calves at both the morphologic and the molecular level using the ligated ileal loop model . Eight 4-5-week-old male Holstein calves underwent laparotomy, and loops were prepared in the ileum . The loops were either inoculated with an S . typhimurium strain pathogenic for cattle or injected with sterile LB broth as control . Samples for histology, transmission and scanning electron microscopy, and RNA extraction were collected at various time points between 5 minutes and 12 hours postinfection . Invasion of both M cells and enterocytes began at 15 minutes postinfection . No specific cell type was the main target for invasion . Intracellular bacteria were observed in the lamina propria after 1 hour postinfection . A severe acute neutrophilic response was associated with invasion of the Peyer's patches . Upregulated expression of CXC chemokines (interleukin {IL}-8, growth-related oncogenes, {GRO} alpha and gamma, and granulocyte chemotactic protein {GCP}2) was detected by reverse transcription polymerase chain reaction beginning at 1 hour postinfection . Expression of proinflammatory (IL-1beta, IL-18, and tumor necrosis factor {TNF}alpha) and anti-inflammatory (IL-10, IL-IRa, and IL-4) cytokines was also assessed . A marked increase in expression of IL-1beta was observed, whereas the profile of expression of IL-18 and TNFalpha did not change after infection . Upregulation of IL-1Ra and IL-4 but not of IL-10 was observed . These findings indicate that infection of bovine ligated ileal loops with S . typhimurium results in an acute neutrophilic inflammatory response that is associated with the upregulation of CXC chemokines (IL-8, GROalpha and gamma, and GCP2), IL-1beta, IL-IRa, and IL-4. Int J Med Microbiol, 2002 Mar, 291(8), 615 - 24 Various bacterial pathogens and symbionts infect the amoeba Dictyostelium discoideum; Skriwan C et al.; The haploid soil amoeba Dictyostelium discoideum is a suitable model organism to study host-pathogen interactions with Legionella pneumophila . In this study we show that D . discoideum AX2 is also susceptible to infection with other important human pathogens and obligate intracellular symbionts . Infection assays demonstrated that Legionella-like amoebal pathogens (LLAP K62), Mycobacterium avium and the obligate intracellular endosymbionts of Acanthamoeba sp . strains TUME1, UWE25 and UWC6 were able to multiply within Dictyostelium . Salmonella typhimurium and Pseudomonas aeruginosa also invaded Dictyostelium, however were degraded shortly after uptake . Comitin-minus host cells were more permissive to infections with L . pneumophila and LLAP K62 . Furthermore, this mutation significantly delayed the degradation of S . typhimurium . Accompanying electron and fluorescence microscopy of infected AX2 cells revealed that L . pneumophila and M . avium replicate within vacuoles, while LLAP K62, TUME1 and UWE25 were tightly enclosed by membranous structures within the cytoplasm . The beta-proteobacterium UWC6 was found to persist in the cytoplasm . The observed subcellular locations which correspond to the locations within the respective natural hosts suggest that D . discoideum is a representative model system for these pathogens and symbionts. J Virol Methods, 2002 May 16, 103(2), 129 - 36 Optimisation of ISO 10705-1 on enumeration of F-specific bacteriophages; Mooijman KA et al.; During the European project 'Bacteriophages in bathing waters' (January 1996-June 1999), research was carried out to optimise the method for detection and enumeration of F-specific (RNA) phages in water . It was evaluated whether further optimisation would be possible/needed for the procedure as described in the standard method of the International Organisation for Standardisation (ISO) 10705-1 . The research focused mainly on optimisation of the different steps for culturing the host strain WG49 Salmonella Typhimurium . It was concluded that all steps described in ISO 10705-1 are necessary and, if followed carefully, using a culture of host strain WG49 Salmonella Typhimurium of good quality, reliable results could be obtained for the enumeration of F-specific RNA phages. J Immunother, 2002 May-Jun, 25(3), 218 - 25 Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium; Rosenberg SA et al.; Salmonella typhimurium genetically modified at the purI and msbB genes to increase dependence on adenine and decrease stimulation of tumor necrosis factor-alpha production were injected intravenously into C57BL/6 mice bearing subcutaneous tumor or lung metastases . Decreased tumor growth and prolonged survival were seen in some, but not all of nine transplantable tumors . Salmonella increased in number in the tumor and reached levels 10,000 times higher than in the normal liver reservoir of these bacteria . Histologic studies revealed Salmonella growth in areas of the tumor although, in all cases, a viable rim of tumor survived and ultimately resulted in progressive tumor growth in all mice . These studies demonstrate that Salmonella can localize to transplantable murine tumors and partially inhibit tumor growth; however, additional modifications of the bacteria may be necessary if this approach is to develop into an effective treatment for patients with cancer. Biosci Biotechnol Biochem, 2002 Feb, 66(2), 363 - 72 Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity; Sun M et al.; Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered . Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of beta-carotene and reactive oxygens, and 17 lipid-radical scavenging products of alpha-tocopherol . The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9 . None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria . Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines . So, we also measured the activity to increase 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) mutagenicity . The products from beta-carotene and alpha-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2 . Thus, the products of beta-carotene and alpha-tocopherol formed after the antioxidant actions were not genotoxic. Dtsch Tierarztl Wochenschr, 2002 Apr, 109(4), 149 - 53 {Will the effectiveness of the immunization of chicks with live Salmonella vaccines be affected by maternal antibodies?}; Methner U et al.; In the progeny of breeder birds which had been vaccinated with live Salmonella Typhimurium and inactivated Salmonella Enteritidis vaccines, the caecal and systemic colonisation by a live Salmonella Enteritidis and a live Salmonella Typhimurium vaccine was studied . The efficacy of the oral immunisation of chicks from vaccinated and non-vaccinated breeders with a live Salmonella Enteritidis vaccine on day 1 of age was studied by an experimental challenge with Salmonella Enteritidis on day 30 of age . Antibody production of isotypes IgG, IgA and IgM was determined in sera and jejunum of the birds . Vaccination of parent birds resulted in an increase of the antibody concentration in sera and jejunum of the chicks . Own antibody production after administration of the live Salmonella vaccine to the day-old chicks was not detected until day 21 of life . Compared to controls, the number of vaccine organisms in the caeca of the progeny of vaccinated breeder birds was reduced by 0.5-1.5 log10 units . The reduction of the Salmonella Enteritidis vaccine was more pronounced than that of the Salmonella Typhimurium vaccine . However, the reduced colonisation by the live Salmonella vaccine strain did not impair the efficacy of the immunisation of the chicks . To ensure efficacy of the active oral immunisation of chicks from vaccinated parent birds with attenuated live Salmonella vaccines also in case where amounts of maternally transferred antibodies are even higher, it should be guaranteed that chicks take in via drinking water the recommended dose of the vaccine strain . In this connection, factors like the low intake of drinking water by very young chicks, the concentration of the vaccine organisms in the water and the survival of the vaccine should also be considered. Mutat Res, 2002 May 22, 502(1-2), 11 - 8 Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine; Motohashi N et al.; Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite . The parent strain TA4107 (oxydelta1{oxydelta(oxyR argH)1}) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively . Therefore, the level of SOS response was monitored via beta-galactosidase activity . A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity . In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity . The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical . Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response . Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite . On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite . Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response . These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response . This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells. Emerg Infect Dis, 2002 May, 8(5), 490 - 5 Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium; Helms M et al.; In a matched cohort study, we determined the death rates associated with drug resistance in Salmonella Typhimurium . We linked data from the Danish Surveillance Registry for Enteric Pathogens with the Civil Registration System and the Danish National Discharge Registry . By survival analysis, the 2-year death rates were compared with a matched sample of the general Danish population, after the data were adjusted for differences in comorbidity . In 2,047 patients with S . Typhimurium, 59 deaths were identified . Patients with pansusceptible strains of S . Typhimurium were 2.3 times more likely to die 2 years after infection than persons in the general Danish population . Patients infected with strains resistant to ampicillin, chloramphenicol, streptomycin, sulfonamide, and tetracycline were 4.8 times (95% CI 2.2 to 10.2) more likely to die, whereas quinolone resistance was associated with a mortality rate 10.3 times higher than the general population. J Immunol, 2002 May 15, 168(10), 5260 - 7 Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis; Gewirtz AT et al.; The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia . In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays . LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed . However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium . A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor . Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha . LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S . typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha . To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis . Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis . Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation. J Exp Med, 2002 May 6, 195(9), 1155 - 66 Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates; Chakravortty D et al.; Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid . A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages . Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S . typhimurium . We studied the effect of Salmonella infection on iNOS activity in macrophages . Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella . Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2 . Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella . The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice . In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style. Mol Microbiol, 2002 May, 44(3), 645 - 61 Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane; Ruiz-Albert J et al.; The Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells . It acts by translocating effector proteins across the membrane of the Salmonella-containing vacuole (SCV) . The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella-induced filaments (Sifs), which are tubular extensions of SCVs . We have investigated the role in S . typhimurium virulence of the putative SPI-2 effector genes sifB, srfJ, sseJ and sseI . An S . typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation . Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs . The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ . Transiently expressed SseJ also inhibited Sif formation by wild-type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA . Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ . Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells. Toxic Rep Ser, 1998 Nov, 66, 1 - G4 NTP Technical Report on the Toxicity Studies of 3,3',4,4'-Tetrachloroazoxybenzene (CAS No . 21232-47-3) Administered by Gavage to F344/N Rats and B6C3F1 Mice; NTP Technical Report on the Toxicity Studies of 3 et al.; 3,3',4,4'-Tetrachloroazobenzene is not commercially manufactured but is formed as an unwanted byproduct in the manufacture of 3,4-dichloroaniline and its herbicidal derivatives Propanil(R), Linuron(R), and Diuron(R) . In addition, environmental contamination by 3,3',4,4'-tetrachloroazobenzene occurs from the degradation of chloranilide herbicides and the photolysis and biolysis of 3,4-dichloroaniline . 3,3',4,4'-Tetrachloroazobenzene was nominated by the United States Environmental Protection Agency for toxicity testing based on concerns over the potential for human exposure, the structural resemblance to 2,3,7,8-tetrachlorodibenzo-p-dioxin, and the reported dioxin-like effects of 3,3',4,4'-tetrachloroazobenzene . The toxicity of 3,3',4,4'- tetrachloroazobenzene was evaluated in 16-day and 13-week gavage studies in male and female F344/N rats and B6C3F1 mice . In addition to histopathology, evaluations included hematology (rats only), clinical chemistry, thyroid hormone analyses (rats only), cytochrome P(450)1A immunohistochemical staining in the liver (rats only), and assessments of male reproductive endpoints and estrous cycle length . Genetic toxicology studies included mutagenicity tests in Salmonella typhimurium and the determination of micronuclei in mouse bone marrow and peripheral blood erythrocytes . In the 16-day studies, groups of five male and five female rats received 3,3',4,4'-Tetrachloroazobenzene in corn oil by gavage 5 days a week at doses of 0, 12.5, 32, 80, 200, or 500 mg per kg body weight . Groups of five male and five female mice received 3,3',4,4'-Tetrachloroazobenzene in corn oil by gavage 5 days a week at doses of 0, 1, 3.2, 10, 32, or 100 mg/kg . Major effects included increases in liver, lung, and spleen weights of rats and liver and heart weights of mice and decreases in thymus weights of rats and mice . No effects were found on survival or mean body weight gains of rats or mice . Incidences of hematopoietic cell proliferation in the spleen were increased in all groups of dosed male rats, in female rats that received 32 mg/kg or greater, and in 100 mg/kg male and female mice . Renal tubule hyaline droplet accumulation in the cytoplasm of renal cortical epithelial cells and chronic nephropathy were observed microscopically in male rats in the 80, 200, and 500 mg/kg groups . Female mice in the 100 mg/kg group had atrophy of the thymus . In the 13-week studies, groups of 10 male and 10 female rats and mice received 3,3',4,4'-Tetrachloroazobenzene in corn oil by gavage 5 days a week at doses of 0, 0.1, 1, 3, 10, or 30 mg/kg . In the 13-week rat study, the major effects included a decrease in the mean body weight gain of 30 mg/kg females and final mean body weights of 30 mg/kg males and females, decreased thymus weights of males and females in the 10 and 30 mg/kg groups accompanied by thymic atrophy observed microscopically, increased incidences of hematopoietic cell proliferation in the spleen in 10 and 30 mg/kg males and females, a responsive anemia in 10 and 30 mg/kg males and females at week 13, and decreased platelet counts in 10 and 30 mg/kg males and females on day 21 and at week 13 . Spleen weights were increased in 10 and 30 mg/kg males and females . Liver weights were increased in males that received 1 mg/kg or greater and in 10 and 30 mg/kg females . Furthermore, hepatic cytochrome P(450)1A staining presence and intensity were increased in 30 mg/kg males and females . Sharp decreases in circulating thyroxine concentrations were observed in males and females at all doses . In spite of this sharp decrease, thyroid-stimulating hormone concentrations were marginally increased . Incidences of hyperplasia of the forestomach were increased in males administered 3 mg/kg or greater and females administered 30 mg/kg . In the 13-week mouse study, the major effects included increases in liver and spleen weights of 10 and 30 mg/kg males and females and increased incidences of hyperplasia of the forestomach in males and females that received 1 mg/kg or greater . Furthermore, a decrease in thymus weight of 30 mg/kg males, an increase in centrilobular hypertrophy of hepatocytes in males that received 3 mg/kg or greater, and an increase in the incidences of hematopoietic cell proliferation in the spleen in males that received 3 mg/kg or greater were observed . A significant decrease in epididymal spermatozoal concentration was observed in 3 and 30 mg/kg males . 3,3',4,4'-Tetrachloroazobenzene was mutagenic in S . typhimurium strain TA97 in the presence of rat liver S9 activation enzymes; no mutagenic activity was detected in strain TA98, TA100, TA1535, or TA1537 with or without S9 . In vivo, the frequency of micronucleated erythrocytes was significantly increased in peripheral blood samples from male and female mice given 3,3',4,4'-Tetrachloroazobenzene by gavage for 13 weeks . However, results of a 3-day exposure of up to 200 mg/kg by intraperitoneal injection did not demonstrate induction of micronuclei in bone marrow erythrocytes of male mice . In summary, 3,3',4,4'-Tetrachloroazobenzene caused typical dioxin-like effects, such as thymic atrophy, an increase in liver weights, induction of hepatic cytochrome P(450)1A, and decreased mean body weight gains . Furthermore, in the 13-week studies, a sharp decrease in circulating thyroxine concentrations was observed even at the lowest dose (0.1 mg/kg) tested in rats . Other effects included a decrease in epididymal spermatozoal concentration in mice, major effects on the hematopoietic system, and increased incidences of hyperplasia of the forestomach in 3 and 30 mg/kg males and 30 mg/kg females . A no-observable-adverse-effect-level (NOAEL) was not reached in rats . The NOAEL in mice was 0.1 mg/kg . Comparison of various dioxin-like effects in these studies with those reported in the literature indicate that 3,3',4,4'-Tetrachloroazobenzene is six to two orders of magnitude less potent than 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxic Rep Ser, 2000 Feb, 57, 1 - F14 NTP Toxicity Studies of Benzyltrimethylammonium Chloride (CAS No . 56-93-9) Administered by Gavage to F344/N Rats, Sprague-Dawley Rats, and B6C3F1 Mice; NTP Toxicity Studies of Carisoprodol (CAS No . 78-44-4) Administered by Gavage to F344/N Rats and B6C3F1 Mice; Carisoprodol is a widely used skeletal muscle relaxant and analgesic and is available as a prescription drug . Comparative studies were conducted to determine the toxicity of carisoprodol administered in corn oil and in 0.5% methylcellulose by gavage . Carisoprodol plasma concentrations of rats and mice were measured at the end of the 13-week studies; single-dose plasma carisoprodol analyses were also performed . Genetic toxicity studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, cultured Chinese hamster ovary cells, and peripheral blood erythrocytes of mice . Groups of 10 male and 10 female F344/N rats received 0, 100, 200, 400, 800, or 1,600 mg carisoprodol per kilogram body weight in corn oil by gavage or 0, 100, 200, 400, or 800 mg/kg carisoprodol in 0.5% methylcellulose by gavage for 13 weeks . Groups of 10 male and 10 female B6C3F1 mice received 0, 75, 150, 300, 600, or 1,200 mg/kg carisoprodol in corn oil by gavage or 0, 600, 1,200, or 1,600 mg/kg carisoprodol in 0.5% methylcellulose by gavage for 13 weeks . Among rats that received carisoprodol in corn oil, survival was similar to that of the vehicle controls . Survival of rats administered carisoprodol in 0.5% methylcellulose was also similar to that of the vehicle controls after adjustment for deaths (two males and one female in the 800 mg/kg group and two females in the 400 mg/kg group) . The final mean body weight gain of males administered 1,600 mg/kg carisoprodol in corn oil was significantly less than that of the vehicle controls; the final mean body weights and body weight gains of female rats in the 800 and 1,600 mg/kg groups were significantly greater . In the carisoprodol in 0.5% methylcellulose study, males in the 200 mg/kg group and females in the 100 and 800 mg/kg groups had significantly greater mean body weights and body weight gains than did the vehicle controls . Clinical findings in rats administered carisoprodol in corn oil or in 0.5% methylcellulose included lethargy, ataxia, diarrhea, and prostration; the incidences were dose-related, and females were more sensitive than males to the effects of carisoprodol . In the carisoprodol in corn oil study, differences in hematology and clinical chemistry parameters occurred with no consistent patterns . The effects of carisoprodol in 0.5% methylcellulose on hematology and clinical chemistry parameters were not studied . In the corn oil study, the kidney and liver weights of male and female rats administered 200 mg/kg carisoprodol or greater were generally significantly greater than those of the vehicle controls . In the 0.5% methylcellulose study, liver weights were significantly greater in male rats administered 400 or 800 mg/kg and in female rats administered 800 mg/kg carisoprodol compared to the vehicle controls; however, a consistent effect on the kidney weights was not observed . Nephropathy was observed in male rats administered 400 mg/kg carisoprodol or greater in corn oil; the livers of four males in the 1,600 mg/kg group had centrilobular hypertrophy of hepatocytes . No lesions were observed histopathologically in female rats administered carisoprodol in corn oil . In the carisoprodol in 0.5% methylcellulose study, the severity of nephropathy in males administered 200 mg/kg or greater was enhanced, and the incidence of nephropathy in female rats in the 800 mg/kg group was slightly greater than that in the vehicle controls . Plasma carisoprodol concentrations at the end of 13 weeks generally increased with increasing dose in rats administered carisoprodol in corn oil or in 0.5% methylcellulose . The plasma carisoprodol concentrations in rats administered a single gavage dose of carisoprodol in corn oil also increased with increasing dose . In the carisoprodol in corn oil mouse study, two females each in the vehicle control and 75 mg/kg groups and one female each in the 150 and 600 mg/kg groups were accidentally killed; all males survived to the end of the study . One male and one female administered 1,600 mg/kg carisoprodol in 0.5% methylcellulose died; seven mice were accidentally killed . The mean body weights and body weight gains of mice administered carisoprodol in corn oil were generally similar to those of the vehicle controls . The final mean body weights and body weight gains of all groups of males and females administered carisoprodol in 0.5% methylcellulose were significantly less . Clinical findings in the carisoprodol in corn oil study included lethargy, ataxia, tremors, and prostration in male and female mice . Ataxia, lethargy, convulsions, and prostration were observed in all dosed groups of males and females administered carisoprodol in 0.5% methylcellulose . In the carisoprodol in corn oil study, liver weights were significantly greater in males administered 300 mg/kg or greater and in females administered 150 mg/kg or greater than in the vehicle controls . In the carisoprodol in corn oil study, no gross or microscopic lesions were considered related to carisoprodol administration . Minimal to mild centrilobular hypertrophy was observed in the liver of all dosed groups of males and in females in the 1,200 and 1,600 mg/kg groups in the carisoprodol in 0.5% methylcellulose study . The testis weights of males administered 1,200 mg/kg carisoprodol in corn oil were significantly less than those of the vehicle controls; the sperm motility of males in this group was also significantly less than that of the vehicle controls . There were no significant differences in vaginal cytology parameters between dosed and vehicle control females . At the end of the carisoprodol in corn oil study, the concentration of carisoprodol was above the limit of detection in the plasma of only one male mouse each in the 300 and 1,200 mg/kg groups and in four females in the 1,200 mg/kg group . In mice administered a single gavage dose of carisoprodol in corn oil, plasma concentrations increased with increasing dose; peak plasma concentrations occurred at 20 to 120 minutes in males and 60 to 120 minutes in females . In the carisoprodol in 0.5% methylcellulose study, plasma carisoprodol concentrations of female, but not male, mice increased with increasing dose; peak plasma carisoprodol concentrations occurred at 30 minutes postdosing in all groups of males and females . Results of proportionality and bioavailability studies indicated that single gavage doses of 200 to 800 mg/kg carisoprodol in 0.5% methylcellulose in rats or 300 to 1,200 mg/kg in mice were dose proportional; absolute bioavailability values increased with increasing dose, ranging from 15% to 32% for rats and from 18% to 38% for mice . For rats, the bioavailability of carisoprodol in 0.5% methylcellulose was approximately fivefold that of carisoprodol in corn oil; the C(max) values of the dose in 0.5% methylcellulose were approximately threefold those of the dose in corn oil . For mice, no significant difference was observed in the bioavailability of carisoprodol between the vehicles; however, the C(max) values of the dose in 0.5% methylcellulose were 1.5 to 1.75 times those of the dose in corn oil . Carisoprodol was not mutagenic in any of four strains of Salmonella typhimurium, with or without S9 metabolic activation . It did induce mutations in L5178Y mouse lymphoma cells in the absence of S9; with S9, no mutagenic activity was noted in this assay . Results of the sister chromatid exchange test with carisoprodol in cultured Chinese hamster ovary cells were considered equivocal with and without S9 . Chromosomal aberrations in cultured Chinese hamster ovary cells were clearly increased by carisoprodol treatment, particularly in the presence of S9 . No significant increases in the frequency of micronucleated erythrocytes were observed in peripheral blood samples from male and female mice administered carisoprodol by gavage for 13 weeks . In conclusion, carisoprodol induced ataxia and prostration in rats and mice, increases in liver weights in rats and mice, and nephropathy in male rats . The bioavailability of carisoprodol in 5% methylcellulose was greater than in corn oil . The no-observed-adverse-effect (NOAEL) level of carisoprodol administered in corn oil or in 0.5% methylcellulose was determined to be 100 mg/kg, compared to the clinical dose of 20 mg/kg per day for adults and 5 to 7.5 mg/kg per day for children. Toxic Rep Ser, 1996 Apr, 50, 1 - E8 NTP Toxicity Studies of Cyclohexanone Oxime Administered by Drinking Water to B6C3F1 Mice (CAS No . 100-64-1); NTP Hepatotoxicity Studies of the Liver Carcinogen Methapyrilene Hydrochloride (CAS No . 135-23-9) Administered in Feed to Male F344/N Rats; Methapyrilene hydrochloride is a histamine H(1)-receptor antagonist that was an active ingredient in many over-the-counter cold and allergy medications . In the mid- to late 1970s, studies in rats suggested that methapyrilene hydrochloride was a hepatocarcinogen, and the drug was removed from these preparations . In most cases, methapyrilene hydrochloride was replaced by pyrilamine maleate, a structurally similar analogue . As part of a program to investigate mechanisms of toxicity whereby structurally similar chemicals produce different toxicities, these chemicals were studied for induction of cell proliferation and protein alterations by two-dimensional gel electrophoresis in the liver of F344/N rats . A complete toxicologic evaluation was not needed for this research-oriented study . Rather, the goal of the present study was to provide retrospective data from subchronic toxicity studies with the known rat carcinogen methapyriline hydrochloride that could then be used to predict the potential carcinogenicity of unknown chemical agents and that could also be compared with similar data on the structural analogue pyrilamine maleate . Pyrilamine maleate differs from methapyrilene hydrochloride in the substitution of the thienyl ring with a paramethoxyphenyl ring . Pyrilamine maleate has been shown to produce an equivocal increase in the incidences of liver neoplasms in rats in 2-year feed studies, but only at 2,000 ppm, indicating that its potency, if any, to produce neoplasms is much less than that of methapyriline hydrochloride . The hepatocarcinogenic peroxisome proliferator Wy-14,643 was included in this study as a positive control that is known to induce cell proliferation, as well as protein alterations, in the liver . In the 14-week study of methapyrilene hydrochloride, groups of 40 male F344/N rats were given 0, 50, 100, 250, or 1,000 ppm methapyrilene hydrochloride, 1,000 ppm pyrilamine maleate (negative control), or 50 ppm Wy-14,643 ( positive control) in feed . Rats in all groups were administered bromodeoxyuridine (BrdU) by osmotic minipump for the assessment of hepatocyte proliferation . Ten rats from each group were evaluated on days 15, 29, and 43 and at 14 weeks . At these times, samples of liver tissue were analyzed for evidence of cell proliferation via BrdU labeling and proliferating cell nuclear antigen (PCNA) labeling . There were no exposure-related deaths . Low mean body weights were generally observed in the 1,000 ppm methapyriline hydrochloride group and in the positive control group . Final mean body weights and mean body weight gains of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group at all time points . The final mean body weights of rats in the positive control group were significantly less than those of the untreated control group for rats evaluated on days 29 and 43 and at week 14; the mean body weight gains of rats in the positive control group were significantly less than those of the untreated control group on day 29 and at week 14 . Feed consumption by rats exposed to 1,000 ppm methapyrilene hydrochloride was significantly less than that by the untreated control group throughout the study . The predominant clinical observation related to methapyrilene hydrochloride exposure was thinness in rats exposed to 1,000 ppm; this finding was first observed on day 29 . On days 29 and 43 and at 14 weeks, the absolute liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride were significantly less than those of the untreated control group . At all time points, the relative liver weights of rats exposed to 1,000 ppm methapyrilene hydrochloride and the absolute and relative liver weights of positive control rats were significantly greater than those of the untreated control group . No significant differences in liver weights were observed between the negative and untreated control groups at any time point . Hepatic lesions were observed predominantly in the 250 and 1,000 ppm methapyrilene hydrochloride groups and in the positive control group . The incidences of bile duct hyperplasia, hepatocyte necrosis, hepatocyte mitosis, and hepatocyte hypertrophy in rats in the 1,000 ppm group were significantly greater than those in the untreated control group at all time points . The severities of hepatocyte hypertrophy and hepatocyte mitosis in 1,000 ppm rats were generally mild to moderate; the lesions occurring in 250 ppm animals were less severe . At each time point, the incidence of bile duct hyperplasia in 250 ppm rats was significantly greater than that in the untreated control group . The incidences of hepatocyte mitosis on days 15 and 29 and the incidences of hepatocyte necrosis on days 29 and 43 in rats in the 250 ppm group were significantly greater than those in the untreated control group . Incidences of pigmentation in the 250 and 1,000 ppm methapyrilene hydrochloride groups were significantly greater than those in the untreated control group on days 29 and 43 and at 14 weeks . In the positive control group, the incidences of granulomatous inflammation were significantly greater than those in the untreated control group on days 15, 29, and 43 . The incidences of hepatocyte hypertrophy and hepatocyte mitosis in the positive control group were significantly greater than those in the untreated control group on days 15, 29, and 43 . The incidence of hepatocyte hypertrophy was also significantly increased in the positive control group at 14 weeks . The severity of hepatocyte hypertrophy in the 1,000 ppm methapyrilene hydrochloride group was generally greater than that in the positive control group at each time point . In general, methapyriline hydrochloride produced a dramatic and sustained increase in hepatic cell proliferation over 14 weeks, whereas pyrilamine maleate at the same concentration produced few if any effects . Wy-14,643 also induced a large increase in cell proliferation which declined over time, as has been observed in previous studies . The mean BrdU labeling indexes of the 250 and 1,000 ppm methapyrilene hydrochloride groups were generally significantly greater than those of the untreated controls at all time points . In the negative control group, the BrdU labeling index was significantly less than that of the untreated control group on day 29 . The BrdU labeling index in the positive control group was significantly greater than that of the untreated control group at all time points . On day 43 and at week 14, the mean PCNA labeling indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group . The mean PCNA labeling indexes of the negative control group were significantly less than those of the untreated control group on days 29 and 43 . On day 29, the mean PCNA labeling index of the positive control group was significantly greater than that of the untreated control group . The mitotic indexes of the 1,000 ppm methapyrilene hydrochloride group were significantly greater than those of the untreated control group at all time points . The mitotic indexes of the 250 ppm group were significantly greater than those of the untreated control group on day 43 and at week 14 . At least 32 proteins underwent significant abundance changes at the highest exposure concentration of methapyrilene hydrochloride, and 39 protein changes were observed in the positive control group . Many, but not all, of the protein changes in the methapyrilene hydrochloride-exposed animals also occurred in the positive control group . Treatment with pyrilamine maleate produced no significant quantitative protein changes, as judged by the same criteria used for methapyrilene hydrochloride and Wy-14,643 . Methapyrilene hydrochloride produced covalent modification of mitochondrial proteins as measured by the charge modification index . PCNA abundance in liver samples from the 250 and 1,000 ppm methapyrilene hydrochloride exposure groups on day 43 was significantly greater than that of the untreated control group . Results of tests for induction of mutagenicity by methapyrilene hydrochloride were negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in L5178Y mouse lymphoma cells, with and without S9 metabolic activation . However, positive responses were obtained in cytogenetic tests with cultured Chinese hamster ovary cells, in which methapyrilene hydrochloride induced sister chromatid exchanges and chromosomal aberrations . The increases in sister chromosome exchanges were obtained with and without S9, but chromosomal aberrations were increased only in the presence of S9 . In summary, the significance of the increased hepatic cell proliferation and the protein alterations observed in this study is not definite, but may be of predictive value for assessing the toxicity and carcinogenicity of chemicals in preclinical assays . A chemical which does not produce an increase in cell proliferation or a large number of protein changes may be considered safer than a similar chemical that produces many such changes. Eur J Biochem, 2002 Apr, 269(8), 2143 - 50 The phosphotransferase system of Streptomyces coelicolor; Kamionka A et al.; We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion . As in E . coli, the crr gene of S . coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I . The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained . Western blot analysis revealed that IIACrr is expressed in vivo . The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr . Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E . coli IIAGlucose, was mutated . The capacity of IIACrr to operate in sugar transport was shown by complementation of the E . coli glucose-PTS . The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E . coli . A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance . These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S . coelicolor. Yi Chuan Xue Bao, 2002 Apr, 29(4), 370 - 6 Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Salmonella typhimurium; Xu B et al.; LT and ST are the main enterotoxins of enterotoxigenic Escherichia coli (ETEC) found in clinical isolates, and CS3 (the common antigen in the CFA/II family of fimbrial antigens) is one of the most prevalent antigens of colonization factors . The genetic determinants encoding CS3 and LT-B/ST fusion toxin were manipulated so that these important antigens are expressed simultaneously in attenuated Salmonella typhimurium oral vaccine strain X4072 . These antigens produced by X4072 (pXZL88) could be recognized with monospecific CS3, LT or ST antibodies respectively . The specific antibodies against CS3, LT and ST could be detected . In the sera of immunized mice via oral route with the live bacteria . Significantly, the antibody to ST was able to neutralize the biological activity of native ST . This prototype construct may be proved to be useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrhea disease. Invest Ophthalmol Vis Sci, 2002 May, 43(5), 1493 - 8 MCP-1 expression in endotoxin-induced uveitis; Tuaillon N et al.; PURPOSE: Monocyte chemoattractant protein (MCP)-1 (CCL-2) is a chemokine with chemoattractant properties for monocytes, memory T cells, natural killer cells, mast cells, and basophils . To delineate the role played by MCP-1 in acute anterior uveitis, a common ocular inflammation, MCP-1(-/-) mice and wild-type matched control mice were analyzed for the development of endotoxin-induced uveitis (EIU) in response to subcutaneous injection of a sublethal dose of lipopolysaccharide (LPS) . METHODS: EIU was induced in MCP-1(-/-) and wild-type control mice by a single subcutaneous injection of Salmonella typhimurium LPS endotoxin at day 0 . Alternatively, MCP-1(-/-) mice were injected subcutaneously with LPS plus recombinant MCP-1 at day 0 and with recombinant MCP-1 6 hours later . Mice were killed at day 1 or 3 after injection . Serum levels of IL-1alpha, IL-1beta, IL-6, IFN-gamma, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1alpha, MIP-2, regulated on activation normal T-cell expressed and secreted (RANTES), and MCP-1 were determined by ELISA . Eyes were collected and analyzed histologically and by RT-PCR for MCP-1, IFN-gamma, IL-6, TNF-alpha, beta-actin, MCP-5, RANTES, KC, inflammatory protein (IP)-10, and toll-like receptor (TLR)-4 . RESULTS: EIU was strongly reduced in MCP-1(-/-) mice compared with wild-type control mice . The number of ocular inflammatory cells was significantly reduced . Moreover, intraocular IFN-gamma transcription was increased . EIU was induced in MCP-1(-/-) mice by co-administration of recombinant rat MCP-1 and LPS . CONCLUSIONS: Data indicate that MCP-1 plays a crucial role in the induction of EIU . MCP-1 may be a new therapeutic strategy for acute anterior uveitis. Mutagenesis, 2002 May, 17(3), 201 - 9 Assessment of the genotoxic potential of ISIS 2302: a phosphorothioate oligodeoxynucleotide; Henry SP et al.; ISIS 2302, a phosphorothioate oligodeoxynucleotide with antisense activity against human ICAM-1 mRNA, was evaluated in a battery of tests to assess genotoxic potential . There was no evidence of genotoxicity in three in vitro studies performed: (i) a bacterial reverse mutation test; (ii) a chromosomal aberration test in Chinese hamster ovary cells; (iii) a mammalian cell gene mutation assay in L5187Y cells . Additionally, there was no in vivo evidence of genetic toxicity in a bone marrow micronucleus study in male and female mice . For all tests, top concentrations or doses assessed met harmonized regulatory guidelines . The cellular uptake of ISIS 2302 into target cells was confirmed using capillary gel electrophoresis and immunohistochemistry . Intracellular uptake into CHO cells, L5187Y cells, Salmonella typhimurium TA98 and bone marrow was concentration- and time-dependent . Consistent with what is known about the physical and chemical properties of phosphorothioate oligodeoxynucleotides, there was no evidence of genotoxicity in any of the assessed end-points . Furthermore, the absence of genotoxicity could not be ascribed to test system insensitivity or to an absence of exposure of the test system to ISIS 2302. Biochemistry, 2002 Apr 30, 41(17), 5493 - 504 Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins; Wood ZA et al.; 2-Cys peroxiredoxins (Prxs) are a large and diverse family of peroxidases which, in addition to their antioxidant functions, regulate cell signaling pathways, apoptosis, and differentiation . These enzymes are obligate homodimers (alpha(2)), utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form two oligomeric states: individual alpha(2) dimers or doughnut-shaped (alpha(2))(5) decamers . Here we characterize both the oligomerization properties and crystal structure of a bacterial 2-Cys Prx, Salmonella typhimurium AhpC . Analytical ultracentrifugation and dynamic light scattering show that AhpC's oligomeric state is redox linked, with oxidization favoring the dimeric state . The 2.5 A resolution crystal structure (R = 18.5%, R(free) = 23.9%) of oxidized, decameric AhpC reveals a metastable oligomerization intermediate, allowing us to identify a loop that adopts distinct conformations associated with decameric and dimeric states, with disulfide bond formation favoring the latter . This molecular switch contains the peroxidatic cysteine and acts to buttress the oligomerization interface in the reduced, decameric enzyme . A structurally detailed catalytic cycle incorporating these ideas and linking activity to oligomeric state is presented . Finally, on the basis of sequence comparisons, we suggest that the enzymatic and signaling activities of all 2-Cys Prxs are regulated by a redox-sensitive dimer to decamer transition. Toxic Rep Ser, 1995 Sep, 42, 1 - D6 Toxicity Studies of 1,3-Diphenylguanidine (CAS No . 102-06-7) Administered in Feed to F344/N Rats and B6C3F1 Mice; NTP Technical Report on the Toxicity Studies of 1 et al.; 1,1,1-Trichloroethane is a widely used solvent in industry and in household products such as cleaning agents, wallpaper and carpet glues, carpets, spray and solid insecticides, and rodenticides . 1,1,1-Trichloroethane was studied because of its widespread use in industry and in the home and the potential for human exposure . Groups of 10 male and 10 female F344/N rats and B6C3F1 mice were given 5,000, 10,000, 20,000, 40,000, or 80,000 ppm microencapsulated 1,1,1,-trichloroethane in feed for 13 weeks . Groups of 10 male and 10 female rats and mice served as untreated controls and received feed without microcapsules; additional groups of 10 male and 10 female rats and mice served as vehicle controls and received feed with empty microcapsules . Animals were evaluated for clinical pathology (rats only), reproductive system effects, and histopathology . Genetic toxicity studies were conducted in Salmonella typhimurium, L5178Y mouse lymphoma cells, and cultured Chinese hamster ovary cells . In addition, peripheral blood slides from the mice in the 13-week study were analyzed for frequency of micronucleated erythrocytes . All rats survived to the end of the study . The final mean body weights of exposed rats were within 10% of those of the untreated and vehicle controls . Feed consumption by exposed groups of male and female rats was similar to that by the control groups, suggesting that the diet was palatable to the animals . Based on average feed consumption values, male rats ingested approximately 300, 600, 1,200, 2,400, or 4,800 mg 1,1,1-trichloroethane/kg body weight per day, and females received 300, 650, 1,250, 2,500, or 5,000 mg/kg per day . In general, changes in clinical pathology parameters were minor, sporadic, and inconsistent between males and females; these differences were not considered to be treatment related or biologically significant . The liver weights of female rats administered 80,000 ppm were significantly less than those of the untreated and vehicle controls . Male rats exposed to 10,000 ppm or greater had a spectrum of nonneoplastic kidney lesions consistent with hyaline droplet nephropathy . No treatment-related gross or microscopic lesions were observed in female rats . There were no exposure-related deaths in mice . Based on average feed consumption values, male mice ingested approximately 850, 1,770, 3,500, 7,370, or 15,000 mg/kg per day, and female mice received 1,340, 2,820, 5,600, 11,125, or 23,000 mg/kg per day . Even though feed consumption by exposed groups was slightly greater than that by the controls, the mean body weights of male and female mice administered 20,000 ppm or greater were significantly less than those of the untreated and vehicle controls . The heart, kidney, and lung weights of the vehicle control male mice were significantly greater than those of the untreated controls . There were no biologically significant differences in organ weights between exposed and control mice . No gross or microscopic lesions in male or female mice were attributed to chemical exposure . Epididymal spermatozoal concentrations of male rats and mice given 80,000 ppm were significantly less than those of the vehicle controls . 1,1,1-Trichloroethane was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537, with or without S9 metabolic activation . In the mouse lymphoma assay for induction of trifluorothymidine resistance in L5178Y cells, 1,1,1-trichloroethane gave a negative response in one test (with and without S9) and an equivocal response in a second test (in the presence of S9) . Results of a sister chromatid exchange test in cultured Chinese hamster ovary cells were considered to be equivocal due to an unrepeated questionable response obtained in the presence of S9 in a single trial; without S9, results were negative . 1,1,1-Trichloroethane induced chromosomal aberrations in cultured Chinese hamster ovary cells in the absence of S9; with S9, the increase in aberrations noted in a single trial was not significant . A small increase in the frequency of micronucleated normochromatic erythrocytes was noted in peripheral blood slides from male mice administered 1,1,1-trichloroethane in feed for 13 weeks; the results were determined to be equivocal, while the female peripheral blood micronucleus test results were negative . In conclusion, 1,1,1-trichloroethane induced nonneoplastic lesions consistent with hyaline droplet nephropathy in male rats . Exposure to 1,1,1-trichloroethane caused decreases in liver weights in female rats and decreases in mean body weights of male and female mice . The no-observed-adverse-effect level (NOAEL) was estimated to be 10,000 ppm for male and female rats and mice. Toxic Rep Ser, 1995 Mar, 39, 1 - D3 NTP Toxicity Studies of Cadmium Oxide (CAS No . 1306-19-0) Administered by Inhalation to F344/N Rats and B6C3F1 Mice; Typhoid fever: pathogenesis and disease; Centre for Molecular Microbiology and Infection, Imperial College of Science Technology and Medicine, London, UKTyphoid fever is an infectious disease of global distribution . Although there is a wealth of data on Salmonella typhimurium infection in the mouse and the interaction of this serovar with human cell lines in vitro, there is a relatively small amount of data on S . typhi and the pathogenesis of typhoid fever . In this review we focus on three areas: adherence to and invasion of gut epithelial cells, dissemination to systemic sites, and survival and replication within host cells . In addition, we attempt to put current salmonella research into the context of typhoid fever. Food Addit Contam, 2002 Apr, 19(4), 323 - 34 Toxicological studies on Polyporus pinsitus laccase expressed by Aspergillus oryzae intended for use in food; Brinch DS et al.; The laccase used in the study was produced by submerged fermentation of Aspergillus oryzae, containing a gene originating from Polyporus pinsitus . Laccase is to be employed as a processing aid in the juice industry to make a clear and stable juice . The enzyme was subject to a series of toxicological tests to document its safety in use . It was not mutagenic in the Salmonella typhimurium reverse mutation assay, and it did not cause chromosomal aberrations in cultured human lymphocytes . No evidence of inhalation toxicity or skin and eye irritation was found . Oral administration to rat of up to 10 ml kg(-1) b.w . day(-1) (equivalent to a total organic solids dosage of 676 mg kg(-1) b.w . day(-1) or a laccase dosage of 2601 LACU kg(-1) b.w . day(-1)) for 13 weeks did not cause any adverse effect . The maximum recommended dosage of laccase used for juice applications is 50 LACU l(-1) juice. Eur Cytokine Netw, 2002 Jan-Mar, 13(1), 104 - 9 Divergent effects of tumor necrosis factor-alpha and lymphotoxin-alpha on lethal endotoxemia and infection with live Salmonella typhimurium in mice; Dharmana E et al.; During septic shock with Gram-negative microorganisms, mortality is determined by two independent factors: high concentrations of circulating proinflammatory cytokines and multiplication of the microorganisms in the organs of the host . We studied the role of endogenous tumor necrosis factor-alpha (TNF) and lymphotoxin-alpha (LT) in the pathogenesis of lethal endotoxemia and infection with viable Salmonella typhimurium . Compared to wild-type control mice, TNF-/-LT-/- knock-out mice were more resistant (100% versus 25% mortality) to a lethal challenge with LPS, due to a significantly decreased production of the proinflammatory cytokines TNF, IL-1alpha and IL-1beta . In contrast, TNF-/-LT-/- mice were highly susceptible to infection with viable S . typhimurium as compared to wild-type mice (100% versus 0% mortality), and this was accompanied by a 100-fold greater bacterial load in their organs . The effect of endogenous TNF and LT during infection was mediated by a defective recruitment of neutrophils at the site of infection, as well as a reduced intracellular killing of S . typhimurium by these cells . These results show that TNF and LT have crucial, yet opposite effects on lethal endotoxemia induced by S . typhimurium LPS and on the infection of mice with live Salmonella microorganisms, and suggest caution when extrapolating results obtained in the lethal endotoxemia model to bacteremia in patients. Vet Microbiol, 2002 May 24, 86(4), 295 - 301 Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization; Yang SJ et al.; Fourteen and 22 each of Salmonella Enteritidis and Salmonella Typhimurium (S . Typhimurium) were isolated from animals from 1983 to 1999 in Korea and tested for their antibiotic resistance patterns, phage types and resistance gene patterns . S . Typhimurium isolates were highly resistant to streptomycin, sulfisoxazole and tetracycline, 95, 95 and 86%, respectively . The incidence of multiple antibiotic resistance (resistant to more than two drugs tested) of S . Typhimurium isolates was extremely high (100%) comparing to S . Enteritidis isolates (21%) . Two of the five ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline) resistant type S . Typhimurium isolates were phage type definitive type 104 (DT104).For the detection of resistance related genes in S . Enteritidis and S . Typhimurium isolates, particularly ACSSuT type S . Typhimurium, antibiotic resistance genes, cmlA/tetR, bla(PSE-1) and bla(TEM), and genus Salmonella specific gene, sipB/C, were amplified using four pairs of primers in a hot-start multiplex polymerase chain reaction (PCR) . Two Korean isolates of S . Typhimurium DT104 showed bla(TEM) amplicons instead of bla(PSE-1) for the ampicillin resistance and they were susceptible to florfenicol . The multiplex PCR used in this study was useful in characterization of multiple drug resistant Salmonella isolates, especially ACSSuT type S . Typhimurium, and identification of beta-lactamase gene distribution among Salmonella isolates. Food Chem Toxicol, 2002 May, 40(5), 617 - 24 Genetic toxicity of a standardized mixture of citrus polymethoxylated flavones; Delaney B et al.; Flavonoids are a ubiquitous family of phytochemicals that display a variety of biological effects, both beneficial and adverse depending on the individual compound . Certain flavonoids are genotoxic while others inhibit the genotoxicity of other mutagens . In the present studies, the mutagenicity of a mixture of polymethoxylated flavones (PMFs) purified from citrus peel oil was evaluated . The mixture consisted of nobiletin (32.5%), 3,3',4',5,6,7,8-heptamethoxyflavone (25.0%), tangeretin (14.0%), trimethylscutellarein (9.1%), sinensetin (3.9%), 5-demethyl-nobiletin (2.8%), hexa-O-methylquercetagetin (3.3%), 5-demethyl-tetramethylscutellarein (0.7%), 5-hydroxy-3,3',4',6,7,8-hexamethoxyflavone (0.7%), and a small quantity of unidentified flavonoid compounds (3.9%) . In vitro addition of the PMF mixture over a concentration range that spanned four log doses (0.0005-5.0 mg/plate) did not reveal any evidence of mutagenicity in five bacterial tester strains (Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537) either in the absence or presence of S9 activation . The PMF mixture exhibited a statistically significant increase in mutagenicity of L5178Y tk(+/-) mouse lymphoma cells at 0.05 (38.5 x 10(-6); P<0.05) and 0.1 mg/ml (61 x 10(-6); P<0.01) compared with vehicle-treated controls (mutation frequency=19.7 x 10(-6)) . However, these responses were within historical values observed in negative control cultures and extremely small compared to the positive control (EMS 0.5 microl/ml; 1685.3 x 10(-6)) . Furthermore, in the presence of S9 there was no indication of genetic toxicity in L5178Y tk(+/-) cells . These results demonstrate that the PMF mixture is not genotoxic in in vitro assay systems. Food Chem Toxicol, 2002 May, 40(5), 599 - 607 Safety evaluation of proanthocyanidin-rich extract from grape seeds; Yamakoshi J et al.; Proanthocyanidins, extracted from grape seeds, are widely used mainly as nutritional supplements . However, there has not been a systematic report to investigate toxicological studies on proanthocyanidins, especially in oral administration . In our studies, proanthocyanidin-rich extract from grape seeds was subjected to a series of toxicological tests to document its safety for use in various foods . The grape seed extract (GSE) was examined for acute and subchronic oral toxicity using Fischer 344 rats and for mutagenic potential by the reverse mutation test using Salmonella typhimurium, the chromosomal aberration test using CHL cells, and the micronucleus test using ddY mice . No evidence of acute oral toxicity at dosages of 2 and 4 g/kg, and no evidence of mutagenicity in the above tests was found . Administration of GSE as a dietary admixture at levels of 0.02, 0.2 and 2% (w/w) to the rats for 90 days did not induce noticeable signs of toxicity . The no-observed-adverse-effect level (NOAEL) of GSE in the subchronic toxicity study was 2% in the diet (equal to 1410 mg/kg body weight/day in males and 1501 mg/kg body weight/day in females) . The results of our studies indicate a lack of toxicity and support the use of proanthocyanidin-rich extract from grape seeds for various foods. Biochemistry, 2002 Apr 23, 41(16), 5093 - 103 Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein; Benton CB et al.; P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium . The 70 kDa subunits include long beta-helix domains . After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts . Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms . The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure . We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins . The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding . Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer . By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge . Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains . These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains . This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer. J Mol Biol, 2002 Apr 5, 317(4), 481 - 92 A cytosolic tRNA with an unmodified adenosine in the wobble position reads a codon ending with the non-complementary nucleoside cytidine; Chen P et al.; Out of more than 500 sequenced cytosolic tRNAs, there is only one with an unmodified adenosine in the wobble position (position 34) . The reason for this rare occurrence of A34 is that it is mostly deaminated to inosine-34 (I34) . I34 is a common constituent in the wobble position of tRNAs and has a decoding capacity different from that of A34 . We have isolated a mutant (proL207) of Salmonella typhimurium, in which the wobble nucleoside G34 has been replaced by an unmodified A in tRNA(Pro)(GGG), which is the only tRNA that normally reads the CCC codon . Thus, this mutant apparently has no tRNA that is considered cognate for the codon CCC . Despite this, the mutant grows normally . As expected, Pro-tRNA selection at the CCC codon in the A-site in a mutant deleted for the proL gene, which encodes the tRNA(Pro)(GGG), was severely reduced . However, in comparison this rate of selection was only slightly reduced in the proL207 mutant with its A34 containing tRNA(Pro)(AGG) suggesting that this tRNA reads CCC . Moreover, measurements of the interference by a tRNA residing in the P-site on the apparent termination efficiency at the A-site indicated that indeed the A34 containing tRNA reads the CCC codon . We conclude that A34 in a cytosolic tRNA is not detrimental to the cell and that the mutant tRNA(Pro)(AGG) is able to read the CCC codon like its wild-type counterpart tRNA(Pro)(GGG) . We suggest that the decoding of the CCC codon by a 5'-AGG-3' anticodon occurs by a wobble base-pair between a protonated A34 and a C in the mRNA . Life Sci Space Res, 1969, 7, 62 - 6 Effects of radiation during space flight on microorganisms and plants on the Biosatellite II and Gemini XI Missions; de Serres FJ; The results of recent experiments with the lysogenic bacteria, Escherichia coli and Salmonella typhimurium, the bread mold Neurospora crassa and the flowering plant Tradescantia on the Biosatellite II and Gemini XI Missions will be summarized . In the lysogenic bacteria experiment (Dr . Rudolf H.T . Mattoni, NUS Corporation) on the Biosatellite II mission significant effects of space flight were found on both growth rate and the induction of prophage . In that part of the Neurospora experiment on both the Biosatellite II and Gemini XI Missions (Dr . J.F . de Serres), utilizing non-dividing and inactive spores, no difference was found in the genetic effects of radiation between the flight and ground samples . In that portion of the Neurospora experiment on the Gemini XI mission utilizing rapidly-metabolizing spores the genetic effects of radiation were less serious in the flight samples than the ground samples . In the Tradescantia experiment (Dr . A.H . Sparrow, Brookhaven National Laboratory) on the Biosatellite II Mission, the irradiated flight material, in general, produced increased rates of cell death, abortion of pollen, loss of reproductive integrity, as well as other abnormalities in cell structure and function . In some of the experiments there were found significantly genetic effects of space flight alone, and the enhancement of various genetic effects of radiation under weightlessness was no more than 2- or 3-fold. Vet Immunol Immunopathol, 2002 May, 86(1-2), 23 - 30 Production and in vivo testing of a recombinant bovine IL-12 as an adjuvant for Salmonella typhimurium vaccination in calves; Takehara K et al.; A recombinant bovine interleukin-12 (boIL-12) that contains a histidine hexamer, rboIL-12His, was produced, purified and administered to calves . We first tried the purification of heterodimer IL-12 from a mixture of p40 homodimer, p40 monomer, and p40-p35 heterodimer with a p35 subunit tagged with a histidine hexamar at its C-terminal (p35His) . A recombinant baculovirus expressing p35His was generated and used for superinfection with a recombinant baculovirus expressing p40 subunit . The expressed subunits, p40 and p35His, were assembled into a 70kDa heterodimer in insect cells, released into culture medium, and then purified using a nickel chelate column . The purified rboIL-12His was bioactive for induction of IFN-gamma in bovine peripheral blood mononuclear cells (PBMCs) in vitro.The purified rboIL-12His was then administered to calves with inactivated Salmonella Typhimurium (ST) . When sera were assayed by ELISA, specific anti-ST IgG1 antibodies were detected in all ST immunized calves, but, specific anti-ST IgG2 antibodies were detected only in calves administered ST along with rboIL-12His, indicating a possible switch to a Th1 response . Administration of commercially available Salmonella vaccine did not elicit IgG2 antibodies in calves . These results suggest that co-administration of IL-12 with inactivated ST cells could induce a Th1-type response in calves. Oncol Res, 2000, 12(11-12), 501 - 8 Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models; Luo X et al.; VNP20009, a genetically modified strain of Salmonella typhimurium with deletions in the msbB and purI loci, exhibited antitumor activities when given systemically to tumor-bearing mice . VNP20009 inhibited the growth of subcutaneously implanted B16F10 murine melanoma, and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA-MB-231 . A single intravenous injection of VNP20009, at doses ranging from 1 x 10(4) to 3 x 10(6) cfu/mouse, produced tumor growth inhibitions of 57-95% . Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP20009 . Using mice with immune system deficiencies, we also demonstrated that the antitumor effects of VNP20009 did not depend on the presence of T and B cells . In addition, VNP20009, given intravenously, inhibited the growth of lung metastases in mice . Only live bacteria showed the antitumor effect. Mutat Res, 2002 Apr 25, 501(1-2), 79 - 98 Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB; Carroll CC et al.; We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene . The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass . Introduction of these plasmids into S . typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP) . Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds . Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98 . In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers. J Environ Pathol Toxicol Oncol, 2002, 21(1), 45 - 56 Modulatory effect of phenolic fractions of Terminalia arjuna on the mutagenicity in Ames assay; Kaur K et al.; We determined the antimutagenicity of phenolic fractions of Terminalia arjuna (soluble and insoluble in chloroform) against two direct-acting mutagens, 4-nitro-o-phenylenediamine (NPD) and sodium azide, and against the S9-dependent mutagen 2-aminofluorene (2AF), in TA98 and TA100 tester strains of Salmonella typhimurium . We found that the phenolic fractions of T . arjuna inhibited revertants induced by the S9-dependent mutagen more remarkably than the direct-acting mutagens . Furthermore, the phenolic fractions showed maximum inhibition of 98% and 101.55%, respectively, in the pre-incubation mode of treatment against the mutations induced by 2AF . Overall, the fractions inhibited the revertants induced by S9-dependent mutagens more effectively than those induced by direct-acting mutagens . The percentage of inhibition was higher in the pre-incubation than with direct acting mutagens . The fraction insoluble in chloroform showed more inhibition than the soluble one, which corresponds to a higher polyphenol content in the insoluble fraction than in the soluble extract. J Environ Pathol Toxicol Oncol, 2002, 21(1), 33 - 44 In vitro protective effects of Terminalia arjuna bark extracts against the 4-nitroquinoline-N-oxide genotoxicity; Pasquini R et al.; We determined the antimutagenic potential of chloroform, acetone, methanol, methanol+HCl, diethyl ether, and ethyl acetate extracts of Terminalia arjuna bark against the model mutagen 4-nitroquinoline-N-oxide (4-NQO) using the Salmonella/microsome, comet, and micronucleus (MN) tests . Salmonella typhimurium TA100 strain and human peripheral white blood cells were coincubated with various concentrations (from 5 to 500 microg) of the six extracts and 4-NQO (from 0.05 to 2 microg) . We found that the 4-NQO mutagenicity was inhibited by more than 70% in the Salmonella/microsome test at the highest nontoxic extract dose of ethyl acetate (50 microg/plate), chloroform (100 microg/plate), acetone, (100 microg/plate), and methanol (500 microg/plate) . A less marked antimutagenicity activity (inhibition of about 40-45%) was observed for the acidic methanol and diethyl ether extracts . The comet assay showed that acetone extract (100 microg/mL) was more effective in reducing the DNA damage caused by 4-NQO (ca . 90%), whereas the chloroform, ethyl acetate, and diethyl ether extracts were cytotoxic . In the MN test, the decrease in 4-NQO clastogenicity was observed by testing the mutagen especially with chloroform and ethyl acetate extracts (inhibition about 40-45%) . The acetone and methanol extracts showed a less marked activity (33% and 37%, respectively) . The results of the present study suggest that T . arjuna bark contains some nonpolar as well as polar compounds with antimutagenic activity against 4-NQO . Several explanations can be suggested, but further investigations are necessary to definitely identify the active compounds. Microbiology, 2002 Apr, 148(Pt 4), 1039 - 48 Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants; Campoy S et al.; It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium . In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S . typhimurium fur mutant than in the wild-type strain . According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an alpha-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S . typhimurium fur cells in comparison with wild-type cells . Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S . typhimurium fur mutant . Likewise, expression of pepE and the E . coli lac operon was the same in the S . typhimurium fur cpdA double mutant and the wild-type strain . Moreover, these results also demonstrate that the S . typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon . This positive control must be mediated by binding of the S . typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay. J Mol Microbiol Biotechnol, 2002 May, 4(3), 287 - 94 Regulation of heme biosynthesis in non-phototrophic bacteria; Schobert M et al.; The biosynthesis of tetrapyrroles like hemes and chlorophylls is essential for most living organisms . In bacteria hemes are integral parts of energy conserving electron transport chains and cofactors of various enzymes . Changes of environmental conditions usually lead to an adaption of the bacterial energy metabolism and often coincide with significant changes of cellular heme levels . This review focuses on the known regulatory mechanisms in non-phototrophic bacteria involved in the control of the formation of the heme biosynthetic apparatus . Species specific differences in the mode of energy generation result in various regulatory strategies . Focusing on the well investigated bacteria Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium the involved environmental stimuli, employed transcriptional regulators and promoter structures as well as the role of protein stability are described . Broad variations of the used regulatory principles were observed. Mol Microbiol, 2002 Feb, 43(3), 809 - 21 Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E . coli; Sperandio V et al.; Quorum sensing is a cell-to-cell signalling mechanism in which bacteria secrete hormone-like compounds called autoinducers . When these auto-inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression . Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E . coli K-12 produces the autoinducer-2 (AI-2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing . Recently, by hybridizing an E . coli K-12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86-24 and its isogenic luxS mutant, we observed that other potential virulence-associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing . The array data also indicated that several genes encoding putative E . coli regulators were controlled by quorum sensing . In this report, we describe a two-component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E . coli regulator B and C (QseBC) . The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E . coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase . We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC . This mutant expressed less flagellin and had reduced motility compared with the wild-type and complemented strains . Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes . A qseC mutant was also generated in E . coli K-12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K-12 . QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA . Motility of a luxS, but not of a qseC, mutant can be restored by providing AI-2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI-2 . However, QseC has no effect on the expression of other quorum sensing-controlled genes such as those encoding for the type III secretion system . These data indicate that QseBC is one component of the quorum-sensing regulatory cascade in both EHEC and K-12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized. Mol Microbiol, 2002 Feb, 43(3), 771 - 82 The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival; Testerman TL et al.; Bacteria must contend with conditions of nutrient limitation in all natural environments . Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation . We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation . Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase . A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress . A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation . Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts. Phytomedicine, 2002 Jan, 9(1), 26 - 32 Antimutagenic and anticarcinogenic effects of Phyllanthus amarus; Sripanidkulchai B et al.; This study aimed to examine the antimutagenic and anticarcinogenic potential of Phyllanthus amarus Schum . et Thonn . using the bacterial preincubation mutation assay and an in-vivo alkaline elution method for DNA single-strand breaks in hamster liver cells . The aqueous extract of the entire plant showed an antimutagenic effect against induction by 2-aminofluorene (AF2), 2-aminoanthracene (2AA) and 4-nitroquinolone-1-oxide (4-NQO) in Salmonella typhimurium strains TA98 and TA100, and in Escherichia coli WP2 uvrA/pKM101 . All the results were dose-dependent; however, inhibition of N-ethyl-N-nitrosoguanidine (ENNG)-induced mutagenesis was observed only with S . typhimurium TA100 . The extract also exhibited activity against 2-nitrofluorene (2NF) and sodium azide-induced mutagenesis with S . typhimurium TA98 and TA100, respectively . Based on the alkaline elution method, the plant extract prevented in vivo DNA single-strand breaks caused by dimethylnitrosamine (DMN) in hamster liver cells . When the extract was administered 30 min prior to the administration of DMN, the elution rate constant decreased more than 2.5 times, compared to that of control . These results indicate that P . amarus possesses antimutagenic and antigenotoxic properties. Avian Dis, 2002 Jan-Mar, 46(1), 137 - 42 Serologic detection of experimental Salmonella enteritidis infections in laying hens by fluorescence polarization and enzyme immunoassay; Gast RK et al.; Detection of infected poultry flocks is essential for controlling eggborne transmission of Salmonella enteritidis to humans . The present study evaluated the detection of antibodies in the sera of experimentally infected chickens by a fluorescence polarization assay with a tracer prepared from the O-polysaccharide of S . enteritidis and an enzyme-linked immunosorbent assay (ELISA) with an S . enteritidis flagellin antigen . In two trials, groups of specific-pathogen-free laying hens were infected orally with either 10(6) or 10(8) colony-forming units (CFU) of S . enteritidis (phage type 13a) or with 10(8) CFU of Salmonella typhimurium . Serum samples were collected before inoculation and at five subsequent weekly intervals . Both assays successfully detected the majority of hens infected with S . enteritidis at either dose level, but they also identified a substantial number of hens infected with S . typhimurium as seropositive . The fluorescence polarization test detected S . enteritidis infection significantly more often and cross-reacted with sera from hens infected with S . typhimurium significantly less often than the ELISA . The fluorescence polarization assay also offered advantages in terms of speed and methodologic simplicity. J Clin Pathol, 2002 Apr, 55(4), 298 - 304 Expression of antimicrobial neutrophil defensins in epithelial cells of active inflammatory bowel disease mucosa; Cunliffe RN et al.; BACKGROUND/AIMS: The normal intestinal epithelium is increasingly being recognised as an important component of the mucosal innate protection against microorganisms . Human neutrophil defensins 1-3 (HNP 1-3) and lysozyme are components of the systemic innate immunity . The aim of this study was to investigate the expression of HNP 1-3 and lysozyme in normal and active inflammatory bowel disease (IBD) mucosa . METHODS: Mucosal tissue sections were studied by immunohistochemistry using antibodies to neutrophil defensins 1-3 and lysozyme . Extracts of purified intestinal epithelial cells were used for immunoblotting studies and antimicrobial activity against the phoP negative strain of Salmonella typhimurium . RESULTS: Surface epithelial cells strongly immunoreactive for neutrophil defensins and lysozyme were seen in active ulcerative colitis and Crohn's disease (but not normal or inactive IBD) mucosal samples . Many of these cells coexpressed both of the antimicrobial proteins . Immunoblotting studies confirmed the expression of neutrophil defensins in extracts of purified ulcerative colitis epithelial cells, which also demonstrated antimicrobial activity . CONCLUSION: HNP 1-3 and lysozyme are expressed in surface enterocytes of mucosa with active IBD and they may play an important role in intestinal host defence against luminal microorganisms. Arch Toxicol, 2002 Mar, 76(2), 122 - 6 Epub 2002 Jan 30. Genotoxicity study of a new tetraalkylammonium derivative of 6-methyluracil (agent No . 547); Karamova NS et al.; Agent No . 547 (1,3-bis{omega-(diethyl-ortho-nitrobenzylammonio)-pentyl}-6-methyluracil dibromide), a newly synthesized inhibitor of mammalian-specific acetyltcholinesterase (EC 3.1.1.7) was investigated for genotoxicity using the DNA-repair test, Ames test and in vivo micronucleus test with mouse peripheral blood erythrocytes . Agent No . 547 did not cause significant changes in growth of repair-deficient Escherichia coli tester strains . The compound was non-mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without rat microsomal activation mixture . However, we observed a marked increase in number of His(+) revertants for both tester strains in preincubation assays . The results obtained in the micronucleus test indicate that agent No . 547 possesses significant clastogenic activity . At the high dose tested (0.5 mg/kg), the compound induced a seven-fold increase in the number of micronuclei over the spontaneous background 48 h after treatment . The results suggest that further work should be promoted to identify the metabolic pathways involved in genotoxicity of agent No . 547 in mammalian cells and to evaluate the real risk of its exposure. BMC Microbiol . 2002 Mar 15;2(1):5. Production of diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011; Rupesh KR et al.; BACKGROUND: Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP) . DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism . Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected . DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP . RESULTS: S . typhimurium PU011, a non-virulent bacterial strain isolated in our lab, was found to produce DAP ammonia lyase enzyme when grown in minimal medium containing DAP . There was a direct correlation between biomass yield and enzyme activity, until 16 h post inoculation in minimal medium containing DAP . Following ammonium sulphate precipitation and passing through Sephadex G100, CM-Sephadex and DEAE-Sephacel for crude enzyme extract preparation, about 68-fold enzyme purity was obtained . The purified enzyme gave maximum activity at pH 8.0 and was stable up to 45 degrees C . The Km value for the substrate was found to be 0.685 mM, calculated from a Line Weaver Burk plot . CONCLUSION: A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated. Immunity, 2002 Mar, 16(3), 325 - 8 Immune response to Salmonella: location, location, location? Hughes EA, Galan JE. Successful immunity against Salmonella infections is dependent on the generation of CD4(+) T helper cells and to a lesser extent on antibody production and CD8(+) T cells . The cells within the lymphatic tissue of the gut are likely to be central for the orchestration of a proper and rapid response . The anatomical restriction of the pathogen may also determine the distribution of effector cells . In this issue of Immunity, McSorley et al . address both of these processes using identifiable CD4 T cells that are specific for Salmonella typhimurium . Such cells localize to the Peyer's patches of the small intestine when the bacteria are delivered orally. Leuk Lymphoma, 2001 Nov-Dec, 42(6), 1367 - 77 CD40 ligand immunotherapy in cancer: an efficient approach; Kuwashima N et al.; Cancer cells do not elicit a clinically sufficient anti-tumor immune response that results in tumor rejection . Recently, many investigators have been trying to enhance anti-tumor immunity and encouraging results have been reported . This review will discuss current anti-cancer immunotherapy; interleukin-2 therapy, tumor vaccine secreting Granulocyte macrophage-colony stimulating factor, dendritic cells fused with tumor cells, and CD40 ligand immunotherapy . Moreover, we introduce our two kinds of CD40 ligand immuno-genetherapy; (1) oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium (published in BLOOD 2000), (2) cancer vaccine transfected with CD40 ligand ex vivo for neuroblastoma (unpublished) . Both approaches resulted in a high degree of protection against the tumor progression and they are simple and safe in the murine system. Microbiol Immunol, 2002, 46(1), 1 - 10 FimZ binds the Salmonella typhimurium fimA promoter region and may regulate its own expression with FimY; Yeh KS et al.; The FimZ protein, an activator of FimA production in Salmonella typhimurium, acts in conjunction with FimY to facilitate the expression of type 1 fimbriae . The predicted amino acid sequence of FimZ suggests that this protein may be a DNA-binding protein related to BvgA, a sensory regulator of virulence gene expression in Bordetella pertussis . Purification of FimZ following overexpression of the protein by a strong inducible promoter and gel mobility shift assays confirm that FimZ is a 25-kDa polypeptide that binds to the promoter region offimA . The region of DNA protected from DNase I digestion by FimZ binding is located between 47 and 98 nucleotides upstream from thefimA transcription initiation site . This region possesses a pair of 7-base pair tandem repeats, of which at least one is necessary for FimZ binding . One copy of the 7-base pair sequence is also located in thefimZ promoter region . In addition, expression from afimZ-lacZ reporter construct confirms that FimZ plays a role in its own expression . Both FimZ and FimY are required for high-level expression of FimZ, which suggests that these two fimbrial proteins are involved in regulating both FimA and FimZ. Mutat Res, 2002 Mar 25, 515(1-2), 181 - 8 Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells treated with aminophenylnorharman formed by norharman with aniline; Ohe T et al.; Aminophenylnorharman (APNH) is a newly identified mutagenic heterocyclic amine formed by coupling of norharman with aniline in the presence of S9 mix . Furthermore, mutagenic amino-3'-methylphenylnorharman (AMPNH) and aminophenylharman (APH) have been identified from a reaction mixture of norharman and o-toluidine and that of harman and aniline, respectively, with S9 mix . Among these three heterocyclic amines, APNH shows most potent mutagenic activity towards Salmonella typhimurium TA98 and YG1024 with S9 mix . In the present study, the induction of sister chromatid exchanges (SCEs) by APNH was examined in Chinese hamster lung (CHL) cells in vitro, comparing it to those of AMPNH and APH . On incubation with rat S9 for 6h, followed by a recovery culture period of 18h, a dose-dependent effect was found at concentrations between 0.00125 and 0.01 microg/ml for APNH and between 0.3125 and 5 microg/ml for AMPNH and APH . The approximate chemical concentrations leading to a three-fold of control SCE levels calculated from slopes of the linear regressions of induced SCEs were 0.005 for APNH, 0.51 for AMPNH and 1.7 microg/ml for APH . Because of the very strong SCE-causing ability of APNH, we further explored its genotoxicity by examining the induction of chromosome aberrations in CHL cells . A dose-dependent effect was found for chromosome aberrations at concentrations between 0.00125 and 0.04 microg/ml of APNH . The aberrations observed were primarily chromatid exchanges (cte) and breaks (ctb) . In conclusion, the potency of SCE induction and clastogenic activity induced by APNH is stronger than Actinomycin D, Mitomycin C (MMC) or 1,8-dinitropyrene which are considered to be the potent clastogens in the literature . Further studies are needed for elucidating mechanisms of the genotoxic actions of these compounds and for evaluating their potential hazards to human health. Mutat Res, 2002 Mar 25, 515(1-2), 125 - 34 Mutagenicity of sediments along the Po River and genotoxicity biomarkers in fish from polluted areas; Vigano L et al.; We monitored the mutagenicity of extracts of sediment fine particles collected, both in the cold season and in the hot season, from 10 reaches along the Po River, the main Italian watercourse . Each sample was representative of several kilometers of river stretch . At sub-toxic doses, the samples were not mutagenic to the Salmonella typhimurium his(-) strains TA98, TA100 and TA102, irrespective of the presence of S9 mix . However, they induced a mutagenic response in YG1024, which is typically reverted by frameshift mutagens that are metabolized in bacteria via acetyl-CoA:N-hydroxylamine O-acetyltransferase . Mutagenicity of sediments was higher during the cold season and had a spatial distribution consistent with the occurrence of pollution sources and confluence with polluted tributaries . Nevertheless, in the final stretch, near the Po delta into the Adriatic Sea, mutagenicity of sediments was low, comparable to that detected in the Po proximal reach, not far away from its springs . Genotoxicity biomarkers were evaluated in three cyprinid species, the "Italian nase" (Chondrostoma soetta), chub (Leuciscus cephalus), and barbel (Barbus plebejus), captured upstream and downstream of the confluence of a polluted tributary (Lambro River) with the Po River . There was no difference between the two areas concerning concentrations of fluorescent aromatic compounds in fish bile while, after metabolic activation, the bile of fish caught from the more polluted area became mutagenic to YG1024 . Moreover, the levels of adducts to liver DNA were significantly higher in L . cephalus caught from the more polluted area, and the increase of micronucleated erythrocyte frequency was borderline to statistical significance, but only in C . soetta . Thus, certain biomarkers of exposure and effect in fish, as assessed under field conditions, correlate with the pollution of river sediments by mutagenic compounds. Mutat Res, 2002 Mar 25, 515(1-2), 15 - 38 Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents . Part II: alkylation far away from the amino function; Glende C et al.; Alkyl and trifluoromethyl derivatives of 4-aminobiphenyl (1) (4ABP) and 2-aminofluorene (7) (2AF) were synthesised and assayed for mutagenicity using Salmonella typhimurium tester strains TA98 and TA100 with and without the addition of S9 mix . Modification of 1 was achieved by attachment of alkyl groups (methyl, ethyl, iso-propyl, n-butyl, tert-butyl) and a trifluoromethyl group (CF(3)) in the 4'-position, the 3'-position (Me, CF(3)) and the 3'-, 5'-positions (DiMe, DiCF(3)) . Compound 7 was modified by introduction of alkyl groups (methyl, tert-butyl, adamantyl) and a trifluoromethyl group (CF(3)) in the 7-position . The derivatives of 1 and 7 show for groups with growing steric demand decreased mutagenic activity . The bulkiest groups (CF