Salmonella typhimurium

Trends Cell Biol, 2005 Jan, 15(1), 2 - 5
Ubiquitination of intracellular bacteria: a new bacteria-sensing system?
Veiga E, Cossart P.
Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation . In a recent article, Perrin et al . reported that the ubiquitination system has a role in the recognition of bacterial pathogens in the cytosol of mammalian cells . They showed that polyubiquitinated proteins accumulate on the surface of cytosolic Salmonella typhimurium . In macrophages, but not epithelial cells, proteasomes become associated with the surface of cytosolic bacteria . The authors proposed that the ubiquitin-proteasome machinery might be implicated indirectly in bacterial clearance.

Int Immunopharmacol, 2005 Feb, 5(2), 359 - 68
Effects of Brazilian and Bulgarian propolis on bactericidal activity of macrophages against Salmonella Typhimurium; Orsi RO et al.; Propolis has been used in folk medicine since ancient times due to its many biological properties, such as antimicrobial, antiinflammatory, antioxidant, immunomodulatory activities, among others . Macrophages play an important role in the early phase of Salmonella infection . In this work, macrophages were prestimulated with Brazilian or Bulgarian propolis and subsequently challenged with Salmonella Typhimurium at different macrophage/bacteria ratio . After 60 min of incubation, cells were harvested with Triton-X to lyse the macrophages . To assess the bactericidal activity, the number of colony-forming units (CFU) of S . typhimurium was determined by plating 0.1 mL in Mueller Hinton agar . After 24 h, CFU were counted, and the percentage of bactericidal activity was obtained . Propolis from Brazil and Bulgaria enhanced the bactericidal activity of macrophages, depending on its concentration . Brazilian propolis seemed to be more efficient than that from Bulgaria, because of their different chemical composition . In Bulgaria, bees collect the material mainly from the bud exudate of poplar trees, while in Brazil, Baccharis dracunculifolia DC . was shown to be the main propolis source . Our data also showed that the increased bactericidal activity of macrophages involved the participation of oxygen (H(2)O(2)) and nitrogen (NO) intermediate metabolites.

Cancer Biother Radiopharm, 2004 Oct, 19(5), 649 - 57
A Combination of flk1-Based DNA Vaccine and an Immunomodulatory Gene (IL-12) in the Treatment of Murine Cancer; Keke F et al.; Aim: The aim of this study was to investigate the antivasculature effects and the antitumor effects of combining attenuated Salmonella typhimurium vaccine strain encoding murine vascular endothelial growth factor (VEGF) receptor-2 (flk1) with plasmid DNA vector encoding the murine IL-12 (mIL-12) gene . Methods: Mouse models of Gl261 glioblastoma were treated with combining orally given attenuated Salmonella typhimurium vaccine strain encoding flk1 with direct intratumoral injection of a nonviral plasmid DNA vector encoding the murine IL-12 (mIL-12) gene . The volumes of tumors were observed . Cytolytic T lymphocyte (CTL) response was measured by a 4-hour(51) Cr release assay, vessle density and tumorcell proliferation were observed by immunostaining, and tumor apoptosis was determined by TUNELstaining . Results: Compared to mice receiving single agent therapy, received either oral immunization flk1-based vaccine only or the therapeutic gene-IL-12 plasmid DNA only or those in the control group, the combination therapy groups developed a strong CTL response and showed more significantly inhibited tumor growth, apoptosis of tumor cells, and reduced neovascularization and cell proliferation in these mice . Conclusions: The therapy of attenuated Salmonella typhimurium vaccine strain encoding flk1 combined with the interleukin-12 gene has significant synergistic effect against tumors.

Ann N Y Acad Sci, 2004 Dec, 1028, 113 - 21
DNA minigene vaccination for adjuvant neuroblastoma therapy; Lode HN et al.; The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine . We demonstrate the induction of protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice following vaccination with tyrosine hydroxylase (TH)-derived antigens . Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mouse tyrosine hydroxylase (mTH) antigens . Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells . These findings were extended by comparing efficacy of mTH minigene vaccines with a minigene vaccine comprising three novel epitopes isolated fom NXS2 neuroblastoma cells . For this purpose, MHC class I was immunoprecipitated from NXS2 cell lysates, and peptides were eluted and examined in tandem-mass spectrometry analysis . This led to the identification of three novel natural MHC class I peptide ligands: TEALPVKLI, from ribonucleotide reductase M2; NEYIMSLI, from Ser/Thr protein phosphatase 2A; and FEMVSTLI, of unknown origin . Two minigenes were constructed, one encoding for the three novel epitopes and the second for three known mTH-derived epitopes with high predicted binding affinity to MHC class I, by cloning them into the mammalian expression vector pCMV-3FUB . Immunized mice showed a reduction in primary tumor growth and the absence of spontaneous liver metastasis in the majority of animals . Importantly, there was no significant difference between the two minigenes, suggesting that, compared with tumor peptide isolation, mTH epitope prediction is similarly effective for designing efficient DNA-minigene vaccines . In summary, these findings establish proof of the concept that disruption of self-tolerance against neuroblastoma-associated epitopes may be an effective adjuvant therapeutic strategy.

Proc Natl Acad Sci U S A . 2005 Jan 11; {Epub ahead of print}
Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium; Zhao M et al.; Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo . The strain grows in tumor xenografts . In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice . S . typhimurium A1 is auxotrophic (Leu/Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally . Whether additional genetic lesions are present is not known . In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction . These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm . In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging . The bacteria, introduced i.v . or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice . By day 15, S . typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing . When the bacteria were injected intratumorally, the tumor completely regressed by day 20 . There were no obvious adverse effects on the host when the bacteria were injected by either route . The S . typhimurium A1 strain grew throughout the tumor, including viable malignant tissue . This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy.

Biochemistry, 2005 Jan 18, 44(2), 675 - 83
Active Site Plasticity of Endonuclease V from Salmonella typhimurium; Feng H et al.; Base deamination is a major type of DNA damage under nitrosative stress . Endonuclease V initiates repair of deaminated base damage by making a nucleolytic incision one nucleotide away from the 3' side of the lesion . Within the endonuclease V family, the substrate specificities are different from one enzyme to another . In this study, we investigated deamination lesion cleavage activities of endonuclease V from the macrophage-residing pathogen, Salmonella typhimurium . Salmonella endonuclease V exhibits limited turnover on cleavage of deoxyinosine- and xanthosine-containing DNA . Binding analysis indicates that this single-turnover property is caused by tight binding to nicked products . The nicking activity is similar between the double-stranded deoxyinosine- and deoxyxanthosine-containing DNA . Cleavage rates are not affected by bases opposite the deoxyinosine or deoxyxanthosine lesions . The enzyme is also active on single-stranded deoxyinosine- and deoxyxanthosine-containing DNA . Unlike endonuclease V from Thermotoga maritima, Salmonella endonucleae V can only turnover deoxyuridine-containing DNA to a limited extent when substrate is in excess . Binding analysis indicates that Salmonella endonuclease V achieves tight binding to deoxyuridine-containing DNA, a property that distinguishes it from Thermotoga endonuclease V . Cleavage analysis on mismatch-containing DNA also indicates that the active site of Salmonella endonuclease V can accommodate pyrimidine-containing mismatches, resulting in more comparable cleavage of pyrimidine- and purine-containing mismatches . This comprehensive DNA cleavage and binding analysis reveals the plastic nature in the active site of Salmonella endonuclease V, which allows the enzyme to enfold both purine and pyrimidine deaminated lesions or base pair mismatches.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2004 Sep, 18(3), 238 - 42
{Salmonella typhimurium as DNA delivery vehicle for DNA-mediated immunization.}; He P et al.; BACKGROUND: To study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMV-beta as report gene . METHODS: Murine peritoneal macrophages were infected with SL7207(pCMV-beta) in vitro, then the expression of the beta-gal were detected by X-gal staining or RT-PCR . After mice were orally immunized with SL7207(pCMV-beta), the expression of beta-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test . RESULTS: The results indicated that the plasmid pCMV-beta could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated S.typhimurium SL7207 harboring plasmid pCMV-beta mice, the expression of beta-gal could be detected in the spleen, mesenteric lymph nodes and Peyers patches of the mice . Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice . Compared with the naked DNA vaccination, SL7207 (pCMV-beta) oral immunization were more efficient in inducing cellular immune responses . CONCLUSIONS: Attenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response.

Phytomedicine, 2004 Nov, 11(7-8), 666 - 72
Antimicrobial and cytotoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent, Sophora flavescens Ait and Echinosophora koreensis Nakai; Sohn HY et al.; Antimicrobial activity of the 18 prenylated flavonoids, which were purified from five different medicinal plants, was evaluated by determination of MIC using the broth microdilution methods against four bacterial and two fungal microorganisms (Candida albicans, Saccaromyces cerevisiae, Escherichia coli, Salmonella typhimurium, Staphylococcus epidermis and S . aureus) . Papyriflavonol A, kuraridin, sophoraflavanone D and sophoraisoflavanone A exhibited a good antifungal activity with strong antibacterial activity . Kuwanon C, mulberrofuran G, albanol B, kenusanone A and sophoraflavanone G showed strong antibacterial activity with 5-30 microg/ml of MICs . Morusin, sanggenon B and D, kazinol B, kurarinone, kenusanone C and isosophoranone were effective to only gram positive bacteria, and broussochalcone A was effective to C . albicans . IC50 values of papyriflavonol A, kuraridin, sophoraflavanone D, sophoraisoflavanone A and broussochalcone A in HepG2 cells were 20.9, 37.8, 39.1, 22.1, and 22.0 microg/ml, respectively . These results support the use of prenylated flavonoids in Asian traditional medicine to treat microbial infection and indicate a high potential for prenylated flavonoids as antimicrobial agents as well as anti-inflammatory agents.

J Immunol, 2005 Jan 15, 174(2), 1020 - 6
Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance; O'brien GC et al.; TLRs are highly conserved pathogen recognition receptors . As a result, TLR4-deficient C3H/HeJ mice are highly susceptible to Gram-negative sepsis . We have previously demonstrated that tolerance induced by bacterial lipoprotein (BLP) protects wild-type mice against polymicrobial sepsis-induced lethality . In this study, we assessed whether pretreatment of C3H/HeJ mice with BLP could induce resistance to a subsequent Gram-negative Salmonella typhimurium infection . Pretreatment with BLP resulted in a significant survival benefit in TLR4-deficient C3H/HeJ mice (p < 0.0002 vs control C3H/HeJ) after challenge with live S . typhimurium (0.25 x 10(6) CFU/mouse) . This survival benefit was associated with enhanced bacterial clearance from the circulation and in the visceral organs (p < 0.05 vs control C3H/HeJ) . Furthermore, pretreatment with BLP resulted in significant increases in complement receptor type 3 (CR3) and FcgammaIII/IIR expression on polymorphonuclear neutrophils (PMNs) and macrophages (p < 0.05 vs control C3H/HeJ) . There was impaired bacterial recognition and phagocytosis in TLR4-deficient mice compared with wild-type mice . However, a significant augmented uptake, ingestion, and intracellular killing of S . typhimurium by PMNs and peritoneal macrophages was evident in BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ) . An up-regulation of inducible NO synthase and increased production of intracellular NO were observed in peritoneal macrophages from BLP-pretreated C3H/HeJ mice (p < 0.05 vs control C3H/HeJ) . Depletion of PMNs did not diminish the beneficial effects of BLP with regard to both animal survival and bacterial clearance . These results indicate that BLP, a TLR2 ligand, protects highly susceptible TLR4-deficient mice from Gram-negative sepsis via enhanced bacterial clearance.

J Rheumatol, 2005 Jan, 32(1), 86 - 92
Outer membrane protein of salmonella is the major antigenic target in patients with salmonella induced reactive arthritis; Saxena S et al.; OBJECTIVE: We previously reported that Salmonella typhimurium was the triggering agent in one-third of our patients with sporadic enteric reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) . The antigens recognized by the synovial T cells in Salmonella induced ReA are not known . We investigated the immunodominant antigens in Salmonella ReA . METHODS: Synovial fluid mononuclear cells (SFMC) from 53 patients with ReA/uSpA were cultured with crude lysate of S . typhimurium . In 20 patients, the triggering agent was found to be Salmonella (stimulation index, SI, > 2.5) . For cell fractionation of S . typhimurium, the sonicated crude lysate was separated by ultracentrifugation into a cytoplasmic supernatant (CYT) and membrane pellet (OMP) . The CYT was further separated on SDS-PAGE and blotted onto nitrocellulose membrane for proliferation assays . SFMC from 20 patients with Salmonella ReA/uSpA were stimulated with OMP, CYT, and cytosolic fractions of S . typhimurium, and proliferation was measured by thymidine incorporation . Quantitation of antigen-specific cells in SF was by intracellular interferon-g staining in 7 patients and paired peripheral blood (PB) in 5 patients after stimulation with crude Salmonella lysate, CYT, and OMP . RESULTS: Out of 20 patients with Salmonella ReA/uSpA, the SFMC showed a significant proliferation to OMP in 19 patients and CYT in 17 patients . The median SI of OMP (8.2, range 2.8-52.5) was significantly higher (p < 0.0005) than for the CYT (4.9, 2.7-18.8) . Fifty percent of the patients showed proliferative response to cytosolic fractions of < 60 kDa . The mean antigen-specific T cell frequency was also higher with OMP (0.68% +/- 0.59%) than CYT (0.53% +/- 0.62%) but this was not statistically significant . Compared to PB, the OMP-specific cells were 7.5 times more numerous in the SF (p < 0.05) . CONCLUSION: The cellular immune response in Salmonella ReA/uSpA is directed predominantly against the OMP and low molecular weight proteins in the cytosolic fraction.

Biofactors, 2004, 22(1-4), 123 - 5
Antimutagenicity of Japanese traditional herbs, gennoshoko, yomogi, senburi and iwa-tobacco; Hiramatsu N et al.; The multistage induction theory is generally regarded as the mechanism of carcinogenesis . In order to prevent the initiation stage of carcinogenesis, it is meaningful to discover the functional components of edible plants . The objective of this research was to test the antimutagenicity of the functional components of several typical traditional herbs used in Japan . The traditional herbs, gennoshoko (Geranium nepalense var . thunbergii), yomogi (Artemisia vulgaris var . indica), senburi (Swertia japonica), iwa-tobacco (Conandron ramondioides), sarunokoshikake (Elfvingia applanata), kanzo (Glycyeehiza uralensis Fisch) and matatabi (Actinidia polygama) were examined by Ames mutagenesis assay test with Salmonella typhimurium TA98 and TA100 against mutagens, Trp-P-1, Trp-P-2 and B(a)P . The water-soluble components or volatile oil of the herbs were extracted in boiling water . The extracts of gennoshoko showed strong antimutagenicity against B(a)P with S . typhimurium TA98 and TA100, as well as Trp-P-1 and Trp-P-2 with S . typhimurium TA98 . Yomogi, senburi and iwa-tobacco were also proved to have good antimutagenicity against Trp-P-1 and Trp-P-2 with S . typhimurium TA98, but weaker antimutagenicity against B(a)P . Other herbs did not show any obvious antimutagenicity against these mutagens . In addition, the volatile oil of yomogi also had remarkable antimutagenic effect against the mutagens we used with S . typhimurium TA98.

Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 1025 - 9
Metabolic epoxidation of trans-4-acetylaminostilbene: a protective mechanism against its activation to a mutagen; Glatt HR et al.; Trans-4-acetylaminostilbene is activated by liver preparations to mutagens for Salmonella typhimurium . Since this compound is metabolized to the trans-alpha,beta-epoxide and since many epoxides are ultimate mutagens, this epoxide was tested for direct mutagenicity . It was, however, found to be non-mutagenic, and, in contrast to the parent compound, the epoxide was no longer activated by liver preparations to mutagens . The same was found for the beta-ketone and for the threo-alpha,beta-dihydrodiol, which are formed metabolically from trans-4-acetylaminostilbene and from its alpha,beta-epoxide . 4-Acetylaminobibenzyl showed a very weak mutagenic activity in the presence of the liver preparation . Thus, it is important to realize that where epoxides are formed from compounds which are known to be metabolized to mutagens, they are not necessarily responsible for the mutagenicity . Epoxidation may even prevent the possibility of bioactivation to mutagens.

Natl Toxicol Program Tech Rep Ser, 2004 Sep, (519), 1 - 274
NTP Toxicology and Carcinogensis Studies of STODDARD SOLVENT IIC (CAS NO . 64742-88-7) in B6C3F(1) Mice (Inhalation Studies); National Toxicology Program; Stoddard solvent (white spirit/mineral spirit) is the most widely used solvent in the paint industry . It is used as a dry cleaning agent; as an extraction, cleaning, and degreasing solvent; and as a solvent in aerosols, paints, wood preservatives, asphalt products, lacquers, and varnishes . Stoddard solvent IIC was nominated by the International Union, United Auto Workers, for carcinogenicity testing because of the large volume used in industrial and other settings . Male and female F344/N rats and B6C3F(1) mice were exposed to Stoddard solvent IIC (greater than 99% pure) by inhalation for 2 weeks, 3 months, or 2 years . Genetic toxicology studies were conducted in Salmonella typhimurium and mouse peripheral blood erythrocytes . 2-Week Study in Rats Groups of five male and five female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m(3), 6 hours per day, 5 days per week for 16 days . All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls . Liver weights of males exposed to 550 mg/m(3) or greater and of females exposed to 275 mg/m(3) or greater were increased . Minimal diffuse cytoplasmic vacuolization of hepatocytes of the liver occurred in all females exposed to 2,200 mg/m(3) . 2-Week Study in Mice Groups of five male and five female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m(3), 6 hours per day, 5 days per week for 17 days . All mice survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls . Liver weights of males and females exposed to 275 mg/m(3) or greater were significantly increased . Cytomegaly of the liver occurred in all males and females exposed to 2,200 mg/m(3) . 3-Month Study in Rats Groups of 10 male and 10 female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m(3), 6 hours per day, 5 days per week for 14 weeks . All rats survived to the end of the study, and the final mean body weight of females exposed to 275 mg/m(3) was greater than that of the chamber controls . The relative kidney, liver, and testis weights of all exposed groups of males and the absolute kidney weights of males exposed to 550 mg/m(3) or greater were increased . The sperm motility of 550 mg/m(3) or greater males was significantly decreased . The incidences of renal tubule granular casts were significantly increased in males exposed to 550 mg/m(3) or greater, and the severities of renal tubule hyaline droplet accumulation, granular casts, and regeneration increased with increasing exposure concentration in males . The incidences of goblet cell hypertrophy of the nasal respiratory epithelium in males and females exposed to 2,200 mg/m(3) were significantly increased . Sperm motility was decreased in males exposed to 550 mg/m(3) or greater . 3-Month Study in Mice Groups of 10 male and 10 female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138, 275, 550, 1,100, or 2,200 mg/m(3), 6 hours per day, 5 days per week for 14 weeks . Mean body weights of exposed groups were similar to those of the chamber controls, but liver weights of males exposed to 2,200 mg/m(3) were significantly increased . The sperm motility of 2,200 mg/m(3) males was significantly decreased . This reduction in sperm motility, while statistically significant, is probably of modest importance as studies in mice have found that fertility is unaffected by motility decreases of less than 40% . The incidences of hematopoietic cell proliferation of the spleen in all exposed groups of females were greater than that in the chamber controls . 2-Year Study in Rats Groups of 50 male and 50 female rats were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 138 (males), 550, 1,100, or 2,200 (females) mg/m(3), 6 hours per day, 5 days per week for 104 to 105 weeks . Additional groups of 10 males and 10 females were exposed to the same concentrations for 3 months for renal toxicity analyses . Survival in the top exposure concentration groups of males and females was significantly less than that of the chamber controls . Mean body weights of exposed males and females were similar to those of the chamber controls . Cell proliferation analyses were performed in the left kidney of males and females after 3 months of exposure . The mean numbers of labeled cells and the labeling indices in males exposed to 550 and 1,100 mg/m(3) were significantly increased . The amount of alpha2u-globulin in the right kidney of males increased with increasing exposure concentration . Also, the incidences of granular casts and cortical tubule degeneration and regeneration were generally increased in exposed males, as was the severity of hyaline droplets . These effects did not occur in females . At 2 years, the incidences of benign and benign or malignant pheochromocytoma (combined) of the adrenal medulla occurred with positive trends in males, and the incidences in the 550 and 1,100 mg/m(3) groups were significantly increased . Due to increased incidences of renal tubule hyperplasia in males at 2 years, extended kidney evaluations were conducted; a slightly increased incidence of renal tubule adenoma occurred in the 1,100 mg/m(3) group . Nonneoplastic lesions related to Stoddard solvent IIC exposure occurred in the kidney of males . 2-Year Study in Mice Groups of 50 male and 50 female mice were exposed to Stoddard solvent IIC by inhalation at concentrations of 0, 550, 1,100, or 2,200 mg/m(3), 6 hours per day, 5 days per week for 105 weeks . Survival of exposed mice was similar to that of the chamber controls . Mean body weights of exposed females were greater than those of the chamber controls . The incidences of hepatocellular adenoma occurred with a positive trend in females, and the incidence of multiple hepatocellular adenoma in females exposed to 2,200 mg/m(3) was significantly increased . However, the incidences of hepatocellular adenoma or carcinoma (combined) and hepatocellular carcinoma alone in exposed males and females were not significantly increased . Genetic Toxicology Stoddard solvent IIC was tested for mutagenicity in Salmonella typhimurium strains TA97, TA98, TA100, and TA1535, with and without S9 metabolic activation enzymes; all results were negative . In vivo, the frequency of micronucleated erythrocytes was assessed in peripheral blood samples from male and female B6C3F(1) mice after 3 months of inhalation exposure to Stoddard solvent IIC, and results were negative . Conclusions Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of Stoddard solvent IIC in male F344/N rats based on increased incidences of adrenal medulla neoplasms; the slightly increased incidences of renal tubule adenoma may have been related to Stoddard solvent IIC exposure . There was no evidence of carcinogenic activity of Stoddard solvent IIC in female F344/N rats exposed to 550, 1,100, or 2,200 mg/m(3) . There was no evidence of carcinogenic activity of Stoddard solvent IIC in male B6C3F(1) mice exposed to 550, 1,100, or 2,200 mg/m(3) . There was equivocal evidence of carcinogenic activity of Stoddard solvent IIC in female B6C3F(1) mice based on increased incidences of hepatocellular adenoma; this slight increase was associated with increased body weight in exposed females . Exposure of male rats to Stoddard solvent IIC resulted in nonneoplastic lesions of the kidney characteristic of alpha2u-globulin accumulation . Synonyms: Medium aliphatic solvent naphtha (petroleum); white spirit.

Natl Toxicol Program Tech Rep Ser, 2004 Dec, (516), 1 - 292
NTP Toxicology and Carcinogensis Studies of 2-METHYLIMIDAZOLE (CAS NO . 693-98-1) in B6C3F(1) Mice (Feed Studies); National Toxicology Program; 2-Methylimidazole is used in the manufacture of pharmaceuticals, photographic chemicals, dyes and pigments, agricultural chemicals, and rubber . It has been identified as a by-product in foods and has been detected in mainstream and sidestream tobacco smoke . 2-Methylimidazole was nominated by the National Cancer Institute for a long-term study because of the high potential for human exposure and a lack of carcinogenicity studies in rodents . Male and female F344/N rats and B6C3F(1) mice were exposed to 2-methylimidazole (99.5% pure) in feed for 2 years . Fifteen-day and 14-week toxicity studies of 2-methylimidazole in F344/N rats and B6C3F(1) mice are reported in NTP Toxicity Report No . 67 (NTP, 2004) . Genetic toxicity studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood . 2-YEAR STUDY IN RATS Groups of 60 male and 60 female rats were fed diets containing 0, 300, 1,000, or 3,000 ppm 2-methylimidazole (males) or 0, 1,000, 2,500, or 5,000 ppm 2-methylimidazole (females) (equivalent to average daily doses of approximately 13, 40, or 130 mg 2-methylimidazole/kg body weight to males and 50, 120, or 230 mg/kg to females) for 106 weeks . Ten male and 10 female rats were necropsied at 6 months . Additional groups of 20 male and 20 female special study rats were exposed to the same concentrations for 8 days or 14 weeks and were evaluated for clinical chemistry, liver enzyme activity, and organ weights . Survival of 2,500 ppm females was significantly less than that of the controls . The mean body weights of 3,000 ppm males and 2,500 and 5,000 ppm females were generally less than those of the controls during most of the study . Feed consumption by 5,000 ppm females was less than that by the control group . The hematology results at 6 months indicated that exposure of rats to 2-methylimidazole induced a decreased erythron that was characterized as microcytic, normochromic, and nonresponsive . The thyroid hormone data indicated that rats administered 2-methylimidazole developed alterations in thyroid hormone concentrations; serum thyroxine and triiodothyronine concentrations were decreased, and thyroid stimulating hormone levels were increased . In general, the thyroid hormone effects were most pronounced early in the study and ameliorated with time . The results for the tissue enzyme content analyses of these 2-year feed studies indicated that exposure of rats to 2-methylimidazole induced an increase in total hepatic UDP-glucuronosyltransferase at all time points evaluated through 6 months . The thyroid gland weights of 3,000 ppm males and 2,500 and 5,000 ppm females were significantly increased at 6 months . At 6 months, two 5,000 ppm female rats had a thyroid gland follicular cell adenoma . The incidences of follicular cell adenoma, follicular cell carcinoma, and adenoma or carcinoma (combined) in the thyroid gland of 5,000 ppm females were significantly greater that those in the controls at 2 years . The incidence of follicular cell adenoma or carcinoma (combined) in the thyroid gland occurred with a positive trend in males . The incidences of follicular cell hyperplasia in all exposed groups of rats were significantly increased at 6 months and 2 years . The incidences of follicle mineralization of the thyroid gland in all exposed groups, except 300 ppm males at 6 months and in 1,000 and 5,000 ppm females at 2 years, were significantly greater than those of the controls . In the liver, the incidences of hepatocellular adenoma or carcinoma (combined) in the two highest exposure groups of males and females exceeded the historical ranges for controls, and the incidences of hepatocellular adenoma in females occurred with a positive trend . The incidences of bile duct hyperplasia and granulomatous inflammation were increased in females, as were those of mixed cell focus in males and females . The incidence of granulomatous inflammation of the spleen in 5,000 ppm females was significantly increased . Lower body weights of female rats exposed to 5,000 ppm likely contributed to the decreased incidences of mammary gland fibroadenoma, pituitary gland adenoma, and clitoral gland adenoma in this group . 2-YEAR STUDY IN MICE Groups of 60 male and 60 female mice were fed diets containing 0, 625, 1,250, or 2,500 ppm 2-methylimidazole (equivalent to average daily doses of approximately 75, 150, or 315 mg/kg to males and 80, 150, or 325 mg/kg to females) for 105 weeks . Ten male and 10 female mice were necropsied at 6 months . Additional groups of 20 male and 20 female special study mice were exposed to the same concentrations for 8 days or 14 weeks and were evaluated for clinical chemistry, liver enzyme activity, and organ weights . Survival of all exposed groups of mice was similar to that of the control groups . The mean body weights of 1,250 and 2,500 ppm males and 2,500 ppm females were less than those of the controls during most of the study . Feed consumption by all exposed groups of mice was similar to that by the control groups . The hematology results at 6 months indicated that exposure of mice to 2-methylimidazole induced a decreased erythron that was characterized as macrocytic, normochromic to hypochromic, and responsive . The thyroid gland weights of 2,500 ppm male and female mice and 1,250 ppm females were increased at 6 months . The incidence of follicular cell adenoma in the thyroid gland of 2,500 ppm males was significantly greater than that in the control group at 2 years . Follicular cell hypertrophy of the thyroid gland occurred in most exposed mice at 6 months, and the incidences of this lesion were significantly increased in the 1,250 and 2,500 ppm groups at 2 years; the incidences of follicular cell hyperplasia were significantly increased in 2,500 ppm males and females at 2 years . The liver weights of 2,500 ppm female mice were significantly increased at 6 months . The incidences of hepatocellular adenoma occurred with positive trends in males and females and the incidences were significantly increased in the 2,500 ppm groups . The incidence of hepatocellular carcinoma was significantly increased in 1,250 ppm males and exceeded the historical control range in 2,500 ppm males . The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in all exposed groups of males . The incidences of hepatocellular karyomegaly in 2,500 ppm males at 6 months and in 1,250 and 2,500 ppm males at 2 years, of hepatocellular cytoplasmic alteration in 1,250 and 2,500 ppm males at 2 years, and Kupffer cell pigmentation in 2,500 ppm males at 2 years were significantly increased . In the spleen, the incidences of hematopoietic cell proliferation in all exposed groups of males and in 2,500 ppm females were significantly increased at 6 months and 2 years . Pigmentation was present in most 1,250 and 2,500 ppm mice at 6 months, and the incidences of this lesion were significantly increased in all exposed groups of males and in 1,250 and 2,500 ppm females at 2 years . The incidences of bone marrow hyperplasia were significantly increased in 1,250 and 2,500 ppm male mice at 2 years . Renal proximal tubule pigmentation was present in most 2,500 ppm male mice at 6 months and 2 years . The responses in the spleen, bone marrow, and kidney were considered to be related to the responsive anemia . In males, the incidences of chronic active inflammation of the epididymis at 1,250 and 2,500 ppm, sperm granuloma at 2,500 ppm, and of germinal epithelial atrophy of the testis at 1,250 and 2,500 ppm were significantly increased at 2 years . GENETIC TOXICOLOGY 2-Methylimidazole was negative in the S . typhimurium mutation assay when tested in strains TA97, TA98, TA100, and TA1535, with and without S9 activation enzymes . Testing of 2-methylimidazole in vivo for induction of chromosomal damage, as measured by micronucleated erythrocyte frequency, produced mixed results . When administered by intraperitoneal injection three times at 24-hour intervals, 2-methylimidazole produced negative results in bone marrow micronucleus tests in rats and mice . However, in the 14-week study of 2-methylimidazole, a significant exposure-related increase in the frequency of micronucleated normochromatic erythrocytes was noted in peripheral blood of male and female mice . Exposure concentration-related increases in the percentage of micronucleated polychromatic erythrocytes in peripheral blood was also seen in male and female mice in the 14-week study . CONCLUSIONS Under the conditions of this 2-year feed study, there was some evidence of carcinogenic activity of 2-methylimidazole in male F344/N rats based on increased incidences of thyroid gland follicular cell neoplasms . The increased incidences of hepatocellular neoplasms in males may have been related to exposure . There was clear evidence of carcinogenic activity of 2-methylimidazole in female F344/N rats based on increased incidences of thyroid gland follicular cell neoplasms . The increased incidences of hepatocellular adenoma in females may have been related to exposure . There was some evidence of carcinogenic activity in male B6C3F(1) mice based on increased incidences of thyroid gland follicular cell adenoma and hepatocellular neoplasms . There was some evidence of carcinogenic activity in female B6C3F(1) mice based on increased incidences of hepatocellular adenoma . Exposure to 2-methylimidazole resulted in nonneoplastic lesions in the thyroid gland and liver of male rats; the thyroid gland, liver, and spleen of female rats; the thyroid gland, liver, spleen, bone marrow, kidney, epididymis and testes of male mice; and the thyroid gland and spleen of female mice . Synonyms: Imidazole,2-methyl; 2-MeI; 2-methylglyoxaline; 2-MI; 2-MZ.

Mutagenesis . 2004 Dec 29; {Epub ahead of print}
Evaluation of the potential genotoxicity of the phosphate binder lanthanum carbonate; Damment SJ et al.; Lanthanum was evaluated for potential genotoxicity using a range of in vitro assays (as the carbonate) in the presence and absence of post-mitochondrial fraction (S9) and in vivo in three independent tests for mutagenicity and clastogenicity (as the carbonate and chloride) . The drug was devoid of mutagenic activity in bacterial assays (maximum concentration 5000 microg/plate) using a range of test strains (Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA and WP2 uvrA pkm101) . No effects were seen in the hgprt gene mutation assay in Chinese hamster ovary cells in the presence of S9 . In the absence of S9, sporadic increases in revertant numbers were not dose-related or reproducible in subsequent experiments and hence were concluded to be chance events . In an in vitro chromosome aberration assay using Chinese hamster ovary cells, chromosome damage in the presence and absence of S9 (concentration 200-5000 microg/ml) was attributed to overt cell toxicity . To confirm this, a comprehensive in vivo evaluation of the drug was performed . Negative results were obtained in two independent rodent micronucleus tests . In the first mice were given oral doses (of carbonate) up to 2000 mg/kg, in the second rats were given a single i.v . bolus injection (of chloride) up to 0.1 mg/kg . Negative results were also obtained in a rat liver unscheduled DNA synthesis assay after treatment for 28 days with i.v . bolus injections (of chloride) up to 0.1 mg/kg/day . In these in vivo studies lanthanum plasma concentrations were >3000 times higher than the steady-state peak plasma concentration observed in dialysis patients given therapeutic doses of lanthanum carbonate . It can be concluded that lanthanum is not genotoxic and that lanthanum carbonate is unlikely to present a latent hazard in therapeutic use.

J Environ Sci Health B, 2004, 39(5-6), 861 - 70
Salmonella enterica serovar Typhimurium hilA-lacZY fusion gene response to iron chelation or supplementation in rich and minimal media; Rishi P et al.; Virulence expression of Salmonella enterica serovar Typhimurium under iron limited condition was measured by beta-galactosidase (beta-gal) assay using a hilA-lacZY fusion strain and calculated as Miller units . hilA-lacZY beta-galactosidase assays were performed in brain heart infusion (BHI) and minimal media (M9), after iron chelation with 2, 2-dipridyl and iron-supplementation respectively . Before performing virulence assays, concentrations of iron in the media were estimated using ferrozine . Iron content was found to be more in BHI (42.6 microg dL(-1)) as compared to M9 (10.03 microg dL(-1)) . beta-gal activity of Salmonella Typhimurium in BHI was generally less than that observed in M9 . After exposure to various combinations of iron chelator in BHI, hilA-lacZY activity only increased at the highest concentration of chelator (2001 microM) but decreased in M9 media for all iron concentrations when compared to controls with no iron amendment . These results indicate that iron availability may influence S . Typhimurium hilA expression.

Drug Metab Pharmacokinet, 2002, 17(1), 1 - 22
Genetically Engineered Bacterial Cells Co-expressing Human Cytochrome P450 with NADPH-cytochrome P450 Reductase: Prediction of Metabolism and Toxicity of Drugs in Humans; Fujita K et al.; Genetically engineered bacterial cells expressing human cytochrome P450 (CYP) have been developed as new tools to predict the metabolism and toxicity of drugs in humans . There are various host cells for the heterologous expression of a form of CYP . Among them, bacterial cells such as Escherichia coli (E . coli) have advantages with regard to ease of use and high yield of protein . CYP protein could be first expressed by the modification of the N-terminal amino acid sequence in E . coli cells in 1991 . Since then, many forms of human CYP have been successfully expressed in E . coli cells . Since the E . coli cells do not possess endogeneous electron transport systems to support the full catalytic activity of CYP, E . coli strains co-expressing both human CYP and NADPH-cytochrome P450 reductase (OR) have been established . Each form of CYP expressed in the E . coli cells efficiently catalyzed the oxidation of a representative substrate at an efficient rate, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP . According to the studies performed so far, the modification of the N-terminal amino acid sequence of CYP did not seem to affect the catalytic properties of CYP . The human CYP expressed in the E . coli cells were applicable for studies to determine a metabolic pathway(s) of drugs and to estimate kinetic parameters of drug metabolism by human CYP . Drug-drug interactions caused by inhibition of the metabolism of drugs by human CYP could also be examined by in vitro inhibition studies with CYP expressed in the E . coli cells . Recently, human CYP was co-expressed with the OR in Salmonella typhimurium (S . typhimurium) cells used for mutation assay (Ames test) by applying the technology for the expression of human CYP and the OR in E . coli cells, to evaluate whether chemicals including drugs are metabolically activated by human CYP and show mutagenicity . These strains of bacteria are considered as useful tools to study the metabolism and the toxicity of drugs in humans.

Vet Immunol Immunopathol, 1980 Aug, 1(3), 277 - 86
Intestinal immunity following a single intraperitoneal immunisation in lambs; Husband AJ; A single intraperitoneal injection of ovalbumin in oil adjuvant in young lambs has been shown to result in the appearance in the intestinal lamina propria of antibody-containing cells, most of which contained antibody of IgA specificity . Intraperitoneal immunisation of lambs with a Salmonella typhimurium vaccine during the suckling period provided protection against postweaning challenge with live organisms . This response was shown to be associated with specific IgA antibody in intestinal secretion.

J Environ Monit, 2005 Jan, 7(1), 60 - 6 Epub 2004 Dec 08.
In vitro genotoxicity of exhaust emissions of diesel and gasoline engine vehicles operated on a unified driving cycle; Liu YQ et al.; Acetone extracts of engine exhaust particulate matter (PM) and of vapor-phase semi-volatile organic compounds (SVOCs) collected from a set of 1998-2000 model year normal emitter diesel engine automobile or light trucks and from a set of 1982-1996 normal emitter gasoline engine automobiles or light trucks operated on the California Unified Driving Cycle at 22 {degree}C were assayed for in vitro genotoxic activities . Gasoline and diesel PM were comparably positive mutagens for Salmonella typhimurium strains YG1024 and YG1029 on a mass of PM extract basis with diesel higher on a mileage basis; gasoline SVOC was more active than diesel on an extracted-mass basis, with diesel SVOC more active on a mileage basis . For chromosomal damage indicated by micronucleus induction in Chinese hamster lung fibroblasts (V79 cells), diesel PM expressed about one-tenth that of gasoline PM on a mass of extract basis, but was comparably active on a mileage basis; diesel SVOC was inactive . For DNA damage in V79 cells indicated by the single cell gel electrophoresis (SCGE) assay, gasoline PM was positive while diesel PM was active at the higher doses; gasoline SVOC was active with toxicity preventing measurement at high doses, while diesel SVOC was inactive at all but the highest dose.

J Agric Food Chem, 2004 Dec 29, 52(26), 8255 - 60
Chemical compositions and antibacterial effects of essential oils of Turkish oregano (Origanum minutiflorum), bay laurel (Laurus nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum vulgare) on common foodborne pathogens; Dadalioglu I et al.; Chemical compositions and inhibitory effects of essential oils of Turkish oregano (Origanum minutiflorum O . Schwarz & P . H . Davis), bay laurel (Laurus nobilis L.), Spanish lavender (Lavandula stoechas subsp . stoechas L.), and fennel (Foeniculum vulgare Mill.) on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus were determined . After the essential oils were applied on the foodborne pathogens at doses of 0 (control), 5, 10, 20, 30, 40, 50, and 80 microL/mL, the resultant numbers of cells surviving were counted . Results revealed that all essential oils exhibited a very strong antibacterial activity against the tested bacteria (P < 0.05) . Gas chromatography-mass spectrophotometry analyses revealed that carvacrol (68.23%), 1,8-cineole (60.72%), fenchone (55.79%), and trans-anethole (85.63%) were the predominant constituents in Turkish oregano, bay laurel, Spanish lavender, and fennel essential oils, respectively.

Chin J Dig Dis, 2004, 5(2), 72 - 5
Therapeutic vaccination against Helicobacter pylori infection with attenuated recombinant Salmonella typhimurium urease B subunit and catalase in mice; Li GQ et al.; OBJECTIVE: To investigate the effects of oral immunization with attenuated recombinant Salmonella typhimurium urease B subunit and catalase vaccines in the treatment of Helicobacter pylori infection in a H . pylori infected mouse model . METHODS: Thirty C57BL/6 mice were randomized into three groups and challenged twice with oral administration of H . pylori within 3 days . Four weeks after the second challenge, the mice were immunized by oral administration of attenuated recombinant S . typhimurium urease B subunit (group A), attenuated recombinant S . typhimurium catalase (group B) or saline (group C), and all mice were killed 4 weeks later . The stomachs were collected for a rapid urease test, modified Giemsa staining and quantitative culture to observe the density of H . pylori, hematoxylin-eosin staining was performed to assess the presence of inflammation and lymphocytes from the spleen were used for the lymphoproliferation assay . RESULTS: The gastric H . pylori density of groups A, B and C was 1.58 x 10(5) c.f.u./g, 4.88 x 10(5) c.f.u./g and 1.92 x 10(6) c.f.u./g, respectively . The H . pylori density was significantly decreased in the therapeutic groups (P < 0.05) . No significant inflammation was found in any group of mice . The lymphoproliferation assays of groups A and B were positive . CONCLUSION: Immunization with oral attenuated recombinant S . typhimurium urease B subunit and catalase vaccines is effective in reducing the density of H . pylori colonization.

Wei Sheng Yan Jiu, 2004 Sep, 33(5), 591 - 4
{Fluoroquinolone resistance mutations in topisomerase genes of Salmonella typhimurium isolates}; Guo Y et al.; OBJECTIVE: Mutations in topisomerase genes were main cause of the resistence of Salmonella typhimurium to fluoroquinolone . METHODS: The MICs of three Salmonella typhimurium isolates X2, X7, X11 to ciprofloxacin were above 32 microg/ml, 0.38 microg/ml and 0.023 microg/ml, respectively . The genetic alterations in four topisomerase genes, gyrA, gyrB, parC, and parE were detected by multiplex PCR amplimer conformation analysis in these three strains . RESULTS: X2 isolate showed both gyrA mutations (Ser83-->Phe, Asp87-->Asn) and parC mutation (Ser80-->Arg) . X7 isolate showed a single gyrA mutation (Ser83-->Phe) and X11 isolate had no changes in all of the four quinolone resistance genes, gyrA, gyrB, parC, and parE . X7 isolate with a single gyrA mutation was less resistant to ciprofloxacin than X2 with double gyrA mutations and an additional parC mutation . CONCLUSION: GyrA and parC genes play important role of the resistence of Salmonella typhimurium to ciprofloxacin.

World J Gastroenterol, 2005 Jan 7, 11(1), 114 - 7
Construction of a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA; Xu C et al.; AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity . METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector . DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions . The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion . Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207 . After that, the recombinant strain was grown in vitro repeatedly . In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot . RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis . It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot . CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Histochem Cell Biol . 2004 Dec 18; {Epub ahead of print}
Salmonella Typhimurium infection in the porcine intestine: evidence for caspase-3-dependent and -independent programmed cell death; Schauser K et al.; The normal intestinal epithelium is renewed with a turnover rate of 3-5 days . During Salmonella infection increased cell loss is observed, possibly as a result of programmed cell death (PCD) . We have, therefore, studied the effects of Salmonella Typhimurium infection on three elements involved in PCD: caspase-3 activation, c-Jun phosphorylation on serine 63 (both detected by immunocytochemistry), and DNA fragmentation (detected by TUNEL reaction), using a pig jejunal loop model . Additionally, we used nuclear staining for detecting signs of classical apoptosis . Activated caspase-3 was detected in scattered epithelial cells and the number of positive cells increased with increasing times of exposure to Salmonella (P<0.0001) . An increase in phospho-c-Jun in epithelial cells was already detectable 5 min after infection and often occurred in cells that appeared not to be invaded by the organism . Changes in caspase-3 activation and c-Jun phosphorylation were most marked in the proximal region of the jejunum . Although rarely observed in the epithelium, proper TUNEL-positive cells were frequently found in the intestinal lumen . Some, but not all, TUNEL-positive cells were also positive for caspase-3, indicating that both caspase-3-dependent and -independent pathways of PCD increased upon infection.

J Mol Biol, 2005 Jan 28, 345(4), 879 - 92
The structure of the oligopeptide-binding protein, AppA, from Bacillus subtilis in complex with a nonapeptide; Levdikov VM et al.; Besides their role as a source of amino acids for Bacillus subtilis, exogenous peptides play important roles in the signalling pathways leading to the development of competence and sporulation . B.subtilis has three peptide transport systems all belonging to the ATP-binding cassette family, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App) with overlapping specificity . These comprise a membrane-spanning channel through which the peptide passes, a pair of ATPases which couple ATP hydrolysis to peptide translocation and a lipid-modified, membrane-anchored extracellular "binding-protein" that serves as the receptor for the system . Here, we present the crystal structure of a soluble form of the peptide-binding protein AppA, which has been solved to 1.6 A spacing by anomalous scattering and molecular replacement methods . The structure reveals a protein made of two distinct lobes with a topology similar to those of DppA from Escherichia coli and OppA from Salmonella typhimurium . Examination of the interlobe region reveals an enlarged pocket, containing electron density defining a nonapeptide ligand . The main-chain of the peptide is well defined and makes a series of polar contacts with the protein including salt-bridges at both its termini . The side-chain density is ambiguous in places, consistent with the interpretation that a population of peptides is bound, whose average electron density resembles the amino acid sequence N-VDSKNTSSW-C.

J Endotoxin Res, 2004, 10(6), 439 - 44
Deacylation and palmitoylation of lipid A by Salmonellae outer membrane enzymes modulate host signaling through Toll-like receptor 4; Kawasaki K et al.; The Salmonella typhimurium virulence gene products, PhoP/PhoQ sense host micro-environments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP . Therefore, deacylation and/or palmitoylation of lipid A could occur in Salmonellae adapted to host environments . The acylation state of lipid A can alter host recognition and signaling by Toll-like receptor (TLR) 4, and may play an important role in host defenses against Salmonellae infection . Deacylated lipid A, deacylated and palmitoylated lipid A, palmitoylated lipid A, and unmodified lipid A species were purified, and the activity was examined using cell lines expressing recombinant human or mouse TLR4 . Compared with unmodified lipid A, the modified lipid A species are 10-100-fold less active . These results suggest that PagL and PagP modify lipid A to reduce TLR4-signaling as part of Salmonellae adaptation to the host environment.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(4), 389 - 91
Anti-angiogenesis effect on glioma of attenuated Salmonella typhimurium vaccine strain with flk-1 gene; Feng K et al.; To investigate the anti-vasculature effects and the anti-glioma effects of attenuated Salmonella typhimurium vaccine strain expressing VEGFR2 (flk-1) gene, plasmid pcDNA3.1-flk1 was constructed and electro-transfected into live attenuated Salmonella typhimurium strain SL7207 . Mouse models of intracranial G1261 glioblastoma were treated with an orally administered attenuated Salmonella typhimurium expressing flk-1 gene . The survival period was recorded and vessel density was observed by immunofluorescence . CTLs activity was measured by MTT assay . Our results showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could significantly inhibit glioblastoma growth, reduce vessel density, prolong the survival period and improve the survival rate in these mice . The flk-1 specific CTLs activity was increased obviously after the vaccination . Our study showed that attenuated Salmonella typhimurium vaccine strain expressing flk-1 gene could break peripheral immune tolerance a in glioma gainst this self-antigen and kill endothelial cells by the orally administered vaccine and can be used for both prophylactic and therapeutic purposes.

J Biol Chem . 2004 Dec 6; {Epub ahead of print}
A cis-spreading nucleoprotein filament is responsible for the gene silencing activity found in the promoter relay mechanism; Chen CC et al.; Transcription-generated DNA supercoiling plays a decisive role in a promoter relay mechanism for the coordinated expression of genes in Salmonella typhimurium ilvIH-leuO-leuABCD gene cluster . A similar mechanism also operates to control expression of the genes in the Escherichia coli ilvIH-leuO-leuABCD gene cluster . However, the mechanism underlying the DNA supercoiling effect remained elusive . A bacterial gene silencer AT8 was found to be important for the repression state of the leuO gene as part of the promoter relay mechanism . In this communication, we demonstrated that the gene silencer AT8 is a nucleation site for recruiting histone-like nucleoid structuring protein (H-NS) to form a cis-spreading nucleoprotein filament that is responsible for silencing of the leuO gene . With a DNA geometric similarity rather than a DNA sequence specificity, the E . coli gene silencer EAT6 was capable of replacing the H-NS nucleation function of the S . typhimurium gene silencer AT8 for the leuO gene silencing . The interchangeability between DNA geometrical elements for supporting the silencing activity in the region is consistent with a previous finding that a neighboring transcription activity determines the outcome of the gene silencing activity . The geometric requirement, which was revealed for this silencing activity explains the decisive role of transcription-generated DNA supercoiling found in the promoter relay mechanism.

Toxicol In Vitro, 2005 Feb, 19(1), 91 - 97
The amoebicidal aqueous extract from Castela texana possesses antigenotoxic and antimutagenic properties; Reyes-Lopez M et al.; Due to long-term treatment toxicity and clinical resistance to drugs commonly used against E . histolytica, new drugs against amoebiasis are urgently needed . Castela texana ("chaparro amargo") is a shrub taken traditionally in teas and capsules of dry plant to treat intestinal amoebic infections . An aqueous extract was prepared and its mutagenic, genotoxic and cytotoxicity properties were evaluated in prokaryotic and eukaryotic systems . This extract was neither mutagenic when evaluated with the Ames test in Salmonella typhimurium strains TA98, TA100 and TA102, nor genotoxic in unscheduled DNA synthesis in hepatocyte cultures, even at the highest concentrations tested . In fact, C . texana extract showed antimutagenic activity on S . typhimurium strains TA98 and TA100 in the Ames test . Furthermore, it was capable of protecting liver cell cultures against unscheduled DNA synthesis induced by 2-acetylaminofluorene at a concentration of 6.77 mug/ml . A free-radical scavenging test was used in order to explore the antioxidant capacity of C . texana extract with S . typhimurium strain TA102 pretreated with norfloxacin, a free radical producer . This extract showed a free radical withdrawal effect . The effective chemoprotective activity of this extract could be due to the antioxidant capacity of the C . texana extract components . In this paper it is shown that the antiamoebic natural product, C . texana, is also antimutagenic and protects against induction of preneoplastic lesions in rat liver . These results justify further studies to extend it use to human beings.

FEBS Lett, 2004 Dec 3, 578(1-2), 128 - 34
Enhancing the first enzymatic step in the histidine biosynthesis pathway increases the free histidine pool and nickel tolerance in Arabidopsis thaliana; Wycisk K et al.; Naturally selected nickel (Ni) tolerance in Alyssum lesbiacum has been proposed to involve constitutively high levels of endogenous free histidine . Transgenic Arabidopsis thaliana expressing a Salmonella typhimurium ATP phosphoribosyl transferase enzyme (StHisG) resistant to feedback inhibition by histidine contained approximately 2-fold higher histidine concentrations than wild type plants . Under exposure to a toxic Ni concentration, biomass production in StHisG expressing lines was between 14- and 40-fold higher than in wild-type plants . This suggested that enhancing the first step in the histidine biosynthesis pathway is sufficient to increase the endogenous free histidine pool and Ni tolerance in A . thaliana.

Med Sci (Paris), 2004 Dec, 20(12), 1119 - 24
{Protagonists of innate immunity during infection with Salmonella}; Salez L et al.; Salmonella are facultative intracellular Gram-negative bacteria that are found ubiquitously in nature and have the ability to infect a wide range of hosts including humans, domesticated, wild mammals, and birds . The principal clinical manifestations associated with Salmonella infection in humans are enteric fever (typhoid and paratyphoid) and a self-limiting gastroenteritis (salmonellosis) . Additionally, silent carriage of this bacterium is frequent and contributes to disease dissemination . Typhoid fever still represents a major public health problem in many developing countries . On the other hand, industrialized countries experience an increased incidence of nontyphoidal Salmonella infections with most cases tracing back to food contamination . Studies using mouse model of infection with a highly virulent Salmonella typhimurium serotype have provided important insight into the complexity of the innate immune response to infection . The players are numerous but emphasis was placed on the genes that were discovered using genetic approaches and in vivo assay with live pathogen and include positional cloning of mouse mutations and manipulation of genes in the context of whole animal either by transgenesis or knockout technologies . Some of the critical genes include those known to play a role in the detection of the bacteria (Cd14, Lbp, Tlr4 and Tlr5) and in microbicidal activity (Slc11a1, Nos2, NADPH oxidase and cryptdins) . These discoveries have already initiated the search for the contribution of particular genetic pathways in the innate immune response of humans to infection with Salmonella and other intracellular microorganisms.

Genetics, 2004 Nov, 168(3), 1119 - 30
Experimental adaptation of Salmonella typhimurium to mice; Nilsson AI et al.; Experimental evolution is a powerful approach to study the dynamics and mechanisms of bacterial niche specialization . By serial passage in mice, we evolved 18 independent lineages of Salmonella typhimurium LT2 and examined the rate and extent of adaptation to a mainly reticuloendothelial host environment . Bacterial mutation rates and population sizes were varied by using wild-type and DNA repair-defective mutator (mutS) strains with normal and high mutation rates, respectively, and by varying the number of bacteria intraperitoneally injected into mice . After <200 generations of adaptation all lineages showed an increased fitness as measured by a faster growth rate in mice (selection coefficients 0.11-0.58) . Using a generally applicable mathematical model we calculated the adaptive mutation rate for the wild-type bacterium to be >10(-6)/cell/generation, suggesting that the majority of adaptive mutations are not simple point mutations . For the mutator lineages, adaptation to mice was associated with a loss of fitness in secondary environments as seen by a reduced metabolic capability . During adaptation there was no indication that a high mutation rate was counterselected . These data show that S . typhimurium can rapidly and extensively increase its fitness in mice but this niche specialization is, at least in mutators, associated with a cost.

Biol Pharm Bull, 2004 Dec, 27(12), 2010 - 3
Experimental analysis of antimicrobial action of dicyclomine hydrochloride; Karaka P et al.; Dicyclomine hydrochloride is an antispasmodic agent . The MIC of dicyclomine against standard strains of Gram positive and Gram negative bacteria were performed by NCCLS broth dilution technique . These drugs showed a rapid killing action on Gram positive bacteria, Staphylococcus aureus NCTC 6571, 8530 and several other reference strains . The killing effect against Gram negative bacteria, Shigella boydii 8 NCTC 254/66 and Salmonella typhimurium NCTC 74 showed that the drug was bacteriostatic with respect to these strains . High rate of killing was achieved for most strains of Gram positive bacteria within 2 h . When administered to Swiss strain of white mice at doses of 30 and 60 microg/g of mouse, the drug could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . According to chi2 test, the in vivo data were highly significant (p<0.001) . Since dicyclomine showed a remarkable inhibitory action against several pathogenic bacteria, in the course of time, it may be developed as a potent antimicrobial agent for many bacterial infections.

Mutat Res, 2004 Dec 31, 565(1), 23 - 34
Influence of extraction parameters on the mutagenicity of soil samples; Courty B et al.; The aim of this study was to investigate the influence of four extraction parameters (type of solvent, temperature, duration of extraction, and soil mass/solvent volume ratio) on the mutagenicity of soil extracts . Four urban soil samples were submitted to the micro-method adaptation of the Ames test on Salmonella typhimurium according to the following sequence: identification of the most sensitive strain (TA98 or TA100), the best solvent(s), the optimum extraction temperature and extraction time, and finally the optimal soil/solvent ratio . Extraction was thus performed using eight different solvents (distilled water, dichloromethane, acetonitrile, acetone, cyclohexane, methanol, hexane, or ethanol), two temperatures (room temperature or 37 degrees C), two durations (4 or 24 h), and two soil mass/solvent volume ratios (1:2 or 1:10) . The results show that strain TA98 was more sensitive than strain TA100, and the observed mutagenicity was expressed as number of TA98 revertants per mg of soil equivalent . No mutagenicity was induced by the distilled water extracts, whereas most of the organic solvent extracts induced a significant mutagenic response . A dichloromethane/acetone mixture appeared to be the best compromise for extraction of mutagens from the urban soils tested . Moreover, the present study showed that a higher mutagenic activity was generally obtained with a temperature of 37 degrees C (compared to room temperature), with an extraction time of 24 h (compared to 4 h), and with a soil mass/solvent volume ratio of 1:10 (compared to 1:2).

Food Chem Toxicol, 2004 Dec, 42(12), 2029 - 35
Mutagenic activity of sweepings and pigments from a household-wax factory assayed with Salmonella typhimurium; Varella SD et al.; The mutagenic activity of garbage originating from a household wax industry was determined by the Salmonella/microsome assay, using the bacterial strains TA100, TA98 and YG1024 . The garbage was obtained by sweeping the floor of the factory at the end of the work shift . Organic compounds were extracted by ultrasound for 30 min in dichloromethane or 70% ethanol . After evaporation of solvent, these extracts (HFS: household-wax factory sweepings) were dissolved in DMSO, and were tested for the mutagenic activity at varying concentrations (HFS-ET: 0.08-0.68 mg/plate, HFS-DCM: 0.60-7.31 mg/plate) . The colouring agents (pigments) used in the production of the wax were also dissolved in DMSO and tested with the assay . The concentrations tested for each pigment were: Amaranth: 0.46-3.65 mg/plate, Auramine: 0.15-1.2 mg/plate and Rhodamine B: 0.22-1.82 mg/plate . Both ET and DCM organic extracts had mutagenic activity, especially in the YG1024 strain . The pigments behaved in a similar way, demonstrating that YG1024 was the most sensitive strain for the detection of mutagenicity, and that metabolization increased the activity . Human exposure (occupational and non-occupational) to industrial residues generated during the household-wax manufacturing and packaging process should be monitored, since this type of garbage is normally deposited in the environment without any control.

J Nat Prod, 2004 Nov, 67(11), 1876 - 8
Biotransformation of nobiletin by Aspergillus niger and the antimutagenic activity of a metabolite, 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone; Okuno Y et al.; Biotransformation of nobiletin (1) by Aspergillus niger has been investigated, and the product obtained was determined as 4'-hydroxy-5,6,7,8,3'-pentamethoxyflavone (2) . Antimutagenic activity of compound 2 was found, which showed suppressive effects on umu gene expression of the SOS response to DNA damage in Salmonella typhimurium TA1535/pSK1002, induced by the chemical mutagens furylfuramide, MeIQ, and Trp-P-1.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Sep-Oct, (5), 16 - 9
{State of Salmonella typhimurium population in water environment under the influence of temperature}; The impact of prebiotics and salmonellosis on apparent nutrient digestibility and Salmonella typhimurium var . Copenhagen excretion in adult pigeons (Columba livia domestica); Laboratory of Animal Nutrition, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium . Geert.Janssens@Ugent.be

The effects of lactose or fructo-oligosaccharide (FOS) supplementation on the excretion of salmonellae, apparent digestibilities and excreta consistency were studied . Thirty-two male pigeons (Columba livia domestica) were randomly divided into 4 equal groups: 3 of 4 groups were orally infected with 10(9) Salmonella Typhimurium var . Copenhagen, after being offered a drinking water supplement of 2% FOS, 2% lactose, or no supplement, respectively, for 2 wk . Pigeons in the fourth group were not challenged with S . Typhimurium and remained unsupplemented . Initially, FOS increased water intake, resulting in more watery excreta . After infection, supplementation showed no major effects on S . Typhimurium excretion, nitrogen retention, or apparent nutrient digestibilities, although lactose--and to a lesser extent FOS--improved apparent fiber digestibility during recovery from the S . Typhimurium infection . The excreta consistency of all pigeons returned to normal when recovering from the Salmonella infection . In this trial, neither FOS nor lactose was successful in tempering the negative aspects of Salmonella infection in pigeons . Nevertheless, it should be stated that future investigations should clarify the importance of duration and level of prebiotic supplementation and infection level.

J Food Prot, 2004 Nov, 67(11), 2403 - 9
A mathematical model for the transmission of Salmonella Typhimurium within a grower-finisher pig herd in Great Britain; Ivanek R et al.; In a study of pigs slaughtered at British abattoirs, approximately 23% carried Salmonella in their cecal (large intestine) contents . The most frequent serotype was Salmonella Typhimurium (STM), which was the second most common cause of human salmonellosis in Great Britain . A pig industry-monitoring program was developed to reduce Salmonella infection on British farms . The control of STM infection on the farm requires an understanding of STM transmission dynamics within the herd, and a mathematical model has been developed for an infected grower-finisher farm . The model estimates the probability of a random pig being infected with STM . There are three broad categories of STM infection in pigs: pigs that are infected but unable to transmit the infection (latent); pigs that are infectious, i.e., able to transmit the infection (shedders); and pigs that have stopped shedding but harbor STM in their internal organs (carriers) . The model estimates that 21.0% (5th and 95th percentiles, 0.05 to 77.5%) of slaughter-age pigs on an infected farm are likely to be shedding STM . Although this range is wide, it is biologically plausible . Sensitivity analysis of the total number of infected pigs revealed that the most significant input parameters are the probability of effective contact between a specific infectious and susceptible pig and the duration of shedding . The model predicted that 11.5% of pigs would be shedding STM at slaughter age . This value is close to the estimate obtained from a British abattoir survey that 11 . 1% of pigs carried STM in their ceca, indicating that the model has reasonable validity.

Vaccine, 2004 Dec 16, 23(5), 595 - 603
Oral vaccination of pigs with an invasive gyrA-cpxA-rpoB Salmonella Typhimurium mutant; Roesler U et al.; The potency to protect pigs against colonization and against clinical salmonellosis was evaluated after oral immunization with a live gyrA-cpxA-rpoB Salmonella (S.) Typhimurium mutant (S . Tm . Nal2/Rif9/Rtt) . Twenty 4-week-old male hybrid piglets were immunized orally, a control group received a placebo . Three weeks postimmunization, all pigs were challenged orally with a highly virulent S . Typhimurium DT104 strain . Clinical investigation revealed that immunization prevented the vaccinated pigs from clinical symptoms of salmonellosis . While all placebo-treated animals showed a 2-4-day episode of moderate to severe clinical symptoms, 90% of immunized pigs did not show any clinical signs at all . The bacteriological results showed a marked beneficial effect of the oral immunization . Vaccinated pigs showed a significantly decreased rate of colonization of the inner organs (42.5% versus 87.5%) when compared to the placebo-treated animals . Furthermore, in comparison to the non-immunized pigs, the vaccines developed a higher specific immunoglobulin (Ig)A antibody activity, but a significant lower IgM antibody activity in serum . The findings underline the ability of an attenuated oral live S . Typhimurium mutant to prevent clinical symptoms of salmonellosis in pigs and to significantly reduce the colonization of tissues and inner organs, as well as the shedding of S . Typhimurium.

Arch Biochem Biophys, 2004 Dec 15, 432(2), 233 - 43
The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-{(2)H}indole; Cash MT et al.; The bacterial tryptophan synthase alpha(2)beta(2) complex catalyzes the final reactions in the biosynthesis of L-tryptophan . Indole is produced at the active site of the alpha-subunit and is transferred through a 25-30 A tunnel to the beta-active site, where it reacts with an aminoacrylate intermediate . Lane and Kirschner proposed a two-step nucleophilic addition-tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-{(2)H}indole reacts with the aminoacrylate intermediate . We have now observed a KIE of 1.4-2.0 in the reaction of 3-{(2)H}indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an alpha-subunit ligand, disodium alpha-glycerophosphate (Na(2)GP), is present . Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-{(2)H}indole with tryptophan synthase preincubated with L-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the L-Trp external aldimine at 423 nm . The addition of Na(2)GP dramatically slows the rate of reaction of indole with the alpha-aminoacrylate intermediate . A primary KIE is not observed in the reaction of 3-{(2)H}indole with the aminoacrylate complex of tryptophan synthase in the presence of Na(2)GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions . The reaction of 2-methylindole does not show a KIE, either in the presence of Na(+) or Na(2)GP . These results support the previously proposed mechanism for the beta-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.

J Biol Chem . 2004 Nov 10; {Epub ahead of print}
Structures of dCTP deaminase from Escherichia coli with bound substrate and product . Reaction mechanism and determinants of mono- and bifunctionality for a family of enzymes; Johansson E et al.; dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium . dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate . Two active site amino acid residues, Arg115 and Glu138, were identified by mutational analysis as important for activity in E . coli dCTP deaminase . None of the mutant enzymes R115A, E138A or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed . The crystal structures of wild type E . coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg2+, and the mutant enzyme also with the substrate dCTP and Mg2+ . The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii . However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases . The nucleotides are well defined as well as Mg2+ that is tridentately coordinated to the nucleotide phosphate chains . We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

Avian Dis, 2004 Sep, 48(3), 595 - 605
Expression of Escherichia coli antigens in Salmonella typhimurium as a vaccine to prevent airsacculitis in chickens; Roland K et al.; Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis . A number of E . coli serotypes are associated with these diseases, although the most prevalent serotype is O78 . Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli . We have been working to develop an effective vaccine to protect chickens against these diseases . We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E . coli strain . In this work, we have constructed an attenuated S . typhimurium that expresses both the O78 lipopolysaccharide and E . coli-derived type 1 fimbriae . In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age . Birds were challenged at 4 wk of age . We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S . typhimurium that did not express any E . coli antigens . In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates . We were not able to demonstrate protection against E . coli O1 or O2 serotype challenge, nor against challenge with wild-type S . typhimurium.

Environ Mol Mutagen, 2004, 44(5), 387 - 93
Antimutagenic activity of spearmint; Yu TW et al.; The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay . Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro-1,2-phenylenediamine (NPD) and 2-hydroxyamino-3-methyl-3H-imidazo{4,5-f}quinoline (N-OH-IQ) using Salmonella typhimurium strain TA98 . Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD . These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ . Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo{4,5-f}quinoline (IQ), in the presence of rat liver S9; however, the concentration for 50% inhibition (IC(50)) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9 . At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro . In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P < 0.05, compared with rats given IQ alone) . Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s) . Environ . Mol . Mutagen., 2004 . (c) 2004 Wiley-Liss, Inc.

Science, 2004 Nov 5, 306(5698), 1040 - 2
Structural insights into the assembly of the type III secretion needle complex; Marlovits TC et al.; Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells . We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope-anchored base . Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base . Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion.

J Vet Med B Infect Dis Vet Public Health, 2004 Oct-Nov, 51(8-9), 389 - 92
Resistance of Salmonella isolates in Germany; Schroeter A et al.; During 2000-2002 the National Veterinary Reference Laboratory for Salmonella (NRL-Salm) in Germany typed 11,911 isolates from animals, food, feed and the environment . All of them were tested for their susceptibility to 17 anti-microbial agents . Sixty-three per cent of all isolates were resistant and 40% were multiresistant (resistant against more than one anti-microbial) . This general resistance level was strongly influenced by those specific serotypes which dominate the Salmonella epidemiology in Germany . Salmonella Typhimurium DT104 isolates from pig and cattle, and their resulting food products, were multiresistant in 98 and 94% of the cases respectively . During the period 2000-2003 an increasing quinolone resistance especially in Salmonella isolates from poultry and poultry meat (to 26%) and in S . Paratyphi B D-tartrate positive isolates (to 64%) could be observed . This increase was accompanied by a shift towards higher minimal inhibitory concentrations for ciprofloxacin.

J Biol Chem, 2005 Jan 7, 280(1), 355 - 60 Epub 2004 Nov 01.
Viability of Escherichia coli topA Mutants Lacking DNA Topoisomerase I; Stupina VA et al.; The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri . This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E . coli . We find that E . coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 degrees C without any compensatory mutations . These topA(-) cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA . In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R . J . (2001) Proc . Natl . Acad . Sci . U . S . A . 98, 9766-9771), E . coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function.

Biol Pharm Bull, 2004 Nov, 27(11), 1840 - 3
Limitation of polymyxin B on suppression of endotoxin shock induced by Salmonella infection in mice; Morita H et al.; The protective effects of an antibiotic polymyxin B (PLB), having lipopolysaccharide (LPS)-binding activity, on infection-induced endotoxin shock in mice were investigated . Infection with 10(8) colony forming units of an attenuated Salmonella typhimurium aroA strain caused lethal endotoxin shock to ddY mice . Treatment with PLB 1 h post infection (p.i.) resulted in significant reduction of mortality and bacterial numbers in livers . In addition, treatment with PLB 1 h p.i . resulted in a transient increase at the early stage and gradual decline in plasma LPS levels . Although plasma levels of sCD14 and high mobility group box chromosomal protein-1 (HMGB-1) increased according with progression of infection, increases in plasma levels of sCD14 and HMGB-1 were downregulated by treatment with PLB 1 h p.i . However, the lethal shock was not blocked by treatment with anti-CD14 monoclonal antibody at 3 h and 6 h p.i . Interestingly, administration of PLB 6 h p.i . did not show any protective activities, indicating that a time window for effective PLB action is present.

J Toxicol Environ Health A, 2004 Dec, 67(23-24), 2037 - 44
Genotoxicity evaluation of Isaria sinclairii (ISE) extract; Ahn MY et al.; The mutagenic potential Isaria sinclairii, a traditional Chinese medicine composed of the fruiting bodies of I . sinclairii and its parasitic host larva, was evaluated using short-term genotoxicity tests, namely, the Ames test, chromosome aberration (CA), and micronuclei (MN) tests . In a Salmonella typhimurium assay, I . sinclairii extract (ISE) did not produce any mutagenic response in the absence or presence of 59 mix with TA98, TA100, TA1535, and TA1537 . In the chromosome aberration (CA) test, ISE induced no significant effect on Chinese hamster ovary (CHO) cells compared with control . In the MN test, no significant change in the occurrence of micronucleated polychromatic erythrocytes was observed in male ICR mice intraperitoneally administered ISE at doses of 15, 150, or 1500 mg/kg . These results indicate that ISE has no mutagenic potential in these in vitro and in vivo systems.

J Environ Pathol Toxicol Oncol, 2004, 23(4), 297 - 302
Mutagenic potential of Mancozeb in Salmonella typhimurium; Shukla Y et al.; Mancozeb, a dithiocarbamate fungicide, was examined for its possible mutagenic activity using Salmonella typhimurium tester strains TA97a, TA98, TA100, and TA102 . We found that Mancozeb exhibited toxic effects at the dose of 40 microg/plate and higher with all tester strains . Mancozeb showed dose-dependent increases in the number of revertants with and without metabolic activation when it was dissolved in DMSO or acetone with strain TA97a; however, the number of revertants at the highest dose was less than two-fold compared to control values . We postulate that the true mutagenic potential of Mancozeb may be masked by its toxic effect to the tester strain used.

J Cell Sci, 2004 Nov 15, 117(Pt 24), 5771 - 80 Epub 2004 Oct 26.
Salmonella typhimurium transcytoses flagellin via an SPI2-mediated vesicular transport pathway; Lyons S et al.; Apical colonization of polarized epithelia by Salmonella typhimurium results in translocation of flagellin to the basolateral membrane domain, thus enabling activation of toll-like receptor 5 (TLR5)-mediated pro-inflammatory gene expression . Such flagellin transcytosis occurred without a change in epithelial permeability to 40 kDa FITC dextran, did not require bacterial motility and was independent of transepithelial movement of intact bacteria . Flagellin transcytosis was blocked at 20 degrees C, suggesting dependence on vesicular transport consistent with results from confocal microscopy that showed flagellin independent of bacteria inside epithelial cells . Furthermore, vesicles isolated from S . typhimurium-infected epithelia were highly enriched in flagellin . Flagellin transcytosis was dependent upon genes of Salmonella pathogenicity island (SPI)-2, which alter vesicular trafficking, but independent of SPI-1 that mediates bacterial invasion . Furthermore, such SPI-2 mutants were unable to mediate the localization of flagellin into intracellular vesicles consistent with flagellin transcytosis mediated by a S . typhimurium take-over of host vesicle trafficking pathways . As a result of their inability to transcytose flagellin, apical colonization by SPI-2 mutants induced substantially less epithelial IL-8 secretion than wild-type strains suggesting that such SPI-2 mediated transcytosis of flagellin plays a role in the pathogenesis of the mucosal inflammation characteristic of human Salmonellosis.

Mutat Res, 2004 Dec 12, 564(2), 149 - 57
Analysis of mutagenic activity of airborne particulate matter, standard reference materials and reference compounds using base pair-specific Salmonella typhimurium tester strains; Erdinger L et al.; The mutagenicity profiles of organic extracts of airborne dust samples from Mannheim, Germany, and two standard reference materials (SRM) as well as eight compounds with different chemical properties were investigated using tester strains Salmonella typhimurium TA700x (Ames II Assay) . Each strain of this series carries a unique missense mutation in the histidine operon and is reverted by only one specific base substitution out of six possible changes . Mutation patterns of eight compounds with different modes of genotoxic action reveal significant differences . Samples of airborne particulate matter (APM) from an industrialized town in Germany (Mannheim) were collected for five consecutive days once a month for 1 year using an automatic high-volume air sampler . Samples taken from Monday to Friday were Soxhlet-extracted and prepared according to standard methods . Although the threshold limit for the least active strains is not triggered by all samples, it can be concluded that mutation patterns of the samples do not vary between different seasons . Standard reference materials (SRMs) were prepared and tested using the same methods . SRMs and APM samples from Mannheim reveal similar mutagenicity profiles in TA700x strains . The comparison of the mutagenicity profiles of air dust extracts from Mannheim and the SRMs, respectively, with reference compounds investigated so far shows some similarities although the patterns do not fit perfectly . Mutagenicity profiles of TA700x-activity of nitro-aromatic compounds published so far are similar to those of APM collected in Mannheim, Germany, as well as to standard reference materials 1648 and 1649.

Mutat Res, 2004 Dec 12, 564(2), 103 - 13
Effects of exposure to diesel exhaust particles (DEP) on pulmonary metabolic activation of mutagenic agents; Zhao HW et al.; Exposure of rats to diesel exhaust particles (DEP) or carbon black (CB) has been shown to induce time-dependent changes in CYP1A1and CYP2B1 in the lung . The present study evaluated the role of these metabolic enzymes on the pulmonary bioactivation of mutagens . Male Sprague-Dawley rats were intratracheally instilled with saline (control), DEP or CB (35 mg/kg body weight) and sacrificed at 1, 3, or 7 days post-exposure . Both control and exposed lung S9 increased the mutagenic activity of 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF), 1-nitropyrene (1-NP), and the organic extract of DEP (DEPE) in Ames tests with Salmonella typhimurium YG1024 in a dose-dependent manner . Lung microsomes prepared form control or particle-exposed S9, but not cytosolic protein, activated 2-AA mutagenicity . Compared to saline controls, CB-exposed S9 was a less potent inducer of 2-AA mutagenicity at all time points, whereas DEP-exposed S9 was less potent than control saline at 3 and 7 days but not 1 day post-exposure . At 3 days post-exposure, DEP- or CB-exposed lung S9 did not significantly affect the mutagenicity of DEPE or 1-NP, when compared to the controls . The mutgenicity of 2-AA, 2-AF, 1-NP, and DEPE were significantly decreased in the presence of inhibitors for CYP1A1 (alpha-naphthoflavone) or CYP2B (metyrapone), but markedly enhanced by CYP1A1 or CYP2B1 supersomes with all the cofactors, suggesting that both CYP1A1 and CYP2B1 were responsible for mutagen activation . These results demonstrated that exposure of rats to DEP or CB altered metabolic activity of lung S9 and S9 metabolic activity dependent mutagen activation . The bioactivation of mutagens are metabolic enzyme- and substrate-specific, and both CYP1A1 and CYP2B1 play important roles in pulmonary mutagen activation.

J Ethnopharmacol, 2004 Dec, 95(2-3), 437 - 45
Mutagenic and antioxidant activities of Croton lechleri sap in biological systems; Lopes MI et al.; The sap of Croton lechleri Muell.-Arg (Euphorbiaceae), called Dragon's blood, is used in folk medicine as a cicatrizant, anti-inflammatory and to treat cancer . In this research, the antioxidant activity of Croton lechleri sap was evaluated against the yeast Saccharomyces cerevisiae and against maize plantlets treated with the oxidative agents apomorphine and hydrogen peroxide . The mutagenic activity of the sap was also analyzed using the Salmonella/microsome assay (Salmonella typhimurium TA97a, TA98, TA100, TA102, TA1535) and in cells of the yeast Saccharomyces cerevisiae . The results showed that Croton lechleri sap possesses significant antioxidant activity against the oxidative damages induced by apomorphine in Saccharomyces cerevisiae under all the conditions studied . However, in the case of hydrogen peroxide, antioxidant activity of the sap was detected only in cells in the stationary phase of growth . The sap was also able to protect cells of the maize plantlets from the toxic effect of apomorphine . This sap showed mutagenic activity for strain TA1535 of Salmonella typhimurium in the presence of metabolic activation and a weak mutagenic activity for strain TA98 . These strains detect base pair substitutions and frameshift mutations, respectively . Mutagenicity was also observed in a haploid Saccharomyces cerevisiae strain XV185-14c for the lys1-1, his1-7 locus-specific reversion and hom3-10 frameshift mutations.

Vet Immunol Immunopathol, 2004 Dec 8, 102(3), 321 - 8
Humoral immune response induced by oral administration of S . typhimurium containing a DNA vaccine against porcine reproductive and respiratory syndrome virus; Jiang P et al.; The ORFs-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major structural proteins of this virus . In this report, we described the induction of a PRRSV GP5-specific immune response by oral vaccination of mice with eukaryotic expression vectors containing the GP5 gene of PRRSV (pcDNA3-GP5), delived by attenuated Salmonella typhimurium aroA . It demonstrated that oral administration of the transformants resulted in expression of the GP5 transcript in the intestinal epithelium . The level of serum neutralization antibodies to PRRSV was not significantly different between the mice immunized with the transformants and the naked plasmid DNA . But the neutralizing antibody titres in sera of the mice immunized with SL7207/pcDNA3-GP5' and pcDNA3-GP5' (resides 2-25 deletion mutant of GP5) were significantly lower than those immunized with the complete GP5 gene . These results show that oral inoculation of the transformants can induce humoral immune response to PRRSV . The signal peptide of the GP5 protein of PRRSV is associated with the neutralizing epitope of the protein . The attenuated S . typhimurium may be used as a delivery system for oral DNA vaccines containing PRRSV GP5 glycoprotein.

FASEB J, 2005 Jan, 19(1), 158 - 9 Epub 2004 Oct 21.
Multidrug resistance-1 (MDR-1): a new target for T cell-based immunotherapy; Niethammer AG et al.; Acquired multidrug resistance (MDR) remains a major challenge in the treatment of cancer with chemotherapeutic drugs . It can be mediated by the up-regulated expression of different proteins within the tumor cell membrane . Here, we used murine multidrug resistance-1 (MDR-1) as a target-antigen for the immunotherapy of cancer . We successfully demonstrated that peripheral T cell tolerance can be broken by oral administration of a DNA vaccine encoding MDR-1 and carried by attenuated Salmonella typhimurium to secondary lymphoid organs . Thus, mice, immunized orally three times at 2-wk intervals and challenged 2 wk thereafter with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells, revealed a significant increase in life span . This was evident, when compared with animals either vaccinated with the empty control vector or challenged with the parental cell lines lacking overexpression of MDR-1 . The immune response induced was antigen-specific and CD8+ T cell-mediated . The presence of the target antigen led to up-regulation of activation markers on CD8+ T cells and resulted in a strong cytotoxic T cell response as well as lysis of tumor target cells in vitro . We furthermore established the vaccine to be an effective treatment for established multi-drug-resistant tumor metastases, resulting in a significantly increased life span of experimental animals . Absence of CD8+ T cells due to in vivo depletion led to abrogation of effectiveness . Taken together, our results demonstrate that T cell tolerance against the MDR-1 self-antigen can be broken . It is anticipated that the combination of such an approach with chemotherapy could lead to more effective treatments of cancer.

Mutat Res, 2004 Nov 22, 556(1-2), 65 - 74
Delineation of antimutagenic activity of catechin, epicatechin and green tea extract; Geetha T et al.; Tea is consumed worldwide as second largest to water in popularity as a beverage . It has been reported that tea extracts have antibacterial, antiviral, antioxidative, antitumor and antimutagenic activities . The protective effect of green tea has been assumed to be due to the powerful scavenging and antioxidative property of high concentrations of unpolymerised catechins and their gallates . In the present proposal green tea extract (GT), (+)-catechin (C) and (-)-epicatechin (EC) were investigated for their antioxidant activity by different in vitro methods like (i) DPPH assay (ii) superoxide anion scavenging and (iii) hydrogen peroxide scavenging activity . Further these agents were also tested against mutagenesis using the well-standardized Ames microsomal test system . The Ames tester strain Salmonella typhimurium TA102, which readily responds to reactive oxygen species, was used and the antimutagenic activity was evaluated against oxidative mutagens tertiary butyl hydroperoxide (ID50-24.41, 29.63 and 113.23 microg for EC, C and GT, respectively) and hydrogen peroxide (ID50-17.3, 18.4 and 88.1 microg for EC, C and GT, respectively) . Ascorbic acid was used as a standard antioxidant in all the experiments . Results indicate that all the three agents possess excellent DPPH free radical scavenging activity (IC50-1.5 microg for EC, 3.45 microg for C and 3.8 microg for GT), good hydrogen peroxide (IC50-11.18 microg for EC, 13.5 microg for C and 11.78 microg for GT) and superoxide anion scavenging (IC50-1.64 microg for EC, 1.74 microg for C and 3.52 microg for GT) activities . Further, they also show antimutagenic activity in the above-mentioned test systems establishing their antioxidant nature to be responsible for such activity . The in vitro antioxidant activity correlates well with the antimutagenic action . (-)-Epicatechin is indicated to be a better agent in comparison to the other two agents (ID50-1.2 times more than C and 5 times more than GT in antimutagenicity studies against t-BOOH and hydrogen peroxide induced mutagenesis) . Ascorbic acid however showed a much less activity (ID50-12.1 mg against t-BOOH and 7.2 mg with hydrogen peroxide induced mutagenesis).

Biochemistry, 2004 Oct 26, 43(42), 13370 - 9
Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain . A key enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance; Gatzeva-Topalova PZ et al.; Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N) . Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin . ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance . ArnA is a bifunctional enzyme . It can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose . We show that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA . This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme . The crystal structure of the ArnA decarboxylase domain from E . coli is presented here . The structure confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family . On the basis of sequence and structure comparisons of the ArnA decarboxylase domain with other members of the short-chain dehydrogenase/reductase (SDR) family, we propose a binding model for NAD(+) and UDP-glucuronic acid and the involvement of residues T(432), Y(463), K(467), R(619), and S(433) in the mechanism of NAD(+)-dependent oxidation of the 4''-OH of the UDP-glucuronic acid and decarboxylation of the UDP-4-keto-glucuronic acid intermediate.

Prev Vet Med, 2004 Oct 14, 65(3-4), 147 - 71
Estimating the number of undetected multi-resistant Salmonella Typhimurium DT104 infected pig herds in Denmark; Rugbjerg H et al.; In Denmark, the detection of multi-resistant Salmonella Typhimurium DT104 (MRDT104)-infected pig herds relies on the national Salmonella surveillance programme at the farm and slaughterhouse levels of production . With the surveillance sampling protocol and the diagnostic methods currently used, some herds might remain undetected . The number of undetected Danish pig herds infected with MRDT104 in the period 1 August 2001-31 July 2002 was estimated and compared with the number of culture-confirmed detected herds . A flow chart was constructed to illustrate where infected herds will go undetected in the surveillance system and Monte Carlo simulation was used to model the actual number of pig herds infected with MRDT1104 . We estimated that 52 (90% CI {28, 178}) finisher herds were infected with MRDT104 compared to 23 (44%) detected . Among sow herds with production of weaners or growers, we estimated that 38 (90% CI {23, 74}) were infected with MRDT104 compared to 7 (18%) actually detected . Among breeder and multiplier herds, we estimated that five (90% CI {3, 8}) herds were infected with MRDT104 compared to three (60%) detected . In total, we estimated that 102 pig herds were infected with MRDT104 from 1 August 2001 till 31 July 2002 (90% CI {63, 228}) . In comparison, 33 (32%) infected herds were detected in this period . The predicted proportion of undetected herds varied considerably with herd type . We infer that the proportion of detected MRDT104 infected herds depended on the intensity of the combined serological and bacteriological testing.

Environ Sci Technol, 2004 Sep 15, 38(18), 4713 - 22
Chemical and biological characterization of newly discovered iodoacid drinking water disinfection byproducts; Plewa MJ et al.; Iodoacid drinking water disinfection byproducts (DBPs) were recently uncovered in drinking water samples from source water with a high bromide/iodide concentration that was disinfected with chloramines . The purpose of this paper is to report the analytical chemical identification of iodoacetic acid (IA) and other iodoacids in drinking water samples, to address the cytotoxicity and genotoxicity of IA in Salmonella typhimurium and mammalian cells, and to report a structure-function analysis of IA with its chlorinated and brominated monohalogenated analogues . The iodoacid DBPs were identified as iodoacetic acid, bromoiodoacetic acid, (Z)- and (E)-3-bromo-3-iodopropenoic acid, and (E)-2-iodo-3-methylbutenedioic acid . IA represents a new class (iodoacid DBPs) of highly toxic drinking water contaminants . The cytotoxicity of IA in S . typhimurium was 2.9x and 53.5x higher than bromoacetic acid (BA) and chloroacetic acid (CA), respectively . A similar trend was found with cytotoxicity in Chinese hamster ovary (CHO) cells; IA was 3.2x and 287.5x more potent than BA and CA, respectively . This rank order was also expressed in its genotoxicity with IA being 2.6x and 523.3x more mutagenic in S . typhimurium strain TA100 than BA and CA, respectively . IA was 2.0x more genotoxic than BA and 47.2x more genotoxic than CA in CHO cells . The rank order of the toxicity of these monohalogenated acetic acids is correlated with the electrophilic reactivity of the DBPs . IA is the most toxic and genotoxic DBP in mammalian cells reported in the literature . These data suggest that chloraminated drinking waters that have high bromide and iodide source waters may contain these iodoacids and most likely other iodo-DBPs . Ultimately, it will be important to know the levels at which these iodoacids occur in drinking water in order to assess the potential for adverse environmental and human health risks.

Lett Appl Microbiol, 2004, 39(5), 401 - 6
Antimicrobial activity of ultrasound-assisted solvent-extracted spices; Thongson C et al.; AIMS: The objective of this research was to determine the antimicrobial activity of conventional and high-intensity ultrasound-assisted (HI-US) solvent-extracted Thai spices, including ginger (Zingiber officinale Rose), fingerroot (Bosenbergia pandurata Holtt) and turmeric (Curouma longa Linn) . METHODS AND RESULTS: Extracts were obtained using hexane, isopropanol and a 7 : 3 isopropanol : hexane mixture as solvents with and without HI-US . The antimicrobial activity of the extracts was assayed against four strains each of Listeria monocytogenes and Salmonella Typhimurium DT 104 using an agar dilution assay . Application of HI-US did not alter antibacterial activity against S . Typhimurium, but antilisterial activity of some HI-US spice extracts decreased . Solvent type affected antimicrobial efficacy of extracts with hexane producing the least antimicrobial activity . Fingerroot extracted with isopropanol-hexane and without HI-US had the best antilisterial effect while HI-US-isopropanol fingerroot extract had the greatest antimicrobial efficacy against S . Typhimurium . CONCLUSIONS: Application of HI-US reduced time of extraction to 5 min, compared with the 24 h required for conventional extraction and maintained antimicrobial activity against Salmonella but slightly reduced activity against Listeria . SIGNIFICANCE AND IMPACT OF THE STUDY: HI-US in combination with proper solvent selection may offer a new tool to optimize extraction of spice essential oil for use as antimicrobial agents, and reduce processing time and costs.

Commun Dis Public Health, 2004 Sep, 7(3), 193 - 9
Antimicrobial resistance and phage types of human and non-human Salmonella enterica isolates in Ireland, 1998-2003; O'Hare C et al.; Between 1998 and 2003, 5,161 isolates (3,182 human) of Salmonella enterica were received by the National Salmonella Reference Laboratory of Ireland . Serotyping, antimicrobial susceptibility testing and phage typing were performed by standard methods . The number of isolates of S . enterica serovar Typhimurium decreased from 579 (80%) in 1998 to 208 (19%) in 2003, while S . enterica serovar Enteritidis increased from 59 (8%) in 1998 to 219 (20%) in 2003 . Definitive (DT) phage types 104 and DT104b accounted for a declining proportion of all Salmonella Typhimurium isolates (from n = 523 {90%} in 1998 to 126 {60%} in 2003) . Numbers of Salmonella Enteritidis phage type 4 declined from 50 (85%) in 1998 to 59 (27%) in 2003 . Twenty-eight isolates of typhoidal Salmonella were received with a history of recent travel in 17 cases . Resistance to multiple (four or more) antimicrobial agents was related to serotype and, where applicable, phage type, and was common in Salmonella Typhimurium . Salmonella Typhimurium predominated among isolates from cattle and pigs (n = 213 {58%}), while Salmonella Livingstone (n = 327) and S . Kentucky (n = 227) were predominant in isolates from poultry (total n = 554 {43%}) . This paper discusses trends, and their implications, in Irish salmonella isolates since the establishment of the Reference Laboratory.

J Appl Microbiol, 2004, 97(5), 964 - 72
Premature Salmonella Typhimurium growth inhibition in competition with other Gram-negative organisms is redox potential regulated via RpoS induction; Komitopoulou E et al.; AIMS: To identify the role of oxidation-reduction (redox) potential in the premature growth inhibition and RpoS induction in Salmonella serotype Typhimurium in competitive growth experiments . METHODS AND RESULTS: Oxidation-reduction potential was measured throughout the growth of a minority population of Salm . Typhimurium in mixed cultures with other Gram-negative and Gram-positive organisms . A lux-based reporter was also used to evaluate RpoS activity in Salm . Typhimurium in competitor studies . In a mixed culture, the multiplication of a minority population of Salm . Typhimurium was inhibited when competing Gram-negative organisms entered the stationary phase . This was not seen when the competing flora was Gram-positive . The change in redox potential during growth in mixed cultures was closely linked to the inhibition of Salm . Typhimurium growth by Gram-negative competitors . An artificially induced drop in redox potential earlier during growth in mixed cultures with Gram-negative organisms reduced the time to RpoS induction in Salm . Typhimurium and thus inhibited its multiplication prematurely . In contrast, RpoS induction and growth inhibition were prevented under high redox potential conditions . CONCLUSIONS: This work shows that the inhibitory activity of competitive organisms can be mediated through their effect on redox potential-regulated RpoS induction . SIGNIFICANCE AND IMPACT OF THE STUDY: Redox potential is shown to be an important determinant of Salm . Typhimurium growth, an observation with practical implications both for its control and detection.

J Appl Microbiol, 2004, 97(5), 916 - 22
Application of antimicrobial ice for reduction of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes) on the surface of fish; Shin JH et al.; AIMS: The efficacy of antimicrobial ice was evaluated for the reduction of foodborne pathogens on the surface of fish . METHODS AND RESULTS: Antimicrobial ice containing chlorine dioxide (ClO2) was utilized to control foodborne pathogens in laboratory media and on fish skin . Escherichia coli O157:H7, Salmonella serotype Typhimurium and Listeria monocytogenes strains were treated with antimicrobial ice for 30 min on plates of selective agar and for 120 min on fish skin at room temperature, and then incubated for enumeration . After treatment with 100 ppm ClO2 for 30 min, 5.4, 4.4 and 3.2 log10 reduction was obtained with E . coli O157:H7, Salm . Typhimurium and L . monocytogenes on laboratory media, respectively . When antimicrobial ice (100 ppm ClO2) was applied to fish skin for 120 min, total reduction of E . coli O157:H7, Salm . Typhimurium and L . monocytogenes was 4.8, 2.6 and 3.3 log10, respectively . CONCLUSION: The initial load of foodborne pathogens was reduced by antimicrobial ice and the lowered microbial level was maintained during treatment . SIGNIFICANCE AND IMPACT OF THE STUDY: The application of antimicrobial ice is a simple and effective method for the safe preservation of fish.

Annu Rev Physiol . 2004 Oct 12; {Epub ahead of print}
Structure and Function of CLC Channels; Chen TY; The CLC family comprises a group of integral membrane proteins whose major action is to translocate chloride (Cl-) ions across the cell membranes . Recently, the structures of CLC orthologues from two bacterial species, Salmonella typhimurium and Escherichia coli, were solved, providing the first framework for understanding the operating mechanisms of these molecules . However, most of the previous mechanistic understanding of CLC channels came from electrophysiological studies of a branch of the channel family, the muscle-type CLC channels in vertebrate species . These vertebrate CLC channels were predicted to contain two identical but independent pores, and this hypothesis was confirmed by the solved bacterial CLC structures . The opening and closing of the vertebrate CLC channels are also known to couple to the permeant ions via their binding sites in the ion-permeation pathway . The bacterial CLC structures can probably serve as a structural model to explain the gating-permeation coupling mechanism . However, the CLC-ec1 protein in E . coli was most recently shown to be a Cl--H+ antiporter, but not an ion channel . The molecular basis to explain the difference between vertebrate and bacterial CLCs, especially the distinction between an ion channel and a transporter, remains a challenge in the structure/function studies for the CLC family . Expected online publication date for the Annual Review of Physiology Volume 67 is February 4, 2005 . Please see for revised estimates.

Drug Chem Toxicol, 2004 Aug, 27(3), 257 - 68
Genotoxicity of aspartame; Rencuzogullari E et al.; In the present study, the genotoxic effects of the low-calorie sweetener aspartame (ASP), which is a dipeptide derivative, was investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test, micronucleus test in human lymphocytes and also Ames/Salmonella/ microsome test . ASP induced CAs at all concentrations (500, 1000 and 2000 microg/ml) and treatment periods (24 and 48 h) dose-dependently, while it did not induce SCEs . On the other hand, ASP decreased the replication index (RI) only at the highest concentration for 48 h treatment period . However, ASP decreased the mitotic index (MI) at all concentrations and treatment periods dose-dependently . In addition, ASP induced micronuclei at the highest concentrations only . This induction was also dose-dependent for 48 hours treatment period . ASP was not mutagenic for Salmonella typhimurium TA98 and TA100 strains in the absence and presence of S9 mix.

J Appl Toxicol, 2004 Sep-Oct, 24(5), 327 - 32
EILATox-Oregon Workshop: blind study evaluation of Vitotox test with genotoxic and cytotoxic sample library; Merilainen J et al.; In order to assess the robustness, sensitivity and specificity of a recently developed Vitotox test, 17 blind coded chemicals and three environmental water samples were tested at the EILATox-Oregon Workshop using the Thermo Electron Vitotox kit . The Vitotox test is a rapid geno- and cytotoxicity test using standard 96- or 384-well microtitre plates . The genotoxicity test is based on two genetically modified Salmonella typhimurium strains containing bacterial luciferase operon from Vibrio fisheri under the SOS inducible promoter . The SOS system is an inducible network in Escherichia coli that responds to DNA damage and activates DNA repair . The Vitotox genotoxicity test bacteria strain carries bacterial luciferase genes under the control of SOS inducible promoter and therefore any DNA damage inside the cells induces the production of bacterial luciferase . The luciferase expression is then followed with a microtitre plate luminometer for 3 h after mixing different dilutions of sample with the test bacteria . The genotoxicity index is calculated for each dilution and the genotoxicity of the sample is interpreted based on kinetic time curves and genotoxicity vs concentration/dilution curves . Cytotoxicity of the sample is determined simultaneously with another test strain containing the same luciferase operon controlled by the constitutive promoter . This bacterium produces constant bioluminescence and any decrease of the bioluminescence production is used as a marker for cytotoxicity . As a miniaturized microtitre plate assay the Vitotox test requires a very small quantity of the sample material . The samples used in the workshop were diluted 1 : 10 or 1 : 100 before testing . Genotoxicity and cytotoxicity data were collected at dilutions of 1 : 10-1 : 2000 . When the samples of the EILATox-Oregon Workshop were tested using the Vitotox test, four coded chemicals out of 17 were determined to be genotoxic . Seven chemicals and one environmental sample were found to be cytotoxic . Three chemical samples were found to be both geno- and cytotoxic.

J Infect Dis, 2004 Nov 1, 190(9), 1652 - 4 Epub 2004 Sep 21.
Quinolone resistance is associated with increased risk of invasive illness or death during infection with Salmonella serotype Typhimurium; Helms M et al.; In a registry-based cohort study, we determined the risk of invasive illness or death associated with infection with quinolone-resistant Salmonella serotype Typhimurium . We linked data from the Danish surveillance registry of enteric pathogens with data from the Danish civil registration system and 2 national health registries . Of 1323 patients infected with Salmonella Typhimurium, 46 (3.5%) were hospitalized due an invasive illness within 90 days of infection, and 16 (1.2%) died within 90 days of infection . After adjustment for age, sex, and comorbidity, infection with quinolone-resistant Salmonella Typhimurium was associated with a 3.15-fold (95% confidence interval, 1.39-7.10-fold) higher risk of invasive illness or death within 90 days of infection, compared with that observed for infection with pansusceptible strains.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 255 - 9
Occurrence of D-histidine residues in antimicrobial poly(arginyl-histidine), conferring resistance to enzymatic hydrolysis; Nishikawa M et al.; The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense . We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides . To elucidate what causes such differences, we here investigated the structural features of the fungal peptides . The acid hydrolysates of the fungal peptide contained d-histidine . When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine) . We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.

Phytother Res, 2004 Aug, 18(8), 628 - 33
Potent antioxidative and antigenotoxic activity in aqueous extract of Japanese rice bran--association with peroxidase activity; Higashi-Okai K et al.; To estimate the preventive potential of Japanese rice bran (Oryza sativa japonica) against the oxygen radical-related chronic diseases such as cardio-vascular diseases and cancer, antioxidative and antigenotoxic activities of the rice bran extracts were analyzed by using assay systems for lipid peroxidation and genotoxin-induced umu gene expression . When effects of the rice bran extracts under different extraction conditions on hydroperoxide generation from auto-oxidized linoleic acid were examined using aluminum chloride method, the water extract showed strong antioxidant activity, but the methanol and acetone extracts did not exhibit significant activity . The water extract of rice bran was divided into the ethanol-precipitable (EP) and supernatant fractions, and EP fraction showed the dominant antioxidant activity, but the supernatant fraction did not exhibit significant antioxidant activity . When the effect of EP fraction on umu C gene expression in SOS response associated with DNA damage in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) was analyzed, it showed a dose-dependent suppressive activity against Trp-P-1-induced umu C gene expression . The bio-chemical analysis of EP fraction indicates that the major antioxidative and antigenotoxic activity of EP fraction is associated with a proteinous component with the molecular weight of more than 30 KDa . As a possible active principle for the antioxidative and antigenotoxic activity in EP fraction, the strong activity of an oxygen radical-scavenging enzyme, peroxidase was detected, and the purified horseradish peroxidase also caused the similar antioxidative and antigenotoxic activities . The significance of this finding is discussed from the viewpoint of the preventive role of rice bran against oxygen radical-related chronic diseases . Copyright (c) 2004 John Wiley & Sons, Ltd.

Environ Mol Mutagen, 2004, 44(4), 329 - 45
Evaluation of the Salmonella umu test with 83 NTP chemicals; Yasunaga K et al.; There is a need for simple rapid tests for evaluating the carcinogenic potential of the thousands of chemical compounds that are developed each year . The DNA-damaging effects of 83 National Toxicology Program (NTP) chemicals, including noncarcinogens and carcinogens, were examined in the umu test by measuring the expression of the umuDC-lacZ genes in Salmonella typhimurium TA1535/pSK1002 . Salmonella were exposed to individual NTP chemicals at 37 degrees C for 2 hr both with and without a rat liver S9 mix; the treated cells were then diluted and incubated for a further 2 hr (posttreatment assay) . O-nitrophenyl-beta-D-galactoside was added to the cultures and the beta-galactosidase activity driven by the Salmonella umuDC-lacZ genes was determined by measurement of the OD(420 nm) and OD(550 nm) of the cultures . Salmonella cell number was simultaneously determined by measurement of OD(600 nm) . The overall concordance between genotoxicity in the umu test and carcinogenicity was 67%, which was similar to the concordance between Ames' test results and carcinogenicity (63%) using the same 83 NTP chemicals . The results of this study indicate that the umu test with a single Salmonella strain is a simple rapid system, with accuracy comparable to existing, more time-consuming assays.

Mutat Res, 2004 Nov 14, 564(1), 39 - 50
Di-epoxides of the three isomeric dicyclopenta-fused pyrenes: ultimate mutagenic active agents; Otero-Lobato MJ et al.; To rationalize the high bacterial mutagenic response recently found for the (di-) cyclopenta-fused pyrene congeners, viz . cyclopenta{cd}-(1), dicyclopenta{cd,mn}-(2), dicyclopenta{cd,fg}-(3) and dicyclopenta{cd,jk}pyrene (4), in the presence of a metabolic activation mixture (S9-mix), their (di-)epoxides at the externally fused unsaturated five-membered rings were previously proposed as the ultimate mutagenic active forms . In this study, cyclopenta{cd}pyrene-3,4-epoxide (5) and the novel dicyclopenta{cd,mn}pyrene-1,2,4,5-di-epoxide (6), dicyclopenta{cd,fg}pyrene-5,6,7,8-di-epoxide (7) and dicyclopenta{cd,jk}pyrene-1,2,6,7-di-epoxide (8) were synthesised from 1 to 4, respectively, and subsequently assayed for bacterial mutagenicity in the standard microsomal/histidine reverse mutation assay (Ames-assay with Salmonella typhimurium strain TA98) . The di-epoxides 6-8 are present as a mixture of their cis- and trans-stereo-isomers in a close to 1:1 ratio ((1)H NMR spectroscopy and ab initio IGLO/III//RHF/6-31G** calculations) . The direct-acting mutagenic activity and the strong cytotoxicity exerted by 5-8 both in the absence or presence of an exogenous metabolic activation system (+/-S9-mix) demonstrate that the ultimate mutagenic active forms are the proposed (di-)epoxides of 1-4.

Cell Microbiol, 2004 Nov, 6(11), 1057 - 70
Identification of Salmonella typhimurium genes responsible for interference with peptide presentation on MHC class I molecules: Deltayej Salmonella mutants induce superior CD8+ T-cell responses; Qimron U et al.; Salmonella-derived epitopes are presented on MHC molecules by antigen-presenting cells, and both CD4+ and CD8+ T cells participate in protective immunity to Salmonella . Therefore, mechanisms that allow Salmonella to escape specific immune recognition are likely to have evolved in this bacterial pathogen . To identify Salmonella genes, which potentially interfere with the MHC class I (MHC-I) presentation pathway, Tn10d transposon mutagenesis was performed . More than 3000 mutants, statistically covering half of the Salmonella genome, were individually screened for altered peptide presentation by infected macrophages . Two mutants undergoing enhanced antigen presentation by macrophages were identified, carrying a Tn10d insertion in the yej operon . This phenotype was validated by specific inactivation and complementation experiments . In accordance with their enhanced MHC-I presentation phenotype, we showed that (i) specific CD8+ T cells were elicited at a higher level in mice, in response to immunization with yej mutants compared to their parental strain in two different experimental settings; and (ii) yej mutants were superior vaccine carriers for heterologous antigens compared to the parental strain in a tumour model.

J Microbiol, 2004 Sep, 42(3), 205 - 10
Molecular cloning and characterization of CMCase gene (celC) from Salmonella typhimurium UR; Yoo JS et al.; The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1 . Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D . The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE . When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin . Optimal temperature and pH for enzyme activity were found to be 50 degrees C and pH 6.5, respectively .

FASEB J, 2004 Dec, 18(15), 1955 - 7 Epub 2004 Dec.
Transcription profiling analysis of the mechanisms of vaccine-induced protection against H . pylori; Walduck A et al.; Development of a vaccine against H . pylori is regarded as desirable alternative to the current antibiotic therapy regimens . Mice immunized with an attenuated recombinant Salmonella typhimurium expressing H . pylori urease subunits A&B have dramatically reduced bacterial loads after a single dose . The mechanism(s) of protection against this largely extra-cellular pathogen are not fully understood . The aim of this study was to identify genes that were regulated specifically in response to immunization, in order to gain a broader picture of the immune response in the immunized gastric epithelium . Gene expression in RNA isolated from the gastric mucosa of immunized and infected Balb/c mice was compared with that in infected only mice at 1, 3, and 14 days after challenge with a mouse-adapted strain of H . pylori . We show that infection with H . pylori causes an immediate reaction in vivo, which was clearly divided into acute and chronic phases, and further that the transcriptional response in the H . pylori infected and immunized gastric mucosa is unique . Analysis of gene expression patterns at day 14 post-infection suggested not only the beginning of a lymphocytic infiltrate, but of an integrated epithelial response characterized by increased expression of genes controlling cell cycle and turnover . This observation was confirmed in independent experiments . The global approach has brought new insights to the effect of immunization on the gastric epithelium and has led us to propose a new multi-factorial model for the mechanisms underlying vaccine-induced protection.

Int J Food Microbiol, 2004 Nov 15, 96(3), 301 - 5
Effectiveness of lemon juice, vinegar and their mixture in the elimination of Salmonella typhimurium on carrots (Daucus carota L.); Sengun IY et al.; Lemon juice, vinegar and the mixture of lemon juice and vinegar (1:1) were tested for their effectiveness in reducing the counts of inoculated Salmonella typhimurium (approximately 6 and 3 log cfu/g) on carrots . Treatment of carrot samples with lemon juice vinegar alone for different exposure times (0, 15, 30 and 60 min) caused significant reductions ranging between 0.79-3.95 and 1.57-3.58 log cfu/g, respectively, while the number of pathogens was reduced to an undetectable level after 30-min treatment by combined used lemon juice vinegar.

J Food Prot, 2004 Sep, 67(9), 1945 - 7
Experimental use of 2-nitropropanol for reduction of Salmonella Typhimurium in the ceca of broiler chicks; Jung YS et al.; The effect of 2-nitropropanol (2NPOH) administration on Salmonella enterica serovar Typhimurium in experimentally infected chicks was determined . Chicks orally challenged with 10(6) CFU/ml of a novobiocin- and naladixic acid-resistant Salmonella Typhimurium at 6 days of age were divided into three groups receiving 0 (control), 6.5, and 13 mg 2NPOH per bird (experiment 1) or four groups receiving 0 (control), 13, 65, and 130 mg 2NPOH per bird (experiment 2) . Treatments were administered orally 1 day post-Salmonella challenge . Cecal contents collected at necropsy 24 and 48 h after treatment were subjected to bacterial and volatile fatty acid (VFA) analysis . In experiment 1, concentrations (mean+/-SD log CFU per g) of Salmonella were reduced (P < 0.05) in the group administered 13 mg 2NPOH per bird at both the 24- and 48-h samplings compared with the controls (2.58+/-2.10 versus 4.64+/-1.79 and 2.88+/-2.78 versus 5.03+/-2.42 at 24 and 48 h, respectively) . In experiment 2, mean+/-SD populations of Salmonella were reduced (P < 0.05) in all groups receiving 2NPOH compared with untreated controls (3.65+/-2.01, 3.39+/-2.42, and 3.47+/-1.55 at 13, 65, and 130 mg, respectively, versus 6.09+/-1.02) . Propionate concentrations were reduced (P < 0.05) by the 13-mg 2NPOH per bird treatment . Total VFA concentrations from the group treated with 13 mg 2NPOH per bird were lower (P < 0.05) by 48, but not 24, hours posttreatment than those from the group treated with 6.5 mg 2NPOH per bird . These results demonstrate the inhibitory activity of 2NPOH against Salmonella Typhimurium in vivo.

J Food Prot, 2004 Sep, 67(9), 1834 - 9
Prevalence and number of Salmonella in irish retail pork sausages; Boughton C et al.; A national Salmonella control program in the pork industry was enacted in Ireland in August 2002 . This study was undertaken as part of a larger project investigating the role of pork as a source of human salmonellosis in Ireland . The objective of this survey was to assess the prevalence of Salmonella in Irish pork sausage at retail level . Samples, comprising branded prepacked sausages and loose sausages from supermarket meat counters and butcher shops, were collected from selected retail sites in four cities from October to December 2001 and from June to August 2002 . A three-tube most-probable-number method was used to enumerate Salmonella in a selected number of samples that were positive by enrichment . Salmonella serotypes were detected in 4.4 and 1.7% of samples at each of the respective sampling periods, a level similar to those reported in other U.S . and U.K . studies . Isolates were characterized by serotype, phage type, and antimicrobial susceptibility . Eighteen (70%) were resistant to at least one antimicrobial, and 15 (58%) were resistant to four or more antimicrobials . Most of the isolates exhibited resistance to tetracycline . Five different phage types were detected . DT104 was the predominant phage type among Salmonella Typhimurium isolates . This study revealed that multidrug-resistant salmonellae are present in a proportion of Irish sausages and that further risk analysis work is necessary in order to quantify the risk posed to public health.

Toxicol Lett, 2004 Nov 2, 153(2), 283 - 92
Antimutagenicity of coriander (Coriandrum sativum) juice on the mutagenesis produced by plant metabolites of aromatic amines; Cortes-Eslava J et al.; Aromatic amines are metabolically activated into mutagenic compounds by both animal and plant systems . The 4-nitro-o-phenylenediamine (NOP) is a well-known direct-acting mutagen whose mutagenic potential can be enhanced by plant metabolism; m-phenylenediamine (m-PDA) is converted to mutagenic products detected by the Salmonella typhimurium TA98 strain, and 2-aminofluorene (2-AF) is the plant-activated promutagen most extensively studied . Plant cells activate both 2-AF and m-PDA into potent mutagens producing DNA frameshift mutations . Coriander (Coriandrum sativum) is a common plant included in the Mexican diet, usually consumed uncooked . The antimutagenic activity of coriander juice against the mutagenic activity of 4-nitro-o-phenylenediamine, m-phenylenediamine and 2-aminofluorene was investigated using the Ames reversion mutagenicity assay (his- to his+) with the S . typhimurium TA98 strain as indicator organism . The plant cell/microbe coincubation assay was used as the activating system for aromatic transformation and plant extract interaction . Aqueous crude coriander juice significantly decreased the mutagenicity of metabolized aromatic amines (AA) in the following order: 2-AF (92.43%) > m-PDA (87.14%) > NOP (83.21%) . The chlorophyll content in vegetable juice was monitored and its concentration showed a positive correlation with the detected antimutagenic effect . Protein content and peroxidase activity were also determined . The concentration of coriander juice (50-1000 microl/coincubation flask) was neither toxic nor mutagenic . The similar shape of the antimutagenic response curves obtained with coriander juice and chlorophyllin (used as a subrogate molecule of chlorophyll) indicated that comparable mechanisms of mutagenic inhibition could be involved . The negative correlation between chlorophyll content and mutagenic response of the promutagenic and direct-acting used amines allows us to deduce that a chemical interaction takes place between the two molecules, leading to the inactivation of mutagenic moiety.

Biochem Biophys Res Commun, 2004 Oct 29, 323(4), 1257 - 64
Structures of wild-type and P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli; Jeong MS et al.; The alpha-subunit of tryptophan synthase (alphaTS) catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is used to yield the amino acid tryptophan in tryptophan biosynthesis . Here, we report the first crystal structures of wild-type and double-mutant P28L/Y173F alpha-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.8A resolution, respectively . The structure of wild-type alphaTS from E . coli was similar to that of the alpha(2)beta(2) complex structure from Salmonella typhimurium . As compared with both structures, the conformational changes are mostly in the interface of alpha- and beta-subunits, and the substrate binding region . Two sulfate ions and two glycerol molecules per asymmetric unit bind with the residues in the active sites of the wild-type structure . Contrarily, double-mutant P28L/Y173F structure is highly closed at the window for the substrate binding by the conformational changes . The P28L substitution induces the exposure of hydrophobic amino acids and decreases the secondary structure that causes the aggregation . The Y173F suppresses to transfer a signal from the alpha-subunit core to the alpha-subunit surface involved in interactions with the beta-subunit and increases structural stability.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 25 - 31
Analysis of integrons in human isolates of Salmonella enterica serovar typhimurium isolated in the Slovak Republic; Majtan V et al.; About 110 sporadic, epidemiologically unrelated Salmonella enterica serovar typhimurium strains isolated in the Slovak Republic were analyzed for the presence of integrons . Of these 110 examined strains, 47 were of definitive phage type DT104 and 63 were strains of various phage type, RDNC and untypeable, designated here as non-DT104 strains . All isolates were also tested for antimicrobial resistance to 10 antibiotics as well as for the presence of virulence plasmid . Of 63 non-DT104 strains, 15 isolates were multiple-resistant, independently from phage type, other strains were resistant to one, two or three drugs . Resistance to ampicillin, streptomycin, tetracycline and sulfisoxazole was most frequently observed . Among the DT104 isolates up 65.9% exhibited characteristic pentaresistance--ACSSuT phenotype . The integron content was studied in PCR experiments using a 5'-CS/3'-CS primer pair . Fourteen non-DT104 strains, independently from phage type, were found to carry integrons with amplicons 650-1900 bp in size . Thirty-six DT104 strains contained integrons of 1000 and 1200 bp and 31 of they exhibited the ACSSuT phenotype . No integron was found in 10 DT104 strains, which included strains mostly resistant only to streptomycin, tetracycline and sulfisoxazole . The majority of non-DT104 strains did not possess any integrons . Our findings show the widespread existence of both resistant and multiple-resistant epidemiologically unrelated Salmonella typhimurium strains and suggest that integrons contribute to this antimicrobial resistance . The presence of 90-kb virulence plasmid in the 54 non-DT104 and in the all DT104 strains was found.

Mutat Res, 2004 Oct 4, 554(1-2), 399 - 406
Analysis of the involvement of human N-acetyltransferase 1 in the genotoxic activation of bladder carcinogenic arylamines using a SOS/umu assay system; Oda Y; Human acetyltransferase genes NAT1 or NAT2 were expressed in a Salmonella typhimurium strain used to detect the genotoxicity of bladder carcinogens . To clarify whether the human and rodent bladder carcinogenic arylamines are activated via either NAT1 or NAT2 to cause genotoxicity, a SOS/umu genotoxicity assay was used, with the strains S . typhimurium NM6001 (NAT1-overexpressing strain), S . typhimurium NM6002 (NAT2-overexpressing strain), and S . typhimurium NM6000 (O-AT-deficient parent strain) . Genotoxicity was measured by induction of SOS/umuC gene expression in the system, which contained both an umuC"lacZ fusion gene and NAT1 or NAT2 plasmids . 4-Aminobiphenyl, 2-acetylaminofluorene, beta-naphthylamine, o-tolidine, o-anisidine, and benzidine exhibited dose-dependent induction of the umuC gene in strain NM6001 . Although the induction of umuC by these chemicals was observed in the NM6002 strain, the induction was considerably lower than in the NM6001 strain . In the parent strain, NM6000, none of these compounds induced umuC gene expression . We also determined activation of these chemicals by recombinant human cytochrome P450 (P450 or CYP) 1A2 enzyme in three S . typhimurium tester strains . The activation of the chemicals was stronger in the NM6001 strain than that in NM6002 . The specific NAT1 inhibitor 5-iodosalicylic acid inhibited umuC gene expression induced by aromatic amines used . These results could provide evidence that the bladder carcinogenic aromatic amines are mainly activated by the NAT1 enzyme to produce DNA damage rather than NAT2 . The NAT1-overexpressing strain can be used to determine the genotoxic activation of bladder carcinogenic arylamines in the umu test and could provide a tool for predicting the carcinogenic potential of arylamines.

Mutat Res, 2004 Oct 4, 554(1-2), 365 - 74
Suppression of chemically induced and spontaneously occurring oxidative mutagenesis by three alleles of human OGG1 gene encoding 8-hydroxyguanine DNA glycosylase; Kim SR et al.; 8-Hydroxyguanine (8-OH-G) is an oxidatively damaged guanine base that causes G:C to T:A transversion mutations . To counteract the mutagenicity of 8-OH-G in DNA, humans possess the hOGG1 gene, which encodes 8-OH-G DNA glycosylase . Interestingly, genetic polymorphisms at codon 326 (hOGG1-Ser326 versus hOGG1-Cys326) and at codon 46 (hOGG1-Arg46 versus hOGG1-Gln46) exist in human populations . hOGG1-Ser326 and -Cys326 have Arg at codon 46, and hOGG1-Gln46 has Ser at codon 326 . In this study, we examined the abilities of three forms of GST-hOGG1 (hOGG1-Ser326, -Cys326 and -Gln46) to suppress chemically induced oxidative mutagenesis using Salmonella typhimurium strains YG3001 and YG3002 . These strains are the mutMST derivatives of Ames tester strains TA1535 (uvrB-) and TA1975 (uvrB+), respectively . The mutMST gene encodes a functional counterpart of the OGG1 gene . Mutations induced by 4-nitroquinoline 1-oxide were by more than 95% suppressed by the expression of any of three forms of GST-hOGG1 in strain YG3002 . Expression of GST-hOGG1 also reduced by 40 and 60%, respectively, the numbers of His+ revertants induced by methylene blue plus visible light and benzo{a}pyrene plus visible light in strain YG3001 . hOGG1-Gln46 displayed a slightly weaker ability to suppress the mutations induced by methylene blue plus visible light than did other two forms although the differences were not statistically significant . About 85 and 95% of spontaneous mutagenesis in strain YG3021 and YG3022, the mutMST mutYST double mutants of strain TA1535 and TA1975, respectively, were suppressed by the expression of any of hOGG1 alleles . hOGG1-Gln46 displayed a weaker suppression than did other two forms in strain YG3022 and the difference was statistically significant . These results suggest that three alleles of the hOGG1 gene efficiently suppress chemically induced and spontaneously occurring oxidative mutagenesis, and that hOGG1-Gln46 may have a weaker ability to suppress the mutations.

Mutat Res, 2004 Oct 4, 554(1-2), 165 - 74
Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002; Motohashi N et al.; We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO-)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes . Mn(III) tetrakis (N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis (N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO- from O2*- and *NO produced through hydrolysis . Mn(III)OBTMPyP became 10-fold more active than the non-brominated form . In the presence of authentic ONOO-, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation . Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO- and SIN-1 were measured . Mn(III)TMPyP is known to be catalytic for ONOO- decomposition in the presence of antioxidants . OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants . Further, in the SIN-1 system, the concentration of NO2- and NO3- were colorimetrically determined by Griess reaction based on the two-step diazotization . NO2- increased by addition of Mn(III) porphyrin and the ratio of NO2- to NO3- was 4-7 times higher than that (1.05) of SIN-1 alone . This result suggests that O2*- from SIN-1 acts as a reductant and *NO cogenerated is oxidized to NO2-, a primarily decomposition product of *NO . Under the pathological conditions where biological antioxidants are depleted and ONOO- and O2*- are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO- decomposition using O2*-.

Acta Biochim Pol, 2004, 51(3), 851 - 6
Antitumour activity of Salmonella typhimurium VNP20047 in B16(F10) murine melanoma model; Jazowiecka-Rakus J et al.; A tumour therapy is proposed based on attenuated Salmonella typhimurium VNP20047 expressing the Escherichia coli cytosine deaminase gene . VNP20047 was administered intravenously to B16(F10) melanoma-bearing C57BL/6 mice . VNP20047 proliferated within tumours and livers regardless of the initial inoculum dose . After 10 days the number of bacteria increased in livers up to 4.2 x 10(6) cfu/g and decreased in tumours down to 5.9 x 10(6) cfu/g . VNP20047 at 1 x 10(5) cfu/mouse, when combined with 5-fluorocytosine, inhibited tumour growth by 85% without prolonging animal survival . Histology studies revealed severe lesions in tumours and livers . These data suggest that S . typhimurium VNP20047 induced inflammatory responses, even though the strain was attenuated.

Biochem J, 2005 Jan 15, 385(Pt 2), 605 - 12
Expression, purification, characterization and structure of Pseudomonas aeruginosa arylamine N-acetyltransferase; Westwood IM et al.; The gene for NAT (arylamine N-acetyltransferase) from Pseudomonas aeruginosa (panat) has been cloned from genomic DNA, and the gene product (PANAT) expressed as an N-terminal histidine-tagged protein in Escherichia coli and purified via nickel ion affinity chromatography . The specific activities of PANAT against a broad range of substrates have been investigated and compared with those of other prokaryotic NAT enzymes . For most arylamine substrates identified, PANAT exhibits in vitro specific activities typically one order of magnitude greater than those of recombinant NAT enzymes from Mycobacterium smegmatis or Salmonella typhimurium . Among the substrates of PANAT so far identified are the anti-tubercular drug isoniazid, 5-aminosalicylate (a drug used in the treatment of inflammatory bowel disease), as well as important environmental pollutants such as 3,4-dichloroaniline and 2-aminofluorene . As well as acetylating common NAT substrates, PANAT is unique among the prokaryotic NATs so far studied in acetylating the folate precursor 4-aminobenzoic acid and the folate catabolite 4-aminobenzoylglutamate . The recombinant protein has been expressed in sufficient quantity to allow protein crystallization, and we have subsequently determined the 1.95 A structure of PANAT by X-ray crystallography.

Mutagenesis, 2004 Sep, 19(5), 365 - 77
Three new consensus QSAR models for the prediction of Ames genotoxicity; Votano JR et al.; Three QSAR methods, artificial neural net (ANN), k-nearest neighbors (kNN), and Decision Forest (DF), were applied to 3363 diverse compounds tested for their Ames genotoxicity . The ratio of mutagens to non-mutagens was 60/40 for this dataset . This group of compounds includes >300 therapeutic drugs . All models were developed using the same initial set of 148 topological indices: molecular connectivity chi indices and electrotopological state indices (atom-type, bond-type and group-type E-state), as well as binary indicators . While previous studies have found logP to be a determining factor in genotoxicity, it was not found to be important by any modeling method employed in this study . The three models yielded an average training/test concordance value of 88%, with a low percentage of false positives and false negatives . External validation testing on 400 compounds not used for QSAR model development gave an average concordance of 82% . This value increased to 92% upon removal of less reliable outcomes, as determined by a reliability criterion used within each model . The ANN model showed the best performance in predicting drug compounds, yielding 97% concordance (34/35 drugs) after the removal of less reliable predictions . The appreciable commonality found among the top 10 ranked descriptors from each model is of particular interest because of the diversity in the learning algorithms and descriptor selection techniques employed in this study . Forty percent of the most important descriptors in any one model are found in one or two other models . Fourteen of the most important descriptors relate directly to known toxicophores involved in potent genotoxic responses in Salmonella typhimurium . A comparison of the validation results with those of MULTICASE and DEREK indicated that the new models presented in this work perform substantially better than the former models in predicting genotoxicity of therapeutic drugs . Substantially higher specificity was achieved with these new models as compared with MULTICASE or DEREK with comparable sensitivities among all models.

Zhonghua Yi Xue Za Zhi, 2004 Jul 17, 84(14), 1152 - 6
{Development of oral vaccine carrying GCPII gene and its role in reducing the dosage of pentobarbital in rat: a primitive research}; Tian SQ et al.; OBJECTIVE: To develop an oral vaccine carrying glutamate carboxypeptidase II (GCP II) and to explore whether it can affect the dosage of pentobarbiturate . METHODS: Polymerase chain reaction, digestion of endonuclease and ligation, blue-white selection were used to construct an expression vector pcDNA3.1-GCP II . HEK293 cells were cultured . The vector pcDNA3.1-GCP II was transfected into the HEK293 cells by Ca(3)(PO(4))(2) coprecipitation method . The transfected HEK293 cells were cultured in HEM liquid culture prepared with G418 . Three weeks after, positive clones, HEK293-GCP II, were identified . Reverse-transcription PCR and immunofluorescence cell staining were used to testify positive cell line; Method of CaCl(2) was used to prepare oral vaccine of attenuated Salmonella typhimurium carrying GCP II (SL-GCP II) . Expression of SL-GCP II in vitro was observed by adding SL-GCP II into the primarily cultured macrophage . Fifty male SD rats were randomly divided into 2 groups of 25 rats: group A, undergoing intragastrical infusion of SL-GCP II, 600 micro l/time, in total 4 times in 4 days; and group B, as control group, undergoing intragastrical infusion of SL3261 . Fifteen days after, 5 g/L pentobarbital sodium was injected intraperitoneally with the first dosage of 1.0 ml and the response was observed in 10 minutes, then 0.1 ml was added every time . The specific dosage of pentobarbital sodium was recorded when anesthesia meeting the requirement of operation was reached . Phenobarbital sodium of this dosage was used to anesthetize the rats to observe the response of the rats . Immunofluorescence method was used to detect the titer of antibody in rat circulation with HEK293 GCP II cells as target cells . RESULTS: An expression vector containing GCP II, pCMV-GCP II, pCDNA3.1-GCP II was constructed . The cell line, HEK 293-GCP II was established . In vitro experiment proved that primarily cultured macrophage phagocytized SL-GCP II and effectively expressed GCP II gene . After infusion of the oral vaccine 22 of the 25 SD rats of the group A produce GCP II antibodies . The dosage of pentobarbiturate used in experimental group was 36.9 mg/kg +/- 1.6 mg/kg; significantly lower than that in the control group (40.8 mg/kg +/- 1.4 mg/kg, P = 0.00) . CONCLUSION: An oral vaccine carrying GCP II gene has been developed that activates the immune response of rat to produce GCP II antibodies and lower the dosage of pentobarbiturate needed.

Exp Toxicol Pathol, 2004 Jul, 55(6), 451 - 4
A modified CULTEX system for the direct exposure of bacteria to inhalable substances; Aufderheide M et al.; Increasing attention is being paid to the impact on human health of inhaled gaseous compounds and complex mixtures such as cigarette smoke . The evaluation of the genotoxicity of such materials is mostly based on experiments with model substances or mixtures and condensates in the standard Ames assay . Due to the methodological difficulties of testing air contaminants in their natural gaseous or aerosolised state, there are no generally accepted concepts and techniques for effective exposure of bacteria under such conditions . Therefore, we established a novel experimental approach using an exposure device based on the cell exposure system CULTEX . This allows us to investigate chemically and physically unchanged atmospheres like mainstream cigarette smoke by exposing bacteria of Salmonella typhimurium strains directly on the surface of culture media . The CULTEX exposure device can be connected to gas or aerosol generating systems . The introduction of this exposure device in the field of inhalation genotoxicology offers new test strategies for the in vitro evaluation of a wide range of inhalable substances in both laboratory and ambient situations . A patent was applied for this technical solution.

Biophys J, 2004 Dec, 87(6), 3703 - 15 Epub 2004 Dec.
Nucleotide-dependent conformational changes in HisP: molecular dynamics simulations of an ABC transporter nucleotide-binding domain; Campbell JD et al.; ATP-binding cassette (ABC) transporters mediate the movement of molecules across cell membranes in both prokaryotes and eukaryotes . In ABC transporters, solute translocation occurs after ATP is either bound or hydrolyzed at the intracellular nucleotide-binding domains (NBDs) . Molecular dynamics (MD) simulations have been employed to study the interactions of nucleotide with NBD . The results of extended (approximately 20 ns) MD simulations of HisP (total simulation time approximately 80 ns), the NBD of the histidine transporter HisQMP2J from Salmonella typhimurium, are presented . Analysis of the MD trajectories reveals conformational changes within HisP that are dependent on the presence of ATP in the binding pocket of the protein, and are sensitive to the presence/absence of Mg ions bound to the ATP . These changes are predominantly confined to the alpha-helical subdomain of HisP . Specifically there is a rotation of three alpha-helices within the subdomain, and a movement of the signature sequence toward the bound nucleotide . In addition, there is considerable conformational flexibility in a conserved glutamine-containing loop, which is situated at the interface between the alpha-helical subdomain and the F1-like subdomain . These results support the mechanism for ATP-induced conformational transitions derived from the crystal structures of other NBDs.

P R Health Sci J, 2004 Jun, 23(2), 95 - 101
Initial interaction of the P22 phage with the Salmonella typhimurium surface; Venza Colon CJ et al.; OBJECTIVES: The goals of these studies were to characterize the interaction of the P22 phage particle with the Salmonella cell surface and to determine the phage elements involved in this interaction by mutational analysis . BACKGROUND: The phage P22 has been characterized extensively . The gene and protein for the phage P22 tailspike, which is the phage adsorption organelle, have been intensively studied . The kinetics of the interaction of the tailspike protein with the cell surface has been studied in detail, surprisingly no mutational analysis has ever been reported that has defined these components and their interaction between themselves and the cell surface . The main and perhaps only component needed for this cell surface interaction is the tailspike protein . METHODS: Adsorption to the cell surface has been measured in the wild type phage and in mutant derivatives, isolated in this study . Phage mutants have been isolated after hydroxylamine mutagenesis . RESULTS: The adsorption of P22 to the cell surface is a temperature-independent event . Forty putative phage adsorption mutants have been isolated . A sample of them have been further analyzed . These divide the adsorption process into at least two stages . One stage contains mutants that absorb with essential wild type phage kinetics to the cell surface while the other stage with delayed adsorption kinetics . CONCLUSIONS: The interaction of the phage P22 with the Salmonella cell surface has been shown to be a complicated one which is temperature-independent and multi-stage . Mutants isolated in this study may help dissect this process even further.

Eur J Immunol, 2004 Nov, 34(11), 3246 - 56
Salmonella typhimurium infection halts development of type 1 diabetes in NOD mice; Zaccone P et al.; Infectious disease has been proposed as an environmental modifier of autoimmunity in both human populations and the NOD mouse . We found that infection of NOD mice with attenuated, but not killed, Salmonella typhimurium can reduce the incidence of type 1 diabetes (T1D), even if infection occurs after the development of a peri-islet pancreatic infiltrate . Functional diabetogenic effector T cells are still present, as demonstrated by the initiation of diabetes in NOD-scid recipients of transferred splenocytes . High levels of IFN-gamma are secreted by splenocytes of infected mice, but there is no evidence of involvement of IL-10 in the protective effect of the infection . Finally, prolonged changes in cell subsets are observed in infected mice involving invariant Valpha14Jalpha281 NuKappaTau and dendritic cells . These data reinforce the idea that prevention of T1D in the NOD mouse cannot be reduced to the simple Th1/Th2 paradigm and that different infections may involve different protective mechanisms.

Microbes Infect, 2004 Jul, 6(9), 813 - 20
Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes; Sanchez-Campillo M et al.; Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs . These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9 . The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium . We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS . Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides . This results in inhibition of intracellular growth of S . typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN) . Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS . These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.

Clin Microbiol Infect, 2004 Oct, 10(10), 904 - 10
Prolonged restaurant-associated outbreak of multidrug-resistant Salmonella Typhimurium among patients from several European countries; Ethelberg S et al.; This report concerns a prolonged restaurant-associated outbreak of infection caused by a multidrug-resistant (ASSuT) strain of Salmonella Typhimurium, phage-type U302, which took place during July and August 2003 and affected people from Denmark and neighbouring countries who had attended a specific restaurant . The outbreak comprised 67 laboratory-verified cases and ten probable cases; however, the actual number of patients was estimated to be more than 390 . The outbreak strain was isolated from a buffet which was probably contaminated by an assistant chef who was found to excrete the epidemic strain . An attack rate of 7.3% was estimated and long incubation periods were observed, including one extreme instance of 27 days . This outbreak underscores the importance of conscientious personal hygiene, including frequent washing of hands, for professionals handling food.

Int J Toxicol, 2004 Jul-Aug, 23(4), 249 - 58
Perfluoro-n-butyl iodide: acute toxicity, subchronic toxicity and genotoxicity evaluations; Dodd D et al.; Perfluoro-n-butyl iodide (PFBI) is a promising alternative to chlorofluorocarbon solvents used in aircraft ground maintenance operations and other military and commercial operations, because it cleans well, has zero ozone depletion potential, and has extremely low global warming properties . Toxicity tests were performed with PFBI to determine and evaluate its health hazard . Using standard testing guidelines (e.g., Organization for Economic Cooperation and Development {OECD}), tests included acute (4-h) and 4-week (6 h/day, 5 days/week) inhalation (nose-only) toxicity studies in rats, acute (10-min) inhalation cardiac sensitization study in dogs, in vitro chromosomal aberrations experiments in human lymphocytes, and in vitro mutagenic experiments in Salmonella typhimurium and Escherichia coli . There were no mortalities in rats (n = 10) exposed for 4 h to 10,000 ppm PFBI, but all rats (n = 10) died within 2 h when exposed to 20,000 ppm PFBI . The 4-h LC50 (95% confidence limits) was 14,000 ppm (13,000 ppm to 16,000 ppm) . Signs (nasal discharge and labored breathing) observed in the rats exposed to 10,000 ppm returned to normal within 48 h . PFBI has the potential to cause cardiac sensitization in epinephrine-challenged dogs at 6200 ppm . A concentration of 3900 ppm was a no-observed-adverse-effect level (NOAEL) in the cardiac sensitization study . In the 4-week inhalation study (5 rats/sex/group), respiratory mucosal hypertrophy/hyperplasia was observed in rats of the 10,000-ppm group . A NOAEL of 1000 ppm was selected for the 4-week study on the basis that the mild increase in T4 observed at 1000 ppm was considered adaptive, not adverse, because of the absence of frank effects in the thyroid . In the in vitro studies, PFBI showed no evidence of either mutagenic or clastogenic activity . The toxicity profile of PFBI was compared to trifluoroiodomethane . In conclusion, the results of these studies indicate a low order of general toxicity and an absence of genotoxicity following PFBI exposure.

Vaccine, 2004 Sep 28, 22(29-30), 4124 - 31
Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers; Mutunga M et al.; Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro . This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven . We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear . However, despite this change, htrA mutants are still attenuated in normal mice . In contrast, they were found to be lethal for gene targeted gp91phox-/- mice deficient in NADPH oxidase, as was a S . typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice . Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/- mice, with more than 60% reduction in cell numbers 2.5h after being infected . The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/- mice, whereas an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages . Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.

Acta Microbiol Immunol Hung, 2004, 51(1-2), 75 - 83
Triflupromazine: a microbicide non-antibiotic compound; Dastidar SG et al.; The antipsychotic phenothiazine triflupromazine, possessing a methyl-thio substituent at position 10 and a fluorine moiety at position 2, exhibited significant antibacterial activity against 279 strains of Gram-positive and Gram-negative bacteria . The minimum inhibitory concentration (MIC) of the drug, according to the agar dilution method, was between 2 and 50 microg/ml for Staphylococcus aureus, and 5 and 100 microg/ml for shigellae and vibrios . Triflupromazine, when injected intraperitoneally into Swiss albino mice at a concentration of 30 microg/mouse (20 g), manifested a significant protection to the mice (p<0.001) when they were challenged with 50 median lethal dose (MLD) of Salmonella typhimurium NCTC 74 . Moreover, there was a statistically significant reduction in the number of viable bacteria in organ homogenates and blood of mice treated with this phenothiazine compound.

J Med Microbiol, 2004 Oct, 53(Pt 10), 1051 - 2
A purulent pericarditis caused by Salmonella typhimurium; Can F et al.; A case of Salmonella typhimurium pericarditis is reported . The diagnosis was based on blood and pericardial effusion cultures.

FEMS Microbiol Lett, 2004 Sep 15, 238(2), 345 - 51
Novel strains of Salmonella typhimurium as potential vectors for gene delivery; Michael A et al.; DNA vaccines are known to induce long-term antigen specific cellular responses . We tested two new strains of Salmonella typhimurium, one carrying a mutation in a SPI-2 gene and the aroC-gene and another carrying mutations in the sifA- and aroC-genes, as potential DNA vaccine delivery vehicles . We compared them with the SL7207 strain and found that the new strains were more invasive, and that they were efficient mediators of gene transfer in vitro using EGFP as reporter gene . We tested the ability of the new strains to survive within the spleen, liver and mesenteric lymph nodes and evaluated their safety in C57/BL/6J mice.

J Microbiol, 2004 Mar, 42(1), 14 - 9
Genomic relationship of Salmonella enterica serovar typhimurium DT104 isolates from Korea and the United States; Kim S et al.; Salmonella enterica serovar Typhimurium DT104 (Salmonella Typhimurium DT104 or DT104) has been emerging as a common pathogen for human in Korea since 1997 . In order to compare the genomic relationship and to search for the dominant strains in Korea, we conducted pulsed-field gel electrophoresis (PFGE) and IS200 fingerprinting of 25 epidemiological unrelated isolates from human and animals from Korea and cattle from America . Two Salmonella Typhimurium DT104 isolates from human in Korea and all 8 isolates from American cattle had indistinguishable patterns from the PFGE and IS200 fingerprinting but multidrug-resistant Salmonella Typhimurium isolates, including DT104, from Korean animals had diverse genetic patterns . The data suggest that a dominant DT104 strain might have circulated between Korean and American cattle and that it had a high level of clonality.

J Immunol, 2004 Sep 15, 173(6), 4091 - 9
Low-dose Salmonella infection evades activation of flagellin-specific CD4 T cells; Srinivasan A et al.; Many pathogens can establish a lethal infection from relatively small inocula, yet the effect of infectious dose upon CD4 T cell activation is not clearly understood . This issue was examined by tracking Salmonella flagellin-specific SM1 T cells in vivo, after i.v . and oral challenge of mice with virulent Salmonella typhimurium . SM1 T cells rapidly expressed activation markers and expanded in response to high-dose infection but remained completely unresponsive in mice challenged with low doses of Salmonella . SM1 T cells, in these mice, remained unresponsive, despite massive bacterial replication in vivo . Naive SM1 T cells in low-dose Salmonella-infected mice were activated rapidly after the injection of flagellin peptide, demonstrating that these T cells were fully capable of responding, ruling out the possibility of a bacterial-induced suppressive environment . The inability of flagellin-specific SM1 T cells to respond to low-dose infection was not due to Ag down-regulation, because flagellin expression was detected using a functional assay . Together, these data suggest that low-dose Salmonella infection can evade flagellin-specific CD4 T cell activation in vivo .

Biosystems, 2004 Aug-Oct, 76(1-3), 249 - 60
Coding theory based models for protein translation initiation in prokaryotic organisms; May EE et al.; Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism . The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook) . We tested the E . coli based coding models on {Formula: see text} untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E . coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50 . The model identified regions on the {Formula: see text} untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most . These regions correspond to the Shine-Dalgarno domain and the non-random domain . Applying the EC coding-based models to B . subtilis, and S . aureus Mu50 yielded results similar to those for E . coli K-12 . Contrary to our expectations, the behavior of S . typhimurium LT2, the more taxonomically related to E . coli, resembled that of the non-translated sequence group.

Peptides, 2004 Aug, 25(8), 1235 - 42
A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean; Wang SY et al.; A non-specific lipid transfer peptide (nsLTP) with antimicrobial activity was isolated from the mung bean (Phaseolus mungo) seeds . The procedure entailed aqueous extraction, ion exchange chromatography on CM-Sephadex and high performance liquid chromatography (HPLC) on POROS-HS-20 . The peptide exhibited a molecular mass of 9.03 kDa in mass spectrometry . It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium . The lipid binding of this peptide was very similar to that of a previously described lipid transfer protein extracted from wheat seeds and maize seeds, indicating that it possessed lipid transfer activity . The present findings add to the scarcity of the literature on leguminous nsLTPs.

Parasitol Res, 2004 Oct, 94(3), 233 - 9 Epub 2004 Sep 01.
Transferrin receptor induction in Toxoplasma gondii-infected HFF is associated with increased iron-responsive protein 1 activity and is mediated by secreted factors; Gail M et al.; Infection with the apicomplexan parasite Toxoplasma gondii results in a significant alteration of the host-cell transcriptional profile . We have previously shown that the transferrin receptor (TfR) is specifically up-regulated in T . gondii-infected human fibroblasts but not in host cells infected with the bacterial pathogens Salmonella Typhimurium and Chlamydia trachomatis . In this report, we describe the prerequisites and physiological conditions that are associated with this pathogen-specific gene induction . Band-shift assays revealed that T . gondii infection resulted in increased activity in the iron response protein IRP1, which, in this state, stabilizes TfR mRNA from degradation . Although T . gondii depends on host-cell iron as demonstrated by sensitivity to deferoxamine, a parasite-induced iron starvation is not responsible for TfR up-regulation . The increased iron availability due to treatment with holotransferrin and FeNTA did not prevent TfR induction nor was the transferrin-independent iron-transporter NRAMP2 up-regulated in infected host cells . In addition, inhibition of parasite replication by drug treatment did not prevent TfR up-regulation . Instead, TfR induction was sensitive to cycloheximide and could be induced by treatment with conditioned media from infected human fibroblasts . Together our findings suggest that the T . gondii-specific TfR up-regulation is not due to a direct interaction of parasitic factors with the iron-uptake machinery of the host cell but is instead mediated indirectly as a result of secreted host cell- or parasite-derived factors.

J Mater Sci Mater Med, 1998 Mar 1, 9(3), 141 - 6
Detection of mutagenic potential of some glass-ionomer cements through Ames testing; Stea S et al.; The mutagenic potential of three commercially available glass-ionomer cements used in dentistry was examined . The cement components were mixed according to the manufacturers indications and set for two defined times: 1 h or, alternatively, 1 wk . Cements B and C set spontaneously; in the case of cement A, the manufacturer suggests the use of a lamp to trigger also a photopolymerization . Photopolymerization, however, was not used . Ames tests were performed on the dimethyl sulphoxide extracts of cements by using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538 and TA 102 . Cement A showed mutagenicity only against TA 1537 strain, either in the presence or absence of metabolic activation with microsomial fraction S9 . The other two cements showed no mutagenic potential . We conclude that glass-ionomer cements are, on the whole, safe materials from the viewpoint of genotoxicity, and hypothesize that the mutagenicity observed in cement A could depend on its polymerization performed without light activation .

Bioinformatics . 2004 Sep 3; {Epub ahead of print}
Detecting overlapping coding sequences with pairwise alignments; Firth AE et al.; MOTIVATION: Overlapping gene coding sequences (CDSs) are particularly common in viruses but also occur in more complex genomes . Detecting such genes with conventional gene-finding algorithms can be difficult for several reasons . If an overlapping CDS is on the same read-strand as a known CDS, then there may not be a distinct promoter or mRNA . Furthermore, the constraints imposed by double-coding can result in atypical codon biases . However these same constraints lead to particular mutation patterns that may be detectable in sequence alignments . RESULTS: In this paper we investigate several statistics for detecting double-coding sequences with pairwise alignments - including a new maximum likelihood method . We also develop a model for double-coding sequence evolution . Using simulated sequences generated with the model, we characterize the distribution of each statistic as a function of sequence composition, length, divergence time and double-coding frame . Using these results, we develop several algorithms for detecting overlapping CDSs . The algorithms are tested on known overlapping CDSs and other overlapping open reading frames (ORFs) in the hepatitis B (HBV), Escherichia coli and Salmonella typhimurium genomes . The algorithms should prove useful for detecting novel overlapping genes - especially short coding ORFs in viruses . AVAILABILITY: Programmes may be obtained from the authors . SUPPLEMENTARY INFORMATION: http://biochem.otago.ac.nz/double.html.

Zh Mikrobiol Epidemiol Immunobiol, 2004 May-Jun, (3), 34 - 9
{Immunostimulating activity of polycomponent vaccine "Immunovac-VP-4" and grippol after their combined administration}; Kurbatova EA et al.; The experimental study of the immunostimulating activity of therapeutic bacterial polycomponent vaccine VP-4 and prophylactic vaccine grippol, introduced both separately and in combination, on mice infected with Salmonella typhimurium, used as a model . Both preparations were found to produce an immunomodulating effect . The combined subcutaneous injection of VP-4 and grippol did not decrease their immunostimulating activity, but their separate administration at an interval of 14 days resulted in essential decrease in the protective activity of each of these two preparations . As shown on the model of Klebsiella infection in mice, challenged 4 weeks after immunization, VP-4 ensured the survival of 78.6% of mice, while after the injection of grippol their survival rate was not different from that of the group of intact animals . The evaluation of the immunostimulating activity of these preparations under the conditions of the prophylaxis of influenza and acute respiratory infections in organized groups of children revealed that the use of VP-4 alone or grippol in combination with VP-4 considerably decreased the number of secondary bacterial complications in children.

J Exp Med, 2004 Sep 6, 200(5), 559 - 68 Epub 2004 Aug 30.
The same IkappaBalpha mutation in two related individuals leads to completely different clinical syndromes; Janssen R et al.; Both innate and adaptive immune responses are dependent on activation of nuclear factor kappaB (NF-kappaB), induced upon binding of pathogen-associated molecular patterns to Toll-like receptors (TLRs) . In murine models, defects in NF-kappaB pathway are often lethal and viable knockout mice have severe immune defects . Similarly, defects in the human NF-kappaB pathway described to date lead to severe clinical disease . Here, we describe a patient with a hyper immunoglobulin M-like immunodeficiency syndrome and ectodermal dysplasia . Monocytes did not produce interleukin 12p40 upon stimulation with various TLR stimuli and nuclear translocation of NF-kappaB was impaired . T cell receptor-mediated proliferation was also impaired . A heterozygous mutation was found at serine 32 in IkappaBalpha . Interestingly, his father has the same mutation but displays complex mosaicism . He does not display features of ectodermal dysplasia and did not suffer from serious infections with the exception of a relapsing Salmonella typhimurium infection . His monocyte function was impaired, whereas T cell function was relatively normal . Consistent with this, his T cells almost exclusively displayed the wild-type allele, whereas both alleles were present in his monocytes . We propose that the T and B cell compartment of the mosaic father arose as a result of selection of wild-type cells and that this underlies the widely different clinical phenotype.

Arch Latinoam Nutr, 2004 Mar, 54(1), 100 - 11
{Oregano: properties, composition and biological activity}; Arcila-Lozano CC et al.; The oregano spice includes various plant species . The most common are the genus Origanum, native of Europe, and the Lippia, native of Mexico . Among the species of Origanum . their most important components are the limonene, gamma-cariofilene, rho-cymenene, canfor, linalol, alpha-pinene, carvacrol and thymol . In the genus Lippia, the same compounds can be found . The oregano composition depends on the specie, climate, altitude, time of recollection and the stage of growth . Some of the properties of this plant's extracts are being currently studied due to the growing interest for substituting synthetic additives commonly found in foods . Oregano has a good antioxidant capacity and also presents antimicrobial activity against pathogenic microorganisms like Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, among others . These are all characteristics of interest for the food industry because they may enhance the safety and stability of foods . There are also some reports regarding the antimutagenic and anticarcinogenic effect of oregano; representing an alternative for the potential treatment and/or prevention of certain chronic ailments, like cancer.

J Food Prot, 2004 Aug, 67(8), 1630 - 3
Effectiveness of a laboratory-scale vertical tower static chamber steam pasteurization unit against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria innocua on prerigor beef tissue; Retzlaff D et al.; A laboratory-scale vertical tower steam pasteurization unit was evaluated to determine the antimicrobial effectiveness of different exposure times (0, 3, 6, 12, and 15 s) and steam chamber temperatures (82.2, 87.8, 93.3, and 98.9 degrees C) against pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria innocua) inoculated onto prerigor beef tissue . Samples were collected and microbiologically analyzed immediately before and after steam treatment to quantify the effectiveness of each time-temperature combination . The 0-s exposure at all chamber temperatures (cold water spray only, no steam treatment) was the experimental control and provided < or = 0.3 log CFU/cm2 reductions . Chamber temperatures of 82.2 and 87.8 degrees C were ineffective (P > 0.05) at all exposure times . At 93.3 degrees C, significant reductions (> 1.0 log CFU/cm2) were observed at exposure times of > or = 6 s, with 15 s providing approximately 1 log cycle greater reductions than 12 s of exposure . The 98.9 degrees C treatment was consistently the most effective, with exposure times of > or = 9 s resulting in >3.5 log CFU/cm2 reductions for all pathogens.

J Ocul Pharmacol Ther, 2004 Aug, 20(4), 345 - 52
Efficacy of low concentrations of ketorolac tromethamine in animal models of ocular inflammation; Waterbury LD et al.; PURPOSE: To determine if topical ophthalmic application of ketorolac tromethamine concentrations below 0.5% can block the biochemical and physiological processes associated with chemically induced ocular inflammation in rabbits . METHODS: Ocular inflammation was induced in rabbits by intravenous (i.v.) injection of endotoxin (2.5 microg/kg) isolated from Salmonella typhimurium, or by a topical application of arachidonic acid (1.0%) . The effect of ketorolac (at concentrations ranging from 0.001%-0.5%) on ocular inflammation was determined by measuring changes in the blood-aqueous barrier, using fluorophotometry (dextran-isothiocyanate-fluorescein; FITC-dextran 2%) and by measuring changes in aqueous humor protein concentrations . Changes in aqueous humor prostaglandin E(2) (PGE(2)) concentrations were also measured . RESULTS: Ketorolac 0.01%-0.5% produced substantial decreases in endotoxin-induced fluorescein leakage into the aqueous humor . The decrease produced by ketorolac 0.1% was comparable to that produced by ketorolac 0.5% . Ketorolac 0.1%-0.5% produced substantial decreases in endotoxin-induced increases in prostaglandin concentrations in the aqueous humor, and in arachidonic acid-induced protein leakage into the aqueous humor . CONCLUSIONS: Topical application of ketorolac concentrations as low as 0.01%-0.1% significantly reduce chemically induced ocular inflammation in rabbits.

Water Sci Technol, 2004, 50(1), 211 - 7
Development and evaluation of methods to detect coliphages in large volumes of water; Sobsey MD et al.; New and improved methods have been developed to detect somatic and male-specific coliphages in large volumes of water by single agar layer (SAL), enrichment and membrane filter methods . Somatic coliphages were detected efficiently on E . coli hosts C and CN13, male-specific coliphages were detected more efficiently on E . coli Famp than on Salmonella typhimurium WG49 and both types of coliphages were detected simultaneously on E . coli C3000 . For water volumes of up to 100 ml, the SAL method was efficient and reliable . For water volumes of <1 L and as many as 10 multiple 1 L volumes, the enrichment method was efficient in detecting very low numbers of coliphages . Membrane filter methods, in which coliphages were adsorbed to and eluted from filters, also were relatively efficient, but they were less efficient than SAL and enrichment methods and were considered to be more cumbersome . For filter adsorption-elution methods, coliphage recoveries were most efficient for cellulose ester filters, less efficient for electropositive 1 MDS filters and least efficient for a direct membrane filter method . Overall, the enrichment method was preferred because of its ability to easily and rapidly detect low levels of coliphages in large sample volumes by either presence-absence or most probable number quantification.

Am J Chin Med, 2004, 32(2), 281 - 90
Antimicrobial activity and cytotoxicity of the essential oil of Curcuma zedoaria; Lai EY et al.; The chemical compositions of the essential oil of Curcuma zedoaria (Berg.) Rosc . were analyzed by gas chromatography-mass spectrometry (GC-MS) and showed a high content of epicurzerenone and curdione representing 46.6% and 13.7% of the total oil, respectively . The essential oil was evaluated for potential antimicrobial activity against Staphylococcus aureus, Escherichia coli, Pseudomonasa aeruginosa, Vibrio parahaemolyticus, Salmonella typhimurium and Bacillus cereus . V . parahaemolyticus was sensitive to the presence of the essential oil, while the most resistant strain appeared to be E . coli . Based on 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay, nitroblue tetrazolium (NBT) reduction and cell morphology, the essential oil of C . zedoaria could inhibit the proliferation of human promyelocytic leukemia HL-60 cells . These results suggest that the essential oil has the antimicrobial activity against some of Gram- positive and negative pathogenic microorganisms and the components of the extract lead to the apoptosis of human cancer cell line.

J Surg Res, 2004 Sep, 121(1), 20 - 4
Preconditioning up-regulates the soluble TNF receptor I response to endotoxin; Wang M et al.; BACKGROUND: Sepsis and endotoxemia frequently complicate the care of surgical patients . Basic and clinical investigations have correlated tumor necrosis factor alpha (TNF) levels with myocardial suppression and lethality after sepsis . Soluble TNF receptor 1 (sTNFRI) is an endogenous mechanism of clearing serum TNF . Elucidating mechanisms of endogenous adaptation may allow the development of novel therapeutic strategies . Endotoxin tolerance (LPS-preconditioning) is associated with a down-regulation of proinflammatory monokine production; thus, similar down-regulation of sTNFRI may be expected . However, it may be equally plausible to hypothesize that the processes which lead to enhanced shedding of these receptors are up-regulated during tolerance . MATERIALS AND METHODS: To study this, sublethal LPS was administered to male rats (Salmonella typhimurium, 500 microg/kg IP in 1 ml bacteriostatic normal saline IP) or an equivalent volume of bacteriostatic normal saline IP (sham) 24 h prior to subsequent LPS challenge . Rats were sacrificed at 0, 1, 2, 4, 6, and 24 h following LPS and serum TNF and TNFRI were measured by ELISAs . RESULTS: LPS induced a significant increase in sTNFRI at 1, 2, 4, and 6 h following LPS . sTNFRI levels returned to baseline by 24 h following LPS treatment . LPS induced a parallel increase in TNF . LPS pretreatment (preconditioning) resulted in a significant increase in TNFRI and a significant decrease in TNF . CONCLUSION: This study constitutes the initial demonstration that tolerance mechanisms: (1) up-regulate sTNFRI, which binds and clears TNF; and (2) reverses the TNF-to-sTNFRI ratio . Safe pharmacologic methods of up-regulating endogenous TNF-clearance mechanisms may ultimately have therapeutic value.

BMC Bioinformatics . 2004 Aug 16;5(1):112.
IdentiCS--identification of coding sequence and in silico reconstruction of the metabolic network directly from unannotated low-coverage bacterial genome sequence; Sun J et al.; BACKGROUND: A necessary step for a genome level analysis of the cellular metabolism is the in silico reconstruction of the metabolic network from genome sequences . The available methods are mainly based on the annotation of genome sequences including two successive steps, the prediction of coding sequences (CDS) and their function assignment . The annotation process takes time . The available methods often encounter difficulties when dealing with unfinished error-containing genomic sequence . RESULTS: In this work a fast method is proposed to use unannotated genome sequence for predicting CDSs and for an in silico reconstruction of metabolic networks . Instead of using predicted genes or CDSs to query public databases, entries from public DNA or protein databases are used as queries to search a local database of the unannotated genome sequence to predict CDSs . Functions are assigned to the predicted CDSs simultaneously . The well-annotated genome of Salmonella typhimurium LT2 is used as an example to demonstrate the applicability of the method . 97.7% of the CDSs in the original annotation are correctly identified . The use of SWISS-PROT-TrEMBL databases resulted in an identification of 98.9% of CDSs that have EC-numbers in the published annotation . Furthermore, two versions of sequences of the bacterium Klebsiella pneumoniae with different genome coverage (3.9 and 7.9 fold, respectively) are examined . The results suggest that a 3.9-fold coverage of the bacterial genome could be sufficiently used for the in silico reconstruction of the metabolic network . Compared to other gene finding methods such as CRITICA our method is more suitable for exploiting sequences of low genome coverage . Based on the new method, a program called IdentiCS (Identification of Coding Sequences from Unfinished Genome Sequences) is delivered that combines the identification of CDSs with the reconstruction, comparison and visualization of metabolic networks (free to download at CONCLUSIONS: The reversed querying process and the program IdentiCS allow a fast and adequate prediction protein coding sequences and reconstruction of the potential metabolic network from low coverage genome sequences of bacteria . The new method can accelerate the use of genomic data for studying cellular metabolism.

Biochemistry, 2004 Aug 24, 43(33), 10619 - 27
Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2; Fresquet V et al.; Cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium (CbiA) is the first glutamine amidotransferase in the anaerobic biosynthetic pathway of vitamin B(12) and catalyzes the ATP-dependent synthesis of cobyrinic acid a,c-diamide from cobyrinic acid using either glutamine or ammonia as the nitrogen source . The cbiA gene was cloned, the overexpressed protein was purified to homogeneity, and the kinetic parameters were determined . CbiA is a monomer with K(m) values of 0.74, 2.7, 53, and 26 200 microM for cobyrinic acid, ATP, glutamine, and ammonia, respectively . Analysis of the glutaminase partial reaction demonstrated that the hydrolysis of glutamine and the synthesis of the cobyrinic acid a,c-diamide product are uncoupled . The time course for the synthesis of the diamide product and positional isotope exchange experiments demonstrate that CbiA catalyzes the sequential amidation of the c- and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate . These results support a model for the catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate . Several conserved residues in the synthetase active site were mutated to address the molecular basis of the amidation order; however, no changes in the order of amidation were obtained . The mutants D45N, D48N, and E90Q have a dramatic effect on the catalytic activity, whereas no effect was found for the mutant D97N . The substitutions by alanine of L47 and Y46 residues specifically decrease the affinity of the enzyme for the c-monoamide intermediate.

Vaccine, 2004 Sep 3, 22(25-26), 3243 - 55
Expression of heterologous antigens in Salmonella Typhimurium vaccine vectors using the in vivo-inducible, SPI-2 promoter, ssaG; McKelvie ND et al.; DNA derived from regions upstream of the Salmonella enterica serovar Typhimurium ssaG gene were used to drive expression of different reporter genes in putative Salmonella vaccine strains . Expression from ssaG was shown to be significantly upregulated once Salmonella had entered murine or human macrophages, and levels of expression were dependent on the length of the ssaG 5' sequence incorporated . S . Typhimurium derivatives harbouring the Escherichia coli heat labile toxin B subunit (LT-B) fused to various lengths of the ssaG promoter region were also constructed as single copy chromosomal integrations . Expression of LT-B by these Salmonella derivatives was detected at significant levels after intra-macrophage survival and mice immunised with these derivatives mounted marked anti-LT-B humoral antibody responses.

Anal Chem, 2004 Aug 15, 76(16), 4806 - 10
Quantum dot biolabeling coupled with immunomagnetic separation for detection of Escherichia coli O157:H7; Su XL et al.; A sensitive, specific, and rapid method for the detection of E . coli O157:H7 was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with immunomagnetic separation . Magnetic beads coated with anti-E . coli O157 antibodies were employed to selectively capture the target bacteria, and biotin-conjugated anti-E . coli antibodies were added to form sandwich immuno complexes . After magnetic separation, the immuno complexes were labeled with QDs via biotin-streptavidin conjugation . This was followed by a fluorescence measurement using a laptop-controlled portable device, which consisted of a blue LED and a CCD-array spectrometer . The peak intensity of the fluorescence emission was proportional to the initial cell concentration of E . coliO157:H7 in the range of 10(3)-10(7) CFU/mL with a detection limit at least 100 times lower than that of the FITC-based method . The total detection time was less than 2 h . Neither E . coli K12 nor Salmonella typhimurium interfered with the detection of E . coli O157:H7.

Ai Zheng, 2004 Aug, 23(8), 869 - 73
{Chemopreventive effect of resveratrol to cancer}; Fu ZD et al.; BACKGROUND & OBJECTIVE: Carcinogenesis is a complex process and at least 3 stages, including initiation, promotion, and progression, have been proposed in the process of carcinogenesis . Resveratrol has attracted considerable attention due to its low toxicity and unique chemical structure . This study was designed to test chemopreventive effect of resveratrol to cancer using various animal models . METHODS:Ames assay and micronucleus formation assay were used to test the antimutagenic activities of resveratrol . Croton oil-induced enhancement of ornithine decarboxylase (ODC) activities of dorsal epidermis cells in mouse and mouse ear edema model were used to investigate the anti-promotion effect of resveratrol . In addition,7,12-dimenthylbenz{a}anthracene (DMBA)/croton oil-induced mouse skin tumor model was used to evaluate chemopreventive effect of resveratrol to cancer in vivo . RESULTS: In Ames test,100 microg/plate of resveratrol exhibited 42.2% of inhibition on the reversion of Salmonella typhimurium TA100 induced by methylmethansulfonate, and 200 microg/plate of resveratrol exhibited 91.8% of inhibition on the reversion induced by benzopyrene.Pretreatment of resveratrol prevented cyclophosphamide (CTX)-induced micronucleus formation of polychromatic erythrocytes of mice bone marrow in dose-dependent manner . Mice treated with 30 mg/kg of resveratrol for 6 days before croton oil exposure have palliative ear edema . Treatment of 180 mg/kg resveratrol for 3 days caused 69.3% decrease of ODC activities in croton oil-induced dorsal epidermis . It was shown that resveratrol could inhibit DMBA/croton oil-induced mouse skin papilloma, which includes prolonging the latent period of tumor occurrence, decreasing the incidence of papilloma, and reducing tumor number per mouse in dose-dependent manner . CONCLUSION: Resveratrol has the ability of anti-mutation and anti-promotion of cancer and merit further studies as a potential cancer chemopreventive agent.

Biochemistry, 2004 Aug 17, 43(32), 10343 - 52
A model for the Bacillus subtilis formylglycinamide ribonucleotide amidotransferase multiprotein complex; Anand R et al.; Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway . PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity . smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood . We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively . PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices . In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer . The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core . We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex . The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ . The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer . Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site . The homology model is consistent with biochemical studies on the reconstituted complex.

Biochemistry, 2004 Aug 17, 43(32), 10328 - 42
Domain organization of Salmonella typhimurium formylglycinamide ribonucleotide amidotransferase revealed by X-ray crystallography; Anand R et al.; Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent conversion of formylglycinamide ribonucleotide (FGAR) and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway . In eukaryotes and Gram-negative bacteria, FGAR-AT is encoded by the purL gene as a multidomain protein with a molecular mass of about 140 kDa . In Gram-positive bacteria and archaebacteria FGAR-AT is a complex of three proteins: PurS, PurL, and PurQ . We have determined the structure of FGAR-AT (PurL) from Salmonella typhimurium at 1.9 A resolution using X-ray crystallography . PurL is the last remaining enzyme in the purine biosynthetic pathway to have its structure determined . The structure reveals four domains: an N-terminal domain structurally homologous to a PurS dimer, a linker region, an FGAM synthetase domain homologous to an aminoimidazole ribonucleotide synthetase (PurM) dimer, and a triad glutaminase domain . The domains are intricately linked by interdomain interactions and peptide connectors . The fold common to PurM and the central region of PurL represents a superfamily for which HypE, SelD, and ThiL are predicted to be members . A structural ADP molecule was found bound to a site related to the putative active site by pseudo-2-fold symmetry and two sulfate ions were found at the putative active site . These observations and the structural similarities between PurM and StPurL were used to model the substrates FGAR and ATP in the StPurL active site . A glutamylthioester intermediate was found in the glutaminase domain at Cys1135 . The N-terminal (PurS-like) domain is hypothesized to form the putative channel through which ammonia passes from the glutaminase domain to the FGAM synthetase domain.

World J Gastroenterol, 2004 Sep 1, 10(17), 2498 - 502
Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity; Bai Y et al.; AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S . typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity . METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB) . Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant . According to the method described by Meacock, stability of the recombinant was evaluated . Semi-lethal capacity test was used to evaluate the safety of recombinant . The immunogenicity of recombinant was evaluated with animal experiments . RESULTS: The attenuated S . typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed . Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor . And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA . The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent . The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally . Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response . CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection .

Acta Crystallogr D Biol Crystallogr, 1995 Jan, 51(Pt 1), 39 - 47
Structure determination of OppA at 2.3 A resolution using multiple-wavelength anomalous dispersion methods; Glover ID; OppA is a 58.8 kDa bacterial transport protein involved in the transport of peptides across the cytoplasmic membrane of Gram-negative bacteria . It binds peptides from two to five residues in length but with little sequence specificity . OppA from Salmonella typhimurium has been cloned and expressed in E . coli and the protein cocrystallized with uranyl acetate, producing two distinct crystal forms with different uranium sites . Multiple-wavelength data collected about the uranium L(III) edge have been collected at the Daresbury Synchrotron Radiation Source (SRS) to a nominal resolution limit of 2.3 A . Maximum-likelihood phasing methods have been used in phase determination from the multiple-wavelength data giving a readily interpretable electron-density map, without any density modification . The electron-density map, calculated at 2.3 A resolution shows OppA to be a bilobal, principally beta-stranded, three-domain protein . The tri-lysine ligand molecule can be clearly seen in the peptide-binding site between the two lobes.

Acta Crystallogr D Biol Crystallogr, 1995 Mar, 51(Pt 2), 145 - 54
The three-dimensional structure of the aspartate receptor from Escherichia coli; Bowie JU; The crystal structure of the periplasmic domain of the aspartate receptor from Escherichia coli has been solved and refined to an R-factor of 0.203 at 2.3 A, resolution . The dimeric protein is largely helical, with four helices from each monomer forming a four-helix bundle . The dimer interface is constructed from four helices, two from each subunit, also packed together in a four-helix bundle arrangement . A sulfate ion occupies the aspartate-binding site . All hydrogen bonds made to aspartate are substituted by direct or water-mediated hydrogen bonds to the sulfate . Comparison of the Escherichia coli aspartate-receptor structure with that of Salmonella typhimurium {Milburn, Prive, Milligan, Scott, Yeh, Jancarik, Koshland & Kim (1991) . Science, 254, 1342-1347; Scott, Milligan, Milburn, Prive, Yeh, Koshland & Kim (1993) . J . Mol . Biol . 232, 555-573} reveals strong conservation in the structure of the monomer, but more divergence in the orientation of the subunits with respect to one another . Mutations that render the Escherichia coli receptor incapable of responding to maltose are either located in spatially conserved sites or in regions of the structures that have high temperature factors and are therefore likely to be quite flexible . The inability of the receptor from Salmonella typhimurium to respond to maltose may, therefore, be because of differences in amino acids located on the binding surface rather than structural differences.

Avian Dis, 2004 Apr-Jun, 48(2), 361 - 9
Effect of a variant infectious bursal disease virus (E/Del) on Salmonella typhimurium infection in commercial broiler chickens; Bautista DA et al.; The effect of infectious bursal disease virus (IBDV) on Salmonella typhimurium (ST) infections in broilers was investigated in terms of Salmonella shedding and persistence, pathogenicity, and isotype specific humoral immune responses . Thirty-six, 1-day-old, straight-run commercial broiler chickens that were Salmonella negative by polymerase chain reaction (PCR) and culture were divided into two groups of 18 chicks each (ST and ST-IBDV) . One group (ST-IBDV) of chicks received the E/Del strain of IBDV (10(5.0) median tissue culture infective dose {TCID50}/ml) through the ocular and cloacal routes divided into doses of 50 microl each at 2 days of age . Both groups were then inoculated with 10(8) colony-forming units (CFU)/ml nalidixic acid-resistant ST in the drinking water at 3 days of age . Environmental Salmonella counts were higher in the ST-IBDV group at 2 and 3 wk postinfection (PI) compared to the ST group . ST carriage in the cecal contents between the ST and ST-IBDV groups was not statistically different . The ST-IBDV group had a single mortality at 10 days postinfection compared to none in the ST group . The ST-IBDV group had significantly lower bursa to body weight ratios at 4 and 6 wk, as well as higher bursal lesion scores than the ST group at 2, 4, and 6 wk PI . The ST group had significant increase in serum IgG from 2 to 6 wk PI in comparison to the ST-IBDV group, which had no significant changes over time . Both IgA and IgM were significantly increased at 4 and 6 wk relative to 2-wk levels . There was an IBDV-induced failure of anti-Salmonella IgG seroconversion over time in ST-IBDV . Both groups continued to shed high levels of Salmonella up to the end of the study despite high antibody levels in the ST group and an unimpaired IgM and IgA production in the ST-IBDV group, indicating a limited influence of humoral immunity on Salmonella clearance.

J Environ Pathol Toxicol Oncol, 1999, 18(3), 191 - 201
Comparison of the phenylenediamine isomers bioactivation by the green alga Chlamydomonas reinhardtii; Vlckova V et al.; The correlation between the chemical structure of arylamines meta-, orto-, para-phenylenediamine (m-PDA, o-PDA, p-PDA), and their mutagenic activity is known . It is accepted that these promutagenic compounds are metabolized to ultimate mutagens in mammals and higher plants . In our previous work, we used the alga Chlamydomonas reinhardtii as the activating organism and the bacteria Salmonella typhimurium and yeast Saccharomyces cerevisiae as the genetic indicators for m-PDA activation . In the present work, we used the same activation system for o-PDA and p-PDA activation . Different responses of the yeast and algal wild-type strain and of the repair-deficient strains to the toxic and mutagenic effects of o-PDA and p -PDA were observed . p-PDA had the most toxic effect on both intact yeast and algal cells and in the algal cell/microbe coincubation assays . Concerning repair-deficient algal strains, the recombination-deficient strain was the most sensitive to both compounds tested, indicating that the recombination process played an important role in the DNA repair of arylamines . The rank order of the PDA isomers mutagenicity (including m-PDA) was o-PDA > m-PDA > p-PDA for revertants in intact yeast and forward mutants in algae; m-PDA > o-PDA > p-PDA in the algal cell/S . typhimurium long-term coincubation assay, the algal cell/S . cerevisiae coincubation assay, and the intact S . cerevisiae assay for gene convertants as well.

Mutat Res, 2004 Aug 8, 562(1-2), 151 - 62
Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens; Yamazaki Y et al.; We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations . Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate . Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S . typhimurium TA1538 . CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) and 2-acetylaminofluorene . Benzo{a}pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538 . These results suggest that the newly developed S . typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.

Mutat Res, 2004 Aug 8, 562(1-2), 143 - 50
Phototoxicity and DNA damage induced by the cosmetic ingredient chemical azulene in human Jurkat T-cells; Wang L et al.; Previous study showed that the cosmetic ingredient chemical azulene and its derivative gauiazulene exhibited photomutagenicity four- to five-fold higher than spontaneous mutation in Salmonella typhimurium TA102 . In this study, phototoxicity including photogenotoxicity of azulene in human Jurkat T-cells is reported . When the cell suspensions are irradiated by light (UVA plus visible light) in the presence of azulene, an azulene dose-dependent cellular DNA damage is observed . At the highest azulene concentration of 50 microM, the average DNA fragmentation is 33 +/- 10%, determined by single cell gel electrophoresis (Comet assay) . Cell viability assay using fluorescein diacetate indicates that the cells could endure the damage and remain viable . Further study revealed that the combination of light and azulene can cause single-strand cleavage on pure PhiX174 plasmid DNA in solution . Studies using scavengers reveal that singlet oxygen and free radicals are involved in causing DNA cleavage . This suggests that the photomutagenicity of azulene in S . typhimurium TA102 could be due to DNA fragmentation caused by the concurrent exposure to azulene and light.

Am J Pathol, 2004 Aug, 165(2), 373 - 81
Degranulation of paneth cells via toll-like receptor 9; Rumio C et al.; The release of antimicrobial peptides and growth factors by Paneth cells is thought to play an important role in protecting the small intestine, but the mechanisms involved have remained obscure . Immunohistochemistry and immunofluorescence showed that Paneth cells express Toll-like receptor 9 (TLR9) in the granules . Injection of mice with oligonucleotides containing CpG sequence (CpG-ODNs) led to a down-modulation of TLR9 and a striking decrease in the number of large secretory granules, consistent with degranulation . Moreover CpG-ODN treatment increased resistance to oral challenge with virulent Salmonella typhimurium . Moreover, our findings demonstrate a sentinel role for Paneth cells through TLR9.

Microbiol Immunol, 2004, 48(7), 553 - 6
Epidemiological characterization of Salmonella Typhimurium DT104 prevalent among food-producing animals in the Japanese veterinary antimicrobial resistance monitoring program (1999-2001); Esaki H et al.; In the course of nationwide investigation on epidemiological characteristics in Salmonella Typhimurium isolates from food-producing animals in Japan between 1999 and 2001, fifty-seven isolates of S . Typhimurium DT104 and 104B obtained from cattle and swine at farm level in Japan between 1999 and 2001 were classified with pulsotype and antimicrobial resistance type . Most of the isolates were resistant to five or more antimicrobials and were genotyped into four groups . The present nationwide investigation shows that at least 11 types of S . Typhimurium related to DT104 are prevalent among food-producing animals across the country.

J Food Prot, 2004 Jul, 67(7), 1389 - 93
Validation of time and temperature values as critical limits for Salmonella and background flora growth during the production of fresh ground and boneless pork products; Mann JE et al.; To provide pork processors with valuable data to validate the critical limits set for temperature during pork fabrication and grinding, a study was conducted to determine the growth of Salmonella serotypes and background flora at various temperatures . Growth of Salmonella Typhimurium and Salmonella Enteritidis and of background flora was monitored in ground pork and boneless pork chops held at various temperatures to determine growth patterns . Case-ready modified atmosphere packaged ground pork and fresh whole pork loins were obtained locally . Boneless chops and ground pork were inoculated with a cocktail mixture of streptomycin-resistant Salmonella to facilitate recovery in the presence of background flora . Samples were held at 4.4, 7.2C, and 10 degrees C and at room temperature (22.2 to 23.3 degrees C) to mimic typical processing and holding temperatures observed in pork processing environments . Salmonella counts were determined at regular intervals over 12 and 72 h for both room and refrigeration temperatures . No significant growth of Salmonella (P < 0.05) was observed in boneless pork chops held at refrigeration temperatures . However, Salmonella in boneless pork chops held at room temperature had grown significantly by 8 h . Salmonella grew at faster rates in ground pork . Significant growth was observed at 6, 24 . and 72 h when samples were held at room temperature, 10 degrees C, and 7.2 degrees C, respectively . No significant growth was observed at 4.4 degrees C . Background flora in ground pork samples increased significantly after 10 h at room temperature and after 12 h for samples held at 10 and 7.2 degrees C . Background flora in samples held at refrigeration temperatures did not increase until 72 h . Background flora in the boneless chops increased significantly after 6 h at room temperature and after 24 h when held at 10 and 4.4 degrees C . These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits to minimize Salmonella growth during production and storage of raw pork products.

J Food Prot, 2004 Jul, 67(7), 1371 - 6
Efficacy of chlorine dioxide gas as a sanitizer of lettuce leaves; Lee SY et al.; Aqueous solutions of sodium hypochlorite or hypochlorous acid are typically used to sanitize fresh fruits and vegetables . However, pathogenic organisms occasionally survive aqueous sanitization in sufficient numbers to cause disease outbreaks . Chlorine dioxide (ClO2) gas generated by a dry chemical sachet was tested against foodborne pathogens on lettuce leaves . Lettuce leaves were inoculated with cocktail of three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and treated with CLO2 gas for 30 min, 1 h, and 3 h in a model gas cabinet at room temperature (22 +/- 2 degrees C) . After treatment, surviving cells, including injured cells, were enumerated on appropriate selective agar or using the overlay agar method, respectively . Total ClO2, generated by the gas packs was 4.3, 6.7, and 8.7 mg after 30 min, 1 h, and 3 h of treatment, respectively . Inoculated lettuce leaves exposed to ClO2 gas for 30 min experienced a 3.4-log reduction in E . coli, a 4.3-log reduction in Salmonella Typhimurium, and a 5.0-log reduction in L . monocytogenes when compared with the control . After 1 h . the three pathogens were reduced in number of CFU by 4.4 . 5.3, and 5.2 log, respectively . After 3 h, the reductions were 6.9, 5.4, and 5.4 log, respectively . A similar pattern emerged when injured cells were enumerated . The ClO2, gas sachet was effective at killing pathogens on lettuce without deteriorating visual quality . Therefore, this product can be used during storage and transport of lettuce to improve its microbial safety.

J Food Prot, 2004 Jul, 67(7), 1353 - 8
Internalization of bacterial pathogens in tomatoes and their control by selected chemicals; Ibarra-Sanchez LS et al.; The effect of different washing or sanitizing agents was compared for preventing or reducing surface and internal contamination of tomatoes by Salmonella Typhimurium and Escherichia coli O157:H7 . The tomatoes were inoculated by dipping them in a bacterial suspension containing approximately 6.0 log CFU/ml of each pathogen and then rinsing them with tap water, hypochlorite solution (250 mg/liter), or lactic acid solution (2%, wt/vol) . All treatments were applied by dipping or spraying, and solutions were applied at 5, 25, 35, and 55 degrees C . With the exception of the lactic acid dip at 5 degrees C, all treatments reduced both pathogens on the surfaces of the tomatoes by at least 2.9 cycles . No significantly different results were obtained (P > 0.05) with the dipping and spraying techniques . For internalized pathogens, the mean counts for tomatoes treated with water alone or with chlorine ranged from 0.8 to 2.1 log CFU/g . In contrast, after lactic acid spray treatment, all core samples of tomatoes tested negative for Salmonella Typhimurium and, except for one sample with a low but detectable count, all samples tested negative for E . coli O157:H7 with a plate count method . When the absence of pathogens was verified by an enrichment method, Salmonella was not recovered from any samples, whereas two of four samples tested positive for E . coli O157:H7 even though the counts were negative . Few cells of internalized pathogens were able to survive in the center of the tomato during storage at room temperature (25 to 28 degrees C) . The average superficial pH of tomatoes treated with tap water, chlorine, or lactic acid was 4.9 to 5.2, 4.1 to 4.3, and 2.5, respectively (P < 0.05), whereas no differences were observed in the internal pH (3.6 to 3.7) of the tomatoes treated with different sanitizers . The general practice in the tomato industry is to wash the tomatoes in chlorinated water . However, chlorine is rapidly degraded by organic matter usually present in produce . Therefore, lactic acid sprays may be a more effective alternative for decontaminating tomato surfaces . The use of warm (55 degrees C) sprays could reduce pathogen internalization during washing.

Indian J Exp Biol, 2003 Mar, 41(3), 216 - 9
Inhibition of mutagenicity of food-derived heterocyclic amines by sulforaphane--a constituent of broccoli; Shishu et al.; Sulforaphane, a constituent of broccoli was investigated for its antimutagenic potential against different classes of cooked food mutagens (heterocyclic amines) . These include imidazoazaarenes such as 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ), 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP); pyridoindole derivatives such as 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2); and, dipyridoimidazole derivative such as 2-amino-6-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1) . Tests were carried out by Ames Salmonella/reversion assay using Salmonella typhimurium TA98 (frame shift mutation sensitive) and TA100 (base pair mutation sensitive) bacterial strains in the presence of Aroclor 1254-induced rat liver S9 . Results of these in vitro antimutagenicity studies strongly suggest that sulforaphane is a potent inhibitor of the mutagenicity induced by imidazoazaarenes such as IQ, MeIQ and MeIQx (approximately 60% inhibition) and moderately active against pyridoindole derivatives such as Trp-P-1 and Trp-P-2 (32-48% inhibition), but ineffective against dipyridoimidazole derivative (Glu-P-1) in TA 100.

Genet Mol Res, 2004 Jun 30, 3(2), 264 - 72
Standardization of conditions for the metabolic activation of N-nitrosodiethylamine in mutagenicity tests; Aiub CA et al.; Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects . This activation involves cytochrome P450s (CYP), which generates unstable metabolites that react with the DNA of cells in the immediate vicinity of metabolite formation . Although NDEA is carcinogenic, it has been considered a weak mutagen in classic genotoxicity assays . We used optimized Salmonella/mammalian microsome genotoxicity assays to assess the mutagenicity and toxicity of low concentrations of NDEA . Using a fixed concentration of NDEA (36.5 mg/ml), we varied the length of preincubation in the presence of different concentrations of an S9 metabolic activation mixture . Salmonella typhimurium strains TA97 and TA102 were resistant to NDEA-induced mutagenesis, even after a preincubation of up to 120 min and the use of different concentrations of the S9 mix . Strain TA98 was susceptible to mutagenesis by NDEA in the absence of the S9 mix and after preincubation with NDEA for 90 min . When bacteria of this strain were preincubated with NDEA for 60 min, mutagenesis was detected at an S9 mix concentration >9.55 mg/ml . NDEA also induced mutagenesis in strain TA100 after preincubation for 90 or 120 min, and this effect was dependent on the S9 concentration . E . coli strain BH990 also showed a concentration-dependent response, with only 60% of the cells surviving after a 120-min preincubation with NDEA in the presence of 19.1 mg S9 mix/ml.

Braz J Med Biol Res, 2004 Jul, 37(7), 1005 - 13 Epub 2004 Jun 22.
Effect of the Escherichia coli EMO strain on experimental infection by Salmonella enterica serovar Typhimurium in gnotobiotic mice; Lima-Filho JV et al.; An experimental infection with Salmonella enterica subsp . enterica serovar Typhimurium was evaluated in gnotobiotic mice previously exposed to a plasmid-free non-pathogenic Escherichia coli (EMO strain) . Mice were exposed to EMO (experimental) or not (control) 10 days before challenge with Salmonella Typhimurium (10(2) colony forming units (CFU)/mouse) . Survival after challenge was higher (P < 0.05) in the experimental group (16%) than in the control animals (0%) . Histopathological examination of the colon and ileum mucosa of the experimental group showed less extensive lesions such as edema, cell inflammatory infiltration and hyperemia . The epithelial cells of the mucosal surface and the production of the mucous layer were also better preserved in the experimental group . The population levels of Salmonella Typhimurium in the feces were initially 10-fold lower (P < 0.05) in the experimental groups . However, 3 days after challenge both experimental and control groups showed similar population levels ranging from 10(8) to 10(9) CFU/g of feces . The intestinal contents of total and anti-Salmonella Typhimurium sIgA were higher in the experimental groups 10 days after inoculation of E . coli EMO strain . Translocation of Salmonella Typhimurium to the spleen was 10-fold lower (P < 0.05) in the experimental group only on day 3 after infection . This was not related to an increase in the bacterial blood clearance of the animals, as shown by experimental venous challenge with E . coli B41 . In conclusion, treatment of mice with E . coli EMO strain promoted a relative protection against experimental infection with Salmonella Typhimurium . This protection was not due to the reduction of the population of pathogens in the intestine but was probably related to stimulation of the immune response.

J Ethnopharmacol, 2004 Sep, 94(1), 43 - 8
The constituents of essential oil and in vitro antimicrobial activity of Micromeria cilicica from Turkey; Duru ME et al.; The chemical composition of the essential oil of Micromeria cilicica (Labiatae) that has been used in folk medicine were analysed by GC, GC-MS, 1H NMR and 13C NMR . The totals of 34 components in hydrodistillation, 30 components in steam distillation were detected . The major component characterized in the essential oils was pulegone (66.55, 64.10%) and other main components were determined as cis-p-menthone (21.71, 25.31%), trans-p-menthone (9.59, 5.59%), nerol (0.35, 2.49%) and 3-octonol (0.81, 0.25%), respectively . Essential oils obtained by hydro and steam distillation and organic solvent extracts of the aerial parts of the plant were investigated for antimicrobial activities on several microorganisms including bacteria and yeast . Moreover, the main constituent of the oil has been tested against the same microorganisms . The extracts and pulegone exhibited a significant antibacterial and antifungal activity . The activities were increased depend on the amount of extracts and pulegone . Pulegone also showed antimicrobial activity, particularly against Candida albicans and Salmonella typhimurium . Furthermore Candida albicans is the most susceptible to pulegone giving two times the effect of nystatin.

Folia Microbiol (Praha), 2004, 49(3), 301 - 5
Synergic activity of selenium and probiotic bacterium Enterococcus faecium M-74 against selected mutagens in Salmonella assay; Belicova A et al.; Concentrated extracts of MRS (De Man-Rogosa-Sharpe) media in which probiotic bacterium Enterococcus faecium strain M-74 was grown exerted different antimutagenic activity against ofloxacin-, N-methyl, N'-nitro-N-nitrosoguanidine- and sodium 5-nitro-2-furylacrylate-induced mutagenicity in Salmonella typhimurium assay depending on the presence (+Se) or absence of disodium selenite pentahydrate (-Se) . The antimutagenicity of MRS(+Se) extract was higher than that of MRS(-Se) extract . Selenium enhanced also the antimutagenic effect of both live and killed cells of E . faecium M-74, respectively . The live bacteria decreased the mutagenicity of selected substances more than killed cells . Synergic activity of selenium with the bacterium was also manifested.

J Clin Periodontol, 2004 Aug, 31(8), 596 - 603
A model of periodontitis in the rat: effect of lipopolysaccharide on bone resorption, osteoclast activity, and local peptidergic innervation; Dumitrescu AL et al.; OBJECTIVE: To establish and characterise a rat model of periodontitis that reiterates the features of human disease . METHODS: Periodontal inflammation was induced by a single injection of 10 microg liposaccharide (LPS) (Salmonella typhimurium) in 1 microl saline into rat mandibular gingiva at the buccomesial aspect of the second molar . Animals were killed after 3, 7 and 10 days, mandibles dissected and sectioned for histological and immunocytochemical analysis . RESULTS: LPS injection resulted in a significant gingival and periodontal inflammation with inflammatory infiltrate, apical migration of the junctional epithelium, interdental bone loss, and activation of osteoclasts at the site of injection 7 and 10 days after injection . At 10 days post injection, there was a significant trend for bone loss on both sides of the mandible . Periodontal inflammation was associated with alteration in the levels of calcitonin gene-related peptide-like immunoreactivity in nerve terminals innervating the inflamed gingival papilla . CONCLUSION: Intragingival injection of LPS in the rat provides an easily induced reproducible experimental model of periodontal inflammation that reiterates features of human disease.

Chem Res Toxicol, 2004 Jul, 17(7), 972 - 82
Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase; Valadez JG et al.; O(6)-Alkylguanine DNA-alkyl transferase (AGT) has been shown to conjugate both 1,2-dibromoethane and dibromomethane, yielding AGT inactivation, DNA-AGT cross-linking, and enhanced mutagenicity . A variety of related chemicals were examined to determine if similar phenomena occur . Among the compounds examined in these systems (histidine operon reversion in Escherichia coli and Salmonella typhimurium tester strains), a strong halide order was generally observed, with increasing activities in the order I > Br >> Cl . At least one Br atom appeared to be required for human AGT-dependent mutations, and compounds with only Cl did not inhibit AGT and were not activated to genotoxins . Of a series of haloforms tested (CHX(3), X = Br or Cl), all were without effect . Among a series of alpha,omega-disubstituted dihaloalkanes (Br or I), the inactivation of AGT increased with methylene chain length (at least up to n = 5) but the most mutagenic activity (AGT-dependent) was seen with n = 1-3 . The effects with n = 1 or 2 were expected from previous results; the mutagenic effect with n = 3 and the reduction with n > 3 may represent a balance between AGT reaction, stability, and reactivity, in the absence of anchimeric assistance . A strong AGT-dependent mutation was observed for 1,3-butadiene diepoxide . We conclude that numerous bis-electrophiles show AGT-dependent activation to mutagenic conjugates . Haloforms and dichloroalkanes are therefore not an issue, but bromohaloalkanes and 1,3-butadiene diepoxide are potential problems . These observations are of relevance in considering toxicity and risks of some chemicals used in industrial applications.

J Antimicrob Chemother, 2004 Aug, 54(2), 321 - 32 Epub 2004 Jul 14.
Pet animals as reservoirs of antimicrobial-resistant bacteria; Guardabassi L et al.; Pet animal numbers have substantially increased in modern society and attention is increasingly devoted to pet welfare . Because of these changes, antimicrobial agents are frequently used in small animal veterinary practice, often including antimicrobial preparations used in human medicine, with heavy use of broad-spectrum agents such as aminopenicillins plus clavulanic acid, cephalosporins and fluoroquinolones . Several longitudinal studies conducted at veterinary hospitals have indicated that resistance to various antimicrobial agents has emerged amongst pet animal isolates of Staphylococcus intermedius, Escherichia coli and other bacteria, including species with a potential for zoonotic transmission and resistance phenotypes of clinical interest, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant Salmonella Typhimurium DT104 . Based on a review of the current literature, the role of pets in the dissemination of antimicrobial resistance has been given little attention when compared with that of food animals . A marked contrast is evident between the current policies on antimicrobial usage in food and companion animals . Apart from a few countries where limited data on antimicrobial usage and occurrence of resistance in bacteria from pet animals are provided, national surveillance programmes only focus on food animals . However, data on pet animals are clearly needed for guiding antimicrobial use policy in small animal veterinary practice as well as for assessing the risk of transmission of antimicrobial resistance to humans.

J Proteome Res, 2004 May-Jun, 3(3), 595 - 603
Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance; Coldham NG et al.; The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR) . Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs) . These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX) . Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry . Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST . A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively . A significant correlation was observed (r2 = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates . A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation . A range of proteins (n = 20) previously associated with the mar locus in E . coli were also found including the key MAR effectors AcrA, TolC and OmpF.

Int J Food Microbiol, 2004 Aug 1, 94(3), 223 - 53
Essential oils: their antibacterial properties and potential applications in foods--a review; Burt S; In vitro studies have demonstrated antibacterial activity of essential oils (EOs) against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereus and Staphylococcus aureus at levels between 0.2 and 10 microl ml(-1) . Gram-negative organisms are slightly less susceptible than gram-positive bacteria . A number of EO components has been identified as effective antibacterials, e.g . carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid, having minimum inhibitory concentrations (MICs) of 0.05-5 microl ml(-1) in vitro . A higher concentration is needed to achieve the same effect in foods . Studies with fresh meat, meat products, fish, milk, dairy products, vegetables, fruit and cooked rice have shown that the concentration needed to achieve a significant antibacterial effect is around 0.5-20 microl g(-1) in foods and about 0.1-10 microl ml(-1) in solutions for washing fruit and vegetables . EOs comprise a large number of components and it is likely that their mode of action involves several targets in the bacterial cell . The hydrophobicity of EOs enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents . Physical conditions that improve the action of EOs are low pH, low temperature and low oxygen levels . Synergism has been observed between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol . Synergy between EO components and mild preservation methods has also been observed . Some EO components are legally registered flavourings in the EU and the USA . Undesirable organoleptic effects can be limited by careful selection of EOs according to the type of food.

BMC Immunol . 2004 Jul 09;5(1):14.
Infection-dependent phenotypes in MHC-congenic mice are not due to MHC: can we trust congenic animals?
McClelland EE, Damjanovich K, Gardner K, Groesbeck ZJ, Ma MS, Nibley M, Richardson KS, Wilkinson M, Morrison LC, Bernhardt P, Potts WK.
BACKGROUND: Congenic strains of mice are assumed to differ only at a single gene or region of the genome . These mice have great importance in evaluating the function of genes . However, their utility depends on the maintenance of this true congenic nature . Although, accumulating evidence suggests that congenic strains suffer genetic divergence that could compromise interpretation of experimental results, this problem is usually ignored . During coinfection studies with Salmonella typhimurium and Theiler's murine encephalomyelitis virus (TMEV) in major histocompatibility complex (MHC)-congenic mice, we conducted the proper F2 controls and discovered significant differences between these F2 animals and MHC-genotype-matched P0 and F1 animals in weight gain and pathogen load . To systematically evaluate the apparent non-MHC differences in these mice, we infected all three generations (P0, F1 and F2) for 5 MHC genotypes (b/b, b/q and q/q as well as d/d, d/q, and q/q) with Salmonella and TMEV . RESULTS: Infected P0 MHC q/q congenic homozygotes lost significantly more weight (p = 0.02) and had significantly higher Salmonella (p < 0.01) and TMEV (p = 0.02) titers than the infected F2 q/q homozygotes . Neither weight nor pathogen load differences were present in sham-infected controls . CONCLUSIONS: These data suggest that these strains differ for genes other than those in the MHC congenic region . The most likely explanation is that deleterious recessive mutations affecting response to infection have accumulated in the more than 40 years that this B10.Q-H-2q MHC-congenic strain has been separated from its B10-H-2b parental strain . During typical experiments with congenic strains, the phenotypes of these accumulated mutations will be falsely ascribed to the congenic gene(s) . This problem likely affects any strains separated for appreciable time and while usually ignored, can be avoided with the use of F2 segregants.

J Antimicrob Chemother, 2004 Aug, 54(2), 429 - 34 Epub 2004 Jul 08.
Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 beta-lactamase; Antunes P et al.; OBJECTIVES: Characterization of the molecular basis for beta-lactam resistance and evaluation of the clonal relatedness among nine isolates of multidrug-resistant Salmonella typhimurium recovered from seven clinical human samples and two pork end products . METHODS: The isolates were examined for susceptibility to antimicrobial agents . The relationships between resistance genes, class 1 integrons, plasmids and isolates were screened by molecular methods such as polymerase chain reaction and restriction fragment length analysis . RESULTS: A bla(OXA-30) gene, located in a class 1 integron, was detected in all isolates . This integron was present on a conjugative plasmid in all but one isolate . By pulsed-field gel electrophoresis, it was determined that all strains share the same chromosomal type . CONCLUSIONS: This study demonstrates the spread of an OXA-30-producing S . typhimurium in Portugal, suggesting dissemination of a resistant clone through the food chain.

Mutat Res, 2004 Jul 11, 561(1-2), 35 - 44
Mutagenicity of different textile dye products in Salmonella typhimurium and mouse lymphoma cells; Jager I et al.; Textile dyes used within the European Union (EU) were examined for available published and unpublished mutagenicity data . Fifty-three dye products that had so far not been investigated for mutagenicity were tested in the bacterial reverse mutation assay with Salmonella typhimurium (Ames test) according to a modification of the OECD 471 guidelines (instead of five strains, only TA98 and/or TA100 were used with and without metabolic activation (S9-mix)) . About 28% (15 out of 53) of the dye samples were positive in the Ames test . Fifteen samples showed positive results with TA98, two were positive in TA100 . The mutagenicity of nine Ames-positive textile dye products was further investigated in the mouse lymphoma assay (MLA) (OECD 476) . Sixty-seven percent (6 out of 9) induced genotoxic effects in the MLA . The induction rates (IR) were between 2.1 and 132 in the bacterial reverse mutation assay and between 2.1 and 15.2 in the MLA . The results confirm previous findings that dye products are marketed that are not sufficiently tested and that show mutagenic effects in in vitro tests.

J Agric Food Chem, 2004 Jul 14, 52(14), 4380 - 7
Antioxidative and antimutagenic activities of 4-vinyl-2,6-dimethoxyphenol (canolol) isolated from canola oil; Kuwahara H et al.; A potent antioxidative compound in crude canola oil, canolol, was recently identified, and reported herein are studies of its scavenging capacity against the endogenous mutagen peroxynitrite (ONOO(-)) . ONOO(-) is generated by the reaction between superoxide anion radical and nitric oxide, both of which are produced by inflammatory leukocytes . Among various antioxidative substances of natural or synthetic origin, canolol was one of the most potent antimutagenic compounds when Salmonella typhimurium TA102 was used in the modified Ames test . Its potency was higher than that of flavonoids (e.g., rutin) and alpha-tocopherol and was equivalent to that of ebselen . Canolol suppressed ONOO(-)-induced bactericidal action . It also reduced intracellular oxidative stress and apoptosis in human cancer SW480 cells when used at a concentration below 20 microM under H(2)O(2)-induced oxidative stress . In addition, canolol suppressed plasmid DNA (pUC19) strand breakage induced by ONOO(-), as revealed by agarose gel electrophoresis.

J Microbiol Methods, 2004 Aug, 58(2), 147 - 68
Detection of biological threats . A challenge for directed molecular evolution; Petrenko VA et al.; The probe technique originated from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, successful staining of tubercle bacilli with synthetic dyes by Paul Ehrlich and discovery of a stain for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram . The technique relies on the principle that pathogens have unique structural features, which can be recognized by specifically labeled organic molecules . A hundred years of extensive screening efforts led to discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria . A new challenge--continuous monitoring of biological threats--requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions . To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed molecular evolution . This review describes how molecular evolution methods are applied for development of peptide, antibody and phage probes, and summarizes the author's own data on development of landscape phage probes against Salmonella typhimurium . The performance of the probes in detection of Salmonella is illustrated by a precipitation test, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy.

Food Chem Toxicol, 2004 Sep, 42(9), 1487 - 95
Genotoxicity and mutagenicity of melanoidins isolated from a roasted glucose-glycine model in human lymphocyte cultures, intestinal Caco-2 cells and in the Salmonella typhimurium strains TA98 and TA102 applying the AMES test; Glosl S et al.; Melanoidins are formed during household cooking procedures and are part of our daily diet, but data on their toxicological potential are still scarce . Therefore, the mutagenic, cytotoxic and genotoxic activity of the water soluble total fraction (sol A), the water soluble high molecular weight fraction (HMW; Molecular weight>12,400 Da) and the remaining water soluble low molecular weight fraction (LMW) isolated from a glucose-glycine model system roasted at 125 degrees C was comprehensively studied in human lymphocytes (genetic end point: sister chromatid exchange (SCE)), Caco-2 cells (SCE, cell viability, cell proliferation) and in the Salmonella typhimurium strains TA98 and TA102 (Ames test) . Tests were performed in a dose- and time-dependent manner . The results indicate a significant increase in SCE formation in human lymphocytes after the exposure to 0.05% and 0.1% of the melanoidin fractions . In Caco-2 cells, only the exposure to LMW increased the SCE formation as a matter of concentration . Cell's proliferation and viability decreased significantly after exposure to melanoidins . In the AMES test, melanoidins did not show a mutagenic potential, neither using the TA98 nor the TA102 strain . These results show that melanoidins isolated from the glucose-glycine mixture exhibited modest but significant genotoxic effects in human lymphocytes and, in particular the LMW, in Caco-2 cells, but they induce neither in low nor in very high concentrations mutagenicity in bacteria strains.

Immunol Rev, 2004 Jun, 199, 181 - 90
DNA vaccines suppress tumor growth and metastases by the induction of anti-angiogenesis; Reisfeld RA et al.; Four novel oral DNA vaccines provide long-lived protection against melanoma, colon, breast, and non-small cell lung carcinoma in mouse model systems . The vaccines are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs and are directed against targets such as carcinoembryonic antigen, tyrosine-related protein, vascular endothelial growth factor receptor-2 {also called fetal liver kinase-1 (FLK-1)}, and transcription factor Fos-related antigen-1 (Fra-1) . The FLK-1 and Fra-1 vaccines are effective in suppressing angiogenesis in the tumor vasculature . All four vaccines are capable of inducing potent cell-mediated protective immunity, breaking peripheral T-cell tolerance against these self-antigens resulting in effective suppression of tumor growth and metastasis . It is anticipated that such research efforts will contribute toward the rational design of future DNA vaccines that will be effective for prevention and treatment of human cancer.

PDA J Pharm Sci Technol, 2004 May-Jun, 58(3), 159 - 68
Adenosine triphosphate bioluminescence analysis for rapid screening of microbial contamination in non-sterile pharmaceutical samples; Jimenez L; An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceutical formulations such as creams, tablets, and capsules . Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else . Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Candida albicans, and Aspergillus niger . Samples were incubated for 24-120 h at 35 degrees C with shaking . Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay . However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was confirmed . After incubation, all microorganisms were detected by the bioluminescence system within 24-72 h . All positive samples were confirmed by using standard plating media . However, to optimize detection of all microorganisms, different enrichment media were developed.

Vet Pathol, 2004 Jul, 41(4), 419 - 23
Expression of cyclooxygenase-2 and nitric oxide synthase 2 in swine ulcerative colitis caused by Salmonella typhimurium; Cho WS et al.; Cyclooxygenase-2 (COX-2) and nitric oxide synthase 2 (NOS2) were detected and localized in 20 pigs with ulcerative colitis caused by natural infection with Salmonella typhimurium . Evidence of NOS2 activity was determined by the formation of nitrotyrosine, a reaction product of peroxynitrite, in NOS2-expressing ulcerative colons by immunohistochemistry . Transcript RNA of COX-2 and NOS2 was consistently detected in colonic tissues from the 20 pigs with ulcerative colitis by using reverse transcription-polymerase chain reaction . Immunohistochemical signals for COX-2 and NOS2 were detected in the ulcerated area of all 20 pigs . Expression of COX-2 and NOS2 was identified continuously within inflammatory intestinal lesions but was minimal in unaffected regions of the colon of S . typhimurium-infected pigs . The immunohistochemistry of serial sections of intestine indicated that the majority of colons containing numerous COX-2-positive cells also had numerous NOS2-positive cells . Localization of NOS2 and a nitrotyrosine antigen was prominent in neutrophils and macrophages in the periphery of the lesions . Simultaneous detection of COX-2 and NOS2 RNA and protein indicated functional activity of prostaglandin and NO production in vivo . This study suggested that COX-2 and NOS2 expression may play a role in the pathophysiologic processes in ulcerative colitis caused by S . typhimurium.

Cancer Detect Prev, 2004, 28(3), 200 - 7
13-cis Retinoic acid ameliorates benzoyl peroxide-induced oxidative stress and hyperproliferative response in murine skin: a chemopreventive study; Sultana S et al.; The present paper assesses the chemopreventive potential of retinoic acid on benzoyl peroxide (BPO)-induced cutaneous tumor promotion response and oxidative stress in murine skin . In this study, we have shown the activities of cutaneous antioxidant enzymes and phase II metabolizing enzymes and the glutathione content were decreased while epidermal ornithine decarboxylase (ODC) activity and DNA synthesis were induced in benzoyl peroxide treated animals . Topical application of retinoic acid resulted in significant inhibition of benzoyl peroxide-induced epidermal ornithine decarboxylase activity and DNA synthesis . Application of retinoic acid at three different doses prior to the application of benzoyl peroxide recovered the depleted level of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, thus resulting in significant inhibition of oxidative stress in dose dependent manner . Enhanced susceptibility of cutaneous microsomal lipid peroxidation and xanthine oxidase activity were significantly reduced (P > 0.05) . The antimutagenic effect of retinoic acid was tested against benzoyl peroxide mediated mutagenicity in Salmonella typhimurium strain TA-98 and TA-100 using 3-methyl cholanthrene-induced murine skin (S9 fraction) as the metabolic activation system . Indeed, with the addition of various concentrations of retinoic acid there was significant reduction in the number of revertants per plate in concentration dependent manner . In summary, our data indicates that retinoic acid may exhibit cancer chemopreventive activity in skin tumorigenesis model.

Mol Microbiol, 2004 Jul, 53(1), 345 - 54
Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression; Ding Y et al.; Hfq is an RNA-binding protein that interacts with both small untranslated RNAs (sRNAs) and mRNAs to modulate gene expression post-transcriptionally . In Escherichia coli and Salmonella typhimurium, Hfq is required for efficient expression of the stationary phase sigma factor sigma(S), and consequently is critical for Salmonella virulence . We have found that Hfq is also essential for the virulence of Vibrio cholerae, as strains lacking hfq fail to colonize the suckling mouse intestine . Deletion of the V . cholerae hfq does not prevent production of sigma(S), nor does it prevent expression of TCP, V . cholerae's primary colonization factor . The expression and activity of the alternative sigma factor sigma(E) are dramatically increased in a V . cholerae hfq mutant . Comparison of the transcriptome of an hfq mutant with that of an rseA mutant, which also overexpresses sigma(E), revealed that sigma(E) controls approximately half the genes found to be upregulated in the hfq mutant . However, increased sigma(E) does not appear to account for this strain's reduced virulence . It is likely that sRNAs, in conjunction with Hfq, are critical regulators of V . cholerae pathogenicity.

Int Immunopharmacol, 2004 Aug, 4(8), 1059 - 66
Anti-inflammatory effects of alpha-melanocyte-stimulating hormone against rat endotoxin-induced uveitis and the time course of inflammatory agents in aqueous humor; Nishida T et al.; PURPOSE: We examined the effects of the immunosuppressive neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) on rat endotoxin-induced uveitis, and to measure the expression of inflammatory cytokines and chemokines with and without the alpha-MSH treatment over the course of the disease . METHODS: We injected Lewis rats once with Salmonella typhimurium lipopolysaccharide (LPS) to induce uveitis . The rats were given intravenous injections of 250, 500 or 1000 microg of alpha-MSH . The eyes were examined over the next 24 h for inflammation . Aqueous humor was collected 6, 12 and 24 h after endotoxin injections and the number of infiltrating cells were counted in anterior chamber . In addition, we assayed the concentration of protein, nitric oxide, TNF-alpha, IL-6, MCP-1 and MIP-2 . RESULTS: Rats injected with alpha-MSH showed a significant decrease in the number of infiltrating cells in anterior chamber . Moreover, alpha-MSH-treated rats with endotoxin-induced uveitis (EIU) showed significantly lower concentrations of protein, nitric oxide, proinflammatory cytokines and chemokines in their aqueous humor . Even the early stages of EIU were suppressed by the injection of alpha-MSH . CONCLUSIONS: Our results demonstrate that the immunosuppressive neuropeptide alpha-MSH inhibits the early induction events of endotoxin-induced inflammation in the eye; therefore, suppresses the subsequent infiltration of cells and intraocular production of inflammatory cytokines and chemokines in eyes . alpha-MSH has a possibility of being a therapeutic strategy for anterior uveitis.

J Food Prot, 2004 Jun, 67(6), 1293 - 8
2-Dodecylcyclobutanone does not induce mutations in the Salmonella mutagenicity test or intrachromosomal recombination in Saccharomyces cerevisiae; Sommers CH et al.; Treatment of foods, such as red meat and poultry, that contain palmitic acid with ionizing radiation leads to the formation of 2-dodecylcyclobutanone (2-DCB), a compound found only in irradiated foods . In this study, the Salmonella mutagenicity test and the yeast DEL assay were used to evaluate the genotoxic potential of 2-DCB . Salmonella Typhimurium tester strains TA98, TA100, TA1535, and TA1537 were exposed to 0, 0.125, 0.25, 0.5, and 1 mg per well of 2-DCB, with and without exogenous metabolic activation (5% S9 fraction), using the microtiter plate-based Miniscreen version of the test . 2-DCB did not induce mutations in the Salmonella mutagenicity test . When Saccharomyces cerevisiae strain RS112, which contains a nonfunctional duplication of the his3 gene that can be induced to form a functional HIS3+ gene by intrachromosomal recombination, was exposed to 0.63, 1.25, 2.5, or 5.0 mg/ml of 2-DCB, no increase in the rate of intrachromosomal (DEL) recombination was observed . The absence of genotoxicity observed in this study using purified 2-DCB agrees with the lack of genotoxic and teratogenic activity observed in previously conducted multigeneration feeding studies of laboratory animals (rats, mice, guinea pigs, and rabbits) that used radiation-sterilized poultry that contained 2-DCB as a unique radiolytic product.

J Food Prot, 2004 Jun, 67(6), 1252 - 6
In vitro antimicrobial activity of essential oils from aromatic plants against selected foodborne pathogens; Rota C et al.; The purpose of this study was to examine the effectiveness of selected essential oils for the control of growth and survival of pathogenic microorganisms of significant importance in food hygiene and to determine whether the antimicrobial effect was due to the major compounds of the oils . MIC and MBC were determined by the tube dilution method . Essential oils from Thymus vulgaris from Spain and France, Salvia sclarea, Salvia officinalis, Salvia lavandulifolia, Lavandula latifolia, Lavandula angustifolia, three hybrids of Lavandula latifolia x Lavandula angustifolia (Lavandin 'Super', Lavandin 'Abrialis', and Lavandin 'Grosso'), Rosmarinus officinalis, Hissopus officinalis, and Satureja montana were evaluated . Inhibition ranged from the strong activity of Satureja montana and Thymus vulgaris (France) to no inhibition with Salvia sclarea and Hissopus officinalis for each of the test strains: Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli O157:H7, Yersinia enterocolitica, Shigella flexneri, Listeria monocytogenes serovar 4b, and Staphylococcus aureus . Because some of the essential oils were highly inhibitory in small quantities to selected pathogenic microorganisms, they may provide alternatives to conventional antimicrobial additives in foods.

J Food Prot, 2004 Jun, 67(6), 1229 - 33
Serological characterization and prevalence of spvR genes in Salmonella isolated from foods involved in outbreaks in Brazil; Geimba MP et al.; Salmonella strains (n = 75) isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, Brazil, during 1999 and 2000 were studied . Strains were serotyped and submitted to PCR analysis to verify the prevalence of Salmonella plasmid virulence (spvR) regulatory gene . Among the 75 isolates, 73 (97%) were classified as Salmonella enterica serovar Enteritidis . All of the Salmonella strains isolated in 1999 were classified as serotype Enteritidis, whereas in 2000 two isolates were serotyped as Salmonella Derby and Salmonella Typhimurium . Regarding the prevalence of spvR gene, 62 strains (82.7%) were PCR positive, and a positive correlation (P < 0.05) between the strains of Salmonella Enteritidis and the presence of spvR gene was demonstrated, which suggests that this gene is a characteristic of the Salmonella Enteritidis analyzed.

Toxicol Ind Health, 2002 Oct, 18(9-10), 425 - 33
Mutagenic effects of cutting fluids and components in the Salmonella typhimurium mutagenicity assay; Kleber M et al.; Emulsions of water-soluble cutting fluids (wsCF) are used in large quantities in the metal industry . In order to reduce the costs for use and disposal of these fluids, new technologies are being introduced . Minimist Lubricant Supply (MLS) uses only minimal amounts of cutting fluids . In contrast to conventional wsCF, which are complex multicompound mixtures, MLS cutting fluids are composed of one or two components only, like fatty alcohols and fatty acid esters . The aim of the study was to identify and compare the mutagenic potential of these cutting fluids as a first indicator of a possible hazard of systemic effects after inhalation or dermal absorption of the fluids at the workplace . The Salmonella typhimurium assay (Ames assay) was used as a screening method to detect mutagenic effects of cutting fluids . Conventional wsCF and MLS cutting fluids were tested in the strains TA 98, TA 100, TA 102 and TA 104, in the presence and absence of an external metabolizing enzyme system (rat liver S9-mix), using a preincubation (20 min) test protocol . For MLS fluids, no mutagenicity was found in a concentration range between 1 and 10 mg/plate in the Ames assay . Among five tested conventional wsCF, two were mutagenic in the Ames assay at concentration ranges between 2.5 and 15 mg/plate . In cooperation with the manufacturer, 18 defined components of cutting fluids were tested separately . The results revealed that formaldehyde generators, derivatives of oxazolidine and hexahydrotriazine used as biocides for preservation of the fluids, were mutagenic . Four components were nonmutagenic but cytotoxic, whereas the remaining components displayed no bacterial mutagenicity . The present results show the potential hazard of biocides for workers handling these fluids . An exposure via inhalation and/or dermal absorption could cause an additional risk due to mutagenic ingredients . Under aspects of workers' safety, a further discussion about the use of specific components in cutting fluids is recommended.

Nucleic Acids Res, 2004 Jul 1, 32(Web Server issue), W394 - 9
BOMP: a program to predict integral beta-barrel outer membrane proteins encoded within genomes of Gram-negative bacteria; Berven FS et al.; This work describes the development of a program that predicts whether or not a polypeptide sequence from a Gram-negative bacterium is an integral beta-barrel outer membrane protein . The program, called the beta-barrel Outer Membrane protein Predictor (BOMP), is based on two separate components to recognize integral beta-barrel proteins . The first component is a C-terminal pattern typical of many integral beta-barrel proteins . The second component calculates an integral beta-barrel score of the sequence based on the extent to which the sequence contains stretches of amino acids typical of transmembrane beta-strands . The precision of the predictions was found to be 80% with a recall of 88% when tested on the proteins with SwissProt annotated subcellular localization in Escherichia coli K 12 (788 sequences) and Salmonella typhimurium (366 sequences) . When tested on the predicted proteome of E.coli, BOMP found 103 of a total of 4346 polypeptide sequences to be possible integral beta-barrel proteins . Of these, 36 were found by BLAST to lack similarity (E-value score < 1e-10) to proteins with annotated subcellular localization in SwissProt . BOMP predicted the content of integral beta-barrels per predicted proteome of 10 different bacteria to range from 1.8 to 3% . BOMP is available at http://www.bioinfo.no/tools/bomp.

Lett Appl Microbiol, 2004, 38(4), 321 - 6
Comparative acid stress response of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at different pH conditions; Koutsoumanis KP et al.; AIMS: The aim of the study was to evaluate the effect of habituation at different pH conditions on the acid resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium, and to identify potential differences between the adaptive responses of the three pathogens . METHODS: Stationary phase cells of L . monocytogenes, E . coli O157:H7 and S . Typhimurium, grown in glucose-free media, were exposed to pH 3.5 broth directly or after habituation for 90 min at various pH conditions from 4.0 to 6.0 . Survivors at pH 3.5 were determined by plating on tryptic soy agar and incubating at 30 degrees C for 48 h . The kinetics (death rate) of the pathogens at pH 3.5 was calculated by fitting the data to an exponential model . RESULTS: Habituation to acidic environments provided protection of the pathogens against lethal acid conditions . This acid protection, however, was found to be pH dependent . For example, for E . coli O157:H7 an increased acid resistance was observed after habituation at a pH range from 4.0 to 5.5, while the maximum acid tolerance was induced at pH 5.0 . Furthermore, the effect of low pH habituation was different among pathogens . For L . monocytogenes, E . coli O157:H7 and S . Typhimurium, the pH range within which habituation resulted to increased acid resistance was 5.0-6.0, 4.0-5.5 and 4.0-5.0, respectively, while the maximum acid tolerance was induced after habituation at pH 5.5, 5.0 and 4.5, respectively . SIGNIFICANCE: Acid stress conditions are common within current food processing technologies . The information on adaptive responses of L . monocytogenes, E . coli O157:H7 and S . Typhimurium after habituation to different pH environments provided in the present study, could lead to a more realistic evaluation of food safety concerns and to a better selection of processes in order to avoid adaptation phenomena and to minimize the potential for food safety risks.

Natl Toxicol Program Tech Rep Ser, 2004 May, (518), 5 - 163
NTP toxicology and carcinogenesis studies of triethanolamine (Cas No . 102-71-6) in B6C3F1 mice (dermal studies); National Toxicology Program et al.; {structure--see text} Triethanolamine is widely used in the manufacturing of household detergents and polishes, textiles, agricultural herbicides, mineral and vegetable oils, paraffin and waxes, pharmaceutical ointments, petroleum demulsifiers, synthetic resins, plasticizers, adhesives, and sealants . It is used as a chemical intermediate for anionic and nonionic surfactants, a vulcanization accelerator, a humectant and softening agent and in many other industrial applications . The National Cancer Institute nominated triethanolamine for study because of its widespread use in cosmetics and other consumer products, its high potential for worker exposure due to its many industrial uses, and its potential for conversion to the carcinogen N-nitrosodiethanolamine . Previous 3-month and 2-year studies of triethanolamine were conducted by the National Toxicology Program in F344/N rats and B6C3F1 mice; results from the 2-year rat study indicated equivocal evidence of carcinogenic activity based on a marginal increase in the incidence of renal tubule adenoma (NTP, 1991) . Interpretation of the results from the 2-year study in mice was complicated by Helicobacter hepaticus infection, prompting a repeat 2-year study in mice . Male and female B6C3F1 mice received triethanolamine (greater than 99% pure) by dermal application for 2 years; a study of absorption, distribution, metabolism, and excretion was performed in additional mice . Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood erythrocytes . 2-YEAR STUDY: Groups of 50 male and 50 female mice received dermal applications of 0, 200, 630, or 2,000 mg/kg (males) and 0, 100, 300, or 1,000 mg/kg (females) triethanolamine in acetone, 5 days per week, for 104 (males) or 104 to 105 (females) weeks . Survival of all dosed groups was similar to that of the vehicle control groups . Body weights of 2,000 mg/kg males were less than those of the vehicle controls from weeks 17 to 37 and at the end of the study; body weights of dosed groups of females were similar to those of the vehicle controls throughout the study . Treatment-related clinical findings included skin irritation at the site of application, which increased with increasing dose and was more severe in males than in females . Gross lesions observed at necropsy included nodules and masses of the liver in dosed females . The incidences of hepatocellular adenoma and hepatocellular adenoma or carcinoma (combined) were significantly increased in all dosed groups of females . The incidence of hemangiosarcoma of the liver in 630 mg/kg males was marginally increased . The incidences of eosinophilic focus in all dosed groups of mice were greater than those in the vehicle controls . Gross lesions observed at necropsy included visible crusts at the site of application in all dosed groups of mice . Treatment-related epidermal hyperplasia, suppurative inflammation, ulceration, and dermal chronic inflammation occurred at the site of application in most dosed groups of mice, and the incidences and severities of these lesions generally increased with increasing dose . GENETIC TOXICOLOGY: Triethanolamine was not mutagenic in any of the in vitro or in vivo tests . It did not induce mutations in Salmonella typhimurium, and no induction of sister chromatid exchanges or chromosomal aberrations was noted in cultured Chinese hamster ovary cells exposed to triethanolamine . These in vitro tests were all conducted with and without S9 metabolic activation . Triethanolamine did not induce sex-linked recessive lethal mutations in germ cells of adult male Drosophila melanogaster exposed by feeding or injection . No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood samples of male or female mice that received dermal applications of triethanolamine for 13 weeks . CONCLUSIONS: Under the conditions of this 2-year dermal study, there was equivocal evidence of carcinogenic activity of triethanolamine in male B6C3F1 mice based on the occurrence of liver hemangiosarcoma . There was some evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of hepatocellular adenoma . Exposure to triethanolamine by dermal application resulted in increased incidences of eosinophilic focus of the liver in males and females . Dosed mice developed treatment-related nonneoplastic lesions at the site of application.

Can J Microbiol, 2004 May, 50(5), 341 - 50
{Effect of previous culture conditions and the presence of the rpoS gene on the survival of Salmonella typhimurium in sea water exposed to sunlight}; Zaafrane S et al.; The effect of sunlight exposure on Salmonella typhimurium isogenic strains harboring an rpoS gene functional (rpoS+) or not functional (rpoS-) was investigated in microcosms of sterile sea water at 20 degrees C . The two strains rapidly lost their ability to produce colonies on solid culture media . The detrimental action of sunlight was more important when the salinity of sea water increased . The survival of stationary phase cells was influenced by RpoS . Bacteria grown in media with high salinity or osmolarity and transferred to sea water in stationary phase were more resistant to irradiation than those grown in media with low salinity . Prior growth under oxidative (0.2 mmol/L of H2O2) or amino acid starved (minimal medium) conditions did not modify the survival of either strain when they were exposed to sunlight . Bacteria were more resistant when cells were incubated in sea water in the dark prior to being exposed to sunlight . The resistance to sunlight irradiation was also greater in clones of both strains isolated from microcosms exposed to sunlight for 90 min, then further inoculated into sea water and reexposed to sunlight.

Bioorg Med Chem, 2004 Jul 15, 12(14), 3791 - 6
Synthesis and properties of bifunctional chloroalkyl nitrosamines with an intercalating moiety; Ishikawa S et al.; Three N-nitroso-N-(arylcarbonyloxymethyl)-3-chloropropylamines were synthesized, and their chemical and biological properties were studied . All arylcarboxylates intercalated with double-stranded DNA, and their mutagenicity and DNA cross-linking activity were affected by their ring structure . The DNA interstrand cross-link formation increased dose dependently after treatment with the acridine analog . The anthraquinone analog showed the highest bacterial mutagenicity among the three nitrosamines in Salmonella typhimurium TA100, while in Salmonella typhimurium TA92, which can detect cross-linking agents, the acridine analog showed the highest mutagenicity . This agreed with the result of a cross-linking assay . These results suggest that the three-ring aromatic moiety gives DNA-intercalating ability to cross-linkable chloropropyl nitrosamine, and the acridine analog is considered as a possible new antitumor lead compound.

Eur J Biochem, 2004 Jul, 271(13), 2624 - 35
The crystal structure of the tryptophan synthase beta subunit from the hyperthermophile Pyrococcus furiosus . Investigation of stabilization factors; Hioki Y et al.; The structure of the tryptophan synthase beta2 subunit (Pfbeta2) from the hyperthermophile, Pyrococcus furiosus, was determined by X-ray crystallographic analysis at 2.2 A resolution, and its stability was examined by DSC . This is the first report of the X-ray structure of the tryptophan synthase beta2 subunit alone, although the structure of the tryptophan synthase alpha2beta2 complex from Salmonella typhimurium has already been reported . The structure of Pfbeta2 was essentially similar to that of the beta2 subunit (Stbeta2) in the alpha2beta2 complex from S . typhimurium . The sequence alignment with secondary structures of Pfbeta and Stbeta in monomeric form showed that six residues in the N-terminal region and three residues in the C-terminal region were deleted in Pfbeta, and one residue at Pro366 of Stbeta and at Ile63 of Pfbeta was inserted . The denaturation temperature of Pfbeta2 was higher by 35 degrees C than the reported values from mesophiles at approximately pH 8 . On the basis of structural information on both proteins, the analyses of the contributions of each stabilization factor indicate that: (a) the higher stability of Pfbeta2 is not caused by either a hydrophobic interaction or an increase in ion pairs; (b) the number of hydrogen bonds involved in the main chains of Pfbeta is greater by about 10% than that of Stbeta, indicating that the secondary structures of Pfbeta are more stabilized than those of Stbeta and (c) the sequence of Pfbeta seems to be better fitted to an ideally stable structure than that of Stbeta, as assessed from X-ray structure data.

Biochem J, 2004 Sep 15, 382(Pt 3), 811 - 9
Incomplete glycosylation and defective intracellular targeting of mutant solute carrier family 11 member 1 (Slc11a1); White JK et al.; Solute carrier family 11 member 1 (Slc11a1, formerly Nramp1) is a highly glycosylated, 12 transmembrane domain protein expressed in macrophages . It resides in the membrane of late endosomes and lysosomes, where it functions as a bivalent cation transporter . Mice susceptible to infection by various intracellular pathogens including Leishmania donovani and Salmonella typhimurium carry a glycine to aspartic acid substitution at position 169 (G169D, Gly(169)-->Asp), within transmembrane domain 4 of Slc11a1 . To investigate the molecular pathogenesis of infectious disease susceptibility, we compared the behaviour of heterologously and endogenously expressed wild-type and mutant Slc11a1 by immunofluorescence, immunoelectron microscopy and Western-blot analysis . We found occasional late endosome/lysosome staining of mutant protein using immunoelectron microscopy, but most of the mutant Slc11a1 was retained within the ER (endoplasmic reticulum) . Using glycosylation as a marker for protein maturation in two independent heterologous expression systems, we found that most mutant Slc11a1 existed as an ER-dependent, partially glycosylated intermediate species . Correct endosomal targeting of wild-type Slc11a1 continued despite disruption of N-glycosylation sites, indicating that glycosylation did not influence folding or sorting . We propose that the G169D mutation causes localized misfolding of Slc11a1, resulting in its retention in the ER and manifestation of the loss of function phenotype.

Emerg Infect Dis, 2004 May, 10(5), 932 - 5
Multidrug-resistant Salmonella Typhimurium infection from milk contaminated after pasteurization; Olsen SJ et al.; An outbreak of multidrug-resistant Salmonella enterica serotype Typhimurium infections occurred in Pennsylvania and New Jersey . A case-control study implicated pasteurized milk from a dairy, and an inspection indicated the potential for contamination after pasteurization . Dairy cattle are the likely reservoir, and milk may be an important vehicle of Salmonella transmission to humans.

Drug Chem Toxicol, 2004 May, 27(2), 157 - 67
A notable antimutagenicity of two nonmutagenic novel oxadiazoles in Salmonella mutagenicity assay; Maslat A et al.; The mutagenic activity of two newly synthesized oxadiazoles: 1,3-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M1) and 1,4-bis(5-benzylthio-1,3,4-oxadiazol-2-yl) benzene (M2) was studied in Salmonella typhimurium strains TA97, TA100, TA102 and TA1537 in the presence and absence of S9mix . The antimutagenicity of M1 and M2 against H2O2, sodium azide (SA) and 4-nitro-o-phenylene diamine (NPD) using the tester strains TA102, TA100 and TA97, respectively, was also investigated . The two compounds were found to be nonmutagenic using the four tester strains . However, they showed high mutagenic repression activity against hydrogen peroxide (95% and 97% for M1 and M2, respectively, at a concentration of 335 micrograms/plate) . Moderate mutagenic repression against NPD (58% and 55% for M1 and M2, respectively, at a concentration of 167.5 micrograms/plate) and low mutagenic repression against SA (21% and 33% for M1 and M2 respectively, at a concentration of 335 micrograms/plate) was detected . The obtained results are very encouraging to test the above mentioned compounds as anticarcinogens.

J Infect Dis, 2004 Jul 1, 190(1), 107 - 14 Epub 2004 Jun 08.
Potent role of vaccines prepared from macrophages infected with live bacteria in protection against Mycobacterium tuberculosis and Salmonella typhimurium infections; Sharma N et al.; The present study describes a novel and simple vaccination strategy that involves the culturing of live Mycobacterium tuberculosis and Salmonella typhimurium in syngeneic, allogeneic, and xenogeneic macrophages, followed by drug treatment and gamma irradiation, to kill the bacteria . Notable observations were that the lymphocytes obtained from the vaccinated mice proliferated and secreted mainly interferon- gamma and IgG2a, but not interleukin-4 and IgG1 . The enumeration of viability of M . tuberculosis indicated a significant level of protection in the vaccinated mice after challenge with live M . tuberculosis . This vaccination strategy worked successfully for tuberculosis but also showed a significant decrease in mortality of mice challenged with live S . typhimurium.

Biochem J, 2004 Sep 1, 382(Pt 2), 589 - 96
Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells; Walia B et al.; Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation . Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface . However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated . In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN . Polarized monolayers of T84 cells were used as an intestinal epithelial model . Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine . Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine . The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide . Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated . Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells . In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine . FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.

Nature, 2004 Jul 8, 430(6996), 213 - 8 Epub 2004 Jun 09.
Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf; Mariathasan S et al.; Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively . The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors . Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function . ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages . Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock . Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages . Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S . typhimurium . Caspase-1 activation was not compromised by loss of RIP2 . These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens . Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.

Epidemiol Infect, 2004 Jun, 132(3), 485 - 93
Risk factors for Salmonella typhimurium DT104 and non-DT104 infection: a Canadian multi-provincial case-control study; Dore K et al.; To identify risk factors for sporadic Salmonella Typhimurium definitive phage-type 104 (DT104) and non-DT104 diarrhoeal illness in Canada, we conducted a matched case-control study between 1999 and 2000 . Cases were matched 1:1 on age and province of residence . Multivariate analysis suggested that recent antibiotic use {odds ratio (OR) 5.2, 95% confidence interval (CI) 1.8-15.3}, living on a livestock farm (OR 4.9, 95% CI 1.9-18.9), and recent travel outside Canada (OR 4.1, 95% CI 1.2-13.8) are independent risk factors for DT104 illness . Similar analyses suggested that recent travel outside North America is a sizable risk factor for non-DT104 illness (OR 66.8, 95% CI 6.7-665.3) . No food exposure was a risk factor in either analysis . Educating health-care providers and the public about appropriate antibiotic use and antimicrobial resistance is important . Appropriate administration of antibiotics to livestock, particularly cattle, and hygienic measures such as handwashing after contact with farm animals may reduce risk . Travel represents an important and probably underestimated risk factor for sporadic illness with S . Typhimurium . Improved national surveillance and detailed investigation of travel-related illness are required.

J Pediatr Gastroenterol Nutr, 2004 Jul, 39(1), 73 - 9
Neutrophil and small intestinal lymphocyte migration after Salmonella typhimurium infection: impact of fermentable fiber; Milo LA et al.; OBJECTIVES: Formula-fed infants have more episodes of acute diarrhea and intestinal infection than do breast-fed infants . Nutrient additions to infant formula that could confer some of the immune benefits of breast milk to formula-fed infants are currently under investigation . This study examined the impact of enteral formulas supplemented with fermentable substrates on small intestinal lymphocyte and neutrophil migration in piglets infected with Salmonella typhimurium . Small intestinal proinflammatory cytokine messenger RNA abundance and in vitro lipopolysaccharide-stimulated interleukin-6 release in whole blood were assessed . METHODS: Piglets were randomized to receive sow milk replacer formula supplemented with methylcellulose (control), soy polysaccharides (SPS) or fructooligosaccharides (FOS) . On day 7, half of the piglets were infected with S . typhimurium . Intestinal lymphocyte, neutrophil and whole blood samples were obtained on day 14 . RESULTS: After infection, there was decreased lymphocyte migration in the control group but not in the SPS and FOS groups . The SPS group had greater neutrophil migration compared with the control and FOS groups, regardless of infection . Small intestinal abundance of proinflammatory cytokine messenger RNA was not significantly changed by either infection or diet . Blood from the FOS group challenged with lipopolysaccharide for 2 hours exhibited decreased interleukin-6 production compared with blood from the control and SPS groups, regardless of infection . CONCLUSIONS: Supplementation of enteral formulas with SPS maintains the migratory function of small intestinal lymphocytes while increasing that of neutrophils.

Biol Pharm Bull, 2004 Jun, 27(6), 883 - 9
Development of TCR alpha beta CD8 alpha alpha intestinal intraepithelial lymphocytes is promoted by interleukin-15-producing epithelial cells constitutively stimulated by gram-negative bacteria via TLR4; Kaneko M et al.; The microbes present in the intestine have a strong influence on the development and maturation of lymphoid organs . The cross-talk mechanisms between intestinal intraepithelial lymphocytes (i-IEL) and noninvasive microbes are still poorly understood . The influence of microbes and lipopolysaccharides on the development of i-IEL, especially the TCR alpha beta(+) CD8 alpha alpha subset, was investigated using the different TLR4-mutant mouse strains C3H/HeJ, BALB/lps(d), and C57BL/10ScCr . Intestinal epithelial cells (i-EC) from TLR4-mutant strains did not express interleukin (IL)-15 mRNA, while IL-15 mRNA expression in i-EC from the corresponding wild-type, C3H/He, BALB/c, and C57BL/10ScSn mice was detected . The development of TCR alpha beta(+) CD8 alpha alpha cells in i-IEL significantly decreased in TLR4-mutant mice compared with the corresponding wild-type mice, while other T cell subsets in i-IEL showed similar percentages in the TLR4-mutant and wild-type mice . Adult thymectomized (ATx-) and lethally irradiated C3H/HeJ mice reconstituted with T cell-depleted bone marrow cells from C3H/He mice showed a significantly lower percentage of TCR alpha beta CD8 alpha alpha i-IEL than ATx-C3H/He mice after transfer of C3H/HeJ BM cells . The percentage of TCR alpha beta CD8 alpha alpha i-IEL and IL-15 mRNA expression in i-EC from BALB/lps(d) mice did not increase during Salmonella typhimurium infection but was significantly enhanced during Listeria monocytogenes infection . Our findings suggest that LPS induces IL-15 production by i-EC, resulting in the development of TCR alpha beta CD8 alpha alpha i-IEL.

Mol Microbiol, 2004 Jun, 52(6), 1827 - 44
The bacterial signal molecule, ppGpp, regulates Salmonella virulence gene expression; Pizarro-Cerda J et al.; Numerous, overlapping global regulatory systems mediate the environmental signalling controlling the virulence of Salmonella typhimurium . With both extra- and intracellular lifestyles, unravelling the mechanisms involved in regulating Salmonella pathogenesis has been complex . Here, we report a factor co-ordinating environmental signals with global regulators involved in pathogenesis . An S . typhimuriumDeltarelADeltaspoT strain deficient in guanosine tetraphosphate (ppGpp) synthesis was found to be highly attenuated in vivo and non-invasive in vitro . The DeltarelADeltaspoT strain exhibited severely reduced expression of hilA and invF, encoding major transcriptional activators required for Salmonella pathogenicity island 1 (SPI-1) gene expression and at least two other pathogenicity islands . None of the growth conditions intended to mimic the intestinal milieu was capable of inducing hilA expression in the absence of ppGpp . However, the expression of global regulators of Salmonella virulence, RpoS and PhoP/Q, and RpoS- and PhoP/Q-dependent, non-virulence-related genes was not significantly different from the wild-type strain . The results indicate that ppGpp plays a central role as a regulator of virulence gene expression in S . typhimurium and implicates ppGpp as a major factor in the environmental and host-dependent regulation of Salmonella pathogenesis.

Tijdschr Diergeneeskd, 2004 May 15, 129(10), 324 - 7
A serological survey for pathogens in old fancy chicken breeds in central and eastern part of The Netherlands; de Wit JJ et al.; To get an impression of the presence of pathogens in multi-aged flocks of old fancy chicken breeds in the Netherlands, plasma samples originating from 24 flocks were examined for antibodies against 17 chicken pathogens . These flocks were housed mainly in the centre and east of the Netherlands, regions with a high poultry density . The owners of the tested flocks showed their chicken at national and international poultry exhibitions . Antibodies against Avian Influenza, Egg Drop Syndrome '76 virus, Pox virus, Salmonella pullorum/gallinarum, Salmonella Enteritidis or Salmonella Typhimurium were not detected . However, antibodies against other Salmonella species, Mycoplasma gallisepticum, infectious bursal disease virus, infectious bronchitis virus, avian encephalomyelitis virus, chicken anaemia virus, infectious laryngotracheitis virus, and avian leukosis virus, subgroups A and B, and subgroup J were detected in a varying proportion of the flocks . This study shows that antibodies against many chicken pathogens are present among the flocks of old fancy chicken breeds that are exhibited at international poultry exhibitions.

J Pediatr Surg, 2004 Jun, 39(6), 937 - 40; discussion 937-40
Preferential proliferation of attenuated Salmonella typhimurium within neuroblastoma; Soto LJ 3rd et al.; BACKGROUND/PURPOSE: Attenuated Salmonella typhimurium, a facultative intracellular parasite that colonizes the liver, has been shown to accumulate within extrahepatic malignancies . The authors sought to define a mechanism for attenuated Salmonella accumulation within cancer cells compared with hepatocytes . METHODS: Invasion and intracellular proliferation of attenuated Salmonella was assessed through an in vitro assay performed on neuroblasoma, osteosarcoma, hepatoma, and colon adenocarcinoma cell lines and compared with freshly isolated mouse hepatocytes . RESULTS: The efficiency of attenuated S typhimurium invasion into hepatocytes was greater than any malignant cell line (3.8 v 0.46; P <.04) . However, the intracellular proliferation of the bacteria was most abundant within neuroblastoma, exceeding the proliferation within hepatocytes (14.3 v 6.2; P <.01) . CONCLUSIONS: Attenuated S typhimurium may prove to be an effective in vivo immunotherapy for the local delivery of therapeutic proteins to neuroblastoma.

J Med Microbiol, 2004 Jul, 53(Pt 7), 691 - 5
Immunocytochemical studies of Salmonella Typhimurium invasion of porcine jejunal epithelial cells; Schauser K et al.; Although infection of pigs with Salmonella Typhimurium represents a serious problem, most studies on Salmonella infection have been carried out in other species . The purpose of the current study was to examine the route(s) of entry of Salmonella Typhimurium in pigs, using a jejunal loop model . The infection process was followed over 240 min using single to triple immunocytochemical detection of Salmonella and intestinal cell markers . Salmonella invasion was observed in both cytokeratin-18-positive and -negative cylindrical absorptive cells within 5-10 min . Subepithelial invasion of ordinary villi was consistently less marked than invasion of the subepithelial layer of Peyer's patches . Our results show that several epithelial cell types were invaded by Salmonella, and that Peyer's patches represent the main portal of entry in early Salmonella infection . Additionally, infection was associated with alterations in the keratin and F-actin cytoskeleton of intestinal epithelial cells, probably reflecting toxin-mediated actions . Such changes were confined to the proximal region of the jejunum, demonstrating a regional heterogeneity of intestinal epithelial cell responses to Salmonella infection.

Comp Immunol Microbiol Infect Dis, 2004 Jul, 27(4), 235 - 46
B cell and macrophage response in chicks after oral administration of Salmonella typhimurium strains; Berndt A et al.; Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed . The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S . typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis . In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age . In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age . Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls . In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms . The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains . Immunised chickens always showed a weaker immune reaction compared to infected animals . Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains . Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.

J Microbiol Methods, 2004 Jul, 58(1), 79 - 86
Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples; Leon-Velarde CG et al.; An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide . This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104 . A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5% . Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates . These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104 . Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively . Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete .

Sci Total Environ, 2004 Jul 5, 327(1-3), 147 - 62
Emission comparison of urban bus engine fueled with diesel oil and 'biodiesel' blend; Turrio-Baldassarri L et al.; The chemical and toxicological characteristics of emissions from an urban bus engine fueled with diesel and biodiesel blend were studied . Exhaust gases were produced by a turbocharged EURO 2 heavy-duty diesel engine, operating in steady-state conditions on the European test 13 mode cycle (ECE R49) . Regulated and unregulated pollutants, such as carcinogenic polycyclic aromatic hydrocarbons (PAHs) and nitrated derivatives (nitro-PAHs), carbonyl compounds and light aromatic hydrocarbons were quantified . Mutagenicity of the emissions was evaluated by the Salmonella typhimurium/mammalian microsome assay . The effect of the fuels under study on the size distribution of particulate matter (PM) was also evaluated . The use of biodiesel blend seems to result in small reductions of emissions of most of the aromatic and polyaromatic compounds; these differences, however, have no statistical significance at 95% confidence level . Formaldehyde, on the other hand, has a statistically significant increase of 18% with biodiesel blend . In vitro toxicological assays show an overall similar mutagenic potency and genotoxic profile for diesel and biodiesel blend emissions . The electron microscopy analysis indicates that PM for both fuels has the same chemical composition, morphology, shape and granulometric spectrum, with most of the particles in the range 0.06-0.3 microm.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Sep, 19(5), 425 - 7
{In-vivo dynamic changes of antigen presenting cells infected with recombinant attenuated Salmonella typhimurium expressing green fluorescent protein}; Wang F et al.; AIM: To clarify in-vivo early dynamic changes of antigen-presenting cells (APCs) infected with recombinant attenuated Salmonella typhimurium . METHODS: BALB/c mice were orally infected with recombinant attenuated Salmonella typhimurium expressing green fluorescent protein (GFP) X4550 (pYAGFP) . Peritoneal macrophages were taken out 3 days after infection, cultured for 24 hours, and observed under the fluorescent microscope . Moreover, BALB/c mice were also infected intravenously with X4550 (pYAGFP) and low density cells (LDCs) were isolated and prepared from mouse spleen and liver 3,6 and 12 hours after infection . Infection rates of macrophages and dendritic cells (DCs) were analyzed by flow cytometry . RESULTS: About 50% peritoneal macrophages were X4550 (pYAGFP) positive . Infection rates of macrophages in spleen and liver were about 20%-40% . As for the DCs, the infection rates in spleen and liver were about 4%-10% and 10%-20%, respectively . CONCLUSION: Recombinant attenuated Salmonella can be captured by APCs in-vivo in early infection, which provides a precondition for inducing effective immune responses.

Mutat Res, 2004 Mar, 566(2), 99 - 130
Genotoxicity of benzene and its metabolites; Whysner J et al.; The potential role of genotoxicity in human leukemias associated with benzene (BZ) exposures was investigated by a systematic review of over 1400 genotoxicity test results for BZ and its metabolites . Studies of rodents exposed to radiolabeled BZ found a low level of radiolabel in isolated DNA with no preferential binding in target tissues of neoplasia . Adducts were not identified by 32P-postlabeling (equivalent to a covalent binding index <0.002) under the dosage conditions producing neoplasia in the rodent bioassays, and this method would have detected adducts at 1/10,000th the levels reported in the DNA-binding studies . Adducts were detected by 32P-postlabeling in vitro and following high acute BZ doses in vivo, but levels were about 100-fold less than those found by DNA binding . These findings suggest that DNA-adduct formation may not be a significant mechanism for BZ-induced neoplasia in rodents . The evaluation of other genotoxicity test results revealed that BZ and its metabolites did not produce reverse mutations in Salmonella typhimurium but were clastogenic and aneugenic, producing micronuclei, chromosomal aberrations, sister chromatid exchanges and DNA strand breaks . Rodent and human data were compared, and BZ genotoxicity results in both were similar for the available tests . Also, the biotransformation of BZ was qualitatively similar in rodents, humans and non-human primates, further indicating that rodent and human genotoxicity data were compatible . The genotoxicity test results for BZ and its metabolites were the most similar to those of topoisomerase II inhibitors and provided less support for proposed mechanisms involving DNA reactivity, mitotic spindle poisoning or oxidative DNA damage as genotoxic mechanisms; all of which have been demonstrated experimentally for BZ or its metabolites . Studies of the chromosomal translocations found in BZ-exposed persons and secondary human leukemias produced by topoisomerase II inhibitors provide some additional support for this mechanism being potentially operative in BZ-induced leukemia.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jul, 19(4), 335 - 337
{Establishment of an attenuated salmonella typhimurium vaccine strain SL3261 expressing Hp-NAP gene}; Jiao ZY et al.; AIM: To develop an attenuated salmonella typhimurium vaccine strain SL3261 expressing Helicobacter pylori neutrophile activation protein(Hp-NAP) . METHODS: The Hp-NAP gene was amplified by PCR and was cloned into prokaryotic expression plasmid pTrc99A to construct recombinant plasmid pTrc99A-NAP . After the pTrc99A-NAP was performed successively by PCR, enzyme digestion analysis and sequencing the homology was compared between the cloned Hp-NAP gene and related genes in GenBank . The vaccine strain SL3261 was transformed by pTrc99A-NAP . After cultivation, positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again . The expression of Hp-NAP protein in the SL3261 bacteria was proved by SDS-PAGE and thin layer scanning . RESULTS: Sequencing and homologous analysis showed that the homology between the cloned Hp-NAP gene and related genes in GenBank reached 98% for their nucleotide and deduced amino acid sequences . The expression of Hp-NAP protein could be detected in the culture fluid of SL3261 bacteria . Relative molecular mass (M(r)) of expression product was about 15 000, and expression amount accounted for 37.5% of total bacterial protein . CONCLUSION: An attenuated salmonella typhimurium vaccine strain SL3261 has been established successfully, which lays the foundation for further developing oral HP vaccine.

Int J Toxicol, 2004 Jan-Feb, 23(1), 41 - 5
Acute and genotoxic profile of a dimeric impurity of cefotaxime; Agarwal SK et al.; The manufacturing and storage of cefotaxime produces different impurities of various concentrations, which may influence the efficacy and safety of the drugs . Because no report of toxicity data is available on the impurities of cefotaxime, the present acute and genotoxicity studies were designed and conducted to provide the information for establishing the safety profile and qualification of the dimeric impurity . Histidine-requiring mutants of Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains, with or without metabolic activation (S-91, were used for point-mutation tests . Neither increase in numbers of revertants, indicative of mutagenic activity, nor inhibition of bacterial growth, indicative of cytotoxicity, was observed when the dimeric impurity of cefotaxime at concentrations of 0.62, 1.85, 5.56, 16.67, and 50 microg/plate was incorporated into plates containing S . typhimurium bacterial strains . Cultures of Chinese hamster ovary (CHO) cells at a cell density of 2 x 10(5) cells per culture were exposed to the dimeric impurity of cefotaxime at the concentration of 11.25, 22.5, and 45 mg per culture, with or without metabolic activation, and harvested at 18 h after exposure . No chromosomal aberrations in the cultured mammalian cells were recorded . Acute intramuscular administration of the dimeric impurity of cefotaxime in Sprague-Dawley rats did not result in any clinical signs and gross pathological changes up to 2000 mg/kg-body weight . The results of these studies indicated that the dimeric impurity of cefotaxime is nonmutagenic in Ames test, nonclastogenic in vitro, and acutely nontoxic in rats.

Anticancer Res, 2004 Mar-Apr, 24(2B), 743 - 5
Plant phenolics protect from bleomycin-induced oxidative stress and mutagenicity in Salmonella typhimurium TA102; Stagos D et al.; Antioxidants are deemed to be important against DNA damage and mutations induced by reactive oxygen species (ROS) . An assay for the ability of plant phenolics to protect against mutations caused by bleomycin treatment in Salmonella typhimurium TA102 cells in concentrations up to 20 microM was developed . Caffeic acid, gallic acid, protocatechuic acid, ferulic acid and rutin hydrate in final concentrations of 0.5 to 20 microM were tested for their ability to protect TA102 cells from mutations caused by oxidative stress from bleomycin . The cut-off concentration of 20 microM was used because as a limit it is biologically meaningful, higher concentrations being unrealistic in vivo . Caffeic acid was very potent at a concentration of 0.5-20 microM . The other four antioxidants were not effective up to 20 microM . The above assay will be helpful to characterize antioxidant molecules.

Fitoterapia, 2004 Jun, 75(3-4), 385 - 8
The in vitro antibacterial/synergistic activities of Withania somnifera extracts; Arora S et al.; The methanol, hexane and diethyl ether extracts from both leaves and roots of Withania somnifera were evaluated for the antibacterial/synergistic activity by agar plate disc-diffusion assay against Salmonella typhimurium and Escherichia coli . Different concentrations of Tibrim, a combination of rifampicin and isoniazid, were tested to find out the minimum inhibitory concentration (MIC), which came out to be 0.1 mg/ml for S . typhimurium and E . coli . From the six extracts tested, only methanol and hexane extracts of both leaves and roots were found to have potent antibacterial activity . A synergistic increase in the antibacterial effect of Tibrim was noticed when MIC of Tibrim was supplemented with these extracts .

Microbes Infect, 2004 May, 6(6), 521 - 8
Interaction of pathogenic bacteria with rabbit appendix M cells: bacterial motility is a key feature in vivo; Marchetti M et al.; Rabbit appendix consists mainly of lymphoid follicles (LF) covered by M cells, the specialized antigen-sampling cells of the mucosal immune system, and surrounded by glandular epithelium . Until now, these M cells have been characterized morphologically and histologically by using cellular markers . Here, the adhesion and transport of pathogenic bacteria were investigated to assess the function of M cells of the appendix . We used the enteroinvasive motile Salmonella typhimurium and the rabbit enteropathogenic non-motile Escherichia coli RDEC-1, which are known to target specifically rabbit M cells of Peyer's patches (PPs) . We found that S . typhimurium efficiently attached and was transported through appendix M cells in vivo . In contrast to S . typhimurium, RDEC-1 targeted M cells only ex vivo, when bacteria were allowed to have direct contact with the surface of the follicle . The difference in interaction of the two bacteria with appendix M cells led us to investigate whether this could be correlated with the lack of motility of RDEC-1 . We used an aflagellate mutant of S . typhimurium and found that it had the same infection phenotype as RDEC-1 . Gene complementation restored the efficiency of infection to that of S . typhimurium wild-type strain . In conclusion, we show that M cells of the appendix display features of the canonical M cells of PP, since they efficiently sample luminal pathogenic bacteria . However, due to the morphology of the appendix, motile bacteria appear to be more potent in their interactions with appendix M cells.

Mutat Res, 2004 Jun 13, 560(2), 199 - 201
6-Dimethylaminopurine (6-DMAP), which is used to produce most cloned animals, is mutagenic in Salmonella typhimurium TA1535; Katoh M et al.; 6-Dimethylaminopurine (6-DMAP) and cycloheximide have recently been used for successful production of cloned mammals . We investigated whether 6-DMAP or cycloheximide are mutagenic agents in the Ames test . Whereas cycloheximide showed no differences compared to the negative control in any of the tester strains, 6-DMAP was clearly mutagenic in Salmonella typhimurium strain TA1535 . Here, we strongly propose that innocuous chemicals be used in the production of cloned animals.

J Vet Med A Physiol Pathol Clin Med, 2003 Dec, 50(10), 484 - 7
Expression of inflammatory cytokines (TNF-alpha, IL-1, IL-6 and IL-8) in colon of pigs naturally infected with Salmonella typhimurium and S . choleraesuis; Cho WS et al.; The expression of mRNA encoding tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6 and IL-8 was studied, by in situ hybridization with a non-radioactive digoxigenin-labelled probe, in formalin-fixed, paraffin wax-embedded colonic tissue from pigs naturally infected with Salmonella typhimurium and S . choleraesuis . By in situ hybridization, a distinct positive signal for TNF-alpha, IL-1, IL-6 and IL-8 was detected in colon from all 12 infected pigs . Hybridization signals for all four inflammatory cytokines were detected primarily inflammatory cells infiltrating the lamina propria and submucosa . In comparison, expression of all four inflammatory cytokines was minimal in non-lesional colon of infected pigs and in normal colon from control pigs . The results suggest that these cytokines play an important role in the pathophysiological processes in porcine salmonellosis.

J Mol Microbiol Biotechnol, 2003, 6(3-4), 211 - 8
Eukaryotic promoters can direct protein synthesis in Gram-negative bacteria; Goussard S et al.; Intracellular bacteria can act as DNA delivery vectors into mammalian cells . Transfer of genetic information can be monitored by screening for cellular expression of a reporter gene under the control of an eukaryotic promoter . However, intracellular bacteria can also efficiently deliver heterologous proteins in the cell cytosol . We have studied the activity of the eukaryotic PCMV promoter in Escherichia coli and Salmonella typhimurium using the lacZ and gfp genes as reporters and determined its strength relative to those of PRSV and PSV40 in E . coli . We found substantial heterologous activity of fragments carrying the PCMV and PRSV promoters, the strength of PRSV being greater than that of PCMV, but did not detect any PSV40 activity in E . coli . The green fluorescent protein (GFP) synthesized in E . coli was transferred to COS-1 cells where it was detectable and stable . Insertion of a transcription terminator or deletion of the bacterial ribosome binding site downstream from PCMV led to the silencing of the promoter in bacteria but not in mammalian cells . These observations should incite to exert caution when interpreting data on the DNA transfer from bacteria to mammallian cells when the genes of interest are under the control of the PCMV or PRSV promoter .

J Food Prot, 2004 May, 67(5), 1014 - 6
Inactivation of pathogenic bacteria by cucumber volatiles (E,Z)-2,6-nonadienal and (E)-2-nonenal; Cho MJ et al.; The effects of (E,Z)-2,6-nonadienal (NDE) and (E)-2-nonenal (NE) on Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium were investigated . A suspension of each organism of 6 to 9 log CFU/ml was incubated for 1 h at 37 degrees C in brain heart infusion solution that contained 0 to 500 or 1,000 ppm of NDE or NE . Depending on concentration, exposure to either NDE or NE caused a reduction in CFU of each organism . Treatment with 250 and 500 ppm NDE completely eliminated viable B . cereus and Salmonella Typhimurium cells, respectively . L . monocytogenes was the most resistant to NDE, showing only about a 2-log reduction from exposure to 500 ppm for 1 h . Conversely, this concentration of NDE caused a 5.8-log reduction in E . coli O157:H7 cells . NE was also effective in inactivating organisms listed above . A higher concentration of NE, 1,000 ppm, was required to kill E . coli O157:H7, L . monocytogenes, or Salmonella Typhimurium compared with NDE . In conclusion, both NDE and NE demonstrated an apparent bactericidal activity against these pathogens.

J Food Prot, 2004 May, 67(5), 952 - 9
Molecular weight of chitosan influences antimicrobial activity in oil-in-water emulsions; Zivanovic S et al.; The objective of this study was to evaluate the antimicrobial efficiency of chitosans in oil-in-water emulsions . Model emulsions were prepared with 20% corn oil, 1.5% Tween 20, 1.5% Trypticase soy broth, 0.58% acetic acid, and chitosan polysaccharide or chitosan oligosaccharide in concentrations of 0, 0.1, 0.2, 0.5, and 0.7% . A control containing HCl was included to determine the role of acetic acid in the overall antibacterial activity . The pH of samples and controls was adjusted to 4.5 . Emulsions were inoculated with Listeria monocytogenes (strains Scott A and 310) or Salmonella Typhimurium DT104 (strains 2486 and 2576) at a level of 10(7) CFU/ml . Inoculated emulsions were incubated at 10 and 25 degrees C for 4 days and analyzed for bacterial count every 24 h . Both tested Salmonella strains were more susceptible to acetic acid than Listeria . However, L . monocytogenes was more affected by chitosan than either Salmonella strain . During the storage at 25 degrees C, initial inoculum in the emulsions with 0.58% acetic acid and 0.1% chitosan polysaccharide was reduced to below the detection limits after 24, 48, 72, or 96 h for L . monocytogenes 310, Salmonella Typhimurium DT104 2576, Salmonella Typhimurium DT104 2486, or L . monocytogenes Scott A, respectively . Chitosan oligosaccharide was less effective against all tested bacteria and showed a concentration-dependent effect . The antimicrobial efficacy of chitosan was reduced at 10 degrees C, and reduction of microbial loads was delayed for approximately 24 h compared with 25 degrees C . Results suggest that addition of 0.1% chitosan polysaccharide would be sufficient to ensure the microbial safety of oil-in-water emulsions regardless of storage temperature.

Natl Toxicol Program Tech Rep Ser, 2004 Feb, (514), 1 - 281
NTP toxicology and carcinogenesis studies of trans-cinnamaldehyde (CAS No . 14371-10-9) in F344/N rats and B6C3F1 mice (feed studies); National Toxicology Program; Cinnamaldehyde is used in foods, beverages, medical products, perfumes, cosmetics, soaps, detergents, creams, and lotions . Cinnamaldehyde has been used as a filtering agent and a rubber reinforcing agent and is used as a brightener in electroplating processes, as an animal repellent, as an insect attractant, and as an antifungal agent . trans-cinnamaldehyde was nominated for study by the Food and Drug Administration based on its widespread use as a flavor and fragrance ingredient and its structural similarity to cinnamyl anthranilate and 3,4,5-trimethoxy cinnamaldehyde, two known rodent carcinogens . Male and female F344/N rats and B6C3F1 mice were exposed to trans-cinnamaldehyde (at least 95% pure) in feed for 3 months or 2 years . Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood erythrocytes . 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were fed diets containing 4,100, 8,200, 16,500, or 33,000 ppm microencapsulated trans-cinnamaldehyde (equivalent to average daily doses of approximately 275, 625, 1,300, or 4,000 mg trans-cinnamaldehyde/kg body weight to males and 300, 570, 1,090, or 3,100 mg/kg to females) for 3 months . Additional groups of 10 male and 10 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls) . All rats survived to the end of the study . Mean body weights of all exposed groups of males and 16,500 and 33,000 ppm females were significantly less than those of the vehicle controls, and 33,000 ppm males lost weight during the study . Feed consumption by exposed groups of males and females was less than that by the vehicle controls throughout the study . Clinical chemistry results of these studies indicated that trans-cinnamaldehyde administration, at the doses selected, induced an increase in serum bile acid concentration that suggests a hepatic effect in both male and female rats . Gross lesions observed at necropsy included multifocal to diffuse white nodules of the forestomach mucosa in 8,200 ppm or greater males and females . Increased incidences of nonneoplastic lesions of the forestomach included squamous epithelial hyperplasia in 8,200 ppm or greater males and females and chronic active inflammation in 33,000 ppm males and 16,500 and 33,000 ppm females . 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 4,100, 8,200, 16,500, or 33,000 ppm microencapsulated trans-cinnamaldehyde (equivalent to average daily doses of approximately 650, 1,320, 2,550, and 5,475 mg/kg to males and 625, 1,380, 2,680, and 5,200 mg/kg to females) for 3 months . Additional groups of 10 male and 10 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls) . One vehicle control male, one 4,100 ppm male, and one 33,000 ppm male died during the first week of the study due to inanition that resulted from difficulty with the feeder . Five 16,500 ppm and eight 33,000 ppm male mice died during weeks 2 and 3 due to unpalatability of the dosed feed . Mean body weights of all exposed groups of males and of females exposed to 8,200 ppm or greater were significantly less than those of the vehicle controls . Feed consumption by 16,500 and 33,000 ppm mice was less than that by the vehicle controls during weeks 1 and 2 . The incidence of squamous epithelial hyperplasia of the forestomach mucosa in 33,000 ppm females was significantly increased, and olfactory epithelial degeneration of the nasal cavity occurred in 16,500 and 33,000 ppm males and females . 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female F344/N rats were fed diets containing 1,000, 2,100, or 4,100 ppm microencapsulated trans-cinnamaldehyde for 2 years . Additional groups of 50 male and 50 female rats received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls) . Dietary concentrations of 1,000, 2,100, or 4,100 ppm delivered average daily doses of approximately 50, 100, or 200 mg/kg to males and females . Survival of 4,100 ppm males was greater than that of the vehicle controls . Mean body weights of 4,100 ppm males and females were generally less than those of the vehicle controls throughout the study . Feed consumption by 2,100 and 4,100 ppm males and 4,100 ppm females was less than that by the vehicle controls at the beginning and end of the study . There were no neoplasms or nonneoplastic lesions that were attributed to exposure to trans-cinnamaldehyde . 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were fed diets containing 1,000, 2,100, or 4,100 ppm microencapsulated trans-cinnamaldehyde for 2 years . Additional groups of 50 male and 50 female mice received untreated feed (untreated controls) or feed containing placebo microcapsules (vehicle controls) . Dietary concentrations of 1,000, 2,100, or 4,100 ppm delivered average daily doses of approximately 125, 270, or 550 mg/kg to males and females . Survival of males in the 2,100 ppm group was less than that of the vehicle control group . Mean body weights of 2,100 and 4,100 ppm males and females were generally less than those of the vehicle controls throughout the study, and mean body weights of 1,000 ppm males were less after week 74 . Feed consumption by exposed mice was similar to that by the vehicle controls . The incidences of olfactory epithelial pigmentation in 4,100 ppm males and in 2,100 and 4,100 females were significantly greater than those in vehicle controls . There were no neoplasms that were attributed to exposure to trans-cinnamaldehyde . GENETIC TOXICOLOGY: trans-cinnamaldehyde was mutagenic in S . typhimurium strain TA100 in the presence of induced mouse liver S9 activation enzymes only . All other strain and activation combinations, including the standard rat and hamster derived liver S9 fractions yielded negative results . trans-cinnamaldehyde induced sister chromatid exchanges in Chinese hamster ovary cells with and without induced rat liver S9 activation . No significant increase in the frequency of chromosomal aberrations occurred in Chinese hamster ovary cells cultured with trans-cinnamaldehyde, with or without induced rat liver S9 . In tests for induction of germ cell genetic damage in male Drosophila melanogaster, trans-cinnamaldehyde induced a significant increase in the frequency of sex-linked recessive lethal mutations when administered by abdominal injection; however, no induction of reciprocal translocations occurred in germ cells of treated males . No increase in the frequency of micronucleated erythrocytes was observed in peripheral blood of male or female mice administered trans-cinnamaldehyde in dosed feed for 3 months . CONCLUSIONS: Under the conditions of this 2-year feed study, there was no evidence of carcinogenic activity of transcinnamaldehyde in male or female F344/N rats exposed to 1,000, 2,100, or 4,100 ppm . There was no evidence of carcinogenic activity of trans-cinnamaldehyde in male or female B6C3F1 mice exposed to 1,000, 2,100, or 4,100 ppm . Exposure to trans-cinnamaldehyde resulted in olfactory epithelial pigmentation in male and female mice.

Toxic Rep Ser, 2004 Apr, (67), 1 - G12
NTP technical report on the toxicity studies of 2- and 4-Methylimidazole (CAS No . 693-98-1 and 822-36-6) administered in feed to F344/N rats and B6C3F1 mice; Chan PC; National Toxicology Program et al.; {Structure-see text} 2-Methylimidazole and 4-methylimidazole are intermediate/starting materials or components in the manufacture of pharmaceuticals, photographic and photothermographic chemicals, dyes and pigments, agricultural chemicals, and rubber; these chemicals have been identified as undesirable by-products in several foods and have been detected in mainstream and sidestream tobacco smoke . The National Cancer Institute nominated 2- and 4-methylimidazole as candidates for toxicity and carcinogenicity studies . Toxicity studies were carried out in male and female F344/N rats and B6C3F1 mice . Animals were exposed to 2- or 4-methylimidazole in feed for 15 days or 14 weeks; clinical pathology studies were conducted in the 14-week studies on days 8, 29, and 86 and at week 14 . Genetic toxicity studies were conducted in Salmonella typhimurium, rat and mouse bone marrow, and mouse peripheral blood . Groups of five male and five female rats and mice were fed diets containing 0, 1,200, 3,300, or 10,000 ppm 2-methylimidazole (equivalent to average daily doses of approximately 115, 290, or 770 mg 2-methylimidazole/ kg body weight to rats; 220, 640, or 2,100 mg/kg to male mice; 300, 800, or 2,400 to female mice) for 15 days . Groups of five male and five female rats and mice were fed diets containing 0, 300, 800, or 2,500 ppm 4-methylimidazole (equivalent to average daily doses of approximately 30, 80, or 220 mg/kg for rats and 65, 170, or 500 mg/kg for mice) for 15 days . In the 15-day 2-methylimidazole studies, all animals survived to the end of the studies . The mean body weights of 10,000 ppm male rats and female mice were significantly less than those of the controls . Feed consumption by 10,000 ppm male and female rats was reduced . Enlarged thyroid glands were observed in 3,300 and 10,000 ppm male and female rats . The incidences of diffuse hyperplasia of follicular cells of the thyroid gland in 3,300 and 10,000 ppm male and female rats and pars distalis hypertrophy of the pituitary gland in 3,300 and 10,000 ppm males and 10,000 ppm females were increased compared to the controls . In all exposed groups of male and female mice, the incidences and severities of follicular cell hypertrophy of the thyroid gland and the severities of hematopoietic cell proliferation of the spleen generally increased with increasing exposure concentration . In the 4-methylimidazole studies, all animals survived to the end of the studies, and there were no significant differences in mean body weights, clinical findings, organ weights, or gross or microscopic lesions between exposed and control groups . Groups of 10 male and 10 female rats and mice were fed diets containing 0, 625, 1,250, 2,500, 5,000, or 10,000 ppm 2- or 4-methylimidazole (equivalent to average daily doses of approximately 40, 80, 160, 300, or 560 mg/kg 2- or 4-methylimidazole to rats; and 100, 165, 360, 780, or 1,740 mg/kg 2-methylimidazole or 100, 240, 440, 915, or 1,840 mg/kg 4-methylimidazole to male mice; and 90, 190, 400, 800, or 1,860 mg/kg 2-methylimidazole or 110, 240, 540, 1,130, or 3,180 mg/kg 4-methylimidazole to females) for 14 weeks . All animals survived to the end of the 14-week 2-methylimidazole studies . Compared to the controls, the mean body weights were significantly decreased in groups of male rats and mice exposed to 2,500 ppm or greater and in 5,000 and 10,000 ppm female rats and mice . In rats, 2-methylimidazole induced a transient erythrocytosis in females and a minimal, exposure concentration-related, microcytic, normochromic, nonresponsive anemia . 2-Methylimidazole increased thyroid-stimulating hormone concentrations and decreased thyroxine and triiodothyronine concentrations of male and female rats in an exposure concentration-related manner . 2-Methylimidazole induced a mild to moderate, exposure concentration-related, macrocytic, hyperchromic, responsive anemia in mice . Triiodothyronine concentrations were increased in exposed male and female mice, and thyroxine concentrations were decreased in exposed females . Relative to the control groups, clinical chemistry evaluations on day 29 and at week 14 identified decreases in alanine aminotransferase concentrations and total protein and albumin concentrations of rats . In the 2-methylimidazole studies, absolute spleen weights were significantly increased in all exposed groups of male rats . The heart and liver weights were increased in all exposed groups of male mice, as were the spleen weights of female mice exposed to 2,500 ppm or greater . Spermatid heads per testis and mean spermatid count were significantly decreased in 10,000 ppm male rats . The estrous cycle of 10,000 ppm female rats was significantly increased . Gross pathology observations included enlarged thyroid glands, small uteri, and mottled spleen in 5,000 and 10,000 ppm mice . The incidences of diffuse follicular cell hyperplasia of the thyroid gland were significantly increased in male rats exposed to 1,250 ppm or greater and female rats exposed to 2,500 ppm or greater . The incidence of testicular degeneration was significantly increased in 10,000 ppm male rats, and two males in the 10,000 ppm group had follicular cell adenoma of the thyroid gland . In mice, there were generally significant increases in the incidences of follicular cell hypertrophy of the thyroid gland, hematopoietic cell proliferation of the spleen, and hemosiderin pigmentation of the renal tubule in males exposed to 1,250 ppm or greater and females exposed to 2,500 ppm or greater . In the 14-week 4-methylimidazole studies, one 10,000 ppm male mouse was found dead during week 4, and seven 10,000 ppm female mice were found dead during weeks 1 and 2 . Mean body weights were significantly less than those of the controls for male rats exposed to 2,500 ppm or greater, 5,000 and 10,000 ppm female rats, male mice exposed to 1,250 ppm or greater, and all exposed groups of female mice . Reduced feed consumption was observed in 5,000 and 10,000 ppm male and female rats . Clinical findings included nasal/eye discharge, ruffled fur, thinness, ataxia, and abnormal breathing in rats, and ruffled fur and dull coats in female mice . On days 29 and 82, functional observations in 5,000 and 10,000 ppm rats included labored or increased respiration, mild tremors, walking on tiptoes, hunched posture, piloerection, crouching over, impaired coordination of movement, ataxia, and pupillary constriction . 4-Methylimidazole induced a transient erythrocytosis and a minimal, exposure concentration-related, microcytic, normochromic, nonresponsive anemia in male and female rats . Clinical chemistry evaluations generally showed a cholestatic effect in exposed male and female rats . At week 14, there was a significant decrease in total protein and albumin concentrations of female rats exposed to 5,000 or 10,000 ppm . In mice, 4-methylimidazole induced a macrocytic, hyperchromic, responsive anemia and, particularly in males, increases in triiododthyronine concentrations and transient decreases in thyroxine concentrations . In the 4-methylimidazole studies, the liver weights of male rats exposed to 2,500 ppm or greater were significantly increased; spleen weights of female rats exposed to 2,500 ppm or greater were decreased . The absolute liver weight was decreased in 10,000 ppm male mice, and relative weights were significantly increased in all exposed groups of mice . In female mice, there was a significant decrease in the absolute weights and increase in the relative weights of the heart, right kidney, and liver in groups exposed to 2,500 ppm or greater . The epididymal spermatozoal concentration was significantly increased in 5,000 ppm male rats . Gross pathology observations included pale livers in male rats exposed to 2,500 ppm or greater and small testes and uteri in 10,000 ppm male and female rats . Microscopic analysis identified significantly increased incidences of cytoplasmic hepatocyte vacuolization of the liver of male rats exposed to 2,500 ppm or greater and 10,000 ppm female rats, hypospermia of the epididymis in 10,000 ppm male rats, atrophy and inflammation of the prostate gland in 10,000 ppm male rats, and degeneration of the testes in 5,000 and 10,000 ppm male rats . 2-Methylimidazole and 4-methylimidazole were negative in the S . typhimurium mutation assay when tested in strains TA97, TA98, TA100, and TA1535, with and without S9 activation enzymes . Testing of 2-methylimidazole in vivo for induction of chromosomal damage, as measured by micronucleated erythrocyte frequency, produced mixed results . When administered by intraperitoneal injection three times at 24-hour intervals, 2-methylimidazole produced negative results in bone marrow micronucleus tests in rats and mice . However, in the 14-week study of 2-methylimidazole, a significant exposure-related increase in the frequency of micronucleated normochromatic erythrocytes was noted in peripheral blood of male and female mice . In vivo, 4-methylimidazole produced uniformly negative results in three-injection bone marrow micronucleus tests in rats and mice and in 14-week peripheral blood micronucleus tests in male and female mice.

Can Vet J, 2004 Apr, 45(4), 321 - 3
Necrotizing hepatitis associated with enteric salmonellosis in an alpaca; Saulez MN et al.; Salmonella typhimurium was isolated from the feces of an alpaca suffering anorexia and weight loss . Multifocal necrotizing and suppurative hepatitis consistent with bacterial infection was found in the liver biopsies . Enteric salmonellosis may be associated with milder physical and clinicopathological changes in camelids than in other large animal species.

Cent Eur J Public Health, 2004 Mar, 12 Suppl, S72 - 5
Mutagenicity of airborne particulate matter PM10; Pastorkova A et al.; Mutagenic activity of extractable organic matter (EOM), from airborne particles collected over winters in four towns of Czech Republic, was investigated by the means of Salmonella typhimurium indicator strains TA98 and YG1041 using the Ames plate incorporation assay . Mutagenicity of all tested samples showed significant dose-related increase in number of revertants per mg of EOM . The direct mutagenic potency detected with TA98 increased further in the presence of external metabolic activation . The mutagenic potency detected with YG1041 was about two orders of magnitude higher than that detected with TA98 . The mutagenicity results correlated with the concentrations of the polycyclic aromatic hydrocarbons (PAHs) determined by GC/MS . Local differences in mutagenicity, expressed as numbers of revertants per m3 of air, were observed with the highest values in Prague air samples . For routine ambient air mutagenicity monitoring, the use of YG1041 and the plate incorporation test are recommendable.

Int J Food Microbiol, 2004 Jun 1, 93(2), 165 - 73
Generation of bactericidal and mutagenic components by pulsed electric field treatment; Reyns KM et al.; Inactivation of stationary phase Escherichia coli, Salmonella Typhimurium and Listeria innocua (10(8) CFU/ml) by high intensity pulsed electric fields (PEF) was studied in water and different buffers at pH 7.0 . The fraction of survivors after PEF treatment with 300 pulses (5 Hz) of 26.7 kV/cm and a pulse width of 2 micros varied between 0.050% and 55%, but was always lower in Tris-HCl buffer than in HEPES-KOH buffer and water . When cell suspensions were stored for 24 h at 25 degrees C after PEF treatment, the survivor fraction further decreased, except for E . coli in water and HEPES-KOH . By following the survival of untreated cells added to water or buffers that were previously PEF treated, this secondary inactivation could be ascribed to the formation of bactericidal components as a result of PEF treatment . Buffers and water containing 10 mM NaCl became bactericidal against all three bacteria upon PEF treatment, and the bactericidal effect could be neutralized by thiosulfate, suggesting that chlorine and/or hypochlorite had been formed . Also in the absence of Cl- ions, PEF treated water and buffers had bactericidal properties, but the specificity of the bactericidal effects against different bacteria differed depending on the buffer used . In the Ames mutagenicity test using His- S . Typhimurium mutant strains, PEF treated Tris buffers containing 10 mM Cl- ions, as well as PEF treated grape juice showed a mutagenic effect . The implications of these findings for the safety of PEF treated foods are discussed .

Int J Food Microbiol, 2004 May 15, 93(1), 63 - 72
Binding interaction studies of the immobilized Salmonella typhimurium with extracellular matrix and muscle proteins, and polysaccharides; Medina MB; Our research attempts to understand the real-time interactions of immobilized Salmonella typhimurium with extracellular membrane proteins (collagen I, fibronectin and laminin) and muscle proteins (actin and myosin) . Salmonella cells were immobilized on the sensor chip of a surface plasmon resonance (SPR) biosensor . Typical results showed that collagen I and myosin had higher binding responses to the S . typhimurium surface but laminin, actin and fibronectin had lower binding responses . The binding kinetics of collagen I and Salmonella cell surface showed an apparent dissociation and association rate constants of 3.90 E-4 s(-1) and 1.07 E+4 mol(-1) s(-1) . Using the model system developed in our laboratory, the interactions of carrageenans and other polysaccharides with collagen and the Salmonella sensor surface were evaluated . The kappa-carrageenans blocked 92-100% binding of collagen to the Salmonella surface, while sodium alginate and low methoxy pectin blocked 50% and 18% binding, respectively . These biosensor studies allowed the rapid evaluation of compounds that may prevent bacterial attachment to poultry skin and carcasses, thus reducing pathogen contamination of poultry foods .

J Microbiol Methods, 2004 Jun, 57(3), 297 - 308
VEX-capture: a new technique that allows in vivo excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments; Wilson JW et al.; We have developed a novel and easily performed procedure for the targeted excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments . This procedure, called Vector-mediated excision and Capture (VEX-Capture), represents a new molecular tool for the convenient manipulation and exchange of large (20-40+ kb) bacterial genomic fragments . VEX-Capture utilizes lox/Cre-mediated site-specific recombination for excision of the targeted genomic segment and homologous recombination for cloning of the excised DNA section onto a self-transmissible, broad-host-range IncP plasmid . The "captured" genomic DNA segment can then be transferred to a wide variety of Gram-negative hosts for basic research and bioengineering purposes . To demonstrate the utility and function of VEX-Capture, we have excised and cloned three separate genomic islands from the Salmonella typhimurium chromosome ranging in size from 26.7 to 40.0 kb . To test the ability of these islands to be established in different bacterial hosts, we transferred them to six other Gram-negative species and monitored their establishment via phenotypic and molecular analysis . RT-PCR was used to assay the expression of selected S . typhimurium island genes in the different species . This analysis led to the discovery that an island-encoded master regulator of S . typhimurium virulence functions is expressed in a species-specific manner . Our results demonstrate the potential for VEX-Capture to be used as a convenient genetic technique for fundamental biological applications in a wide variety of bacterial species.

Biol Pharm Bull, 2004 May, 27(5), 679 - 83
A citrus flavonoid hesperidin suppresses infection-induced endotoxin shock in mice; Kawaguchi K et al.; Administration of a citrus flavonoid hesperidin (HES) to mice before LPS challenge significantly reduced tumor necrosis factor (TNF)-alpha production in a dose-dependent manner . Treatment of HES 3 h before intraperitoneal (i.p.) infection with 10(8) CFU Salmonella typhimurium aroA resulted in rescue from lethal shock as similar to LPS-nonresponder mice . Not only bacterial numbers in livers and spleens but also plasma LPS levels significantly decreased by pretreatment with HES . In addition, HES markedly suppressed plasma levels of TNF-alpha and high mobility group box chromosomal protein 1 (HMGB-1), decreased the number of apoptotic cells in livers and normalized the activated states of blood coagulation factors such as prothrombin time and platelet numbers caused by infection . Pretreatment of LPS with HES suppressed the chromogenic Limulus reaction.

Vet Immunol Immunopathol, 2004 Mar, 98(1-2), 31 - 41
Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5; Abdalla SA et al.; Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily . In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5 . The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)) . The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II . Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius . The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly . The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line . With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5 . Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24 . Thus, both genes were expressed in the same tissues and cell line among examined materials . These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.

Best Pract Res Clin Gastroenterol, 2004 Apr, 18(2), 405 - 19
Defensin-mediated innate immunity in the small intestine; Ouellette AJ; Epithelial cells contribute to innate immunity by releasing antimicrobial peptides (AMPs) onto mucosal surfaces . In the small bowel, Paneth cells at the base of the crypts of Lieberkuhn secrete alpha-defensins and additional AMPs at high levels in response to cholinergic stimulation and when exposed to bacterial antigens . The release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells that renew the epithelial cell monolayer from colonization by potentially pathogenic microbes and to confer protection from enteric infection . The most compelling evidence for a Paneth cell role in enteric resistance to infection is evident from studies of mice transgenic for a human Paneth cell alpha-defensin, HD-5, which are completely immune to infection and systemic disease from orally administered Salmonella typhimurium . Alpha-defensins in Paneth cell secretions may also interact with bacteria in the intestinal lumen above the crypt-villus boundary and influence the composition of the enteric microbial flora, but that remains to be demonstrated.

J Infect Dis, 2004 May 15, 189(10), 1914 - 20 Epub 2004 Apr 27.
Helicobacter pylori flagellin evades toll-like receptor 5-mediated innate immunity; Gewirtz AT et al.; Helicobacter pylori colonizes the human stomach for decades unless pharmacologically eradicated . We hypothesized that this flagellated pathogen escapes immune clearance, in part, by avoiding detection by the flagellin receptor Toll-like receptor 5 (TLR5) . In contrast to other gram-negative microbes, H . pylori did not release flagellin . Furthermore, recombinant H . pylori flagellin (FlaA) was significantly less potent (1000-fold) than Salmonella typhimurium flagellin in activating TLR5-mediated interleukin (IL)-8 secretion . TLR5 can mediate flagellin-induced IL-8 secretion via p38 mitogen-activated protein kinase signaling; however, compared with potent induction by S . typhimurium flagellin, H . pylori FlaA-dependent p38 activation was substantially attenuated . In addition, disruption of H . pylori flaA decreased motility but had no effect on H . pylori-induced IL-8 secretion, which indicates that H . pylori flagellin plays no role in activating epithelial orchestration of inflammation . We conclude that H . pylori evades TLR5-mediated detection, which may contribute to its long-term persistence in individual hosts.

Curr Biol, 2004 May 4, 14(9), 806 - 11
Recognition of bacteria in the cytosol of Mammalian cells by the ubiquitin system; Perrin AJ et al.; Recent studies have suggested the existence of innate host surveillance systems for the detection of bacteria in the cytosol of mammalian cells . The molecular details of how bacteria are recognized in the cytosol, however, remain unclear . Here we examined the fate of Salmonella typhimurium, a gram-negative bacterial pathogen that can infect a variety of hosts, in the cytosol of mammalian cells . These bacteria typically occupy a membrane bound compartment, the Salmonella-containing vacuole (SCV), in host cells . We show that some wild-type bacteria escape invasion vacuoles and are released into the cytosol . Subsequently, polyubiquitinated proteins accumulate on the bacterial surface, a response that was witnessed in several cell types . In macrophages but not epithelial cells, the proteasome was observed to undergo a dramatic subcellular relocalization and become associated with the surface of bacteria in the cytosol . Proteasome inhibition promoted replication of S . typhimurium in the cytosol of both cell types, in part through destabilization of the SCV . Surprisingly, the cytosol-adapted pathogen Listeria monocytogenes avoided recognition by the ubiquitin system by using actin-based motility . Our findings indicate that the ubiquitin system plays a major role in the recognition of bacterial pathogens in the cytosol of mammalian cells.

Br Poult Sci, 2004 Feb, 45(1), 41 - 8
Morphology of the small intestinal mucosal surface of broilers in relation to age, diet formulation, small intestinal microflora and performance; van Leeuwen P et al.; 1 . Three experiments were performed to relate morphological characteristics of the small intestinal mucosal surface to age, dietary factors, small intenstinal microflora and performance of broilers . Characterisation of the small intestinal mucosal surface using a dissecting microscope was based on the orientation of the villi, villus shape and the presence of convoluted villi . 2 . In Trial 1, the morphological changes of the mucosal surface were studied weekly in the period from 7 to 28 d of age . At d 7 mainly tongue- and leaf-shaped villi together with some ridge-shaped ones were observed in the middle section of the small intestine, displaying a regular zigzag pattern on 53% of the mucosal surface . During the period from d 7 to 14, the area with ridge-shaped villi increased from 7 to 63% and did not change significantly over the next 2 weeks . 3 . In Trial 2, three protein sources, soy isolate (SI), wheat gluten (WG), hydrolysed wheat gluten (HWG) and SI with added L-glutamine (SI + Gln), were studied with respect to their effect as dietary components on villus morphology in the mid-small intestine and performance . Diets were fed with (0 to 14 d) and without pectin (14 to 21 d) . Feed conversion ratio on the HWG diet improved in comparison to the native WG diet . During the period 0 to 14 d of age the mucosal area with zigzag-oriented villi increased when the pectin diet was supplemented with Gln . Moreover, weight gain of birds fed the SI + Gln diet increased in the period 41 to 21 d . 4 . In Trial 3, a study was made of the morphological response of the villi to a stimulation of microbial activity in the digesta after addition of highly methylated pectin to the soybean meal (SBM) diet . This was performed with and without inoculation of a non-virulent Salmonella typhimurium on d 7 . By d 21 the birds fed the pectin diet showed impaired weight gain and higher feed conversion . The pectin affected the mucosal surface by decreasing the area with the zigzag pattern and increasing the area with convoluted, mainly ridge-shaped villi . The Salmonella typhimurium infection increased the effects of pectin on performance and mucosal morphology.

FEBS Lett, 2004 Apr 30, 564(3), 229 - 33
Structural basis for ion conduction and gating in ClC chloride channels; Dutzler R; Members of the ClC family of voltage-gated chloride channels are found from bacteria to mammals with a considerable degree of conservation in the membrane-inserted, pore-forming region . The crystal structures of the ClC channels of Escherichia coli and Salmonella typhimurium provide a structural framework for the entire family . The ClC channels are homodimeric proteins with an overall rhombus-like shape . Each ClC dimer has two pores each contained within a single subunit . The ClC subunit consists of two roughly repeated halves that span the membrane with opposite orientations . This antiparallel architecture defines a chloride selectivity filter within the 15-A neck of a hourglass-shaped pore . Three Cl(-) binding sites within the selectivity filter stabilize ions by interactions with alpha-helix dipoles and by chemical interactions with nitrogen atoms and hydroxyl groups of residues in the protein . The Cl(-) binding site nearest the extracellular solution can be occupied either by a Cl(-) ion or by a glutamate carboxyl group . Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays . These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl(-) ion.

Eur J Pharmacol, 2004 Apr 26, 491(1), 61 - 8
Gastric oxidative stress and hemorrhagic ulcer in Salmonella typhimurium-infected rats; Hung CR et al.; Infection of Salmonella typhimurium (Salmonella typhi) can lead to various organ diseases . This research first proposed that Salmonella typhi-infection could result in gastric oxidative stress and hemorrhagic ulcers that were ameliorated by ofloxacin, lysozyme chloride and several antioxidants, including exogenous glutathione (GSH), allopurinol and dimethylsulfoxide (DMSO) . Male Wistar rats were given intrajejunally the live culture of Salmonella typhi {1 x 10(10) colony-forming unit (CFU)/rat} and followed by deprivation of food for 36 h . Age-matched control rats received vehicle only . Rat stomachs were irrigated for 3 h with either normal saline or a simulated gastric juice containing 100 mM HCl, 17.4 mM pepsin and 54 mM NaCl . Infection of Salmonella typhi produced an aggravation of ulcerogenic factors, including enhancing gastric acid back-diffusion, mucosal lipid peroxide generation and hemorrhagic ulcer as well as an attenuation of mucosal GSH level . Intragastric irrigation of gastric juice caused further aggravation of these gastric biochemical parameters . This exacerbation of ulcerogenic factors was abolished by pretreatment of ofloxacin and lysozyme chloride . Antioxidants, such as reduced GSH, allopurinol and DMSO also produced significant (P<0.05) amelioration of gastric damage in Salmonella typhi-infected rats . In conclusion, infection of Salmonella typhi substantially caused gastric oxidative stress and disruption of gastric mucosal barriers, consequently resulted in gastric hemorrhagic ulcerations that were effectively ameliorated by ofloxacin, lysozyme chloride and various antioxidants.

Int Immunol, 2004 Jun, 16(6), 819 - 29 Epub 2004 Apr 19.
Functional comparison of the mouse DC-SIGN, SIGNR1, SIGNR3 and Langerin, C-type lectins; Takahara K et al.; The mouse (m) DC-SIGN family consists of several homologous type II transmembrane proteins located in close proximity on chromosome 8 and having a single carboxyl terminal carbohydrate recognition domain . We first used transfected non-macrophage cell lines to compare the polysaccharide and microbial uptake capacities of three of these lectins--DC-SIGN, SIGNR1 and SIGNR3--to another homologue mLangerin . Each molecule shares a potential mannose-recognition EPN-motif in its carbohydrate recognition domain . Using an anti-Tag antibody to follow Tag-labeled transfectants, we found that each molecule could be internalized, although the rates differed . However, mDC-SIGN was unable to take up FITC-dextran, FITC-ovalbumin, zymosan or heat-killed Candida albicans . The other three lectins showed distinct carbohydrate recognition properties, assessed by blocking FITC-dextran uptake at 37 degrees C and by mannan binding activity at 4 degrees C . Furthermore, only SIGNR1 was efficient in mediating the capture by transfected cells of Gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, while none of the lectins tested were competent to capture Gram-positive bacteria, Staphylococcus aureus . Interestingly, transfectants with SIGNR1 lacking the cytoplasmic domain were capable of binding FITC-zymosan in a manner that was abolished by EDTA or mannan, but not laminarin . In addition, resident peritoneal CD11b+ cells expressing SIGNR1 bound zymosan at 4 degrees C in concert with a laminarin-sensitive receptor . Therefore these homologous C-type lectins have distinct recognition patters for microbes despite similarities in the carbohydrate recognition domains .

Biosens Bioelectron, 2004 Jun 15, 19(11), 1497 - 504
Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium; Oh BK et al.; An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium . A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface . The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy . The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR . The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively . The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM) . Consequently, an immunosensor based on SPR for the detection of S . typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml . The current fabrication technique of a SPR immunosensor for the detection of S . typhimurium could be applied to construct other immnosensors or protein chips.

Environ Pollut, 1989, 58(4), 313 - 23
Carbaryl-A selective genotoxicant; Grover IS et al.; Mutagenic effects of carbaryl, a contact insecticide with slight systemic properties, have been investigated employing histidine reversion assay in Salmonella typhimurium strains and in vivo chromosomal aberrations in root meristems of Allium cepa . A detailed investigation revealed that carbaryl did not enhance significantly the frequency of histidine revertants in any of the strains of Salmonella i.e . frameshift mutagen tester (TA98), base pair substitution tester strain (TA1535) and ochre mutant strain (TA102) . The supplementation with S9 mix did not have any appreciable effect . S14 prepared from wheat seedlings also did not enhance the reversion frequency significantly . However, carbaryl induced both clastogenic and physiological types of chromosomal aberration . The spectrum of chromosomal aberrations included c-mitosis, stickiness, vagrant chromosomes, polyploidy multi-polarity, delayed anaphases, end to end joining of chromosomes, chromosome breaks, ring chromosomes and anaphase bridges . The frequency of chromosomal aberrations was reduced by transferring the carbaryl treated bulbs to distilled water for 24 and 48 h . Similarly, recovery in the mitotic index was noticed by such transfer . The differences between the two assays may be attributed to differences in the metabolism of the test organisms.

J Food Prot, 2004 Apr, 67(4), 698 - 705
Characterization of antimicrobial-resistant phenotypes and genotypes among Salmonella enterica recovered from pigs on farms, from transport trucks, and from pigs after slaughter; Gebreyes WA et al.; The main objectives of this study were to determine antimicrobial resistance patterns among Salmonella serotypes and to evaluate the role of transport trucks in dissemination of antimicrobial-resistant strains of Salmonella . Salmonella from groups of nursery and finishing pigs on farms, from trucks, and from pigs after slaughter were compared using serotyping, patterns of antimicrobial resistance, and pulsed-field gel electrophoresis patterns . The five farms included in the study yielded 858 isolates representing 27 Salmonella serovars . The most common resistance observed (80% of all isolates) was to tetracycline; resistance to ampicillin (42%), chloramphenicol (31%), amoxicillin/clavulanic acid (30%), and piperacillin (31%) also were common . We found a correlation between serovar and antimicrobial resistance . High correlation was found between Salmonella Typhimurium var . Copenhagen and chloramphenicol resistance (Spearman rank correlation, rho = 0.7) . Multidrug resistance was observed primarily in Salmonella Typhimurium var . Copenhagen (94%) and Salmonella Typhimurium (93%) and was much less common in the other common serovars, including Salmonella Derby (7%) and Salmonella Heidelberg (8%) . Of the 225 isolates exhibiting the most common pentaresistance pattern in this study, amoxicillin/clavulanic acid-ampicillin-chloramphenicol-piperacillin-tetracycline, 220 (98%) were Salmonella Typhimurium var . Copenhagen, and 86% of the isolates of this serovar had this pattern . Isolates from the trucks were similar, based on pulsed-field gel electrophoresis patterns, to those from the cecum and mesenteric lymph nodes of pigs on two of the farms, suggesting the probable infection of pigs during transport . Class I integrons were also common among various serovars.

J Appl Microbiol, 2004, 96(5), 903 - 12
Cellular effects of monohydrochloride of L-arginine, N-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus; Rodriguez E et al.; AIMS: Here we study the effect of monohydrochloride of L-arginine, N(alpha)-lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 microg ml(-1), respectively . METHODS AND RESULTS: Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane . Cell integrity was altered mainly in the outer membrane of S . typhimurium, but there was no significant change in the cytoplasm . However, in Staph . aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm . Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis . In S . typhimurium the proportion of damaged cells after 24 h was 97% and in Staph . aureus 56.3% . LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7.7 and 3.34 microg ml(-1) for Staph . aureus and S . typhimurium, respectively . Membrane disruption was detected by measuring the proton flow across the membrane . CONCLUSIONS: Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted . SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time the cellular effects of LAE on bacterial cells were studied.

Pharmazie, 2004 Mar, 59(3), 217 - 21
Anti-genotoxic activity of the mushroom Lactarius vellereus extract in bacteria and in mammalian cells in vitro; Mlinaric A et al.; In a previous study we screened a range of mushroom species growing in Slovenia for their anti-genotoxic potential and found Lactarius vellereus to be the most effective . In this study genotoxic and anti-genotoxic activities of methanol extracts of Lactarius vellereus (Fr.: Fr.) Fr . were evaluated in the bacterial reverse mutation test with Salmonella typhimurium TA98 and, in the mammalian cell test with human hepatoma (HepG2) cells, using the comet assay to measure DNA damage . The extract induced no mutations in S . typhimurium TA98 and no DNA damage in HepG2 cells . Against the indirect acting mutagen 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) the extract showed significant, dose dependent antimutagenic activity, while it did not counteract the direct acting mutagen 4-nitroquinoline oxide (4-NQO) . The extract also exerted a protective effect against IQ induced genotoxicity in mammalian cells of human origin . Treatment of HepG2 cells with the L . vellereus extract (125-500 microg/ml) together with IQ, reduced the genotoxic effect of the latter in a dose dependent manner . Our findings show that a methanol extract of L . vellereus is highly protective against IQ induced DNA damage in human derived cells and L . vellereus can be considered as a natural source of antimutagens with potential pharmacological applications in cancer prevention.

Microbiology, 2004 Apr, 150(Pt 4), 775 - 83
Bile-salt-mediated induction of antimicrobial and bile resistance in Salmonella typhimurium; Prouty AM et al.; By DNA microarray, the Salmonella typhimurium marRAB operon was identified as being bile-activated . Transcriptional assays confirm that marRAB is activated in the presence of bile and that this response is concentration-dependent . The bile salt deoxycholate is alone able to activate transcription, while there was no response in the presence of other bile salts tested or a non-ionic detergent . Deoxycholate is able to interact with MarR and interfere with its ability to bind to the mar operator . In addition, incubation of salmonellae in the presence of sublethal concentrations of bile is able to enhance resistance to chloramphenicol and bile, by means of both mar-dependent and mar-independent pathways . To further characterize putative marRAB-regulated genes that may be important for the resistance phenotype, acrAB, which encodes an efflux pump, was analysed . In S . typhimurium, acrAB is required for bile resistance, but while transcription of acrAB is activated by bile, this activation is independent of marRAB, as well as Rob, RpoS or PhoP-PhoQ . These data suggest that bile interacts with salmonellae to increase resistance to bile and other antimicrobials and that this can occur by marRAB- and acrAB-dependent pathways that function independently with respect to bile activation.

J Biol Chem, 2004 Jun 25, 279(26), 26803 - 6 Epub 2004 Apr 08.
Structure and mechanism of O-acetylserine sulfhydrylase; Rabeh WM et al.; The O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium catalyzes a beta-replacement reaction in which the beta-acetoxy group of O-acetyl-L-serine (OAS) is replaced by bisulfide to give L-cysteine and acetate . The kinetic mechanism of OASS is ping-pong with a stable alpha-aminoacrylate intermediate . The enzyme is a homodimer with one pyridoxal 5'-phosphate (PLP) bound per subunit deep within the protein in a cleft between the N- and C-terminal domains of each of the monomers . All of the active site residues are contributed by a single subunit . The enzyme cycles through open and closed conformations as it catalyzes its reaction with structural changes largely limited to a subdomain of the N-terminal domain . The elimination of acetic acid from OAS is thought to proceed via an anti-E2 mechanism, and the only catalytic group identified to date is lysine 41, which originally participates in Schiff base linkage to PLP . The transition state for the elimination of acetic acid is thought to be asynchronous and earlier for Cbeta-O bond cleavage than for Calpha-H bond cleavage.

Cancer Sci, 2004 Apr, 95(4), 290 - 9
Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish; Sugimura T et al.; Research leading to the discovery of a series of mutagenic and carcinogenic heterocyclic amines (HCAs) was inspired by the idea that smoke produced during cooking of food, especially meat or fish, might be carcinogenic . More than ten kinds of HCAs, actually produced by cooking or heating of meat or fish, have now been isolated and their structures determined, most being previously unregistered compounds . They are highly mutagenic towards Salmonella typhimurium in the presence of S9 mix and are also mutagenic in vitro and in vivo toward mammalian cells . HCAs have now been chemically synthesized in quantity and subjected to long-term animal testing . When HCAs were fed in the diet, rodents developed cancers in many organs, including the colon, breast and prostate, and one HCA produced hepatomas in monkeys . The lesions exhibited alteration in genes including Apc, beta-catenin and Ha-ras, and these changes provide clues to the induction mechanisms . The HCAs are oxidized to hydroxyamino derivatives by cytochrome P450s, and further converted to ester forms by acetyltransferase and sulfotransferase . Eventually, they produce DNA adducts through the formation of N-C bonds at guanine bases . There are HCA-sensitive and resistant strains of rodents and a search for the responsible genes is now under way . While the content of HCAs in dishes consumed in ordinary life is low and not sufficient in itself to explain human cancer, the coexistence of many other mutagens/carcinogens of either autobiotic or xenobiotic type and the possibility that HCAs induce genomic instability and heightened sensitivity to tumor promoters suggest that avoidance of exposure to HCAs or reduction of HCAs' biological effects as far as possible are to be highly recommended . Usage of microwave ovens for cooking and supplementation of the diet, for example with soy-isoflavones, which have been found to suppress the occurrence of HCA-induced breast cancers, should be encouraged . Advice to the general public about how to reduce the carcinogenic load imposed by HCAs would be an important contribution to cancer prevention.

Phytomedicine, 2004 Feb, 11(2-3), 230 - 4
Antimicrobial activity in methanolic extracts of several plant species from northern Argentina; Salvat A et al.; Thirty-nine native plant species were collected from the provinces of Chaco and Formosa, in northern Argentina, and were screened for antimicrobial activity . The plants were dried and extracted thoroughly with methanol . The dry extracts, dissolved in dimethylsulfoxide, were tested for inhibition of microbial growth via microplate assay with an oxidation-reduction dye . The test organisms were: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecium . Inhibition of respiratory activities in some of these microbial species was produced by the extracts of Astronium balansae, Geoffroea decorticans, Peltophorum dubium, Geoffroea spinosa, Lantana balansae, Prosopis kuntzei, Prosopis ruscifolia and Bulnesia sarmientoi, with minimal inhibitory concentrations (MIC) ranging from 0.08 to 0.5 mg dry matter/ml . Further in vitro experiments measuring the growth of S . aureus in liquid culture confirmed that all of the above extracts at 2 x MIC were able to inhibit bacterial growth effectively, and that some of them (A . balansae, G . decorticans, P . dubium, G . spinosa, P . kuntzei and B . sarmientoi) were able to reduce the initial number of viable counts by at least one order of magnitude in 10 hours, indicating that these extracts should be investigated further for the possible presence of bactericidal components.

Vaccine, 2004 Apr 16, 22(13-14), 1692 - 9
Comparative analysis using a mouse model of the immunogenicity of artificial VLP and attenuated Salmonella strain carrying a DNA-vaccine encoding HIV-1 polyepitope CTL-immunogen; Karpenko LI et al.; Two systems have been examined for delivery of DNA-vaccine encoding a HIV-1 polyepitope CTL-immunogen (TCI) . One is intended for i.m . injection and is in the form of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate . The other is intended for mucosal immunization and is based on attenuated Salmonella typhimurium strain 7207, which can deliver pcDNA-TCI directly into professional antigen-presenting cells (APC) . After immunization, the artificial VLP and recombinant Salmonella induced an enhanced HIV specific serum antibody, proliferative and CTL responses compared to those induced by naked pcDNA-TCI . The most significant responses were produced when pcDNA-TCI was delivered by Salmonella.

Mutat Res, 2004 Apr 11, 559(1-2), 177 - 87
Inhibitory effects of beer on heterocyclic amine-induced mutagenesis and PhIP-induced aberrant crypt foci in rat colon; Nozawa H et al.; Anti-mutagenic and anti-carcinogenic effects of beer on heterocyclic amine (HCA)-induced carcinogenesis were studied in vitro and in vivo . Four commercial beers (two pilsner-type, black, and stout) showed inhibitory effects against five HCAs, 2-amino-3,8-dimethylimidazo {4,5-f}quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP), 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), 2-amino-6-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1) and 2-amino-3-methylimidazo{4,5-f}-quinoline (IQ), in the Ames assay using Salmonella typhimurium TA98 in the presence of rat S9 mix . The inhibitory effects of dark-colored beers (stout and black beer) were greater than those of pilsner-type beers . Dark-colored beers suppressed CYP1A2 activity in a dose-dependent manner, suggesting that inhibition of HCA activation is partly responsible for their strong anti-mutagenic effects . Anti-mutagenic effects were also observed when the pooled human S9 mix or activated IQ was used in the assay . The micronucleus test using Chinese hamster lung CHL/IU cells showed that the addition of freeze-dried samples of pilsner-type and stout beer to the culture medium significantly reduced the number of cells with micronuclei induced with PhIP or Trp-P-2 . Single-cell gel electrophoresis assay (comet assay) revealed that oral ingestion of pilsner-type and stout beers for 1 week significantly inhibited DNA damage in the liver cells of male ICR mice exposed to MeIQx (13 mg/kg, i.p.) . A decrease in the formation of DNA adducts was also observed using a 32P-postlabeling method . Male Fischer 344 rats orally received PhIP (75 mg/kg, five times a week for 2 weeks) and aberrant crypt foci (ACF) formation in the colon was analyzed after 5 weeks . The number of ACF was significantly reduced in rats fed a diet containing freeze-dried beer . These results suggest that beer inhibits the genotoxic effects of HCAs and may reduce the risk of carcinogenesis caused by food borne carcinogens.

Mutat Res, 2004 Apr 11, 559(1-2), 105 - 19
Bacterial mutagenicity of the three isomeric dicyclopenta-fused pyrenes: the effects of dicyclopenta topology; Otero-Lobato MJ et al.; Cyclopenta{cd}pyrene (1) and its congeners dicyclopenta{cd,mn}- (2), dicyclopenta{cd,fg}- (3), dicyclopenta{cd,jk}pyrene (4), which were all identified as constituents of combustion exhausts, as well as their partially hydrogenated derivatives 3,4-dihydrocyclopenta{cd}- (5), 1,2,4,5-tetrahydrodicyclopenta{cd,mn}- (6), 5,6,7,8-tetrahydrodicyclopenta{cd,fg}- (7) and 1,2,6,7-tetrahydrodicyclopenta{cd,jk}pyrene (8), were assayed for mutagenicity in the Salmonella typhimurium strain TA98 using different concentrations of microsomal protein in the metabolic activation system (S9-mix, with S9-fraction from liver of Aroclor-1254-treated rats: 2, 4 and 10% (v/v), respectively) . Whereas a positive mutagenic response is found for 1-4 in the presence of S9-mix, 5-8 exert no mutagenicity either with or without S9-mix . Since for 1-4 the highest response is observed with S9-mix 2% (v/v) instead of the standard 4% (v/v), a one-step activation pathway, i.e . epoxidation of the five-membered ring olefinic bonds, appears to be operational . Surprisingly, 3 and, to a lesser extent, 2 (11.7 versus 4.2 His revertants/nmol) also give a positive response in the absence of S9-mix . Hence, 2 and 3 are expected to contribute to the direct-acting mutagenicity of the non-polar fraction of combustion exhausts . Presumably for the direct-acting mutagenicity one-electron transfer processes play a role in bioactivation . The experimental observations are supported by semi-empirical AM1 calculations on the possible ultimate metabolites, i.e . mono-epoxides (2a-4a), cis-di-epoxides (2b-4b) and trans-di-epoxides (2c-4c) and the related mono-hydroxy carbocations (2d-4d and 2e-4e), and the radical anions 1*(-)-4*(-).

Mutat Res, 2004 Apr 11, 559(1-2), 83 - 95
In vivo mutagenicity of benzo{f}quinoline, benzo{h}quinoline, and 1,7-phenanthroline using the lacZ transgenic mice; Yamada K et al.; Phenanthrene, a simplest angular polycyclic aromatic hydrocarbon with a bay-region in its molecule, is reported to be non-mutagenic, although most angular (non-linear) polycyclic aromatic hydrocarbons, such as benzo{a}pyrene and chrysene, are known to show genotoxicity after metabolic transformation into a bay-region diol epoxide . On the other hand, benzo{f}quinoline (BfQ), benzo{h}quinoline (BhQ), and 1,7-phenanthroline (1,7-Phe), which are all aza-analogs of phenanthrene, are mutagenic in the Ames test using Salmonella typhimurium TA100 in the presence of a rat liver S9 fraction . In this report, we undertook to investigate the in vivo mutagenicity of BfQ, BhQ and 1,7-Phe by an in vivo mutation assay system using the lacZ transgenic mouse (Muta Mouse) . BfQ and BhQ only slightly induced mutation in the liver and lung, respectively . BfQ- and BhQ-induced cII mutant spectra showed no characteristics compared with that of the control . These results suggest that the in vivo mutagenicities of BfQ and BhQ were equivocal . On the other hand, 1,7-Phe induced a potent mutation in the liver and a weak mutation in the lung . Furthermore 1,7-Phe depressed the G:C to A:T transition and increased the G:C to C:G transversion in the liver like quinoline, a hepatomutagen possessing the partial structure of 1,7-Phe, compared with the spontaneous mutation spectrum . These results suggest that the in vivo mutagenicity of 1,7-Phe might be caused by the same mechanism as that of quinoline, which induced the same mutational spectrum change (G:C to C:G transversion).

Environ Mol Mutagen, 2004, 43(3), 159 - 68
Comparative in vitro and in vivo genotoxicities of 7H-benzo{c}fluorene, manufactured gas plant residue (MGP), and MGP fractions; Cizmas L et al.; Manufactured gas plant residue (MGP) is a complex mixture of polycyclic aromatic hydrocarbons (PAHs) that is tumorigenic in the lungs of mice . This study compared the relative genotoxicity of 7H-benzo{c}fluorene (BC), a PAH component of MGP, with MGP and MGP fractions in order to assess the contribution of BC to the genotoxicity of MGP . An MGP sample was separated into seven fractions (F1-F7) using silica gel column chromatography with petroleum ether (PE) followed by PE:acetone (99:1 v/v, then 98:2) . PAHs were quantified using gas chromatography/mass spectrometry . An aliquot of F2, the fraction with the highest BC concentration and highest weighted mutagenic activity in Salmonella typhimurium strain TA98, was further separated using silica gel thin-layer chromatography with hexane . The first F2 subfraction, sF2-a, was enriched in BC and coeluting compounds and contained 35,000 ppm BC and 216,109 ppm carcinogenic PAHs (cPAHs, the sum of seven PAHs categorized by the U.S . EPA as class B2 carcinogens) . The second F2 subfraction, sF2-b, contained a ninefold lower concentration of BC, with 3,900 ppm BC and 45,216 ppm cPAHs . Female ICR mice received topical application of crude MGP, crude MGP spiked with analytical-grade BC, F2, sF2-a, sF2-b, or analytical-grade BC . DNA adduct levels were analyzed by nuclease P1-enhanced (32)P-postlabeling . In lung DNA of mice receiving 0.48 or 3.0 mg/mouse, net total RAL x 10(9) values were F2, 30.8 and 87.2; sF2-a, 24.8 and 106.7; and sF2-b, 19.6 and 151.0, respectively . Mice dosed with 0.10 mg analytical-grade BC (the mass of BC in 3.0 mg sF2-a) exhibited a net total RAL x 10(9) value of 7.03 in lung DNA . This was equal to approximately 7% of the total RAL x 10(9) value produced by 3.0 mg sF2-a . Thus, although BC appears to make an appreciable contribution to pulmonary adduct formation, the results suggest that MGP components other than BC play an important role in lung DNA adduct formation following topical MGP administration .

Mol Biol Cell, 2004 Jun, 15(6), 2954 - 64 Epub 2004 Apr 02.
Dynein-mediated vesicle transport controls intracellular Salmonella replication; Marsman M et al.; Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV) . In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes . The exact mechanism of the inhibition of phagolysosomal fusion is not understood . Rab GTPases control several proteins involved in membrane fusion and vesicular transport . The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV . We show that the Rab7 GTPase cycle is not affected on the SCV . We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella . Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV . Subsequently, SCV fuse with lysosomes . As a result, the intracellular replication of Salmonella is inhibited . Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.

Epidemiol Infect, 2004 Apr, 132(2), 263 - 72
Molecular characterization of Salmonella enterica subsp . enterica serovar Typhimurium DT1 isolates; Lindqvist N et al.; Salmonella Typhimurium DT1 is endemic to Finland and has caused human outbreaks since the 1960s . Domestic DT1 isolates (n=235) from 1972 to 1999 from human cases, animals and other sources, as well as foreign DT1 isolates from human cases (n=20) were analysed by molecular methods . Pulsed-field gel electrophoresis (PFGE) yielded 38 XbaI profiles . Of these, XbaI profile 10 was seen in 49% (125/255) of the isolates . Twelve IS200 profiles were obtained; the most common IS200 profile D was seen in 64% (33/52) of the isolates . Two clusters were formed by compilation of the XhaI-, BlnI- and SpeI-PFGE and IS200 profiles and possession of the serovar-specific virulence plasmid . The major cluster contained eight IS200 profiles, including IS200 profile D and XhaI profile 10, and had no virulence plasmid, and can be regarded as typical of the endemic Typhimurium DT1 infection.

Lett Appl Microbiol, 2004, 38(5), 410 - 4
Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I; Kehoe SC et al.; AIMS: The mortality and morbidity rate caused by Shigella dysenteriae type I infection is increasing in the developing world each year . In this paper, the possibility of using batch process solar disinfection (SODIS) as an effective means of disinfecting drinking water contaminated with Sh . dysenteriae type I is investigated . METHODS: Phosphate-buffered saline contaminated with Sh . dysenteriae type I was exposed to simulated solar conditions and the inactivation kinetics of this organism was compared with that of Sh . flexneri, Vibrio cholerae and Salmonella typhimurium . SIGNIFICANCE: Recovery of injured Sh . dysenteriae type I may be improved by plating on medium supplemented with catalase or pyruvate . Sh . dysenteriae type I is very sensitive to batch process SODIS and is easily inactivated even during overcast conditions . Batch process SODIS is an appropriate intervention for use in developing countries during Sh . dysenteriae type I epidemics.

J Appl Toxicol, 2004 Mar-Apr, 24(2), 83 - 91
Mutagenicity of textile dye products; Schneider K et al.; Within an EU-funded research project, 281 textile dye products in use at nine textile finishing companies from eight European countries were assessed for potential mutagenic properties . Most of the dyes belonged to the so-called existing substances . Data sources considered were data published in the literature, unpublished industrial data provided by dye producing companies, and laboratory testing . Data on mutagenicity are virtually absent for many of the dyes . Unpublished test results performed on behalf of the dye manufacturing industry proved to be an important data source that is not accessible under usual circumstances . Four dye stuffs contained in seven dye products in use at the textile finishing companies were judged to be mutagenic, based on published data from the literature . Mutagenicity testing using Salmonella typhimurium, strains TA98 and TA100, revealed positive results for about 28% (15 out of 53) of the dye products investigated . Upon further testing with the mouse lymphoma assay (L5178Y/TK(+/-)) 67% (6 out of 9) of Ames-positive dyes proved to be mutagenic in this mammalian cell test . All data sources combined led to an overall assessment of 14 dye products out of 281 being mutagenic . For 16 there is a suspicion of mutagenicity due to positive responses in one test but 71 of the dye products are without any data on mutagenicity . This paper describes the data aggregation process, evaluation criteria and the overall assessment, and exemplifies controversial evaluations .

Microbes Infect, 2004 Apr, 6(4), 350 - 9
Poor survival but high immunogenicity of IL-2-expressing Salmonella typhimurium in inherently resistant mice; al-Ramadi BK et al.; An IL-2-expressing, attenuated strain of Salmonella typhimurium (strain GIDIL2) was previously shown to survive poorly and to have lower immunogenicity in susceptible mice than its parental, non-cytokine-expressing strain (designated BRD509) . In the present study, we compared the immune responses induced by both bacterial strains in inherently Salmonella-resistant C3H/HeN mice . Analysis of the bacterial loads in the peritoneum and spleen revealed that colony-forming units (CFUs) of GIDIL2 were consistently lower than the corresponding BRD509 CFUs . As early as 48 h after inoculation, there were 60-fold lower CFUs of GIDIL2 than BRD509 organisms in the peritoneal cavity . Similarly, the differences in splenic CFUs of GIDIL2 were 20- to 50-fold lower than those of BRD509 over a period of 3-21 days post-injection . This rapid rate of clearance of the GIDIL2 organisms correlated with significantly decreased infection-induced splenomegaly and nitric oxide production by spleen cells . However, despite the poor survival of GIDIL2 organisms in vivo, they could activate peritoneal NK cells efficiently . As early as 48 h after immunization, equivalent levels of NK-mediated cellular cytotoxicity were induced by BRD509 and GIDIL2 strains . Direct evidence for priming of the immune response was shown by demonstrating increased production of IFN-gamma in a recall response by spleen memory T cells obtained 3 weeks after immunization . Finally, mice inoculated with a single dose of either BRD509 or GIDIL2 organisms were fully protected against a challenge of >100-fold the LD50 dose of virulent Salmonella . Taken together, our data demonstrate that despite their rapid clearance from the reticuloendothelial system, IL-2-expressing Salmonella are immunogenic and fully capable of affording excellent protection against virulent challenge in Salmonella-resistant C3H/HeN mice.

Biomaterials, 2004 Aug, 25(18), 4019 - 27
Mutagenic potentials of dental cements as detected by the Salmonella/microsome test; Kaplan C et al.; The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test . The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C . Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver . Poscal and Aqualox elicited mutagenic effects on S . typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S . typhimurium TA 98 . No mutagenic effects were detected for Argion . The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1) . We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.

Biosens Bioelectron, 2004 May 15, 19(10), 1139 - 47
Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella typhimurium; Yang L et al.; Interdigitated microelectrodes (IMEs) were used as impedance sensors for rapid detection of viable Salmonella typhimurium in a selective medium and milk samples . The impedance growth curves, impedance against bacterial growth time, were recorded at four frequencies (10Hz, 100Hz, 1kHz, and 10kHz) during the growth of S . typhimurium . The impedance did not change until the cell number reached 10(5)-10(6) CFUml(-1) . The greatest change in impedance was observed at 10Hz . To better understand the mechanism of the IME impedance sensor, an equivalent electrical circuit, consisting of double layer capacitors, a dielectric capacitor, and a medium resistor, was introduced and used for interpreting the change in impedance during bacterial growth . Bacterial attachment to the electrode surface was observed with scanning electron microscopy, and it had effect on the impedance measurement . The detection time, t(D), defined as the time for the impedance to start change, was obtained from the impedance growth curve at 10Hz and had a linear relationship with the logarithmic value of the initial cell number of S . typhimurium in the medium and milk samples . The regression equations for the cell numbers between 4.8 and 5.4 x 10(5) CFUml(-1) were t(D) = -1.38 log N + 10.18 with R(2) = 0.99 in the pure medium and t(D) = -1.54 log N + 11.33 with R(2) = 0.98 in milk samples, respectively . The detection times for 4.8 and 5.4 x 10(5) CFUml(-1) initial cell numbers were 9.3 and 2.2 h, respectively, and the detection limit could be as low as 1 cell in a sample.

Mol Biol Evol, 2004 Jun, 21(6), 1152 - 9 Epub 2004 Mar 24.
Evolution of prokaryotic DNA: intragenic and extragenic divergences observed with orthologs from three related species; Fuglsang A; This study compared orthologous gene pairs from Escherichia coli K12, E . coli O157:H7 EDL933, Salmonella typhimurium LT2, and Yersinia pestis CO92 using only homologs of equal length, and differing nucleotides were counted and mapped . The data showed very clearly how the rates of divergence change with intragenic and extragenic position . The rate of synonymous mutation is lowest near start codons and near stop codons, and, a little surprisingly, the opposite seemed to be true for nonsynonymous substitutions . Analysis outside genes reveals that nucleotide divergences occur less frequently upstream of start codons than downstream of stop codons, and a remarkable drop in divergences is seen for two of the data sets around N = 9 nucleotides upstream of start codons; that is, the Shine-Dalgarno region changes at a lower rate . The explanation is likely to be the link between expressivity and sequence complementarity to the 3' end of 16S ribosomal rRNA . The latter is highly conserved across many bacterial and archaebacterial species.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 97 - 105
Genome-wide cloning and characterization of microbial esterases; Ro HS et al.; We have isolated putative esterase genes from various bacterial chromosomes . Thirty open reading frames predicted to encode esterases were randomly selected from 13 sequenced bacterial chromosomes and were cloned into an expression vector . The esterase activity of the resulting clones was tested on a tributyrin plate at different pH values and temperatures . Nine out of thirty tested clones exhibited significant tributyrin hydrolyzing activity . The enzyme S5 from the gene b0494 of Escherichia coli, the enzyme S12 from the gene STM0506 of Salmonella typhimurium, and the enzyme S28 from the gene AF1716 of Archaeoglobus fulgidus exhibited high activity at an alkaline pH range . The esterase S11 encoded by the gene PA3859 of Pseudomonas aeruginosa PAO1 and the esterase S21 from the gene SMc01033 of Sinorhizobium meliloti 1021, both showed a sharp increase in enzyme activity above pH 8.0 . Furthermore, the enzymes S5, S12, S21, and S28 retained the esterase activity when they were incubated at 50 degrees C, suggesting that these enzymes are thermostable . Subsequent pH vs . activity and temperature vs . activity experiments with selected enzymes in a solution assay system confirmed the validity of the above data . The genome-wide exploration strategy of proteins provided valuable information on the esterases by revealing subtle biochemical differences between the esterases of different sources.

Nutrition, 2004 Apr, 20(4), 372 - 6
Translocation of Salmonella typhimurium in rats on total parenteral nutrition correlates with changes in intestinal morphology and mucus gel; Sakamoto K et al.; OBJECTIVE: We tested whether alterations in intestinal morphology and mucus gel correlate with differences in Salmonella typhimurium translocation between rats treated with total parenteral nutrition (TPN) and rats given a diet of chow . METHODS: Twenty-seven male Wistar rats were assigned to one of two groups: one received TPN for 14 d and the other (control) received standard rat chow and water ad libitum . Salmonella typhimurium (5 x 10(8) cells; GIFU 12142) was injected into a closed ileal loop . Portal venous blood (PVB), inferior vena cava blood (IVCB), and mesenteric lymph nodes (MLNs) were sampled for evaluation of bacterial translocation . Sections of the loop were prepared and stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and fluorescein isothiocyanate-labeled Ulex europaeus agglutinin I (FITC-UEA-I) for image analysis . Perimeter, mucosal thickness, villus area, and positively stained mucus area were measured . A fluorescent antibody study was also done . RESULTS: Organisms were found in cultures of 1 in 13 control rats and 9 in 14 TPN rats . There were more bacteria in MLNs than in PVB or IVCB . There was no increase in the number of bacteria over time in PVB, IVCB, or MLNs . Perimeter and villus area (P < 0.001) and mucosal thickness (P < 0.01) were significantly smaller in the TPN group than in the control group . The positively stained mucus area was significantly smaller in the TPN group than in the control group (P < 0.05 with PAS, P < 0.01 with FITC-UEA-I) . Salmonella typhimurium invaded specifically through Peyer's patches . In all culture-negative samples, bacteria were trapped by the mucous layer, with a very small number attached to the epithelial surface . CONCLUSION: Significant villous atrophy and reduction of mucus play an important role in the rapid translocation of S . typhimurium through Peyer's patches in rats after 2 wk of TPN.

Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 709 - 11 Epub 2004 Mar 23.
Purification, crystallization and preliminary X-ray analysis of uridine phosphorylase from Salmonella typhimurium; Dontsova MV et al.; The structural udp gene encoding uridine phosphorylase (UPh) was cloned from the Salmonella typhimurium chromosome and overexpressed in Escherichia coli cells . S . typhimurium UPh (StUPh) was purified to apparent homogeneity and crystallized . The primary structure of StUPh has high homology to the UPh from E . coli, but the enzymes differ substantially in substrate specificity and sensitivity to the polarity of the medium . Single crystals of StUPh were grown using hanging-drop vapor diffusion with PEG 8000 as the precipitant . X-ray diffraction data were collected to 2.9 A resolution . Preliminary analysis of the diffraction data indicated that the crystal belonged to space group P6(1(5)), with unit-cell parameters a = 92.3, c = 267.5 A . The solvent content is 37.7% assuming the presence of one StUPh hexamer per asymmetric unit.

Curr Opin Microbiol, 2004 Feb, 7(1), 51 - 7
Living in the danger zone: innate immunity to Salmonella; Wick MJ; Phagocytic cells, including macrophages, neutrophils and dendritic cells, are critical components of the innate immune response to bacterial pathogens such as Salmonella typhimurium . These cells can have several roles during the early stage of an infection including controlling bacterial replication and producing cytokines and chemokines that activate and recruit additional cells . Macrophages, neutrophils and dendritic cells increase in number early after oral Salmonella infection and produce cytokines important in host survival such as tumor necrosis factor alpha (TNF-alpha) . All three phagocytic cell types also harbor bacteria during infection . Natural killer cells, natural killer T cells and T cell receptor alpha beta T cells also respond rapidly to infection and are early sources of interferon-gamma during infection with Salmonella . Studies using infection models with Salmonella are providing a picture of the innate response to bacteria and insight into the role of defined cell types and cytokines important in the transition from innate to adaptive immunity.

Mutat Res, 2004 Mar 14, 558(1-2), 155 - 67
Genotoxic and mutagenic activity of environmental air samples in Flanders, Belgium; Du Four VA et al.; Atmospheric pollution is assumed to play a role in the incidence of respiratory diseases and cancers . Airborne particles are able to penetrate deep into the lung and are composed of complex chemical mixtures, including mutagens and carcinogens such as polycyclic aromatic compounds (PACs) . The present study reports mutagenic and genotoxic activities associated with ambient air collected near a busy street in Borgerhout, at an industrial site in Hoboken and in Peer, a rural community 70 km east of Antwerp in Flanders, Belgium . Airborne particulates (PM10) and semi-volatile organic compounds were sampled during winter and summer . Samples were collected with a high-volume sampler using quartz filters (QF) and polyurethane foam (PUF) cartridges . The mutagenic and genotoxic activity of the organic extracts was determined using the Salmonella test/standard plate-incorporation assay and the Vitotox assay . Concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) in the extracts were determined by reversed-phase high-performance liquid chromatography (HPLC) . The mutagenicity assay, using Salmonella typhimurium strain TA98, demonstrated direct mutagenicity of up to 58 revertants/m3 for the QF extracts and low or no mutagenic activity in the PUF extracts . Metabolic activation of the samples resulted in high indirect mutagenicity for both QF and PUF extracts: up to 96 revertants/m3 were found in QF samples and 62 revertants/m3 in PUF samples . Genotoxic effects of the filter extracts were assessed with the Vitotox assay: some direct genotoxic effects were noted, i.e . without metabolic activation, but almost no effects were observed after metabolic activation . Without activation, most PUF extracts were bacteriotoxic . With metabolic activation this toxicity disappeared, but genotoxic effects were not observed . Statistical analysis showed that the observed biological effects correlated well with the PAH concentrations.

Mutat Res, 2004 Mar 14, 558(1-2), 93 - 110
Evaluation of the genotoxicity of cis-bis(3-aminoflavone)dichloroplatinum(II) in comparison with cis-DDP; Kosmider B et al.; Short-term tests that detect genetic damage have provided information needed for evaluating carcinogenic risks of chemicals to man . The mutagenicity of cis-bis(3-aminoflavone)dichloroplatinum(II) (cis-{Pt(AF)2Cl2}) in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) was evaluated in the standard plate-incorporation assay in four strains of Salmonella typhimurium: TA97a, TA98, TA100 and TA102, in experiments with and without metabolic activation . It was shown that cis-{Pt(AF)2Cl2} acts directly and is mutagenic for three strains of S . typhimurium: TA97a, TA98 and TA100 . In comparison with cis-DDP this compound showed a weaker genotoxicity . Contrary to cis-DDP it has not shown toxic properties in the tester bacteria . The genotoxicity of both tested compounds was evaluated using chromosomal aberration, sister chromatid exchange and micronucleus assays, without and with metabolic activation, in human lymphocytes in vitro . The inhibitory effects of both compounds on mitotic activity, cell proliferation kinetics and nuclear division index were also compared . In all test systems applied, cis-{Pt(AF)2Cl2} was a less effective clastogen and a weaker inducer of both sister chromatid exchanges and micronuclei in comparison with cis-DDP, with and without metabolic activation . cis-{Pt(AF)2Cl2} has a direct mechanism of action and is less cytostatic and cytotoxic than the other compound . These results provide important data on the genotoxicity of cis-{Pt(AF)2Cl2} and indicate its beneficial properties as a potential anticancer drug, especially in comparison with cis-DDP.

J Chromatogr B Analyt Technol Biomed Life Sci, 2004 Mar 25, 802(1), 217 - 23
Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline; Boyce A et al.; Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes . It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed . We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells) . We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) at micromolar concentrations . Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity . Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity . Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.

J Food Prot, 2004 Mar, 67(3), 579 - 82
Decontamination of cattle hides prior to slaughter using washes with and without antimicrobial agents; Mies PD et al.; Two trials were conducted to determine the efficacy of cattle wash treatments in reducing pathogens on hides of cattle before slaughter . In trial I, live cattle (n = 120) were washed in an automated, commercial cattle wash system with one of four treatments (single water wash, double water wash, water wash with 0.5% L-lactic acid, or water wash with 50 ppm chlorine) . Samples were collected at three locations (brisket, belly, and inside round) pre- and posttreatment to evaluate the effectiveness of treatments on the reduction of aerobic plate counts, coliforms, Escherichia coli and the incidence of Salmonella . For all three locations, bacterial numbers increased from 0.1 to 0.8 log CFU/cm2 posttreatment . In trial II, hide samples were inoculated in the laboratory with 6.0 log CFU/cm2 of rifampicin-resistant Salmonella serotype Typhimurium . Hide wash treatments included higher concentrations of chlorine (100, 200, and 400 ppm) and L-lactic acid (2, 4, and 6%), as well as other antimicrobial agents such as ethanol (70, 80, and 90%), acetic acid (2, 4, and 6%), and Oxy-Sept 333 (0.5, 2, and 4%) . Spray wash treatments with ethanol and 4 to 6% concentrations of lactic acid had greater (P < 0.05) mean log reductions than 2% solutions of acetic or lactic acid, as well as 100, 200, and 400 ppm chlorine and the control water wash treatment . Spray wash treatments with Oxy-Sept 333 and 100, 200, or 400 ppm chlorine were not effective (P > 0.05) in reducing Salmonella Typhimurium compared to the (control) distilled water spray wash treatment . Several effective cattle hide interventions were identified in a controlled laboratory setting, but the high concentrations required for effectiveness would likely present problems from an animal welfare standpoint.

J Food Prot, 2004 Mar, 67(3), 448 - 55
Antibiotic resistance of Salmonella isolated from hog, beef, and chicken carcass samples from provincially inspected abattoirs in Ontario; Larkin C et al.; The emergence of antimicrobial-resistant Salmonella organisms, especially Salmonella Typhimurium DT104, has been reported in many countries, including the United States and Canada . The purposes of this study were to determine the antimicrobial resistance patterns of Salmonella isolated from hog, beef, and chicken carcasses from provincially inspected abattoirs in Ontario and to determine the agreement between the agar dilution method and the microbroth dilution method for measurement of antimicrobial resistance of the isolates . Antimicrobial resistance of Salmonella isolates from hogs (n = 71), beef (n = 24), and chicken (n = 295) to amikacin, ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, sulfamethoxazole, and tetracycline was determined using the two methods . None of the 390 isolates were resistant to ciprofloxacin at levels of 0.125 microg/ml . All chicken and hog isolates were sensitive to amikacin, whereas all beef isolates were sensitive to both amikacin and gentamicin . Multiple antimicrobial resistance (resistance to more than one antimicrobial) was found in 29% of bovine isolates and 42% of porcine isolates using both methods for testing and in 42% by the agar dilution and 33% by the microbroth dilution methods in the chicken isolates . Overall, there was good agreement between the two test methods for resistance to most of the antimicrobials, with disagreement found in the results in 1.3% of the isolates for ampicillin and sulfamethoxazole, 8.2% for streptomycin, 5.6% for cephalothin, and 1.0% of the isolates for tetracycline . The lack of agreement between the two test methods was found mostly among the chicken isolates.

Antonie Van Leeuwenhoek, 2004 Jan, 85(1), 53 - 62
Anti-stress activity of Propionibacterium freudenreichii : identification of a reactivative protein; Vorobjeva L et al.; Propionibacterium freudenreichii subsp . shermanii is known to prevent mutations caused by various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, 4-nitro-quinoline-1-oxide and by UV radiation in both prokaryotic and eukaryotic cells . It was also shown to prevent or repair damage caused by H(2)O(2) or UV radiation in Salmonella typhimurium and Escherichia coli, a characteristic previously designated as reactivative effect . In order to characterise this effect at the molecular level, we have purified the active component from a P . freudenreichii cell-free extract using a combination of ammonium sulfate precipitation, anion-exchange and size-exclusion chromatography . The isolated 35 kDa protein was then identified using both N-terminal and internal peptide sequencing as a cysteine synthase . The latter was localised in the P . freudenreichii proteomic map . It is constitutively expressed but also clearly induced during adaptation to detergent and heat, but not acid, stresses . The biological meaning of cysteine synthase in the context of adaptation to oxidative and non-oxidative stresses is discussed.

Antonie Van Leeuwenhoek, 2004 Jan, 85(1), 45 - 51
Influence of short chain fatty acids and lysine on Salmonella typhimurium cadA expression; Zabala Diaz IB et al.; In Salmonella typhimurium, cadA has a role in virulence expression and is an inducible gene that responds to external lysine concentration . In this study, a strain of S . typhimurium carrying a cadA: lacZ fusion was used to determine if the induction of cadA occurred under different lysine concentrations and mildly acid conditions in the presence of short chain fatty acids . Aliquots of an 18-h culture of S . typhimurium were placed on fresh media containing different lysine concentrations at pH 5.8 adjusted by addition of HCl or by 1 M short chain fatty acids (SCFA, acetic, propionic and butyric acid) stock solution . After an induction period of 2 h, beta -galactosidase activities were assayed . Expression of cadA in rich medium was significantly higher than that of minimal medium at neutral pH and different lysine concentrations . In contrast, at pH 5.8, there was a significant increase in cadA expression, particularly when pH was adjusted using HCl at all lysine levels . Addition of a mixture of organic acids yielded an overall lower cadA expression at all lysine levels studied when compared to HCl . However, each SCFA challenge (individual or as a mixture) caused a high level of expression, both at neutral and acidic pH . Based on these results it is apparent that in the presence of external lysine, SCFA and nutrient availability can influence cadA expression in S . typhimurium.

Chem Res Toxicol, 2004 Mar, 17(3), 416 - 23
Nitrosation of glycine ethyl ester and ethyl diazoacetate to give the alkylating agent and mutagen ethyl chloro(hydroximino)acetate; Zhou L et al.; Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test . We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate {ClC(=NOH)COOEt, ECHA} and a 9% yield of ethyl chloroacetate . The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA . Nitrosation of ethyl diazoacetate also yielded ECHA . Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA . ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate {PhN=C(NHOH)CO2Et} . This product was different from ethyl {(phenylamino)carbonyl}carbamate {PhNHC(=O)NHCO2Et}, which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline . ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative . ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM) . In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.

J Environ Sci Health B, 2004 Jan, 39(1), 199 - 207
The combination of zinc compounds and acidic pH limits aerobic growth of a Salmonella typhimurium poultry marker strain in rich and minimal media; Park SY et al.; The objective of the present study was to examine the combined effects of zinc compounds with different acidic pH levels on the aerobic growth of a S . typhimurium poultry isolate in either rich or minimal media . When overall main effects of pH levels of medium or concentrations of Zn compounds were compared, growth rates of the S . typhimurium poultry isolate were significantly (p < 0.05) decreased by stepwise increase of pH levels of medium (pH 4, 5, 6, and 7) or concentrations (0.67, 3.35, and 6.03%) of Zn compounds (Zn acetate and Zn sulfate) . In general growth rates of S . typhimurium poultry isolate appeared to be more reduced by Zn acetate than by Zn sulfate and more reduced in minimal media compared to rich media.

Inflamm Res, 2004 Mar, 53(3), 107 - 10 Epub 2004 Feb 16.
Mast cell density during initiation and progression of the local Shwartzman reaction; Ramirez-Hernandez C et al.; OBJECTIVE AND DESIGN: To quantify the number of mast cells in the skin of rabbits during initiation and progression of the local Shwartzman reaction . MATERIALS: Thirty New Zealand rabbits were divided in two groups (n = 15/group) . One group was subjected to the Shwartzman reaction and the other group served as control . Subsequently, animals were further subdivided in six groups of five animals each according to time of euthanasia . TREATMENTS: The local Shwartzman reaction was induced by two inoculations of Salmonella typhimurium lipopolysaccharide . Preparatory inoculation was given intradermally and, 24 h later, the provocative injection was administered intravenously . Controls were subjected to the same procedure but received saline . After provocative injection animals were killed at 1, 8, and 15 days . METHODS: Skin samples were fixed in Carnoy's solution and mast cells were identified employing a low pH toluidine blue stain . Numbers of mast cells were determined per square millimetre and, subsequently, those cells degranulated were identified and quantified to obtain absolute values . A Student's t test was initially used to compare Shwartzman versus controls at each time point . Subsequently, an ANOVA test employing a factorial experiment was used to assess a possible interaction between time of euthanasia and treatments . RESULTS: The values were transformed (natural logarithms) for appropriate statistical comparisons . Independent comparisons at each time point showed that Shwartzman groups had higher numbers of mast cells than controls at 1 and 8 days, but not at 15 days (5.71 +/- 1.00 Vs . 2.40 +/- 0.58, P < 0.005; 3.77 +/- 0.90 Vs . 2.33 +/- 0.56, P < 0 . 025, and 2.61 +/- 0.25 Vs . 2.39 +/- 0.39, P > 0.05, respectively) . Degranulated cells were numerous in Shwartzman groups, particularly at day 1 (3.48 +/- 0.78) and less obvious at day 8 (0.72 +/- 0.50), but were scarce by day 15 (-0.67 +/- 0.99) as well as controls (-0.68 +/- 0.91) . The factorial experiment demonstrated that the Shwartzman reaction and time of euthanasia were independently significant (P < 0.005) but their interaction at day 1 was the major contributor (P < 0.005) . Tukey's w pairwise comparisons of means confirmed that the Shwartzman group killed at day 1 was significantly different from the others (P < 0.05) . CONCLUSIONS: Mast cells significantly increase in the early stages of the local Shwartzman reaction . Thus, mast cells are a highly dynamic cell population, which have a prominent role during the acute phase of this lipopolysaccharide-induced inflammatory reaction but not during healing.

Toxic Rep Ser, 2004 Jan, 69, 1 - F10
NTP technical report on the toxicity studies of Butanal Oxime (CAS No . 110-69-0) administered in drinking water and by gavage to F344/N rats and B6C3F1 mice; Burka L; Butanal oxime is used as a volatile antiskinning agent in paints, inks, and similar products . Butanal oxime was chosen for toxicology testing as a representative of the aldoxime class . Male and female F344/N rats and B6C3F1 mice received butanal oxime (99 percent pure) in drinking water for 15 days or by gavage in 0.5 percent methylcellulose for 14 weeks . Animals were evaluated for clinical pathology, reproductive system effects, and histopathology . Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes . In the 15-day studies, groups of five male and five female rats and mice received 0, 312, 625, 1,250, 2,500, or 5,000 ppm butanal oxime in drinking water, resulting in average daily doses of approximately 40, 70, or 100 mg butanal oxime/kg body weight to male and female rats; 45, 90, 130, 200, or 300 mg/kg to male mice; and 45, 85, 100, 130, or 170 mg/kg to female mice . Due to body weight loss and lack of water consumption, all male and female rats receiving 2,500 or 5,000 ppm were removed from the study on day 9; average daily doses were not calculated for these groups . All other rats and mice survived until the end of the studies . Mean body weights of 1,250 ppm male and female rats and 2,500 and 5,000 ppm male and female mice were significantly less than those of the controls . Male mice receiving 5,000 ppm and females receiving 2,500 or 5,000 ppm lost weight during the study . Water consumption by rats and mice receiving 1,250 ppm or greater was less than that by the controls . Thinness in 2,500 and 5,000 ppm rats and mice was the only clinical finding of toxicity . Spleen weights were significantly decreased in 2,500 and 5,000 ppm female mice . No chemical-related lesions were observed grossly; histologic examinations were not performed . In the 14-week studies, groups of 10 male and 10 female rats and mice received butanal oxime by gavage at doses of 0, 25, 50, 100, 200, or 600 mg/kg, 5 days per week for 14 weeks . All 600 mg/kg rats died or were killed moribund during the first week of the study; in the 600 mg/kg mouse groups, seven males and nine females died, were killed moribund, or were killed accidentally before the end of the study . Mean body weights of 100 and 200 mg/kg male rats, 600 mg/kg male mice, and female mice administered 50 mg/kg or greater were less than those of the controls . Clinical findings of toxicity in 600 mg/kg rats included loss of coordination, wobbly gait, shaking, blinking, constant grooming and scratching of the face, head weaving, burying of the face in bedding, lethargy, and prostration; in 600 mg/kg mice, clinical findings included ataxia, loss of balance after rearing, squinting, and burying of the face in the bedding . Hematology results of the 14-week gavage studies indicate that butanal oxime induces a methemoglobinemia and a responsive anemia in rats and mice . Spleen weights of 100 and 200 mg/kg male rats, female rats administered 50 mg/kg or greater, and 200 and 600 mg/kg male mice were increased, as were the liver weights of 200 mg/kg female rats and mice . In animals that died early due to butanal oxime administration, hepatocellular necrosis was the primary pathologic finding . Degeneration of the nasal olfactory epithelium was observed in dosed rats and mice that died early as well as in animals that survived to the end of the studies . Additional chemical-related nasal findings were respiratory epithelial changes in male rats and suppurative exudate in male and female mice . Increased incidences and/or severities of splenic hematopoietic cell proliferation and pigmentation (hemosiderin) as well as bone marrow hyperplasia were also observed in dosed groups, particularly in the 200 and 600 mg/kg groups, and were indicative of erythrocyte damage . Butanal oxime (3 to 10,000 ug/plate) was mutagenic in S . typhimurium strain TA1535 in the presence of 5 percent or 10 percent rat liver S9; an equivocal response was seen in TA100 with 30 percent rat S9, and no mutagenic activity was seen in TA98, with or without rat or hamster liver S9 . Butanal oxime induced chromosomal aberrations in cultured Chinese hamster ovary cells, with and without S9 . Significant increases in the frequencies of micronucleated normochromatic erythrocytes were observed in vivo in peripheral blood of male and female mice administered 25 to 600 mg/kg butanal oxime for 14 weeks by gavage . Synonyms: Butanaloxime; butylaldoxime; butyraldehyde oxime; n-butyraldehyde oxime; butyraldoxime; n-butyraldoxime Trade names: Exkin 1, Exkin No . 1 Anti-Skinning Agent, Skino #1, Troykyd Anti-Skin BTO

J Biol Chem, 2004 May 7, 279(19), 20044 - 8 Epub 2004 Mar 10.
3-O-deacylation of lipid A by PagL, a PhoP/PhoQ-regulated deacylase of Salmonella typhimurium, modulates signaling through Toll-like receptor 4; Kawasaki K et al.; Toll-like receptor 4 (TLR4)-mediated responses, which are induced by the lipid A portion of lipopolysaccharide, are important for host defense against Salmonellae infection . A variety of different data indicate that the acylation state of lipid A can alter TLR4-mediated responses . The S . typhimurium virulence gene product PhoP/PhoQ signals the presence of host microenvironments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP . We now demonstrate that 3-O-deacylation and palmitoylation of lipid A decreases its ability to induce TLR4-mediated signaling . Deacylated lipid A, deacylated and palmitoylated lipid A, palmitoylated lipid A, and unmodified lipid A species were purified from Escherichia coli heterologously expressing PagL and/or PagP . The purified lipid A preparations showed spectra of a single lipid A species on mass spectrometry and gave a single band on thin layer chromatography . The activity of purified lipid A species was examined using human and mouse cell lines that express recombinant human TLR4 . Compared with unmodified lipid A, the modified lipid A species are 30-100-fold less active in the ability to induce NF-kappaB-dependent reporter activation . These results suggest that the lipid A modifications reduce TLR4-signaling as part of Salmonellae adaptation to host environments.

Ann Trop Paediatr, 2004 Mar, 24(1), 75 - 80
Salmonella bacteraemia in Turkish children: 37 cases seen in a university hospital between 1993 and 2002; Ciftci E et al.; The aim of the study was to evaluate the clinical pattern of Salmonella bacteraemia in Turkish children . From 1993 to 2002, all children with a blood culture positive for Salmonella were retrospectively evaluated in the Division of Paediatric Infectious Diseases in Ankara University School of Medicine . All Salmonella isolates were serotyped and investigated for antimicrobial susceptibility . During the 10-year study period, 40 patients with Salmonella bacteraemia were identified . Of 37 eligible children, eight had enteric fever and 29 had non-typhoidal salmonellosis . Salmonella typhimurium was the most common serotype in the group with non-typhoidal salmonellosis . No significant differences were found between the enteric fever and non-typhoidal salmonellosis groups with regard to clinical features, laboratory findings and outcome, except in mean platelet counts and mean serum alanine aminotransferase (ALT) levels . In vitro resistance rates of Salmonella strains were low . Outcome was excellent in all but one child with hydrocephalus and gross neurological sequelae owing to meningitis . Salmonella bacteraemia is relatively uncommon in Turkish children . Differentiating between enteric fever and non-typhoidal Salmonella bacteraemia on clinical grounds is difficult.

Acta Pol Pharm, 2003 Sep-Oct, 60(5), 357 - 62
Evaluation of the mutagenic activity of phenolics from the bark of Yucca schidigera Roezl; Czeczot H et al.; The mutagenic activity of yuccaols A, B, and C, trans-resveratrol and trans - 3.3',5.5'-tetrahydroxy -4'-methoxystilbene was tested by the Ames method with Salmonella typhimurium strains TA97, TA98, TA100, TA102 in the absence and presence of metabolic activation (S9 fraction) . These phenolic compounds have been isolated and identified from the hark of Yucca schidigera . All of them were found to be non-toxic and non-mutagenic for testing doses in any of the S . typhimurium strains.

Proc Natl Acad Sci U S A, 2004 Mar 16, 101(11), 3945 - 50 Epub 2004 Mar 04.
Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export; Thomas J et al.; Bacterial type III protein export underlies flagellum assembly and delivery of virulence factors into eukaryotic cells . The sequence of protein interactions underlying the export pathway are poorly characterized; in particular, it is not known how chaperoned substrates in the cytosol are engaged by the membrane-localized export apparatus . We have identified a stalled intermediate export complex in the flagellar type III export pathway of Salmonella typhimurium by generating dominant-negative chaperone variants that are export-defective and arrest flagellar assembly in the wild-type bacterium . These chaperone variants bound their specific export substrates strongly and severely reduced their export . They also attenuated export of other flagellar proteins, indicating that inhibition occurs at a common step in the pathway . Unlike the cytosolic wild-type chaperone, the variants localized to the inner membrane, but not in the absence of the flagellar type III export apparatus . Membrane localization persisted in fliOPQR, flhB, flhA, fliJ, and fliH null mutants lacking specific flagellar export components but depended on the presence of the membrane-associated ATPase FliI . After expression of the variant chaperones in Salmonella, a stalled intermediate export complex, which contained chaperone, substrate, and the FliI ATPase with its regulator FliH, was isolated . Neither chaperone nor substrate alone was able to interact with liposome-associated FliI, but the chaperone-substrate-FliI(FliH) complex was assembled when chaperone was prebound to its substrate . Our data establish a key event in the type III protein export mechanism, docking of the cytosolic chaperone-substrate complex at the ATPase of the membrane-export apparatus.

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