Natl Toxicol Program Tech Rep Ser, 2003 Oct, (509), 1 - 290 NTP toxicology and carcinogensis Studies of 2,4-hexadienal (89% trans,trans isomer, CAS No . 142-83-6; 11% cis,trans isomer) (Gavage Studies); National Toxicology Program; 2,4-Hexadienal, a colorless to yellow liquid with a pungent "green" or citrus odor, is used as a food additive for flavor enhancement, as a fragrance agent, as a starting material or intermediate in synthetic reactions in the chemical and pharmaceutical industries, as a fumigant, and as a corrosion inhibitor for steel . 2,4-Hexadienal was nominated for study by the National Cancer Institute because of the potential for carcinogenicity based on its alpha,beta-unsaturated aldehyde structure and the potential link between exposure to lipid peroxidation products in the diet and human malignancies . The commercial product is a mixture containing chiefly trans,trans-2,4-hexadienal in equilibrium with cis,trans-2,4-hexadienal . Male and female F344/N rats and B6C3F1 mice received 2,4-hexadienal (89% trans,trans; 11% cis,trans) in corn oil by gavage for 16 days, 14 weeks, or 2 years . Tissues and plasma from dosed rats were examined for malondialdehyde and glutathione concentrations, and DNA adducts were characterized in liver and forestomach samples from dosed rats and mice . Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes . 16-DAY STUDY IN RATS: Groups of five male and five female rats were administered 0, 3, 9, 27, 80, or 240 mg 2,4-hexadienal/kg body weight in corn oil by gavage, 5 days per week, for 16 days . Three male and three female 240 mg/kg rats died before the end of the study . Mean body weight gains of 240 mg/kg rats were significantly less than those of the vehicle controls . Clinical findings included diarrhea, ataxia, lethargy, and nasal/eye discharge in males, and lethargy, paleness, and abnormal breathing in females in the 240 mg/kg groups . Liver weights of 240 mg/kg females were significantly greater than those of the vehicle controls . Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in most 240 mg/kg rats, and forestomach epithelial hyperplasia was microscopically evident in most 80 mg/kg rats . 16-DAY STUDY IN MICE: Groups of five male and five female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 3, 9, 27, 80, or 240 mg/kg, 5 days per week, for 16 days . Chemical-related deaths occurred in one male and one female in the 240 mg/kg groups . Female mice in the 240 mg/kg group lost weight during the study . Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in all 240 mg/kg mice, and forestomach epithelial hyperplasia and hyperkeratosis were microscopically evident in 80 mg/kg mice . 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks . All rats survived to the end of the study . Mean body weights of 30, 60, and 120 mg/kg males were significantly less than those of the vehicle controls . The only clinical finding attributed to 2,4-hexadienal administration was hypersalivation in 30 and 120 mg/kg males and females . The incidences of forestomach hyperplasia and nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg rats . Nasal lesions occurred in most 120 mg/kg male rats . 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks . No deaths were attributed to administration of 2,4-hexadienal . Mean body weights of males and females were similar to those of the vehicle controls throughout the study . Clinical findings included salivation and anal wetness in males and females . Kidney weights of 60 and 120 mg/kg males and liver weights of 60 mg/kg males and females were significantly greater than those of the vehicle controls . The incidences of forestomach hyperplasia and/or nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg mice . 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 2,4-hexministered 2,4-hexadienal in corn oil by gavage at doses of 0, 22.5, 45, or 90 mg/kg, 5 days per week, for up to 105 weeks . Survival of all dosed groups of rats was similar to that of the vehicle control groups . The mean body weights of 90 mg/kg males were generally less than those of the vehicle controls throughout the study . The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female rats . This neoplasm was found in 58% of males and 34% of females in the 90 mg/kg groups . In the forestomach of male rats, papilloma multiplicity was increased in the 90 mg/kg group, and squamous cell carcinomas were found in one 45 mg/kg male and two 90 mg/kg males . Epithelial hyperplasia of the forestomach occurred in most 45 and 90 mg/kg rats . 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 30, 60, or 120 mg/kg, 5 days per week, for up to 105 weeks . Survival of dosed mice was similar to that of the vehicle controls . The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study . The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female mice; squamous cell carcinomas were present in 120 mg/kg males and females . Epithelial hyperplasia of the forestomach occurred in many 120 mg/kg mice . Two 120 mg/kg males had uncommon squamous cell carcinoma of the oral cavity (tongue) . GENETIC TOXICOLOGY: 2,4-Hexadienal was mutagenic in S . typhimurium strain TA100 with and without induced hamster or rat liver enzymes; no mutagenic activity was detected with strains TA1535 or TA98, with or without S9 . Results of bone marrow tests in male rats and male mice given intraperitoneal injections of 2,4-hexadienal showed a small increase in the induction of micronucleated erythrocytes . However, neither test was repeated, and the test results were judged to be inconclusive . Results of peripheral blood micronucleus tests in male and female mice treated with 2,4-hexadienal by gavage for 14 weeks were negative . CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity* of 2,4-hexadienal in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of squamous cell neoplasms of the forestomach . The occurrence of squamous cell carcinoma of the oral cavity (tongue) in male B6C3F1 mice may have been related to the administration of 2,4-hexadienal . Hyperplasia of the forestomach in male and female rats and mice was associated with administration of 2,4-hexadienal . Synonyms: Hexa-2,4-dienal; 2,4-hexadienal; 2,4-hexadien-1-al; 2,4-Hx; 1,3-pentadiene-1-carboxaldehyde; 2-propylene acrolein; sorbaldehyde; sorbic aldehyde Toxicol Appl Pharmacol, 2004 Mar 1, 195(2), 166 - 81 In vitro biotransformation and genotoxicity of the drinking water disinfection byproduct bromodichloromethane: DNA binding mediated by glutathione transferase theta 1-1; Ross MK et al.; The drinking water disinfection byproduct bromodichloromethane (CHBrCl(2)) was previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione transferase theta 1-1 (GSTT1-1) . Several experimental approaches were undertaken in this study to investigate the DNA covalent binding potential of reactive intermediates generated by GSTT1-1-mediated metabolism of CHBrCl(2) . First, rodent hepatic cytosol incubations containing {(14)C}CHBrCl(2), supplemented glutathione (GSH), and calf thymus DNA resulted in approximately 3-fold (rat liver cytosol) and 7-fold (mouse liver cytosol) greater amounts of total radioactivity (RAD) associated with the purified DNA as compared to a control (absence of rodent cytosol) following liquid scintillation counting (LSC) of isolated DNA . The relative increase in DNA labeling is consistent with the conjugation activity of these rodent cytosols toward CHBrCl(2) . Second, exposure of GSTT1-1-expressing S . typhimurium to {(14)C}CHBrCl(2) resulted in a concentration-dependent increase of bacterial DNA-associated total radioactivity . Characterization of DNA-associated radioactivity could not be assigned to a specific deoxynucleoside adduct(s) following enzymatic hydrolysis of DNA and subsequent HPLC analysis . A possible explanation for this observation was formation of a 'transient' adduct that was unstable in the DNA isolation and hydrolysis procedures employed . To circumvent problems of adduct instability, reactions of {(14)C}CHBrCl(2) with GSH catalyzed by recombinant rat GSTT1-1 were performed in the presence of calf thymus DNA or, alternatively, the model nucleophile deoxyguanosine . Hydroxyapatite chromatography of {(14)C}-labeled DNA or HPLC chromatography of {(14)C}-labeled deoxyguanosine derivatives demonstrated the covalent binding of {(14)C}CHBrCl(2)-derived metabolites to DNA and deoxyguanosine in low yield (approximately 0.02% of {(14)C}CHBrCl(2) biotransformed by GSTT1-1 resulted in DNA adducts) . Cytochrome P450 (CYP)- and GST-catalyzed biotransformation of CHBrCl(2) in rat tissues (kidney and large intestine) that develop tumors following chronic CHBrCl(2) exposure were compared with rat liver (a nontarget tissue) . Rat liver had a significant capacity to detoxify CHBrCl(2) (to carbon dioxide) compared with kidney and large intestine as a result of CYP-catalyzed oxidation, liver was approximately 16-fold more efficient than kidney and large intestine when intrinsic clearance values (V(max)/K(m)) were compared . In contrast, the efficiency of GST-mediated GSH conjugation of CHBrCl(2) in kidney and large intestine was only slightly lower than liver (approximately 2- to 4-fold lower), thus, the relative amounts of reactive intermediates that are produced with the capacity to covalently modify DNA may be enhanced in these extrahepatic tissues . The significance of these findings is that conjugation of CHBrCl(2) with GSH can result in the covalent modification of DNA and that cancer target tissues in rats have a much reduced detoxification capacity, but only a modest decrease in bioactivation capacity, as compared to the liver (a nontarget tissue in rats). Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3398 - 403 Epub 2004 Mar 01. Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome; Deng S et al.; Transcription and replication both influence and are influenced by superhelical changes in DNA . Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years . Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion . Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins . Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA . Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains . High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module . Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome . In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval . A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span . These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes. Mol Cell, 2004 Feb 27, 13(4), 453 - 4 Actin lessons from pathogens; Le Clainche C et al.; Salmonella typhimurium secretes proteins that co-opt the host actin cytoskeleton to induce membrane ruffling, leading to the uptake of the bacterium . New information about the biochemical activities of the Salmonella protein SipA suggests that this protein might inhibit host cell actin dynamics by competing with ADF/cofilin and gelsolin, two key proteins that promote the turnover of actin filaments. Environ Mol Mutagen, 2004, 43(2), 128 - 33 Mutagenicity of carbon tetrachloride and chloroform in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, using a gas exposure method; Araki A et al.; The volatile solvents carbon tetrachloride and chloroform are carcinogens that are often reported as nonmutagenic in bacterial mutagenicity assays . In this study, we evaluated the mutagenicity of these compounds in Salmonella typhimurium TA98, TA100, TA1535, and TA1537, and Escherichia coli WP2uvrA/pKM101 and WP2/pKM101, with and without S9 mix, using a gas exposure method . Tests were also conducted with a glutathione-supplemented S9 mix . Carbon tetrachloride was mutagenic in TA98 without S9 mix, and in WP2/pKM101 and WP2uvrA/pKM101 with and without S9 mix; carbon tetrachloride was not mutagenic in TA100, TA1535 or TA1537 . Chloroform was mutagenic in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix . Chloroform was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 with or without S9 mix, and was not mutagenic in TA98, TA100, TA1535, TA1537, or WP2uvrA/pKM101 in the presence of glutathione-supplemented S9 mix . The data indicate that carbon tetrachloride and chloroform are bacterial mutagens when adequate exposure conditions are employed and suggest that a genotoxic mode of action could contribute to the carcinogenicity of these compounds . Nat Biotechnol, 2004 Mar, 22(3), 313 - 20 Epub 2004 Feb 08. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins; Yu YA et al.; We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases . The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases . Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated . Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging . These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication . We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors . Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer. Int Arch Allergy Immunol, 2004 Mar, 133(3), 295 - 304 Epub 2004 Feb 25. DNA vaccines designed to inhibit tumor growth by suppression of angiogenesis; Reisfeld RA et al.; The development of new blood vessels, i.e . angiogenesis, is a rate-limiting step in the development of tumors since tumor growth is generally limited to 1-2 mm3 in the absence of a blood supply . Thus, the inhibition of tumor growth by attacking the tumor's vascular supply offers a primary target for antiangiogenic intervention by DNA-based vaccines . Here, we describe two novel orally delivered DNA vaccines which suppress tumor angiogenesis and induce a robust cell-mediated immune response that provides for long-lived protection against melanoma, colon, breast and non-small-cell lung carcinoma in mouse model systems . These vaccines, which are delivered by attenuated Salmonella typhimurium to secondary lymphoid organs, are directed against such targets as vascular endothelial growth factor receptor 2 (FLK-1) and transcription factor Fos-related antigen 1 (Fra-1) . Both vaccines break peripheral T cell tolerance against these self-antigens and induce a robust T cell-mediated immune response leading to suppression of tumor angiogenesis and resulting in effective suppression of tumor growth and metastases . Such research efforts may open up new possibilities for the rational design of future DNA vaccines effective for the prevention and treatment of cancer . Nat Prod Res, 2004 Apr, 18(2), 189 - 95 Analysis of essential oil of Satureja thymbra by hydrodistillation, thermal desorber, and headspace GC/MS techniques and its antimicrobial activity; Goren AC et al.; The essential oil composition of Satureja thymbra was analyzed by direct thermal desorber and Headspace GC/MS analysis methods . Its constituents were determined to be mainly carvacrol (40.15%), gamma-terpinene (26.56%), p-cymene (16.39%), and thymol (13.16%) . The other techniques, thermal desorber and Headspace GC/MS, were used for the plant leaves at three different temperature, which showed similar results . The thermal desorber GC/MS gave better and more sensitive results than Headspace GC/MS . The essential oil was found to be active against the bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Staphylococcus aureus and the yeast Candida albicans. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2422 - 7 Interplay between antibacterial effectors: a macrophage antimicrobial peptide impairs intracellular Salmonella replication; Rosenberger CM et al.; Antimicrobial peptides have established an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated . Macrophage expression of the murine cathelicidin-related antimicrobial peptide (CRAMP) was increased after infection by the intracellular pathogen Salmonella typhimurium, and this increase required reactive oxygen intermediates . By using CRAMP-deficient mice or synthetic CRAMP peptide, we found that CRAMP impaired Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria . This impaired bacterial cell division also depended on intracellular elastase-like serine protease activity, which can proteolytically activate cathelicidins . Macrophage serine protease activity induced filamentation and enhanced the activity of CRAMP in vitro . A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment . Although cationic peptides have been hypothesized to have activity against pathogens within macrophages, this work provides experimental evidence that the antimicrobial arsenal of macrophages includes cathelicidins . These results show that intracellular reactive oxygen intermediates and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen, and they highlight the cooperativity between macrophage antibacterial effectors. Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2404 - 9 Evolutionary comparisons suggest many novel cAMP response protein binding sites in Escherichia coli; Brown CT et al.; The cAMP response protein (CRP) is a transcription factor known to regulate many genes in Escherichia coli . Computational studies of transcription factor binding to DNA are usually based on a simple matrix model of sequence-dependent binding energy . For CRP, this model predicts many binding sites that are not known to be functional . If they are indeed spurious, the underlying binding model is called into question . We use a species comparison method to assess the functionality of a population of such predicted CRP sites in E . coli . We compare them with orthologous sites in Salmonella typhimurium identified independently by CLUSTALW alignment, and find a dependence of mutation probability on position in the site . This dependence increases with predicted site binding energy . The positions where mutation is most strongly suppressed are those where mutation would have the biggest effect on predicted binding energy . This finding suggests that many of the novel sites are functional, that the matrix model correctly estimates their binding strength, and that calculated CRP binding strength is the quantity that is conserved between species . The analysis also identifies many new E . coli binding sites and genes likely to be functional for CRP. Mutagenesis, 2004 Mar, 19(2), 149 - 56 Activation of 3-nitrobenzanthrone and its metabolites to DNA-damaging species in human B lymphoblastoid MCL-5 cells; Arlt VM et al.; 3-Nitrobenzanthrone (3-NBA) is one of the most potent mutagens in the Ames Salmonella typhimurium assay and a suspected human carcinogen recently identified in diesel exhaust and in airborne particulate matter . 3-Aminobenzanthrone (3-ABA), 3-acetylaminobenzanthrone (3-Ac-ABA) and N-acetyl-N-hydroxy-3-aminobenzanthrone (N-Ac-N-OH-ABA) have been identified as 3-NBA metabolites . In the present study we investigated the genotoxic effects of 3-NBA and its metabolites in the human B lymphoblastoid cell line MCL-5 . DNA strand breaks were measured using the Comet assay, chromosomal damage was assessed using the micronucleus assay and DNA adduct formation was determined by 32P-post-labelling analysis . DNA strand-breaking activity was observed with each compound in a concentration-dependent manner (1-50 microM, 2 h incubation time) . At 50 microM median comet tail lengths (CTLs) were 25.0 microm for 3-NBA, 48.0 microm for 3-ABA, 54.5 microm for 3-Ac-ABA and 65.0 microm for N-Ac-N-OH-ABA . Median CTLs in control incubations were in the range 7.7-13.1 micro m . Moreover, the strand-breaking activity of 3-NBA was more pronounced in the presence of a DNA repair inhibitor, hydroxyurea . Depending on the concentration used (1-20 microM, 24 h incubation time), 3-NBA and its metabolites also showed clastogenic activity in the micronucleus assay . 3-NBA and N-Ac-N-OH-ABA were the most active at low concentrations; at 1 microM the total number of micronuclei per 500 binucleate cells was 4.7 +/- 0.6 in both cases, compared with 1.7-3.0 for controls (P < 0.05) . Furthermore, multiple DNA adducts were detected with each compound (1 microM, 24 h incubation time), essentially similar to those found recently in vivo in rats treated with 3-NBA or its metabolites . DNA adduct levels ranged from 1.3 to 42.8 and from 2.0 to 39.8 adducts/10(8) nt using the nuclease P1 and butanol enrichment procedures, respectively . DNA binding was highest for N-Ac-N-OH-ABA, followed by 3-NBA, and much lower for 3-ABA and 3-Ac-ABA . All major 3-NBA-derived DNA adducts produced in MCL-5 cells were found to be formed from reductive metabolites bound to purine bases and lacked an N-acetyl group . These results demonstrate that 3-NBA and its metabolites are effectively activated to DNA-damaging species in human MCL-5 cells, which may reflect the genotoxic potential of 3-NBA in humans . Environmental exposure to 3-NBA may be a health hazard for large sections of the population and the risks associated with such exposure require further assessment. Bioorg Med Chem, 2004 Mar 1, 12(5), 853 - 7 Synthesis and antimicrobial properties of imidazolium and pyrrolidinonium salts; Demberelnyamba D et al.; For the purpose of developing new disinfectants and antiseptics, we searched for compounds having high bactericidal activity against gram-positive bacteria, gram-negative bacteria, and fungi . Three different series of quaternary imidazolium and pyrrolidinonium salts were synthesized: series A (1-alkyl-3-methylimidazolium chlorides and bromides); series B (1-alkyl-2-methyl-3-hydroxyethylimidazolium chlorides); and series C (N-alkyl-N-hydroxyethylpyrrolidinonium) . Series B and C were newly designed . These three series were tested to evaluate their antibacterial and antifungal properties for the first time . Seven microbial strains were used in the study: Escherichia coli KCTC1924, Salmonella typhimurium KCTC1926, Staphylococcus aureus 209 KCTC1916, Staphylococcus aureus R209 KCTC1928, Bacillus subtilis KCTC1914, Candida albicans KCTC1940, and Chlorella regularis . The antimicrobial efficiency was measured by bacterial and fungal growth inhibition expressed as minimal inhibitory concentration (MIC) values . Series A and B imidazolium salts had very good antimicrobial activity against the examined Gram-negative bacteria, Gram-positive bacteria, and fungi . Also the pyrrolidinonium salt was found to have low MIC for some of tested microorganisms . The antibacterial and antifungal active properties of the salts depend upon the structure of functional groups and the alkyl chain length in the imidazolium and pyrrolidinonium ring . Among the synthesized quaternary imidazolium and pyrrolidinonium salts, the imidazolium salts containing a long alkyl chain and the introduction of a hydroxyethyl chain and methyl group into the imidazolium ring structure leads to broad spectrum active antimicrobial agents which not only have bacteriostatic properties but could be powerful bactericides. J Agric Food Chem, 2004 Feb 25, 52(4), 781 - 7 Volatile constituents from the leaves of Callicarpa japonica Thunb . and their antibacterial activities; Kim YS et al.; Volatile substances of Callicarpa japonica Thunb . were examined for their antibacterial activities against six foodborne microorganisms using the optical densitometer Bioscreen C . Extracts of C . japonica were obtained by simultaneous steam distillation and solvent extraction (SDE), and those extracted for 1.5 and 2.0 h at pH 6.0 strongly inhibited the growth of Bacillus cereus and Salmonella typhimurium; the content of the volatile substances of leaves at these pH levels were 543.1 and 706.7 mg/kg, respectively . All foodborne microorganisms tested were strongly inhibited by the addition of >8% (v/v) of the SDE extracts to broth medium . The major volatile components of the SDE extracts obtained at 1.5 h and pH 6.0 were gamma-caryophyllene, 1-octen-3-ol, 2-hexenal, germacrene B, and aromadendrene II, with corresponding peak areas of 44.14, 15.6, 9.86, 5.24, and 4.01%, respectively, and major antibacterial components were 1-octen-3-ol and 2-hexenal . Among the 32 materials identified as volatile flavor components, 2-hexenal, 2,4-hexadienal, 1-octen-3-ol, 2,4-heptadienal, and epiglobulol strongly inhibited microorganism growth . In particular, 2-hexenal (107.52 mg/L) and 1-octen-3-ol (678.64 mg/L) inhibited the growth of most microorganisms tested by >90%. J Food Prot, 2004 Feb, 67(2), 371 - 7 Inactivation of Salmonella typhimurium and Escherichia coli O157:H7 in apple juice by a combination of nisin and cinnamon; Yuste J et al.; Pasteurized apple juice with nisin (0, 25, 50, 100, and 200 ppm, wt/vol) and cinnamon (0 and 0.3%, wt/vol) was inoculated with Salmonella Typhimurium and Escherichia coli O157:H7 at 10(4) CFU/ml and stored at 5 and 20 degrees C . Counts on tryptic soy agar (TSA), selective medium (xylose Lysine desoxycholate agar for Salmonella Typhimurium, and MacConkey sorbitol agar for E . coli O157:H7), and thin agar layer (TAL) were determined at 1 h and 1, 3, 7, and 14 days . The TAL method (selective medium overlaid with TSA) was used for recovery of sublethally injured cells . The pathogens were gradually inactivated by the acidic pH of apple juice . Nisin and cinnamon greatly contributed to the inactivation . The killing effect was more marked at 20 degrees C, with counts in all treated samples being undetectable by direct plating in 3 days for Salmonella Typhimurium and 7 days for E . coli O157:H7 . Thus, several factors influenced the decrease in counts: low pH, addition of nisin and cinnamon, and storage temperature . The TAL method was as effective as TSA in recovering injured cells of the pathogens . The combination of nisin and cinnamon accelerates death of Salmonella Typhimurium and E . coli O157:H7 in apple juice and so enhances the safety of the product. Int J Food Microbiol, 2004 Feb 15, 91(1), 91 - 8 Viability loss and morphology change of foodborne pathogens following exposure to hydrostatic pressures in the presence and absence of bacteriocins; Kalchayanand N et al.; Cell suspensions of three pathogens were exposed to hydrostatic pressure (HP), bacteriocin mixture (nisin and pediocin) or a combination of HP+bacteriocins and changes in colony forming units (cfu) and cell-morphology by scanning electron microscopy (SEM) were studied . Cell viability loss, as determined from the reduction in cfu before and after a treatment, occurred in Listeria monocytogenes by all three treatments and in Salmonella typhimurium and Escherichia coli O157:H7 by HP and HP+bacteriocin combination . Cell wall and cell membrane collapse and cell lysis was indicated in L . monocytogenes exposed to bacteriocin or HP+bacteriocin and in Salmonella and E . coli exposed to HP or HP+bacteriocin. Life Sci, 2004 Mar 5, 74(16), 2023 - 36 Role of histamine and acid back-diffusion in modulation of gastric microvascular permeability and hemorrhagic ulcers in Salmonella typhimurium-infected rats; Hung CR et al.; Documentation concerning the pathogenesis of gastric hemorrhagic ulcer in Salmonella typhimurium (Salmonella typhi)-infective disease is lacking . This research first proposed that alterations of mast cell histamine release, gastric acid back-diffusion and mucosal microvascular permeability are important in modulating gastric ulcer and hemorrhage in Salmonella typhi-infected rats . Additionally, effects of several histamine-related drugs on this ulcer model were evaluated . Male Wistar rats were deprived food for 36 h . Live cultures of Salmonella typhi (OU 5045, 1 x 10(10) CFU in 1.0 mL of sterilized phosphate buffer saline) were challenged, intrajejunally to rats just before withdrawal of food . Control rats received the same volume of sterilized vehicle only . Rat stomachs were irrigated for 3 h with either normal saline or simulated gastric juice . Gastric acid back-diffusion, mucosal histamine concentration, microvascular permeability as well as luminal hemoglobin content and ulcer areas were determined . Severe gastric hemorrhage and mucosal ulcerations, particularly in acidic stomachs, were observed in Salmonella typhi-infected rats . A positive correlation of histamine to gastric hemorrhage and ulcer was found in those rats with Salmonella typhi-infection . This hemorrhagic ulcer in Salmonella typhi-infected rats was effectively ameliorated by intraperitoneal ketotifen, diphenhydramine and ranitidine but was worsen by exogenous histamine or diamine oxidase . In conclusion, enhancement of acid back-diffusion, mast cell histamine release and microvascular permeability is important in modulating gastric hemorrhage and ulcer in Salmonella typhi-infected rats. Rev Esp Quimioter, 2003 Dec, 16(4), 398 - 402 {Different antibiotic resistance mechanisms associated with integrons in clinical isolates of Salmonella typhimurium}; Gallardo F et al.; Antibiotic resistance in clinical isolates of Salmonella typhimurium has steadily risen in recent years . Some of the resistance genes may be carried into integrons . In this study, integrons, both from 10 epidemiologically related and unrelated S . typhimurium clinical isolates, were characterized, showing that epidemiologically different strains can carry the same integron, and that epidemiologically related strain can carry different integrons . Among the resistance genes detected in this study were genes encoding b-lactamases (bla(oxa-30) in two strains, and bla(pse-1) in five strains, one of which was carrying this cassette in two different integrons); aminoglycoside-modifying enzymes (aadA2 in four strains, one of which was carrying this cassette in two different integrons, and aadA1 in six strains); as well dihydrofolate reductases (dfrAI in three strains). Autoimmun Rev, 2004 Jan, 3(1), 61 - 9 Is there a role for viruses in triggering autoimmune hepatitis? Vento S, Cainelli F. A role for viruses in autoimmune hepatitis (AH) has been repeatedly proposed but convincing evidence links only two viruses, hepatitis A and Epstein-Barr virus, to the type 1 form of the disease, and only in those rare cases where a genetic predisposition exists and the viral infection occurs at the right time, i.e . when other unknown factors are cooperating . In spite of an impressive amount of information conclusively showing molecular mimicry between cytochrome P450IID6 (the target autoantigen of autoantibodies characteristic of AH type 2) sequences and viral (hepatitis C virus, herpes simplex virus 1, cytomegalovirus, human T lymphotropic viruses 1 and 2) or bacterial (Salmonella typhimurium) antigens, no infectious agent is clearly able to induce this second form of the disease . In conclusion, the molecular mimicry theory has so far found little clinical evidence in its support and many more clinical observations are needed in order to unreveal possible links between viruses and AH. Planta Med, 2004 Jan, 70(1), 17 - 22 Suppression of infection-induced endotoxin shock in mice by a citrus flavanone naringin; Kawaguchi K et al.; The protective effect of the Citrus flavanone naringin was demonstrated in an endotoxin shock model based on Salmonella infection . Intraperitoneal ( i . p.) infection with 10 (8) CFU Salmonella typhimurium aroA caused lethal shock in lipopolysaccharide (LPS) -responder but not LPS-non-responder mice . Administration of 1 mg naringin 3 h before infection resulted in protection from lethal shock, similar to LPS-non-responder mice . The protective effect of naringin was time- and dose-dependent . Treatment with naringin resulted not only in a significant decrease in bacterial numbers in spleens and livers, but also in a decrease in plasma LPS levels . In addition, naringin markedly suppressed TNF-alpha and normalized the activated states of blood coagulation factors such as prothrombin time, fibrinogen concentration and platelet numbers caused by infection . Interestingly, treatment with naringin suppressed high levels of soluble CD14 and high mobility group-1 molecule caused by infection. Microb Ecol, 2004 Feb, 47(2), 175 - 85 Epub 2004 Feb 09. A portable array biosensor for detecting multiple analytes in complex samples; Taitt CR et al.; The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation . This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument . These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested . Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology . Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading . This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis . J Biol Chem, 2004 Apr 23, 279(17), 17054 - 62 Epub 2004 Feb 02. Thiamine biosynthesis in Escherichia coli: in vitro reconstitution of the thiazole synthase activity; Leonardi R et al.; The biosynthesis of thiamine in Escherichia coli requires the formation of an intermediate thiazole from tyrosine, 1-deoxy-d-xylulose-5-phosphate (Dxp), and cysteine using at least six structural proteins, ThiFSGH, IscS, and ThiI . We describe for the first time the reconstitution of thiazole synthase activity using cell-free extracts and proteins derived from adenosine-treated E . coli 83-1 cells . The addition of adenosine or adenine to growing cultures of Aerobacter aerogenes, Salmonella typhimurium, and E . coli has been shown previously to relieve the repression by thiamine of its own biosynthesis and increase the expression levels of the thiamine biosynthetic enzymes . By exploiting this effect, we show that the in vitro thiazole synthase activity of cleared lysates or desalted proteins from E . coli 83-1 cells is dependent upon the addition of purified ThiGH-His complex, tyrosine (but not cysteine or 1-deoxy-d-xylulose-5-phosphate), and an as yet unidentified intermediate present in the protein fraction from these cells . The activity is strongly stimulated by the addition of S-adenosylmethionine and NADPH. Mol Microbiol, 2004 Jan, 51(2), 483 - 95 Salmonella type III secretion-associated chaperones confer secretion-pathway specificity; Lee SH et al.; Type III protein secretion systems (TTSSs) are ancestrally related to the flagellar export system and are essential for the virulence of many bacteria pathogenic for humans, animals and plants . Most proteins destined to travel the TTSS pathway possess at least two domains that specifically target them to the secretion apparatus . One of the domains is located within the amino terminal first approximately 20 amino acids and the second domain, located within the first approximately 140 amino acids, serves as a binding site for specific chaperones . It has been previously proposed that these two secretion signals are capable of operating independently of one another to facilitate secretion into the extracellular environment . We have found that in the absence of their chaperone-binding domains, the Salmonella typhimurium TTSS-secreted proteins SptP and SopE are no longer targeted for secretion through their cognate TTSS and, instead, are secreted through the flagellar export pathway . These results indicate the existence of an 'ancestral' flagellar secretion signal within TTSS-exported proteins that is revealed in the absence of the chaperone-binding domain . Furthermore, we found that secretion into culture supernatants as well as translocation into host cells by the cognate TTSS require both, the amino terminal and chaperone-binding domains . We conclude from these studies that a critical function for the TTSS-associated chaperones is to confer secretion-pathway specificity to their cognate secreted proteins. Genome Biol . 2004;5(2):R9 . Epub 2004 Jan 29. In silico identification and experimental validation of PmrAB targets in Salmonella typhimurium by regulatory motif detection; Marchal K et al.; BACKGROUND: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence . PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages . The PmrAB system also confers resistance to Fe3+-mediated killing . New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets . RESULTS: We performed an in silico analysis of possible targets of the PmrAB system . Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes . To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure . As well as the known targets, we detected additional targets with unknown functions . Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ) . Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements . CONCLUSIONS: We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally . We also pinpointed directions for further research that could help elucidate the S . typhimurium virulence pathway. Carcinogenesis, 2004 May, 25(5), 779 - 86 Epub 2004 Jan 30. Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of sulfotransferases and acetyltransferases in nitrofen mutagenicity; Glatt H et al.; Nitrofen had been used as a herbicide, until its carcinogenic and teratogenic activity in rodents was detected . A food contamination occurring in 2002 in Germany led to the initiation of new studies in order to better understand the potential risk for humans . Nitrofen is a nitroarene and as such might be activated to a mutagen via reduction to the corresponding hydroxylamine and subsequent formation of a reactive acetic or sulfuric acid ester . Therefore, we have investigated the mutagenicity of nitrofen in Salmonella typhimurium strains engineered for the expression of all human xenobiotic-metabolizing sulfotransferases (SULTs) and acetyltransferases (NATs) identified . Nitrofen was inactive in the parental strains TA1538, TA98 and TA100, but was mutagenic even at low doses when human sulfotransferase SULT1A1 (the major broad-spectrum phenol SULT) was expressed in these strains, but not when it was expressed in a TA1538-derived strain deficient in an endogenous nitroreductase . Several other human SULTs (in particular 1A3 and 1C1) as well as human NAT2 (unlike NAT1) also activated nitrofen, but were markedly less efficient than SULT1A1 . Likewise, expression of rat and mouse SULT1A1 led to weaker mutagenic activity of nitrofen than expression of the corresponding human enzyme . An endogenous acetyltransferase only activated nitrofen to a mutagen when it was strongly over-expressed in the TA98-derived strain YG1024 . Thus, humans might be more susceptible to the carcinogenic effects of nitrofen than mice and rats, which have been used in long-term studies . The fact that several SULTs show particular high expression in fetal tissues suggests that this activation pathway may also play a role in the teratogenic effects observed. Int J Food Microbiol, 2004 Feb 1, 90(3), 331 - 9 Studies to determine the critical control points in pork slaughter hazard analysis and critical control point systems; Pearce RA et al.; Aerobic mesophilic counts (AMC), coliform (CC) and coliform resuscitation counts (CRCs) were obtained by swabbing 50 cm(2) areas at three sites (ham, belly and neck) on pig carcasses, after each of seven stages of the slaughter/dressing process (bleeding, scalding, dehairing, singeing, polishing, evisceration and chilling) . In most cases, there were no statistical differences (P>0.05) among the counts derived by these three methods . Reductions in counts at individual sites were observed after scalding (3.5 log(10) cfu cm(-2)), and singeing (2.5 log(10) cfu cm(-2)) . Increases in counts at individual sites were observed after dehairing (2.0 log(10) cfu cm(-2)) and polishing (1.5 log(10) cfu cm(-2)) . The incidence of Salmonella on pig carcasses was also obtained by swabbing the outside surfaces of 100 half carcasses . Information on the incidence of Salmonella in scald tank water (108 samples) was also investigated . Carcass swabs and scald tank water were examined for the presence of Salmonella using standard enrichment methods . Salmonella were detected on 31% of carcasses immediately after bleeding, 7% of carcasses immediately after dehairing and evisceration, and 1% of carcasses immediately after scalding . Serovars included Salmonella Typhimurium, Salmonella Hadar, Salmonella Infantis and Salmonella Derby . No Salmonella were recovered from samples of scald tank water . The impact of pig slaughter/dressing processes on carcass microbiology and their potential use as critical control points (CCPs) during pork production are discussed. Nucleic Acids Res, 2004 Jan 26, 32(2), 522 - 34 Print 2004. Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives; Sibley MH et al.; A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species . We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli . This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there . To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C . Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2 . To discuss this variation, we adopt the term 'framework' to refer to genes that are stable components of genomes within related lineages, while 'migratory' genes are transient inhabitants of the genome . Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined . The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation . The patterns observed are consistent with the action of a site-specific recombinase . Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked. Biochemistry, 2004 Feb 3, 43(4), 837 - 42 Structural elements, mechanism, and evolutionary convergence of Rho protein-guanine nucleotide exchange factor complexes; Erickson JW et al.; Rho GTPases act as key regulators of cellular biochemistry by determining the timing, direction, and amplitude of signal transduction in a number of important pathways . The rate of activation of a GTPase-controlled reaction is limited by the rate of GTP binding to the Rho protein, and this, in turn, depends on the rate that GDP dissociates from the GTPase . The latter is controlled by the action of guanine nucleotide exchange factors (GEFs) that catalyze GDP-GTP exchange by increasing the rate of GDP dissociation . Here, the recently reported structural information for Rho GTPase-GEF complexes and the molecular basis for the specificity of their interactions are discussed . Underscoring the importance of regulating the Rho GTPase activation pathway, genetically unrelated proteins have evolved which complement or mimic the Dbl homology-Pleckstrin homology (DH-PH) domain-containing family of proteins in their ability to catalyze GDP-GTP exchange . In particular, the structure of the mammalian Cdc42 protein bound to the SopE protein from Salmonella typhimurium illustrates how two unrelated protein folds are able to carry out guanine nucleotide exchange by a remarkably similar mechanism . It will be interesting to see if this conservation of mechanism extends to a newly recognized class of GEFs related to the DOCK180 family. Acta Microbiol Pol, 2003, 52(3), 285 - 92 Amlodipine: a cardiovascular drug with powerful antimicrobial property; Kumar KA et al.; Ten cardiovascular drugs were procured in pure form from their manufacturers in India and screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive and gram-negative types . These bacteria were inhibited by the common antibiotics at 1-5 mg ml(-1) level through our earlier studies . Since most of the bacteria were moderate to highly responsive to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera of gram-positive and 15 genera of gram-negative bacteria . Most of these were inhibited by the drug at 50-200 microg ml(-1) level and few strains were sensitive even at lower concentrations (10 microg ml(-1)) . The bacteria could be arranged in the decreasing order of sensitivity towards amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella spp . and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the drug . Amlodipine was found to be bactericidal in nature when its mode of action was studied against S . aureus 6571, V . cholerae 14035 and Sh boydii 8 NCTC 254/66 . The antibacterial activity of amlodipine could also be confirmed in vivo . When it was given to Swiss strain of white mice at different dosages (30 and 60 microg/mouse), it could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . According to Chi square test the in vivo data were highly significant (p<0.001). Environ Mol Mutagen, 2004, 43(1), 10 - 9 Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter; Oda Y et al.; In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S . typhimurium strain NM2009 to detect genotoxic activity in small volume samples . The assay was used to test the genotoxicity of several standard mutagens and environmental samples . Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9 . The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity . The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain . Induction of umuC gene expression was detected more readily with CPRG than ONPG . The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan . The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons . Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples . J Mol Biol, 2004 Feb 6, 336(1), 81 - 92 Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily; Zaremba M et al.; The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence . Unlike other restriction enzymes, it functions without metal ions . The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily . Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically . BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer . On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer . The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second . Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus . It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily . BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence. J Mol Biol, 2004 Feb 6, 336(1), 25 - 42 Purine and pyrimidine-specific repression of the Escherichia coli carAB operon are functionally and structurally coupled; Devroede N et al.; Transcription of the carAB operon encoding the sole carbamoylphosphate synthetase of Escherichia coli proceeds from a tandem pair of promoters . P2, downstream, is repressed by arginine and the ArgR protein, whereas P1 is submitted to pyrimidine-specific regulation and as shown here to purine-specific control exerted by binding of the PurR protein to a PUR box sequence centered around nucleotide -128.5 with respect to the start of P1 transcription . In vivo analyses of the effects of trans and cis-acting mutations on the regulatory responses and single round in vitro transcription assays indicated that ligand-bound PurR is by itself unable to inhibit P1 promoter activity . To exert its effect PurR relies on the elaborated nucleoprotein complex that governs P1 activity in a pyrimidine-specific manner . Thus we reveal the existence of an unprecedented functional and structural coupling between the modulation of P1 activity by purine and pyrimidine residues that appears to result from the unique position of the PUR box in the carAB control region, far upstream of the promoter . Missing contact and premethylation binding interference studies revealed the importance of base-specific groups and of structural aspects of the PUR box sequence in complex formation . Permutation assays indicated that the overall PurR-induced bending of the carAB control region is slightly less pronounced than that of the purF operator . The PUR boxes of the carAB operon of E.coli and Salmonella typhimurium are unique in that they have a guanine residue at position eight . Interestingly, guanine at this position has been proposed to be extremely unfavorable on the basis of modeling and binding studies, as its exocyclic amino group would enter into a steric clash with the side-chain of lysine 55 . To analyze the effect of guanine at position eight in the upstream half-site of the carAB operator we constructed the adenine derivative and assayed in vivo repressibility of P1 promoter activity and in vitroPurR binding to the mutant operator, and constructed a molecular model for the unusual lysine 55-guanine 8 interaction. Am J Physiol Gastrointest Liver Physiol, 2004 Jun, 286(6), G1024 - 31 Epub 2004 Jan 22. Salmonella typhimurium SipA-induced neutrophil transepithelial migration: involvement of a PKC-alpha-dependent signal transduction pathway; Silva M et al.; Salmonella typhimurium elicits an intense proinflammatory response characterized by movement of polymorphonuclear neutrophils (PMN) across the epithelial barrier to the intestinal lumen . We previously showed that S . typhimurium, via the type III secretion system effector protein SipA, initiates an ADP-ribosylation factor-6- and phospholipase D-dependent lipid-signaling cascade that directs activation of protein kinase C (PKC) and subsequent transepithelial movement of PMN . Here we sought to determine the specific PKC isoforms that are induced by the S . typhimurium effector SipA in model intestinal epithelia and to link the functional consequences of these isoforms in the promotion of PMN transepithelial migration . In vitro kinase PKC activation assays performed on polarized monolayers of T84 cells revealed that S . typhimurium and recombinant SipA induced activation of PKC-alpha, -delta, and -epsilon . To elucidate which of these isoforms play a key role in mediating epithelial cell responses that lead to the observed PMN transepithelial migration, we used a variety of PKC inhibitors with different isoform selectivity profiles . Inhibitors selective for PKC-alpha (Go-6976 and 2,2',3,3',4,4'-hexahydroxyl-1,1'-biphenyl-6,6'-dimethanoldimethyl ether) markedly reduced S . typhimurium- and recombinant SipA-induced PMN transepithelial migration, whereas inhibitors to PKC-delta (rottlerin) or PKC-epsilon (V1-2) failed to exhibit a significant decrease in transepithelial movement of PMN . These results were confirmed biochemically and by immunofluorescence coupled to confocal microscopy . Our results are the first to show that the S . typhimurium effector protein SipA can activate multiple PKC isoforms, but only PKC-alpha is involved in the signal transduction cascade leading to PMN transepithelial migration. Vet Microbiol, 2004 Jan 14, 98(1), 37 - 43 Intestinal colonisation-inhibition and virulence of Salmonella phoP, rpoS and ompC deletion mutants in chickens; Methner U et al.; Administration of live Salmonella strains to day-old chicks provides profound protection against superinfection with a related strain within a matter of hours by a colonisation-inhibition mechanism, which is primarily a bacterial physiological process . Although currently available, commercial, live attenuated Salmonella vaccines induce protection by adaptive immunity, none of them is able to induce protection against Salmonella organisms by colonisation-inhibition and, therefore, they are unable to protect newly-hatched birds immediately after oral vaccination . In this study, mutants of Salmonella Typhimurium and Enteritidis with deletions in phoP and rpoS, either alone or in combination with ompC, were characterised and tested for their level of attenuation and their ability to inhibit the intestinal colonisation of the isogenic parent strains in chickens . Mutants with deletions only in rpoS demonstrated an unaffected potential to inhibit the intestinal colonisation of the challenge strain but were still fully virulent for the chickens . Mutants with deletions in phoP, either alone or in combination with rpoS, resulted in a high level of attenuation, unimpaired ability to colonise the gut and a nearly unaffected potential to inhibit the challenge strain from caecal colonisation . Mutants with an additional deletion in ompC revealed a reduced capacity of intestinal colonisation-inhibition when compared to the control strains and both the single rpoS and the phoP deletion mutants . Mutations in phoP- or phoP-regulated genes may therefore be used for the development of live attenuated Salmonella vaccines possessing these novel characteristics. J Endotoxin Res, 2003, 9(6), 381 - 4 The dual role of LBP and CD14 in response to Gram-negative bacteria or Gram-negative compounds; Heumann D et al.; Innate immunity initiates protection of the host organism against invasion of micro-organisms by specific recognition mechanisms . This article reviews the dual role of LBP/CD14 in innate immunity, focusing mostly on experiments performed in mice by the authors . LPS induces uncontrolled pro-inflammatory response that kills the host and is LBP- and CD14-dependent, as neutralization of LBP and CD14 prevents lethal shock . However, surprisingly, the synthetic Pam3CysSerLys4 bacterial lipoprotein from Escherichia coli (BLP), which is well tolerated in mice, kills the mice upon LBP or CD14 blockade . Furthermore, after blockade of LBP and CD14, the mice succumb to a challenge with virulent Klebsiella pneumoniae or Salmonella typhimurium . Therefore, host responses to Gram-negative bacteria are not identical to that of LPS or BLP . When the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted . Under these conditions, LBP, CD14, and likely Toll-like receptors (TLRs) are a prerequisite to trigger a pro-inflammatory response of macrophages, which is crucial for keeping an infection under control . These studies indicate that we are very far from understanding how the innate system works and more work needs to be done concerning LBP, CD14 or TLRs . Therefore, caution should be the rule about the use of therapeutic approaches to block the pro-inflammatory response in Gram-negative infections. Int J Antimicrob Agents, 2004 Jan, 23(1), 99 - 102 Studies on the antibacterial potentiality of isoflavones; Dastidar SG et al.; The isoflavonoid compounds 'YS11-YS21' were screened for possible antimicrobial property against 12 known Gram-positive and Gram-negative sensitive bacteria . YS11 and YS16 failed to show antimicrobial activity and YS12, 13, 14, 15, 17, 18 and 20 had moderate antimicrobial action . Compounds YS19 and YS21 showed pronounced antimicrobial property . YS19 and YS21 were then tested in vitro against 214 strains of bacteria from one Gram-positive and six Gram-negative genera . The minimum inhibitory concentration (MIC) of YS19 and YS21 was determined by agar dilution method and ranged from 25 to 200 mg/l in most strains . At concentrations of 30 and 60 microg/mouse these compounds offered significant protection to mice challenged with 50 median lethal dose (MLD) of a virulent strain of Salmonella Typhimurium. J Antimicrob Chemother, 2004 Feb, 53(2), 266 - 70 Epub 2004 Jan 16. Antimicrobial susceptibility of Salmonella isolated from cattle, swine and poultry (2001-2002): report from the Japanese Veterinary Antimicrobial Resistance Monitoring Program; Esaki H et al.; OBJECTIVES: The Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) Program was established in 1999 to examine the susceptibility of bacteria from food-producing animals to antimicrobial agents . This study tested the susceptibility of Salmonella isolates collected during 2001-2002 to 20 antimicrobials.Materials and methods: MICs of antimicrobial agents were determined using the NCCLS agar dilution method, and interpreted according to breakpoints obtained from the bimodal MIC distributions . RESULTS: A total of 82 Salmonella were isolated from food-producing animals and tested for antimicrobial susceptibility . Isolates resistant to ampicillin, dihydrostreptomycin, kanamycin, oxytetracycline, chloramphenicol, bicozamycin, nalidixic acid, oxolinic acid and trimethoprim were obtained from healthy animals and diagnostic sample submissions . Salmonella Dublin was isolated only from cattle and showed resistance to older quinolones . Resistance to ampicillin, dihydrostreptomycin, kanamycin and oxytetracycline was common across all serotypes . Fluoroquinolone-resistant Salmonella Choleraesuis was isolated from swine and was the first Japanese report on this type of resistance in Salmonella from an animal origin . Most Salmonella Typhimurium isolates showed resistance to ampicillin, chloramphenicol, dihydrostreptomycin and oxytetracycline . S . Typhimurium DT104 accounted for 40.7% of S . Typhimurium isolates and was more often multi-drug resistant . Most Salmonella Infantis isolates from poultry showed resistance to dihydrostreptomycin, oxytetracycline, trimethoprim or kanamycin . In Salmonella Enteritidis, the major serotype isolated from food-poisoning in Japan, only resistance to dihydrostreptomycin was observed . CONCLUSIONS: This is the first JVARM report of Salmonella isolates, and continuous investigations at the national level on antimicrobial resistance in Salmonella isolated from food-producing animals will be important in the JVARM Program. J Biol Chem, 2004 Apr 2, 279(14), 13555 - 63 Epub 2004 Jan 16. Infection-induced up-regulation of the costimulatory molecule 4-1BB in osteoblastic cells and its inhibitory effect on M-CSF/RANKL-induced in vitro osteoclastogenesis; Saito K et al.; Bacterial infection sometimes impairs bone metabolism . In this study, we infected the osteoblastic cell line MC3T3-E1 with Mycobacterium bovis bacillus Calmette-Guerin (BCG) and identified genes that were up-regulated in the BCG-infected cells by the suppression subtractive hybridization method . A gene encoding 4-1BB (CD137), a member of the tumor necrosis factor-alpha receptor family, was found to be one of the up-regulated genes . Up-regulation of 4-1BB was also observed by infection with Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, and by treatment with lipopolysaccharides and heat-killed BCG . Bone marrow cells and the macrophage-like cell lines J774 and RAW264.7 were found to express 4-1BB ligand (4-1BBL) . Recombinant 4-1BB (r4-1BB) that was immobilized on culture plates strongly inhibited macrophage colony stimulating factor (M-CSF)/receptor activator of nuclear factor-kappaB ligand (RANKL)-induced in vitro osteoclast formation from bone marrow cells . Anti-4-1BBL antibody also inhibited osteoclast formation to a lesser extent, indicating involvement of reverse signaling through 4-1BBL during inhibition of osteoclast formation . A casein kinase I (CKI) inhibitor markedly suppressed the inhibitory effect of r4-1BB on M-CSF/RANKL-induced osteoclast formation, suggesting that CKI might be involved in 4-1BB/4-1BBL reverse signaling . r4-1BB showed no effects on M-CSF- or RANKL-induced phosphorylation of I-kappaB, ERK1/2, p38, or JNK, whereas RANKL-induced phosphorylation of Akt, a downstream target of phosphatidylinositol 3-kinase (PI3K), was completely abolished by r4-1BB, suggesting that 4-1BB/4-1BBL reverse signaling may interfere with PI3K/Akt pathway . r4-1BB also abolished RANKL-mediated induction of nuclear factor of activated T cells-2 . This study may elucidate a novel role of 4-1BB in cell metabolism, especially osteoclastogenesis. Carcinogenesis, 2004 May, 25(5), 801 - 7 Epub 2004 Jan 16. Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido {2,3-b}indole (MeAalphaC) in recombinant test systems expressing human xenobiotic-metabolizing enzymes; Glatt H et al.; 2-Amino-3-methyl-9H-pyrido{2,3-b}indole (MeAalphaC) and some metabolites were investigated for mutagenicity in mammalian cell lines and bacterial strains engineered for the expression of human enzymes . MeAalphaC induced gene mutations (studied at the hprt locus) in Chinese hamster V79-derived cells co-expressing cytochrome (CYP) 1A2 and sulphotransferase (SULT) 1A1 even at a concentration of 30 nM, but was inactive in cells co-expressing CYP1A2 and N-acetyltransferase (NAT) 1 or 2 . MeAalphaC, tested in the presence of rat liver post-mitochondrial fraction, showed strongly enhanced mutagenicity in a Salmonella typhimurium strain expressing human SULT1A1 compared with the control (recipient) strain TA1538/1,8-DNP (deficient in endogenous acetyltransferase) . Mutagenicity was also enhanced, although to a lesser extent, when NAT2 was expressed in the latter strain . The metabolite, 2-hydroxylamino-3-methyl-9H-pyrido{2,3-b}indole (N-OH-MeAalphaC) was a direct mutagen to strains TA1538 and TA1538/ 1,8-DNP . This mutagenicity was strongly enhanced in corresponding strains expressing SULT1A1 . A moderate enhancement was observed when SULT1A2, SULT1B1, SULT1C2 or NAT2 were expressed in strain TA1538 . The remaining enzymes studied (SULT1A3, 1C1, 1E1, 2A1, 2B1a, 2B1b, 4A1 and NAT1) did not indicate any activation of N-OH-MeAalphaC . Preliminary mutagenicity experiments in SULT-expressing S.typhimurium strains were conducted with other hydroxylated metabolites of MeAalphaC . The phenols, 6- and 7-hydroxy-MeAalphaC, were inactive under the conditions studied . The benzylic alcohol, 2-amino-3-hydroxymethyl-9H-pyrido{2,3-b}indole, was mutagenic in a strain expressing SULT1A1, but its activity was much weaker than that of N-OH-MeAalphaC . Thus, N-hydroxylation (e.g . mediated by CYP1A2) and sulpho conjugation (primarily mediated by SULT1A1) was the dominating activation pathway of MeAalphaC in model systems engineered for human enzymes . Some other SULT forms as well as NAT2 were also capable of activating N-OH-MeAalphaC, although with much lower efficiency than SULT1A1 . Another minor activation pathway involved benzylic hydroxylation followed by sulpho conjugation by SULT1A1. Mutat Res, 2004 Feb 14, 557(2), 137 - 49 Effects of paving asphalt fume exposure on genotoxic and mutagenic activities in the rat lung; Zhao HW et al.; Asphalt fumes are complex mixtures of aerosols and vapors containing various organic compounds, including polycyclic aromatic hydrocarbons (PAHs) . Previously, we have demonstrated that inhalation exposure of rats to asphalt fumes resulted in dose-dependent induction of CYP1A1 with concomitant down-regulation of CYP2B1 and increased phase II enzyme quinone reductase activity in the rat lung . In the present study, the potential genotoxic effects of asphalt fume exposure due to altered lung microsomal enzymes were studied . Rats were exposed to air or asphalt fume generated under road paving conditions at various concentrations and sacrificed the next day . Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and examined for DNA damage using the comet assay . To evaluate the systemic genotoxic effect of asphalt fume, micronuclei formation in bone marrow polychromatic erythrocytes (PCEs) was monitored . Lung S9 from various exposure groups was isolated from tissue homogenates and characterized for metabolic activity in activating 2-aminoanthracene (2-AA) and benzo{a}pyrene (BaP) mutagenicity using the Ames test with Salmonella typhimurium YG1024 and YG1029 . This study showed that the paving asphalt fumes significantly induced DNA damage in AM, as revealed by DNA migration in the comet assay, in a dose-dependent manner, whereas the micronuclei formation in bone marrow PCEs was not detected even at a very high exposure level (1733 mg h/m3) . The conversion of 2-AA to mutagens in the Ames test required lung S9-mediated metabolic activation in a dose-dependent manner . In comparison to the controls, lung S9 from rats exposed to asphalt fume at a total exposure level of 479+/-33 mg h/m3 did not significantly enhance 2-AA mutagenicity with either S . typhimurium YG1024 or YG1029 . At a higher total asphalt fume exposure level (1150+/-63 mg h/m3), S9 significantly increased the mutagenicity of 2-AA as compared to the control . However, S9 from asphalt fume-exposed rats did not significantly activate the mutagenicity of BaP in the Ames test . These results show that asphalt fume exposure, which significantly altered both phases I and II metabolic enzymes in lung microsomes, is genotoxic to AM and enhances the metabolic activation of certain mutagens through altered S9 content. J Mol Biol, 2004 Jan 30, 335(5), 1151 - 71 Genomic analysis of bacteriophages SP6 and K1-5, an estranged subgroup of the T7 supergroup; Scholl D et al.; We have determined the genome sequences of two closely related lytic bacteriophages, SP6 and K1-5, which infect Salmonella typhimurium LT2 and Escherichia coli serotypes K1 and K5, respectively . The genome organization of these phages is almost identical with the notable exception of the tail fiber genes that confer the different host specificities . The two phages have diverged extensively at the nucleotide level but they are still more closely related to each other than either is to any other phage currently characterized . The SP6 and K1-5 genomes contain, respectively, 43,769 bp and 44,385 bp, with 174 bp and 234 bp direct terminal repeats . About half of the 105 putative open reading frames in the two genomes combined show no significant similarity to database proteins with a known or predicted function that is obviously beneficial for growth of a bacteriophage . The overall genome organization of SP6 and K1-5 is comparable to that of the T7 group of phages, although the specific order of genes coding for DNA metabolism functions has not been conserved . Low levels of nucleotide similarity between genomes in the T7 and SP6 groups suggest that they diverged a long time ago but, on the basis of this conservation of genome organization, they are expected to have retained similar developmental strategies. J Appl Microbiol, 2004, 96(2), 271 - 8 Oxidation-reduction potential regulates RpoS levels in Salmonella Typhimurium; Komitopoulou E et al.; AIMS: The aim of this work was to investigate the connection between oxidation-reduction (redox) potential and stationary phase induction of RpoS in Salmonella Typhimurium . METHODS AND RESULTS: A lux-based reporter was used to evaluate RpoS activity in S . Typhimurium pure cultures . During growth of S . Typhimurium, a drop in the redox potential of the growth medium occurred at the same time as RpoS induction and entry into stationary phase . An artificially induced decrease in redox potential earlier during growth reduced the time to RpoS induction and Salmonella entered the stationary phase prematurely . In contrast, under high redox conditions, Salmonella grew unaffected and entered the stationary growth phase as normal, although RpoS induction did not occur . As a consequence, stationary phase cells grown in the high redox environment were significantly more heat sensitive (P < 0.05) than those grown under normal conditions . CONCLUSIONS: This work suggests that redox potential can regulate RpoS levels in S . Typhimurium and can thus, control the expression of genes responsible for thermal resistance . SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to manipulate RpoS induction and control stationary phase gene expression can have important implications in food safety . Early RpoS induction under low redox potential conditions can lead to enhanced resistance in low cell concentrations to inimical processes such as heat stress . Inhibition of RpoS induction would abolish stationary phase protective properties making cells more sensitive to common food control measures. Indian J Med Res, 2003 Nov, 118, 192 - 6 Antibacterial potential of an antispasmodic drug dicyclomine hydrochloride; Karak P et al.; BACKGROUND & OBJECTIVES: Several compounds are known to possess antimicrobial activity in addition to their predesignated pharmacological actions . In the present study, dicyclomine hydrochloride, an antispasmodic drug, was tested for possible antimicrobial property in vitro and in vivo . METHODS: The minimum inhibitory concentration (MIC) of dicyclomine against the bacteria was determined by agar and broth dilution methods in vitro . The antibacterial activity of dicyclomine was confirmed by animal experiments . Toxicity and protective efficacy of the drug were tested in vivo . RESULTS: Dicyclomine inhibited most of the bacterial isolates tested at 25-100 microg/ml concentration, and a few were sensitive even at a lower concentration (10 microg/ml) . Dicyclomine was found to be bacteriostatic in nature against Shigella dysenteriae 7, and bactericidal against S . aureus NCTC 6571, 8530, and 8531 . When administered to Swiss white mice at doses of 30 and 60 microg/mouse, dicyclomine protected the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . INTERPRETATION & CONCLUSION: Dicyclomine showed inhibitory action against several pathogenic bacteria . It also offered significant protection to mice against the bacterial challange . As dicyclomine is in routine therapeutic use, it may be developed as a potent antimicrobial agent in many infections. Poult Sci, 2003 Dec, 82(12), 1886 - 97 Effect of intravenous endotoxin on blood cell profiles of broilers housed in cages and floor litter environments; Wang W et al.; Commercial broilers are constantly exposed to airborne microorganisms and endotoxin (lipopolysaccharide, LPS) . It has been shown that microbial contamination of the air was higher in broiler houses using floor litter than in broiler houses using netting-type floors . The current study evaluated the effect of housing conditions on blood leukocyte profiles and tested the hypothesis that, when compared to broilers reared in clean stainless steel cages (Cage group), broilers raised on floor litter (Floor group) should experience a higher environmental challenge and have a desensitized immune system that may exhibit better tolerance/resistance to subsequent intravenous LPS challenge . Hematological parameters were evaluated prior to and following i.v . administration of 1 mg/kg BW Salmonella typhimurium LPS (dissolved at 1 mg/0.25 mL in PBS) or i.v . injection of 0.25 mL/kg BW PBS alone . The results showed that prior to LPS/PBS injection, broilers in the cage group had higher heterophil and monocyte concentrations, a higher B cell percentage within the lymphocyte population, and a higher heterophil to lymphocyte (H:L) ratio in the blood . The i.v . LPS injection resulted in 25% mortality in the cage group and 42% mortality in the floor group within 8 h post-injection . LPS reduced the concentrations of total white blood cells (WBC) and all differential WBC except eosinophils and increased thrombocyte concentrations within 1 h post-injection in both groups . All of these values returned to their respective pre-injection levels within 48 h post-injection in the surviving birds . The two groups exhibited similar overall hematological changes after LPS injection except that the cage group showed a higher H:L ratio at 8 h post-injection and a lower B-cell percentage within the lymphocyte population at 48 h post-injection when compared with the floor group . We concluded that the immune systems of broilers reared on floor litter were desensitized and exhibited less pronounced leukocyte responses to i.v . LPS when compared with those of broilers reared in clean stainless steel cages . However, such desensitization of the immune system did not help broilers survive subsequent i.v . LPS challenge. J Food Prot, 2004 Jan, 67(1), 178 - 80 Salmonella in sesame seed products; Brockmann SO et al.; In the context of an international outbreak of multiresistant Salmonella Typhimurium DT 104 that was correlated to the consumption of halvah ("helva," an Asian candy made from sesame seed), we examined several sesame seed products for the occurrence of Salmonella . Of 117 ready-to-eat food items containing sesame, we isolated salmonellae from 11 (9.4%) samples . In addition to finding Salmonella Typhimurium DT 104 in the halvah involved in the outbreak, we also isolated different Salmonella Typhimurium strains out of halvah from other manufacturers and countries of origin, as well as Salmonella Offa, Salmonella Tennessee, and Salmonella Poona from sesame paste (tahini) and sesame seed, which is sold for raw consumption in cereals. J Food Prot, 2004 Jan, 67(1), 156 - 61 Antigenotoxic effects of water extract from Korean fermented soybean paste (doen-jang); Kim JG; Aflatoxin B1 is a major metabolite of the toxigenic molds Aspergillus flavus and Aspergillus parasiticus . In this study, a bacterial reverse mutation assay with Salmonella Typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 and an in vitro chromosome aberration test with Chinese hamster lung (CHL) cells were used to investigate the genotoxicity of water extract from Korean soybean paste (doen-jang {dwen-jahng}) and its antigenotoxic activity against aflatoxin B1 . The water extract itself did not exhibit cytotoxicity or mutagenicity . The extract significantly reduced the numbers of revertants when it was added to the assay system with Salmonella Typhimurium TA100 (P < 0.05) . The extract also exhibited significant inhibitory effects on chromosome aberration in CHL cells (P < 0.05) . Dose-response relationships were observed between the concentration of the water extract and both its antimutagenic effect and its suppression of chromosome aberration . The results of this work indicate that water extract from Korean soybean paste could have potential as an antigenotoxic substance. J Food Prot, 2004 Jan, 67(1), 148 - 55 Antimicrobial and antioxidant activities of natural extracts in vitro and in ground beef; Ahn J et al.; Inhibition of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes by grape seed extract (ActiVin) and pine bark extract (Pycnogenol) and the effect of these natural extracts on the oxidative stability of raw ground beef were studied . In an agar dilution test, the MICs of ActiVin and Pycnogenol were determined to be 4.0 mg/ml for 4.43 log CFU per plate of E . coli O157:H7 and 4.0 mg/ml for 4.38 log CFU per plate of L . monocytogenes . In an inhibition curve test, populations of E . coli O157:H7, Salmonella Typhimurium, and L . monocytogenes fell to below the detection limit (10 CFU/ml) after 16 h of incubation . The numbers of E . coli O157:H7, L . monocytogenes, and Salmonella Typhimurium declined by 1.08, 1.24, and 1.33 log CFU/g, respectively, in raw ground beef treated with 1% Pycnogenol after 9 days of refrigerated storage . ActiVin (1%) and oleoresin rosemary (1%) resulted in an approximately 1-log CFU/g reduction in the populations of all three pathogens after 9 days . The addition of 1% ActiVin and Pycnogenol contributed to the maintenance of an acidic pH of 5.80 and 5.58, respectively, in raw ground beef . Compared to the control, all treatments increased in L* (lightness), with the exception of ActiVin . ActiVin and oleoresin rosemary had the highest a* (redness) and b* (yellowness) values, respectively . ActiVin most effectively retarded lipid oxidation, followed by Pycnogenol . The results suggest that these natural extracts have potential to be used with other preservative methods to reduce pathogenic numbers, lipid oxidation, and color degradation in ground beef. J Food Prot, 2004 Jan, 67(1), 53 - 9 Modeling the boundaries of growth of Salmonella Typhimurium in broth as a function of temperature, water activity, and pH; Koutsoumanis KP et al.; The growth limits of a mixture of five strains of Salmonella Typhimurium in tryptic soy broth were examined at different environmental conditions . The response of the pathogen was monitored in a total of 350 combination treatments of temperature (10 to 35 degrees C), pH (3.76 to 6.44), and water activity (aw, 0.913 to 0.990) for 62 days . No growth/growth (turbidity) data were modeled by logistic polynomial regression . The concordance index of the logistic model was 99.8%, indicating a good fit to the observed data . The minimum pH and aw values that permitted growth were 3.94 and 0.942, respectively, and occurred in the temperature range of 25 to 35 degrees C . At temperatures below this range, the minimum pH and aw allowing growth increased as the temperature decreased . The results showed an abrupt change in the probability of growth close to the boundary with minor changes of the environmental factors . The probabilities predicted by the model were compared with published data on the actual response of Salmonella Typhimurium or other salmonellae serotypes in 50 cases of food products, including salad dressing, mayonnaise, meat, cheese, vegetables, and fruits . The model predicted successfully the response of the pathogen in 90% of the tested cases . The results of the study indicated that the developed model predicts satisfactorily the growth/no growth interface of Salmonella Typhimurium in foods and can provide useful quantitative data for the development of safer food products and processes. J Food Prot, 2004 Jan, 67(1), 46 - 52 A rapid and automated fiber optic-based biosensor assay for the detection of Salmonella in spent irrigation water used in the sprouting of sprout seeds; Kramer MF et al.; Recent outbreaks of foodborne illness have been linked to the consumption of contaminated sprouts . The spent irrigation water used to irrigate sprouts can carry many microorganisms, including pathogenic strains of Escherichia coli and Salmonella enterica . These pathogens are believed to originate from the seeds . The U.S . Food and Drug Administration recommends that sprout producers conduct microbiological testing of spent irrigation water from each production lot at least 48 h after seeds have germinated . Microbial analysis for the detection of Salmonella is labor-intensive and takes days to complete . A rapid and automated fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water was developed in this study . Alfalfa seeds contaminated with various concentrations of Salmonella Typhimurium were sprouted . The spent irrigation water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research International, Monroe, Wash.), an automated fiber optic-based detector . Salmonella Typhimurium could be positively identified in spent irrigation water when seeds were contaminated with 50 CFU/g . Viable Salmonella Typhimurium cells were also recovered from the waveguides after the assay . This biosensor assay system has the potential to be directly connected to water lines within the sprout-processing facility and to operate automatically, requiring manual labor only for preventative maintenance . Therefore, the presence of Salmonella Typhimurium in spent irrigation water could be continuously and rapidly detected 3 to 5 days before the completion of the sprouting process. J Food Prot, 2004 Jan, 67(1), 27 - 33 Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR; Li Y et al.; With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella . Under the optimized conditions, the assay yielded a 252-bp product from E . coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively . When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed . In the specificity test, 10 E . coli O157:H7 strains and one E . coli O157:NM strain showed the expected 252-bp amplicon . Seven other E . coli strains yielded no signal . Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species . No nonspecific amplification was observed with DNA from 48 other bacterial strains . Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts . The whole procedure can be easily completed within 30 h . The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E . coli O157:H7, Salmonella, and/or Shigella. J Food Prot, 2004 Jan, 67(1), 4 - 11 An assessment of the microbiological risks involved with egg washing under commercial conditions; Hutchison ML et al.; The potential benefits of washing eggs is offset by a historical perception in the European Union that wetted eggs are prone to spoilage and water loss . This study describes the effects of spray jet washing under various processing conditions to shell surface counts of Salmonella and the presence of bacteria in egg contents . Experiments used eggs that were contaminated with Salmonella Enteritidis PT4 or Salmonella Typhimurium DT104 before cuticle hardening . Washing of contaminated eggs under optimum conditions resulted in a more than 5-log reduction of Salmonella counts from the shell surface . Salmonella was not isolated from the yolk or albumen of any egg washed by the optimal protocol, suggesting that when properly controlled, egg washing did not cause Salmonella to enter the contents . However, contamination did arise if strict control was not maintained over the wash and rinse water temperatures . Both Salmonella Enteritidis and Salmonella Typhimurium were shown to enter the egg contents when water temperatures were lowered, indicating that strict temperature control must be maintained in order to prevent the ingress of Salmonella into egg contents . Other washing machine parameters that were investigated did not significantly affect Salmonella entry into the egg contents but influenced shell surface kill levels to varying degrees. Am J Physiol Gastrointest Liver Physiol, 2004 Jun, 286(6), G1050 - 8 Epub 2004 Jan 08. Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice; Clarke LL et al.; Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen . Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice . On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine . Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion . Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution . Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates . Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine . Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis . Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens. Avian Dis, 2003 Oct-Dec, 47(4), 1474 - 80 Infection models for Salmonella typhimurium DT110 in day-old and 14-day-old broiler chickens kept in isolators; Bjerrum L et al.; A series of experiments was undertaken to investigate the infection dynamics of various doses of S . typhimurium in day-old and 14-day-old broiler chickens kept in isolators . The infections were followed quantitatively in ceca and ileum by enumerating the colony forming units (cfu) of the challenge strain . It was found that the inoculation of 10(7) cfu of S . typhimurium to day-old chickens established stable cecal infection in all the animals for 35 days . For 14-day-old chickens, stable and lasting infections were seen with inoculation of 10(9) cfu . Lower doses yielded more variable results, and the bacteria were rapidly eliminated from most birds, especially in 14-day-old inoculated chickens . Salmonella was found in spleen and liver 2-3 days postinoculation . Salmonella was cleared from both organs or reduced to very low numbers within 3 weeks. Mutat Res, 2004 Jan 10, 557(1), 99 - 108 Photomutagenicity of 16 polycyclic aromatic hydrocarbons from the US EPA priority pollutant list; Yan J et al.; The photomutagenicity of 16 polycyclic aromatic hydrocarbons (PAHs), all on the United States Environmental Protection Agency (US EPA) priority pollutant list, was studied . Concomitant exposing the Salmonella typhimurium bacteria strain TA102 to one of the PAHs and light (1.1 J/cm2 UVA+2.1 J/cm2 visible) without the activation enzyme S9, strong photomutagenic response is observed for anthracene, benz{a}anthracene, benzo{ghi}perylene, benzo{a}pyrene, indeno{1,2,3-cd}pyrene, and pyrene . Under the same conditions, acenaphthene, acenaphthylene, benzo{k}fluoranthene, chrysene, and fluorene are weakly photomutagenic . Benzo{b}fluoranthene, fluoranthene, naphthalene, phenanthrene, and dibenz{a,h}anthracene are not photomutagenic . These results indicate that PAHs can be activated by light and become mutagenic in Salmonella TA102 bacteria . At the same time, the mutagenicity for all the 16 PAHs was examined with the standard mutagenicity test with 10% S9 as the activation system . Benzo{b}fluoranthene, benzo{k}fluoranthene, chrysene, acenaphthylene, and fluorene are weakly mutagenic, while the rest of the PAHs are not . In general, the photomutagenicity of PAHs in TA102 does not correlate with their S9-activated mutagenicity in either TA102 or TA98/TA100 since they involve different activation mechanisms. Mutat Res, 2004 Jan 10, 557(1), 85 - 97 Genotoxicity studies with pure trans-capsaicin; Chanda S et al.; Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin . However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles . Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances . These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays . In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range . trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range . Limited evidence for very weak activity was also obtained in the absence of S9 mix . trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen . Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes . Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear . Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations. Nucleic Acids Res, 2004 Jan 02, 32(1), 143 - 50 Print 2004. Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes; Nahvi A et al.; Recent studies have begun to reveal that numerous fundamental metabolic pathways in bacteria are regulated by riboswitches residing within certain messenger RNAs . These riboswitches selectively bind metabolites and modulate gene expression in response to changing ligand concentrations . Previously, we provided evidence that the btuB mRNAs of Escherichia coli and Salmonella typhimurium each carry a coenzyme B12-dependent riboswitch that causes repressed translation of the encoded cobalamin-transport protein at elevated coenzyme concentrations . Herein, we use a phylogenetic analysis to define a consensus sequence and secondary structure model for the ligand- binding domain of this riboswitch class . RNA structures that conform to this model are widespread in both Gram-positive and Gram-negative organisms . In addition, we find that the 5'-untranslated region (5'-UTR) of the cobalamin biosynthesis (cob) operon of S.typhimurium carries an RNA motif that matches this consensus sequence . Biochemical and genetic characterization of this motif confirms that the RNA directly binds coenzyme B12, and that it likely serves as a genetic control element for regulating expression of the 25-gene operon for cobalamin production in this pathogen. J Am Soc Mass Spectrom, 2004 Jan, 15(1), 1 - 11 Characterization of acylphosphatidylglycerols from Salmonella typhimurium by tandem mass spectrometry with electrospray ionization; Hsu FF et al.; Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization . The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)) . This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule . The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., {M - H - R'(2)CH=CO}(-) > {M - H - R(2)CO(2)H}(-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., {M - H - R(1)CO(2)H}(-) > {M - H - R'(1)CH=CO}(-); {M - H - R(3')CO(2)H}(-) > {M - H -R'(3')CH=CO}(-)) . The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS) . However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of {M - H - R'(2)CH=CO - R(3)CO(2)H - 74}(-) > {M - H - R'(2)CH=CO - R(1)CO(2)H - 74}(-) z.Gt; {M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74}(-) . This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents . Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated . The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined. Mutat Res, 2004 Jan 12, 545(1-2), 37 - 47 Biphasic effects of the flavonoids quercetin and naringenin on the metabolic activation of 2-amino-3,5-dimethylimidazo{4,5-f}quinoline by Salmonella typhimurium TA1538 co-expressing human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and cytochrome b5; Kang IH et al.; Heterocyclic amines (HCAs) produced by cooking meat products at high temperatures are promutagens that are activated by cytochrome P450 (CYP) lA2 . Using a newly developed Salmonella typhimurium TA1538/1A2bc-b5 strain, we tested the effect of quercetin and naringenin on the mutagenicity of 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) . TA1538/1A2bc-b5 bears two plasmids, one expressing human CYP1A2 and NADPH-P450 reductase (NPR), and the other plasmid which expresses human cytochrome b5 (cyp b5) . TA1538/1A2bc-b5 cells showed high activities of 7-ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) associated with CYP1A2 and are very sensitive to mutagenesis induced by several HCAs . MeIQ was found to be the strongest mutagen among the HCAs tested in this system . Mutagenicity of MeIQ was enhanced 50 and 42% by quercetin at 0.1 and 1 microM, respectively, but suppressed 82 and 96% at 50 and 100 microM . Naringenin also increased the MeIQ-induced mutation about 37 and 22% at 0.1 and 1 microM, but suppressed it 32 and 63% at 50 and 100 microM concentrations, respectively, in TA 1538/1A2bc-b5 cells . Thus, they stimulated the MeIQ induced mutation at low concentrations, but strongly suppressed it at high concentrations . This biphasic effect of flavonoids was due to the stimulation or the inhibition of CYP1A2 activity in a dose-dependent manner judging by the activities of EROD or MROD in the Salmonella cells . These results indicate that quercetin and naringenin can exhibit inhibitory or stimulating effects on CYP1A2 mediated mutagenesis by MeIQ, depending on their concentrations. Mutat Res, 2004 Jan 12, 545(1-2), 11 - 21 Mutagenicity of benzo{b}phenanthro{2,3-d}thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT; Kumar S et al.; Benzo{b}phenanthro{2,3-d}thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S . typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006 . Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9 . In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9 . Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9 . BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9 . In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation . In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9 . Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide . The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S . typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products . Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer. Teratog Carcinog Mutagen, 2003, Suppl 2, 31 - 41 Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents; Harrington DJ et al.; Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells . However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity . For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102) . DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes . The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells . The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis . Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele . However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells . Lett Appl Microbiol, 2004, 38(1), 8 - 12 The effect of copper on the death rate of Salmonella typhimurium DT104:30 in food substrates acidified with organic acids; Beal JD et al.; AIMS: The objective of this study was to investigate the effect of copper ions on the survival of Salmonella typhimurium DT104:30 in acidified liquid food substrates . METHODS AND RESULTS: The decimal reduction time (Dvalue) of Salm . typhimurium DT104:30 was determined in acidified liquid pig feed (LPF) and skimmed milk (SM) containing a range of copper concentrations (0-50 ppm) . As copper concentration increased, the death rate of Salm . typhimurium DT104:30 increased . In LPF acidified with 150 mmol l-1 lactic acid the presence of 50 ppm copper resulted in a 10-fold increase in the death rate . CONCLUSIONS: The presence of copper salts in acidic liquid food substrates significantly increases the death rate of Salm . typhimurium DT104:30 . SIGNIFICANCE AND IMPACT OF THE STUDY: This finding could influence policy on levels of copper in pig feed. J Gastroenterol Hepatol, 2003 Dec, 18(12), 1384 - 91 Helicobacter pylori-induced enlarged-fold gastritis is associated with increased mutagenicity of gastric juice, increased oxidative DNA damage, and an increased risk of gastric carcinoma; Nishibayashi H et al.; BACKGROUND AND AIM: The severe inflammation, increased cell proliferation and marked acid inhibition observed in subjects with Helicobacter pylori-associated enlarged-fold gastritis suggest that enlarged-fold gastritis may be a risk factor for gastric carcinoma . The purpose of the present study was to determine whether a relationship exists between enlarged-fold gastritis and gastric carcinoma . METHODS: One hundred and thirty-five H . pylori-positive patients with early gastric carcinoma and 141 age- and sex-matched H . pylori-positive controls without gastric carcinoma were involved in the study . The widths of gastric body folds were measured by double-contrast radiographs . The mutagenicity of gastric juice was assayed using the Ames test and Salmonella typhimurium TA-98 or TA-100 with S9-mix . Levels of 8-hydroxydeoxyguanosine (8-OHdG) in gastric mucosa were examined using high-performance liquid chromatographic-electrochemical detection . RESULTS: An upward shift in the distribution of gastric fold widths in H . pylori-positive patients with early gastric carcinoma was found . Enlarged-fold gastritis (fold width >/=5 mm) was observed in 81% of the patients with gastric carcinoma, compared with 46% of H . pylori-positive controls . The odds ratio for gastric carcinoma increased with increasing fold width to a maximum of 35.5 in persons with fold width >/=7 mm . The prevalence of diffuse-type early gastric carcinoma in the body region increased with increasing fold width . The mutagenicity of gastric juice from the patients with enlarged-fold gastritis was significantly higher than that in H . pylori-negative controls and H . pylori-positive patients without enlarged folds . Mucosal 8-OHdG levels in the body region of patients with enlarged-fold gastritis were significantly higher than in H . pylori-negative controls and H . pylori-positive patients without enlarged-fold gastritis . Eradication of H . pylori significantly decreased the mutagenicity of gastric juice and 8-OHdG levels in the gastric mucosa from patients with enlarged-fold gastritis . CONCLUSION: A significant association is suggested between enlarged-fold gastritis and gastric carcinoma. Environ Mol Mutagen, 2003, 42(4), 233 - 42 Genetic toxicity of methamphetamine in vitro and in human abusers; Li JH et al.; Methamphetamine (METH) is a widely abused psychomotor stimulant . Although numerous studies have examined METH-induced neurotoxicity, its ability to produce genotoxic effects has not been evaluated . In this article, we report on the genotoxicity of METH in vitro and in human METH abusers . METH induced his(+) revertants in Salmonella typhimurium strains TA98 and TA100, and increased the frequency of hprt mutants, micronuclei, and sister chromatid exchange (SCE) in cultured Chinese hamster ovary K1 (CHO-K1) cells . These METH-induced genotoxic effects were eliminated if METH exposure was conducted in the presence of rat liver S9, indicating that the genotoxicity was caused by METH, and not by metabolites of METH . In addition, reactive oxygen species (ROS) scavengers inhibited the METH-induced micronuclei in CHO-K1 cells . Further investigation with 76 human long-term METH abusers and 98 unexposed controls demonstrated that total METH exposure correlated with micronucleus and SCE frequencies in cultured lymphocytes . The results of this study indicate that METH is a genotoxic agent and that ROS may play a role in METH-induced genotoxicity . BMC Mol Biol . 2003 Dec 13;4(1):11. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E . coli; Murphy KC et al.; BACKGROUND: The lambda Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E . coli such as EHEC and EPEC . Our early experiments with the use of lambda Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E . coli . RESULTS: In this report, we have identified conditions that optimize the use of lambda Red for recombineering in EHEC and EPEC . Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes . In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors . We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (approximately 1 kb of flanking homology); both work well and each have their own advantages . The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam . This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin . Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC . CONCLUSIONS: The lambda Red recombineering technology has been optimized for use in pathogenic species of E . coli, namely EHEC and EPEC . As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA . These results should encourage the use of lambda Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development. J Food Prot, 2003 Dec, 66(12), 2302 - 6 A rapid most-probable-number-based enzyme-linked immunosorbent assay for the detection and enumeration of Salmonella Typhimurium in poultry wastewater; Goodridge C et al.; The rapid and accurate detection and enumeration of low levels of Salmonella Typhimurium in food processing facilities are critical components of an effective hazard analysis critical control point program . The objective of this study was to develop a rapid (8 h) most probable number (MPN)-enzyme-linked immunosorbent assay (ELISA) for the detection and enumeration of Salmonella Typhimurium in wastewater . The specific objectives were to (i) characterize poly- and monoclonal Salmonella Typhimurium-specific antibodies in order to select the most specific and sensitive antibody for Salmonella Typhimurium detection, and (ii) validate the MPN assay through a correlation between the 8-h MPN-ELISA and the traditional 48-h Salmonella Typhimurium MPN method in poultry scald water . Poultry scald water samples were spiked with 10 and 50 CFU/ml of Salmonella Typhimurium . The traditional MPN method used a 48-h enrichment period followed by an analysis, while the MPN-ELISA used a 5-h enrichment period followed by a 3-h ELISA analysis . No differences (P < 0.05) were found between the traditional MPN and the MPN-ELISA, indicating the promise of the MPN-ELISA for the rapid detection and enumeration of Salmonella Typhimurium within an 8-h shift . This abbreviated assay will permit increased product sampling and more rapid movement of food between production and processing, resulting in reduced spoilage and quality losses. Trends Microbiol, 2003 Dec, 11(12), 562 - 9 Translating tissue culture results into animal models: the case of Salmonella typhimurium; Hurley BP et al.; Investigators use both in vitro and in vivo models to better understand infectious disease processes . Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relev