Salmonella typhimurium

J Nutr, 2002 Mar, 132(3), 472 - 7
Dietary oligofructose and inulin protect mice from enteric and systemic pathogens and tumor inducers; Buddington KK et al.; Prebiotics induce changes in the population and metabolic characteristics of the gastrointestinal bacteria, modulate enteric and systemic immune functions, and provide laboratory rodents with resistance to carcinogens that promote colorectal cancer . There is less known about protection from other challenges . Therefore, mice of the B6C3F1 strain were fed for 6 wk a control diet with 100 g/kg cellulose or one of two experimental diets with the cellulose replaced entirely by the nondigestible oligosaccharides (NDO) oligofructose and inulin . From each diet, 25 mice were challenged by a promoter of colorectal cancer (1,2-dimethylhydrazine), B16F10 tumor cells, the enteric pathogen Candida albicans (enterically), or were infected systemically with Listeria monocytogenes or Salmonella typhimurium . The incidences of aberrant crypt foci in the distal colon after exposure to dimethylhdrazine for mice fed inulin (53%) and oligofructose (54%) were lower than in control mice (76%; P < 0.05), but the fructans did not reduce the incidence of lung tumors after injection of the B16F10 tumor cells . Mice fed the diets with fructans had 50% lower densities of C . albicans in the small intestine (P < 0.05) . A systemic infection with L . monocytogenes caused nearly 30% mortality among control mice, but none of the mice fed inulin died, with survival intermediate for mice fed oligofructose . Mortality was higher for the systemic infection of S . typhimurium (>80% for control mice), but fewer of the mice fed inulin died (60%; P < 0.05), with mice fed oligofructose again intermediate . The mechanistic basis for the increased resistance provided by dietary NDO was not elucidated, but the findings are consistent with enhanced immune functions in response to changes in the composition and metabolic characteristics of the bacteria resident in the gastrointestinal tract.

Biochemistry, 2002 Mar 12, 41(10), 3520 - 8
Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase; Cao H et al.; Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes the formation of nicotinate mononucleotide, carbon dioxide, and pyrophosphate from 5-phosphoribosyl 1-pyrophosphate (PRPP) and quinolinic acid (QA, pyridine 2,3-dicarboxylic acid) . The enzyme is the only type II PRTase whose X-ray structure is known . Here we determined the kinetic mechanism of the enzyme from Salmonella typhimurium . Equilibrium binding studies show that PRPP and QA each form binary complexes with the enzyme, with K(D) values (53 and 21 microM, respectively) similar to their K(M) values (30 and 25 microM, respectively) . Although neither PP(i) nor NAMN products bound well to the enzyme, 130-fold tighter binding of PP(i) (K(D) = 75 microM) and NAMN (K(D) = 6 microM) in a ternary complex was observed . Phthalic acid (K(D) = 21 microM) and PRPP each caused a 2.5-fold tightening of the other's binding . Isotope trapping experiments indicated that the E.QA complex is catalytically competent, whereas the E.PRPP complex could not be trapped . Pre-steady-state kinetics gave a linear rate of NAMN formation, indicating that on-enzyme phosphoribosyl transfer chemistry is rate-determining . Isotope trapping from the steady state revealed that nearly all QA and about one-third of PRPP in ternary enzyme.QA.PRPP complexes could be trapped as the product . Substrate inhibition by PRPP was observed . These data demonstrate a predominantly ordered kinetic mechanism in which productive binding of quinolinic acid precedes that of PRPP . An E.PRPP complex exists as a nonproductive side branch.

Zhonghua Yu Fang Yi Xue Za Zhi, 1999 Jan, 33(1), 34 - 6
{Expression and stability of fragment of Plasmodium merozoite major surface protein 1 in recombinant attenuated Salmonella typhimurium}; Qian F et al.; OBJECTIVE: To determine the invasive ability of recombinant attenuated Salmonella typhimurium X4064 (pQEM1) strain containing gene fragment of Plasmodium falciparium merozoite surface protein 1 (MSP1), the stability of its plasmid, and its re-expression . METHODS: BALB/c mice were fed with recombinant attenuated S . typhimurium containing No . 1 gene fragment of P . falciparium MSP1 by gastric tube . Plate incubation, plasmid endonuclease analysis and Western blot were used to identify the recombinant attenuated S . typhimurium strains isolated from mice and the ability of re-expression, and its growth were determined in vitro . RESULTS: The attenuated strain X4064 of S . typhimurium isolated from mice contained recombinant plasmid pQEM1, no . 1 MSP1 fragment of P . falciparium was expressed in vitro in S . typhimurium X4064 (pQEM1) strain, and its growth curve of X4064 (pQEM1) strain in mice was basically similar to that of X4064 . CONCLUSION: The recombinant plasmid pQEM1 could steadily exist in the X4064 strain of S . typhimurium, without influence on its invasion into host cells . X4060 (pQEM1) strain isolated from infected mice still had the ability to re-express M1 protein.

J Immunol, 2002 Mar 1, 168(5), 2415 - 23
Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog; Hamerman JA et al.; After i.p . infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guerin, macrophages recovered from the peritoneal cavity display classical signs of immune activation . We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guerin infection . Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products . The cytokine IFN-gamma also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products . We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages . The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15 . Whereas spi2a was induced by either bacterial products or IFN-gamma, ISG15 was induced only by bacterial products . Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported . Macrophage activation is accompanied by the activation of a variety of proteases . It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.

Eur J Med Chem, 2002 Feb, 37(2), 127 - 33
Quantitative structure--activity relationships of antimutagenic benzalacetones and 1,1,1-trifluoro-4-phenyl-3-buten-2-ones; Yamagami C et al.; The antimutagenic activities (IC(50)) of benzalacetones (BZ) and 1,1,1-trifluo-4-phenyl-3-buten-2-ones (TF) against UV-induced mutagenesis in Escherichia coli WP2s(uvrA trpE) were quantitatively analyzed in terms of physicochemical parameters by regression analyses . Structural requirements for maximal potency were derived from the results of quantitative structure--activity relationship (QSAR) analyses: (1) ring substituents should be electron-withdrawing; (2) 2-OH substituents incapable of intramolecular hydrogen-bonding notably increase the potency; and (3) replacement of CH(3) group by CF(3) in the side chain enhances the activity . Contrary to our expectations, the best correlation lacked hydrophobic effects . Antimutagenic activities against gamma-induced mutagenesis in Salmonella typhimurium TA2638 were also studied for some derivatives in the BZ series, where, in addition to electronic and hydrogen-bonding factors, a hydrophobic term was also significant . Physicochemical meanings of the derived correlations are discussed.

Eur J Biochem, 2002 Jan, 269(2), 443 - 50
Structure of peptidase T from Salmonella typhimurium; Hakansson K et al.; The structure of peptidase T, or tripeptidase, was determined by multiple wavelength anomalous dispersion (MAD) methodology and refined to 2.4 A resolution . Peptidase T comprises two domains; a catalytic domain with an active site containing two metal ions, and a smaller domain formed through a long insertion into the catalytic domain . The two metal ions, presumably zinc, are separated by 3.3 A, and are coordinated by five carboxylate and histidine ligands . The molecular surface of the active site is negatively charged . Peptidase T has the same basic fold as carboxypeptidase G2 . When the structures of the two enzymes are superimposed, a number of homologous residues, not evident from the sequence alone, could be identified . Comparison of the active sites of peptidase T, carboxypeptidase G2, Aeromonas proteolytica aminopeptidase, carboxypeptidase A and leucine aminopeptidase reveals a common structural framework with interesting similarities and differences in the active sites and in the zinc coordination . A putative binding site for the C-terminal end of the tripeptide substrate was found at a peptidase T specific fingerprint sequence motif.

Drug Chem Toxicol, 2002 Feb, 25(1), 93 - 107
Lack of DNA binding in the rat nasal mucosa and other tissues of the nasal toxicants roflumilast, a phosphodiesterase 4 inhibitor, and a metabolite, 4-amino-3,5-dichloropyridine, in contrast to the nasal carcinogen 2,6-dimethylaniline; Jeffrey AM et al.; The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats . The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the 32P-postlabeling assay . For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4'-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF) . In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines . 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP . Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis . Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent . N-hydroxy-ADCP was essentially inactive in this assay . For the 32P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days . Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled . The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P1 and butanol enhancement methods . Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies . No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs . In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.

Prev Vet Med, 2002 Jan 22, 52(3-4), 251 - 65
Qualitative and quantitative risk assessment for human salmonellosis due to multi-resistant Salmonella Typhimurium DT104 from consumption of Danish dry-cured pork sausages; Alban L et al.; Salmonella Typhimurium DT104 (DT104) is unwanted in products for human consumption due to its antibiotic resistance and ability to cause disease . We intended to set up an improved monitoring and management program to aid in deciding when to use pork contaminated with DT104 for production of sausages without jeopardizing consumer safety . We started by carrying out two assessments of the risk for human health associated with consumption of sausages produced by: (1) Danish pork from average slaughter days; (2) imported pork (IMP) with average prevalence of DT104 . The assessments showed that, if Salmonella is present, it is usually in lower numbers (< or =50 per 400 cm(2) surface) . Additionally, during processing, the numbers will be reduced by at least 2 log-units . In Danish (DK) pork, DT104 constitutes 0.2-1.0% of the Salmonella isolates reported, while in imported pork (IMP), 18% . We estimated that out of one million, 25 g servings of DK dry-cured sausages, up to two DT104 bacteria could be found in each of 245 servings . Out of one million servings of 25 g IMP dry-cured sausages, up to two DT104 bacteria would occur in each of 19,260 servings.

Mol Microbiol, 2002 Jan, 43(1), 95 - 106
A potential role for periplasmic superoxide dismutase in blocking the penetration of external superoxide into the cytosol of Gram-negative bacteria; Korshunov SS et al.; Superoxide is a key component of the antibacterial weaponry of phagocytes . Presumably, for this reason, strains of Salmonella typhimurium express a periplasmic superoxide dismutase (SOD) that is essential for full virulence . Because most anions cannot easily penetrate lipid membranes, it is thought that the phagosomal superoxide either damages an unknown target on the bacterial surface or reacts with nitric oxide to form peroxynitrite (HOONO), a toxic oxidant that can freely enter bacteria . However, in this study, we tested whether superoxide itself could penetrate membranes . Superoxide that was generated at high pH (>7.5) very slowly reduced cytochrome c that was encapsulated inside lipid vesicles . It did so much more quickly at lower pH (<7) . Under the latter conditions, more superoxide was protonated and uncharged (HO2*), and the penetrance of superoxide was proportional to the concentration of this species . The permeability coefficient of HO2* was determined to be 9 x 10(-4) cm sec(-1), just slightly lower than that of water and far higher than the value of the anionic form (O2-, <10(-7) cm sec(-1) . When Escherichia coli mutants that lack periplasmic SOD were exposed to super-oxide at pH 6.5, cytosolic fumarase B was damaged . Damage was minimal at higher pH or in strains that contained periplasmic SOD . Thus, in the acidic phagolysosome, superoxide may be able to penetrate and attack cytosolic targets of captive bacteria . This process may contribute to the potency of the oxidative burst . One role of periplasmic SOD may be to avert this damage . In contrast, periplasmic SOD was ineffective at lowering the extracellular super-oxide concentration and, therefore, may have little impact upon HOONO formation.

Lett Appl Microbiol, 2002, 34(1), 62 - 6
Development of membrane filter holder (MFH) method for recovery of heat-injured Escherichia coli O157:H7 and Salmonella typhimurium; Kang DH; AIMS: A method of recovering sublethally heat-injured bacteria was developed with specific apparatus (membrane filter holder; MFH) which was originally used for Iso-Grid Hydrophobic membrane . filter holder . METHODS AND RESULTS: The procedure used a non-selective agar underlayed with a selective medium with a MFH . A non-selective agar was poured on upper part (compartment A) of MFH, and then injured foodborne pathogens were inoculated on the non-selective medium . After 3-h repair incubation period, selective agar was added to the bottom of the chamber (compartment B) of the MFH and further incubated . By diffusing through the non-selective top agar, selective agents from the underlay medium impart selectivity to the system . CONCLUSIONS: Using the MFH method, recovery of heat-injured foodborne pathogens (Escherichia coli O157:H7 and Salmonella typhimurium) were not different (P > 0.05) from recoveries with non-selective media (TSA) . However, the recoveries of foodborne pathogens on MFH were significantly higher (P < 0.05) than those of direct plating on selective medium such as SMAC (MacConkey Sorbitol Agar) or XLD (Xylose Lysine Desoxycholate) . SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the MFH method is a simple and convenient method for recovery of heat-injured foodborne pathogens.

Chem Res Toxicol, 2002 Feb, 15(2), 198 - 208
Characterization of DNA adducts derived from syn-benzo{ghi}fluoranthene-3,4-dihydrodiol-5,5a-epoxide and comparative DNA binding studies with structurally-related anti-diolepoxides of benzo{ghi}fluoranthene and benzo{c}phenanthrene; Chang HF et al.; This paper reports structural characterization of the adducts and tetraols formed from syn-benzo{ghi}fluoranthene-3,4-dihydrodiol-5,5a-epoxide (syn-B{ghi}FDE, 3) and comparative DNA-binding and mutagenicity studies involving 3, anti-B{ghi}FDE (2), and anti-benzo{c}phenanthrene-11,12-dihydrodiol-13,14-epoxide (anti-BcPDE, 5) . The structures of nine DNA adducts and two racemic tetraols derived from 3 have been determined spectroscopically . Similar characterization of adducts obtained from the anti-isomer 2 was described in the preceding paper in this issue {Chang et al . (2002) Chem . Res . Toxicol . 15, 187-197} . The majority of DNA adducts with 3 are those from the trans- or cis-opening of the epoxide at C5a by the exocyclic amino groups of dG, dA, and dC . The diolepoxides 2 and 3 are rigid structure analogues of anti- and syn-BcPDE (5 and 6), respectively, thus serving as models for probing molecular deformity and diol conformation in diolepoxide-DNA interaction . Comparative DNA binding experiments indicate that 57% of 2 and 33% of 3 were converted into DNA adducts, whereas a 71% conversion was observed for 5 . In general, lower percentages were observed with denatured calf-thymus DNA . As for base selectivity, 2 showed a greater affinity for dA relative to dG (dA/dG ratio, 0.79) than 3 (0.56) when reacted with native calf-thymus DNA . A much higher dA/dG ratio (1.41) was obtained for 5 . The overall dA/dG ratios were lower with denatured DNA, indicating the importance of the secondary structure of DNA for both adduct formation and chemical selectivity . The T-shape pseudo-diaxial diols of 3 appears to have favorable electrostatic interactions with the nearby phosphate backbone in the minor groove of DNA, thereby yielding greater amounts of dG adducts than the pseudo-diequatorial 2 . The anti-isomer 2 was found to be seven times more mutagenic than 3, but they are significantly less mutagenic than the nonplanar analogue 5 when tested in Salmonella typhimurium TA 100.

Chem Res Toxicol, 2002 Feb, 15(2), 140 - 52
Structure of the 1,N(2)-propanodeoxyguanosine adduct in a three-base DNA hairpin loop derived from a palindrome in the Salmonella typhimurium hisD3052 gene; Weisenseel JP et al.; The solution structure of the 1,N(2)-propanodeoxyguanosine (PdG) adduct was determined in a 3-base hairpin loop formed by d(CGCGGTXTCCGCG) (X = PdG) . This sequence is contained within the Salmonella typhimurium hisD3052 gene, a hotspot for frameshift mutagenesis . PdG provides a structural model for the primary adduct induced in DNA by malondialdehyde, the 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido{1,2-a}-purin-10(3H)-one (M(1)G) lesion . The solution structure of the PdG-containing hairpin was refined by molecular dynamics calculations restrained by a combination of NMR-derived distances and dihedral angles, using a simulated annealing protocol . The structure of the PdG-modified hairpin consisted of a five-base-pair stem and a three-base loop consisting of T(6), X(7), and T(8) . T(6) projected into the minor groove of the stem adjacent to G(4) . The modified base X(7) stacked on top of the duplex stem and wedged between bases T(8) and C(9) . The PdG moiety was oriented such that the imidazole proton was facing the minor groove of the stem and the exocyclic protons projected into the major groove . The structure of the adducted hairpin was compared with the structure of the corresponding unmodified oligodeoxynucleotide, and was found to be similar . There was a minor difference in the backbone angles of the G and PdG Hairpins at the phosphate linkage between G(5) and T(6) involving the G(5) epsilon angle and T(6) alpha and beta angles . The PdG-modified hairpin exhibited an increase in T(m) of approximately 2 degrees C compared to the unmodified hairpin . The structural and thermodynamic similarities suggested that PdG does not stabilize this hairpin and thus may not promote its extrusion in duplex DNA . The structural results are correlated with the results of site-specific mutagenesis experiments in the same sequence, which do not show evidence of frameshift mutations associated with hairpin loop formation . The geometry of this three-base loop is similar to that of other DNA hairpins containing three-base loops, and suggests a common motif for the folding of these loops.

Chem Res Toxicol, 2002 Feb, 15(2), 127 - 39
Structure of an oligodeoxynucleotide containing a 1,N(2)-propanodeoxyguanosine adduct positioned in a palindrome derived from the Salmonella typhimurium hisD3052 gene: Hoogsteen pairing at pH 5.2; Weisenseel JP et al.; The structure of the 1,N(2)-Propanodeoxyguanosine (PdG) adduct was determined at pH 5.2 in the oligodeoxynucleotide duplex 5'-d(CGCGGTXTCCGCG)3'.5'-d(CGCGGACACCGCG)-3' (X = PdG) . This sequence, referred to as the -TXT- sequence, is contained within the Salmonella typhimurium hisD3052 gene and contains a palindrome, representing a potential hotspot for frameshift mutagenesis . PdG provides a model for the primary adduct induced in DNA by malondialdehyde, the 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido{1,2-a}-purin-10(3H)-one (M(1)G) lesion . The solution structure was refined by molecular dynamics calculations restrained by a combination of NMR-derived distances and dihedral angles, using a simulated annealing protocol . PdG introduced a localized perturbation into the sequence at base pair X(7).C(20), which was pH-dependent . At neutral pH, conformational exchange resulted in spectral line broadening, and it was not possible to determine the structure . A stable structure was observed at pH 5.2 in which PdG rotated about the glycosyl bond into the syn conformation . This placed the exocyclic moiety into the major groove of the duplex . PdG formed a protonated Hoogsteen pair with nucleotide C(20) in the complementary strand . The pseudorotation of the deoxyribose at C(20) was altered to an approximately equal blend of C2'-endo and C3'-endo structures . However, these made little difference in the overall structure of the modified oligodeoxynucleotide . The structure was compared to that of PdG in the 5'-d(CGCXCGGCATG)-3'.5'-(CATGCCGCGCG)-3' sequence (the -CXC- sequence) at pH 5.8 {Singh, U . S., Moe, J . G., Reddy, G . R., Weisenseel, J . P., Marnett, L . J., and Stone, M . P . (1993) Chem . Res . Toxicol . 6, 825-836} . A sequence effect was observed . When PdG was placed into the -TXT- sequence at low pH, the structural perturbation was limited to the X(7).C(20) base pair . In contrast, when PdG was placed into the -CXC- sequence at low pH, both the modified base pair and its 3'-neighbor base pair were disrupted . The results are discussed in the context of differential outcomes for site-specific mutagenesis and replication bypass experiments when PdG was placed in the -TXT- and -CXC- sequences, respectively.

J Food Prot, 2002 Feb, 65(2), 403 - 7
Occurrence of Salmonella enterica serotype typhimurium DT104A in retail ground beef; Zhao T et al.; Surveillance data of cattle and human isolates of Salmonella enterica serovar Typhimurium DT104 indicate that this pathogen emerged worldwide in the 1980s, particularly in cattle . Studies were conducted to determine the prevalence of Salmonella Typhimurium DT104 in ground beef . Samples were also tested for the presence of generic Escherichia coli . A total of 404 fresh ground beef samples obtained at retail stores from New York, San Francisco, Philadelphia, Denver, Atlanta, Houston, and Chicago were shipped overnight to Georgia for processing . Salmonella spp . were isolated from 14 (3.5%) samples . Eight different serotypes were identified among the isolates, including Salmonella Typhimurium (5), Salmonella Lille (3), Salmonella Montevideo (1), Salmonella Hadar (1), Salmonella Meleagridis (1), Salmonella Cerro (1), Salmonella Kentucky (1), and Salmonella Muenster (1) . Antibiotic resistance profiles indicated that all five Salmonella Typhimurium isolates were resistant to ampicillin, streptomycin, sulfamethoxazole, ticarcillin, and tetracycline but that they were sensitive to chloramphenicol . Phage typing revealed that all five Salmonella Typhimurium isolates were DT104A, a subtype of DT104 . All five Salmonella Typhimurium DT104A isolates were obtained from ground beef sampled from retail outlets in San Francisco . Pulsed-field gel electrophoresis (PFGE) genomic DNA profiles of the five Salmonella Typhimurium DT104A isolates from ground beef were indistinguishable from those of four control Salmonella Typhimurium DT104 penta-resistant isolates from cattle that were used for comparison . A total of 102 generic E . coli isolates were obtained, only three of which were multiresistant to antibiotics . In addition, three E . coli isolates were recovered from samples that were positive for Salmonella Typhimurium DT104A . No correlation of antibiotic resistance profiles was observed between Salmonella Typhimurium DT104A and generic E . coli, as two of the three E . coli isolates were susceptible to all of the antibiotics tested, and the third isolate was resistant only to cephalothin . These data indicate that Salmonella Typhimurium DT104A can be isolated from retail ground beef, and because there was little overlap in antibiotic resistance patterns between Salmonella Typhimurium DT104A and E . coli isolates from the same ground beef samples, these limited data suggest that the transfer of antibiotic resistance genes among enteric bacteria in ground beef may not be common . This latter observation is further supported by the limited isolation of multiantibiotic-resistant E . coli from retail ground beef.

J Food Prot, 2002 Feb, 65(2), 284 - 90
Prevalence and antibiotic susceptibility of Salmonella isolated from beef animal hides and carcasses; Bacon RT et al.; This study determined the prevalence of Salmonella on beef animal hides and carcasses and antimicrobial susceptibility profiles against a panel of 13 antibiotics . In each of the eight commercial packing facilities, of which five processed primarily heifers and steers and the remaining three processed primarily cows and bulls, hide and carcass sponge swab samples were obtained immediately before hide removal and before carcass chilling, respectively . Overall, prevalence of Salmonella on external surfaces (hides) of cattle was 15.4% (49 of 319), whereas prevalence after dehiding and other slaughtering/dressing processes, including the application of decontamination treatments, was, as expected, reduced (P < 0.05) to 1.3% (4 of 320) on carcass surfaces . From 53 total Salmonella-positive hide and carcass samples, 526 biochemically confirmed isolates were obtained to determine antimicrobial susceptibility profiles . Of 53 Salmonella-positive samples, individually, 24 (45.3%), 17 (32.1%), 17 (32.1%), 11 (20.8%), 8 (15.1%), 8 (15.1%), 8 (15.1%), 4 (7.5%), and 2 (3.8%) samples yielded at least one isolate resistant to amoxicillin/clavulanic acid, tetracycline, streptomycin, sulfonamides, ampicillin, ampicillin/sulbactam, chloramphenicol, gentamicin, and trimethoprim/sulfamethoxazole, respectively . None of the Salmonella-positive samples yielded an isolate resistant to ceftriaxone, ciprofloxacin, enrofloxacin, or levofloxacin . Although none of the samples yielded an isolate simultaneously resistant to three or four antimicrobials, a total of eight samples yielded at least one isolate resistant to five or more antimicrobials tested . Included among the 18 group B-positive samples were three samples that, individually, yielded at least one Salmonella Typhimurium var . Copenhagen DT104 isolate resistant to at least six antimicrobials tested . Results from this study support current prudent therapeutic and subtherapeutic antimicrobial use recommendations.

Structure (Camb), 2002 Feb, 10(2), 225 - 35
Crystal structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella typhimurium at 2.3 A resolution; Cheng G et al.; The crystal structures of Salmonella typhimurium 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase (HMPP kinase) and its complex with substrate HMP have been determined . HMPP kinase catalyzes two separate ATP-dependent phosphorylation reactions and is an essential enzyme in the thiamin biosynthetic pathway . HMPP kinase is a homodimer with one active site per monomer and is structurally homologous to members of the ribokinase family . A comparison of the structure of HMPP kinase with other members of the ribokinase family suggests an evolutionary progression . Modeling studies suggest that HMPP kinase catalyzes both of its phosphorylation reactions using in-line displacement mechanisms . We propose that the active site accommodates the two separate reactions by providing two different binding modes for the phosphate group of HMP phosphate.

Cell Mol Biol (Noisy-le-grand), 2001 Nov, 47(7), 1115 - 20
Salmonella-mediated mucosal cell-mediated immunity; Lillard JW Jr et al.; Oral immunization with the recombinant Salmonella typhimurium strain (BRD 847) expressing the C fragment of tetanus toxin (TT) induces brisk Ag-specific mucosal S-IgA and serum Ab responses characterized by strong IgG2a Abs to the encoded antigen . We have constructed an attenuated Salmonella typhimurium (aroA- aroD-) strain that expresses chicken egg albumin (OVA) to further elucidate the role of Salmonella-induced Th1 cell phenotype on mucosal cell-mediated immunity (CMI) . Peyer's patches and spleen lymphocytes from mice that received the oral Salmonella-OVA vaccine showed dramatic increases in the percent cell lysis of the H-2b restricted EG7.OVA tumor cell line . These results indicate that a single dose of rSalmonella vaccine antigen vector is required to illicit systemic and mucosal Th1-type responses and CTLs . These results also support the existence of a highly regulated relationship between specific cell-mediated immunity and a branch of the humoral immune system, i.e . mucosal IgA responses.

Teratog Carcinog Mutagen, 2002, 22(2), 113 - 28
Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium; Kargalioglu Y et al.; We analyzed the cytotoxicity and mutagenicity of the drinking water disinfection by-products (DBPs) bromoform (BF), bromoacetic acid (BA), dibromoacetic acid (DBA), tribromoacetic acid (TCA), chloroform (CF), chloroacetic acid (CA), dichloroacetic acid (DCA), trichloroacetic acid (TCA), 3-chloro-4-(dichloromethyl)-5-hydroxy-2{5H}-furanone (MX), and potassium bromate (KBrO3) in Salmonella typhimurium strains TA98, TA100, and RSJ100 +/- S9 . Solvent controls of DMSO and ethanol and a positive control of ethylmethanesulfonate (EMS) were also analyzed . We developed a rapid microplate-based method to determine the cytotoxicity of the DBPs and we determined their mutagenic potencies . The distributions of the rank order for the cytotoxicity and mutagenicity of these DBPs were compared and the structure-function relationships were identified . TA100 -S9 was the most sensitive strain for these DBPs . The rank order of the mutagenic potency adjusted with a cytoxicity factor was MX > BA > EMS > DBA > DCA > CA with TBA, TCA, BF, and CF not mutagenic . From a structure-function perspective, the brominated acetic acids were more cytotoxic and mutagenic than their chlorinated analogs . BA was 150x more mutagenic than CA . The mutagenic potency of the haloacetic acids was inversely related to the number of halogen atoms of the molecule . BA was 36x more mutagenic than DBA . The differential cytotoxicity expressed by the DBPs indicated that a cytotoxicity analysis enhanced the sensitivity of the mutagenicity data, which resulted in an enhanced precision for comparing their relative mutagenic strengths . This information is critical when conducting quantitative structure-function analysis of these hazardous agents .

Ann Rheum Dis, 2002 Mar, 61(3), 264 - 6
Reactive arthritis following an outbreak of Salmonella typhimurium phage type 193 infection; Hannu T et al.; OBJECTIVES: To determine the occurrence and the clinical picture of reactive arthritis (ReA) following an outbreak of Salmonella typhimurium . METHODS: An outbreak of S typhimurium phage type DT 193 occurred in several municipalities in Finland in 1999 . A questionnaire which had a specific emphasis on musculoskeletal symptoms was mailed to all 78 subjects with a positive stool culture . Based on the answers, all subjects with recent joint complaints were clinically examined or interviewed by telephone . RESULTS: Sixty three of 78 subjects (81%) returned the questionnaire . Of these 63 subjects, five (8%) fulfilled the criteria for ReA . All the five subjects with ReA were adults with oligo- or polyarthritis . The antigen HLA-B27 was positive in two of the four subjects tested . In two of five subjects with ReA, the duration of acute arthritis was over six months . Subjects who had received antimicrobial drugs developed acute musculoskeletal symptoms significantly (p=0.013) less often than those without such treatment . None of the subjects with ReA had received antimicrobial drugs before the onset of joint symptoms . CONCLUSIONS: The occurrence of ReA following an outbreak of S typhimurium was at the same level as in outbreaks due to other salmonella serotypes reported previously by us, indicating that the frequency of ReA after various outbreaks is approximately 10% . Early use of antimicrobial drugs may prevent the development of musculoskeletal symptoms.

Biochem Biophys Res Commun, 2002 Feb 15, 291(1), 116 - 23
3D model of human arylamine N-acetyltransferase 2: structural basis of the slow acetylator phenotype of the R64Q variant and analysis of the active-site loop; Rodrigues-Lima F et al.; The human arylamine N-acetyltransferase NAT2 is responsible for the biotransformation of numerous arylamine drugs and carcinogens . A common polymorphism of the NAT2 gene has been associated with susceptibility to drug toxicity and various malignancies . In this study, we used the crystal structure of the Salmonella typhimurium NAT (StNAT) to construct a high-quality model of a catalytic N-terminal region of NAT2 (residues 34-131) . We show that this region has a similar structure in StNAT and the human isoforms NAT1 and NAT2 . Comparison of the structures of these three molecules suggests that NATs have an active-site loop with a conserved structure, which is involved in substrate recognition . Our model is consistent with previous experimental data and provides the first plausible structural basis of the effects of a common genetic polymorphism (Arg(64)-->Gln) on NAT2 activity . (c)2002 Elsevier Science (USA).

J Mol Biol, 2002 Feb 1, 315(5), 975 - 94
Bacteriophage p22 portal vertex formation in vivo; Moore SD et al.; Bacteriophage with double-stranded, linear DNA genomes package DNA into pre-assembled icosahedral procapsids through a unique vertex . The packaging vertex contains an oligomeric ring of a portal protein that serves as a recognition site for the packaging enzymes, a conduit for DNA translocation, and the site of tail attachment . Previous studies have suggested that the portal protein of bacteriophage P22 is not essential for shell assembly; however, when assembled in the absence of functional portal protein, the assembled heads are not active in vitro packaging assays . In terms of head assembly, this raises an interesting question: how are portal vertices defined during morphogenesis if their incorporation is not a requirement for head assembly? To address this, the P22 portal gene was cloned into an inducible expression vector and transformed into the P22 host Salmonella typhimurium to allow control of the dosage of portal protein during infections . Using pulse-chase radiolabeling, it was determined that the portal protein is recruited into virion during head assembly . Surprisingly, over-expression of the portal protein during wild-type P22 infection caused a dramatic reduction in the yield of infectious virus . The cause of this reduction was traced to two potentially related phenomena . First, excess portal protein caused aberrant head assembly resulting in the formation of T=7 procapsid-like particles (PLPs) with twice the normal amount of portal protein . Second, maturation of the PLPs was blocked during DNA packaging resulting in the accumulation of empty PLPs within the host . In addition to PLPs with normal morphology, smaller heads (apparently T=4) and aberrant spirals were also produced . Interestingly, maturation of the small heads was relatively efficient resulting in the formation of small mature particles that were tailed and contained a head full of DNA . These data suggest that incorporation of portal vertices into heads occurs during growth of the coat lattice at decision points that dictate head assembly fidelity .

Microbes Infect, 2002 Jan, 4(1), 75 - 82
Assembly of the type III secretion needle complex of Salmonella typhimurium; Kimbrough TG et al.; The type III secretion needle complex (NC) of Salmonella typhimurium is a complex secretory system that functions to translocate virulence proteins into eukaryotic cells . Evolutionarily it is related to bacterial flagella . Assembly of the NC occurs through ordered secretion, polymerization, and assembly, and requires the coordinated expression and association of over 20 different proteins . Recent progress in the understanding of the assembly and architecture of the NC is reviewed.

J Biol Chem, 2002 Apr 12, 277(15), 13346 - 53 Epub 2002 Jan 30.
The Salmonella typhimurium flagellar basal body protein FliE is required for flagellin production and to induce a proinflammatory response in epithelial cells; Reed KA et al.; During apical colonization by Salmonella typhimurium, intestinal epithelial cells orchestrate a proinflammatory response that involves secretion of chemoattractants, predominantly interleukin-8, which coordinate neutrophil trans-epithelial migration at the site of infection . This host-pathogen interaction requires several S . typhimurium genes . To identify novel genes that participate in this pathogen-induced proinflammatory response, we created S . typhimurium Tn-10 transposon mutants and identified a single mutant with Tn-10 insertional inactivation within the fliE flagellar locus that was able to adhere to and invade intestinal epithelial cells normally but was unable to induce interleukin-8 secretion in host cells . The fliE-deficient mutant failed to secrete flagellin and lacked any surface assembly of flagellae . Unlike wild-type S . typhimurium, the fliE-deficient mutant did not activate the IkappaBalpha/NF-kappaB signaling pathway or induce the coordinated trans-epithelial migration of isolated human neutrophils . Transcomplementation of the fliE-deficient mutant with a wild-type fliE-harboring plasmid restored all defects and produced a wild-type S . typhimurium phenotype . Furthermore, functional down-regulation of basolateral TLR5 completely inhibited the monolayers' ability to respond to both wild-type S . typhimurium and purified flagellin but had no affect on tumor necrosis factor alpha-induced responses . We therefore conclude that S . typhimurium fliE is essential for flagellin secretion, flagellar assembly, and S . typhimurium-induced proinflammatory responses through basolateral TLR5 and is consistent with the emerging model of S . typhimurium flagellin-induced inflammation.

J Biol Chem, 2002 May 24, 277(21), 18753 - 62 Epub 2002 Jan 30.
Macrophages inhibit Salmonella typhimurium replication through MEK/ERK kinase and phagocyte NADPH oxidase activities; Rosenberger CM et al.; Host responses during the later stages of Salmonella-macrophage interactions are critical to controlling infection but have not been well characterized . After 24 h of infection, nearly half of interferon-gamma-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria . Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses . To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-gamma-primed RAW 264.7 cell gene expression following infection . We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period . Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units . Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation . Either MEK kinase or phagocyte oxidase inhibitors could be added 4-8 h after infection and still significantly decrease bacterial filamentation . Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology . In summary, Salmonella Typhimurium infection of interferon-gamma-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication . These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens.

J Biol Chem, 2002 Apr 12, 277(15), 12770 - 6 Epub 2002 Jan 30.
Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells; Scott CC et al.; Salmonella typhimurium invades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons . The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes . We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells . Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV . Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV . Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1 . Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells . We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV . The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7 . Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells . These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella.

Diagn Microbiol Infect Dis, 2002 Jan, 42(1), 17 - 20
Susceptibility of human isolates of Salmonella typhimurium DT 104 to antimicrobial agents used in human and veterinary medicine; Beaudin BA et al.; Multiple antibiotic resistance is frequently observed among strains of Salmonella typhimurium DT104 . We examined the antibiotic resistance patterns of 240 human isolates submitted from central and northern Alberta to our laboratory for confirmatory testing during 1996-1999 . Broth microdilution MIC panels included antibiotics proposed by the Canadian National Enteric Disease Surveillance Committee for human and animal isolates . Seven different susceptibility patterns were observed . The two most common patterns accounted for 83% of isolates; 48% were susceptible to all antibiotics tested and 35% were resistant to ampicillin, tetracycline, chloramphenicol and amoxicillin-clavulanate . All strains were susceptible to enrofloxacin and trovafloxacin with variable resistance to kanamycin and chloramphenicol . There were more susceptible isolates observed in 1996 and 1997 than in 1998 and 1999, but multiple resistant isolates were found throughout the study period.

Mutat Res, 2002 Feb 15, 514(1-2), 133 - 46
Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests; Pfuhler S et al.; The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives . They were tested for genotoxicity in three short-term test systems, i.e . in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains . The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S . typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix) . In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate) . FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l) . However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes . By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9 . In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential . Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l . FA at 6 mmol/l completely inhibited the tubulin assembly . These results could clearly be confirmed by electron microscopy examination . The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro . On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.

Mutat Res, 2002 Feb 15, 514(1-2), 19 - 27
Parthenin, a sesquiterpene lactone of Parthenium hysterophorus L . is a high toxicity clastogen; Ramos A et al.; The in vitro and in vivo genotoxicity of parthenin, a sesquiterpene lactone from Parthenium hysterophorus L . with allergenic and irritant action, was assessed in three short-term tests: bacterial reversion in Salmonella typhimurium and Escherichia coli, in vitro chromosomal aberrations in peripheral blood lymphocytes and micronuclei in mouse peripheral blood . Parthenin was not mutagenic in S . typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 but a weak response was observed in TA 102 (+S9) from 0.19 to 1.22 micromole per plate . Concentrations of 7.62 micromole per plate or higher were toxic, but the effect was reduced when S9 was present . Screening of oxidative mutagenesis with E . coli strains IC 188 and IC 203 gave negative results . Parthenin induced chromosomal aberrations, mainly chromatid breaks, in blood lymphocytes exposed to 10-60 microM during 20 h . An association was found with cytotoxicity, since concomitant nuclear alterations such as pycnosis, micronuclei and karyorrhexis were observed . Sister chromatid exchanges (SCEs) in lymphocytes were not influenced by exposure to parthenin; rather a decrease was observed at 60 microM . On the other hand, a minor increment in polyploid metaphases was found at 40 microM . When a single intraperitoneal (i.p.) dose of 4-31 mg/kg of parthenin was administered to mice, a positive increase in the micronucleated reticulocyte (RET) frequency was observed at 48 h for both sexes at the highest dose.

FEMS Microbiol Lett, 2002 Jan 10, 206(2), 229 - 34
Salmonella typhimurium DsbA is growth-phase regulated; Goecke M et al.; Northern blot analyses, transcription assays using a dsbA::lacZ transcriptional fusion, and primer extension mapping were used to characterize the promoter region of dsbA from Salmonella typhimurium . Transcription assays measuring promoter activity of a 258-bp segment of DNA immediately upstream of the dsbA translational start site showed strong growth-phase dependence, with maximal expression in stationary phase and high levels of expression maintained for at least 72 h . This expression was not RpoS-dependent . Two transcripts initiating in the dsbA promoter region were mapped by primer extension analysis and their levels were monitored by Northern blot analysis . Growth conditions such as pH and O(2) levels affected dsbA transcription independently of growth phase . The data suggests that the promoter region of S . typhimurium is not constitutively activated . Its regulation may reflect a requirement for DsbA during conditions resulting in stationary-phase-like growth in the environment.

Environ Mol Mutagen, 2002, 39(1), 43 - 8
Mutagenic activity and mutational specificity of antiprotozoal drugs with and without nitrite treatment; Ono-Ogata T et al.; We examined the mutagenic activities of six antiprotozoal drugs (three diaminopyrimidine compounds {pyrimethamine, diaveridine, and trimethoprim} and three 8-aminoquinoline derivatives {primaquine, pentaquine, and pamaquine}) in Escherichia coli WP2uvrA/pKM101 and Salmonella typhimurium TA100 and TA98 with and without nitrite treatment . The diaminopyrimidine compounds showed no mutagenic activity under any condition in any strain . The 8-aminoquinoline derivatives after nitrite treatment at 5-20 mM for 5 min at pH 3, on the contrary, showed clear mutagenicity in TA100 and WP2uvrA/pKM101 in the presence and absence of S9 mix . We concluded that 8-aminoquinoline derivatives became mutagenic following nitrite treatment . In the Lac(+) reversion assay with E . coli WP3101P-WP3106P, these nitrite-treated compounds induced G:C --> A:T transitions and G:C --> T:A transversions in the absence of S9 mix . On the other hand, A:T --> T:A transversions were induced only in the presence of S9 mix, suggesting a different kind of products may be responsible for the mutagenicity .

Protein Expr Purif, 2002 Feb, 24(1), 138 - 51
Expression and purification of the rifamycin amide synthase, RifF, an enzyme homologous to the prokaryotic arylamine N-acetyltransferases; Pompeo F et al.; The assembly of the polyketide backbone of rifamycin B by the type I rifamycin polyketide synthase, encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam . The sequence of the RifF protein from Amycolatopsis mediterranei shows 26% identity and 40% homology with the members of the arylamine N-acetyltransferase (NAT) family of proteins . Based on the homology of the primary structures and the similarity of the reactions catalyzed by the two enzymes, we have compared the RifF protein with members of the NAT family . We have modeled the three-dimensional (3D) structure of RifF using NAT from Salmonella typhimurium and Mycobacterium smegmatis as a template . Proteolytic digestions of RifF revealed accessible regions in the protein which are in agreement with the modeled structure . We have expressed the whole protein and individual domains of the protein based on comparison with NAT from S . typhimurium and have purified the proteins by affinity chromatography using a hexahistidine tag . RifF has been further purified using ion-exchange (Mono Q) chromatography . An antiserum has been generated using the C-terminal nona- and tridecapeptides of RifF and has been shown to recognize RifF uniquely . It does not cross-react with any other member of the NAT family .

Food Addit Contam, 2002 Jan, 19(1), 62 - 9
Antimutagenic activity of natural phenolic compounds present in the common bean (Phaseolus vulgaris) against aflatoxin B1; Cardador-Martinez A et al.; Polyphenols with antimutagenic and anticarcinogenic properties are present in fruits, vegetables and legumes . In this study, the Salmonella typhimurium tester strains TA98 and TA100 were used in the microsuspension assay to examine the antimutagenic effect of phenolic compounds extracted from the common bean (Phaseolus vulgaris) against mutagenicity induced by aflatoxin B1 (AFB1) . A dose-response curve was constructed for AFB1; from which a level of 40 ng AFB1/tube was selected for all antimutagenicity assays . The AFB1 and phenolic extract (PE) were not toxic to the bacteria at concentrations tested . In the case of PE, results were similar to the number of spontaneous revertants for TA98 and TA100 . The inhibitory effect of PE against AFB1 mutagenicity was dose-dependent at the lower concentrations tested (2.5, 5, 10, 12.5, 15 and 25 microgram-equivalent (+)-catechin/tube for TA98; 0.5, 1, 1.5, 2.5, 5, 10 and 25 microgram-equivalent (+)-catechin/ tube for TA100) . Further, a two-stage incubation procedure was used to investigate the potential interaction between PE and AFB1 . The greatest inhibitory effect of the PE on AFB1 mutagenicity occurred when PE and AFB1 were incubated together . When the bacteria were first incubated with PE followed by a second incubation with AFB1, lower inhibition was observed . Lower inhibition was also observed when the bacteria were first incubated with AFB1 followed by a second incubation with PE . The results suggest that the mechanism of inhibition could involve the formation of a chemical complex between of PE and AFB1.

J Infect Chemother, 1999 Dec, 5(4), 196 - 200
Diffusion of macrolide antibiotics through the outer membrane of Moraxella catarrhalis; Tsujimoto H et al.; We reported previously that the high susceptibility of Moraxella catarrhalis to macrolide antibiotics and other hydrophobic antimicrobial agents was related to the hydrophobicity of the cell surface . Electrophoretic analysis of lipopolysaccharide (LPS) extracted from M . catarrhalis revealed a deep rough-type profile similar to that of an LPS Re type mutant of Salmonella typhimurium, which also exhibits high susceptibility to macrolides . Moreover, treatment of 32P-labeled cells of M . catarrhalis by phospholipase C induced the release of radioactive materials . These results suggested that hydrophobic agents such as macrolides readily access the cell surface exposed by the deep rough-type LPS and phospholipids, and permeate into the cell interior through the lipid bilayer . In fact, M . catarrhalis cells rapidly accumulated large amounts of the macrolide antibiotics, erythromycin and rokitamycin, whereas no accumulation of the macrolides was observed in cells having smooth-type or Rc type LPS under the same conditions.

J Food Prot, 2002 Jan, 65(1), 196 - 8
Growth of Escherichia coli O157:H7, Salmonella typhimurium DT104, and Listeria monocytogenes in dark cutting beef at 10 or 22 degrees C; Hooper-Kinder CA et al.; An experiment was conducted to determine the effects of the dark, firm, and dry (DFD) condition of beef on growth of the foodborne pathogens Escherichia coli O157:H7, Salmonella Typhimurium DT104, and Listeria monocytogenes Scott A in ground beef . Longissimus muscles from a DFD carcass (pH = 6.45) and normal carcass (N; pH = 5.64) were ground and samples obtained (100 and 0% DFD, respectively) . Equal amounts of the 0 and 100% DFD ground samples were mixed to obtain 50% DFD samples . Inoculated 0, 50, and 100% DFD samples were packaged into oxygen-permeable overwrap and stored at 10 degrees C for E . coli O157:H7, Salmonella Typhimurium DT104, and L . monocytogenes Scott A or at 22 degrees C for E . coli O157:H7 . Growth characteristics of E . coli O157:H7, Salmonella Typhimurium DT104, and L . monocytogenes Scott A did not differ (P > 0.05) between 0 and 100% DFD . Results indicated that the DFD beef used in this study was no more susceptible to growth of E . coli O157:H7, Salmonella Typhimurium, or L . monocytogenes Scott A than N beef.

J Appl Toxicol, 2002 Jan-Feb, 22(1), 45 - 60
Genetic toxicology studies with glutaraldehyde; Vergnes JS et al.; Glutaraldehyde (GA; CAS no . 111-30-8) has a wide spectrum of industrial, scientific and biomedical applications, with a potential for human exposure particularly in its biocidal applications . The likelihood for genotoxic effects was investigated in vitro and in vivo . A Salmonella typhimurium reverse mutation assay showed no evidence for mutagenic activity with strains TA98, TA1535, TA1537 and TA1538, with or without metabolic activation . However, there was a weak mutagenic response (1.9-2.3-fold at the highest non-toxic concentration) with TA100 in the presence of metabolic activation . In a Chinese hamster ovary (CHO) forward gene mutation assay (HGPRT locus) there were no consistent, statistically significant, reproducible or dosage-related increases in the frequency of 6-thioguanine resistant cells . There were no reproducible or dosage-related increases in sister chromatid exchanges in an in vitro test in CHO cells . An in vitro cytogenetics study in CHO cells showed no evidence for an increase in chromosomal aberrations on treatment with GA, either in the presence or absence of metabolic activation . In vivo, a mouse peripheral blood micronucleus test showed no increase in micronucleated polychromatophils at sampling times of 30, 48 and 72 h after acute gavage dosing with GA at 40, 80 and 125 mg kg(-1) (corresponding to 25, 50 and 85% of the LD(50)) . The absence of an in vivo clastogenic potential was confirmed by no increase in chromosomal aberrations in a rat bone marrow cytogenetics study with sampling at 12, 24 and 48 h after acute gavage dosing with GA (12.5, 30 or 60 mg kg(-1) with males, and 7.5, 20 or 40 mg kg(-1) with females) . Thus, in this series of tests, GA produced genotoxic effects in vitro only in a bacterial reverse mutation assay with no evidence for in vivo genotoxicity .

Genome Biol . 2002;3(1):RESEARCH0004 . Epub 2001 Dec 14.
Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants; Xie G et al.; BACKGROUND: Tryptophan synthase consists of two subunits, alpha and beta . Two distinct subgroups of beta chain exist . The major group (TrpEb_1) includes the well-studied beta chain of Salmonella typhimurium . The minor group of beta chain (TrpEb_2) is most frequently found in the Archaea . Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies . RESULTS: Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the alpha chain) are absent in TrpEb_2 . Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2 . In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes . CONCLUSIONS: TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction . However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional beta chain, as TrpEb_1 is absent . The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1 . We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase beta chains . A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.

Ter Arkh, 2001, 73(11), 70 - 3
{Priming-phenomenon of neutrophils in patients with Flexner infection}; Gorobchenko AN et al.; AIM: To study reproducibility of priming phenomenon of neutrophils in patients with acute Flexner's dysentery and its realization manifestation . MATERIAL AND METHODS: A chemiluminescent response of peripheral blood neutrophils was studied in patients with acute mild and moderate dysentery in the presence of luminol . Lipopolysaccharide Salmonella typhimurium at a final concentration of 20 ng/ml served as a priming substance of S-chemotype . Neutrophils were stimulated with phorbol myristate acetate in concentration 10(-6) M and isolated on Histopaque double gradient (Sigma reagents, USA) . RESULTS: In reproduction of priming-phenomenon in vitro on neutrophils of patients with acute Flexner's dysentery chemiluminescence amplitude increased 1.29-1.69-fold vs control . CONCLUSION: Priming-phenomenon on neutrophils in acute dysentery is reproducible . This confirms modulating properties of lipopolysaccharides in conditions of systemic nonphysiological endotoxinemia . Priming phenomenon may be involved in both maintenance of homeostasis and pathophysiological processes.

J Agric Food Chem, 2002 Jan 30, 50(3), 633 - 41
Decontamination of aflatoxin-forming fungus and elimination of aflatoxin mutagenicity with electrolyzed NaCl anode solution; Suzuki T et al.; Electrolysis of a 0.1% (17.1 mM) solution of NaCl using separate anode and cathode compartments gives rise to solutions containing active chemical species . The strongly acidic "anode solution" (EW+) has high levels of dissolved oxygen and available chlorine in a form of hypochlorous acid (HOCl) with a strong potential for sterilization, which we have investigated here . Exposing Aspergillus parasiticus at an initial density of 10(3)spores in 10 microL to a 50-fold volume (500 microL) of EW+ containing ca . 390 micromol HOCl for 15 min at room temperature resulted in a complete inhibition of fungal growth, whereas the cathode solution (EW-) had negligible inhibitory effects . Moreover, the mutagenicity of aflatoxin B(1) (AFB(1)) for Salmonella typhimurium TA-98 and TA-100 strains was strongly reduced after AFB(1) exposure to the EW+ but not with the EW- . In high-performance liquid chromatography analysis, the peak corresponding to AFB(1) disappeared after treatment with the EW+, indicating decomposition of the aflatoxin . In contrast, the routinely used disinfectant sodium hypochlorite, NaOCl, of the same available chlorine content as that of EW+ but in a different chemical form, hypochlorite (OCl-) ion, did not decompose AFB(1) at pH 11 . However, NaOCl did decompose AFB(1) at pH 3, which indicated that the principle chemical formula to participate in the decomposition of AFB(1) is not the OCl- ion but HOCl . Furthermore, because the decomposition of AFB(1) was suppressed by pretreating the EW+ with the OH radical scavenger thiourea, the chemical species responsible for the AFB(1)-decomposing property of the EW+ should be at least due to the OH radical originated from HOCl . The OH in EW+ was proved by electron spin resonance analysis.

Toxic Rep Ser, 2000 May, (47), 1 - 56, A1-E6
NTP technical report on the toxicity studies of methacrylonitrile (CAS No . 126-98-7) . Administered by gavage to F344/N rats and B6C3F1 mice; Ghanayem BI; Methacrylonitrile is an aliphatic nitrile used extensively in the preparation of homo- and copolymers, elastomers, and plastics and as a chemical intermediate in the preparation of acids, amides, esters, and other nitriles . This aliphatic nitrile is also used as a replacement for acrylonitrile in the manufacture of an acrylonitrile/butadiene/styrene-like polymer . Methacrylonitrile was nominated for toxicity and carcinogenicity testing by the National Cancer Institute due to its high production volume and extensive use, the lack of chronic or carcinogenicity data, and its structural resemblance to the known rat carcinogen acrylonitrile . The current 13-week studies were conducted as part of an overall effort by the NTP to assess the toxicity and carcinogenicity of methacrylonitrile . During the 13-week studies, groups of 20 male and 20 female F344/N rats were administered 0, 7.5, 15, 30, 60, or 120 mg methacrylonitrile/kg body weight in deionized, purified water by gavage . Groups of 20 male and 20 female B6C3F1 mice were administered 0, 0.75, 1.5, 3, 6, or 12 mg/kg methacrylonitrile . Ten male and ten female rats and mice from each group were evaluated on day 32 . The results of these studies clearly revealed that male rats are more sensitive than females to methacrylonitrile treatment . In the rat study, 19 males and one female administered 120 mg/kg and two males administered 60 mg/kg died during the first week of the study . Males in the 60 mg/kg group at the 32-day interim evaluation and at 13 weeks and females in the 120 mg/kg group at 13 weeks had significantly lower final mean body weights and body weight gains than did the vehicle controls; the surviving male in the 120 mg/kg group also weighed less than the controls at the 32-day interim evaluation . Clinical findings of toxicity were dose dependent and included lethargy, lacrimation, tremors, convulsions, ataxia, and abnormal breathing . There was hematologic evidence indicating that administration of methacrylonitrile induced minimal, normocytic, normochromic anemia . At the 32-day interim evaluation, a minimal dose-related anemia was evidenced by decreases in hematocrit values, hemoglobin concentrations, and erythrocyte counts in male and female rats . The anemia ameliorated by week 13 . Administration of methacrylonitrile resulted in dose-related increases in serum thiocyanate and blood cyanide concentrations of male and female rats . These changes were expected and would be consistent with the in vivo metabolism of methacrylonitrile to cyanide . Blood cyanide concentrations were generally higher in males than in females, which may explain the higher sensitivity of males to the lethal effect of methacrylonitrile . There was also biochemical evidence of increased hepatocellular leakage and/or altered function in dosed male rats, suggesting that the liver may be a target organ for toxic effects of methacrylonitrile . Minimal, but significant, decreases in absolute right kidney and thymus weights (32-day interim evaluation) and increases in liver and stomach weights (week 13) occurred in male rats that received 60 mg/kg compared to the vehicle controls . In female rats, stomach weights of the 60 and 120 mg/kg groups were significantly greater and thymus weights of the 120 mg/kg group were significantly less than those of the controls on day 32 and at week 13; liver weights were also significantly greater in females in the 120 mg/kg group than in the vehicle controls on day 32 . Male and female rats administered 60 mg/kg and females administered 120 mg/kg had significantly greater incidences of metaplasia of the nasal olfactory epithelium on day 32 and at the end of the study than did the vehicle controls; incidences of olfactory epithelial necrosis were also significantly greater in females in the 60 and 120 mg/kg groups than in the vehicle controls on day 32 . Incidence and/or severity increased with increasing dose in females; however, the mortality in male rats administered 120 mg/kg made it difficult to assess the dose-response relationship in males . The no-observed-adverse-effect level for the nasal cavity of rats was 30 mg/kg . Female rats administered 60 or 120 mg/kg methacrylonitrile had significantly longer estrous cycles than did the vehicle controls . Females in the 60 mg/kg group spent more time in diestrus than the vehicle controls . One male and one female mouse in the 12 mg/kg groups died early . Methacrylonitrile administration caused no significant differences in final mean body weights or body weight gains . Clinical findings included lethargy, tremors, ataxia, convulsions, and abnormal breathing . At the 32-day interim evaluation, stomach weights of males administered 3 mg/kg or greater were significantly greater and thymus weights of males in the 12 mg/kg group were significantly less than those of the vehicle controls . At week 13, however, the stomach weights of only males in the 12 mg/kg group were increased relative to the vehicle controls . No treatment-related histopathologic lesions occurred in mice . Methacrylonitrile did not induce mutations in any of several strains of Salmonella typhimurium, with or without S9 activation, and did not induce sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster fed methacrylonitrile during the larval stage . Results of in vivo bone marrow micronucleus tests with methacrylonitrile in male rats and mice were also negative . In summary, gavage administration of methacrylonitrile to rats and mice resulted in dose-dependent lethargy, tremors, lacrimation, convulsions, and abnormal breathing . However, these effects were more pronounced in rats than mice; these differences may be attributed to the higher doses of methacrylonitrile administered to rats . Body weight gain and survival data of rats demonstrated that males are more sensitive to methacrylonitrile dosing than females . There is an apparent correlation between blood cyanide concentrations and survival rates, with males having greater cyanide concentrations and lower survival rates than female rats administered methacrylonitrile . Microscopically, the only target of methacrylonitrile toxicity was the olfactory epithelium of the nasal cavity . Necrotic and metaplastic effects were induced in male and female rats that received 60 or 120 mg/kg per day . No similar lesions were observed in mice administered methacrylonitrile . The no-observed-adverse-effect level for olfactory epithelial lesions in male and female rats administered methacrylonitrile for 13 weeks was 30 mg/kg per day . No clear chemical-related effects were observed in male or female mice administered methacrylonitrile for 13 weeks by gavage at doses up to 12 mg/kg per day.

Toxic Rep Ser, 1996 Mar, (52), 1 - 91, A1-9, B1-9 passim
NTP technical report on toxicity studies of urethane in drinking water and urethane in 5% ethanol administered to F344/N rats and B6C3F1 mice; Chan PC; Urethane, a byproduct of fermentation found in alcoholic beverages, is carcinogenic in rodents and is classified by the International Agency for Research on Cancer as a possible human carcinogen . The United States Food and Drug Administration nominated urethane for study because of the widespread exposure of humans through the consumption of fermented foods and beverages and because of a lack of adequate dose-response data about the carcinogenicity of urethane with and without the coadministration of ethanol . Comparative studies of urethane in drinking water and in 5% ethanol were conducted to investigate possible effects of ethanol on urethane toxicity . Toxicokinetic studies of urethane in drinking water and in 5% ethanol and genetic toxicity studies of urethane in vivo and in vitro were also conducted . Groups of 10 male and 10 female F344/N rats and B6C3F1 mice, 6 weeks of age, received 0, 110, 330, 1,100, 3,300, or 10,000 ppm urethane in drinking water or in 5% ethanol for 13 weeks . Toxicokinetic evaluations were performed for urethane in the plasma of male mice after 13 weeks of administration in drinking water or 5% ethanol . The mutagenicity of urethane in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without S9 was tested at doses up to 16,666 micrograms/plate; urethane was also tested for induction of sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells and sex-linked recessive lethal mutations and chromosomal reciprocal translocations in Drosophila melanogaster . The frequency of micronucleated erythrocytes induced in peripheral blood and bone marrow cells of mice by urethane in drinking water and in 5% ethanol was also evaluated . In rats that received urethane in drinking water, seven males and four females administered 10,000 ppm and one female administered 3,300 ppm died before the end of the study; body weight gains were reduced at these concentrations . Two males and all females given 10,000 ppm urethane in 5% ethanol died during the study, and the body weight gains of males and females that received 3,300 ppm were lower than those of the controls . Relative right kidney, liver, and lung weights of males and females and relative right testis weights of males administered 1,100 ppm or greater were generally higher than those of the controls in each study . Leukopenia and lymphopenia were observed in rats receiving urethane in either drinking water or ethanol and occurred in males receiving 330 ppm or greater and females receiving 110 ppm or greater . Other differences in hematology and clinical chemistry variables were not considered to be biologically significant . Lymphoid depletion of the spleen, lymph nodes, and thymus was observed in male and female rats receiving 1,100, 3,300, or 10,000 ppm urethane in drinking water . Cellular depletion of the bone marrow occurred in males and females in the 10,000 ppm groups . Hepatocellular fatty changes and clear cell foci of alteration were noted in the liver of males and females that received 3,300 or 10,000 ppm . The incidences of nephropathy were significantly increased in female rats that received 1,100 ppm or greater; the severity of this lesion in exposed males and females was greater than that in the controls . Females that received 330 ppm or greater had higher incidences of cardiomyopathy than the controls; the severity of this lesion was greater in males in the 10,000 ppm group and females in the 3,300 and 10,000 ppm groups than in the controls . In rats that received urethane in 5% ethanol, lymphoid depletion occurred in males and females in the 3,300 and 10,000 ppm groups . Cellular depletion of the bone marrow was observed in males and females in the 10,000 ppm groups . Only males in the 10,000 ppm group had hepatocellular fatty change (8/10) and clear cell foci (1/10); the incidence and severity of nephropathy in males and females and cardiomyopathy in males were similar to those in rats administered urethane in drinking water; however, no cardiomyopathy was observed in females receiving urethane in ethanol . The estrous cycle length of females receiving urethane in ethanol appeared to be longer than that of females receiving urethane in drinking water . Because cycle length was longer in the 10,000 ppm groups than in the controls in both the drinking water and ethanol vehicle studies, this difference may represent an exacerbation of the toxicity of urethane . A longer estrous cycle may be a sign of reproductive impairment and correlates with a decrease in female fecundity . All mice administered 10,000 ppm urethane in either vehicle died . All mice that received 3,300 ppm urethane in drinking water died, while only one male and four females receiving 3,300 ppm urethane in 5% ethanol died . Body weight gains of males and females in all 1,100 ppm groups were less than those of the respective controls, but the weight gains of mice receiving 1,100 ppm urethane in 5% ethanol were greater than those of mice receiving urethane in drinking water . The mean body weights of the lower exposure groups were similar to those of the respective controls, and there were no other differences between the body weights of mice receiving urethane in drinking water and those receiving urethane in 5% ethanol . Fluid consumption, and therefore total urethane intake, appeared lower in mice receiving the 5% ethanol vehicle than in those receiving the water vehicle . The relative right kidney, liver, and lung weights of males and females administered urethane in drinking water or ethanol were generally greater than those of the controls . Clearance of urethane from the plasma of male mice was complete within 2 hours after urethane was administered in water, but urethane was not cleared 12 hours after administration in 5% ethanol . At the end of 13 weeks of urethane administration, the plasma urethane elimination half-life was 0.8 hours; the kinetics were similar for concentrations of 110, 330, and 1,100 ppm urethane in water and in ethanol . However, at each exposure level, the plasma urethane concentration was four times greater for urethane administered in 5% ethanol than for urethane administered in drinking water, indicating a possible inhibition of urethane metabolism by ethanol . Kinetic measurements for elimination by female mice could not be obtained from the data collected . In mice administered urethane in drinking water, lung inflammation occurred in males and females that received 1,100 ppm or greater . Alveolar epithelial hyperplasia occurred in the lungs of males in the 330 and 1,100 ppm groups and females in the 1,100 ppm group; one male mouse in the 330 ppm group had an alveolar/bronchiolar adenoma (see the following summary table) . Mice receiving urethane in 5% ethanol had lower incidences and severity of lung inflammation but generally greater incidences and severity of alveolar epithelial hyperplasia than mice receiving the same concentrations of urethane in drinking water . Alveolar/bronchiolar adenomas occurred in four males and one female administered urethane in ethanol . {table: see text} Nephropathy was observed in males and females that received urethane in either vehicle, and the lesions in female mice were more severe than those in male mice; ethanol did not appear to increase the incidence or severity of nephropathy . Cardiomyopathy occurred in males and females that received 1,100 or 3,300 ppm urethane in drinking water and in females that received 3,300 ppm urethane in ethanol . Lymphoid depletion occurred in mice that received 3,300 or 10,000 ppm urethane; 5% ethanol did not appear to enhance these effects . However, urethane in 5% ethanol induced ovarian atrophy; the incidence of this lesion was lower in females receiving urethane in drinking water . A concentration of 1,100 ppm urethane in either drinking water or ethanol effectively stopped estrous cycling . Urethane is clearly genotoxic in vitro and in vivo . In vitro, urethane induced mutations in Salmonella typhimurium strain TA1535 in the presence of liver S9 enzymes . Sister chromatid exchanges were induced in cultured Chinese hamster ovary (CHO) cells with and without S9 . However, no induction of chromosomal aberrations was observed in CHO cells treated with urethane, with or without S9 . In vivo, urethane induced sex-linked recessive lethal mutations and reciprocal translocations in germ cells of adult male Drosophila melanogaster fed urethane . Significantly increased frequencies of micronucleated erythrocytes were observed in peripheral blood obtained from male and female mice after 45 days of exposure and in bone marrow and peripheral blood obtained after 13 weeks of exposure to urethane in drinking water . There appeared to be no significant difference in the magnitude of the response in the peripheral blood micronucleus test between mice administered urethane in drinking water and mice administered urethane in 5% ethanol . In summary, concentrations of 1,100 ppm urethane or greater in drinking water caused lymphoid and bone marrow cell depletion and hepatocellular lesions and increased the severity of nephropathy and cardiomyopathy in male and female rats . The lethal effects of 10,000 ppm urethane were slightly exacerbated by 5% ethanol in female rats . Urethane administered in drinking water induced lung inflammation, alveolar and bronchiolar hyperplasia, alveolar/bronchiolar adenomas, nephropathy, cardiomyopathy, lymphoid and bone marrow cell depletion, seminiferous tubule degeneration, and ovarian atrophy and follicular degeneration in mice . In female mice, 5% ethanol appeared to exacerbate ovarian atrophy . Mice administered urethane in 5% ethanol consumed less fluid, and therefore less urethane, than mice receiving urethane in drinking water . Coadministration of urethane and ethanol inhibited the clearance of urethane from plasma . (ABSTRACT TRUNCATED)

Natl Toxicol Program Tech Rep Ser, 2001 Nov, (497), 1 - 225
Toxicology and carcinogenesis studies of methacrylonitrile (CAS No . 126-98-7) in F344/N rats and B6C3F1 mice (gavage studies); NTP technical report on the toxicity and metabolism studies of chloral hydrate (CAS No . 302-17-0) . Administered by gavage to F344/N rats and B6C3F1 mice; Chloral hydrate is widely used as a sedative and a hypnotic in pediatric medicine . It is also a byproduct of water chlorination . Chloral hydrate has been shown to be genotoxic in numerous prokaryotic and eukaryotic assay systems including human lymphocytes in vitro . One of its metabolites, trichloroacetic acid, has demonstrated hepatocarcinogenic activity in mice . Trichloroethylene and perchloroethylene, both of which are metabolized to chloral hydrate, have been shown to be carcinogenic in rats and/or mice . Because of this evidence of carcinogenicity and because of the wide-spread use of chloral hydrate, 16- or 17-day range-finding toxicity studies and separate 16- or 17-day metabolism studies were performed in F344/N rats and B6C3F1 mice in preparation for further long-term rodent studies . In addition, in vitro studies of the metabolism and DNA-binding capacity of chloral hydrate and its metabolites were performed . Genetic toxicity studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse bone marrow cells . For the range-finding studies, groups of eight male and eight female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were administered 0, 50, 100, 200, 400, or 800 mg chloral hydrate per kg body weight in water by gavage 5 days per week for 17 days (rats) or 16 days (mice) for a total of 12 doses . One male rat receiving 800 mg/kg died after five doses . Two 800 mg/kg female rats died after dosing ended but before study termination . One male mouse in each group except the 400 mg/kg group died before the end of the study . Two 800 mg/kg female mice also died before the end of the study . The final mean body weight of 800 mg/kg male rats and the mean body weight gains of 400 and 800 mg/kg males were significantly less than those of the vehicle controls . The mean body weight gains of all groups of dosed male mice were significantly greater than that of the vehicle control group . The only clinical finding in rats and mice attributed to chloral hydrate treatment was light sedation in the 400 mg/kg groups and heavy sedation in the 800 mg/kg groups; sedation subsided within 30 minutes or 3 hours, respectively . The liver weights of 400 mg/kg male mice and 800 mg/kg male and female mice were significantly greater than those of the vehicle control groups . No chemical-related lesions were observed in rats or mice . Male and female rats and mice were administered a single dose of 50 or 200 mg chloral hydrate per kg body weight in water by gavage, or 12 doses of 50 or 200 mg/kg over 17 days (rats) or 16 days (mice) . Plasma concentrations of chloral hydrate and its metabolites were determined 15 minutes, 1, 3, 6, and 24 hours, and 2, 4, 8, and 16 days after receiving 1 or 12 doses . Maximum concentrations of chloral hydrate were observed at the initial sampling point of 15 minutes . By 1 hour, the concentrations had dropped substantially, and by 3 hours, chloral hydrate could not be detected in rats or mice . Trichloroacetic acid was the major metabolite detected in the plasma . In rats, the concentrations rose slowly, with the peaks occurring between 1 and 6 hours after treatment . In mice, the peak concentrations were found 1 hour after dosing . The concentrations then slowly decreased such that by 2 days the metabolite could no longer be detected in rats or mice . Trichloroethanol was assayed both as the free alcohol and its glucuronide . In rats, the maximum concentrations of free trichloroethanol occurred at 15 minutes, while the peak concentrations of trichloroethanol glucuronide were found at 1 hour; by 3 hours, concentrations of both metabolites approached background levels . In mice, the maximum concentrations of both metabolites occurred at 15 minutes, and by 1 to 3 hours concentrations approached background levels . The plasma concentrations of chloral hydrate and its metabolites were dose dependent in rats and mice . In mice, plasma concentrations of trichloroacetic acid were significantly higher after a single dose than after 12 doses . None of the metabolic parameters appears to account for species differences that may exist in hepatocarcinogenicity . The data from the study of metabolism and DNA adduct formation indicated that in vitro metabolism of 200 microM to 5 mM chloral hydrate by male B6C3F1 mouse liver microsomes (control microsomes) generated free radical intermediates that resulted in endogenous lipid peroxidation, forming malondialdehyde, formaldehyde, acetaldehyde, acetone, and propionaldehyde . Similar concentrations of trichloroacetic acid and trichloroethanol, the primary metabolites of chloral hydrate, also generated free radicals and induced lipid peroxidation . Lipid peroxidation induced by trichloroacetic acid nearly equaled that induced by chloral hydrate, while that from trichloroethanol was three- to fourfold less . Metabolism of 200 microM to 5 mM chloral hydrate, trichloroacetic acid, and trichloroethanol by liver microsomes of B6C3F1 mice pretreated with pyrazole (pyrazole-induced microsomes) yielded lipid peroxidation products at concentrations two- to threefold greater than those from liver microsomes of untreated mice . Additionally, chloral hydrate-induced lipid peroxidation catalyzed by control and pyrazole-induced microsomes was reduced significantly by 2,4-dichloro-6-phenylphenoxyethylamine, a general cytochrome P450 inhibitor . Human lymphoblastoid transgenic cells expressing cytochrome P(450)2E1 metabolized 200 to 5,000 micrograms/mL chloral hydrate to reactants inducing mutations, whereas the parental cell line was inactive . The malondialdehyde-modified DNA adduct, 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido{1,2 alpha}purin-10(3H)-one (MDA-MG-1), formed from the metabolism of 1 mM chloral hydrate, trichloroacetic acid, and trichloroethanol by control B6C3F1 mouse liver microsomes, mouse pyrazole-induced microsomes, male F344/N rat liver microsomes, and human liver microsomes in the presence and absence of calf thymus DNA was also determined . When incubated in the absence of calf thymus DNA, the amount of malondialdehyde formed from metabolism by pyrazole-induced mouse microsomes was twice that from rat or human liver microsomes . Amounts of chloral hydrate-induced and trichloroacetic acid-induced lipid peroxidation products formed from metabolism by rat and human liver microsomes were similar, and these quantities were about twice those formed from the metabolism of trichloroethanol . The quantity of MDA-MG-1 formed from the metabolism of chloral hydrate, trichloroacetic acid, and trichloroethanol by mouse, rat, and human liver microsomes exhibited a linear correlation with the quantity of malondialdehyde formed under incubation conditions in the absence of calf thymus DNA . Chloral hydrate was shown to be mutagenic in vitro and in vivo . At doses from 1,000 to 10,000 micrograms/plate, it induced mutations in S . typhimurium strain TA100, with and without S9 activation; an equivocal response was obtained in S . typhimurium strain TA98 in the absence of S9, and no mutagenicity was detected with strain TA1535 or TA1537 . Chloral hydrate at doses from 1,700 to 5,000 micrograms/mL induced sister chromatid exchanges; at doses from 1,000 to 3,000 micrograms/mL, chromosomal aberrations were induced in cultured Chinese hamster ovary cells, with and without S9 . Results of a sex-linked recessive lethal test in D . melanogaster were unclear; administration of chloral hydrate by feeding produced an inconclusive increase in recessive lethal mutations, results of the injection experiment were negative . An in vivo mouse bone marrow micronucleus test with chloral hydrate at doses from 125 to 500 mg/kg gave a positive dose trend . In summary, due to the absence of chloral hydrate-induced histopathologic lesions in rats and mice, no-observed-adverse-effect levels (NOAELs) were based on body weights of rats and liver weights of mice . The NOAELs for rats and mice were 200 mg/kg . Chloral hydrate was rapidly metabolized by rats and mice, with trichloroacetic acid occurring as the major metabolite . Peak concentrations of trichloroacetic acid occurred more quickly in mice . Plasma concentrations of chloral hydrate were dose dependent, but metabolic rates were unaffected by dose or sex . Chloral hydrate was mutagenic in vitro and in vivo . Metabolism of chloral hydrate and its metabolites produced free radicals that resulted in lipid peroxidation in liver microsomes of mice, rats, and humans . Induction of cytochrome P(450)2E1 by pyrazole increased the concentrations of lipid peroxidation products; inhibition of cytochrome P(450)2E1 by 2,4-dinitrophenylhydrazine reduced these concentrations . Metabolism of chloral hydrate and its metabolites by mouse, rat, and human liver microsomes formed malondialdehyde, and in the presence of calf thymus DNA formed the DNA adduct MDA-MG-1.

Toxic Rep Ser, 2000 Apr, (61), 1 - 53, A1-13
NTP technical report on the toxicity studies of benzophenone (CAS No . 119-61-9) . Administered in feed to F344/N rats and B6C3F mice; Chhabra RS; Benzophenone is used as a photoinitiator, a fragrance enhancer, an ultraviolet curing agent, and, occasionally, as a flavor ingredient; it is also used in the manufacture of insecticides, agricultural chemicals, and pharmaceuticals and is an additive for plastics, coatings, and adhesives . In 14-week studies conducted to determine the toxicity of benzophenone, groups of 10 male and 10 female F344/N rats and B6C3F1 mice were given 0, 1,250, 2,500, 5,000, 10,000, or 20,000 ppm benzophenone in feed . These exposure concentrations resulted in the following average daily doses: 75, 150, 300, 700, or 850 mg benzophenone per kilogram body weight for male rats; 80, 160, 300, 700, or 1,000 mg/kg for female rats; 200, 400, 800, 1,600, or 3,300 mg/kg for male mice; and 270, 540, 1,000, 1,900, or 4,200 mg/kg for female mice . Animals were evaluated for clinical pathology, reproductive system effects, liver cytochrome P450 effects, and histopathology . Genetic toxicity studies were conducted in Salmonella typhimurium and mouse bone marrow polychromatic erythrocytes . Benzophenone was unpalatable at 20,000 ppm . All 20,000 ppm rats had significant body weight loss and were terminated for humane reasons before the end of studies . All male mice and four female mice in the 20,000 ppm group died . There was no exposure-related mortality in the remaining groups . Significantly decreased body weights relative to the controls were observed in all exposed groups of female rats and all exposed groups of male rats except the 1,250 ppm group . Lower body weights were apparent in 10,000 ppm male mice and in 5,000 ppm or greater female mice . In rats, the liver and kidney were identified as target organs of benzophenone toxicity . Treatment-related increases in liver weights were attributed to hypertrophy and/or cytoplasmic vacuolization of hepatocytes . Increased kidney weights were associated with a spectrum of renal changes in exposed males and females . Unique lesions observed in animals that died early as well as in survivors were well demarcated, wedge-shaped areas of prominent tubule dilatation . The lesion occurred in 2,500 ppm or greater males and in 10,000 and 20,000 ppm females . Foci of tubule regeneration were increased relative to the controls in exposed males and females . In exposed mice, significant microscopic findings were limited to centrilobular hypertrophy in the liver that corresponded to increased liver weights . The severity of hepatocyte hypertrophy was exposure-concentration dependent, with marked severity in all 20,000 ppm animals . Clinical chemistry analyses confirmed liver toxicity . In rats, increases in serum bile salt concentrations indicated cholestatic liver disease . On day 22, a 15-fold increase was evident in the 20,000 ppm groups, and at week 14, a twofold increase was seen in the 10,000 ppm groups . Increases in alanine aminotransferase and sorbitol dehydrogenase activities were mild in mice; however, more convincing of liver damage were increased alkaline phosphatase activities and serum bile salt concentrations, especially in 20,000 ppm females . Biochemical data indicated that benzophenone was a relatively potent inducer of the phenobarbital-type (2B) cytochrome P450 enzymes . Overall, induction was greater in rats than in mice . The gross (increased organ weights) and microscopic (hepatocellular hypertrophy) liver changes associated with benzophenone administration in rats and mice accompanied benzophenone-induced increases in pentoxyresorufin dealkylase activity . Benzophenone was not mutagenic in S . typhimurium strain TA98, TA100, TA1535, or TA1537, with or without S9 activation, and it did not induce micronuclei in bone marrow erythrocytes of male mice administered benzophenone by intraperitoneal injection . In conclusion, the liver is the primary target organ of benzophenone toxicity in rats and mice based on increases in liver weights, hepatocellular hypertrophy, clinical chemistry changes, and induction of liver microsomal cytochrome P450 2B isomer . The kidney was also identified as a target organ of benzophenone toxicity in rats only, based on exposure concentration-related increases in kidney weights and microscopic changes . The no-observed-adverse-effect level for benzophenone was not achieved in these studies.

Bull Group Int Rech Sci Stomatol Odontol, 2000 Jan-Apr, 42(1), 23 - 9
Mouthrinses: a comparative microbiological study; Gautier G et al.; This study was performed in order to evaluate the efficacy of different mouthrinses whose use is extended in Spain . Six different antiseptic mouthrinses were studied by means of determination of Minimal Inhibitory Concentration (MIC) values against Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Bacillus subtilis, Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans . Also in vivo experiments were carried out in volunteers by the use of mouthrinses and evaluation of bacterial populations before and after the treatment . Finally, the kinetics of bacterial death was determined . Results suggested that the determination of MIC values is not a reliable method to evaluate the antibacterial effect of such products . On the other hand those rinsing solutions based on the effect of oxygen, such as those containing carbamide peroxide have a greater efficacy against anaerobic bacteria compared with rinses whose active molecule is a disinfectant . Finally, the kinetics of bacterial death demonstrates that the essential oil rinse kills bacteria much faster . All tested mouthrinses were active as antibacterial although those based on oxygen production or essential oils were more active than solutions based on chlorhexidine and Triclosan.

J Biol Chem, 2002 Apr 5, 277(14), 12175 - 81 Epub 2002 Jan 17.
The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity; Mushtaq A et al.; Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms . NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans . The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases . The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed . To investigate the function of the first two domains of full-length NAT from S . typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing . Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S . typhimurium are toxic when overexpressed intracellularly in Escherichia coli . Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate . Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S . typhimurium.

Zhonghua Yi Xue Za Zhi, 2001 May 25, 81(10), 613 - 6
{Construction of attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori catalase and observation on its protective immunity}; Liao W et al.; OBJECTIVE: To investigated the effect of attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori (Hp) catalase (KatA) on protection against Hp infection . METHODS: Recombinant plasmid expressing KatA was constructed . Expression of KatA was induced with IPTG, and analyzed by SDS-PAGE and Western blot . The recombinant plasmid were introduced into attenuated Salmonella typhimurium SL3261 strain to construct live oral vaccine strain . C57BL/6 mice was orally immunized with this vaccine strain and then given orogastric challenge with live Hp Sydney strain . The stomach samples were submitted to a rapid urease test and quantitative culture . RESULTS: The SDS-PAGE showed a dominant additional protein with molecular weight of 79kDa which could specially react with antibody against GST and accounted for 19% of total bacteria proteins . Animal experiment indicated that this vaccine strain could protect mice against Hp infection . CONCLUSION: Attenuated Salmonella typhimurium vaccine strain expressing KatA could induce effective immune response in protection against Hp infection,which may play an important role in preventing and treating Hp infection and related diseases.

Immunopharmacol Immunotoxicol, 2001 Nov, 23(4), 519 - 30
Immunization of mice against Salmonella typhimurium using different DNA preparations; Kalfayan LH et al.; Groups of female BALB/c mice were given primary and booster injections of whole genomic DNA extracted from S . typhimurium, P . aeruginosa, or S . aureus . Other groups of mice were immunized in a similar manner with the 1.57kb fragment of the mouse virulence gene (mviA), pTargeT vector (plasmid DNA)/1.57kb construct, pTargeT vector, or saline . Mice in all groups were challenged intraperitoneally with 100 LD50 of S . typhimurium . The bacterial genomic DNA was extracted using the Pure Gene extraction kit . Specific primers were used to amplify the 1.57kb fragment by PCR . The pTargeT Mammalian Expression Vector System was used to prepare the plasmid/ 1.57kb construct . Bacterial genomic DNA extracted from P . aeruginosa and S . aureus appeared to induce non-specific resistance in mice . Specific, in addition to non-specific resistance appeared to be induced when genomic DNA from S . typhimurium was used . There was a prolongation of survival in the groups of mice that received either the 1.57kb fragment or the pTargeT vector/1.57kb construct and 16.67% and 33.34% respectively, of mice in each group survived at 40 days post challenge . None of the mice in the saline control group survived by day 7 post challenge . It is suggested that the non-specific resistance observed in this study might have been due to the adjuvant effect of the non-methylated CpG and other immunostimulatory motifs in bacterial DNA . Specific resistance obtained when genomic DNA from S . typhimurium was used might have been due to minute antigenic contamination, or virulence factor genes other than the mviA gene, might have been expressed in the host, which induced specific immunity.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 235 - 44
Chitosan disrupts the barrier properties of the outer membrane of gram-negative bacteria; Helander IM et al.; The mode of antimicrobial action of chitosan (polymeric beta-1,4-N-acetylglucosamine) on gram-negative bacteria was studied with special emphasis on its ability to bind to and weaken the barrier function of the outer membrane (OM) . Chitosan (250 ppm) at pH 5.3 induced significant uptake of the hydrophobic probe 1-N-phenylnaphthylamine (NPN) in Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium . The effect was reduced (E . coli, salmonellae) or abolished (P . aeruginosa) by MgCl2 . No NPN uptake was observed during exposure of the salmonellae to chitosan at pH 7.2 . Chitosan also sensitized P . aeruginosa and the salmonellae to the lytic effect of sodium dodecyl sulfate (SDS); such sensitization was not blocked by MgCl2 and was reversible by washing chitosan-treated cells prior to SDS exposure . Chemical and electrophoretic analyses of cell-free supernatants of chitosan-treated cell suspensions showed that interaction of chitosan with E . coli and the salmonellae involved no release of lipopolysaccharide (LPS) or other membrane lipids . However, chitosan rendered E . coli more sensitive to the inhibitory action of dyes and bile acids used in selective media . Highly cationic mutants of S . typhimurium were more resistant to chitosan than the parent strains . Electron microscopy showed that chitosan caused extensive cell surface alterations and covered the OM with vesicular structures . Chitosan thus appeared to bind to the outer membrane, explaining the loss of the barrier function . This property makes chitosan a potentially useful indirect antimicrobial for food protection.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 211 - 8
Kinetic analysis of the bactericidal action of heated scallop-shell powder; Sawai J et al.; Shell powder of scallop (Patinopecten yessoensis) was exposed to heat treatment at between 200 and 1000 degrees C, and the bactericidal action of the powder slurry was investigated . Shell powder heated at 700 degrees C or higher exhibited bactericidal action against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus subtilis (vegetative cells) . The death of bacteria in the shell powder slurry followed first-order reaction kinetics, and the apparent death rate constant (k) was determined . An increase in exposure temperature enhanced the bactericidal action . The bactericidal action is due to calcium oxide that is converted from calcium carbonate, which is the main component of the shell powder, by heat treatment . The slurry temperature is found to significantly affect the bactericidal action of the shell powder . The slope of the Arrhenius plot of k for E . coli and S . aureus that were grown at 37 degrees C exhibited a discontinuous point at approximately 22 degrees C, at which the values of activation energy for the death of bacteria in the powder slurry changed . This temperature corresponds to that of the phase transition of cell membrane lipids . The bactericidal action of the shell powder is greater than that of a NaOH solution of identical pH . Although the pH of the shell powder slurry is high, the slurry was considered to possess other antibacterial mechanisms in addition to that of alkalinity.

Proteomics, 2002 Jan, 2(1), 85 - 93
Proteome study of Francisella tularensis live vaccine strain-containing phagosome in Bcg/Nramp1 congenic macrophages: resistant allele contributes to permissive environment and susceptibility to infection; Kovarova H et al.; The phagocytosis of pathogens by macrophages classically initiates maturation of the phagosome that involves a dynamic exchange of phagosomal components with intracellular compartments of the endocytic pathway . The intracellular microorganisms have developed sophisticated mechanisms to sense environmental conditions and respond to them by phenotypic alterations that ensure their adaptation, survival and proliferation inside the cell . They have learned also to utilise host cellular components to ensure own survival . Recent results suggest that the Bcg locus/Nramp1 gene (natural resistance-associated macrophage protein 1) controls natural resistance to infection by Francisella tularensis LVS (live vaccine strain) and its effect is opposite to that observed for other Bcg/Nramp1-controlled pathogens such as several mycobacterial species, Leischmania donovani, and Salmonella typhimurium . In the case of F . tularensis LVS infection, the mutant allele of the Bcg locus (Bcg(s)/Nramp1(s)) is associated with natural resistance and, inversely, the wild type allele (Bcg(r)/Nramp1(r)) confers susceptibility . To determine whether differential allelic expression of the Bcg locus/Nramp1 gene modifies the composition of F . tularensis LVS-containing phagosomes (FCP), we have utilised an approach where we isolated FCP from infected Bcg congenic B10R (Bcg(r)/Nramp1(r)) and B10S (Bcg(s)/Nramp1(s)) macrophages of susceptible and resistant phenotype, respectively . Comparative proteomic analysis of the two phagosomal compartments with subsequent mass spectrometric analysis allowed identification of several proteins typical for FCP from B10R macrophages . They include a bacterial hypothetical 23 kDa protein, 60 kDa chaperonin GroEL, and host putative proteins that appeared to be mitochondrial ATP synthase beta-chain and NADH-ubiquinone oxidoreductase based on high cross-species homology . High abundance of the hypothetical 23 kDa protein correlates with the susceptible phenotype and, possibly, pathogenicity of F . tularensis LVS . The results demonstrate that F . tularensis LVS can exploit ion transport function of Bcg/Nramp1 to its own advantage.

FEMS Microbiol Lett, 2002 Jan 2, 206(1), 93 - 7
Aminoguanidine renders inducible nitric oxide synthase knockout mice more susceptible to Salmonella typhimurium infection; Zhou X et al.; Aminoguanidine (AG), a nitric oxide synthase (NOS) inhibitor, has been widely used to study the role of inducible NOS (iNOS) in host defense against infections caused by various pathogens including Salmonella typhimurium . iNOS has been reported to play an important role in host defense against S . typhimurium infection both in vitro and in vivo . In this report we show those AG treatment lead to weight loss in both wild-type and iNOS knockout mice, and rendered them more susceptible to Salmonella infection . These results suggest that AG may have side effects other than the inhibition of iNOS, and that data obtained from studies using AG should be interpreted with caution.

Avian Dis, 2001 Oct-Dec, 45(4), 962 - 71
Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars; Okamura M et al.; In Experiment 1, mature laying hens were inoculated intravaginally with 10(6) colony-forming units of Salmonella enterica serovar enteritidis (S . enteritidis), Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to compare their abilities to colonize the reproductive organs of chickens and to contaminate eggs . Salmonella enteritidis was more frequently recovered (from 11 of 40 eggs, 27.5%) than the other serovars, and especially the inner shell was contaminated with these organisms in 10 of 40 eggs (25.0%) . The contamination rates and the viable counts in cloaca were significantly (P < 0.05) higher in hens inoculated with S . enteritidis than in those inoculated with the other serovars at 4 days postinoculation (PI) . In the vagina, the positive rates were 90%-100% in hens inoculated with S . enteritidis, and the viable counts of the organisms in this portion were significantly (P < 0.05) higher than those of the other serovars at 2, 4, and 7 days PI . The ceca were colonized similarly by each serovar at 7 days PI . The spleen and ovary were infected with S . enteritidis in three and one hen, respectively . No Salmonella was recovered from liver and peripheral blood in any hen . Salmonella enteritidis was recovered from other oviductal portions than the vagina (10%-20%), whereas no forming egg was contaminated in the oviduct . In Experiment 2, the in vitro adherence of these six serovars to the vaginal epithelium was compared with vaginal explants . The mean number of S . enteritidis attaching to the secondary villi in the vaginal lumen was significantly (P < 0.05) higher than those of the other serovars . These results suggest that S . enteritidis has a specific advantage over the other Salmonella serovars by its capacity to colonize the vaginal tissues of hens, and this higher affinity of S . enteritidis to the vagina may play a significant role in the production of many S . enteritidis-contaminated eggs.

Avian Dis, 2001 Oct-Dec, 45(4), 922 - 37
Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens; Roy P et al.; The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks . Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks . Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S . typhimurium (one source), S . heidelberg (four sources), S . montevideo (two sources), S . hadar (one source), S . kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage . Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum . Another group of 24 chicks was kept as an uninoculated control group . The chicks were observed daily for clinical signs and mortality . Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI) . All the chicks were weighed individually at 7, 14, 21, and 28 DPI . Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI . Selected tissues were taken for histopathology at 7 and 14 DPI . Dead chicks were examined for gross lesions and tissues were collected for histopathology . Chicks inoculated with S . pullorum had the highest mortality (66.66%), followed by S . typhimurium (33.33%) . Chicks inoculated with S . heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a . Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs . Mean body weight reduction ranged from 0.67% (inoculated with S . kentucky 00-926-2) to 33.23% (inoculated with S . typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls . Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S . typhimurium, S . heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S . pullorum.

Microbiology, 2002 Jan, 148(Pt 1), 123 - 31
Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium; Quan JA et al.; Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known . The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined . cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium . Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities . Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level . Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB . Comparable effects were seen for E . coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant . Adenylate cyclase thus appears to be responsive to sulfur deprivation . These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.

Vet Immunol Immunopathol, 2002 Jan 15, 84(3-4), 191 - 207
Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds; Dil N et al.; The purpose of this study was to examine iNOS gene expression and activity in macrophages from different chicken genetic lines against various bacterial LPS . Furthermore, the possible involvement of surface LPS receptors as candidates for differential iNOS gene induction in these genetic lines of chicken was also examined . Sephadex-elicited abdominal macrophages (1 x 10(6)) as well as iNOS hyper-responder macrophages from a transformed chicken macrophage cell line, MQ-NCSU, were exposed to 5 microg/ml LPS from E . coli, Shigella flexneri, Serratia marcensces, and Salmonella typhimurium . Nitrite levels were quantitated in the culture supernatant fractions of macrophages after 24h by the Griess method . The results showed that macrophages from K-strain (B(15)B(15)) (range from two separate trials: 31-89 microM) and MQ-NCSU (22-81 microM) were high responders whereas macrophages from both GB1 (B(13)B(13)) (15-38 microM) and GB2 (B(6)B(6)) (7-15 microM) chickens were low responders against all LPSs used . Northern blot analysis revealed that K-strain macrophages expressed higher intensity of 4.5Kb iNOS mRNA (iNOS/beta-actin ratio) than macrophages from GB2 regardless of the LPS source . To elucidate possible molecular mechanism(s) involved in iNOS gene expression in these two strains of chickens, the constitutive expression of LPS-related macrophage cell surface receptors, CD14, Toll-like receptor-2 (TLR2), and Toll-like receptor-4 (TLR4), was examined via flow cytometry using anti-human CD14, TLR2 and TLR4 antibodies . CD14 surface expression and intensity was not different between macrophages from K-strain or GB2 chickens . In contrast, while the overall percentage of TLR4-positive macrophages was the same (K-strain, trial 1=92%, trial 2=62%; GB2, trial 1=91%, trial 2=64%), the mean fluorescence intensity (MFI), an indicator of receptor number, was significantly higher (P=0.05) in K-strain macrophages (MFI: trial 1=145; trial 2=131) than GB2 macrophages (MFI: trial 1=101; trial 2=98) . Furthermore, TLR2 (a previously thought candidate as LPS signaling molecule) positive cell numbers were higher in K-strain than the GB2 macrophages in one of the two trials with no difference in the intensity of TLR2 expression in either trial . These findings suggest that the observed differences in iNOS expression and activity among the K-strain (hyper-responder) and GB2 (hypo-responder) chickens are, at least in part, due to differential expression of TLR4 (an LPS signaling molecule), leading to more intense LPS-mediated activation of K-macrophages.

Environ Mol Mutagen, 2001, 38(4), 339 - 46
Predicting the mutagenicity of tobacco-related N-nitrosamines in humans using 11 strains of Salmonella typhimurium YG7108, each coexpressing a form of human cytochrome P450 along with NADPH-cytochrome P450 reductase; Fujita K et al.; Tobacco, including snuff and chewing tobacco, contains N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosodiethylamine (NDEA), N-nitrosopyrrolidine (NPYR), N-nitrosopiperidine (NPIP), N-nitrosomorpholine (NMOR), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NABS), and N-nitrosoanatabine (NATB) . The role of human cytochrome P450 (CYP) in the metabolic activation of these tobacco-related N-nitrosamines was examined by a Salmonella mutation test using genetically engineered Salmonella typhimurium (S . typhimurium) YG7108 cells each expressing a form of human CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase . Mutagen production from NNK was catalyzed by CYP in the following order: CYP1A2, CYP1A1, CYP1B1, CYP2A6, CYP2C19, CYP3A4 . The metabolic activation of one of the N-alkylnitrosamines, NDEA, was mediated by CYP2A6, followed by CYP2E1 . Cyclic N-nitrosamines such as NPYR, NPIP, and NMOR were also primarily activated by CYP2A6, and to a lesser extent by CYP2E1 . NNN, a pyridine derivative of NPYR, was activated by CYP1A1 at an efficiency similar to that of CYP2A6 . NABS, a pyridine derivative of NPIP, was mainly activated by CYP3A4, followed by CYP1A1 and CYP2A6 . Thus, the addition of a pyridine ring to NPYR or NPIP altered the forms of CYP primarily responsible for mutagenic activation . NATB was metabolically activated solely by CYP2A6, whereas the genotoxicity of NATB was much lower than that of NNN or NPYR . Based on these data, we conclude that CYP2A6 was responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines tested in the present study .

Environ Mol Mutagen, 2001, 38(4), 329 - 38
Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase; Fujita K et al.; A series of Salmonella typhimurium (S . typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established . The parental S . typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents . The expression levels of CYP holo-protein in the genetically engineered S . typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4 . The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture . Each form of CYP expressed in the S . typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate . The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP . These S . typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved .

J Clin Oncol, 2002 Jan 1, 20(1), 142 - 52
Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma; Toso JF et al.; PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice . These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer . PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009 . Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects . RESULTS: The maximum-tolerated dose was 3 x 10(8) cfu/m(2) . Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia . VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12 . Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2) . None of the patients experienced objective tumor regression, including those patients with colonized tumors . CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed . No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.

Vasa, 2001 Nov, 30(4), 293 - 6
{Single intervention for treatment of Salmonella typhimurium-induced symptomatic abdominal aortic aneurysm with spondylitis}; Tautenhahn J et al.; Simultaneous treatment of Salmonella typhimurium-induced symptomatic abdominal aortic aneurysm with associated spondylitis . Bacterially infected aneurysms associated with local spondylitis, while representing a potentially fatal clinical picture, are an operative challenge for vascular surgeons and orthopaedic surgeons alike . In this context, the concurrent occurrence of an infection with Salmonella typhimurium as a causative agent is a rare observation . The case report gives an outline of the simultaneous vascular and orthopaedic surgical procedure . The subrenal mycotic aneurysm was removed in a first step . The continuity of the aorta was restored centrally through an autogenic aortic graft with caudal anastomosis to a dacron vascular prosthetic tube . Initially, the latter was chosen of excessive length so as to facilitate the orthopaedic surgeon's approach . Upon completion of stabilising surgery of the vertebral column, the dacron tube was reduced in length as necessary and the surgical area was enclosed with an omentum majus plastic mesh . No complications were noted during the 18-month follow-up period.

J Food Prot, 2001 Dec, 64(12), 2071 - 4
Efficacy of cetylpyridinium chloride in immersion treatment for reducing populations of pathogenic bacteria on fresh-cut vegetables; Wang H et al.; The efficacy of cetylpyridinium chloride (CPC) immersion to reduce the numbers of three pathogenic bacteria (Listeria monocytogenes, Salmonella Typhimurium, and Escherichia coli O157:H7) on three different fresh-cut vegetables (broccoli, cauliflower, and radishes) was studied . The fresh-cut vegetables were inoculated with one of the three pathogenic bacteria at a concentration of 10(5) CFU/ml for 1 h at room temperature and then treated with 0.1 or 0.5% CPC immersion for 1 min . Both Salmonella Typhimurium and E . coli O157:H7 plates were incubated from 48 to 72 h at 37 degrees C, and L . monocytogenes plates were incubated from 72 to 96 h before being counted . The results of three experiments showed that for the average of the three vegetables treated with 0.1 and 0.5% CPC, L . monocytogenes was reduced by 2.85 and 3.70 log CFU/g, Salmonella Typhimurium by 2.37 and 3.15 log CFU/g, and E . coli O157:H7 by 1.01 and 1.56 log CFU/g, respectively, in comparison with the vegetables treated with water only . The 0.5% CPC treatment was significantly different (P < 0.05) from the 0.1% CPC treatment on reduction of L . monocytogenes, Salmonella Typhimurium, and E . coli O157:H7 . The CPC residual on the treated vegetables and their washing solutions were evaluated by using high-performance liquid chromatography.

Indian J Pediatr, 2001 Nov, 68(11), 1079 - 80
Neonatal Salmonella typhimurium meningitis; Totan M; Meningitis due to Salmonella is a very rare sign of Salmonellosis . A 10-day-old female premature neonate with Salmonella typhimurium meningitis is presented in this report . The clinical features, outcome and antibiotic treatment are discussed . Although it is extremely rare, Salmonella meningitis should be considered in differential diagnosis of neonatal meningitis.

Inflamm Res, 2001 Nov, 50(11), 534 - 43
Modulation of leukocyte-endothelium interaction by nitric oxide synthase inhibitors: effects on leukocyte adhesion in endotoxin-induced uveitis; Baatz H et al.; OBJECTIVE AND DESIGN: To examine the effects of the nitric oxide synthase (NOS) inhibitors aminoguanidine (AG) and L-NAME on leukocyte adhesion in endotoxin-induced uveitis (EIU) . MATERIAL: Uveitis was induced in Lewis rats (n = 124) by LPS injection (Salmonella typhimurium) . TREATMENT: Rats either (1) did not receive any LPS or other treatments (controls), received (2) only subcutaneous saline injections with LPS administration, (3) a single s.c . dose of AG (100 mg/kg body weight) at the time of LPS administration, (4) a single s.c . injection of AG 8 h after LPS injection, (5) s.c . injections of AG at the time of LPS administration and 8 h after LPS injection or (6) received a single dose of L-NAME (75 mg/kg body weight) at the time of LPS administration . METHODS: Intravital microscopy (IVM) of iris vessels was performed at 2, 4, 8, 16, 24 and 48 h after endotoxin injection . Aqueous humor analysis for protein concentration and cell count was performed after IVM . RESULTS: At 2 h after the induction of uveitis, significantly more rolling leukocytes were detected in the AG and L-NAME-treated group than in untreated EIU (4.8 +/- 0.31 and 9.83 +/- 0.64 vs . 2.85 +/- 0.37%, mean +/- SEM, p < 0.01) . However, at 16 h the percentage of rolling leukocytes was significantly reduced in all groups which had received AG (LPS: 8.08 +/- 0.37%; LPS/AG 0 h: 3.78 +/- 0.25%; LPS/AG 8 h: 5.34 +/- 0.3%; LPS/AG 0+8h: 3.86 +/- 0.31%) . L-NAME enhanced leukocyte rolling even at 24 h after LPS (12.38 +/- 0.64%) . Early treatment of EIU with AG significantly reduced the number of sticking leukocytes at 4, 8 and 24 h (306 +/- 13 vs . 571 +/- 41, 228 +/- 12 vs . 345 +/- 19 and 240 +/- 14 vs . 469 +/- 23 cells/mm2, respectively) . L-NAME inhibited LPS-induced sticking of leukocytes at all observed time points and this effect was most pronounced at 24 h (147 +/- 10 vs . 469 +/- 23 cells/mm2) . CONCLUSIONS: In EIU, administration of AG or L-NAME causes enhanced leukocyte rolling in the early inflammatory response . However, firm adhesion of leukocytes to the vascular endothelium decreases and this effect prevails, ameliorating leukocyte infiltration.

Berl Munch Tierarztl Wochenschr, 2001 Nov-Dec, 114(11-12), 433 - 7
{Laboratory-based surveillance of salmonellosis of humans in Germany--safety of Salmonella typhimurium and Salmonella enteritidis live vaccines}; Rabsch W et al.; The Paul Ehrlich Institute, Langen, in Germany has been licensed different live vaccines of S . Typhimurium and S . Enteritidis for use in the veterinary medicine since I the 90s . The Robert Koch Institute has established a lab-based surveillance system for these live vaccine strains for an evaluation of recent public health safety . Since 2000 all strains of S . Typhimurium and S . Enteritidis from humans were investigated in respect to their phage types and other vaccine markers . 3676 S . Typhimurium strains and 4489 S . Enteritidis strains mainly from Salmonellose patients were investigated after phage typing according to their auxotrophic or antibiotic resistance markers . The live vaccine strains of Zoosaloral, TAD Salmonella vacT or TAD Salmonella vacE and Salmovac SE could not be found from infections in humans.

Vet Q, 2001 Nov, 23(4), 199 - 201
Salmonella typhimurium DT104 septicaemia with meningitis in neonatal piglets; van der Wolf PJ et al.; Clinical salmonellosis in pigs in the Netherlands usually manifests itself as diarrhoea . In finishing pigs this is sometimes accompanied by peracute mortality, mainly in the last month of the finishing period . This is the first report describing Salmonella Typhimurium DT104 infection of 1-week-old suckling piglets in the Netherlands . The piglets showed nervous symptoms and died . The clinical symptoms, gross pathology, histopathological, bacteriological and phagetyping results are presented as well as the antimicrobial resistance pattern . This case is not only important as an extension of the clinical syndrome of salmonellosis in pigs in the Netherlands, but also because of the risk of human infection after consumption of pork or pork products contaminated with this pathogenic and multiple resistant Salmonella clone.

Adv Exp Med Biol, 2001, 500, 513 - 6
Structure of the malondialdehyde deoxyguanosine adduct M1G when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene; Schnetz-Boutaud NC et al.; Malondialdehyde (MDA) is a toxic and mutagenic metabolite produced by lipid peroxidation, and prostaglandin biosynthesis . MDA induces frameshift mutations in tester strains of Salmonella typhimurium . It reacts with DNA, and at physiological pH the major adduct is a pyrimidopurinone formed by reaction with guanine: M1G {3-(2'-deoxy-beta-D-erythropentofuranosyl)pyrimido{1,2-alpha}-purin-10(3H)-one} . When site-specifically incorporated into a duplex oligodeoxynucleotide containing a frameshift-prone (CG)3 repeat derived from the Salmonella typhimurium hisd3052 gene, spontaneous opening of M1G to the N2-(3-oxo-1-propenyl)-dG species occurred . In this work d(ATCGCMCGGCATG), (M=M1G) was annealed to d(CATGCCGCGAT) to model the putative strand slippage intermediate which would precede a two base deletion in the (CG)3 iterated repeat . 1H NMR studies indicate that in contrast to the duplex DNA structure, M1G remains intact . A single bulge conformation exists . M1G and its 3'-neighbor cytosine are unpaired . The M1G is intrahelical and stacked, whereas the unpaired cytosine is poorly stacked and appears to be extrahelical.

Int J Food Microbiol, 2001 Nov 8, 70(3), 243 - 54
Salmonella in slaughter pigs: prevalence, serotypes and critical control points during slaughter in two slaughterhouses; Swanenburg M et al.; The purpose of this study was to show the distribution of Salmonella in slaughtered pigs and the environment of the slaughterhouse . 1,114 samples of slaughtered pigs (six different samples for Salmonella isolation and one serum sample for ELISA on antibodies per pig) and 477 samples of the slaughterhouse environment were collected in two slaughterhouses on two sampling days per slaughterhouse . Salmonella was isolated from one or more samples of 47% of the pigs . The highest prevalence of Salmonella was observed in rectal content samples (25.6%), whereas the lowest prevalence of Salmonella was observed on the carcasses (1.4%) . The prevalence of Salmonella in other samples was: 19.6% in tonsils, 9.3% on livers, 9.3% on tongues, and 9.3% in mesenterial lymphnodes . The prevalence of Salmonella in environmental samples was high in the drain water samples in both slaughterhouses (61%) and on the carcass splitter in one slaughterhouse (33%) . Salmonella typhimurium was the most frequently isolated serotype in pig samples and environmental samples in both slaughterhouses: 43% of the Salmonella isolates from pigs and 33% of the Salmonella isolates from the environment was S . typhimurium . The results of this study show that Salmonella prevalences in pigs differ a lot, depending on which part of the pig is sampled . Not all different samples of the pig will become available for human consumption, but collecting more than one sample per pig showed that Salmonella can be found in almost the whole pig . The result of surface samples of carcass and liver gives information about hygiene during the slaughter process; the result of tonsils, lymphnodes and rectal contents, combined with the serological result, gives information about infection of the pig before the slaughter process (on the farm, during transport or in lairage) . It can be concluded that results of Salmonella isolation of slaughter pigs should always be carefully interpreted, depending on the type of sample that has been collected.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1327 - 34
Salmonella typhimurium outer membrane remodeling: role in resistance to host innate immunity; Ernst RK et al.; Resistance to innate immunity is essential for salmonellae pathogenesis . The salmonellae PhoP/PhoQ regulators sense host environments to promote remodeling of the bacterial envelope . This remodeling includes enzymes that modify lipopolysaccharide (LPS) . Modified LPS promotes bacterial survival by increasing resistance to cationic antimicrobial peptides and by altered host recognition of LPS.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1261 - 9
Bacterial virulence, proinflammatory cytokines and host immunity: how to choose the appropriate Salmonella vaccine strain?
Raupach B, Kaufmann SH.
Salmonella infection in its mammalian host can be dissected into two main components . The co-ordinate expression of bacterial virulence genes which are designed to evade, subvert or circumvent the host response on the one hand, and the host defence mechanisms which are designed to restrict bacterial survival and replication on the other hand . The outcome of infection is determined by the one which succeeds in disturbing this equilibrium more efficiently . This delicate balance between Salmonella virulence and host immunity/inflammation has important implications for vaccine development or therapeutic intervention . Novel Salmonella vaccine candidates and live carriers for heterologous antigens are attenuated strains with defined genetic modifications of metabolic or virulence functions . Although genetic defects of different gene loci can lead to similar degrees of attenuation, effects on the course of infection may vary, thereby altering the quality of the elicited immune response . Studies with gene-deficient animals indicate that Salmonella typhimurium strains with mutations in aroA, phoP/phoQ or ssrA/ssrB invoke different immune responses and that a differential repertoire of pro-inflammatory cytokines is required for clearance . Consequently, Salmonella mutants defective in distinct virulence functions offer the potential to specifically modulate the immune response for defined medical applications.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1249 - 59
The involvement of class Ib molecules in the host response to infection with Salmonella and its relevance to autoimmunity; Soloski MJ et al.; Class I molecules with limited polymorphism have been implicated in the host response to infectious agents . Following infection with Salmonella typhimurium, mice develop a CD8+ CTL response that specifically recognizes bacteria infected cells . An immunodominant component of the CTL response recognizes a peptide epitope derived from the Salmonella GroEL molecule that is presented by the non-polymorphic MHC class Ib molecule Qa-1 . T cells recognizing the bacterial peptide also cross-recognize a homologous peptide from the mammalian hsp60 molecule . Since Qa-1 has a functional equivalent in humans, this observation may be relevant not only to the host response involved in clearing infection but also in understanding the link between infection with Gram-negative pathogens and autoimmune disease.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1239 - 48
Antigen-presenting cells and anti-Salmonella immunity; Yrlid U et al.; The present article summarizes studies aimed at addressing the role of antigen-presenting cell populations, particularly dendritic cells (DC), in the immune response to Salmonella typhimurium . Data from in vitro studies shed light on presentation of antigens expressed in Salmonella on major histocompatibility complex class I and class II molecules by infected DC and macrophages, and the activation state of DC following infection . Finally, data from in vivo studies addressing the role of DC and defined DC subsets during the host response to Salmonella using a murine infection model are discussed.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1213 - 22
Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria; Freudenberg MA et al.; Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host . This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Microbes Infect, 2001 Nov-Dec, 3(14-15), 1201 - 12
Salmonella-induced macrophage death: the role of caspase-1 in death and inflammation; Monack DM et al.; Salmonella typhimurium invades host macrophages and can induce either an almost immediate cell death or establish an intracellular niche within the phagocytic vacuole . Rapid cell death depends on the Salmonella pathogenicity island SPI1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases . Caspase-1-dependent cell death leads to the activation of the potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 to produce bioactive cytokines . Animal studies indicate that the activation of these cytokines is necessary for efficient colonization of the mouse gastrointestinal tract . Salmonella that reside in the phagocytic vacuole do not cause this early cell death and can trigger a macrophage death at a much later time point . This late-phase cell death is dependent on SPI2-encoded genes and ompR.

Biochem Pharmacol, 2001 Dec 15, 62(12), 1653 - 60
Protective effects of baicalein and wogonin against benzo{a}pyrene- and aflatoxin B(1)-induced genotoxicities; Ueng YF et al.; To evaluate the protective effects of baicalein and wogonin against benzo{a}pyrene- and aflatoxin (AF) B(1)-induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo{a}pyrene and AFB(1) were studied in C57BL/6J mice . Baicalein and wogonin reduced benzo{a}pyrene and AFB(1) genotoxicities as monitored by the umuC gene expression response in Salmonella typhimurium TA1535/pSK1002 . Baicalein added in vitro decreased liver microsomal benzo{a}pyrene hydroxylation (AHH) activity with an ic(50) of 33.9 +/- 1.4 microM at 100 microM benzo{a}pyrene . Baicalein also inhibited AFQ(1) and AFB(1)-epoxide formation from AFB(1) (50 microM) oxidation (AFO) with ic(50) values of 22.8 +/- 1.4 and 5.3 +/- 0.8 microM, respectively . However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 microM wogonin was only about 51-65% . Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively . Baicalein treatment resulted in 39 and 32% decreases in AFQ(1) and AFB(1)-epoxide formation from liver microsomal AFO, respectively . Wogonin treatment resulted in 39 and 47% decreases in AFQ(1) and AFB(1)-epoxide formation, respectively . A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo{a}pyrene via gastrogavage . These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo{a}pyrene- and AFB(1)-induced hepatic toxicities and that wogonin had a stronger protective effect in vivo.

J Antimicrob Chemother, 2002 Jan, 49(1), 169 - 72
Plasmid-mediated TEM-3 extended-spectrum beta-lactamase production in Salmonella typhimurium in Casablanca; AitMhand R et al.; Isolates of extended-spectrum beta-lactamase (ESBL)-producing Salmonella typhimurium were recovered from children admitted to the IbnRochd University Hospital of Casablanca in 1994 . These isolates produced TEM-3 as shown by PCR, isoelectric focusing and sequencing . Production of TEM-3 and resistance to gentamicin were encoded by a 10 kb plasmid that could be transferred by conjugation and transformation . This report extends the list of ESBLs produced by S . typhimurium and stresses the need for continuous surveillance of non-typhoidal Salmonella to adapt antibiotic treatment and preventive measures.

Carcinogenesis, 2001 Dec, 22(12), 2033 - 8
Induction of cytochrome P450 1B1 in lung, liver and kidney of rats exposed to diesel exhaust; Hatanaka N et al.; We have shown previously that diesel exhaust particle (DEP) extracts (DEPE) and 1-nitropyrene were genotoxically activated by human cytochrome P450 1B1 in SOS/umu assay . In this study, the in vivo induction of P450 family 1 enzymes in rats by exposure to diesel exhaust was investigated with regard to mRNA levels, P450 enzyme content, drug oxidation activities in the microsomes and umu gene expression of typical P450 substrates and DEPE itself catalyzed by the microsomes . Male Fischer 344 rats (4 weeks old) were exposed to 0.3 and 3.0 mg/m(3) DEP for 12 h per day for 4 weeks; the former dose corresponded to the typical daily airborne particle concentration . The levels of mRNA of rat P450 1B1 and P450 1A1 in the lung and liver were significantly increased 1.1-1.4-fold by exposure to 0.3 mg/m(3) DEP . Diesel exhaust particle extracts induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of a functional P450 system and were further activated by human recombinant P450 1B1 . Using an O-acetyltransferase overexpressing Salmonella strain, genotoxic activation of P450 1B1 marker chemicals (1-nitropyrene, 1-aminopyrene and DEPE) by lung, liver and kidney microsomes was increased 1.7-4.2-, 1.4-1.5- and 1.0-1.3-fold, respectively, by exposure to 0.3 mg/m(3) DEP . Activation of 3-amino-1,4-dimethyl-5H-pyrido {4,3-b}indole (Trp-P-1; marker for P450 1A1) by lung microsomes and the P450 1A2 content in liver microsomes were slightly increased by exposure to 3.0 mg/m(3) DEP . This is the first report to suggest that typical daily contaminant levels (0.3 mg particle/m(3)) of diesel exhaust can induce P450 1B1 in rats and that the induced P450 1B1 may catalyze the genotoxic activation of DEP.

FEMS Immunol Med Microbiol, 2001 Dec, 32(1), 73 - 83
Alcohol consumption is associated with alterations in macrophage responses to interferon-gamma and infection by Salmonella typhimurium; Sibley DA et al.; Abuse of ethanol (EtOH) by human beings and administration of EtOH to experimental animals has been shown to be associated with a suppression of the immune system . Consumption of EtOH has also been associated with an increased incidence and severity of infections of human beings and experimental animals, which has been attributed to the immunosuppression associated with EtOH consumption . It has been shown that EtOH also affects the function of macrophages (MO), which are important effector cells in the innate and adaptive immune responses to infectious agents . The present studies were designed to investigate the effects of EtOH on MO function with an animal model of EtOH consumption . The experiments reported in this paper were done with inflammatory MO and were designed to determine the effects of EtOH on the ability of inflammatory MO to respond to interferon-gamma (IFN-gamma) to control the intracellular growth of Salmonella typhimurium, as well as the production of proinflammatory cytokines and nitric oxide . The ability of MO from EtOH-fed mice to respond to bacterial endotoxin (lipopolysaccharide (LPS)) and IFN-gamma was also evaluated . MO isolated from EtOH-fed mice did not respond as well to IFN-gamma as MO isolated from control mice as measured by control of S . typhimurium, as well as tumor necrosis factor (TNF) and nitric oxide production . Interleukin (IL)-6 production was not affected . Activation of MO from EtOH-fed mice with LPS and IFN-gamma produced levels of nitric oxide and TNF only slightly less than the levels seen in MO from control mice, but a significant decrease in IL-6 was seen when MO from EtOH-fed mice were stimulated with this combination . Flow cytometric analyses showed that IFN-gamma receptor expression was not affected by EtOH . Together the data presented in this paper show that consumption of EtOH is associated with changes in inflammatory MO responses to IFN-gamma.

Immunol Lett, 2002 Feb 1, 80(2), 89 - 96
Oral DNA vaccination: antigen uptake and presentation by dendritic cells elicits protective immunity; Cochlovius B et al.; Melanoma differentiation antigens, such as glycoprotein 100 (gp100), have been shown to induce both cellular and humoral immune responses against melanoma in mouse and man . They are therefore considered as potential targets for melanoma immunotherapy . In this study, we have used the attenuated auxotrophic mutant strain SL7207 of Salmonella typhimurium as vehicle for a human gp100 (hgp100) DNA vaccine against melanoma . In vitro studies indicate that Salmonella/pCMV-hgp100 is efficiently scavenged by dendritic cells, resulting in the expression of the hgp100 transcription unit in the DC . In addition, oral administration of Salmonella/pCMV-hgp100 results in the expression of hgp100 RNA and protein by cells exhibiting DC-morphology in mesenteric lymph nodes as soon as 3 days after vaccination . Analysis of the efficacy of the Salmonella/pCMV-hgp100 vaccine in the B16/hgp100 model demonstrated the induction of strong anti-hgp100 CTL responses and protective immunity in 70% of the vaccinated mice, but not in control mice . Based on these data, we consider S . typhimurium as a useful vehicle for the design of recombinant DNA based anti-cancer vaccines.

Mutat Res, 2001 Dec 12, 484(1-2), 95 - 102
Study of adaptive mutations in Salmonella typhimurium by using a super-repressing mutant of a trans regulatory gene purR; Yang Z et al.; Salmonella typhimurium purR encodes a transcriptional repressor regulating gene expression of de novo purine nucleotide biosynthesis . It represses purD gene transcription by binding to the 16-base pair purD operator (PUR box) . A S . typhimurium strain carrying a super-repressing mutant of purR, purR(s), has been used as an experimental system to study adaptive mutation . Escherichia coli lac genes were genetically engineered into S . typhimurium chromosome and repressed by purR(s) so that they could be used as an indicator of adaptive mutations in purR(s) or in the purD operator . Mutations in purR(s) or in the purD operator accumulated when the mutant strain was placed on a minimal lactose plate supplemented with 10 microg/ml of adenine during prolonged incubation . These specific mutations reverted the mutant strain from lac(-) to lac(+) phenotype . The lac(+) strains were categorized into the early- and late-arising mutants according to the time for colony appearance . Our genetic studies indicate that (i) Poisson distributed mutations accumulated in the chromosomal regulatory gene purR or the purD operator in very slowly dividing cells under selection; (ii) after about 8 days of selection, the frequency of mutations in purD operator reached the high value of about two mutations per 10(8) cells; (iii) the mutational spectrum in the purD operator during growth was not significantly different from that during selection; (iv) defects in mutL or mutS appeared to have a stronger effect on growth-dependent mutations than on adaptive mutations.

Biochemistry, 2001 Dec 25, 40(51), 15638 - 49
The exocyclic 1,N2-deoxyguanosine pyrimidopurinone M1G is a chemically stable DNA adduct when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene; Schnetz-Boutaud NC et al.; The pyrimidopurinone adduct M1G {3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido{1,2-a}-purin-10(3H)-one}, formed in DNA upon exposure to malondialdehyde or base propenals, was incorporated into 5'-d(ATCGCMCGGCATG)-3'-5'-d(CATGCCGCGAT)-3', where M = M1G . This duplex contained a two-nucleotide bulge in the modified strand, and was named the M1G-2BD oligodeoxynucleotide . It provided a model for -2 bp strand slippage deletions associated with the (CpG)3-iterated repeat hotspot for frameshift mutations from the Salmonella typhimurium hisD3052 gene . M1G was chemically stable in the M1G-2BD duplex at neutral pH . The two-base bulge in the M1G-2BD oligodeoxynucleotide was localized and consisted of M1G and the 3'-neighbor deoxycytosine . The intrahelical orientation of M1G was established from a combination of NOE and chemical shift data . M1G was in the anti conformation about the glycosyl bond . The 3'-neighbor deoxycytosine appeared to be extruded toward the major groove . In contrast, when M1G was placed into the corresponding fully complementary (CpG)3-iterated repeat duplex at neutral pH, spontaneous and quantitative ring-opening to N(2)-(3-oxo-1-propenyl)-dG (the OPG adduct) was facilitated {Mao, H., Reddy, G . R., Marnett, L . J., and Stone, M . P . (1999) Biochemistry 38, 13491-13501} . The structure of the M1G-2BD duplex suggested that the bulged sequence lacked a cytosine amino group properly positioned to facilitate opening of M1G and supports the notion that proper positioning of deoxycytosine complementary to M1G is necessary to promote ring-opening of the exocyclic adduct in duplex DNA . The structure of the M1G-2BD duplex was similar to that of the structural analogue 1,N(2)-propanodeoxyguanosine (PdG) in the corresponding PdG-2BD duplex {Weisenseel, J . P., Moe, J . G., Reddy, G . R., Marnett, L . J., and Stone, M . P . (1995) Biochemistry 34, 50-64} . The fixed position of the bulged bases in both instances suggests that these exocyclic adducts do not facilitate transient bulge migration.

J Magn Reson Imaging, 2001 Dec, 14(6), 779 - 88
Lack of mutagenic and co-mutagenic effects of magnetic fields during magnetic resonance imaging; Schreiber WG et al.; Mutagenic and co-mutagenic effects of static, pulsed bipolar gradient, and high-frequency magnetic fields, as well as combinations of them, were examined using the Ames test . The Ames test using Salmonella typhimurium bacteria, wild-type strain RTA, preincubation assay, without metabolic activation, was performed . All combinations of magnetic fields were tested with and without co-exposure to N-methyl-N'-nitro-N-nitrosoguanidine and benzo{a}pyrene-4,5-oxide, ethylene oxide, carboplatin, or cisplatin . As expected, chemical mutagens caused a clear-cut increase of the revertants in the Ames test . However, neither the static fields nor a combination of a static magnetic field with the time-varying bipolar gradient field or a pulsed high-frequency magnetic field caused an alteration in the number of revertants in the Ames test . No co-mutagenic effect of any magnetic field combination was observed . In conclusion, magnetic fields used during clinical magnetic resonance imaging (MRI) were neither mutagenic nor co-mutagenic .

Teratog Carcinog Mutagen, 2001, 21(5), 349 - 59
Effect of hydrogen peroxide on nitric oxide (NO)-induced mutagenicity in Salmonella typhimurium; Saliim ET et al.; Nitric oxide (NO) has been reported to impart, alone or in combination with reactive oxygen species (ROS), the cytotoxicity and putative genotoxicity associated with the immunological response . The present study examined the change in the mutagenic activity profile of the NO-donor spermine NONOate (SperNO) as a result of introduction of hydrogen peroxide (H(2)O(2)) to the Ames assay . The aim was to determine whether the assay could detect H(2)O(2)-induced co- or anti-mutagenic effects on NO-induced mutagenesis, and the Salmonella typhimurium base-pair substitution tester strain TA1535 provided an appropriate tool . While TA1535 was shown by the authors and others to be strongly sensitive to NO-induced mutagenesis, it has also been shown to be insensitive to H(2)O(2)-induced mutagenicity {1,2} . When H(2)O(2) (0.25-4.0 micromol/pl) was added directly to cells treated with SperNO (0.01-1.0 micromol/pl), co-mutagenicity was not detected, but a drop in reversion count and detectable toxicity was observed, especially at doses > 0.1 micromol/pl . When glucose/glucose oxidase (GOX) or reduced glutathione (GSH) were used as H(2)O(2)-generation systems the results varied . Reversion induced by SperNO (1 micromol/pl) was moderately enhanced by GOX (10-20 mUnits/pl), but the increase albeit reproducible did not reach a doubling (co-mutagenicity) . GOX (40 micromol/pl) induced a reduction in reversion count, but no visible toxicity . On the other hand, GSH (20- 80 micromol/pl) gave a strong co-mutagenic effect . Co-mutagenicity was highest (> 5x) at 80 micromol/pl GSH and 0.1 micromol/pl SperNO . Based on these findings, it could be concluded that a) H(2)O(2), when steadily generated in the cell, has a modulatory effect on NO-mutagenicity, and such a conclusion is not inconsistent with the wide range of responses reported for the two chemicals, and/or b) the observed co-mutagenic effects of GSH may not be attributable solely to H(2)O(2) generation .

J Appl Toxicol, 2001 Nov-Dec, 21(6), 449 - 60
In vitro and in vivo genetic toxicology studies with diethylene glycol monohexyl ether; Ballantyne B et al.; Diethylene glycol monohexyl ether (DEGHE; CAS no . 112-59-4), an industrial chemical, was investigated for the potential to produce genotoxic effects using three in vitro and two in vivo tests . No mutagenic activity occurred in either the absence or presence of metabolic activation with a Salmonella typhimurium reverse assay using strains TA98, TA100, TA1535, TA1537 and TA1538 . In a Chinese hamster ovary (CHO) forward gene mutation test (HGPRT locus) there was an increase in the mutation frequencies, which were relatively small compared with the solvent control values, somewhat inconsistent between duplicate cultures and occurred particularly in the presence of metabolic activation . Linear regression analysis indicated a marginally significant trend for dosage versus mutation frequency, suggesting that DEGHE was weakly positive in this test . A sister chromatid exchange test in CHO cells showed no significant dosage-related effects in the presence or absence of metabolic activation . A peripheral blood micronucleus test in mice by dosing with an intraperitoneal injection of DEGHE did not show any potential for DEGHE to increase the incidence of micronucleated polychromatophilic erythrocytes . In a first femoral bone marrow chromosome aberration test in the rat by peroral dosing, DEGHE did not cause any increase in aberrations for 12-h and 24-h samples with males and females or with females at 48-h sampling . However, with males at 48 h the two lowest doses showed an increased number of aberrations, but not at the high doses . A repeat study in males with a larger number of doses and 24-h and 48-h samples did not replicate this finding . It is concluded that DEGHE may have limited weak mutagenic activity in vitro but is devoid of clastogenic potential .

Int J Cancer, 2001 Nov 1, 94(3), 438 - 43
Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium; Yuhua L et al.; An attenuated strain of Salmonella typhimurium was used as a vehicle for oral gene therapy against murine tumor . Eukaryotic expression vectors containing genes of human interleukin-12 (hIL-12), human granulocyte/macrophage colony-stimulating factor (hGM-CSF), mouse (m)IL-12, mGM-CSF and green fluorescent protein (GFP) were used to transform attenuated Salmonella (SL3261), and such transformants were administered orally to BALB/c and C57BL/6 mice . As a reporter gene, GFP expression in murine liver, spleen, tumor, intestine and kidney was confirmed by confocal and flow cytometry . Soluble cytokines were detected in murine sera, and the concentrations were much higher than those of the control, which contributed to the increased number of cytotoxic T cells and prolongation of survival . Oral cytokine gene therapy using live attenuated Salmonella demonstrated a significant protection against the development of two unrelated murine tumors . These results suggest that such gene therapy has the potential to be simple, effective and (above all) safe against tumor .

Protein Eng, 2001 Nov, 14(11), 891 - 6
Increasing the hydrophobic interaction between terminal W-motifs enhances the stability of Salmonella typhimurium sialidase . A general strategy for the stabilization of beta-propeller protein fold; Witarto AB et al.; Protein engineering of the beta-propeller protein aimed at enhancing the structural stability of the protein was carried out using a monomeric single domain beta-propeller protein, Salmonella typhimurium sialidase, as a model . Ala53 and Ala69 each located at strands B and C of the W1 motif were mutated to Leu and Val, respectively, to increase the hydrophobic interaction between W1 and W6 motifs . The mutants showed enhanced stability towards guanidine hydrochloride and thermal unfolding . Ala53Leu showed higher stability, probably owing to the capability of the mutated Leu to interact extensively with more residues involved in the hydrophobic interactions between the terminal W-motifs . The mutations, which are located far from the active site, have no significant effect on the enzymatic properties . The strategy to enhance the stability proposed here might be applied to the other beta-propeller proteins.

Cell Microbiol, 2001 Dec, 3(12), 825 - 37
Salmonella pathogenicity island 2-dependent macrophage death is mediated in part by the host cysteine protease caspase-1; Monack DM et al.; Salmonella typhimurium invades host macrophages and can either induce a rapid cell death or establish an intracellular niche within the phagocytic vacuole . Rapid cell death requires the Salmonella pathogenicity island (SPI)1 and the host protein caspase-1, a member of the pro-apoptotic caspase family of proteases . Salmonella that do not cause this rapid cell death and instead reside in the phagocytic vacuole can trigger macrophage death at a later time point . We show here that the human pathogen Salmonella typhi also triggers both rapid, caspase-1-dependent and delayed cell death in human monocytes . The delayed cell death has previously been shown with S . typhimurium to be dependent on SPI2-encoded genes and ompR . Using caspase-1(-/-) bone marrow-derived macrophages and isogenic S . typhimurium mutant strains, we show that a large portion of the delayed, SPI2-dependent death is mediated by caspase-1 . The two known substrates of activated caspase-1 are the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18, which are cleaved to produce bioactive cytokines . We show here that IL-1beta is released during both SPI1- and SPI2-dependent macrophage killing . Using IL-1beta(-/-) bone marrow-derived macrophages and a neutralizing anti-IL-18 antibody, we show that neither IL-1beta nor IL-18 is required for rapid or delayed macrophage death . Thus, both rapid, SPI1-mediated killing and delayed, SPI2-mediated killing require caspase-1 and result in the secretion of IL-1beta, which promotes inflammation and may facilitate the spread of Salmonella beyond the gastrointestinal tract in systemic disease.

Mol Cell Probes, 2001 Oct, 15(5), 267 - 74
Sample preparation methods for PCR detection of Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes on beef chuck shoulder using a single enrichment medium; Bhaduri S et al.; To improve the utility of the polymerase chain reaction (PCR) for food samples, methods for preparing template DNA were developed to remove PCR inhibitors . Beef chuck shoulder medallions, artificially contaminated, individually or in combination, with Escherichia coli serotype O157:H7 strain FSIS 45753-35, Salmonella typhimurium DT104 strain 13HP, or Listeria monocytogenes strain Scott A at concentrations of 10, 1 and 0.5 cfu/cm(2)were swabbed with a sponge, and the sponges were enriched for 18 h at 37 degrees C in universal pre-enrichment broth (UPB) . Enriched broth cultures (EBC), cell pellets (CP), or phosphate-buffered saline-washed cell pellets (PBSCP) from enriched sponge samples were compared for detection of E . coli O157:H7, S . typhimurium DT104, or L . monocytogenes by the PCR using the BAX(TM)system . Recovery of the three organisms was effective for detection of each pathogen at initial levels of 10, 1 and 0.5 cfu/cm(2)when inoculated separately, or in combination, onto the beef samples . Use of EBC, CP, or PBSCP of sponge-swabbed samples eliminated problems associated with inhibition of the PCR by food components, time-consuming extraction of DNA, and inhibition due to large amounts of non-target DNA derived from the food . The procedure involving enrichment of sponge-swabbed beef samples in UPB followed by PCR amplification using EBC with the BAX system is the most efficient and simple method for detection of E . coli O157:H7, S . typhimurium DT104, and L . monocytogenes .

J Pediatr Surg, 2001 Dec, 36(12), 1849 - 52
Acute abdomen caused by Salmonella typhimurium infection in children; Arda IS et al.; Salmonella spp . infections can be particularly challenging when they manifest as acute abdominal problems and lead to emergency surgery . Examples of such serious conditions are Salmonella-related intestinal perforation, gallbladder involvement, salpingitis, and peritonitis . Mesenteric lymphadenitis associated with Salmonella typhimurium mimics acute appendicitis and can make it difficult to establish a timely and definitive diagnosis in young patients who present with right lower abdominal pain . Paralytic ileus is a fairly common manifestation of Salmonella infection at all ages, but complete intestinal obstruction requiring surgical intervention is very rare . Because of the nature of the diagnostic process, a significant number of patients with Salmonella infection present with acute abdomen and undergo needless operations . This report describes the cases of 2 pediatric patients who underwent surgery to address persistent pain in the right lower abdominal quadrant and complete intestinal obstruction, respectively . The first patient had inflamed mesenteric lymph nodes that caused appendicitislike symptoms, and the second had dense adhesions between the mesentery and the terminal segments of the ileum that led to intestinal blockage . Serology results showed that both patients' titers for BO ("B and O agglutinating {BO}") antibodies rose to 1:640 in the week after their admission to hospital, a pattern and level that is indicative of S typhimurium infection . J Pediatr Surg 36:1849-1852 .

Mutat Res, 2001 Dec 12, 484(1-2), 61 - 8
Cytochrome b(5) coexpression increases the CYP2E1-dependent mutagenicity of dialkylnitrosamines in methyltransferase-deficient strains of Salmonella typhimurium; Cooper MT et al.; Addition of cytochrome b(5) to recombinant cytochrome P450 2E1 systems has been shown to enhance the metabolism of dialkylnitrosamines in vitro . To determine if this effect could be observed with recombinant expression systems in vivo, we have constructed mutagenicity tester strains that coexpress full-length human cytochrome P450 2E1 (CYP2E1), rat cytochrome P450 reductase, and human cytochrome b(5) in Salmonella typhimurium lacking ogt and ada methyltransferases (YG7104, ogt(-); and YG7108, ogt(-), ada(-)) . These new recombinant strains exhibit a four- to five-fold greater mutagenic response to dimethylnitrosamine, diethylnitrosamine, and dipropylnitrosamine than strains that contain only CYP2E1 and reductase, and are over 100-fold more sensitive to nitrosamines than the parental strains in the presence of an exogenous activating system (S9 fraction) . The four-fold increase in mutagenicity in the presence of cytochrome b(5) was consistent with increasing alkyl chain length up to dibutylnitrosamine, which was poorly activated by CYP2E1 . The greatest enhancement was obtained with a tricistronic construct in which the b(5) cDNA preceded the P450 and reductase cDNAs; placing the b(5) cDNA after the reductase cDNA was substantially less effective . These new, highly sensitive strains may prove useful in the detection of nitrosamine contamination of food and environmental samples.

Biochim Biophys Acta, 2001 Nov 7, 1568(1), 1 - 6
A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate; He M et al.; Glyphosate or Roundup is the most extensively used herbicide for broad-spectrum control of weeds . Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants . Applying the staggered extension process, we randomly mutated and recombined the aroA genes of Salmonella typhimurium and Escherichia coli to obtain four variants that exhibit significantly enhanced tolerance to glyphosate . All four mutants are chimeras of the two parental genes and, in addition, three of them carry one or more de novo point mutations . None of the amino acid substitutions in the mutants was in a position previously known to be important for catalysis or substrate binding . Kinetic analysis of EPSPS activity from these mutants indicated that the tolerance was attributed to a 2-10-fold increased specific activity, 0.4-8-fold reduced affinity to glyphosate, and 2.5-19-fold decreased K(m) for phosphoenolpyruvate . Such mutants will be instrumental for the structural and function study of the enzyme and for the generation of transgenic crops resistant to the herbicide.

J Food Prot, 2001 Nov, 64(11), 1817 - 9
Reduction of Escherichia coli O157:H7 and Salmonella typhimurium in artificially contaminated alfalfa seeds and mung beans by fumigation with ammonia; Himathongkham S et al.; Sprouts eaten raw are increasingly perceived as hazardous foods because they have been vehicles in outbreaks of foodborne disease, often involving Escherichia coli O157:H7 and Salmonella Typhimurium . Although the source of these pathogens has not been established, it is known that the seeds usually are already contaminated at the time sprouting begins . Earlier studies had shown that ammonia was lethal to these same pathogens in manure, so it seemed reasonable to determine whether ammonia was effective against them when associated with seeds to be used for sprouting . Experimentally contaminated (10(8) to 10(9) CFU/g) and dried seeds, intended for sprouting, were sealed in glass jars in which 180 or 300 mg of ammonia/liter of air space was generated by action of ammonium sulfate and sodium hydroxide . Samples were taken after intervals up to 22 h at 20 degrees C . Destruction of approximately 2 to 3 logs was observed with both bacteria associated with alfalfa seeds, versus 5 to 6 logs with mung beans . Greater kills are apparently associated with lower initial bacterial loads . Germination of these seeds was unaffected by the treatment . It appears that this simple treatment could contribute significantly to the safety of sprout production from alfalfa seeds and mung beans.

J Food Prot, 2001 Nov, 64(11), 1751 - 5
Evaluation of universal preenrichment broth for growth of heat-injured pathogens; Zhao T et al.; Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen . Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined . Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser . Typhimurium, Salmonella enterica ser . Enteritidis . and Listeria monocytogenes in UPB . Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca . 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples . Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media . Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays . Cells often required 3 to 4 h for recovery before growth was initiated . Salmonella Typhimurium, Salmonella Enteritidis, E . coli O157:H7, or L . monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml . At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml . Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E . coli O157:H7, or L . monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml . These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.

Natl Toxicol Program Tech Rep Ser, 2000 Dec, (500), 1 - 173
Toxicology and carcinogenesis studies of naphthalene (cas no . 91-20-3) in F344/N rats (inhalation studies); Salmonella Typhimurium infections transmitted by chlorine-pretreated clover sprout seeds; Epidemic Intelligence Service, Division of Applied Public Health Training, Epidemiology Program Office, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA . zud4@cdc.gov

Raw seed sprouts have caused numerous outbreaks of enteric infections . Presoaking seeds in a 20,000 mg/liter (ppm) calcium hypochlorite solution before sprouting is recommended to reduce bacterial contamination and infection risk . In 1999, the authors investigated an outbreak of Salmonella serotype Typhimurium infections in Colorado . In a case-control study, they matched 20 cases with 58 controls by age, sex, and telephone prefix; 10 (52%) of 19 cases and no controls recalled eating raw alfalfa-style sprouts in the 5 days before the patient's illness (p < 0.00001) . Traceback implicated clover sprouts grown from seeds shared by two sprouters . The time period and region over which these sprouts were sold matched the occurrences of 112 culture-confirmed illnesses . Only one of the sprouters presoaked seeds as recommended, and fewer infections were attributable to this sprouter (0.29 vs . 1.13 culture-confirmed infections/50-pound (110.1-kg) bag of seed) . After recall of the implicated sprouts and seed, S . Typhimurium illnesses declined . Contaminated raw clover sprouts can cause outbreaks of enteric illness . Presoaking contaminated seeds in a 20,000 mg/liter calcium hypochlorite solution reduces, but does not eliminate, the risk of infection . Until safer production methods are developed, persons eating raw sprouts continue to risk developing potentially serious gastrointestinal illness.

Mol Microbiol, 2001 Nov, 42(3), 777 - 93
The LuxS-dependent autoinducer AI-2 controls the expression of an ABC transporter that functions in AI-2 uptake in Salmonella typhimurium; Taga ME et al.; In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers . A novel autoinducer called AI-2, originally discovered in the quorum-sensing bacterium Vibrio harveyi, is made by many species of Gram-negative and Gram-positive bacteria . In every case, production of AI-2 is dependent on the LuxS autoinducer synthase . The genes regulated by AI-2 in most of these luxS-containing species of bacteria are not known . Here, we describe the identification and characterization of AI-2-regulated genes in Salmonella typhimurium . We find that LuxS and AI-2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)-type transporter . We have named this operon the lsr (luxS regulated) operon . The Lsr transporter has homology to the ribose transporter of Escherichia coli and S . typhimurium . A gene encoding a DNA-binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI-2 detection to operon expression . This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI-2 . Mutations in the lsr operon render S . typhimurium unable to eliminate AI-2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI-2 into the cells . It is intriguing that an operon regulated by AI-2 encodes functions resembling the ribose transporter, given recent findings that AI-2 is derived from the ribosyl moiety of S-ribosylhomocysteine.

J Appl Microbiol, 2001 Nov, 91(5), 780 - 5
Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy; De Cesare A et al.; AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated . METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively . Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD . CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm . typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886) . SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.

Environ Microbiol, 2001 Oct, 3(10), 638 - 48
Oxygen tension and nutrient starvation are major signals that regulate agfD promoter activity and expression of the multicellular morphotype in Salmonella typhimurium; Gerstel U et al.; Expression of multicellular behaviour (rdar morphotype) is a characteristic of wild-type Salmonella typhimurium strains . The key target for the regulation of rdar morphotype expression is the agfD promoter . The regulation of two rdar morphotypes, regulated and semi-constitutive (the latter differs from the former by the insertion of A after position -17), by various environmental conditions was studied using transcriptional fusions to the regulated and semi-constitutive agfD promoters by Western blot analysis and phenotypic analysis of the rdar morphotype . AgfD promoter activities were strongly dependent on oxygen tension . Expression maxima were observed in rich medium under microaerophilic conditions and in minimal medium under aerobic conditions . The regulated rdar morphotype was only expressed under conditions of maximal promoter activity . Glucose did not influence rdar morphotype expression, and the two promoters showed no consistent response to pH . In the stationary phase of growth, nitrogen and phosphate depletion were found to be signals that switch on the agfD promoters . In the logarithmic phase of growth, ethanol was the stress signal that enhanced rdar morphotype expression . The results indicate that, although the regulated and semi-constitutive agfD promoters are key factors in the grade of expression of the multicellular behaviour, common signals such as oxygen tension, depletion of nutrients and ethanol vary their levels of expression significantly.

Toxicol Sci, 2001 Dec, 64(2), 185 - 91
Inhibition of human cytochrome P450 2E1 by nicotine, cotinine, and aqueous cigarette tar extract in vitro; Van Vleet TR et al.; Cigarette smoke is a complex mixture containing, among other chemicals, pyridine alkaloids and N-nitrosamines . Carcinogenic tobacco-specific N-nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are both activated by cytochrome P450 (CYP) 2E1 in rats . Previous reports indicate that nicotine and the main nicotine metabolite, cotinine, reduce the mutagenicity of both NNK and NDMA in Salmonella typhimurium . To study the mechanism of this effect, we examined inhibition of CYP 2E1 activity, as assessed by p-nitrophenol (pNP) hydroxylation, by nicotine, cotinine, and an aqueous cigarette tar extract (ACTE) in human 2E1-expressing microsomes . At all substrate concentrations (0-1.25 mM) nicotine was a significantly more potent inhibitor of CYP 2E1 activity compared to cotinine . Estimated Ki values for nicotine and cotinine (both at 10 mM) were 13 mM (2 mg/ml) and 308 mM (54 mg/ml) respectively . The Ki for ACTE was 0.2 mg/ml at a concentration of 0.32 mg/ml . This rank order for inhibition was also seen when the data was expressed as IC(50) . When compared on a mass/vol basis, ACTE was a significantly more potent CYP 2E1 inhibitor relative to nicotine and cotinine . Double-reciprocal plots indicated that nicotine and ACTE inhibited by a competitive, while cotinine inhibited CYP 2E1 by an uncompetitive mechanism . Although the contribution of nicotine to ACTE-mediated 2E1 inhibition is probably modest, pyridine alkaloid-mediated CYP 2E1 inhibition is a possible mechanism for the observed inhibition of NNK and NDMA mutagenicity by nicotine and cotinine in vitro.

J Biol Chem, 2002 Feb 1, 277(5), 3708 - 17 Epub 2001 Nov 14.
Structural model of MalK, the ABC subunit of the maltose transporter of Escherichia coli: implications for mal gene regulation, inducer exclusion, and subunit assembly; Bohm A et al.; We are presenting a three-dimensional model of MalK, the ABC subunit of the maltose transporter from Escherichia coli and Salmonella typhimurium . It is based on the recently published crystal structure of the closely related Thermococcus litoralis MalK . The model was used to identify the position of mutations affecting the different functions of the ABC subunit . Six malK point mutations were isolated specifically affecting the interaction with MalT, the transcriptional regulator of the maltose system . They were mapped on the structural model and define a MalT interaction site that is located on an exposed surface of the C-terminal regulatory domain . Published point mutations that confer an inducer exclusion insensitive phenotype form a patch adjacent to and oriented perpendicularly to the MalT interaction site . Three sequence motifs were identified and visualized that are highly conserved among ABC subunits with extended C termini . They form a subdomain between the regulatory and ATPase domain and might play an important role in signal transduction events between these two domains . Mutations in this domain remain fully active in MalT regulation but cause transport defects . In addition, amino acids that have previously been shown to be involved in the interaction with the transmembranous subunits MalF and MalG and that fall into the highly conserved N-terminal ATPase domain were visualized . The validity of the modeled MalK structure was verified by structure-directed mutagenesis of amino acids located within the proposed MalK-MalT interaction site.

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 137 - 42
Directed evolution of alpha-aspartyl dipeptidase from Salmonella typhimurium; Kong X et al.; Model-free approaches (error-prone PCR to introduce random mutations, DNA shuffling to combine positive mutations, and screening of the resultant mutant libraries) have been used to enhance the catalytic activity and thermostability of alpha-aspartyl dipeptidase from Salmonella typhimurium, which is uniquely able to hydrolyze Asp-X dipeptides (where X is any amino acid) and one tripeptide (Asp-Gly-Gly) . Under double selective pressures of activity and thermostability, through two rounds of error-prone PCR and three sequential generations of DNA shuffling, coupled with screening, a mutant pepEM3074 with approximately 47-fold increased enzyme activity compared with its wild-type parent was obtained . Moreover, the stability of pepEM3074 is increased significantly . Three amino acid substitutions (Asn89His, Gln153Glu, and Leu205Arg), two of them are near the active site and substrate binding pocket, were identified by sequencing the genes encoding this evolved enzyme . The mechanism of the enhancement of activity and stability was analyzed in this paper .

J Biol Chem, 2002 Jan 25, 277(4), 2886 - 96 Epub 2001 Nov 08.
Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli . Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose; Breazeale SD et al.; Addition of the 4-amino-4-deoxy-l-arabinose (l-Ara4N) moiety to the phosphate groups of lipid A is implicated in bacterial resistance to polymyxin and cationic antimicrobial peptides of the innate immune system . The sequences of the products of the Salmonella typhimurium pmrE and pmrF loci, both of which are required for polymyxin resistance, recently led us to propose a pathway for l-Ara4N biosynthesis from UDP-glucuronic acid (Zhou, Z., Lin, S., Cotter, R . J., and Raetz, C . R . H . (1999) J . Biol . Chem . 274, 18503-18514) . We now report that extracts of a polymyxin-resistant mutant of Escherichia coli catalyze the C-4" oxidation and C-6" decarboxylation of {alpha-(32)P}UDP-glucuronic acid, followed by transamination to generate {alpha-(32)P}UDP-l-Ara4N, when NAD and glutamate are added as co-substrates . In addition, the {alpha-(32)P}UDP-l-Ara4N is formylated when N-10-formyltetrahydrofolate is included . These activities are consistent with the proposed functions of two of the gene products (PmrI and PmrH) of the pmrF operon . PmrI (renamed ArnA) was overexpressed using a T7 construct, and shown by itself to catalyze the unprecedented oxidative decarboxylation of UDP-glucuronic acid to form uridine 5'-(beta-l-threo-pentapyranosyl-4"-ulose diphosphate) . A 6-mg sample of the latter was purified, and its structure was validated by NMR studies as the hydrate of the 4" ketone . ArnA resembles UDP-galactose epimerase, dTDP-glucose-4,6-dehydratase, and UDP-xylose synthase in oxidizing the C-4" position of its substrate, but differs in that it releases the NADH product.

Mol Microbiol, 2001 Oct, 42(2), 469 - 81
IpaC from Shigella and SipC from Salmonella possess similar biochemical properties but are functionally distinct; Osiecki JC et al.; Invasion plasmid antigen C (IpaC) is secreted via the type III secretion system (TTSS) of Shigella flexneri and serves as an essential effector molecule for epithelial cell invasion . The only homologue of IpaC identified thus far is Salmonella invasion protein C (SipC/SspC), which is essential for enterocyte invasion by Salmonella typhimurium . To explore the biochemical and functional relatedness of IpaC and SipC, recombinant derivatives of both proteins were purified so that their in vitro biochemical properties could be compared . Both proteins were found to: (i) enhance the entry of wild-type S . flexneri and S . typhimurium into cultured cells; (ii) interact with phospholipid membranes; and (iii) oligomerize in solution; however, IpaC appeared to be more efficient in carrying out several of the biochemical properties examined . Overall, the data indicate that purified IpaC and SipC are biochemically similar, although not identical with respect to their in vitro activities . To extend these observations, complementation analyses were conducted using S . flexneri SF621 and S . typhimurium SB220, neither of which is capable of invading epithelial cells because of non-polar null mutations in ipaC and sipC respectively . Interestingly, both ipaC and sipC restored invasiveness to SB220 whereas only ipaC restored invasiveness to SF621, suggesting that SipC lacks an activity possessed by IpaC . This functional difference is not at the level of secretion because IpaC and SipC are both secreted by SF621 and it does not appear to be because of SipC dependency on this native chaperone as coexpression of sipC and sicA in SF621 still failed to restore detectable invasiveness . Taken together, the data suggest that IpaC and SipC differ in either their ability to be translocated into host cells or in their function as effectors of host cell invasion . Because IpaB shares significant sequence homology with the YopB translocator of Yersinia species, the ability for IpaC and SipC to associate with this protein was explored as a potential indicator of translocation function . Both proteins were found to bind to purified IpaB with an apparent dissociation constant in the nanomolar range, suggesting that they may differ with respect to effector function . Interestingly, whereas SB220 expressing sipC behaved like wild-type Salmonella, in that it remained within its membrane-bound vacuole following entry into host cells, SB220 expressing ipaC was found in the cytoplasm of host cells . This observation indicates that IpaC and SipC are responsible for a major difference in the invasion strategies of Shigella and Salmonella, that is, they escape into the host cell cytoplasm . The implications of the role of each protein's biochemistry relative to its in vivo function is discussed.

Toxicol Lett, 2001 Dec 15, 125(1-3), 39 - 49
Metabolic activation of three arylamines and two organophosphorus insecticides by coriander (Coriandrum sativum) a common edible vegetable; Cortes-Eslava J et al.; Organophosphorus insecticides and arylamines, widely distributed in the environment, can be activated into mutagens by plants . Plant activation of three aromatic amines, 4-nitro-o-phenylenediamine (NOP), m-phenylenediamine (m-PDA) and 2-aminofluorene (2AF), and two organophosphorus insecticides, dimethoate and methyl parathion has been the focus of this study . The plant cell/microbe coincubation assay was used employing coriander (Coriandrum sativum) suspended cell cultures as the activating system . Interestingly, this vegetable is included in the Mexican diet and ingested generally uncooked and could have epidemiological consequences . As a genetic end point, the Salmonella typhimurium tester strain TA98 was used . Protein contents, as well as peroxidase activity and peroxidase activity inhibited by diethyldithiocarbamate (DEDTC) of coriander cultures were determined after the coculture . Coriander cells highly activated three aromatic amines, NOP, m-PDA and 2-AF to mutagenic products detected in Salmonella . On the other hand, insecticides were only lightly activated, probably because peroxidase activity of coriander cells was inhibited, corroborated by DEDTC peroxidase inhibition . In all the assays, NOP was the more potent mutagenic compound . The results demonstrated that coriander cells were metabolically competent and suitable for a plant cell microbe coincubation assay, developed to analyze the promutagen activation by plant systems and can be used as a indicator of potential genetic effects.

J Biol Chem, 2002 Jan 18, 277(3), 2258 - 65 Epub 2001 Nov 07.
Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages; Cuellar-Mata P et al.; Salmonella survive and replicate within mammalian cells by becoming secluded within specialized membrane-bound vacuoles inaccessible to the host defense mechanisms . Delayed acidification of the vacuole and its incomplete fusion with lysosomes have been implicated in intracellular Salmonella survival . Nramp1 confers to macrophages resistance to a variety of intracellular pathogens, including Salmonella, but its precise mode of action is not understood . We investigated whether Nramp1 affects the maturation and acidification of Salmonella-containing vacuoles (SCV) . A mouse-derived macrophage line (RAW/Nramp1(-)) devoid of Nramp1 and therefore susceptible to infection was compared with isogenic clones stably transfected with Nramp1 (RAW/Nramp1(+)) . Intravacuolar pH, measured in situ, was similar in Nramp1-expressing and -deficient cells . SCV acquired LAMP1 and fused with preloaded fluid-phase markers in both cell types . In contrast, although few vacuoles in RAW/Nramp1(-) acquired mannose 6-phosphate receptor, many more contained M6PR in RAW/Nramp1(+) cells . Shortly after closure, SCV in RAW/Nramp1(-) became inaccessible to extracellular markers, suggesting inability to fuse with newly formed endosomes . Expression of Nramp1 markedly increased the access to extracellularly added markers . We propose that Nramp1 counteracts the ability of Salmonella to become secluded in a compartment that limits access of bactericidal agents, allowing the normal degradative pathway of the macrophage to proceed.

Rev Inst Med Trop Sao Paulo, 2001 Sep-Oct, 43(5), 247 - 50
Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat; dos Santos LR et al.; The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat . Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass) . The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol . As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Food Chem Toxicol, 2001 Dec, 39(12), 1253 - 61
Using base-specific Salmonella tester strains to characterize the types of mutation induced by benzidine and benzidine congeners after reductive metabolism; Claxton LD et al.; Although benzidine (Bz), 4-aminobiphenyl (ABP), 3,3'-dichlorobenzidine HCl (DCBz), 3,3'-dimethylbenzidine (DMBz), 3,3'-dimethoxybenzidine (DMOBz) and the benzidine congener-based dye trypan blue (TB) produce primarily frameshift mutations in Salmonella typhimurium, the base-substitution strain TA100 also responds to these compounds when S9 is present . Performing DNA sequence analysis, other investigators have shown that ABP induces frameshift, base-pair and complex mutations . Also, it was found that an uninduced hamster liver S9 preparation with glucose-6-phosphate dehydrogenase, FMN, NADH and four times glucose 6-phosphate gave a stronger mutagenic response than the conventional plate incorporation with rat S9 activation mixture for all the compounds tested . Using the base-specific tester strains of S . typhimurium (TA7001-TA7006) with the above reductive metabolic activation system, we surveyed these compounds for the ability to produce specific base-pair substitutions after reductive metabolism . Bz was weakly mutagenic in TA7005 (0.04 revertants/microg) . ABP was mutagenic in TA7002 (1.4 revertants/microg), TA7004 (0.6 revertants/microg), TA7005 (2.98 revertants/microg) and TA7006 (0.4 revertants/microg) . DCBz was weakly mutagenic in TA7004 (0.01 revertants/microg) . It was concluded that benzidine induced some CG->AT transversions in addition to frameshift mutations . ABP induced TA->AT, CG->AT, and CG->GC transversions as well as GC->AT transitions . DCBz induced only GC->AT transitions . Because DMBz, DMOBz and TB were not mutagenic in this base-substitution mutagen detection system, their mutagenic activity was attributed strictly to frameshift mechanisms.

Cell Microbiol, 2001 Nov, 3(11), 731 - 44
A role for the PhoP/Q regulon in inhibition of fusion between lysosomes and Salmonella-containing vacuoles in macrophages; Garvis SG et al.; After uptake by murine macrophages, Salmonella typhimurium is able to survive and replicate within specialized phagosomes called Salmonella-containing vacuoles (SCVs), which are segregated from the late endocytic pathway . The molecular basis of this process and the virulence factors required are not fully understood . In this study, we used confocal fluorescence microscopy to evaluate interactions between the endocytic pathway of the murine macrophage cell line RAW 264.7 and different S . typhimurium strains . The analysis was carried out using the fluid-phase marker Texas red-ovalbumin and antibodies against the lysosomal enzyme cathepsin D, the late endosomal lipid lysobisphosphatidic acid and the adaptor proteins AP-1 and AP-3 . Less than 10% of wild-type SCVs were associated with these markers at 24 h after uptake by macrophages . A similar low level of association was observed for vacuoles containing mutant strains affected in the function of the Salmonella pathogenicity island (SPI)-2 type III secretion system or the virulence plasmid spv operon . However, at this time point, the proportion of vacuoles containing phoP-mutant bacteria that were associated with each of the markers ranged from 25% to 50% . These results show that the regulon controlled by the PhoP/Q two-component system makes a major contribution to trafficking of the SCV in macrophages . Segregation of SCVs from the endocytic pathway was also found to be dependent on bacterial proteins synthesized between 15 min and 4 h after uptake into macrophages . However, after this time, protein synthesis was not required to maintain the segregation of SCVs from late endosomes and lysosomes.

Int J Antimicrob Agents, 2001 Oct, 18(4), 403 - 6
Trifluoperazine: a broad spectrum bactericide especially active on staphylococci and vibrios; Mazumder R et al.; Trifluoperazine showed some significant antimicrobial activity when tested against 293 strains from two Gram-positive and eight Gram-negative genera . Minimum inhibitory concentrations of the drug were measured using an agar dilution technique . Forty six of 55 strains of Staphylococcus aureus were inhibited by 10-50 microg/ml of trifluoperazine . This drug also inhibited strains of Shigella spp., Vibrio cholerae and V . parahaemolyticus at a concentration of 10-100 microg/ml . Other bacteria including Pseudomonas spp . were moderately sensitive to trifluoperazine . In the in vivo studies this compound offered significant protection to Swiss albino mice at a concentration of 30 microg/mouse (P<0.001) when challenged with 50 median lethal dose of Salmonella typhimurium NCTC 74.

Curr Microbiol, 2001 Dec, 43(6), 418 - 23
The product of the cysK gene of Bacillus stearothermophilus V mediates potassium tellurite resistance in Escherichia coli; Vasquez CC et al.; The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined . Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment . E . coli cells harboring a recombinant plasmid containing the Ter determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF . This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms . Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K2TeO3 that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421 . Introduction of the B . stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance.

Mutagenesis, 2001 Nov, 16(6), 523 - 8
S9 induction by the combined treatment with cyclohexanol and albendazole; Escobar-Garcia D et al.; Cyclohexanol (CH) is an industrial solvent capable of inducing cytochrome P450 (CYP) enzymes including the CYP2E and CYP2B subfamilies . S9 from CH treated rats is able to activate several N-nitrosamines that are poorly activated by Aroclor 1254, phenobarbital/beta-naphthoflavone (PB/NF) or 3-methylcholanthrene S9 fractions into mutagens detected by the Salmonella typhimurium Ames test . Additionally, albendazole (ABZ) is a widely used anthelmintic drug and a potent inducer of the CYP1A subfamily . Since CYP1A, -2B and -2E subfamilies are implicated in the activation of several environmental mutagens/carcinogens, we studied CYP induction in the rat liver by the combined effect of these two compounds, and used S9 derived from it in the Salmonella/microsome assay to compare with S9 obtained from Aroclor or PB/NF treated rats . Total CYP content in hepatic microsomes was induced by Aroclor, but not by any of the other chemical combinations . Western blot and enzymatic activity analysis revealed quantitative but not qualitative differences in the CYP subfamilies present in the different microsomal fractions; all of the chemicals used increased the levels of CYP1A1/2, CYP2B1/2 and CYP2E1 with respect to control microsomes . CYP3A was not modified by the different treatments . When tested in the Ames test, Aroclor S9 and PB/NF S9 were the most effective in the activation of benzo{a}pyrene and 3-methylcholanthrene which are metabolized mainly by CYP1A1; additionally, the highest mutagenic potency of 2-aminofluorene and N-nitrosodipropylamine, which are activated by CYP1A2 and CYP2B, respectively, were obtained with PB/NF S9 . All these compounds were also activated when CH/ABZ S9 was used as the exogenous source of metabolism . Mutagens like N-nitrosopyrrolidine and N-nitrosodimethylamine, activated by CYP2E1, were detected only when CH/ABZ S9 was used, and the effectiveness of the different S9 fractions in activating cyclophosphamide decreased in the following order: Aroclor = PB/NF > CH/ABZ > control . From these experiments we can conclude that the individual CYP- inducing properties of ABZ and CH complement each other when the two compounds are administered in conjunction and that the resulting S9 fraction is able to activate several known mutagens in the Ames test.

Nature, 2001 Oct 25, 413(6858), 848 - 52
Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18; Parkhill J et al.; Salmonella enterica serovar Typhi (S . typhi) is the aetiological agent of typhoid fever, a serious invasive bacterial disease of humans with an annual global burden of approximately 16 million cases, leading to 600,000 fatalities . Many S . enterica serovars actively invade the mucosal surface of the intestine but are normally contained in healthy individuals by the local immune defence mechanisms . However, S . typhi has evolved the ability to spread to the deeper tissues of humans, including liver, spleen and bone marrow . Here we have sequenced the 4,809,037-base pair (bp) genome of a S . typhi (CT18) that is resistant to multiple drugs, revealing the presence of hundreds of insertions and deletions compared with the Escherichia coli genome, ranging in size from single genes to large islands . Notably, the genome sequence identifies over two hundred pseudogenes, several corresponding to genes that are known to contribute to virulence in Salmonella typhimurium . This genetic degradation may contribute to the human-restricted host range for S . typhi . CT18 harbours a 218,150-bp multiple-drug-resistance incH1 plasmid (pHCM1), and a 106,516-bp cryptic plasmid (pHCM2), which shows recent common ancestry with a virulence plasmid of Yersinia pestis.

Gastroenterology, 2001 Nov, 121(5), 1158 - 66
Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV-specific CTL in a transgenic mouse model; Wedemeyer H et al.; BACKGROUND & AIMS: The ability to induce cytotoxic T cells is considered an important feature of a candidate hepatitis C virus (HCV) vaccine . We used an oral immunization strategy with attenuated HCV-NS3-transformed Salmonella typhimurium to deliver DNA directly to the gut-associated lymphoid tissue . METHODS: HLA-A2.1 transgenic mice were immunized once with transformed attenuated Salmonella . HCV-specific CD8+ T cells were analyzed in vitro as well as in vivo by challenge of mice with recombinant HCV-NS3 vaccinia virus . RESULTS: Salmonella (10(8) colony-forming units; 20 microg plasmid DNA) induced cytotoxic and IFN-gamma-producing CD8+ T cells specific for the immunodominant epitope NS3-1073 in 26 of 30 mice (86%) that persisted for at least 10 months . A second epitope (NS3-1169) was also recognized by cytotoxic and IFN-gamma-producing T cells, whereas a third one (NS3-1406) stimulated IFN-gamma production without cytotoxicity . The minimal amount of plasmid DNA required to induce CTLs was 2 ng . Upon challenge with recombinant HCV-NS3-expressing vaccinia virus, vaccinia titers were significantly lower in mice immunized with Salmonella-NS3 than in mice immunized with control Salmonella, demonstrating the in vivo function of CTLs . CONCLUSIONS: Oral immunization with attenuated Salmonella typhimurium as a carrier for HCV DNA induces long-lasting T-cell responses.

Mutat Res, 2001 Nov 15, 498(1-2), 207 - 17
Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk; Pfau W et al.; Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins . Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts . Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays . Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9 . A seventh extract, tested only in the absence of S9, was inactive . Extracts were either active or inactive in at least three of the four tests applied . Four extracts were active or inactive in all four tests . The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.

Mutat Res, 2001 Nov 15, 498(1-2), 107 - 15
Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan; Watanabe T et al.; We previously determined the chemical structures of four 2-phenylbenzotriazole mutagens (PBTA-1, -2, -3 and -4) in blue rayon-adsorbed material from the Nishitakase River in Kyoto prefecture and the Nikko River in Aichi prefecture in Japan . On the basis of a synthesis study, these four PBTA derivatives were deduced to have originated from corresponding dinitrophenylazo dyes by reduction and chlorination . 2-{(2-Bromo-4,6-dinitrophenyl)azo}-5-{bis(2-acetoxyethyl) amino}-4-methoxyacetanilide (Color Index Name, Disperse Blue 79:1; CAS Registry Number, 75497-74-4) is a very common dinitrophenylazo dye used in textile dyeing factories . In the present study, we synthesized 2-{4-{bis(2-acetoxyethyl)amino}-2-(acetylamino)-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-5) from Disperse Blue 79:1 by reduction with sodium hydrosulfite and subsequent chlorination with sodium hypochlorite . On hydrolysis of PBTA-5 with alkali, 2-{2-(acetylamino)-4-{bis(2-hydroxyethyl)amino}-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) was obtained . Both PBTA-5 and -6 were potent mutagens, inducing 723,000 revertants and 485,000 revertants per microgram of Salmonella typhimurium YG1024, respectively, in the presence of S9 mix . To clarify whether PBTA-5 and -6 exist in the environment, water samples were collected from five rivers flowing through regions where textile dyeing industries are developed . PBTA-6 was detected at levels of 3-134 ng/g blue rayon in all water samples that were examined . On the other hand, the amount of PBTA-5 in the samples was less than the detection limit.

Mutat Res, 2001 Nov 15, 498(1-2), 99 - 105
Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines; Ferrer M et al.; Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties . This plant extract is being studied for treatment of viral diseases in animals and humans . Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property . Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S . typhimurium) YG1024, in different co-treatment approaches . This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA>2-AA>2-AF>9-AP>1-AP . Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis . Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay . Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic . In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay . Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.

Mutat Res, 2001 Nov 15, 498(1-2), 19 - 37
Transformation of mutagenic aromatic amines into non-mutagenic species by alkyl substituents . Part I . Alkylation ortho to the amino function; Glende C et al.; Alkyl-substituted derivatives of 2-aminonaphthalene (2-AN) 1, 2-aminofluorene (2-AF) 6 and 4-aminobiphenyl (4-ABP) 11 were synthesized and the mutagenic activity of these compounds determined in Salmonella typhimurium strains TA98 and TA100 with and without S9 mix . In the case of the ortho-substituted 4-aminobiphenyls 12-15 (3-alkyl=ethyl, iso-propyl, n-butyl, tert-butyl) the substituent with the strongest steric demand (3-tert-butyl) shows the strongest influence on the decrease of mutagenicity if compared with the parent compound . In the series of the bis-ortho-disubstituted compounds 16-18 (3,5-dimethyl-, 3,5-diethyl- and 3,5-diisopropyl-4-aminobiphenyl) generation of non-mutagenic species occurs already with the introduction of two ethyl groups . For the 4-aminobiphenyl derivatives 12-15 and 16-18, as well as for the 1-alkylated 2-aminofluorenes 7-10 and the 1-alkylated 2-aminonaphthalenes 2-5 a smaller mutagenicity was observed if compared with predicted mutagenicities as calculated by the QSAR equations of Debnath et al . (Environ . Mol . Mutagen . 19 (1992) 37) . The largest differences resulted in the cases of the tert-butyl substituted compounds . Only with smaller alkyl groups like ethyl the QSAR predictions and the experimentally determined mutagenicities come close to each other . Thus, these results show that appropriate alkyl substitution reduces (eliminates) mutagenicity, secondly, it is necessary to introduce steric parameters to predict the mutagenicity of such compounds correctly.

Vaccine, 2001 Nov 12, 20(3-4), 577 - 85
Induction of specific CD8+ memory T cells and long lasting protection following immunization with Salmonella typhimurium expressing a lymphocytic choriomeningitis MHC class I-restricted epitope; Shams H et al.; Numerous studies have shown the potential of Salmonella typhimurium as a vector for delivery of heterologous proteins for vaccination against other pathogens . Earlier studies showed that the inefficient elicitation of MHC class I-restricted responses could limit the use of S . typhimurium as a heterologous antigen delivery vector for vaccination . We recently developed an approach to overcome this limitation by using a bacterial-encoded specialized protein secretion system, termed type III, to deliver proteins into the class I antigen presenting pathways . Thus, peptides of interest fused to proteins bearing the type III secretion signal, which can elicit protective CTL responses . Because protective immunity is usually assessed a few weeks after vaccination, there is a paucity of information regarding duration of protective immunity induced by this system . We show here that mice immunized orally with S . typhimurium vectors expressing a MHC class I-restricted epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein developed specific antiviral CTL responses . CD8+ T cells were found to be necessary for this CTL activity against targets presenting the LCMV epitope . The survival of mice challenged with lethal doses of LCMV 60 or 135 days after vaccination was as complete as the survival of mice challenged 2 weeks after immunization with the same vectors . By demonstrating their ability to induce prolonged protective immunity after oral delivery, S . typhimurium vectors have met an essential requirement in support of their development as vectors for heterologous vaccination.

Vaccine, 2001 Nov 12, 20(3-4), 421 - 9
An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice; Niethammer AG et al.; A DNA vaccine encoding human carcinoembryonic antigen (CEA) broke peripheral T-cell tolerance toward this tumor self-antigen expressed by Lewis lung carcinoma stably transduced with CEA in C57BL/6J mice transgenic for CEA . This vaccine, delivered by oral gavage with an attenuated strain of Salmonella typhimurium (SL7207), and boosted with an antibody-IL2 fusion protein, induced tumor-protective immunity mediated by MHC class I antigen-restricted CD8(+) T cells, resulting in eradication of subcutaneous tumors in 100% of mice and prevention of experimental pulmonary metastases in 75% of experimental animals . Both CTL and antigen-presenting dendritic cells were activated as indicated by a decisive increase in their respective activation markers CD2, CD25, CD28 as well as CD48 and CD80 . The antitumor effects of this CEA-based DNA vaccine obtained in prophylactic settings, suggest that this approach could lead to the rational design of effective treatment modalities for human lung cancer.

Vaccine, 2001 Nov 12, 20(3-4), 413 - 20
Vaccination of mice with live recombinant Salmonella typhimurium aroA against H . pylori: parameters associated with prophylactic and therapeutic vaccine efficacy; Koesling J et al.; Previously we described a recombinant attenuated Salmonella typhimurium aroA strain (SL3261{pYZ97}) with constitutive expression of plasmid encoded Helicobacter pylori urease subunits A and B (UreAB) . Single dose oral vaccination effectively induced prophylactic immunity against bacterial challenge in BALB/c mice . Here we successfully extended this approach to several mouse strains with allelic differences in NRAMP-1 and H-2 genes . The respective host determinants are known to influence the immune response against S . typhimurium . A comparative analysis of the vaccine efficacy in C57BL/6 and BALB/c mice showed that the live vaccine confers long lasting immunity in both strains (>18 weeks) . In C57BL/6 mice, protection was still observed 54 weeks while not all vaccinated BALB/c were immune when challenged after this time . BALB/c mice also needed higher doses of SL3261{pYZ97} for full protection . We also demonstrate a therapeutic potential of SL3261{pYZ97} in H . pylori infected BALB/c and C57BL/6 mice . Urease- and carrier-specific serum antibody responses as well as the level of colonization by the Salmonella were analyzed in both mouse strains after immunization with low (4 x 10(7)CFU) or high (1 x 10(9)CFU) vaccine doses . The results are discussed in the context of inoculum size and the mode of antigen supply required for effective vaccination with recombinant Salmonella.

Ugeskr Laeger, 2001 Oct 8, 163(41), 5677 - 8
{Five cases of gastroenteritis with multiresistant Salmonella enterica serovar Typhimurium DT104 related to farm animals in Denmark}; Schiellerup P et al.; Whereas the overall incidence of human Salmonella infections in Denmark has fallen during the past three years, the number of infections with multidrug-resistant Salmonella Typhimurium definitive type 104 (DT104) has risen . We report five cases of human infection with DT104 in patients living on farms, in which cattle and mixed herds of cattle and pigs were infected with DT104 . Transmission from the animals to the patients in the cases described is likely to have occurred . These cases emphasize the risky of infection through contact with animals infected with DT104.

Can Vet J, 2001 Oct, 42(10), 788 - 92
Emergence of Salmonella typhimurium definitive type 104 (DT104) as an important cause of salmonellosis in horses in Ontario; Weese JS et al.; Salmonella Typhimurium definitive type 104 (DT104) has emerged as a common cause of salmonellosis in humans and cattle, yet previous reports involving horses are sparse . This study reports the emergence of DT104 as an important pathogen in horses in Ontario . The first clinical case of DT104 infection at the Ontario Veterinary College was identified in 1997 . Seventeen cases of DT104-associated salmonellosis were identified between 1997 and 2000 . In 2000, 12 of 13 cases of salmonellosis were due to DT104 . Salmonellosis in horses due to DT104 is of concern, since the organism is multiresistant to antibiotics and poses increased zoonotic risk . Phage type distribution of Salmonella isolates should be monitored to determine whether DT104 will remain a prevalent equine pathogen.

J Biol Chem, 2001 Dec 21, 276(51), 48431 - 9 Epub 2001 Oct 18.
Regulation of Salmonella-induced neutrophil transmigration by epithelial ADP-ribosylation factor 6; Criss AK et al.; Salmonella typhimurium elicits an acute inflammatory response in the host intestinal epithelium, characterized by the movement of polymorphonuclear leukocytes (PMN) across the epithelial monolayer to the intestinal lumen . It was recently shown that SipA, a protein secreted by S . typhimurium, is necessary and sufficient to drive PMN transmigration across model intestinal epithelia (Lee, C . A., Silva, M., Siber, A . M., Kelly, A . J., Galyov, E., and McCormick, B . A . (2000) Proc . Natl . Acad Sci . USA 97, 12283-12288) . However, the epithelial factors responsible for this process have not been identified . Here, for the first time, we demonstrate that S . typhimurium-induced PMN transmigration across Madin-Darby canine kidney-polarized monolayers is regulated by the GTPase ARF6 . Apically added S . typhimurium promoted the translocation of ARF6 and its exchange factor ARNO to the apical surface . Overexpression of a dominant-negative mutant of ARF6 inhibited Salmonella-induced PMN transmigration, which was due to a reduction in apical release of the PMN chemoattractant PEEC (pathogen-elicited epithelial chemoattractant), without affecting bacterial internalization . Furthermore, ARF6 and its effector phospholipase D (PLD) were both required for bacteria-induced translocation of protein kinase C (PKC) to membranes . These results describe a novel signal transduction pathway, in which Salmonella initiates an ARF6- and PLD-dependent lipid signaling cascade that, in turn, directs activation of PKC, release of PEEC, and subsequent transepithelial PMN movement.

PDA J Pharm Sci Technol, 2001 Sep-Oct, 55(5), 286 - 9
PCR detection of Salmonella typhimurium in pharmaceutical raw materials and products contaminated with a mixed bacterial culture using the BAX system; Jimenez L et al.; The BAX system, a PCR-based assay, was evaluated for detecting Salmonella typhimurium in pharmaceutical raw materials and products contaminated with mixed bacterial cultures of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhimurium . Artificially contaminated samples were preenriched in lactose broth with and without Tween 20 . After preenrichment, samples were analyzed by PCR and standard methods . Ten of 25 samples did not show presence of the specific Salmonella spp . 740-base pair DNA fragment . However, S . typhimurium was isolated and identified by standard methods from all 25 samples . To optimize S . typhimurium detection in PCR negative samples, lactose broth was replaced by buffered peptone water (BPW) as the preenrichment broth . When BPW was used, all 10 samples were PCR positive . BPW enrichments increased S . typhimurium growth resulting in rapid PCR detection . The presence of non-Salmonella bacteria influenced the performance of the PCR-based assay . Optimization of S . typhimurium PCR detection in mixed culture required the use of different preenrichment broths . However, the BAX system detected S . typhimurium within 27 hours while standard methods required 5-7 days.

Int J Food Microbiol, 2001 Sep 28, 69(3), 217 - 25
Inhibitory activity of honey against foodborne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power; Taormina PJ et al.; Antimicrobial activity of honey has been attributed to hydrogen peroxide, which is produced by naturally occurring glucose oxidase, and phenolic compounds, although lethality of and inhibition by these and other components against microorganisms vary greatly, depending on the floral source of nectar . This study was undertaken to compare honeys from six floral sources for their inhibitory activity against Escherichia coli O157:H7, Salmonella typhimurium, Shigella sonnei, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus . A disc assay revealed that development of zones of inhibition of growth depends on the type and concentration of honey, as well as the test pathogen . Growth of B . cereus was least affected . The inhibition of growth of S . sonnei, L . monocytogenes, and S . aureus in 25% solutions of honeys was reduced by treating solutions with catalase, indicating that hydrogen peroxide contributes to antimicrobial activity . Darker colored honeys were generally more inhibitory than light colored honeys . Darker honeys also contained higher antioxidant power . Since antimicrobial activity of the darker colored test honeys was not eliminated by catalase treatment, non-peroxide components such as antioxidants may contribute to controlling the growth of some foodborne pathogens . The antibacterial properties of honeys containing hydrogen peroxide and characterized by a range of antioxidant power need to be validated using model food systems.

Mutat Res, 2001 Nov 1, 483(1-2), 35 - 41
Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase; Fujita K et al.; The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium (S . typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR) . The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated . Eleven strains of S . typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed . Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined . Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result . Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP . These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1 . N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity . With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased . CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA . Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.

Reprod Fertil Dev, 1998, 10(3), 225 - 31
Construction and immunological assessment of Salmonella typhimurium expressing fox sperm LDH-C4; Bird P et al.; This study examined immune responses of foxes to oral doses of recombinant Salmonella typhimurium expressing fox sperm-specific lactate dehydrogenase (fLDH) . The cDNA for fLDH was cloned into the expression plasmid pKK233.2 (pKKfLDH) . Salmonella typhimurium aroA- (SL3261) was transformed with either the pKK233.2 plasmid alone (SpKK) or the pKKfLDH construct (SpKfLDH) . The fLDH expressed by SpKfLDH retained enzymatic activity and was recognized by human LDH-C4-specific antibody . Male European red foxes (Vulpes vulpes) were given an initial oral dose of 1 x 10(11) cfu of either SpKK (control, n = 3) or SpKfLDH (test, n = 6), followed four weeks later with a further dose of 1 x 10(11) cfu . Antibodies to Salmonella lipopolysaccharide (LPS-04) and fLDH were measured in plasma and saliva for eight consecutive weeks after the initial doses . Both LPS-04 IgG- and IgA-specific antibodies as well as fLDH-specific IgG antibodies were detected in plasma and saliva . However, there was a marked fLDH-specific IgA response in saliva consistent with induction of the common mucosal immune system . The antibody measurements demonstrated the feasibility of using recombinant Salmonella as an oral vaccine to elicit gamete antigen-specific mucosal immune responses in foxes.

Int J Parasitol, 2001 Nov, 31(13), 1441 - 9
Immunisation with Salmonella typhimurium-delivered glyceraldehyde-3-phosphate dehydrogenase protects mice against challenge infection with Echinococcus multilocularis eggs; Muller-Schollenberger V et al.; Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium . The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E . multilocularis eggs was evaluated . Oral or intraperitoneal immunisation with live attenuated S . typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E . multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8% . The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E . multilocularis . By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E . multilocularis antibodies were detectable in the sera, immunisation with E . coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E . multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections . These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.

J Immunol, 2001 Oct 15, 167(8), 4560 - 5
A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice; Xiang R et al.; A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells . Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2) . These conclusions are supported by four lines of evidence . First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2 . Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1 . Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12 . Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69 . Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.

J Endotoxin Res, 2001, 7(3), 211 - 7
Porins from Salmonella typhimurium accelerate human blood coagulation in vitro by selective stimulation of thrombin activity: implications in septic shock DIC pathogenesis; Di Micco B et al.; The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium, on human blood coagulation was investigated . It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro . Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin . A possible binding of porins with thrombin has been suggested to be the basis of this effect . The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.

Ceska Slov Farm, 2001 Sep, 50(5), 238 - 42
{Cytotoxic and genotoxic activity of certain preservative agents in cosmetics}; Jantova S et al.; Cytotoxic effects of the preservative compounds for cosmetics JMAC TD, Bronopol, CA 24, and Euxyl K100 were studied . Bronopol demonstrated the highest cytotoxic effect on the proliferation of V79 and VH10 fibroblast cell lines--the IC100 values being 10 mg/l during the whole experiment . The preservatives CA 24 and Euxyl K100 showed 4-times and 5-times smaller cytotoxic activity than Bronopol IC100 = 42 or 50.3 mg/l) . The preservative compounds on silver chloride ions JMAC TD manifested the lowest cytotoxicity of the preservatives tested (IC100 = 150 mg/l); 15-times smaller than Bronopol, 3.5-times smaller than CA 24 and 3-times smaller than Euxyl K100 . The biocide JMAC TD did not exhibit mutagenic effects on the bacteria Salmonella typhimurium TA 98 and TA 100.

Microbiology, 2001 Oct, 147(Pt 10), 2705 - 15
Genomic analysis and growth-phase-dependent regulation of the SEF14 fimbriae of Salmonella enterica serovar Enteritidis; Edwards RA et al.; Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe . Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars . SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently . Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes . Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location . Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon . The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes . Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase . The regulation was coordinated by the RpoS sigma factor.

J Mol Biol, 2001 Sep 28, 312(4), 807 - 21
Unusual molecular architecture of the Yersinia pestis cytotoxin YopM: a leucine-rich repeat protein with the shortest repeating unit; Evdokimov AG et al.; Many Gram-negative bacterial pathogens employ a contact-dependent (type III) secretion system to deliver effector proteins into the cytosol of animal or plant cells . Collectively, these effectors enable the bacteria to evade the immune response of the infected organism by modulating host-cell functions . YopM, a member of the leucine-rich repeat protein superfamily, is an effector produced by the bubonic plague bacterium, Yersinia pestis, that is essential for virulence . Here, we report crystal structures of YopM at 2.4 and 2.1 A resolution . Among all leucine-rich repeat family members whose atomic coordinates have been reported, the repeating unit of YopM has the least canonical secondary structure . In both crystals, four YopM monomers form a hollow cylinder with an inner diameter of 35 A . The domain that targets YopM for translocation into eukaryotic cells adopts a well-ordered, alpha-helical conformation that packs tightly against the proximal leucine-rich repeat module . A similar alpha-helical domain can be identified in virulence-associated leucine-rich repeat proteins produced by Salmonella typhimurium and Shigella flexneri, and in the conceptual translation products of several open reading frames in Y . pestis.

Can J Microbiol, 2001 Aug, 47(8), 777 - 81
Effects of antecedent fermentative and respiratory growth on the detection of chloramine-stressed Escherichia coil and Salmonella typhimurium; Thunberg RL et al.; In vitro laboratory studies were performed to assess the effects of antecedent growth conditions on the recovery of Escherichia coli ATCC 25922 and Salmonella typhimurium ATCC 14028 following chloramine disinfection . Six- and 18-h cultures of each organism were grown under aerobic, fermentative, and nitrate-reducing conditions prior to disinfection . At predetermined time intervals during a 10-min exposure to chloramine, survivors were surface plated on nonselective recovery media to determine C(n)t values . It was observed that nitrate-reducing growth predisposed the test organisms towards an increased sensitivity to chloramine stress over cells grown under fermentation or aerobic conditions (p < 0.01).

Can J Microbiol, 2001 Aug, 47(8), 711 - 21
Isolation and characterization of a chromosomally encoded disulphide oxidoreductase from Salmonella enterica serovar Typhimurium; Turcot I et al.; In this study, the chromosomally encoded disulphide oxidoreductase dsbA from Salmonella typhimurium was cloned and characterized . A survey of a number of serovars of Salmonella subspecies I showed that dsbA is highly conserved in most, but not all members of this subclass of Salmonella species . Using motility, beta-galactosidase, and alkaline phosphatase assays as indirect indicators of disulphide oxidoreductase activity, we demonstrated that DsbA from S . typhimurium LT2 can only partially complement an Escherichia coli dsbA-null strain . This is surprising considering the high degree of conservation between these two DsbA proteins (87% amino acid identity) . To determine the contribution of DsbA to the proper folding and assembly of proteins of S . typhimurium, deletion mutants were created in the avirulent strain LT2 and in the virulent strain SL1344 . These null alleles were constructed by partial deletion of the dsbA-coding region and then insertion of an antibiotic resistance marker in the gene . Mutants no longer expressing a functional disulphide oxidoreductase exhibit pleitropic effects, including an increase in colony mucoidy, a dramatic decrease in motility, and an increased susceptibility to the cationic peptide protamine sulphate . The disruption of disulphide bond formation was also shown to specifically affect the stability of several proteins secreted into the extracellular environment.

Cancer Gene Ther, 2001 Aug, 8(8), 599 - 611
Gene delivery by attenuated Salmonella typhimurium: comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination; Weth R et al.; Using the murine B16F1 melanoma, we compared a CTL- versus helper T cell (TH)-directed vaccination approach . Mice were either orally vaccinated with attenuated Salmonella typhimurium (SL) or subcutaneously with dendritic cells (DCs) loaded with gp100 peptides predicted to bind to H2-Kb/H2-Db molecules . SL were transformed with the murine gp100 cDNA (SL-gp100) or with a fusion construct of gp100 and a fragment of invariant chain cDNA (SL-gp100/Ii) . Transcription of these genes in vivo has been readily observed in monocytes and DC . Retardation of B16F1 growth was more efficiently achieved by vaccination with SL-gp100 than with DC . Vaccination with SL-gp100/Ii aiming at preferential presentation by MHC II molecules provided some further improvement due to a stronger expansion of TH and CTL . The importance of help was further sustained by a prolongation of the survival time when mice concomitantly received IL2 . Notably, prophylactic, compared to therapeutic, vaccination had no additional impact on survival time/rate . This was due to a striking decrease in frequencies of gp100-specific TH, CTL, and cytokine-expressing cells during tumor growth . Thus, the efficacy of vaccination was limited by tumor-induced immunosuppression . Our data demonstrate the oral route of vaccination via Salmonella as a most convenient transfer regimen and confirm the superiority of protocols aiming at preferential activation of TH.

Boll Chim Farm, 2001 Jul-Aug, 140(4), 238 - 42
Synthesis and antibacterial activity of pyridyl thioureas and arylthiosemicarbazones; Kumar KS et al.; {N-(2-pyridyl)-N'-(4-(un) substituted} thioureas and (substitutedaryl)thiosemicarbazones were synthesised and evaluated for their antibacterial activity . All aryl thiosemicarbazones showed good activity against Aeromonas hydrophilia and Salmonella typhimurium . But none of the pyridyl thioureas showed any prominent activity against tested bacteria.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 401 - 3
Improvement of an invA-based PCR for the specific detection of Salmonella typhimurium in organs of pigs; Scholz HC et al.; The aim of this study was to investigate the suitability of the invA-based polymerase chain reaction (PCR) assay for the specific detection of Salmonella in organs of experimentally infected pigs and to compare these results to classical bacterial culture . While the PCR conditions specified in the "Deutsche Industrie Norm", DIN 10135 (section 35 LMBG, 1999), cutle based on the publication of Rahn et al . 1992, revealed various unspecific amplification products, modifications of the PCR conditions allowed the specific amplification of the invA fragment from inner organs . The modified PCR assay correlates exactly with cultivation results (as required by DIN Norm 6579) and enables the detection of Salmonella within 48 hours with equal sensitivity compared to routine cultivation.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 393 - 6
Feasibility of fluorescent detection of pathogens on pork carcasses; Fravalo P et al.; The direct immunofluorescent detection of pathogens on pork skin is evaluated . Calibrate contamination of pork skin with Salmonella Typhimurium (ST) and Listeria monocytogenes (Lm) is developed in 2 h at 4 degrees C . Then a specific indirect immunofluorescent staining protocol is optimized in order to obtain specific and intensive signals able to be detected by electronic cameras (deported microscopy) . Despite the individual staining of ST and Lm is possible on pork skin and is specific and bright, the deported microscopy failed to detect these particles . After respectively 3 and 6 h, we obtain micro-colonies of ST and Lm . Due to the limited power of the video camera used, only the microscope permits the detection on the skin . However, our work gives standard conditions to mime the pathogens contamination and staining directly on a biological matrix such as pork skin . This work is a first step in the development of direct and rapid detection of pathogens on biological matrix.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 382 - 4
Experimental rapid infection in market swine following exposure to a Salmonella contaminated environment; Hurd HS et al.; The objective of these experiments was to evaluate the possibility of swine becoming infected with Salmonella Typhimurium after a short time interval in a contaminated environment . Two experiments were conducted . Experiment 1 consisted of five trials with eight market weight swine . Pigs were necropsied at 2 (n = 10), 3 (n = 10) and 6 (n = 5) hours after continuous exposure to an environment contaminated with feces shed by swine intranasally inoculated with nalidixic acid-resistant Salmonella Typhimurium (chi 4232) . In Experiment 2, pigs were necropsied after 30 minutes (n = 6), 60 minutes (n = 6), 2 hours (n = 6), and 6 hours (n = 3) . In addition, control animals with no exposure were also necropsied in both experiments . At necropsy, the superficial inguinal, ileocecal, and mandibular lymph nodes, as well as cecal contents, distal ileum portion, and feces were evaluated . All samples were cultured for the presence of the nalidixic acid-resistant Salmonella . Feces deposited on the floor by intranasally inoculated swine were mixed with water to form slurry with a resulting load of 10(3)-10(5) Salmonella Typhimurium CFU per gram . In Experiment 1, 80% percent of animals with a 2-hour, 60% of animals with a 3-hour, and 100% of animals with a 6-hour exposure to this slurry had at least one sample test positive for the marked Salmonella Typhimurium strain . In Experiment 2, 50% of the 30 minute, 50% of the 60 minute, and 33% of the 2-hour exposed pigs had at least one sample test positive . These experiments show that market swine can become infected during routine resting or holding periods when exposed to relatively low levels (10(3) CFU) of Salmonella in the simulated pre-slaughter environment, and that exposure times as short as 30 minutes are sufficient to produce contaminated gastrointestinal tracts . They also demonstrate the high risk of holding pigs longer than six hours . Intervention at this step in the swine production process may have a significant impact on the safety of pork products.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 378 - 81
A bioreactor system to study survival of Salmonella Typhimurium in pig gut content; Naughton PJ et al.; The batch culture system included six bioreactors . Three bioreactors containing stomach slurry were maintained at pH 4.5 and 6 respectively . Bioreactors containing small intestine slurry were maintained at pH 5.6 and 7 respectively . The bioreactors were inoculated with 10 ml of viable Salmonella . The bioreactors were maintained for 6 hours . Samples of 10 ml were taken at 0 time and at 1, 2, 4 and 6 hours . The samples were analysed for the presence of Salmonella and SCFA . In the stomach samples Salmonella numbers increased at pH 6 but fell at pH 4 . In the small intestine sample Salmonella numbers increased at pH 6 and 7 . In terms of SCFA production, in the stomach, with samples at pH 6 there was little change in the amounts of lactate, succinate and formate to that detected at 0 time, however levels of acetate did increase slightly . In the small intestine samples levels of succinate and formate increased slightly up to 4 hours, levels of acetate increased significantly from 0 to 6 hours . In terms of the specific growth rates of the individual strains, both strains grew at pH 6 in the stomach content and to a greater extent in the small intestinal content . A bactericidal effect was observed at pH 4 in the stomach content while neither killing nor growth occurred at pH 5 either in the stomach or the small intestine content . Both strains grew well in the small intestine content at pH 7, showing generation times of up to 24 min.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 366 - 9
Assessment of the human risk associated with use of pork with possible presence of Salmonella typhimurium DT104 for dry-cured sausages; Alban L et al.; We examined whether pork with suspected content of Salmonella Typhimurium DT104 (DT104) could be used for production of dry-cured sausages without jeopardizing consumer safety . The results of the risk assessment showed, that if Salmonella is present in raw pork, it is usually in low numbers . Additionally, during processing, an eventual presence of Salmonella will be reduced with at least two log units . The simulations showed that only 1-2 DT104 would be present in dry-cured sausages made by Danish pork, and this extremely seldom . Likewise, up to 4 DT104 would be present in dry-cured sausages made by foreign pork . It is not clear whether these low numbers of DT104 are capable of producing disease at all . However, if higher numbers are present, disease might occur . Therefore, we set up a monitoring and managing program, including a list with demands to processing in order to achieve minimum two-log reduction of any DT104 bacteria . The suggested scheme implies a far better and more systematic monitoring than the current system, ensuring the consumer a higher degree of food safety.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 363 - 5
Effect of separate transport, lairage, and slaughter on occurrence of Salmonella typhimurium on slaughter carcasses; Boes J et al.; The study aimed to reduce cross-contamination between finishers from Salmonella-positive and Salmonella-negative herds during transport, lairage, and slaughter, thereby reducing the prevalence of Salmonella Typhimurium on slaughter carcasses . In Phase 1 of the study, pigs from Salmonella-negative herds were kept in lairage for 2-4 hours either in clean pens (intervention group) or pens contaminated with Salmonella-infected faeces (control group) . All pigs were slaughtered on the same slaughterline, and carcass swabs 24 hours after slaughter revealed a low degree of cross-contamination in the pens: there was no difference in Salmonella-positive carcasses between intervention (1.7%) and control groups (0.8%) . In Phase 2, control pigs from Salmonella-negative herds were mixed with pigs from Salmonella-positive herds during lairage for 2-4 hours, while the intervention group still consisted of pigs from Salmonella-negative herds . All pigs were slaughtered on the same line: first intervention, then control . Carcass swabs taken 24 hours after slaughter failed to show a reduction in Salmonella-positive carcasses in the intervention group (4.5%) compared with the originally Salmonella-negative pigs in the control group (3.6%) . In pigs from Salmonella-positive herds the occurrence of Salmonella was substantially higher at 10.4% . When the results were corrected for 6 carcass samples found positive with S . Heidelberg on the same day, which was attributed to a transient hygiene failure, only 2.2% of the carcasses in the intervention group were Salmonella-positive . We conclude that even though cross-contamination occurs in the abattoir pens, its importance on the slaughter line may be greater . However, the final results of this study should be awaited to conclude whether separate slaughter of pigs from Salmonella-positive and Salmonella-negative herds should be recommended.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 360 - 2
Prevalence of Salmonella serotypes on pig carcasses from high- and low-risk herds slaughtered in three abattoirs; Quirke AM et al.; The aim of this study was to compare the prevalence of Salmonella serotypes at two different sites on pig carcasses from herds classified as high-risk or low-risk and to elucidate the relationship between carcass contamination levels and serological status . Caecal samples and carcass surface swabs were cultured for Salmonella from a total of 210 pigs from low risk herds (< 19% of pigs in herd Salmonella seropositive) and 209 pigs from high risk herds (> 32% of pigs in herd Salmonella seropositive) in three abattoirs . Meat juice samples were collected for analysis by ELISA . The prevalence of Salmonella in the caecal contents of "low-risk" pigs was 10%, which was significantly lower than the 19% prevalence in "high-risk" pigs (p < 0.01) . The corresponding figures for skin samples collected immediately post-evisceration were 2% and 12% . The predominant Salmonella serotype in the caecal contents of both the low-risk and high-risk pigs was Salmonella Typhimurium . Salmonella Kentucky and Salmonella Derby were the most frequent isolates from the carcass surface swabs of low- and high-risk pigs respectively . There was a positive association between seropositivity of pigs from high-risk herds and caecal carriage (p < 0.05) . Results showed that herd categorisation based on serological results was useful in predicting Salmonella isolation rates from caecal samples and surface swabs of slaughtered pigs.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 350 - 2
Bacteriological and serological examination and risk factor analysis of Salmonella occurrence in sow herds, including risk factors for high Salmonella seroprevalence in receiver finishing herds; Kranker S et al.; A strong association between the seroprevalence in sows and the occurrence of Salmonella Typhimurium among weaners has been shown . As shown several times for finisher herds, the risk-factors, ready mixed pelleted feed and health status also apply to sow herds . Risk factors on the sow level, for high seroprevalence in finishers have been quantified . It has been shown, that isolating Salmonella in weaners is a risk factor for high seroprevalence in finishers . Feed factors; ready-mixed pelleted feed for both sows and finishers, dry feed for sows, have been shown to have a significant effect on high seroprevalence, monitored by meat juice samples at slaughter . The etiological fraction of ready-mixed pelleted feed for sows and for finishers is of the same magnitude, indicating that intervention on the sow level could prove to contribute considerably to the effect of intervention programs.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 342 - 5
Investigation of the efficacy of a genetically-stabile live Salmonella typhimurium vaccine for use in swine; Springer S et al.; Hybrid swine (Landrace x Pietrain) aged 3-4 weeks were immunized twice at an interval of 3 weeks solely by the oral route and by the oral/parenteral route to evaluate the efficacy of a live S . Typhimurium vaccine . In each experiment a control group was run without vaccination . The animals were challenged at the age of 8-10 weeks by oral test infection with a labelled S . Typhimurium DT 104 strain . An ELISA was used to establish the presence of antibodies to S . Typhimurium in serum samples, coupled with clinical investigation . The presence of the challenge strain in the ileal and caecal mucosa and in the ileocolic lymph nodes was investigated quantitatively using the Koch plating method to determine the degree of colonization of those organs at the time of slaughter . The clinical course of disease was used to assess the success of vaccination . However, it was not possible to trigger, in a reproducible manner, clinical signs of disease in unvaccinated animals through infection . The vaccinated animals had a significantly lower (p < 0.05) colonization of the ileal and caecal mucosa than the unvaccinated animals . This was also seen to a lesser degree for the ileocolic lymph nodes.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 323 - 6
A new Salmonella surveillance and control programme in Danish pig herds and slaughterhouses; Nielsen B et al.; The Danish Salmonella Surveillance and Control Programme for pigs operates at all stages of the production chain and has been applied nationally since 1995 . Due to the program the level of Salmonella in Danish pork has declined from 3.5% in 1993 to 0.7% in the year 2000 . Simultaneously, the number of human cases with salmonellosis due to pork has declined from approximately 1,144 in 1993 to 166 in 2000 . In year 2001, the programme has been improved at a number of stages . A new classification scheme for the serological surveillance of finisher herds has been developed . The individual test cut-off in the mix-ELISA has been reduced to 20 OD% . Only herds producing more than 200 finishers/year are sampled . Based on the serological result from the last 3 months a new weighted salmonella index is calculated: The Danish Bacon and Meat Council has agreed on a new stricter penalty system . Level 2 and 3 herds get a penalty of 2% and 4% of the value per slaughter carcass, respectively . A new method of Salmonella testing on carcasses has been introduced; 5 carcasses per slaughter day are swabbed at 3 defined areas at 100 cm2 for each sample . This method is more sensitive than the one used previously . Herds infected with multiresistant Salmonella Typhimurium DT104 have to follow special restrictions . These include a requirement for a herd intervention plan, restriction on livestock trade, and a requirement for special slurry handling . Carcasses from DT 104 herds must be heat-treated or decontaminated with hot water.

Avian Dis, 2001 Jul-Sep, 45(3), 631 - 8
Combination of competitive exclusion and immunization with an attenuated live Salmonella vaccine strain in chickens; Methner U et al.; To use the advantages of both the competitive exclusion (CE) technique and immunization with a live Salmonella vaccine, the combination of these methods was studied . Specific-pathogen-free chickens were pretreated by combined or single administration of a CE culture and a commercial live Salmonella typhimurium vaccine on days 1 and 2 of life and challenged with Salmonella typhimurium on day 3 to study the exclusion effect by both the CE preparation and the Salmonella vaccine . The exclusion effect by the CE culture combined with the immunologic effect by the live vaccine was studied after challenge of the birds on day 43 of age . The number of challenge organisms in ceca was used to evaluate the efficacy of the pretreatment . The protective exclusion effect of the CE culture was substantial in very young chicks and still detectable in 6-wk-old birds . The attenuated Salmonella typhimurium vaccine produced only an initially occurring exclusion effect . Because the exclusion effect of the CE culture was considerably stronger than the exclusion effect of the attenuated Salmonella typhimurium vaccine, the combination of both did not result in an additive protective effect . In order to exploit the exclusion potential between Salmonella strains and to attain an additive exclusion effect by a CE culture and a vaccine strain, live Salmonella vaccines are needed that are sufficiently attenuated without affecting genes essential for colonization exclusion of other Salmonella organisms . In 6-wk-old birds, the exclusion effect by the CE culture combined with the immunologic effect by the live Salmonella vaccine resulted in a degree of protection considerably beyond that generated by the exclusive use of the two methods . The administration of the live Salmonella vaccine strain prior to or simultaneously with the CE culture revealed the best protective effect because such combinations ensure an adequate persistence of the vaccine strain as prerequisite for the expression of an exclusion effect in very young chicks and the development of a strong immune response affording protection in older birds.

Vaccine, 2001 Oct 12, 20(1-2), 140 - 7
A crucial role of macrophages in the immune responses to oral DNA vaccination against hepatitis B virus in a murine model; Zheng B et al.; In the previous study, we had shown that live oral vaccination with Salmonella typhimurium delivering plasmid DNA-HBsAg (oral DNA vaccine) evoked a vigorous T cell response and a weak antibody response with predominant subclass IgG2a in mice, suggesting a significant involvement by professional antigen presenting cells (APC) . In the present study, this possibility was further studied by infecting peritoneal macrophages (MPhi) with the oral DNA vaccine . Although, the infected cells could only express low level of the viral antigen, they nevertheless stimulated a vigorous lymphocyte proliferation of splenocytes from immune mice, induced these cells to elaborate interferon-gamma and stimulated development of HBV-specific cytotoxicity against target cells expressing the viral antigen . Infusion of the infected MPhi evoked a vigorous Th 1 and cytotoxic T lymphocyte (CTL) response and a weak IgG2a antibody response in mice, which was essentially the same as response to the oral DNA vaccine . In contrast, recombinant protein vaccine evoked a vigorous IgG1 antibody response and a weak T cell response . While, given intramuscularly, the same plasmid DNA vaccine as that contained in the oral DNA vaccine evoked a vigorous IgG1 antibody response and a moderate T cell response in these animals . It was concluded that professional APC may orchestrate the immune response to live oral DNA vaccine and it was of interest to note that different vaccine formulation and routes of administration evoke distinct immune response to HBV.

Biochem J, 2001 Oct 1, 359(Pt 1), 17 - 22
Lipid modification of the Cu,Zn superoxide dismutase from Mycobacterium tuberculosis; D'orazio M et al.; The leader sequence of Mycobacterium tuberculosis Cu,Zn superoxide dismutase (Cu,ZnSOD) contains a prokaryotic membrane lipoprotein attachment site . In the present study, we have found that the protein, which exhibits detectable SOD activity, is lipid-modified and associated with the bacterial membrane when expressed either in M . tuberculosis or in Escherichia coli . These results provide the first demonstration of lipid modification of a Cu,ZnSOD . An analysis of the sodC genes present in available databases indicates that the same signal for lipid modification is also present in the sodC gene products from other mycobacteria and Gram-positive bacteria and, uniquely, in two distinct sodC gene products from the Gram-negative bacterium Salmonella typhimurium . Evidence is also provided for an up-regulation of M . tuberculosis sodC in response to phagocytosis by human macrophages, suggesting that Cu,ZnSOD is involved in the mechanisms that facilitate mycobacterial intracellular growth.

J Food Prot, 2001 Sep, 64(9), 1435 - 8
Incidence of Salmonella in minced meat produced in a European Union-approved cutting plant; Stock K et al.; Contamination of minced meat with Salmonella is still considered a major problem in food hygiene . Therefore, in this study the Salmonella incidence in minced meat produced in a European Union-approved slaughtering and cutting plant was investigated in detail . Throughout 21 months, 297 pool samples (1,485 individual samples) of mixed minced meat (beef and pork) were examined according to Council Directive 94/65/EC and to ISO 6579 . Salmonellae were detected in 47 (15.8%) of the pool samples . After separation of the positive pools, 93 individual samples were determined to be Salmonella positive, representing 6.3% of the total 1,485 samples . Serotyping resulted in most isolates (69.6%) being identified as Salmonella Typhimurium . It was further shown that the incidence of Salmonella isolations varied during the year and that the isolation rate was higher on some days of the week compared with others.

Poult Sci, 2001 Sep, 80(9), 1293 - 8
Effects of tannic acid on cecal volatile fatty acids and susceptibility to Salmonella typhimurium colonization in broiler chicks; Kubena LF et al.; Young chickens are more susceptible to Salmonella colonization than older chickens that have developed resistance with age as native microflora become established . Elevated concentrations of cecal propionic acid and total volatile fatty acids (VFA) have been observed by many researchers to be indicators of establishment of anaerobic microflora and protection against Salmonella colonization of the ceca . Disruption of the native microflora or competitive exclusion (CE) cultures by components of diets, such as tannic acid (TA), could alter the concentrations of propionic acid and total VFA and possibly affect Salmonella colonization . Two experiments were conducted using day-of-hatch, mixed-sex broiler chicks to evaluate the effects of TA on cecal VFA and the susceptibility to Salmonella colonization . All chicks in both experiments were challenged orally with 10(4) cfu of Salmonella typhimurium (ST) on Day 3 (Experiment 1) or Day 4 (Experiment 2) . One-half of the chicks were orally gavaged on the day of hatch with a CE culture (PREEMPT) and were fed diets containing 0, 0.75, or 1.5% TA for up to 12 d of age . Chicks were maintained in batteries in separate rooms for the experimental period . There were some alterations in concentrations of cecal propionic acid or total VFA in chicks fed diets containing 0.75 or 1.5% TA in non CE-treated chicks and in CE-treated chicks . No significant differences were observed for numbers of Salmonella cecal culture-positive chicks or in the numbers of ST in the cecal contents due to dietary content of TA . With minor exceptions, the chicks treated with the CE culture had higher cecal concentrations of propionic acid and were less susceptible to Salmonella colonization than the non CE-treated chicks . Further research is necessary to determine the biological significance of these changes.

J Vet Med Sci, 2001 Aug, 63(8), 943 - 4
An occurrence of salmonella infection in cranes at the Izumi Plains, Japan; Maeda Y et al.; Ten thousand or more cranes migrate from Siberia and stay at the Izumi Plains, in the northern part of Kagoshima prefecture, Japan, every winter season . Four hundred and twenty samples of cranes feces were obtained 1995 to 1997 and investigated for Salmonella . As a result, twenty-nine of Salmonella strains were isolated . All isolates were determined to be identical, Salmonella Typhimurium (04:i: 1,2) . since all of them indicated the same patterns of plasmid profiling and antibiotic sensitive spectrums . The isolates showed a high pathogenicity to chicken, and most of them were isolated in the latter half of the winter season; therefore the cranes were infected with the isolates during the winter season.

Am J Physiol Gastrointest Liver Physiol, 2001 Oct, 281(4), G890 - 8
EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption; Savkovic SD et al.; Enteropathogenic Escherichia coli (EPEC) alters many functions of the host intestinal epithelia . Inflammation is initiated by activation of nuclear factor (NF)-kappaB, and paracellular permeability is enhanced via a Ca2+- and myosin light-chain kinase (MLCK)-dependent pathway . The aims of this study were to identify signaling pathways by which EPEC triggers inflammation and to determine whether these pathways parallel or diverge from those that alter permeability . EPEC-induced phosphorylation and degradation of the primary inhibitor of NF-kappaB (IkappaBalpha) were tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta independent . In contrast to Salmonella typhimurium, EPEC-stimulated IkappaBalpha degradation and IL-8 expression did not require Ca2+ . Instead, extracellular signal-regulated kinase (ERK)-1/2 was significantly and rapidly activated . ERK1/2 inhibitors attenuated IkappaBalpha degradation and IL-8 expression . Although ERK1/2 can activate MLCK, its inhibition had no impact on EPEC disruption of the tight junction barrier . In conclusion, EPEC-induced inflammation 1) is TNF-alpha and IL-1beta receptor independent, 2) utilizes pathways differently from S . typhimurium, 3) requires ERK1/2, and 4) employs signals that are distinct from those that alter permeability . This is the first time that EPEC-activated signaling cascades have been linked to independent functional consequences.

Traffic, 2001 Sep, 2(9), 643 - 53
Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments; Brumell JH et al.; Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection . A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs) . In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers . The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter . Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules . Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs . The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern . Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation . Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes . Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event.

Vet Rec, 2001 Aug 25, 149(8), 227 - 32
Observations on the distribution and control of Salmonella species in two integrated broiler companies; Davies R et al.; The effectiveness of cleaning and disinfecting broiler farms and the persistence of Salmonella species in two integrated broiler companies was investigated for two years . Both companies used a cleaning and disinfection regime which included the application of a spray of phenolic disinfectant followed by fogging with formaldehyde solution, and this was highly effective in preventing carry-over of infection in the broiler houses . The disinfection of service areas and areas outside the houses was less effective but it had no influence on the Salmonella status of later flocks . Both companies had persistent problems with the contamination of pellet cooling systems in their feedmills with Salmonella 4, 12:d:- in company A, and with Salmonella binza and Salmonella ohio in company B . The hatcher incubators of both companies were also persistently contaminated with Salmonella livingstone and Salmonella thomasville in company A and with Salmonella senftenberg in company B . At both companies sites Salmonella enteritidis and Salmonella typhimurium Tr104 were also isolated occasionally from various locations.

Cell Microbiol, 2001 Sep, 3(9), 587 - 97
Intracellular replication of Salmonella typhimurium strains in specific subsets of splenic macrophages in vivo; Salcedo SP et al.; We used flow cytometry and confocal immunofluorescence microscopy to study the localization of Salmonella typhimurium in spleens of infected mice . Animals were inoculated intragastrically or intraperitoneally with S . typhimurium strains, constitutively expressing green fluorescent protein . Independently of the route of inoculation, most bacteria were found in intracellular locations 3 days after inoculation . Using a panel of antibodies that bound to cells of different lineages, including mononuclear phagocyte subsets, we have shown that the vast majority of S . typhimurium bacteria reside within macrophages . Bacteria were located in red pulp and marginal zone macrophages, but very few were found in the marginal metallophilic macrophage population . We have demonstrated that the Salmonella SPI-2 type III secretion system is required for replication within splenic macrophages, and that sifA(-) mutant bacteria are found within the cytosol of these cells . These results confirm that SifA and SPI-2 are involved in maintenance of the vacuolar membrane and intracellular replication in vivo.

Biochem Pharmacol, 2001 Sep 15, 62(6), 685 - 92
Comparison of the mutant frequencies and mutation spectra of three non-genotoxic carcinogens, oxazepam, phenobarbital, and Wyeth 14,643, at the lambdacII locus in Big Blue transgenic mice; Singh VK et al.; Oxazepam (OX), a widely used benzodiazepine anxiolytic, phenobarbital (PHE), a drug used for convulsive disorders, and Wyeth 14,643 (WY; {4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio}acetic acid), a hypolipidemic agent, are all hepatocarcinogenic in B6C3F1 mice . They have been classified as "non-genotoxic" carcinogens since they are non-DNA reactive in in vitro assays and are either negative or weakly positive in Salmonella typhimurium (Ames assay) . Male B6C3F1 Big Blue(R) transgenic mice were fed 2500 ppm of OX or PHE or 500 ppm of WY in their diet, while a control group of mice received diet alone for 180 days . The mutant frequency (MF) of cII in the control mice, after correction for clonality, was 6.2 +/- 2.8 x 10(-5) . The MF values for mice fed OX, PHE, and WY were 10.0 +/- 3.6 x 10(-5) (P < 0.05), 7.9 +/- 1.3 x 10(-5) (P = 0.1) and 17.4 +/- 4.2 x 10(-5) (P < 0.01), respectively . The mutation spectrum (MS) at cII from the PHE-fed mice was significantly different (P < 0.05) from that of the control mice even though the MF was not, whereas the MS spectra of mice fed OX (P = 0.4) and WY (P = 0.7) were not significantly different . The PHE-derived spectrum differed from the spontaneous spectrum in the lower occurrence of G:C>C:G transversions (17 vs 1.6%) and the higher incidence of A:T>T:A transversions (3.4 vs 9.5%) . Prior to correction for clonal expansion, each treated group exhibited a high incidence of frameshift mutations at the homopolymeric run of guanines at bp 179-184 (OX 21%, PHE 21%, WY 16% of the total mutations); this was not the case with the control group (6%) . Even after clonal correction, more than 10% of the mutations were frameshifts in the treated mice, while 5% were frameshifts in the control mice . Despite this hypersensitive region of the gene, our findings suggest that the cII locus is less sensitive than the lacI locus to mutation induction by non-DNA reactive carcinogens.

Mutat Res, 2001 Sep 20, 496(1-2), 83 - 8
The superiority of organically cultivated vegetables to general ones regarding antimutagenic activities; Ren H et al.; We found organically cultivated (OC) vegetables, using a water-soluble chitosan as a soil improvement agent and leaf surface spray, had much longer shelf life and better taste than that of generally cultivated (GC) vegetables . The purpose of this study is to determine the relative antimutagenic activity between OC and GC vegetables . Eleven OC vegetables were harvested in March and April in 1999 and 2000, and GC ones were supplied as a control from nearby farms on the same date . The former vegetables were planted on the field where no pesticide had been used for the last 3 years . Forward mutation test with Salmonella typhimurium TM677 and 8-azaguanine as a detection agent was used to determine the antimutagenic activity of juices prepared from OC and GC vegetables against authentic mutagens, such as 4-nitroquinoline oxide (4NQO), benzo(a)pyrene (BaP), and 3-amino-1-methyl-5H-pyrido{4,3-b}indole acetate (Trp-P-2) . This microbiological test is a convenient method to use for the food samples containing free histidine . Antimutagenic activity was evaluated by the difference of mutagenic activities between mutagenecity of authentic compounds and that observed upon incubation at 37 degrees C for 2h with each vegetable juice . OC Chinese cabbage, carrot, Welsh onion, and Qing-gen-cai suppressed 37-93% of the mutagenic activity of 4NQO, while the GC ones were held down to 11-65% . Against BaP, three species of OC vegetables showed 30-57% antimutagenecity, while GC ones did only 5-30% . Similarly, the OC spinach decreased the activity of Trp-P-2 to 78%, and the GC suppressed it by 49%.

Mutat Res, 2001 Sep 20, 496(1-2), 75 - 81
Catechins are not major components responsible for anti-genotoxic effects of tea extracts against nitroarenes; Ohe T et al.; The anti-genotoxic properties of tea leaf extracts were examined in a Salmonella umu-test . Seven non-fermented teas (green tea), one semi-fermented tea (oolong tea), two fermented teas (black tea and Chinese pu er tea) and two other teas were examined for their anti-genotoxic abilities and for their catechins contents . This was to study the relationship between catechins contents and anti-genotoxic activity of various tea leaf extracts . All types of tea extracts showed more potent suppressive effects against umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK 1002 induced by four nitroarenes (1-nitropyrene, 2-nitrofluorene, 3-nitrofluoranthene and a mixture of 1,6- and 1,8-dinitropyrene) rather than 4-NQO, AF-2 and MNNG . The anti-genotoxic effect of 12 tea leaf extracts on 1-NP, 2-NF, 3-NF and DNP decreased in the order: oolong tea (semi-fermented tea)>black tea (fermented tea)>sencha (non-fermented tea, an ordinary grade green tea)>tocyucya (other tea)>Chinese pu er tea (fermented tea) . The amount of catechins (EGC, C, EGCG, EC and ECG) in various teas in decreasing order was non-fermented tea>semi-fermented tea>fermented tea>other tea . A remarkable feature was the effectiveness of black tea and Chinese pu er tea in suppressing the genotoxicity induced by nitroarenes, in spite of the fact that these fermented teas do not have high catechins contents . Statistical analysis showed that no significant (P<0.01) correlation was found between the anti-genotoxicity of tea extracts against nitroarenes and the catechins contents in tea leaf extracts . In further experiment, fractionation of sencha extract by HPLC revealed that anti-genotoxicity of the peak fraction corresponding to catechins accounted for <10% of the total anti-genotoxic activity of sencha extract against for 1-nitropyrene . These results suggest that catechins are not major components responsible for the anti-genotoxic effects of tea leaf extracts against direct-acting nitroarenes.

Acta Astronaut, 1995 Aug, 36(3), 177 - 81
Separation of bacterial cells by free flow electrophoresis under microgravity: a result of the SpaceLab-Japan project on Space Shuttle flight STS-47; Akiba T et al.; We demonstrated free flow electrophoresis (FFE) of charged cells under microgravity, where gravitational effects are almost eliminated . Separation of a mixture of three bacterial strains (mutants of Salmonella typhimurium LT2) by FFE was conducted on NASA Space Shuttle flight STS-47 (September 1992) . The experiment was designed to differentiate three strains having different lipopolysaccharide core structures in the cell membrane . The results were compared to those of ground experiments, in order to examine whether or not FFE in a weightless environment provides distinct advantages . Smooth strain SL1027 and rough strain SL3749 migrated to two separated fractions . The quality (viability) and the yields of the separated samples were sufficient to show the advantage of microgravity . Another rough strain, SL1102, exhibited unexpected electrophoretic behavior, which prevented the complete resolution of the three strains . All the strains were recovered as viable cells after 8 days of flight . The present study suggests that electrophoretic separation of bacterial cells is much more effective under microgravity conditions with relatively good resolution in comparison with the ground operation.

Eur J Immunol, 2001 Sep, 31(9), 2529 - 38
Lethal Escherichia coli and Salmonella typhimurium endotoxemia is mediated through different pathways; Netea MG et al.; Despite the differences in the molecular structure between lipopolysaccharides (LPS) isolated from Escherichia coli, Klebsiella pneumoniae or Salmonella typhimurium, the potential differences in their biological effects in vivo have not been investigated . In the present study, TNF and LT double knock-out (TNF-/-LT-/-) mice were almost as susceptible as TNF+/+LT+/+ controls to S . typhimurium LPS, but they were significantly more resistant to lethal endotoxemia induced by E . coli or K . pneumoniae LPS . The effect was not due to endotoxin-associated proteins . In the knock-out mice, this difference in lethality was accompanied by decreased interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) production after challenge with E . coli LPS, whereas after S . typhimurium LPS more IL-1 and IFN-gamma were produced . In contrast, more IL-10 was produced after challenge of mice with E . coli LPS than with S . typhimurium LPS . The hypothesis that a combination of pro-inflammatory cytokines is responsible for the mortality after S . typhimurium LPS was suggested by experiments in mice deficient in IL-1beta-converting enzyme (ICE-/- mice) . ICE-/-mice, lacking mature IL-1beta and IL-18, but also defective in IFN-gamma and TNF production, were completely protected against both E . coli and S . typhimurium LPS . Experiments in Toll-like receptor (TLR)-4 defective mice suggested that the difference is not due to differential activation of TLR4 . In conclusion, TNF and LT play a central role in the lethality due to E . coli LPS, whereas the lethal effects of S . typhimurium LPS are mediated through mechanisms also involving other cytokines such as IFN-gamma, IL-1 and IL-18.

J Biol Chem, 2001 Nov 16, 276(46), 43132 - 44 Epub 2001 Sep 04.
Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm; Trent MS et al.; Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A . We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E . coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N . Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid . Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species {M - H}(-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41) . (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage . Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit . One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside . Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue . The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety . The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

J Biol Chem, 2001 Nov 16, 276(46), 43122 - 31 Epub 2001 Sep 04.
An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: induction on polymyxin-resistant mutants and role of a novel lipid-linked donor; Trent MS et al.; Attachment of the cationic sugar 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A is required for the maintenance of polymyxin resistance in Escherichia coli and Salmonella typhimurium . The enzymes that synthesize l-Ara4N and transfer it to lipid A have not been identified . We now report an inner membrane enzyme, expressed in polymyxin-resistant mutants, that adds one or two l-Ara4N moieties to lipid A or its immediate precursors . No soluble factors are required . A gene located near minute 51 on the S . typhimurium and E . coli chromosomes (previously termed orf5, pmrK, or yfbI) encodes the l-Ara4N transferase . The enzyme, renamed ArnT, consists of 548 amino acid residues in S . typhimurium with 12 possible membrane-spanning regions . ArnT displays distant similarity to yeast protein mannosyltransferases . ArnT adds two l-Ara4N units to lipid A precursors containing a Kdo disaccharide . However, as shown by mass spectrometry and NMR spectroscopy, it transfers only a single l-Ara4N residue to the 1-phosphate moiety of lipid IV(A), a precursor lacking Kdo . Proteins with full-length sequence similarity to ArnT are present in genomes of other bacteria thought to synthesize l-Ara4N-modified lipid A, including Pseudomonas aeruginosa and Yersinia pestis . As shown in the following article (Trent, M . S., Ribeiro, A . A., Doerrler, W . T., Lin, S., Cotter, R . J., and Raetz, C . R . H . (2001) J . Biol . Chem . 276, 43132-43144), ArnT utilizes the novel lipid undecaprenyl phosphate-alpha-l-Ara4N as its sugar donor, suggesting that l-Ara4N transfer to lipid A occurs on the periplasmic side of the inner membrane.

J Biol Chem, 2001 Nov 16, 276(46), 43111 - 21 Epub 2001 Sep 04.
Lipid A modifications in polymyxin-resistant Salmonella typhimurium: PMRA-dependent 4-amino-4-deoxy-L-arabinose, and phosphoethanolamine incorporation; Zhou Z et al.; Lipid A of Salmonella typhimurium can be resolved into multiple molecular species . Many of these substances are more polar than the predominant hexa-acylated lipid A 1,4'-bisphosphate of Escherichia coli K-12 . By using new isolation methods, we have purified six lipid A subtypes (St1 to St6) from wild type S . typhimurium . We demonstrate that these lipid A variants are covalently modified with one or two 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties . Each lipid A species with a defined set of polar modifications can be further derivatized with a palmitoyl moiety and/or a 2-hydroxymyristoyl residue in place of the secondary myristoyl chain at position 3' . The unexpected finding that St5 and St6 contain two l-Ara4N residues accounts for the anomalous structures of lipid A precursors seen in S . typhimurium mutants defective in 3-deoxy-d-manno-octulosonic acid biosynthesis in which only the 1-phosphate group is modified with the l-Ara4N moiety (Strain, S . M., Armitage, I . M., Anderson, L., Takayama, K., Quershi, N., and Raetz, C . R . H . (1985) J . Biol . Chem . 260, 16089-16098) . Phosphoethanolamine (pEtN)-modified lipid A species are much less abundant than l-Ara4N containing forms in wild type S . typhimurium grown in broth but accumulate to high levels when l-Ara4N synthesis is blocked in pmrA(C)pmrE(-) and pmrA(C)pmrF(-) mutants . Purification and analysis of selected compounds demonstrate that one or two pEtN moieties may be present . Our findings show that S . typhimurium contains versatile enzymes capable of modifying both the 1- and 4'-phosphates of lipid A with l-Ara4N and/or pEtN groups . PmrA null mutants of S . typhimurium produce lipid A species without any pEtN or l-Ara4N substituents . However, PmrA is not needed for the incorporation of 2-hydroxymyristate or palmitate.

Plasmid, 2001 Jul, 46(1), 65 - 70
The plasmid-stabilizing ytl2 protein coats DNA in a sequence-independent manner; Wong DK et al.; Plasmids carrying the ytl2 gene from the large resident plasmid pSLT of Salmonella typhimurium were stabilized >10(5)-fold (compared to control ytl2-free plasmids) in S . typhimurium cells . Purified Ytl2 protein was localized in the cell cytosol and bound to DNA in a sequence-independent manner to form a high-molecular-weight complex, suggesting cooperative binding to the DNA . A mutant ytl2 gene, with a modified C-terminus, did not mediate plasmid stabilization and the mutant Ytl2 protein did not bind cooperatively to DNA . In vivo, while a plasmid carrying the ytl2 gene was stabilized, another plasmid (lacking ytl2) coexisting in the same cell was not . This result suggests that the Ytl2 protein, newly synthesized in a transcription-translation complex, binds preferentially to DNA of the replicon which encodes it and that this binding initiates subsequent cooperative DNA coating by more Ytl2 molecules . Plasmid-encoded newly synthesized Ytl2 protein is thus unavailable to stabilize coresident plasmid DNA, which does not contain the ytl2 gene .

Chemosphere, 2001 Sep, 44(8), 1703 - 9
A study of 2,4,6-trinitrotoluene inhibition of benzo{a}pyrene uptake and activation in a microbial mutagenicity assay; Washburn KS et al.; A number of in vitro and in vivo studies have determined that binary and complex mixtures may interact to produce a toxicity that could not be predicted based on the individual chemicals . The present study was conducted with a binary mixture of model compounds to investigate possible interactions affecting their mutagenicity . The compounds included Benzo{a}pyrene (BAP), a polycyclic aromatic hydrocarbon that is an indirect-acting mutagen of great environmental concern, and 2,4,6-Trinitrotoluene (TNT), a nitro-aromatic compound that is a direct-acting mutagen frequently found as a soil contaminant at munitions sites . This study indicated that a binary mixture of BAP and TNT failed to induce the positive mutagenic response in Salmonella typhimurium strain TA98 characteristic of either compound alone . Spectrofluorometric analysis of BAP, and kinetic analyses of 3HBAP uptake in the presence or absence of TNT using TA98 cells that were treated or untreated with activated rat liver microsomes were performed . In cells preloaded with BAP, cellular BAP fluorescence was rapidly suppressed in the presence of TNT . Mass spectroscopy of BAP and TNT mixtures revealed a number of products, believed to be the result of complexation and nitration, that may account for the antagonistic action of TNT on BAP-induced mutagenicity in TA98 cells . Further, kinetic studies indicated that TNT inhibited the incorporation of BAP into cells.

Chemosphere, 2001 Sep, 44(8), 1673 - 83
The chemical and biogenotoxic characterization of organic xenobiotics in aquatic sediment materials 1 . The application and comparison of chemically non-specific and biogenotoxic methods; Picer M et al.; The aim of this work was to evaluate the Ames assay and mixed function oxidase (MFO)-Induct Test used in parallel with chemical group tests (ECD fingerprint and PAH estimation) for the characterization of the organic pollution of water sediment materials . Sediment materials were collected from "clean" and relatively heavily polluted locations in the Middle Adriatic Sea, and from some locations in continental Croatia polluted with wastewaters from different enterprises . Characterization of the organic extracts of the sediment materials investigated was performed chemically using UV spectrofluorometry for the determination polyaromatic hydrocarbons (PAH) and gas chromatography for the determination of volatile EC detector sensitive materials . Genotoxic analysis of the extracts was performed using the MFO-Induct Test and mutagenicity testing using the Standard Plate Incorporation Test as described by Maron and Ames with Salmonella typhimurium TA 98 . Measurement of the BaPMO enzyme activity in the livers of carp treated i.p . with total extracts of the sediment investigated confirmed that the methanol extracts generally contained more inducing matter than the petroleum ether extracts . Ames assay showed that for all the samples following the elimination of the sulfur, there was an increase in the number of revertants in comparison to the control number, which indicates that the samples contained mutagenic substances . The larger doses of extracts generally demonstrated cytotoxicity, as evidenced by a reduced number of spontaneous revertants in the SalmonellalMicrosome Test . Investigation of the correlation of the chemical parameters with the biological parameter showed that the induction of BaPMO exhibited a statistically significant correlation with the level of the ECD fingerprint of the petroleum ether sediment extract.

Mol Gen Mikrobiol Virusol, 2001, (3), 8 - 12
{Differential gene expression in culturable and non-culturable Salmonella typhimurium}; Boshnakov RKh et al.; Differential gene expression in culturable and non-culturable forms of Salmonella typhimurium was studied by the molecular display method . Six fragments of differentially expressed gene cDNA, depending on culturable or non-culturable state of the cultures, were isolated, cloned, and sequenced . Identification of corresponding S . typhimurium differentially expressed genes was carried out by comparing the sequences of cDNA fragments with the bacterial genome data base.

Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 2000, (118), 1 - 20
{Development of novel genotoxicity assays by genetic engineering methods}; Nohmi T; Novel genotoxicity assays have been developed to efficiently detect the genotoxicity of various environmental chemicals, and to evaluate the potential hazards to man . Salmonella typhimurium strains harboring plasmids carrying the genes encoding drug metabolizing enzymes, such as acetyl Co-A O-acetyltransferase, or the strains lacking DNA repair enzymes, such as O6-methylguanine DNA methyltransferase, are highly sensitive to the mutagenicity of particular classes of mutagens . Thus, they are widely used for the efficient detection of genotoxicity of complex mixtures . Transgenic mice, named gpt delta, have been established for the detection and molecular analysis of mutations in various organs of rodents induced by chemicals . Future perspective of the genotoxicity assays using genetically engineered organisms is discussed.

Int J Food Microbiol, 2001 Sep 1, 68(3), 187 - 97
Microstructural effects on microbial survival: phase-separating dextran solutions; Hills BP et al.; Evolving microstructure in a model dextran solution is shown to exert a major influence on the survival of Escherichia coli K-12 frag 1 and Salmonella typhimurium LT2 . The microstructure results from microscopic phase separation, which develops over several hours resulting in hardening of the solution into a glassy state . The microstructure is characterized by an array of physical methods including image analysis, electron spin resonance and bulk rheology, and it is shown that bacterial survival depends on the formation of microscopic . water-rich domains and not primarily on bulk water activity or hardness.

Food Chem Toxicol, 2001 Nov, 39(11), 1045 - 53
Evaluation of the cytotoxicity, mutagenicity and antimutagenicity of emerging edible plants; Yen GC et al.; This study evaluates the toxic, mutagenic and antimutagenic effects of emerging edible plants that are consumed as new leafy vegetables in Taiwan . Among eight plant extracts, only the extracts of Sol (Solanum nigrum L.) showed cytotoxicity to Salmonella typhimurium TA100 in the absence of S9 mix . The toxicity of extracts from different parts of the Sol plant, such as leaf and stem, immature fruit and mature fruit, towards S . typhimurium TA100 and human lymphocytes was also assayed . The immature fruit extracts of Sol exhibited strong cytotoxicity with dose dependence and induced significant DNA damage in human lymphocytes based on the comet assay . However, no mutagenicity was found in eight plant extracts to TA98 or TA100 either with or without the S9 mixture . Sol and Sec {Sechium edule (Jacq.) Swartz} extracts showed the strongest inhibitory effect towards the mutagenicity of 2-amino-3-methyl-imidazo{4,5-f}quinoline (IQ) in S . typhimurium TA98 and TA100; the ID(50) was less then 1 mg/plate . Cra {Crassocephalum creidioides (Benth.) S . Moore} extracts also expressed moderate antimutagenic activities towards IQ and benzo{a}pyrene (B{a}P) either in TA98 or in TA100; the ID(50) was 1.63-2.41 mg/plate . The extracts from Bas (Basella alba L.), Bou (Boussingaultia gracilis Miers var . pseudobaselloides Bailey), Cen (Centella asiatica L . Urban), Cor (Corchorus olitorius L.) and Por (Portulaca oleracea L.) showed weak to moderate inhibition of mutagenicity of IQ . However, the potential antimutagenicity of these plant extracts towards B{a}P was weaker than that towards IQ . For a direct mutagen, 4-nitroquinoline-N-oxide (NQNO), only the Sol extracts showed strong inhibitory effects in the TA100 system . The antimutagenic activity of water extracts of Sec was partly reduced by heating at 100 degrees C for 20 min . The heat-stable antimutagens in Sec extracts could be produced in the plant extract preparation process . Fractions with molecular weights above 30,000 showed the strongest antimutagenicity and peroxidase activity in all the fractions of the Sec extracts.

Proc Natl Acad Sci U S A, 2001 Sep 11, 98(19), 10578 - 83 Epub 2001 Aug 28.
In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae; Li YF et al.; S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits . The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity . With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E . coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes . Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation . A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak . AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit . Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product . Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E . coli AdoMetDC reported by Diaz and Anton {Diaz, E . & Anton, D . L . (1991) Biochemistry 30, 4078-4081}.

Mutat Res, 2001 Oct 18, 497(1-2), 223 - 33
Metabolic activation of carcinogenic 1-nitropyrene by human cytochrome P450 1B1 in Salmonella typhimurium strain expressing an O-acetyltransferase in SOS/umu assay; Hatanaka N et al.; Metabolic activation of 1-nitropyrene (1-NP) by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes was investigated . 1-NP induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 in the absence of any P450 system, but the activities were influenced by the levels of bacterial O-acetyltransferase (OAT) and nitroreductase . Metabolic activation of 1-NP by human P450 1B1/NPR membranes was observed and was influenced by the levels of OAT levels in tester strains . Metabolic activation of 1-NP (0.3microM) by P450 1B1 was 750 umu units/min/nmol P450 1B1 in an OAT-overexpressing strain NM2009 . The metabolic activation of 1-NP (3-30microM) was similar (approximately 300 umu units/min/nmol P450 1B1) using TA1535/pSK1002 or OAT-deficient strain NM2000 . P450 1B1 had the highest catalytic activities among P450 family 1 enzymes for the activation of 1-aminopyrene (1-AP) in the OAT-overexpressing strain NM2009, suggesting nitrenium ion formation via N-hydroxylation/O-acetylation . High-performance liquid chromatography (HPLC) analyses revealed the formation of 1-nitropyrene-6-ol and also 1-nitropyrene-3-ol, 1-nitropyrene-8-ol, and trans-4,5-dihydroxy-4,5-diol-1-nitropyrene from 1-NP (10microM), catalyzed by P450 1B1 . These results indicate that 1-NP can be activated by human P450 1B1 to a genotoxic agent by nitroreduction/O-acetylation at low substrate concentrations and probably by epoxidation (independent of OAT) at high concentrations.

Mutat Res, 2001 Oct 18, 497(1-2), 111 - 21
The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S . typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity; Abril N et al.; Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)) . In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE) . No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy . Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy . At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.

Mutat Res, 2001 Oct 18, 497(1-2), 1 - 9
Anomalous mutagenicity profile of cyclohexanone oxime in bacteria: cell survival in background lawns; Prival MJ; The basis for the observed mutagenicity of cyclohexanone oxime in the presence of hamster liver S9 in Salmonella typhimurium strain TA1535, but not in TA100, was explored . While the chemical had no effect on the appearance of the background lawn in either strain, it did cause a reduction in mutant colony counts in strain TA100, raising the possibility of selective toxicity to this strain . Viability of the two strains was determined directly by titering the cells in background lawns over a 3 day period . In order to do this, cells embedded in top agar overlays were released by extruding agar plugs through small holes in the bottoms of centrifuge tubes, followed by vigorous vortexing . Viable cell counts in background lawns of strain TA100, but not strain TA1535, were greatly reduced in the presence of cyclohexanone oxime . Most of the loss of viable TA100 cells occurred on days 2 and 3 following plating, after the cells had exhausted the histidine in the medium and stopped growing . Therefore, the observed loss of background lawn viable cells is unlikely to be the cause of the non-mutagenicity of cyclohexanone in strain TA100 . Analysis of reversion spectra showed that cyclohexanone oxime-induced C-->T transitions in the second position of the CCC triplet at the his mutation site in strain TA1535, but had no significant effect on any transition or transversion in strain TA100.

J Am Chem Soc, 2001 Sep 5, 123(35), 8564 - 72
Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase; Warncke K et al.; The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state . The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine . Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H . The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz . The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz . The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine . This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact . The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.

Mol Genet Genomics, 2001 Jul, 265(5), 905 - 12
Transcriptional profile of Toxoplasma gondii-infected human fibroblasts as revealed by gene-array hybridization; Gail M et al.; To investigate the host-cell response to infection with the obligate intracellular pathogen Toxoplasma gondii, the transcriptional profiles of infected and uninfected human fibroblasts (HFF) were determined by hybridization to gene arrays representing nearly 600 genes . Transcripts that displayed a greater than five-fold increase in level relative to uninfected controls were also examined by RT-PCR and Northern analysis, resulting in the identification of 13 genes that were strongly up-regulated after infection with T . gondii . Comparisons with the transcriptional profiles of fibroblasts infected with Salmonella typhimurium and Chlamydia trachomatis allowed the identification of genes which are specifically induced in T . gondii-infected cells . While most of the up-regulated genes were induced on infection with all three pathogens, the genes for the transferrin receptor and MacMARCKS were up-regulated in Toxoplasma-infected fibroblasts only . Expression of the transferrin receptor protein was examined by Western analysis and found to be specifically elevated in Toxoplasma-infected fibroblasts . Genes which are specifically induced in T . gondii-infected cells are particularly interesting for further studies, since they might be used to dissect specific interactions of this pathogenic parasite with its host cell.

J Endotoxin Res, 2001, 7(2), 157 - 63
TNF-alpha hyper-responses to Gram-negative and Gram-positive bacteria in Propionibacterium acnes primed or Salmonella typhimurium infected mice; Merlin T et al.; IFN-gamma-dependent hypersensitivity to LPS is inducible in mice by infection or pre-treatment with killed bacteria . Hypersensitive mice exhibit enhanced inflammatory responses to LPS, including the overproduction of TNF-alpha . Using Lps(n) BALB/c and Lps(d) BALB/c/l mice, primed with Propionibacterium acnes or infected with Salmonella typhimurium, we show that concurrently to hypersensitivity to LPS, a hypersensitivity to other constituents of killed Gram-negative or Gram-positive bacteria and to staphylococcal enterotoxin B (SEB) develops . The TNF-alpha hyper-responses in sensitized mice induced by different Gram-positive bacteria, are generally weaker than those by Gram-negative bacteria and vary significantly, due to the absence of a common, LPS-equivalent component . Using IFN-gamma R(-/-) and the respective wild-type mice, we demonstrate that although sensitization to LPS and killed Listeria monocytogenes is exclusively IFN-gamma-dependent, an IFN-gamma-independent, moderate sensitization to certain TNF-alpha-inducing constituents in bacteria may develop in parallel.

Chem Biol Interact, 2001 Jul 31, 137(1), 89 - 99
On the role of alkylating mechanisms, O-alkylation and DNA-repair in genotoxicity and mutagenicity of alkylating methanesulfonates of widely varying structures in bacterial systems; Eder E et al.; The Ames test and the SOS-chromotest are widely used bacterial mutagenicity/genotoxicity assays to test potential carcinogens . Though the molecular mechanisms leading to backmutations and to the induction of SOS-repair are in principle known the role of alkylation mechanisms, of different DNA-lesions and of DNA-repair is in parts still unknown . In this study we investigated 14 monofunctional methanesulfonates of widely varying structures for mutagenicity in Salmonella typhimurium strain TA 1535 sensitive for O(6)-guanine alkylation for comparison with strain TA 100 in order to obtain additional information on the role of alkylation mechanisms, formation of the procarcinogenic DNA-lesion O(6)-alkylguanine and the role of DNA-repair in induction of backmutation . The substances were also tested in the SOS-chromotest with Escherichia coli strain PQ 37 and strain PQ 243 lacking alkyl base glycosylases important for base excision repair in order to examine the role of alkylation mechanisms, of base excision repair and the role of O-alkyl and N-alkyl DNA-lesions on the induction of SOS-repair . The secondary methanesulfonates with very high S(N)1-reactivity isopropyl methanesulfonate and 2-butyl methanesulfonate showed highest mutagenicities in both strains . The higher substituted methanesulfonates with very high S(N)1-reactivity had lower mutagenic activities because of reduced half lives due to their high hydrolysis rates . A clear increase in mutagenicities in strain TA 100 was observed for the primary compounds methyl methanesulfonate and allyl methanesulfonate with very high S(N)2-reactivity . The primary compound phenylethyl methanesulfonate has a relatively high mutagenicity in both Salmonella strains which can be explained by an increased S(N)1-reactivity and by low repair of the O(6)-phenylethylguanine . Highest SOSIPs (SOS inducing potency) in strains PQ 37 and PQ 243 were found for methyl methanesulfonate and for the secondary compounds with high S(N)1-reactivity . The ratios in the SOSIPs between strain PQ 243 and PQ 37, indirectly indicative for the role of O- and N-alkylation in the induction of SOS-repair, was high for the primary methanesulfonates and lower for the secondary, indicating that the SOS-repair is, to a certain extent, also induced by other lesions than O(6)-alkylation . The results indicate that O(6)-alkylation is also a predominant lesion for backmutation in strain TA 100 and that in the case of monofunctional alkylating agents high S(N)2-reactivities are required to induce error prone repair mediated backmutations . The O(6)-alkylguanine lesion is also important for induction of SOS-repair in the SOS-chromotest, however, other sites of alkylation which are repaired by the base pair excision repair system can also efficiently contribute to the induction of SOS-repair.

Kaohsiung J Med Sci, 2001 May, 17(5), 230 - 8
Antimutagenicity of extracts of Hericium erinaceus; Wang JC et al.; Hericium erinaceus is valuable in the diet and in medical treatment . It contains water-soluble polysaccharides that have been found to enhance immunity and which show anti-artificial pulmonary metastatic tumor effects . In this study, water and ethanol extracts of the mycelium and fruiting body of Hericium erinaceus were examined by the Ames test using Salmonella typhimurium TA98 to screen for antimutagenic effects against 5 mutagens: AFB1, B{a}P, Glu-P-1, NQNO, and Trp-P-1 . We found that both extracts have the strongest antimutagenic activity against Trp-P-1, followed by Glu-P-1, B{a}P-1, AFB1, and finally NQNO . In addition, the antimutagenicity of the extracts was produced in a concentration-dependent manner . At a concentration of 200 ppm, both extracts showed the highest inhibitory action . However, the linear correlation indicated that concentration-activity relationship was not significant (p > 0.05) . In addition, extracts showed less antimutagenicity after heat treatment (p < 0.05) . This suggests that the antimutagenicity of the extracts is heat-labile . The ethanol extract from mycelium or fruiting body had better antimutagenic effects than did the water extract (p < 0.05) . Also, the extract from the fruiting body had better antimutagenic effects than did that from the mycelium.

Mutat Res, 2001 Jun 27, 493(1-2), 115 - 26
Structures of mutagens produced by the co-mutagen norharman with o- and m-toluidine isomers; Hada N et al.; Norharman, abundantly present in cigarette smoke and cooked foods, is not mutagenic to Salmonella typhimurium strains . However, norharman shows mutagenicity to S . typhimurium TA98 and YG1024 in the presence of S9 mix when coexisting with aromatic amines, including aniline, o- and m-toluidines . We previously reported that the mutagenicity from norharman and aniline in the presence of S9 mix was due to the formation of a mutagenic compound, 9-(4'-aminophenyl)-9H-pyrido{3,4-b}indole (aminophenylnorharman) . In the present study, we analyzed the mutagens produced by norharman with o- or m-toluidine in the presence of S9 mix . When norharman and o-toluidine were reacted at 37 degrees C for 20 min, two mutagenic compounds, which were mutagenic with and without S9 mix, respectively, were produced, and these were isolated by HPLC . The former mutagen was deduced to be 9-(4'-amino-3'-methylphenyl)-9H-pyrido{3,4-b}indole (amino-3'-methylphenylnorharman) on the basis of various spectral data, and this new heterocyclic amine was confirmed by its chemical synthesis . The latter mutagen was identified to be the hydroxyamino derivative . Amino-3'-methylphenylnorharman induced 41,000 revertants of TA98, and 698,000 revertants of YG1024 per microg with S9 mix . Formation of the same DNA adducts was observed in YG1024 when amino-3'-methylphenylnorharman or a mixture of norharman plus o-toluidine was incubated with S9 mix . These observations suggest that norharman reacts with o-toluidine in the presence of S9 mix to produce amino-3'-methylphenylnorharman, and this compound is metabolically activated to yield its hydroxyamino derivative . After activation by O-acetyltransferase, it might bind to DNA and exert mutagenicity in S . typhimurium TA98 and YG1024 . When norharman and m-toluidine were reacted in the presence of S9 mix, 9-(4'-amino-2'-methylphenyl)-9H-pyrido{3,4-b}indole (amino-2'-methylphenylnorharman) was identified as a mutagen . Thus, the mutagenicity of norharman with m-toluidine may follow a mechanism similar to that with o-toluidine.

Mutat Res, 2001 Jun 27, 493(1-2), 31 - 8
Cytotoxic effect of three arsenic compounds in HeLa human tumor and bacterial cells; Abdullaev FI et al.; Numerous epidemiological studies suggest that arsenic (As) compounds are carcinogens, however, recent data have renewed the interest in their anticarcinogenic properties . The cytotoxic effects of three arsenic compounds were assessed: sodium arsenite, sodium arsenate and sodium cacodylate, representing the trivalent and pentavalent species of arsenic, along with a dimethylated pentavalent arsenic species . HeLa cells and Salmonella typhimurium (strains TA98 and TA100) were exposed to As compounds and the cytotoxic effects were evaluated . Alterations on RNA and DNA synthesis in HeLa cells were also examined . All arsenic compounds produced a dose-dependent inhibition on colony formation and DNA synthesis in HeLa cells, yet any of them significantly influenced RNA synthesis in these cells . No evidence of arsenic-induced mutagenicity or antimutagenicity was observed using the Ames assay . In bacterial cells, only sodium arsenite caused a dose-dependent inhibition of colony formation.Collectively, these results indicate that in both, HeLa and S . typhimurium cell systems, only trivalent sodium arsenite can act as an effective inhibitor of cell growth . The possible mechanism(s) of the cytotoxic effect of arsenite in these two different cell systems might be due to its reactivity with intracellular sulfhydryl groups.

Toxicology, 2001 Jun 21, 163(2-3), 213 - 8
Different levels of Schistosoma mansoni infection increased the mutagenicity of benzo(a)pyrene, the activity of aryl hydrocarbon hydroxylase and the formation of hepatic microsomal hydrogen peroxide; Awney HA et al.; The present study investigates the influence of different levels of Schistosoma mansoni infection (60, 120, 180, 300, 600 cercariae per mice) after 33 days on the activity of aryl hydrocarbon hydroxylase (AHH) and the formation of hepatic hydrogen peroxide (H(2)O(2)) during the metabolic activation of benzo(a)pyrene {B(a)p} . Also, it shows the mutagenic effect of B(a)p at different levels of S . mansoni infection using Salmonella typhimurium TA 98 and TA 102 as a tester strains . High levels of H(2)O(2) production (222 nmol/mg protein) and AHH activity (240 pmol 3-OH B(a)p per mg protein) were seen at 300 cercariae per mice . Increasing histidine revertant colonies at TA98 and TA102 were detected at different levels of S . mansoni infection . These data clearly demonstrate that S . mansoni infection changes the mutagenicity of B(a)p, AHH activity, as well as enhancing the formation of hepatic H(2)O(2) generated during the metabolic activation of B(a)p in infected mice.

Biosci Biotechnol Biochem, 2001 Jul, 65(7), 1652 - 5
Antimutagenicity of deacylated anthocyanins in purple-fleshed sweetpotato; Yoshimoto M et al.; The antimutagenicity of the 3-sophoroside-5-glucoside of cyanidin and 3-sophoroside-5-glucoside of peonidin, the anthocyanin derivatives deacylated from the 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of peonidin (YGM-6) which had been purified from the sweetpotato with purple-colored flesh, was investigated by using Salmonella typhimurium TA 98 . A comparison of the antimutagenicity between YGM-3 and YGM-6 and the deacylated derivatives showed that the activity of cyanidin was stronger than that of peonidin . Deacylation of the peonidin-type pigment markedly decreased this antimutagenicity . Caffeic acid showed the strongest antimutagenicity of the constituent organic acids of the anthocyanin pigments, caffeic acid, ferulic acid, and p-hydroxybenzoic acid . These results suggest that the cathecol structure plays an important role in the strong antimutagenicity of anthocyanin pigments.

J Exp Med, 2001 Aug 20, 194(4), 379 - 91
Maturation of dendritic cells is accompanied by rapid transcriptional silencing of class II transactivator (CIITA) expression; Landmann S et al.; Cell surface expression of major histocompatibility complex class II (MHCII) molecules is increased during the maturation of dendritic cells (DCs) . This enhances their ability to present antigen and activate naive CD4(+) T cells . In contrast to increased cell surface MHCII expression, de novo biosynthesis of MHCII mRNA is turned off during DC maturation . We show here that this is due to a remarkably rapid reduction in the synthesis of class II transactivator (CIITA) mRNA and protein . This reduction in CIITA expression occurs in human monocyte-derived DCs and mouse bone marrow-derived DCs, and is triggered by a variety of different maturation stimuli, including lipopolysaccharide, tumor necrosis factor alpha, CD40 ligand, interferon alpha, and infection with Salmonella typhimurium or Sendai virus . It is also observed in vivo in splenic DCs in acute myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalitis . The arrest in CIITA expression is the result of a transcriptional inactivation of the MHC2TA gene . This is mediated by a global repression mechanism implicating histone deacetylation over a large domain spanning the entire MHC2TA regulatory region.

J Agric Food Chem, 2001 Aug, 49(8), 4019 - 25
Suppression of chemical mutagen-induced SOS response by alkylphenols from clove (Syzygium aromaticum) in the Salmonella typhimurium TA1535/pSK1002 umu test; Miyazawa M et al.; A methanol extract from clove (Syzygium aromaticum) showed a suppressive effect of the SOS-inducing activity on the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) in the Salmonella typhimurium TA1535/pSK1002 umu test . The methanol extract was re-extracted with hexane, dichloromethane, ethyl acetate, butanol, and water . The hexane fraction showed a suppressive effect . Suppressive compounds in the hexane fraction were isolated by silica gel column chromatography and identified as trans-isoeugenol (1) and eugenol (2) by GC, GC-MS, IR, and (1)H and (13)C NMR spectroscopy . Compounds 1 and 2 suppressed the furylfuramide-induced SOS response in the umu test . Compounds 1 and 2 suppressed 42.3 and 29.9% of the SOS-inducing activity at a concentration of 0.60 micromol/mL . These compounds were assayed with other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . In addition, compounds 1 and 2 were assayed with aflatoxin B(1) (AfB(1)) and 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which require liver metabolizing enzymes . These compounds showed suppressive effects of the SOS-inducing activity against furylfuramide, 4NQO, AfB(1), and Trp-P-1 . To research the structure-activity relationship, methyl esters of 1 and 2 (1Me and 2Me) and o-eugenol (3), as compounds similar to 2, were also assayed with all mutagens . Compounds 1Me, 2Me, and 3 showed weak suppressive effects of the SOS-inducing activity against furylfuramide.

Chem Res Toxicol, 2001 Aug, 14(8), 1118 - 27
Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: mono- and dihalomethanes; Wheeler JB et al.; A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation . Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO . Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases . Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH(3)Cl and using in vitro assays (HCHO formation) and the S . typhimurium mutagenesis assay with the dihalomethanes CH(2)Cl(2), CH(2)Br(2), CH(2)BrCl, CH(2)ICl, CH(2)I(2), and CH(2)ClF . GSTs 5-5 and T1 had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested . In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested . A similar pattern was observed for the conjugation of CH(3)Cl . The reversion tests with S . typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5 . Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic . The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue {Wheeler, J . B., Stourman, N . V., Armstrong, R . N., and Guengerich, F . P . (2001) Chem . Res . Toxicol . 14, 1107-1117} . The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps.

Chem Res Toxicol, 2001 Aug, 14(8), 1107 - 17
Conjugation of haloalkanes by bacterial and mammalian glutathione transferases: mono- and vicinal dihaloethanes; Wheeler JB et al.; Glutathione (GSH) transferases are generally involved in the detoxication of xenobiotic chemicals . However, conjugation can also activate compounds and result in DNA modification . Activation of 1,2-dihaloethanes (BrCH(2)CH(2)Br, BrCH(2)CH(2)Cl, and ClCH(2)CH(2)Cl) was investigated using two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and a bacterial dichloromethane dehalogenase (DM11) . Although the literature suggests that the bacterial dehalogenase does not catalyze reactions with CH(3)Cl, ClCH(2)CH(2)Cl, or CH(3)CHCl(2), we found a higher enzyme efficiency for DM11 than for the mammalian GSH transferases in conjugating CH(3)Cl, CH(3)CH(2)Cl, and CH(3)CH(2)Br . Enzymatic rates of activation of 1,2-dihaloethanes were determined in vitro by measuring S,S-ethylene-bis-GSH, the major product trapped by nonenzymatic reaction with the substrate GSH . Salmonella typhimurium TA 1535 systems expressing each of these GSH transferases were used to determine mutagenicity . Rates of formation of S,S-ethylene-bis-GSH by the GSH transferases correlated with the mutagenicity determined in the reversion assays for the three 1,2-dihaloethanes, consistent with the view that half-mustards are the mutagenic products of the GSH transferase reactions . Half-mustards {S-(2-haloethyl)GSH} containing either F, Cl, or Br (as the leaving group) were tested for their abilities to induce revertants in S . typhimurium, and rates of hydrolysis were also determined . GSH transferases do not appear to be involved in the breakdown of the half-mustard intermediates . A halide order (Br > Cl) was observed for both GSH transferase-catalyzed mutagenicity and S,S-ethylene-bis-GSH formation from 1,2-dihaloethanes, with the single exception (both assays) of BrCH(2)CH(2)Cl reaction with DM11, which was unexpectedly high . The lack of substrate saturation seen for conjugation of dihalomethanes with GSTs 5-5 and T1 was also observed with the mono- and 1,2-dihaloethanes {Wheeler, J . B., Stourman, N . V., Thier, R., Dommermuth, A., Vuilleumier, S., Rose, J . A., Armstrong, R . N., and Guengerich, F . P . (2001) Chem . Res . Toxicol . 14, 1118-1127}, indicative of an inherent difference in the catalytic mechanisms of the bacterial and mammalian GSH transferases.

J Food Prot, 2001 Aug, 64(8), 1255 - 60
Antibacterial activity in extracts of Camellia japonica L . petals and its application to a model food system; Kim KY et al.; The potential presence of naturally occurring antimicrobials in petals of Camellia japonica L., a member of the tea family, was investigated against foodborne pathogens in microbiological media and food . Petals of the camellia flower (C . japonica L.) were extracted with methanol and fractionated into basic, acidic, and neutral fractions . The acidic fraction (equivalent to 1.0 g of raw sample per disk) produced an inhibitory zone of 14 to 19 mm (diameter) in a disk assay against the pathogens Salmonella Typhimurium DT104, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on agar plates . Silica gel adsorption column chromatography, Sephadex LH-20 column chromatography, and preparative purification by high-pressure liquid chromatography were used to purify compounds in the fraction . The mass spectrum of the antibacterial compound isolated had a molecular ion (M+) of m/z 116 and showed good conformity with the spectrum of fumaric acid (HOOC-CH=CH-COOH) . An aqueous extract from the petals of C . japonica L . had an inhibitory effect on growth of all pathogens at 37 degrees C in microbiological media by increasing the lag phase . None of the microorganisms was inhibited completely . Milk was used as a model food system . Aqueous extract at a concentration of 100 mg/ml was bacteriostatic against all the foodborne pathogens in the milk stored at 25 degrees C for up to 4 days.

J Nutr Sci Vitaminol (Tokyo), 2001 Apr, 47(2), 126 - 31
Acute toxicity and mutagenicity study on branched corn syrup and evaluation of its laxative effect in humans; Kishimoto Y et al.; We developed a branched corn syrup (BCS, average molecular weight: 500, content of indigestible portion: 45%) by heat treatment of indigestible dextrin with hydrochloric acid . To confirm the safety of BCS, we conducted both an acute toxicity test and a mutagenicity test . Moreover, we observed gastroenteric effects of BCS in fifty healthy humans . The results are summarized as follows . 1) There was no death observed after oral administration of BCS in Sprague-Dawley-strain rats . Lethal dose (LD)50, value was estimated to be more than 10 g/kg body weight . 2) No mutagenicity was observed in Salmonella typhimurium TA98, TA100, TA1535, TA1537, or Escherichia coli WP2uvrA . 3) Fifty adults were divided into five groups often (five of each sex) and orally administered BCS at 0.2, 0.3, 0.4 . 0.5 and 0.6 g/kg body weight as indigestible portion . Although no diarrhea was observed in females, BCS at 0.6 g/kg as indigestible portion caused diarrhea in two out of five males . The maximum non-effective dose of indigestible portion of BCS was estimated to be 0.5 g/kg in males and more than 0.6 g/kg in females.

Mutagenesis, 2001 Sep, 16(5), 401 - 6
Genotoxicity of human breast milk from different countries; Martin FL et al.; Dietary and/or environmental factors appear to play a key role in the international variations that exist in breast cancer incidence . The genotoxicity of breast milk extracts is being examined as a possible indicator of in vivo exposure of mammary epithelial cells to DNA-damaging agents . Breast milk samples were obtained from the UK (n = 32), a high risk country, and from Hong Kong (n = 10), India (n = 20) and Singapore (n = 20), countries of lower breast cancer incidence . The abilities of breast milk extracts to induce DNA damage detected as single-strand breaks (SSBs) in the alkaline Comet assay and to induce micronuclei in MCL-5 cells and mutations in Salmonella typhimurium YG1019 were investigated . In the Comet assay 18 of 32 (56%) UK samples induced significant increases in DNA SSBs compared with 2 of 10 (20%), 5 of 20 (25%) and 8 of 20 (40%) of the samples from Hong Kong, India and Singapore, respectively . The proportion of positive samples was significantly higher in the UK group than in the combined low breast cancer incidence group and significantly higher than in the Indian group (P < 0.05, Fisher's exact test) . In the micronucleus assay 9 of 32 (28%) UK samples showed significant activity compared with 0 of 10 (0%), 2 of 20 (10%) and 3 of 20 (15%) of the samples from Hong Kong, India and Singapore, respectively . Extracts of all the aforementioned milk samples were also tested for bacterial mutagenicity . Nine of 32 (28%) UK samples induced significant activity with a dose-response effect . Although activity was detected in samples from the other countries, comparable dose-response data could not be obtained because of a lack of material . This pilot study suggests that genotoxic components occur more frequently in UK breast milk than in milk from some other countries with a lower incidence of cancer . More work is required to confirm these initial findings and to examine their relevance to variations in breast cancer incidence.

Cancer Res, 2001 Aug 15, 61(16), 6178 - 84
Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma; Niethammer AG et al.; We demonstrate that a mouse-human chimeric anti-ganglioside GD2-interleukin (IL)-2 fusion protein (ch14.18-IL2) substantially amplifies tumor-protective immunity against murine melanoma induced by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to murine melanoma peptide epitopes gp100(25-35) and TRP-2(181-188) . This combination therapy led to the complete rejection of a lethal challenge with B78D14 murine melanoma cells in six of eight mice and a marked suppression of s.c . tumor growth in the two remaining animals . The tumor-protective immunity was mediated by MHC class I antigen- restricted CD8(+) T cells together with CD4(+) T cell help, which was required only for tumor cell killing in the effector phase of the immune response . A single oral vaccination with the DNA vaccine, which was carried by attenuated Salmonella typhimurium, was equally as effective as three such vaccinations applied at 2-week intervals . The immunological mechanisms involved in this antitumor effect were suggested by a decisively increased secretion of tumor necrosis factor alpha TNFTnTNa and IFN-gamma from CD4(+) and CD8(+) T cells and a markedly up-regulated expression on CD8(+) T cells of high-affinity IL-2 receptor alpha chain (CD25), costimulatory molecule CD28, and adhesion molecule lymphocyte function-associated antigen-2 (LFA-2/CD2) . Additionally, the combination therapy induced increased expression of costimulatory molecules B7.1 and CD48 on murine antigen-presenting cells . Taken together, our results suggest that IL-2 targeted to the tumor microenvironment by a specific antibody-IL-2 fusion protein is a potent enhancer of tumor-protective immunity induced by an oral DNA vaccine that may ultimately enhance the chances of success in its clinical application.

Mutat Res, 2001 Sep 1, 480-481, 55 - 69
The antimutagenic effect of vanillin and cinnamaldehyde on spontaneous mutation in Salmonella TA104 is due to a reduction in mutations at GC but not AT sites; Shaughnessy DT et al.; Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that, when added to assay plates, reduced the spontaneous mutant frequency in Salmonella typhimurium strain TA104 (hisG428, rfa, uvrB, pKM101) by 50% . To date, no study has demonstrated whether or not the antimutagenic effects of an agent are due to a reduction in all classes of mutations or to a reduction in selective classes of mutations . To explore this issue, we have determined the spontaneous mutation spectrum in TA104 as well as the mutation spectrum after treatment of cells with antimutagens at concentrations that produced approximately a 50% reduction in mutant frequency but only a 10% reduction in survival . Statistical analysis revealed no significant difference between the mutation spectra of VAN- and CIN-treated cells . Relative to untreated cells, treatment with either VAN or CIN produced a significant reduction in mutations at GC sites, whereas neither compound produced a significant reduction in mutations at AT sites . Antimutagenesis experiments in hisG428 strains of Salmonella with varying DNA repair backgrounds showed that VAN and CIN require SOS repair genes to produce an antimutagenic effect against spontaneous mutagenesis . Studies evaluating the effect of VAN and CIN on growth rate showed that neither compound suppressed growth relative to untreated cells . To our knowledge, this is the first study to examine if an antimutagen reduced all or just some classes of mutations that were available for reduction.

Mutat Res, 2001 Sep 1, 480-481, 23 - 35
Antimutagenesis and anticarcinogenesis, from the past to the future; Weisburger JH; Observations on cancer causation are some 150 years old, but actual detailed research on elements bearing on cancer started at the beginning of the twentieth century . Rapid progress, however, is only some 40 years old . Studies in humans documented certain lifestyle related factors to lead to cancer, and research in animal models strengthened this information . With the realization that there are carcinogens that in a metabolically activated attack DNA, in contrast to other agents that act by promoting, enhancing processes through totally distinct mechanisms, it became possible to develop and apply tests for DNA reactivity, in a prokaryotic organism, the widely used Salmonella typhimurium test by Ames and in a eukaryotic system, namely freshly explanted liver cells displaying evidence of DNA repair by Williams . A battery of these two tests are over 90% accurate in defining genotoxicity . Virtually all documented human carcinogens are genotoxic . With advances in molecular biology, mutational events are traced to changes in tumor suppressor genes or in oncogenes, that can serve as markers of risk . In addition, reactive oxygen systems (ROS) are involved in both the early steps in cancer and in the developmental aspects . Thus, foods containing antioxidants such as vegetables, fruits, soy products, cocoa and tea that counteract ROS are protective in cancer causation and development . Worldwide application of current knowledge and mechanisms to cancer prevention, the definitive means of cancer control, is likely to lower not only cancer but also heart disease risk in the current century.

Drug Metab Dispos, 2001 Sep, 29(9), 1176 - 82
Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P450 1B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009; Shimada T et al.; A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells . Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz{a}anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo{a,l}pyrene, benzo{c}phenanthrene, and dibenz{a,h}anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo{a}pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz{a}anthracene-3,4-diol, dibenzo{a,l}pyrene-11,12-diol, benzo{b}fluoranthene-9,10-diol, benzo{c}chrysene, 5,6-dimethylchrysene-1,2-diol, benzo{c}phenanthrene-3,4-diol, 7,12-dimethylbenz{a}anthracene, benzo{a}pyrene, 5-methylchrysene, and benz{a}anthracene . We also determined activation of these procarcinogens by recombinant (T . ni) human P450 enzymes in S . typhimurium NM2009 . There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E . coli and T . ni cells, although basal activities with three lots of CYP1B1 in T . ni cells were very high without substrates and NADPH in our assay system . Using 14 forms of human P450s (but not CYP1B1) (in T . ni cells), we found that CYP1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Folia Microbiol (Praha), 2001, 46(2), 113 - 7
Antibacterial and mutagenic activities of new isothiocyanate derivatives; Sovcikova A et al.; Nine newly synthesized isothiocyanate derivatives were demonstrated to posses antibacterial and genotoxic activities in vitro . 4-Hydroxybutyl isothiocyanate exhibited a broad antibacterial effect, with MIC values of 762 mumol/L for Staphylococcus aureus and Escherichia coli . Ethyl 4-methylsulfoxidobutanoate had the highest antibacterial activity in Gram-positive bacteria, the MIC value being 425 mumol/L for S . aureus . The highest tested concentrations of ethyl 4-isothiocyanatobutanoate and 4-hydroxybutyl isothiocyanate produced a bacteriocidal effect in Gram-positive bacteria . The compounds showed no mutagenic effects on Salmonella typhimurium tester strains TA 98 and TA 100, either in the absence or in the presence of a metabolically active microsomal S9 fraction from rat liver using standard Ames test.

Poult Sci, 2001 Aug, 80(8), 1190 - 200
Effects of vitamin E and C supplementation on performance, in vitro lymphocyte proliferation, and antioxidant status of laying hens during heat stress; Puthpongsiriporn U et al.; Vitamin E (dl-alpha-tocopheryl acetate) was evaluated for its effects on performance, lymphocyte proliferation, and antioxidation in layers during heat stress . In Trial 1, 25, 45, or 65 IU of vitamin E/kg were fed to four replicated pens (five hens/cage) of DeKalb Delta or Hy-Line W-36 per treatment starting at 20 wk of age . At 34 wk of age, hens were heat-stressed at diurnal temperature ranging from 21 C to 35 C for 3 wk . The performances of hens not exposed to heat stress were not influenced by supplemental vitamin E . Supplemental vitamin E did not affect egg production; however, egg mass was greater (P < 0.05) with supplementation of 65 IU of vitamin E/ kg during heat stress . Egg yolk was significantly increased (P < 0.04) when hens were fed 45 and 65 lU/kg compared with the control vitamin E level (25 lU/kg) . Haugh units were higher (P < 0.01) for hens fed 65 IU of vitamin E/kg compared to 25 and 45 lU/kg . Lymphocyte proliferative responses to concanavalin A (Con A) and Salmonella typhimurium lipopolysaccharide (LPS) were greater (P < 0.0001) in hens fed 45 and 65 IU of vitamin E/kg during heat stress . Strain had no effect on any of the parameters measured . In Trial 2, a 2 x 2 factorial was designed to test effects of vitamin C in drinking water (0 and 1,000 ppm) and dietary vitamin E (25 and 65 IU/kg) . Eight replications per treatment with four hens per replication cage were heat-stressed at constant temperature of 35 C for 3 wk . Egg production and egg mass were higher when hens were fed 65 IU of vitamin E/kg than when hens were fed 25 lU/kg (81.5 vs . 75.9%, P < 0.03 and 48.2 vs . 44.6 g, P < 0.03, respectively) . Yolk solids weight for the 65 IU vitamin E/kg group was higher (P < 0.01) compared to the 25 IU/kg group . ConA and LPS mitogenic responses were greater in hens fed 65 IU of vitamin E (P < 0.001 or P < 0.003, respectively) or 1,000 ppm of vitamin C (P < 0.001 or P < 0.002, respectively) . The combination of 65 IU vitamin E/kg and 1,000 ppm vitamin C showed the highest ConA and LPS mitogenic responses among the treatments . No interaction effects of the two vitamins on production measurements or lymphocyte proliferative responses were observed . TBA values in egg yolk and plasma of hens fed 65 IU of vitamin E/kg were lower (P < 0.0001) than those of hens that received 25 IU of vitamin E/kg . These results suggest that vitamin E supplementation at 65 IU/kg diet may enhance production, induction of in vitro lymphocyte proliferation by ConA and LPS, and antioxidant properties of egg yolks and plasma of White Leghorn hens during heat stress and that supplementation of 1,000 ppm vitamin C may further enhance in vitro lymphocyte proliferative responses of hens during heat stress.

Poult Sci, 2001 Aug, 80(8), 1164 - 70
Effects of dietary polyunsaturated fatty acids on in vivo splenic cytokine mRNA expression in layer chicks immunized with Salmonella typhimurium lipopolysaccharide; Sijben JW et al.; Effects of dietary polyunsaturated fatty acids (PUFA) on immune responses in poultry have been reported . However, effects on the underlying mechanisms, such as the role of cytokines, have not been documented because the necessary tools were lacking . Recently, primer sets for chicken interleukin (IL)-1beta, IL-2, interferon-gamma (IFN-gamma), myelomonocytic growth factor (MGF), and transforming growth factor (TGF)-beta2 have become available . Therefore, in the present study we first examined the in vivo effects of an inflammatory challenge with Salmonella typhimurium lipopolysaccharide (LPS) on cytokine profiles in growing laying-type chicks . Second, we examined whether dietary fat sources affected the observed cytokine profiles . Two hundred forty chicks were assigned in a 2 x 4 factorial design of treatments, with injection with LPS or saline and dietary fat source as factors . Factors were i.v . injection with S . typhimurium LPS or saline (control) and four dietary fat sources: corn oil, linseed oil, menhaden oil, and tallow . Two hours after injection, birds were killed, and their spleens were removed for RNA extraction . Reverse transcription polymerase chain reactions with primer sets for chicken IL-1beta, IL-2, IFN-gamma, MGF, TGF-beta2, and beta-actin were performed with RNA samples pooled by pen . The expression of cytokine mRNA was expressed relative to the level of beta-actin mRNA . Interleukin-1 (P < 0.001), MGF (P < 0.0001), IL-2 (P < 0.001), and IFN-gamma (P < 0.001) mRNA expressions were enhanced by challenge with LPS . Immunization treatment had no effect on TGF-beta2 or beta-actin expression . Dietary treatment did not affect mRNA expression of IL-1, MGF, IFN-gamma, TGF-beta2, or beta-actin . Interleukin-2 expression in LPS-injected birds that were fed the fish-oil-enriched diet was enhanced (P = 0.05) . The present study indicates that in vivo effects of immune challenge on cytokine mRNA expression can be measured in poultry . The observation that mRNA level of IL-2, but not the mRNA levels of IFN-gamma or MGF, is enhanced by dietary fish oil at 2 h suggests that dietary PUFA at this moment initially affected naive T lymphocytes.

J Control Release, 2001 Jul 6, 74(1-3), 313 - 5
Salmonella-based tumor-targeted cancer therapy: tumor amplified protein expression therapy (TAPET) for diagnostic imaging; Tjuvajev J et al.; In preclinical studies, genetically engineered Salmonella have the ability to localize, selectively accumulate, and persist within transplantable murine tumors, spontaneous murine tumors and human tumor xenographs, and can express therapeutic proteins at high levels . These strains of engineered non-virulent Salmonella typhimurium display the capacity to accumulate and grow selectively in a variety of tumor types and to inhibit the growth of primary and metastatic tumors following intravenous injection into tumor-bearing mice . One strain of the bacteria (VNP20009) which has endogenous antitumor activity is currently in Phase I clinical trials . The bacteria are highly attenuated and genetically stable . The combination of the lipid mutation and the purine auxotrophy attenuate the virulence of the bacteria by greater than 10000-fold and enhance the specificity of the bacteria for tumor tissue . These bacteria have been found to be safe in mice, pigs and monkeys when administered intravenously . Second-generation Salmonella vectors will be developed to include transgenes that will express therapeutic agents and reporter transgenes for non-invasive imaging . We have performed a preliminary study to demonstrate localization of {(14)C}FIAU in tumored mice pretreated with Salmonella expressing HSV1-TK . The {(14)C}FIAU radioactivity and bacterial count data strongly support a Salmonella(TK)-dependent {(14)C}FIAU accumulation of at least 30-fold higher in tumor tissue compared to muscle tissue . These data warrant further investigation on the use of genetically engineered Salmonella as a systemically administered tumor-specific agents for tumor therapy and delivery of diagnostic imaging markers.

Adv Drug Deliv Rev, 2001 Aug 23, 50(1-2), 81 - 106
Exploiting M cells for drug and vaccine delivery; Clark MA et al.; The specialised antigen sampling M cells represent an efficient portal for mucosal drug and vaccine delivery . Delivery may be achieved using synthetic particulate delivery vehicles including poly(DL-lactide-co-glycolide) microparticles and liposomes . M cell interaction of these delivery vehicles is highly variable, and is determined by the physical properties of both particles and M cells . Delivery may be enhanced by coating with reagents including appropriate lectins, microbial adhesins and immunoglobulins which selectively bind to M cell surfaces . Live attenuated microorganisms are also suitable as vaccines and mucosal vectors and many, including Salmonella typhimurium, innately target to M cells . After cell surface adhesion, delivery vehicles are rapidly transported across the M cell cytoplasm to underlying lymphoid cells and may subsequently disseminate via the lymphatics . Further definition of M cell development and function should permit exploitation of their high transcytotic capacity for safe and reliable mucosal delivery.

Cell Microbiol, 2001 Aug, 3(8), 567 - 77
Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella; Meresse S et al.; Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) . Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles . This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane . We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice . Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium . Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles . We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

J Ethnopharmacol, 2001 Sep, 77(1), 103 - 9
Antimutagenicity and induction of anticarcinogenic phase II enzymes by basidiomycetes; Shon YH et al.; Extracts from Phellinus linteus, Phellinus igniarius, and Agrocybe cylindracea have been tested for their antimutagenic properties against direct-acting mutagens {4-nitro-o-phenylenediamine (NPD) and sodium azide (NaN(3))} and indirect-acting mutagens {2-aminofluorene (2-AF) and benzo{a}pyrene (B{a}P)}, using the Salmonella typhimurium tester strains TA 98 and TA 100 . In addition, the chemopreventive potentials of these extracts to induce NAD(P)H:quinone oxidoreductase (QR) and glutathione S-transferase (GST) activities and glutathione (GSH) level extracts from the filtrate of the cultured broth of P . linteus, polysaccharide extracts from the cultured broth (PI I) and mycelia (PI II) and water extract of fruiting bodies (PI II) of P . igniarius, and polysaccharide extracts from the cultured broth (AC I) and mycelia (AC II) of A . cylindracea showed inhibitory effects on the mutagenic activities induced by the direct-acting mutagens, NPD and NaN(3), and the indirect-acting mutagens, 2-AF and B{a}P . QR was induced with PI I, PI II, AC I, and AC II, and GST activity was induced with PL I, PL II, PI I, PI II, PI III and AC I in murine Hepa1c1c7 cell culture . In addition, PL I, PL II, PI I, PI II, PI III and AC II increased glutathione level . These results suggest that P . linteus, P . igniarius, and A . cylindracea have antimutagenic activities and may play a role in the prevention of cancer by inducing QR and GST activities and increasing GSH level.

Sci Total Environ, 2001 Jul 25, 275(1-3), 95 - 108
A comparative assessment of Boise, Idaho, ambient air fine particle samples using the plate and microsuspension Salmonella mutagenicity assays; Claxton LD et al.; The primary objective of this study is to characterize the genotoxic potential of the ambient air aerosols collected within an air shed impacted primarily by wood smoke and automotive emissions . The study also examines the relative merits of a microsuspension assay and the standard plate assay for monitoring the presence of airborne particle-bound mutagens . Wintertime ambient air particulate samples collected from Boise, Idaho, USA, were shown to contain extractable organic matter that is mutagenic in the Salmonella typhimurium microsuspension and plate-incorporation assays . Differences in the results from the primary sites, auxiliary sites and the background site demonstrate that the particle-bound mutagens are not evenly distributed within the air shed and are more associated with the location of sampling than with the time of sampling or the type of bioassay used to evaluate the samples . This study also demonstrates that the bioassay protocol used in such studies should depend upon the characteristics of the air shed's mutagens and the purpose of the study . For example, the microsuspension assay gave somewhat more variable results between samples but was approximately threefold more sensitive than the plate assay . When strain TA98 was used in the microsuspension assay, the mutagenic response was greater without an exogenous activation system . The reverse was true for the plate assay in which the use of an exogenous activation system increased the mutagenicity response . TA100 in the microsuspension assay provided results comparable to those with TA98 . This is important because TA100 can also be used to bioassay semivolatile and volatile organics associated with ambient air mutagenicity . This, in turn, allows a comparison of the mutagenicity of organics collected by differing methods due to their volatility . Future studies should be directed toward correlation of mutagenicity results with other analytical results in order to further develop methods for better characterization of the genotoxicity of ambient air.

Invest Ophthalmol Vis Sci, 2001 Aug, 42(9), 2022 - 30
The effects of intraocular injection of interleukin-13 on endotoxin-induced uveitis in rats; Lemaitre C et al.; PURPOSE: Interleukin (IL)-13 is a strong immunomodulatory cytokine that inhibits macrophages from secreting proinflammatory mediators . This study was conducted to investigate the effect of intraocular injection of IL-13 on the development of endotoxin-induced uveitis (EIU) in the Lewis rat . METHODS: One injection into the anterior chamber of recombinant human IL-13 (6 ng in 10 microl saline) was performed either simultaneously with a single injection of lipopolysaccharide (LPS) from Salmonella typhimurium into the footpad or 6 hours before the IL-13 injection . EIU was evaluated by slit lamp examination at 6, 16, and 24 hours after LPS injection . Counts of inflammatory cells were performed on cryostat sections after specific immunostaining . Anterior chamber paracentesis was performed, and kinetic analysis of the IL-13 injected in the anterior chamber was performed by ELISA . Cytokine and chemokine gene expression in the iris-ciliary body and the retina was evaluated by reverse transcription-polymerase chain reaction . RESULTS: A significant inhibition of ocular inflammation was observed in IL-13-treated rats at 16 and 24 hours after LPS injection . Unilateral injection of IL-13 inhibited EIU only in the injected eye . High levels of IL-13 were detected in the aqueous humor at 2 hours after local IL-13 injection to remain high up to 18 hours . In contrast, IL-13 was not detected in the corresponding sera . Quantitative analysis of inflammatory cells in ocular tissues showed a significant decrease in OX-42(+) cells (microglia, activated macrophages, dendritic cells, and polymorphonuclear leukocytes) and ED1(+) cells (monocytes-macrophages and dendritic cells) in treated rats . A decreased expression of TNF-alpha, IL-1 beta, IL-6, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 mRNAs was observed in the iris-ciliary body and the retina from IL-13-treated rats, whereas IFN-gamma was upregulated in the iris-ciliary body . CONCLUSIONS: Injection of IL-13 into the anterior chamber may inhibit the ocular inflammation induced by LPS injection by reducing intraocular cytokine and chemokine mRNA expression in ocular tissues.

Environ Mol Mutagen, 2001, 38(1), 69 - 79
Genetic toxicology testing of the antimalarial drugs chloroquine and a new analog, AQ-13; Riccio ES et al.; AQ-13 ({N1-(7-chloro-quinolin-4yl)-3-(N3,N3-diethylamino)propylamine} dihydrochloride trihydrate) is an aminoquinoline antimalarial drug that is effective against chloroquine-resistant strains of Plasmodium falciparum . It is structurally similar to the widely used chloroquine diphosphate (CQ) . We evaluated these drugs in the three assays currently recommended by the International Conference on Harmonization (ICH): bacterial mutagenesis in Salmonella typhimurium and Escherichia coli, mammalian cell mutagenesis in L5178Y mouse lymphoma cells, and micronucleus induction in rat bone marrow . A small but statistically significant increase in revertant colonies was produced by CQ with Salmonella tester strain TA98 without metabolic activation (MA) and by AQ-13 with strain TA1537 both with and without MA . In L5178Y cells, testing of CQ and AQ-13 up to cytotoxic concentrations with and without MA produced no increase in mutant colonies and no increase in the numbers of small colonies . Slight decreases in the ratio of polychromatic erythrocytes (PCE) to red blood cells (RBC) were observed in male and female rats treated with CQ and in females only treated with AQ-13; however, none of these changes was statistically significant . No increases in the frequency of micronucleated PCE were observed at any dose level of CQ or AQ-13 . Although both CQ and AQ-13 showed weak bacterial mutagenicity, this mutagenic effect was not confirmed in either the mouse lymphoma mutagenesis assay or the micronucleus assay . These results indicate that CQ and AQ-13 should pose minimal risk of genotoxic damage in human populations being administered these drugs .

Mutat Res, 2001 Aug 8, 479(1-2), 197 - 206
Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450; Muto S et al.; Catechins, major polyphenol constituents of green tea, are potent chemopreventive agents against cancers caused by chemical carcinogens in rodents . The effects of four epicatechin derivatives, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC), on the metabolic activation of benzo{a}pyrene (B{a}P), 2-amino-1-methyl-6-phenylimidazo-{4,5-b}pyridine (PhIP) and aflatoxin B(1) (AFB(1)) by human cytochrome P450 (CYP) were examined . B{a}P, PhIP and AFB(1) were activated by respective human CYP1A1, CYP1A2 and CYP3A4 expressed in the membrane fraction of genetically engineered Salmonella typhimurium (S . typhimurium) TA1538 cells harboring the human CYP and human NADPH-CYP reductase (OR), when the membrane fraction was added to S . typhimurium TA98 . Galloylated catechins, ECG and EGCG inhibited the mutagenic activation potently, while EGC and EC showed relatively weak inhibitory effects . Catechins also inhibited the oxidations of typical substrates catalyzed by human CYPs, namely ethoxycoumarin O-deethylation by CYP1A1, ethoxyresorufin O-deethylation by CYP1A2 and midazolam 1'-hydroxylation by CYP3A4 . The IC(50) values of catechins for the inhibition of human CYP were roughly the same as those seen in the mutagenic activation . EGCG inhibited other forms of human CYP such as CYP2A6, CYP2C19 and CYP2E1, indicating the non-specific inhibitory effects of EGCG toward human CYPs . Furthermore, EGCG inhibited human NADPH-cytochrome CYP reductase (OR) with a K(i) value of 2.5 microM . These results suggest that the inhibition of the enzyme activity of CYP is accounted for partially by the inhibition of OR.

Arthritis Rheum, 2001 Jul, 44(7), 1677 - 88
Minimal alterations in the HLA-B27-bound peptide repertoire induced upon infection of lymphoid cells with Salmonella typhimurium; Ramos M et al.; OBJECTIVE: To characterize putative changes in the HLA-B27-bound peptide repertoire following infection of lymphoid cells with Salmonella typhimurium, a bacterium known to trigger reactive arthritis in HLA-B27-positive individuals . METHODS: A protocol was developed for efficient large-scale infection of lymphoblastoid cell transfectants expressing HLA-B*2705 . HLA-B27-bound peptide pools were isolated from noninfected and infected B*2705+ cells and comparatively analyzed by high-performance liquid chromatography . Peptide-containing chromatographic fractions from noninfected and infected cells were systematically compared by mass spectrometry (MS) to look for putative differences at the level of individual peptides . RESULTS: The presence of B*2705 did not influence S typhimurium invasion, since this was equally efficient in nontransfected or B27-transfected cells . The chromatographic profiles of B*2705-bound peptides from noninfected and infected cells were virtually identical . A total of 808 molecular species were compared by MS . Of these, 807 were present in both infected and noninfected cells . Only one molecular species from infected cells lacked a detectable counterpart in noninfected cells . CONCLUSION: Intracellular infection of lymphoid cells by S typhimurium induces minimal alterations in the HLA-B27-bound peptide repertoire . Minor changes detectable by cytotoxic T lymphocytes, but not easily amenable to direct biochemical analysis, are not ruled out.

Vaccine, 2001 Jul 20, 19(30), 4167 - 74
Nasal immunisation with Salmonella typhimurium producing rotavirus VP2 and VP6 antigens stimulates specific antibody response in serum and milk but fails to protect offspring; Coste A et al.; Rotavirus specifically infects the small intestine of young infants resulting in severe diarrhoea . Mucosal antibody responses are required to cure the infection, and mucosal administration of rotavirus-like particles induces protective immunity without requiring a mucosal adjuvant such as cholera toxin . In addition, the rotavirus protein VP6 has been defined as a protective antigen in an adult mouse rotavirus infection model . Salmonella typhimurium is an epithelium-invasive bacterium that induces specific immune responses in mucosal tissues against itself and carried antigens . In this work, we investigated the capacity of a live recombinant S . typhimurium vaccine to stimulate antibody responses against rotavirus . We constructed an attenuated S . typhimurium strain simultaneously producing VP6 and VP2 rotavirus proteins in the cytoplasm . In contrast to expression in eukaryotic cells, VP6 and VP2 did not form virus-like particles in our bacterial system . After nasal administration of female mice, the live recombinant Salmonella were able to elicit an antibody response specific to both VP2 and VP6 in serum and milk . However, these antibodies failed to passively protect the offspring against rotavirus-induced diarrhoea.

J Am Chem Soc, 2001 Feb 28, 123(8), 1730 - 9
Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross-link in a 5'-(CpG) motif: model of a malondialdehyde cross-link; Dooley PA et al.; Malondialdehyde (MDA), a known mutagen and suspected carcinogen, is a product of lipid peroxidation and byproduct of eicosanoid biosynthesis . MDA can react with DNA to generate potentially mutagenic adducts on adenine, cytosine, and particularly guanine . In addition, repair-dependent frame shift mutations in a GCGCGC region of Salmonella typhimurium hisD3052 have been attributed to formation of interstrand cross-links (Mukai, F . H . and Goldstein, B . D . Science 1976, 191, 868--869) . The cross-linked species is unstable and has never been characterized but has been postulated to be a bis-imino linkage between N(2) positions of guanines . An analogous linkage has now been investigated as a stable surrogate using the self-complementary oligodeoxynucleotide sequence 5'-d(AGGCG*CCT)(2,) in which G* represents guanines linked via a trimethylene chain between N(2) positions . The solution structure, obtained by NMR spectroscopy and molecular dynamics using a simulated annealing protocol, revealed the cross-link only minimally distorts duplex structure in the region of the cross-link . The tether is accommodated by partially unwinding the duplex at the lesion site to produce a bulge and tipping the guanine residues; the two guanines and the tether attain a nearly planar conformation . This distortion did not result in significant bending of the DNA, a result which was confirmed by gel electrophoresis studies of multimers of a 21-mer duplex containing the cross-link.

J Bioenerg Biomembr, 2001 Apr, 33(2), 79 - 92
Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease; Ames GF et al.; The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ . We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP {HisQMP2(his6)} . The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity . Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid . HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS) . HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded . Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP . The effect of various ATP analogs was analyzed . Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer . The permeability properties of two kinds of reconstituted PLS preparations are described.

J Food Prot, 2001 Jul, 64(7), 958 - 63
Survival of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes in and on vacuum packaged Lebanon bologna stored at 3.6 and 13.0 degrees C; Chikthimmah N et al.; Escherichia coli O157:H7, Salmonella Typhimurium, or Listeria monocytogenes was spread onto the surface of Lebanon bologna luncheon slices using sterile glass rods . The inoculated slices were stacked and vacuum packaged . The packages were stored at 3.6 or 13 degrees C . The foodborne pathogens . E . coli O157:H7, Salmonella Typhimurium, or L . monocytogenes were reduced in Lebanon bologna during storage at 3.6 or 13 degrees C . The higher storage temperature (13.0 degrees C) resulted in significantly faster destruction of E . coli O157:H7 and L . monocytogenes, compared to storage at refrigeration temperature (3.6 degrees C) (P < 0.005) . E . coli O157:H7 was the most resistant to destruction among the three foodborne pathogens . A linear destruction of E . coli O157:H7 occurred only after an initial lag period . Storage temperature did not have a significant effect on the rate of destruction of Salmonella Typhimurium . Foodborne pathogens inoculated prior to fermentation did not show any enhanced survival compared to control cells (inoculated after fermentation) during storage of the Lebanon bologna at 3.6 degrees C.

J Food Prot, 2001 Jul, 64(7), 950 - 7
Fate of Escherichia coli O157:H7, Salmonella typhimurium DT 104, and Listeria monocytogenes in fresh meat decontamination fluids at 4 and 10 degrees C; Samelis J et al.; Bacterial pathogens may colonize meat plants and increase food safety risks following survival, stress hardening, or proliferation in meat decontamination fluids (washings) . The objective of this study was to evaluate the ability of Escherichia coli O157:H7, Salmonella Typhimurium DT 104, and Listeria monocytogenes to survive or grow in spray-washing fluids from fresh beef top rounds sprayed with water (10 or 85 degrees C) or acid solutions (2% lactic or acetic acid, 55 degrees C) during storage of the washings at 4 or 10 degrees C in air to simulate plant conditions . Inoculated Salmonella Typhimurium DT 104 (5.4 +/- 0.1 log CFU/ml) died off in lactate (pH 2.4 +/- 0.1) and acetate (pH 3.1 +/- 0.2) washings by 2 days at either storage temperature . In contrast, inoculated E . coli O157:H7 (5.2 +/- 0.1 log CFU/ml) and L . monocytogenes (5.4 +/- 0.1 log CFU/ml) survived in lactate washings for at least 2 days and in acetate washings for at least 7 and 4 days, respectively; their survival was better in acidic washings stored at 4 degrees C than at 10 degrees C . All inoculated pathogens survived in nonacid (pH > 6.0) washings, but their fate was different . E . coli O157:H7 did not grow at either temperature in water washings, whereas Salmonella Typhimurium DT 104 failed to multiply at 4 degrees C but increased by approximately 2 logs at 10 degrees C . L . monocytogenes multiplied (0.6 to 1.3 logs) at both temperatures in water washings . These results indicated that bacterial pathogens may survive for several days in acidic, and proliferate in water, washings of meat, serving as potential cross-contamination sources, if pathogen niches are established in the plant . The responses of surviving pathogens in meat decontamination waste fluids to acid or other stresses need to be addressed to better evaluate potential food safety risks.

J Food Prot, 2001 Jul, 64(7), 1067 - 71
Evaluation of thin agar layer method for recovery of acid-injured foodborne pathogens; Wu VC et al.; The thin agar layer (TAL) method of Kang and Fung was used to enumerate acid-injured foodborne pathogens . This method involves overlaying 14 ml of nonselective medium (tryptic soy agar {TSA}) onto a prepoured and solidified pathogen-specific, selective medium in a petri dish . After surface plating, injured cells resuscitated and grew on TSA during the first few hours of incubation; then, the selective agents from the selective medium diffused to the top layer, interacted with the recovered microorganisms, and started to produce typical reactions . Foodborne pathogens were exposed to 2% acetic acid for 1, 2, or 4 min, and the recovery rate with the TAL method was compared with the rate of TSA and pathogen-specific, selective media . No significant difference occurred between TSA and TAL (P > 0.05) for enumeration of acid-injured Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica, and both recovered significantly higher numbers than the selective medium for each respective pathogen (P < 0.05) . For recovery of acid-injured Listeria monocytogenes, no difference (P > 0.05) occurred among TSA, TAL, and selective media . However, fewer cells were recovered in the selective media . The TAL method is a one-step, convenient procedure for recovery of acid-injured cells.

Microb Pathog, 2001 Aug, 31(2), 91 - 102
Salmonella typhimurium-induced cytokine production and surface molecule expression by murine macrophages; Svensson M et al.; The influence of Salmonella enterica serovar Typhimurium (S . typhimurium) on co-stimulatory molecule expression, cytokine production and induction of nitric oxide synthase (iNOS) by murine macrophages (Mphi), as well as the influence of the phoP locus on these aspects of S . typhimurium-Mphi interaction, was characterized . Pulsing Mphi with S . typhimurium resulted in increased surface expression of MHC-I, MHC-II, CD86 and CD54 . Furthermore, co-incubating S . typhimurium with Mphi resulted in interleukin (IL)-12p40, IL-6 and tumor necrosis factor-alpha production as well as iNOS induction while IL-12p70 was not detectable . Finally, although phoP did not influence the level of surface molecule expression or cytokine production by S . typhimurium-pulsed Mphi phoP did influence the level of iNOS induction . Together these data show that S . typhimurium interaction with Mphi activates these cells in ways that may enhance their ability to productively stimulate Salmonella-specific T cells following phagocytic processing and presentation of Salmonella antigens .

Scand J Infect Dis, 2001, 33(6), 420 - 2
Drug resistance of Salmonella strains isolated from community infections in Ankara, Turkey, 1993-99; Aysev AD et al.; 160 Salmonella strains were isolated from children at the paediatrics department of Ankara University . 48.1% of the isolates were Salmonella enteritidis, 41.9% Salmonella typhimurium and 10% other serotypes . For the analysis of data, the study period was divided into 2 periods: 1993-95 and 1996-99 . A decline in the isolation rate of S . typhimurium (from 63.1% to 30.1%) and rapid rise in S . enteritidis (from 31.6% to 57.3) was observed during the review period . However, for S . typhimurium isolates, the 5-drug (ampicillin, chloramphenicol, streptomycin, tetracycline and sulfonamides) pattern of resistance was increased from 13.5% to 38.7% in the second period . Since S . enteritidis and 5-drug-resistant S . typhimurium have also increased in other countries, their pandemic spread in humans indicates the continuing importation and exportation of these pathogens.

Mutat Res, 2001 Aug 22, 495(1-2), 97 - 102
Effect of various alkyl and unsaturated substituents on the mutagenicity of some nitrophenyl thioethers; Juneja TR et al.; A variety of nitro-substituted phenyl alkyl/aryl thioethers and nitroso-substituted phenyl alkyl/aryl thioethers have been synthesized and tested for their mutagenicity towards Salmonella typhimurium strain TA100, TA98, TA98NR and TA98/1,8-DNP(6) in the absence of S9 mix . The relative order of mutagenicity in TA98 and TA100 among p-nitrophenyl thioethers having alkyl or aryl substituents is allyl>phenyl>benzyl>butyl>propyl>ethyl>methyl . Compounds having an alkyl chain C(6) to C(12) were found to be non-mutagenic . Among the various positional isomers (ortho, meta and para) of nitro-substituted diphenyl thioethers only the compounds having the -NO(2) function at the para position is mutagenic, whereas compounds having a -NO(2) function at ortho and meta are non-mutagenic . However, the reduced intermediate, ortho-nitroso derivative was found to be mutagenic in all the four strains but the meta-nitroso derivative was found to be non-mutagenic . All mutagens were found to be non-mutagenic when tested in nitroreductase deficient strain TA98NR, whereas their nitroso intermediates are found to be mutagenic . A substantial fall in the mutagenic activity is observed when some mutagens are tested in O-acetyltransferase deficient strain TA98/1,8-DNP(6).

Fukushima J Med Sci, 2000 Dec, 46(1-2), 13 - 23
Opsonic function and concentration of human serum ficolin/P35; Taira S et al.; Collectins, C-type (Ca2+-dependent) animal lectins with both collagenous and carbohydrate recognition domains, function as opsonins against pathogens . We previously described an N-acetylglucosamine (GlcNAc)-binding lectin (ficolin/P35) with a collagen- and a fibrinogen-like sequence present in human serum . In this report we show that ficolin/P35 can serve as an opsonin and enhance the clearance of pathogens having surface GlcNAc . Ficolin/P35 bound to an Ra chemotype strain of Salmonella typhimurium (TV119) which has an exposed GlcNAc at the non-reducing termini of the polysaccharide . On the other hand, ficolin/P35 did not bind to LT2, a smooth type strain of S . typhimurium with additional O-polysaccharides covering GlcNAc . Ficolin/P35 enhanced the uptake of TV119 by monocytes or polymorphonuclear leukocytes but had no opsonic activity towards LT2 . These results suggest that, like collectins, ficolin/P35 is a collagenous lectin which has a role in innate immunity against certain pathogenic organisms by acting as an opsonin . We prepared monoclonal antibodies against ficolin/P35 and developed an enzyme-linked immunosorbent assay (ELISA) for measuring ficolin/P35 concentrations in humans . The mean serum concentration of ficolin/P35 from 130 normal individuals was estimated to be 13.7 microg/ml.

Bioorg Khim, 2001 May-Jun, 27(3), 184 - 90
{Protein engineering of uridine phosphorylase from Escherichia coli K-12 . II . Comparative study of hybrid and mutant forms of uridine phosphorylases}; Chebotarev DV et al.; Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed . Producing strains of the corresponding proteins were genetically engineered . Mutant forms of the E . coli K-12 UPase were produced by site-directed mutagenesis . A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed . It was shown that Asp27 unlike Asp5 and Asp29 residues of the E . coli UPase forms part of the active site of the protein . A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.

J Mol Evol, 2001 Jun, 52(6), 540 - 2
The closest BLAST hit is often not the nearest neighbor; Koski LB et al.; It is well known that basing phylogenetic reconstructions on uncorrected genetic distances can lead to errors in their reconstruction . Nevertheless, it is often common practice to report simply the most similar BLAST (Altschul et al . 1997) hit in genomic reports that discuss many genes (Ruepp et al . 2000; Freiberg et al . 1997) . This is because BLAST hits can provide a rapid, efficient, and concise analysis of many genes at once . These hits are often interpreted to imply that the gene is most closely related to the gene or protein in the databases that returned the closest BLAST hit . Though these two may coincide, for many genes, particularly genes with few homologs, they may not be the same . There are a number of circumstances that can account for such limitations in accuracy (Eisen 2000) . We stress here that genes appearing to be the most similar based on BLAST hits are often not each others closest relative phylogenetically . The extent to which this occurs depends on the availability of close relatives present in the databases . As an example we have chosen the analysis of the genomes of a crenarcheaota species Aeropyrum pernix, an organism with few close relatives fully sequenced, and Escherichia coli, an organism whose closest relative, Salmonella typhimurium, is completely sequenced.

Mol Microbiol, 2001 Jun, 40(6), 1289 - 99
AraC/XylS family members, HilC and HilD, directly bind and derepress the Salmonella typhimurium hilA promoter; Schechter LM et al.; During infection, Salmonella enterica serovar Typhimurium colonizes the small intestine of its hosts . This process requires a type III secretion system encoded by several genes on Salmonella pathogenicity island 1 (SPI1), a 40 kb region of DNA near centisome 63 of the Salmonella chromosome . SPI1 gene expression is controlled by a complex regulatory cascade . HilA, a member of the OmpR/ToxR family of transcriptional regulators, directly activates the expression of two SPI1 operons encoding type III apparatus components . hilA transcription is repressed by many environmental conditions and regulatory mutations . This repression requires an upstream repressing sequence (URS) located between -314 and -68 relative to the hilA transcription start site . The repressing activity of the URS is counteracted by two AraC/XylS family members named HilC and HilD . We show that HilC and HilD bind directly to the hilA promoter region in vitro . We also provide evidence that HilC and HilD bind to the same or overlapping sites within the URS . Our data are consistent with a model in which HilC and HilD derepress hilA expression by binding directly to the URS and counteracting its repressing effect in vivo.

J Biol Chem, 2001 Sep 7, 276(36), 34035 - 40 Epub 2001 Jul 05.
SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell; Friebel A et al.; The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion . Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell . However, it has been a puzzle why S . typhimurium might employ two toxins with redundant function . We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases . In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1 . Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1 . We conclude that the translocated effector proteins SopE and SopE2 allow S . typhimurium to specifically activate different sets of RhoGTPase signaling cascades.

Comp Immunol Microbiol Infect Dis, 2001 Jul, 24(3), 151 - 64
Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide; Chelmonska-Soyta A et al.; We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS) . After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes . However, it did not change NO synthesis by the resting monocytes . Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS . Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan . We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.

Cell Microbiol, 2001 Jul, 3(7), 473 - 86
Expression of constitutively active Rab5 uncouples maturation of the Salmonella-containing vacuole from intracellular replication; Baldeon ME et al.; The enteric bacterial pathogen Salmonella typhimurium enters and proliferates within both phagocytic and non-phagocytic host cells . Upon entry, the bacteria reside in membrane-bound vacuoles (SCVs) that mature with time, as evidenced by the sequential loss of early endosomal markers, followed by the selective recruitment of a number of lysosomal membrane glycoproteins (LAMPs) . This remodelling process renders the SCVs non-fusogenic with lysosomes and is also thought to create a vacuolar environment permissive for replication . We demonstrate that disruption of the endocytic pathway by the expression of a constitutively active form of the small GTPase rab5 (rab5Q79L) significantly altered the biogenesis of the SCVs without inhibiting bacterial replication in HeLa cells . Expression of rab5Q79L caused the retention of early endosomal markers on SCVs and early acquisition of LAMP2, and led to an increase in the kinetics of intracellular replication . We also demonstrate that a significant fraction of LAMP2 in SCVs is derived from the cell surface via endocytosis rather than via the biosynthetic route . Further, in fibroblasts lacking a functional AP3 adaptor complex, in which all newly synthesized LAMP is delivered to the cell surface, recruitment of LAMP to the SCVs remained unaffected . These findings raise the possibility that all the SCV-associated LAMP could be derived by endocytosis from the cell surface.

J Hum Virol, 2001 Mar-Apr, 4(2), 103 - 8
Mucosal immunization with Salmonella typhimurium expressing Lassa virus nucleocapsid protein cross-protects mice from lethal challenge with lymphocytic choriomeningitis virus; Djavani M et al.; OBJECTIVES: Lassa fever virus (LAS) is transmitted to man by rodent carriers and is fatal in a third of untreated cases . Our goal is to provide immune protection from Lassa fever by mucosal vaccination . STUDY DESIGN/METHODS: Mice were vaccinated intragastrically with control vectors or with vectors (vaccinia or Salmonella) expressing LAS nucleocapsid protein (NP) . Mice were challenged intracranially with a lethal dose of the related arenavirus, lymphocytic choriomeningitis virus (LCMV), as a measure of the vaccine's ability to elicit cross-protection . RESULTS: Salmonella and vaccinia vectors expressing LAS NP each protected a third of the mice from lethal challenge with LCMV . All mice vaccinated with a vector expressing LCMV NP were protected as expected . CONCLUSIONS: The LAS recombinant Salmonella vector is comparable to the LAS recombinant vaccinia vector in its ability to cross-protect mice from lethal challenge . Nucleocapsid protein is an inadequate immunogen on its own, but provides sufficient cross-protection to make it a useful component of a broadly reactive arenavirus vaccine.

Mol Plant Microbe Interact, 2001 Jul, 14(7), 857 - 66
Knockout of an azorhizobial dTDP-L-rhamnose synthase affects lipopolysaccharide and extracellular polysaccharide production and disables symbiosis with Sesbania rostrata; Gao M et al.; A nonpolar mutation was made in the oac2 gene of Azorhizobium caulinodans . oac2 is an ortholog of the Salmonella typhimurium rfbD gene that encodes a dTDP-L-rhamnose synthase . The knockout of oac2 changed the lipopolysaccharide (LPS) pattern and affected the extracellular polysaccharide production but had no effect on bacterial hydrophobicity . Upon hot phenol extraction, the wild-type LPS partitioned in the phenol phase . The LPS fraction of ORS571-oac2 partitioned in the water phase and had a reduced rhamnose content and truncated LPS molecules on the basis of faster migration in detergent gel electrophoresis . Strain ORS571-oac2 induced ineffective nodule-like structures on Sesbania rostrata . There was no clear demarcation between central and peripheral tissues, and neither leghemoglobin nor bacteroids were present . Light and electron microscopy revealed that the mutant bacteria were retained in enlarged, thick-walled infection threads . Infection centers emitted a blue autofluorescence under UV light . The data indicate that rhamnose synthesis is important for the production of surface carbohydrates that are required to sustain the compatible interaction between A . caulinodans and S . rostrata.

J Biol Chem, 2001 Aug 31, 276(35), 32984 - 9 Epub 2001 Jul 02.
Location of the receptor-interaction site on CheB, the methylesterase response regulator of bacterial chemotaxis; Barnakov AN et al.; Sensory adaptation in bacterial chemotaxis is mediated by covalent modification of chemoreceptors, specifically methylation and demethylation of glutamates catalyzed by methyltransferase CheR and methylesterase CheB . The methylesterase is a two-domain response regulator in which phosphorylation of the regulatory domain enhances activity of the catalytic domain . In Escherichia coli and Salmonella typhimurium, a crucial determinant of efficient methylation and demethylation is a specific pentapeptide sequence at the chemoreceptor carboxyl terminus, a position distant from sites of enzymatic action . Each enzyme binds pentapeptide, but the site of binding has been located only for CheR . Here we locate the pentapeptide-binding site on CheB by assessing catalytic activity and pentapeptide binding of CheB fragments, protection of CheB from proteolysis by pentapeptide, and interference with pentapeptide-CheB interaction by a CheB segment . The results place the binding site near the hinge between regulatory and catalytic domains, in a segment spanning the carboxyl-terminal end of the regulatory domain and the beginning of the linker that stretches to the catalytic domain . This location is quite different from the catalytic domain location of the pentapeptide-binding site on CheR and is likely to reflect the rather different ways in which pentapeptide binding enhances enzymatic action for the methyltransferase and the methylesterase.

Microbiology, 2001 Jul, 147(Pt 7), 1897 - 907
A chromosomal region surrounding the ompD porin gene marks a genetic difference between Salmonella typhi and the majority of Salmonella serovars; Santiviago CA et al.; In this work it is shown that the majority of Salmonella serovars most frequently associated with the systemic infection of vertebrate hosts produce a major outer-membrane porin, OmpD . However, OmpD is absent from the outer-membrane protein profiles of Salmonella typhi strain Ty2 and 26 clinical isolates of S . typhi examined by SDS-PAGE . To determine whether the ompD gene is present in S . typhi, primers internal to the ompD coding sequence were used to amplify the gene by PCR . With the exception of S . typhi strains, the ompD gene was amplified from the genomes of all Salmonella serovars tested . Consistently, a specific ompD probe did not hybridize with DNA isolated from the S . typhi strains . Taken together, these results demonstrate that S . typhi does not produce OmpD due to the absence of the ompD gene . Furthermore, it was investigated whether the deletion of ompD extended to smvA . This gene is adjacent to ompD in the Salmonella typhimurium chromosome and encodes a protein involved in the resistance to methyl viologen, a superoxide-generating agent . Although PCR failed to amplify the smvA gene from the S . typhi strain Ty2 genome, it was possible to amplify it from the chromosome of the clinical strains . On the other hand, hybridization analyses showed that the smvA gene is present in all the S . typhi strains tested . In contrast to the other Salmonella serovars, S . typhi strain Ty2 and the clinical isolates showed sensitivity to methyl viologen, suggesting that smvA gene is inactive in S . typhi . In conclusion, the ompD-smvA region is variable in structure among Salmonella serovars . It is hypothesized that the absence of ompD may suggest a role in host specificity.

Vaccine, 2001 Jul 16, 19(28-29), 4028 - 35
Protective efficacy against tuberculosis of ESAT-6 secreted by a live Salmonella typhimurium vaccine carrier strain and expressed by naked DNA; Mollenkopf HJ et al.; We have constructed a recombinant (r) attenuated Salmonella typhimurium strain which secretes ESAT-6 of Mycobacterium tuberculosis via the hemolysin secretion system of E . coli . Additionally, we have ligated ESAT-6 to different commercially available mammalian expression systems for use as naked DNA vaccines . We studied protection against M . tuberculosis induced by vaccination with each of these constructs alone or in combination in mice . Vaccination with a single dose of r S . typhimurium secreting ESAT-6 reduced numbers of tubercle bacilli in the lungs throughout the course of infection . The combined prime-boost vaccination did not considerably enhance protection.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 229 - 33
Lipid modification of prelipoproteins is dispensable for growth in vitro but essential for virulence in Streptococcus pneumoniae; Petit CM et al.; A Deltalgt (Lgt, lipoprotein diacylglyceryl transferase) isogenic mutant was obtained which indicates that lgt is not essential for cell growth in vitro, like in the Gram-positive bacterium Bacillus subtilis, but unlike in the proteobacteria Escherichia coli and Salmonella typhimurium . The mutation was transduced to a virulent strain . A 5 log attenuation was observed in a respiratory tract model of infection . Metabolic labeling by {U-14C}palmitate revealed the presence of eight to ten lipoproteins in the wild-type strain only, with molecular masses between 15 and 80 kDa . Our findings suggest a major difference in the role of lipoproteins in Gram-positive bacteria versus the proteobacteria.

Environ Mol Mutagen, 2001, 37(4), 324 - 8
Mutagenicity of 2-alkylpropenals in Salmonella typhimurium strain TA 100: structural influences; Eder E et al.; alpha,beta-Unsaturated aldehydes are a class of mutagenic and carcinogenic compounds that form promutagenic 1,N(2)-propanodeoxyguanosine adducts . They are important industrial and environmental compounds, are formed endogenously, and are found in food . We recently published structure-mutagenicity relationships for 3-alkyl substituted alpha,beta-unsaturated aldehydes (beta-alkylacroleins) and here we present structural influences on the mutagenicity of the 2-alkyl substituted alpha,beta-unsaturated aldehydes (alpha-alkylacroleins), 2-methylacrolein, 2-ethylacrolein, 2-propylacrolein, and 2-butylacrolein, in Salmonella typhimurium TA 100 . All four alkylacroleins are mutagenic without S9-mix; however, the results are strongly influenced by bacterial toxicity of the alkylacroleins . In general, toxicity increases with increasing length of the alkyl substituent . The increasing toxicity with increasing alkyl groups can be explained by increasing lipophilicity that allows the compounds to better penetrate into the bacterial cell . Other structural effects, such as steric hindrance of the deoxyguanosine binding (DNA-adduct formation) and the positive inductive effect of the alkyl groups, have only a slight effect on mutagenesis . Addition of S9-mix leads to an increase in the absolute revertant peak values but a decrease in mutagenic activities, as expressed by revertants per micromol . This effect is also observed with heat-inactivated S9-mix and does not depend on metabolic activation . The effect of S9-mix can be explained by partial detoxication of the substances by nucleophilic components of the S9-mix such as glutathione .

Mutat Res, 2001 Jul 25, 494(1-2), 41 - 53
Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate; Clarke JJ et al.; Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion . Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA . Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use . Such toxicology studies include mutagenicity assays . Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays . In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S . typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation . QH was not mutagenic in S . typhimurium tester strains TA100 and TA1535 with and without S9-activation . QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation . QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation . QH was negative for polyploidy in the same chromosome aberration test . Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay . These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

Cell Microbiol, 2001 Jun, 3(6), 417 - 26
Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells; Jepson MA et al.; Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1) . Several protein targets of the SPI1-encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence . We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin-Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min) . The invasion defect associated with sipA was no longer apparent after 15 min of infection . Confocal microscopy of F-actin in tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin-stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild-type and sipA mutant strains of S . typhimurium . Time-lapse phase-contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild-type bacteria . These studies also revealed that, after ruffle propagation, individual sipA mutant S . typhimurium either invaded more slowly than wild-type bacteria or failed to invade at all . Furthermore, although wild-type S . typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it . A wild-type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid . Together, these data indicate that loss of sipA significantly decreases the efficiency of S . typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles . It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.

Eur J Surg, 2001 May, 167(5), 366 - 70
Effect of granulocyte-macrophage colony stimulating factor on bacterial translocation after experimental obstructive jaundice; Unal AE et al.; OBJECTIVE: To investigate the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on bacterial translocation promoted by obstructive jaundice . DESIGN: Controlled animal study . SETTING: University hospital, Turkey . ANIMALS: 30 male Wistar albino rats . INTERVENTIONS: The first group (n = 10) was the sham operation (control) group, and the second and the third (n = 10 each) had common bile duct (CBD) ligation and division under sterile conditions . The third group were also treated with GM-CSF 200 ng subcutaneously daily between the fifth and ninth postoperative days . All animals were killed on the tenth day, and evaluated biochemically and histopathologically . Mesenteric lymph nodes were cultured under aerobic conditions . MAIN OUTCOME MEASURES: Biochemical analysis, histopathological evaluation, and aerobic cultures . RESULTS: There was no bacterial translocation in either the control or GM-CSF groups, whereas Escherichia coli and Salmonella typhimurium were found in 4 and 2 animals, respectively in the ligation group . Although no aerobic bacteria was found in controls and the GM-CSF groups, bacterial translocation was 6/10 in the ligation alone group (p <0.01) . CONCLUSION: Activation of inflammatory response with GM-CSF is highly effective in prevention of bacterial translocation in obstructive jaundice.

J Immunol, 2001 Jul 1, 167(1), 357 - 65
Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing hybrid Yersinia type III proteins; Russmann H et al.; In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium . Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes . In vitro experiments showed that S . typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion . Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides . Mice orally vaccinated with a single dose of attenuated S . typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-gamma-producing cells reactive with listeriolysin O 91-99 or p60 217-225, respectively . This CD8 T cell response protected mice against a challenge with L . monocytogenes . In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains.

Biochim Biophys Acta, 2001 Jul 2, 1506(1), 1 - 11
The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium; Keyhani E et al.; This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding . Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra . V(max) of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0+/-2.0 pmol/min/mg protein (prot.) in intact cells, 37.0+/-3.5 pmol/min/mg prot . in spheroplasts, 125.0+/-6.0 pmol/min/mg prot . in membrane fragments, and 538.0+/-8.5 pmol/min/mg prot . in solubilized cytochrome d . The pseudo-first order rate constants were 0.004 s(-1) for intact cells, 0.005 s(-1) for spheroplasts, 0.007 s(-1) for membrane fragments and 0.025 s(-1) for the solubilized enzyme . The V(max) value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments . The K(s) of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d . This difference between the K(s) values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme . The K(s) value was nearly the same at all pH investigated (pH 5-10) . Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments (K(s)=18+/-3 mM, V(max)=377+/-28 pmol/min/mg prot . at pH 7) and the solubilized enzyme (K(s)=18+/-1.2 mM, V(max)=649+/-45 pmol/min/mg prot . at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme . Pseudo-first order rate constants were 0.025 s(-1) and 0.042 s(-1) respectively for membrane fragments and solubilized enzyme . The value of V(max) for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6-10) while the K(s) value dropped significantly with increasing pH . The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction . Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.

J Agric Food Chem, 2001 Jun, 49(6), 3046 - 50
Structural analysis of a novel antimutagenic compound, 4-Hydroxypanduratin A, and the antimutagenic activity of flavonoids in a Thai spice, fingerroot (Boesenbergia pandurata Schult.) against mutagenic heterocyclic amines; Trakoontivakorn G et al.; Six compounds were isolated from fresh rhizomes of fingerroot (Boesenbergia pandurata Schult.) as strong antimutagens toward 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) in Salmonella typhimurium TA98 . These compounds were 2',4',6'-trihydroxychalcone (pinocembrin chalcone; 1), 2',4'-dihydroxy-6'-methoxychalcone (cardamonin; 2), 5,7-dihydroxyflavanone (pinocembrin; 3), 5-hydroxy-7-methoxyflavanone (pinostrobin; 4), (2,4,6-trihydroxyphenyl)-{3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl}methanone (5), and (2,6-dihydroxy-4-methoxyphenyl)-{3'-methyl-2'-(3' '-methylbut-2' '-enyl)-6'-phenylcyclohex-3'-enyl}methanone (panduratin A; 6) . Compound 5 was a novel compound (tentatively termed 4-hydroxypanduratin A), and 1 was not previously reported in this plant, whereas 2-4 and 6 were known compounds . The antimutagenic IC(50) values of compounds 1-6 were 5.2 +/- 0.4, 5.9 +/- 0.7, 6.9 +/- 0.8, 5.3 +/- 1.0, 12.7 +/- 0.7, and 12.1 +/- 0.8 microM in the preincubation mixture, respectively . They also similarly inhibited the mutagenicity of 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . All of them strongly inhibited the N-hydroxylation of Trp-P-2 . Thus, the antimutagenic effect of compounds 1-6 was mainly due to the inhibition of the first step of enzymatic activation of heterocyclic amines.

J Agric Food Chem, 2001 Jun, 49(6), 2767 - 73
Isolation and characterization of structurally novel antimutagenic flavonoids from spinach (Spinacia oleracea); Edenharder R et al.; Thirteen compounds, isolated from spinach (Spinacia oleracea), acted as antimutagens against the dietary carcinogen 2-amino-3-methylimidazo{4,5-f}quinoline in Salmonella typhimurium TA 98 . The antimutagens were purified by preparative and micropreparative HPLC from a methanol/water (70:30, v/v) extract of dry spinach (commercial product) after removal of lipophilic compounds such as chlorophylls and carotenoids by solid-phase extraction (SPE) . Pure active compounds were identified by instrumental analysis including FT-IR, (1)H and (13)C NMR, UV-vis spectroscopy, and mass spectrometry . All of these compounds were flavonoids and related compounds that could be attributed to five groups: (A, methylenedioxyflavonol glucuronides) 5,3'-dihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 1), 5,2',3'-trihydroxy-4'-methoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 2), 5-hydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavonol 3-O-beta-glucuronide (compound 3); (B, flavonol glucuronides) 5,6,3'-trihydroxy-7,4'-dimethoxyflavonol 3-O-beta-glucuronide (compound 4), 5,6-dihydroxy-7,3',4'-trimethoxyflavonol 3-O-beta-glucuronide (compound 5); (C, flavonol disaccharides) 5,6,4'-trihydroxy-7,3'-dimethoxyflavonol 3-O-disaccharide (compound 6), 5,6,3',4'-tetrahydroxy-7-methoxyflavonol 3-O-disaccharide (compounds 7 and 8); (D, flavanones) 5,8,4'-trihydroxyflavanone (compound 9), 7,8,4'-trihydroxyflavanone (compound 10); (E, flavonoid-related compounds) compounds 11, 12, and 13 with incompletely elucidated structures . The yield of compound 1 was 0.3%, related to dry weight, whereas the yields of compounds 2-13 ranged between 0.017 and 0.069% . IC(50) values (antimutagenic potencies) of the flavonol glucuronides ranged between 24.2 and 58.2 microM, whereas the flavonol disaccharides (compounds 7 and 8), the flavanones (compounds 9 and 10), and the flavonoid-related glycosidic compounds 11-13 were only weakly active . The aglycons of compounds 7 and 8, however, were potent antimutagens (IC(50) = 10.4 and 13.0 microM, respectively).

Chem Res Toxicol, 2001 Jun, 14(6), 686 - 93
Metabolic activation of benzo{c}phenanthrene by cytochrome P450 enzymes in human liver and lung; Baum M et al.; The environmentally occurring polycyclic aromatic hydrocarbon (PAH) benzo{c}phenanthrene (B{c}PH) is a weak carcinogen in rodents . In contrast, the dihydrodiol-epoxides of B{c}PH are among the most carcinogenic PAH metabolites tested so far . In rodents, B{c}PH is predominantly metabolized to B{c}PH-5,6-dihydrodiol (B{c}PH-5,6-DH) and only to a minor extent to B{c}PH-3,4-DH, the proximate precursor of the highly potent ultimate carcinogen, B{c}PH-3,4-DH-1,2-epoxide . This might explain why in rodents B{c}PH is a weak carcinogen . However, little is known about human metabolism of B{c}PH . Using microsomal preparations from human liver and lung, we investigated the metabolic activation of B{c}PH . In contrast to the findings in experimental animals, human liver microsomes predominantly generated B{c}PH-3,4-DH and only to a minor extent B{c}PH-5,6-DH . Only one lung tissue sample was found to be metabolically active, producing B{c}PH-5,6-DH together with small amounts of B{c}PH-3,4-DH . Catalytic activities known to be associated with specific cytochrome P450 (P450) enzyme activities were determined and correlated with the spectrum of B{c}PH metabolites . The results indicate that B{c}PH-DH formation in human liver is mainly mediated by P450 1A2 . Studies with P450 enzyme selective inhibitors confirmed these findings . Further support was obtained using preparations of the respective human recombinant P450 enzymes expressed in Escherichia coli and yeast . In addition to P450 1A2, P450 1B1 effectively mediated B{c}PH-metabolism . The umu-assay for induction of SOS repair response in Salmonella typhimurium TA 1535 pSK 1002 containing a umuC-lacZ reporter gene was used to study metabolic generation of genotoxic metabolites from B{c}PH-DHs in human microsomal preparations . B{c}PH-3,4-DH was activated by human liver microsomes to a potent genotoxic agent . Taken together, the results clearly demonstrate that human liver microsomes can effectively catalyze the biotransformation of B{c}PH into highly genotoxic metabolites . The results provide evidence that B{c}PH should be considered a potentially potent carcinogen in humans, and that rodent models may underestimate the risk.

Chem Res Toxicol, 2001 Jun, 14(6), 661 - 71
An evaluation of the mutagenicity, metabolism, and DNA adduct formation of 5-nitrobenzo{b}naphtho{2,1-d}thiophene; King LC et al.; Thioarenes, sulfur-containing polycyclic aromatic compounds, are environmental contaminants suspected of posing human health risks . In this study, 5-nitrobenzo{b}naphtho{2,1-d}thiophene (5-nitro-BNT), a nitrated-thioarene, was examined for its mutagenicity, metabolism and subsequent formation of DNA adducts . 5-Nitro-BNT was weakly mutagenic in Salmonella typhimurium strains TA98 and TA100 without Aroclor-1254-induced rat liver S9 (S9), and its activity was increased in the presence of S9 . Anaerobic metabolism of 5-nitro-BNT by S9 or xanthine oxidase (XO) produced one major metabolite, identified as 5-amino-BNT by NMR, MS, and UV spectroscopy and by comparison with an authentic standard . Aerobic S9 metabolism of 5-nitro-BNT produced a major metabolite, identified as trans-9,10-dihydroxy-9,10-dihydro-5-nitro-BNT (5-nitro-BNT-9,10-diol) . Also present was a minor amount of 5-amino-BNT and trans-9,10-dihydroxy-9,10-dihydro-5-amino-BNT (5-amino-BNT-9,10-diol) . DNA adduct analyses were performed using the (32)P-postlabeling assay and reversed-phase HPLC . Three major XO-derived calf thymus DNA adducts were detected . On the basis of their chromatographic mobilities, two adducts were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one adduct with 2'-deoxyadenosine . Incorporation of allopurinol (a specific XO inhibitor) in the incubation mixture resulted in loss of all three adducts, confirming enzymatic mediation by XO . Aerobic S9 activation of 5-nitro-BNT with calf thymus DNA produced three adducts . On the basis of their chromatographic mobilities, two were identified as reaction products of 5-nitro-BNT with 2'-deoxyguanosine and one with 2'-deoxyadenosine . Incorporation of 1-aminobenzotriazole (a P450 inhibitor) in the incubation mixture resulted in a loss of these adducts, confirming enzymatic mediation by P450 . Aerobic S9-catalyzed metabolism of 5-nitro-BNT-9,10-diol produced the same DNA adducts as observed with 5-nitro-BNT . Aerobic S9-catalyzed metabolism of 5-amino-BNT-9,10-diol produced the same deoxyadenosine-derived DNA adducts as observed with 5-nitro-BNT and 5-nitro-BNT-9,10-diol . These results provide additional information that both ring oxidation and nitroreduction are involved in the metabolism, DNA adduct formation and mutagenicity of 5-nitro-BNT.

J Biol Chem, 2001 Aug 24, 276(34), 32230 - 9 Epub 2001 Jun 18.
Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane . Functional analysis of IpaC; Kuwae A et al.; Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death . The bacterial strategy of macrophage infection, however, remains largely speculative . Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria . When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000 . J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester . Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli . The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining . The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence . Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.

Biol Chem, 2001 Apr, 382(4), 533 - 41
Bacteria-mediated transfer of eukaryotic expression plasmids into mammalian host cells; Weiss S et al.; Invasive intracellular bacteria are able to transfer eukaryotic expression plasmids into mammalian host cells in vitro and in vivo . This can be used to induce immune responses toward protein antigens encoded by the plasmid or to complement genetic defects . Plasmid transfer takes place when the recombinant bacterium dies within the host cell, either due to metabolic attenuation or induction of autolysis . Alternatively, antibiotics can be used and spontaneous transfer has also been observed, indicating that this phenomenon might also occur under physiological conditions . Plasmid transfer has been reported for Shigella flexneri, Salmonella typhimurium and S . typhi, Listeria monocytogenes and recombinant Escherichia coli, but other invasive bacteria should also share this property . In vivo attempts were mainly directed toward vaccination using shigella and salmonella as carrier . So far a wide variety of antigens have been used succesfully in mice . Often this type of immunization was superior over direct application of antigen or using the same bacterium as a heterologous carrier expressing the antigen via a prokaryotic promoter . Characterization of the host cells revealed that macrophages and dendritic cells might be responsible for immune stimulation by either expressing the antigen or cross-presenting the antigen after uptake of apoptotic antigen expressing cells.

J Acquir Immune Defic Syndr, 2001 May 1, 27(1), 7 - 13
A novel recombinant marker virus assay for comparing the relative fitness of hiv-1 reverse transcriptase variants; Lu J et al.; A novel recombinant marker virus assay (RMVA) has been developed to perform growth competition assays for assessing fitness of HIV-1 . This assay allowed the generation of replication-competent viruses by homologous recombination of polymerase chain reaction (PCR)-derived reverse transcriptase (RT) coding sequences in RT-deleted proviral clones of HIV-1 in which the nef gene was replaced by the Salmonella typhimurium histidinol dehydrogenase (hisD) or human heat-stable placental alkaline phosphatase (PLAP) gene (pHIVDeltaRTBalIDeltanefhisD and pHIVDeltaRTBalIDeltanefPLAP, respectively) . The proportion of a given RT species in a mixed culture was determined by quantifying the linked hisD or PLAP marker gene using real-time PCR . The RMVA was tested by comparing the relative fitness of wild-type and lamivudine-resistant recombinant viruses . The RMVA reproducibly detected differences in the fitness of these two viruses in growth competition assays . With appropriate modification of the recombination vectors, the RMVA should be useful for analyzing the fitness of viruses resistant to protease, integrase, or fusion inhibitors and should be applicable in clinical research.

Bioorg Med Chem, 2001 Jun, 9(6), 1509 - 15
Synthesis and structure--mutagenicity relationship of benzo-annulated cyclopentaphenanthrenes; Marrocchi A et al.; The synthesis of 2,3-dihydro-1H-indeno{5,4-a}anthracene (2), the fluoreno{a}anthracenes 3 and 4, 2,3-dihydro-1H-cyclopenta{a}chrysene (6), 3,4-dihydro-2-vinylphenanthrene (10) and cyclopenta{c}chrysenes 11, 12 has been described . Structure analysis of the new products by (1)H and (13)C NMR spectroscopy is presented . Estimates of the mutagenic activity of compounds 2--4, 6 and 11--14 in Salmonella typhimurium determined by Ames' test indicate that all products are inactive for both TA 98 and TA 100 strains except 4,5-dihydro-3H-cyclopenta{c}chrysene (12) . The mutagenic properties of these compounds have been compared with those shown by previously studied benzo{g}cyclopenta{a}phenanthrenes and cyclopenta{c}phenanthrenes and discussed . Some conclusions have been drawn about the effects of benzoannulation and of the carbonyl function on the mutagenicity of this class of compounds.

Phytother Res, 2001 Jun, 15(4), 360 - 3
Mutagenicity and antioxidant assessment of Stachitarpheta jamaicensis (L.) Vahl; Ramos A et al.; Stachitarpheta jamaicensis (L.) Vahl . is a member of the Verbenaceae commonly used in Cuba, mainly as vermifugue and against diarrhoea . The mutagenic potential of a hydroalcohol extract of its aerial parts was assessed in vitro using the Salmonella/microsome assay and in vivo in the mouse bone marrow micronucleus test . No positive response was observed in a battery of four Salmonella typhimurium strains employed: TA 1535, TA 1537, TA 98 and TA 100, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation . In the same way, no increase in the micronucleus frequency in mitotic erythropoietic tissue was observed when animals were administered the extract orally in doses of 500, 1000 and 2000 mg/kg . The extract inhibited lipid peroxidation in the rat liver microsomal fraction (IC(50) = 3.6 microg/mL) but it does not seem to be an effective.OH radical scavenger (IC(50) = 76.7 microg/mL) . Noteworthy, it increased in a dose dependent way the level of revertant colonies in E . coli IC 203, a strain sensitive to oxidative mutagenesis, when assayed together with hydrogen peroxide and ferrous sulphate, which suggests a pro-oxidant action .

Pharmazie, 2001 May, 56(5), 412 - 4
Evaluation of mutagenic activity of several antimalarial extracts from Eupatorium inulaefolium; Pabon A et al.; Eupatorium inulaefolium is used as an antimalarial agent by traditional healers of the Tumaco region (Narino-Colombia) . Several extracts of this plant have been tested by our laboratory and in vitro antimalarial activity against the FCB-2 strain of Plasmodium falciparum has been confirmed . For this reason, the mutagenic effect of the methanol, dichloromethane, and hexane extracts of Eupatorium inulaefolium (number 83377 university of Antioquia herbarium) were evaluated using the Ames test . None of the extracts evaluated had mutagenic effects on TA-98 or TA-100 strains of Salmonella typhimurium.

J Toxicol Sci, 2001 May, 26 Suppl 1, 243 - 54
{Genotoxicity studies of cefmatilen hydrochloride hydrate (S-1090)}; Kondo K et al.; Cefmatilen hydrochloride hydrate (S-1090), a new non-ester type of orally active cephem antibiotic synthesized in Shionogi Research Laboratories, was evaluated for its genotoxic potential using three assay systems . In a reverse mutation test with bacteria of Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2uvrA using the preincubation method, the number of revertant colonies in the S-1090 treated plates was almost equal to that in the negative control plates in all strains with and without metabolic activation system with S9 mix (maximum dose, 100 micrograms/plate in TA98) . In a chromosomal aberration test with cultured Chinese hamster lung cells (CHL/IU), S-1090 did not induce structural chromosome aberrations or polyploid cells either in the absence or presence of S9 mix up to the 50% growth inhibition doses . The potential of inducing clastogenicity and/or disruption of mitotic apparatus in vivo by S-1090 was evaluated by a micronucleus test with bone marrow cells of male Jc1:ICR mice . S-1090 suspended in 0.5% aqueous methylcellulose was administered by oral gavage up to 2000 mg/kg/day in single and double dosing groups . No induction of micronucleated polychromatic erythrocytes was observed 24 hr after the last dosing in each group . As all three genotoxicity tests showed negative responses, S-1090 is thought to have no genotoxic potential.

Chem Biol Interact, 2001 Jun 1, 135-136, 703 - 13
In vitro genotoxicity testing of (1-chloroethenyl)oxirane, a metabolite of beta-chloroprene; Himmelstein MW et al.; (1-Chloroethenyl)oxirane (CEO) is a metabolite of beta-chloroprene (2-chloro-1,3-butadiene, CD) . The purpose of this study was to evaluate the in vitro mutagenic and clastogenic (chromosome breaking) potential of CEO . For comparative purposes, the study also included an evaluation of the racemic compounds, 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB) . Mutagenicity was evaluated in a bacterial reverse mutation test (Ames), using the pre-incubation method in the presence and absence of an exogenous metabolism system (Aroclor)-induced rat liver S9) . Four Salmonella typhimurium tester strains, TA97a, TA98, TA100 and TA1535 were used . The exposure concentrations in the sealed incubation vials ranged from 0 to 69 mM for CEO, 0 to 102 mM for EB, and 0 to 83 mM for DEB . All three compounds showed signs of toxicity, with DEB being substantially more toxic than either CEO or EB . Mutagenic activity was observed with all three chemicals in primarily the base pair substitution strains (S . typhimurium TA100 and TA1535), but some activity was also seen in the frameshift elimination strains (S . typhimurium TA97a and TA98) . The observed mutagenic responses after exposure with CEO or EB were greater than the observed response for DEB, most likely because of the higher toxicity of DEB . Generally, the mutagenic responses were unchanged in the frameshift strains and base pair substitution strains in the presence of S9 metabolism . In vitro clastogenicity was evaluated using the cytochalasin-B blocked micronucleus test in cultured Chinese hamster V79 cells . The test was conducted without S9 metabolism because of the absence of substantial changes in the Ames test . Exposure concentrations ranged from 0 to 0.943 mM for CEO, 0 to 3.0 mM for EB, and 0 to 0.035 mM for DEB, with the upper exposure concentrations dictated by cytotoxicity . Cytotoxicity, measured as a reduction in the proportion of binucleated cells and altered cell morphology, was observed for CEO at concentrations > or =0.175 mM . Exposure to EB led to a reduced proportion of binucleated cells at concentrations > or =2.0 mM, and cell death was observed after DEB exposure at concentrations > or =0.025 mM . No clastogenicity was observed in the V79 cells when tested up to cytotoxic concentrations of CEO, whereas an elevated frequency of micronuclei was observed after exposure to either EB (> or =1.0 mM) or DEB (> or =0.0125 mM) . These results suggest that CEO-induced mutagenicity, but not clastogenicity, may contribute to the observed beta-chloroprene-induced carcinogenicity in the rodent bioassay studies.

Chem Biol Interact, 2001 Jun 1, 135-136, 65 - 80
Genetic and reproductive toxicity of butadiene and isoprene; Anderson D; Butadiene (BD) and its 2-methyl analogue, isoprene, have been extensively studied in animals and BD in population studies . Both chemicals are metabolised by liver cytochrome P450 dependent monogenases to monoepoxide and diepoxide intermediates . The diepoxide intermediates of both compounds were mutagenic in Salmonella typhimurium . However, unlike the monoepoxide of BD, the monoepoxides of isoprene were not mutagenic . It appears that they have no alkylating capacity . BD did not induce somatic cell mutation and recombination or sex-linked recessive lethal mutation in Drosophila melanogaster and isoprene produced no increase in chromosomal aberrations in CHO cells in vitro . Comparative concentrations of haemoglobin adducts in the blood of mice and rats after exposure to BD indicated that reaction with blood may decrease the levels of reactive intermediates available to tissues in rats, but not in mice contributing to greater potency of BD in the mouse . For isoprene, the adducts reach approximately the same concentrations in both species . DNA adducts have also been detected in testicular and lung cells of mice after BD exposure . The level of epoxybutene haemoglobin adducts was significantly elevated in BD-exposed workers, but lower than in rats and mice . In conjunction with the toxicology and carcinogenesis studies for BD and isoprene, additional mice were included for the evaluation of cytogenetic effects . Both chemicals produced increases in sister chromatid exchanges in bone marrow cells and in the frequency of micronuclei in normochromatic and polychromatic erythrocytes, but only BD produced an increase in the percent of bone marrow cells with chromosomal aberrations . At similar doses, the effects with BD were 2-3 times larger than with isoprene . There were also increased hprt mutation frequencies in rats and mice after BD exposure . Biomonitoring studies with hprt mutations in lymphocytes showed conflicting results, with both positive and negative findings . BD has been shown to be positive in one human cytogenetic biomonitoring study and not in three others, but chromosomal aberrations were increased in BD-exposed workers after challenge with gamma rays . Re-analysis of GSTTI null individuals showed positive results . There was an increase in spermatid micronuclei in mice by BD and its metabolites and in rats only by its metabolites . The cytotoxic response of germ cells in mice is greater than in rats . Dominant lethal mutations have been induced by BD and diepoxybutane, but not by epoxybutene . There was some evidence of congenital malformations in mice after BD exposure and there was a linear concentration-related induction of heritable translocations in mice . There was no induction of dominant lethal mutations or congenital malformations in rats . Using the heritable translocation data in mice, it has been determined that if a worker is continually exposed over 5 or 6 weeks to 20-25 ppm of BD, the risk of producing a child with a balanced reciprocal translocation is twice as high as the background risk . Since genetic damage cannot be measured directly in human germ cells, risk to such cells can also be estimated from germ cells and somatic cells of the mouse and human somatic cells using the parallelogram approach . Using doubling doses, the fourth corner of the parallelogram was calculated as a doubling dose for human germ cells of 4390 ppm/h . However, it is still questioned if man is more like rat than mouse in terms of sensitivity to exposure . Similar germ cell data do not exist for isoprene . In conventional developmental studies, where rats and mice were exposed to BD, maternal toxicity was shown in rats but there was no evidence of developmental toxicity or teratogenic effects and there was a small effect on sperm morphology . After exposure to isoprene, there was no adverse effect on rat dams or other reproductive indices . In mice, there was reduced foetal body weight and decreased maternal weight gain and isoprene also affected ovarian follicles . There was a reduction in testicular function parameters such as testicular weight and sperm motility.

Acta Biol Hung, 2001, 52(1), 171 - 8
Evaluation of genotoxity and mutagenicity of DL-p-chlorophenylalanine, its methyl ester and some N-acyl derivatives; Straukas J et al.; DL-p-chlorophenylalanine (PCPA) and its derivatives were evaluated for genotoxic effects using Escherichia coli and Bacillus subtilis strains lacking various DNA-repair mechanisms in spottest and in suspension test . The mutagenic activity of studied compounds was determined by the Ames test . Reverse mutation test was performed with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 without S9 mix . 0.02 M nitrosomethylurea (NMU) standard mutagen was used as a positive control . The results showed that the parent nonessential amino acid PCPA had no detectable genotoxic and mutagenic activities in bacteria . The methyl ester of this amino acid and its N-phenylacetyl derivative possessed weak genotoxicity . Meanwhile N-sec-butyloxycarbonyl, N-benzyloxycarbonyl, N-(p-nitrophenylacetyl) and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine exhibited appreciable genotoxicity . Among the seven tested compounds only N-benzyloxycarbonyl and N-(p-nitrophenoxyacetyl) derivatives of DL-p-chlorophenylalanine have been found to be mutagenic . Only parent PCPA possessed antimutagenic properties in respect of nitrosomethylurea . The structural modification, which strongly affects genotoxicity and mutagenicity perhaps may be due to steric hydrance of the substituents, causing interference with enzyme and DNA interactions.

J Biol Chem, 2001 Aug 10, 276(32), 30521 - 6 Epub 2001 May 31.
Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells; Ogushi K et al.; Antimicrobial peptides are crucial for host defense at mucosal surfaces . Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined . Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells . Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S . enteritidis was isolated . The supernatant factor was heat-stable and proteinase-sensitive . After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S . enteritidis . Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells . In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody . In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences . We conclude that S . enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

J Vet Med A Physiol Pathol Clin Med, 2001 Apr, 48(3), 153 - 63
Effects of indomethacin on Salmonella typhimurium- and cholera toxin-induced fluid accumulation in the porcine small intestine; Unmack MA et al.; The effect of the cyclooxygenase and prostaglandin E2 (PGE2) synthesis inhibitor, indomethacin, on the secretory responses induced by Salmonella serotype Typhimurium (ST) and cholera toxin (CT), in the porcine small intestine was investigated . ST (10(10) colony-forming units) and CT (56 micrograms) were instilled in tied-off intestinal loops in young anaesthetized pigs receiving intravenous indomethacin in a total dose of 7.5 mg/kg, or saline . The accumulated fluid in the loops and the luminal content of endogenous secretagogues PGE2 and 5-hydroxytryptamine (5-HT) were measured . ST induced fluid accumulation in the jejunum, whereas CT induced fluid accumulation in the jejunum and ileum . Indomethacin had no effect on the secretory responses . Indomethacin had a significant effect on the luminal content of PGE2 in jejunal ST and CT loops, whereas no effect of indomethacin was observed on the luminal content of 5-HT in ST and CT loops . In ST and CT loops, an increased content of PGE2 and 5-HT compared with test loops infused with Ringer's solution was observed . These results indicate that the porcine jejunal secretory response to ST and CT does not involve prostaglandins although indomethacin has an influence on the luminal release of PGE2 but not of 5-HT.

FEMS Microbiol Lett, 2001 May 30, 199(2), 215 - 9
Genetic variability among archival cultures of Salmonella typhimurium; Edwards K et al.; The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies . In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage . Considerable genetic variability was observed in these populations . Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.

Mutat Res, 2001 May 31, 492(1-2), 81 - 90
Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase; Oda Y et al.; We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S . typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT) . These strains were established by introducing two plasmids into S . typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes . Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested . O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein . All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo {4,5-f}quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains . 3-Amino-1,4-dimethyl-5H-pyrido{4,3-b}-indole and 3-amino-1-methyl-5H-pyrido{4,3-b}-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains . Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains . beta-Naphthylamine and benzo{a}pyrene did not exhibit genotoxicity in any of the strains . These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.

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