Salmonella typhimurium

Mutat Res, 2001 May 31, 492(1-2), 73 - 80
Identification of 2-{2-(acetylamino)-4-amino-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) as a potent mutagen in river water in Kyoto and Aichi prefectures, Japan; Nukaya H et al.; We have previously isolated five mutagens in blue rayon-adsorbed substances from water at a site below sewage plants in the Nishitakase River, in Kyoto, Japan, and identified two of them as 2-phenylbenzotriazole derivatives, 2-{2-(acetylamino)-4-{bis(2-methoxyethyl)amino}-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) and 2-{2-(acetylamino)-4-{(2-cyanoethyl)ethylamino}-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2) . In the present study, we collected adsorbed materials on blue cotton (3 kg x 9 times) at the same location, and isolated a sufficient amount (97 microg) of one of the remaining three mutagens other than PBTA-1 and PBTA-2, for structural analysis, by multiple column chromatography . The structure of mutagen, accounting for 12% of the total mutagenicity of the blue rayon-adsorbed substances, was determined to be a PBTA-1 analogue, 2-{2-(acetylamino)-4-amino-5-methoxyphenyl}-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-4) . PBTA-4 is a potent mutagen, inducing 190,000 and 7,800,000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix . In addition to the water of the Nishitakase River, PBTA-4 was detected in water samples from two rivers that flow through other regions where textile-dyeing industries have been developed . Like other PBTA analogues, PBTA-4 might also be produced from azo dyes during industrial processes in dyeing factories and treatment at sewage plants.

Mutat Res, 2001 May 31, 492(1-2), 7 - 11
Mutagenicity of bay-region amino-substituted cyclopenta{a}phenanthrenes and 2- and 5-aminochrysene; Catterall FS et al.; The relative mutagenic potentials of 11-amino-16,17-dihydro-15H-cyclopenta{a}phenanthrene, its 17-keto derivative, and 2- and 5-aminochrysene have been compared in Salmonella typhimurium TA98 and TA100 in the presence of a postmitochondrial liver preparation from Aroclor 1254 induced rats . The 11-amino hydrocarbon is a very weak mutagen (0.27 revertants/nmol), whereas the 11-amino-17-ketone is much more active (129 revertants/nmol) . 2-Aminochrysene is the most mutagenic arylamine ( approximately 500 revertants/nmol) among these compounds, but its 5-amino isomer is much less active (0.9 revertants/nmol) . Possible reasons for these marked differences are suggested.Use of TA98 with over-expressing O-acetyltransferase (YG 1024) and deficient in this enzyme (TA98/l,8-DNP(6)) with the 11-amino-17-ketone and with 5-aminochrysene clearly indicates the importance of this enzyme in their bioactivation, implying oxidation of the amino group to the hydroxylamine in both these compounds.

J Pharm Biomed Anal, 2001 Jun, 25(3-4), 589 - 97
Photostability and phototoxicity studies on diltiazem; Andrisano V et al.; The photostability of diltiazem was studied in aqueous solutions at different pH values . Firstly, the hydrolysis of the drug to desacetyldiltiazem in alkaline medium was evaluated and then the drug photodegradation under exposure to UVA-UVB radiation (solar simulator) was monitored by HPLC methods . The main photoproduct was isolated and characterized as diltiazem-S-oxide on the basis of the NMR and mass spectra . The HPLC method was also applied to the selective analysis of diltiazem in commercial formulations . Tests on mutagenicity and photomutagenicity of the drug were also carried out using Salmonella typhimurium TA 102 strain . In this testing the drug neither was mutagenic nor toxic.

Carcinogenesis, 2001 Jun, 22(6), 943 - 50
The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines; Yueh MF et al.; The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest . These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs) . Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event . In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2 . Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP) . To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene . DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity . Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity . In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase . CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9 . These results indicate that UGT1A9 can control the outcome of a genotoxic response . The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

Acta Pol Pharm, 2001 Jan-Feb, 58(1), 31 - 4
Tofisopam--evaluation of mutagenic and genotoxic properties; Chlopkiewicz B et al.; The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains . The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique . Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.

Antiviral Res, 2001 May, 50(2), 139 - 45
Evaluation of the mutagenic and genotoxic activities of anti-hepatitis B analogs of beta-L-adenosine by the Ames test and the Comet assay; Placidi L et al.; beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro . None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation . In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction . None of the four compounds induced DNA strand breakage with and without metabolic activation . In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.

Biochem J, 2001 Jun 1, 356(Pt 2), 327 - 34
Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop; Rodrigues-Lima F et al.; Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics . The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad . In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT {Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat . Struct . Biol . 7, 560-564}, is demonstrated . Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29-131) in which 37% of the residues are conserved . Homology modelling provided a good quality model of the corresponding region in human NAT1 . The location of the catalytic triad was found to be identical in StNAT and NAT1 . Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122-131 in NAT1 model and residues 122-133 in StNAT) . This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity . Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

Vet Res, 2001 Mar-Apr, 32(2), 119 - 29
Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs; Malorny B et al.; The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates . German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing . A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e . plasmid profiling and PFGE with all three enzymes . The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one . Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S . Typhimurium DT104 isolates are highly clonal.

J Mol Microbiol Biotechnol, 2001 Jul, 3(3), 381 - 4
Regulation of galactoside transport by the PTS; Kuroda M et al.; Inducer exclusion, regulation of activity of transporter, is mediated by phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) . To elucidate the molecular mechanism of the inducer exclusion, numerous biochemical and genetic studies have been performed . It is now well known that non-phosphorylated IIA(Glc) inhibits the transport via direct binding to the transporter . Analysis of inducer exclusion resistant mutants of lactose transporter and melibiose transporter in Escherichia coli and Salmonella typhimurium revealed amino acid residues that are involved in the interaction with IIA(Glc) . It is concluded that there are multiple interaction sites for IIA(Glc) in these transporters.

J Immunol, 2001 Jun 1, 166(11), 6802 - 11
Differential involvement of dendritic cell subsets during acute Salmonella infection; Kirby AC et al.; Within murine CD11c(+) dendritic cells (DC), CD8alpha+, CD8alpha-CD4+, and CD8alpha-CD4- subsets are defined . This study characterized the localization, number, and function of these subsets during acute Salmonella typhimurium infection . Immunohistochemical and flow cytometric analyses of spleens from mice orally infected with virulent S . typhimurium revealed that in situ redistribution and alteration in the absolute number and function of DC occurred in a subset-specific manner during infection . CD8alpha-CD4+ DC present at B cell follicle borders in the spleen of naive mice were absent 5 days post-Salmonella infection, despite no overall change in the absolute number of CD8alpha-CD4+ splenic DC . CD8alpha+ and CD8alpha-CD4- DC were prominently associated with the red pulp, and the frequency of these cells increased strikingly 5 days post-Salmonella infection . Significant quantitative increases in both CD8alpha+ and CD8alpha-CD4- subsets were associated with the in situ redistribution . Examination of Salmonella-infected TAP1(-/-)/beta(2)-microglobulin(-/-) mice, which lack CD8alpha+ T cells, confirmed the differential subset-specific modulations in the DC populations both in situ and quantitatively . Ex vivo intracellular cytokine analysis showed significantly increased frequencies of CD8alpha(+) DC producing TNF-alpha at days 2 and 5 postinfection . In contrast, CD4+ DC producing TNF-alpha were transiently increased followed by a significant reduction . No significant increase in IL-12p40 or IL-10 production by splenic DC was detected during the first 5 days post-S . typhimurium infection . Together these data reveal differential modulation of splenic DC subsets with regard to organization, number, and cytokine production during the course of acute Salmonella infection.

Microbes Infect, 2001 Mar, 3(3), 237 - 47
Non-typhoidal salmonellosis: emerging problems; Rabsch W et al.; Two major changes in the epidemiology of non-typhoidal salmonellosis have occurred during the second half of the 20th century . First, Salmonella typhimurium strains resistant to multiple antibiotics have emerged and spread within populations of food animals . Secondly, Salmonella enteritidis has emerged as a major egg-associated pathogen . This article reviews available data on the origins of the human epidemics.

FEMS Microbiol Lett, 2001 May 15, 199(1), 125 - 9
Bacterial swimming speed and rotation rate of bundled flagella; Magariyama Y et al.; Swimming speed (v) and flagellar-bundle rotation rate (f) of Salmonella typhimurium, which has peritrichous flagella, were simultaneously measured by laser dark-field microscopy (LDM) . Clear periodic changes in the LDM signals from a rotating bundle indicated in-phase rotation of the flagella in the bundle . A roughly linear relation between v and f was observed, though the data points were widely distributed . The ratio of v to f (v-f ratio), which indicates the propulsive distance during one flagellar rotation, was 0.27 microm (11% of the flagellar pitch) on average . The experimental v-f ratio was twice as large as the calculated one on the assumption that a cell had a single flagellum . A flagellar bundle was considered to propel a cell more efficiently than a single flagellum.

Arch Toxicol, 2001 Apr, 75(2), 118 - 22
Genotoxic effects of N-nitrosodicyclohexylamine in isolated human lymphocytes; Westphal GA et al.; Dicyclohexylamine x nitrite is classified as an "experimental equivocal tumorigenic agent" by the National Toxicology Program . Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine x nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine x nitrite . Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes . Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography . Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H- and 13C-NMR . More than 97% purity was achieved . Stability and availability in the solvent were checked by GC/MS . N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15-100 micrograms/ml (= 71.4-476.2 microM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments . For the Ames test, arochlor-1254-, beta-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104 . No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 micrograms (= 1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5 . In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes . With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.

Equine Vet J, 2001 May, 33(3), 311 - 8
Pulmonary and systemic effects of inhaled endotoxin in control and heaves horses; Pirie RS et al.; To investigate whether inhaled endotoxin contributes to airway inflammation and dysfunction in stabled horses, control (n = 6) and asymptomatic heaves (previously termed chronic obstructive pulmonary disease)-susceptible (n = 7) horses were given inhalation challenges with 20, 200 and 2,000 microg of soluble Salmonella typhimurium Ra60 lipopolysaccharide (LPS) . LPS inhalation induced a dose-dependent neutrophilic airway inflammatory response in both groups . Inhalation with 2,000 microg of LPS also induced detectable lung dysfunction in the heaves group . LPS inhalation did not alter clinical score, tracheal secretion volume or airway reactivity in either group . The no-response thresholds were lower for the heaves group (<20 microg for airway inflammation; 200 to 2,000 microg for lung dysfunction) than for the control group (20 to 200 microg for airway inflammation; >2,000 microg for lung dysfunction) . To enable comparison of these threshold levels with airborne endotoxin concentrations in stables, horses also received a 5 h duration hay/straw challenge, during which the total and respirable airborne endotoxin concentrations were determined . Comparison of the effects of acute LPS inhalation and hay/straw challenges suggest that inhaled endotoxin is not the sole cause of heaves . However, it is likely that it contributes to airway inflammation, both in heaves horses in concert with other inhalants, and in normal horses when they are exposed to high levels in poor stable environments.

Br J Pharmacol, 2001 May, 133(2), 237 - 42
Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin; Irie K et al.; Anti-inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)-induced microvascular permeability change . Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 microg site-1) from Salmonella typhimurium . Dye leakage in the skin was significantly increased 2 h after injection of LPS . This LPS-induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg-1), milrinone (5 - 10 mg kg-1), rolipram (0.5 - 10 mg kg-1) and zaprinast (5 - 10 mg kg-1) . The dye leakage was also inhibited by beta-adrenoceptor agonists, including isoproterenol (0.5 - 5 mg kg-1) and salbutamol (0.05 - 5 mg kg-1), an adenylate cyclase activator, forskolin (5 mg kg-1), and a cell permeable cyclic AMP analogue, 8-bromo-cyclic AMP (8-Br-cAMP, 10 mg kg-1) . LPS caused a transient increase in serum TNF-alpha level peaking at 1 h after the injection . This increase in serum TNF-alpha was completely blocked by a pretreatment with pentoxifylline (160 mg kg-1), milrinone (5 mg kg-1), rolipram (1 mg kg-1), zaprinast (10 mg kg-1), salbutamol (0.5 mg kg-1), forskolin (1 mg kg-1) and 8-Br-cAMP (10 mg kg-1) . LPS caused an increase in serum IL-1alpha level peaking at 3 h after injection . This increase in serum IL-1alpha was not significantly suppressed by the cyclic AMP elevating agents . Our study suggests that cyclic AMP elevating agents attenuate LPS-induced microvascular permeability change by suppressing TNF-alpha up regulation.

Regul Toxicol Pharmacol, 2001 Apr, 33(2), 157 - 64
Safety evaluation of lipase produced from Candida rugosa: summary of toxicological data; Flood MT et al.; The toxicity of lipase AY, an enzyme preparation used in lipid hydrolysis to produce flavors, was evaluated in a series of studies . A 13-week dietary toxicity study in Sprague-Dawley (Crj:CD) rats was conducted in which animals received lipase AY in the feed at concentrations of 0, 625, 1250, or 2500 mg/kg body wt . No adverse treatment-related effects were observed . Lack of genotoxic potential was demonstrated by the results of an in vitro reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli strain WP2 uvrA, by an in vitro forward mutation assay in L5178Y mouse lymphoma cells, and by an in vitro chromosome aberration test in CHL/IU cells derived from fibroblasts of the lungs of Chinese hamsters . Finally, the particular strain of Candida rugosa, the yeast strain used to prepare lipase AY, has been shown to be nonpathogenic upon a single injection into the tail vein of rats of viable spores at doses up to 1.5x10(7) colony-forming units per animal . The results of these studies demonstrate that the enzyme preparation may be considered safe to workers and consumers when employed in the production of flavors from fats .

Br J Nutr, 2001 Apr, 85(4), 431 - 40
Biochemical behaviour of norbixin during in vitro DNA damage induced by reactive oxygen species; Kovary K et al.; Naturally occurring antioxidants such as carotenoids are extensively studied for their potential in reducing the risk for cancer and other chronic diseases . In the present study, the radical-scavenger activity of the food additive norbixin, a water-soluble carotenoid extracted from Bixa orellana seeds and commercialized as annatto, was evaluated under conditions of DNA damage induced by reactive oxygen species, particularly by hydroxyl radicals . The cell-free scavenger activity of norbixin was evaluated using plasmid DNA as target molecule and Sn2+ or Fe2+ as oxidant . The addition of H2O2 enhanced DNA breakage induced by metal ions, particularly Fe2+ . Under these conditions, norbixin started to protect plasmid DNA against single- and double-strand breakage at a metal:norbixin ratio of 1:1 (Sn2+) and 1:10 (Fe2+) . However, at lower ratios to Sn2+, norbixin enhanced Sn2+-induced DNA breakage (P < 0.05) . The ability of norbixin to protect genomic DNA against oxidative damage was assessed in murine fibroblasts submitted to H2O2-induced oxidative stress and the results were evaluated by the comet assay . Under low serum conditions (2 % fetal bovine serum (FBS)), a protective effect of norbixin against H2O2-induced DNA breakage was inversely related to its concentration, a protection ranging from 41 % (10 microm) to 21 % (50 microm) . At higher concentrations of norbixin, however, oxidative DNA breakage was still enhanced, even in the presence of a high serum concentration (10 % FBS) . Under normal conditions, norbixin per se has no detectable genotoxic or cytotoxic effects on murine fibroblasts . The antimutagenic potential of norbixin against oxidative mutagens was also evaluated by the Salmonella typhimurium assay, with a maximum inhibition of 87 % against the mutagenicity induced by H2O2 . Although plasmid DNA and Ames data indicated that norbixin can protect DNA against oxidative damage, it seems to be a risky guardian of genomic DNA as it can also increase the extent of oxidative damage.

Eur J Clin Microbiol Infect Dis, 2001 Mar, 20(3), 206 - 9
Antibiotic resistance patterns and plasmid profiles of Salmonella typhimurium isolates in Turkey; Otkun M et al.; To understand the resistance mechanisms present in 75 isolates of Salmonella typhimurium derived from clinical infections in Turkey, antimicrobial resistance patterns and associated plasmids were investigated . Among the 22 strains that produced extended-spectrum beta-lactamase (ESBL), 20 were resistant to aminoglycosides and 12 to trimethoprim-sulfamethoxazole . Strains that did not produce ESBL did not express aminoglycoside or trimethoprim-sulfamethoxazole resistance, although 27 of them were ampicillin resistant . None of the strains were resistant to imipenem or fluoroquinolones . Nineteen strains producing ESBL carried a plasmid of >100 MDa . Seven ESBL-producing strains conjugally transferred their ESBLs and trimethoprim-sulfamethoxazok resistance . No correlation was found between the resistance patterns and plasmids in non-ESBL-producing strains.

J Exp Med, 2001 May 7, 193(9), 1097 - 104
The Exocytosis-regulatory protein synaptotagmin VII mediates cell invasion by Trypanosoma cruzi; Caler EV et al.; The intracellular protozoan parasite Trypanosoma cruzi causes Chagas' disease, which affects millions of people in Latin America . T . cruzi enters a large number of cell types by an unusual mechanism that involves Ca(2+)-triggered fusion of lysosomes with the plasma membrane . Here we show that synaptotagmin VII (Syt VII), a ubiquitously expressed synaptotagmin isoform that regulates exocytosis of lysosomes, is localized on the membranes of intracellular vacuoles containing T . cruzi . Antibodies against the C(2)A domain of Syt VII or recombinant peptides including this domain inhibit cell entry by T . cruzi, but not by Toxoplasma gondii or Salmonella typhimurium . The C(2)A domains of other ubiquitously expressed synaptotagmin isoforms have no effect on T . cruzi invasion, and mutation of critical residues on Syt VII C(2)A abolish its inhibitory activity . These findings indicate that T . cruzi exploits the Syt VII-dependent, Ca(2+)-regulated lysosomal exocytic pathway for invading host cells.

Environ Toxicol, 2001, 16(2), 151 - 7
Assessment of toxicity and mutagenicity in air particulate matter from an urban industrial area in the coast of the Rio de la Plata; Muller A et al.; Chemical characterization and effects assessment of semivolatile organic compounds in organic extracts from air particulate matter from the region of the greater La Plata area was undertaken . Effects covered the study of mutagenicity with the Ames test (Salmonella typhimurium TA100 and TA98 strains with metabolic activation by S9) and cytotoxicity using the Tetrahymena pyriformis test system (growth rate, cell volume, and cell respiration) . Chemical analysis of organic extracts was done using gas chromatography . Results demonstrate the presence of polycyclic aromatic hydrocarbons (PAH) in the matrix, high mutagenicity, and cytotoxicity . A higher mutagenic activity detected with TA98 and TA100 strains is associated with an increment of total PAH and total five or six ring PAH content, respectively . Linked with it, a PAH dependent toxicity on Tetrahymena pyriformis has been observed . This cell system proved to be very sensitive . From the results obtained with the cell respiration assay with T . pyriformis it appears that uncoupling agents are present in the samples . The results of this study indicate that air particulate matter from the Rio de La Plata area contains significant genotoxic and cytotoxic activity probably due to the presence of PAHs.

Mikrobiologiia, 2001 Jan-Feb, 70(1), 39 - 44
{Extracellular protein of propionic acid bacteria inhibits induced mutation in strains of Salmonella typhimurium}; Vorob'eva LI et al.; A culture of propionic acid bacteria grown in a glucose-containing minimal medium, as well as the culture liquid and logarithmic-phase cells obtained from this culture, were found to inhibit the base pair substitution mutations induced by 4-nitroquinoline N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, and sodium azide and the frameshift mutations induced by 9-aminoacridine . The antimutagenic activity of the culture liquid (CL) was presumably due to the presence of an extracellular thermolabile protein with a molecular mass of no more than 12 kDa based on the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92 degrees C, and its dialysis in a cellulose sack, which retains substances with molecular masses greater than 12 kDa . The residual antimutagenic activity of the dialyzed culture liquid was probably related to the interaction of the mutagen with thiols, rather than to the presence of organic acids (acetic or propionic) . Thiols may also contribute to the antimutagenic activity of the P . shermanii cells.

Environ Toxicol Chem, 2001 May, 20(5), 960 - 4
Isomeric effects on thiosulfate transformation and detoxification of 1,3-dichloropropene; Wang Q et al.; The fumigant 1,3-dichloropropene (1,3-D) is one of the most heavily used pesticides but also a suspected carcinogen . Previous research has shown that 1,3-D was rapidly transformed and detoxified by ammonium thiosulfate (ATS), a sulfur and nitrogen fertilizer . As common formulations contain cis and trans isomers at roughly equivalent ratios, this study was conducted to understand isomeric differences in thiosulfate transformation and detoxification of 1,3-D . Under the same conditions, reaction of cis-1,3-D with thiosulfate was more than three times faster than trans-1,3-D, which was correlated with a lower reaction activation energy for the cis isomer . The trans isomer was considerably more toxic to the luminescent bacteria Vibrio fisheri than the cis isomer, but the toxicity was reduced by 14 times after thiosulfate transformation . Mutagenic activity to strains of Salmonella typhimurium was observed for trans-1,3-D but was not detected after thiosulfate transformation . These results suggest that thiosulfate transformation detoxifies 1,3-D primarily by deactivating the trans isomer, and the reaction is toxicologically beneficial, as it negates the potential harmful effects of 1,3-D to the environment and human health.

Microbes Infect, 2001 Apr, 3(4), 259 - 65
Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide; Hofer S et al.; Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses . Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2) . In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium . We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S . typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced . Four out of five members, i.e . p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics . Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation . This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology.

Avian Dis, 2001 Jan-Mar, 45(1), 83 - 91
Vaccination against Salmonella enteritidis in Dutch commercial layer flocks with a vaccine based on a live Salmonella gallinarum 9R strain: evaluation of efficacy, safety, and performance of serologic Salmonella tests; Feberwee A et al.; This study describes a field trial in which 80 commercial layer flocks, with an increased risk of Salmonella enteritidis (SE) infection and placed on farms with a certified Standardized Biosecurity Programme (SBP) or a request for a SBP certificate, were vaccinated with a vaccine based on a live attenuated Salmonella gallinarum (SG) 9R strain . An evaluation is presented of the efficacy of the vaccine against SE infections, the effect on the performance of serologic Salmonella tests, and the spread of the vaccine strain to the egg content . For the efficacy study, assessment of the flock level occurrence of SE infections in the vaccinated group of 80 flocks was compared with that of a nonvaccinated group of 1854 flocks hatched in the same period . This control group was examined according to the compulsory control programme in The Netherlands . An evaluation was done of the performance of serologic Salmonella tests and the spread of the vaccine strain to the inner egg content of five of the vaccinated flocks . Findings demonstrated the flock level occurrence of SE infections in the vaccinated group (2/80 = 2.5%) to be significantly (P = 0.01) lower than that of the nonvaccinated group (214/1854 = 11.5%) . Vaccination resulted in 59.0% positive test results in lipopolysaccharide BD enzyme-linked immunosorbent assay (ELISA) for detecting antibodies against Salmonella serogroups B and D and 0% positive test results in the rapid plate agglutination test for detecting antibodies against S . pullorum (SP)/SG . The mean specificities of two blocking ELISAs (gm- and i-double antibody sandwich ELISAs) based on the flagellar antigen of SE and Salmonella typhimurium (ST) on the same sera were 99.6% and 96.1%, respectively . The vaccine strain could not be isolated from any of the 450 pools of 10 eggs . On the basis of these results, we concluded that vaccination with a vaccine based on an attenuated SG 9R strain contributes to the reduction of SE infections in commercial layer flocks . Furthermore, serologic monitoring of SE, ST, and SP/SG can still be carried out on flocks vaccinated with an attenuated SG 9R strain . Additionally, we found no indication of the spread of the vaccine strain to the egg content.

Avian Dis, 2001 Jan-Mar, 45(1), 61 - 9
Differences among six Salmonella serovars in abilities to colonize reproductive organs and to contaminate eggs in laying hens; Okamura M et al.; The abilities of Salmonella serovars to colonize the reproductive organs of chickens and to contaminate eggs were compared . Mature laying hens were inoculated intravenously with 10(5) colony-forming units of Salmonella enteritidis, Salmonella typhimurium, Salmonella infantis, Salmonella hadar, Salmonella heidelberg, or Salmonella montevideo to cause the systemic infection . Salmonella enteritidis was recovered from three yolks of the laid eggs (7.0%), suggesting egg contamination from the transovarian transmission of S . enteritidis . The liver, spleen, and cecum were colonized by each serovar similarly at 4 or 7 days postinoculation (PI), whereas the ovary and preovulatory follicles were colonized by S . enteritidis with significantly (P < 0.05) higher levels than by the other serovars at 4 and 7 days PI . Salmonella enteritidis was recovered from the cloaca and vagina at 2, 4, and 7 days PI and from the other portions of the oviduct at 4 and 7 days PI . In addition, S . enteritidis had been persistent in the peripheral blood for 7 days PI . These results suggest that S . enteritidis is the predominant serovar to colonize the reproductive organs of mature laying hens among six serovars used in this study, reflecting the field situatibn in which the predominant outbreaks of human salmonellosis were caused by S . enteritidis-contaminated eggs recently . The ability of S . enteritidis to colonize the reproductive organs may be one of the reasons that egg contamination with S . enteritidis has increased.

Avian Dis, 2001 Jan-Mar, 45(1), 245 - 50
Isolation of Newcastle disease virus and Salmonella typhimurium from the brain of double-crested cormorants (Phalacrocorax auritus); Clavijo A et al.; Avian paramyxovirus type 1 (Newcastle disease virus) and Salmonella typhimurium were isolated from the brain and lung tissues of double-crested cormorants (Phalacrocorax auritus) from Lac Canard, Alberta, Canada . More than 100 birds died during this outbreak in 1999 . Affected birds presented signs of central nervous system disease characterized by unilateral wing and leg paralysis . Other geographic locations in the provinces of Alberta and Saskatchewan have reported cases of cormorants suffering from diseases with signs compatible with Newcastle disease . The virus isolated in the 1999 outbreak was characterized as mesogenic . These findings suggest that other pathogens, like S . typhimurium, may influence the clinical presentation of disease caused by mesogenic strains of Newcastle disease virus in cormorants.

Biochemistry, 2001 Feb 20, 40(7), 1903 - 12
The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition; Gill HS et al.; Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS) . The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods . The structure suggests a noncovalent, dead-end mechanism of inhibition . Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme . One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327 . The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket . The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin . The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands . The inhibition of GS by methionine sulfoximine can be explained by the same mechanism . These models of inhibited GS further illuminate its catalytic mechanism.

Lett Appl Microbiol, 2001 May, 32(5), 293 - 7
Screening of some plants from Northern Argentina for their antimicrobial activity; Salvat A et al.; AIMS: Screening of antimicrobial activity in 25 plant species from Northern Argentina . METHODS AND RESULTS: Inhibition of microbial growth was measured by a microplate assay with an oxidation-reduction indicator (Alamar Blue) . Test organisms were: Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecium . Weak inhibitory activities (MIC=0.5 mg dry matter ml(-1)) were found in methanolic extracts of Rivina humilis, Crateva tapia, Funastrum claucum and Schinopsis balansae . Stronger bacteriostatic power was detected in Vassobia breviflora (MIC=0.25 mg ml(-1) against Staphylococcus aureus, and 0.5 mg ml(-1) against Enterococcus faecium) . This activity was purified five-fold by extraction with dichloromethane, and it was found equally effective against susceptible or antibiotic-resistant strains of Staph . aureus . In addition, the purified extract was synergistic with gentamicin, and it was bactericidal at 24 h, with a concentration of 0.25 mg ml(-1) . CONCLUSION: There is a significant antimicrobial activity in Vassobia breviflora . SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies will be required to disclose the potential importance of these findings.

Virus Genes, 2001 Mar, 22(2), 151 - 8
Multiple copies of the upstream regulatory region of the major capsid protein gene of bacteriophage MB78 inhibit phage morphogenesis; Sharma R et al.; The 2.311 kb EcoRI F fragment of bacteriophage MB78 has been cloned in multicopy vectors pUC19 and pCR90 . Salmonella typhimurium strains carrying such plasmids cannot support development of phage MB78 while other Salmonella phages like P22 and 9NA grow normally . Most of the phage MB78 induced functions are normal in such transformed hosts but proper maturation of the phage particles does not take place . Deletion of 138 bp from the 3' end of the cloned fragment reverses the inhibitory effect . Analysis of nucleotide and the deduced amino acid sequence of a 1.2 kb HindIII-SalI fragment of the phage genome which overlaps the 138 bp confirms that this part contains the upstream regulatory region of the major structural protein gene . It seems that in presence of multiple copies of the upstream regulatory region (which includes a number of promoter like sequence) of the coat protein gene, the maturase gene is down regulated and this is effective only in cis, a situation quite similar to that of Qbeta RNA phages.

Nature, 2001 Apr 26, 410(6832), 1099 - 103
The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5; Hayashi F et al.; The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host . Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity . Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals . Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production . TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry . Expression of L . monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity . All known TLRs signal through the adaptor protein MyD88 . Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin . Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

Plasmid, 2001 Mar, 45(2), 101 - 13
Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors; Takamatsu D et al.; pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors . In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli . pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers . These vectors could be introduced into S . suis, E . coli, Salmonella typhimurium, S . pneumoniae, and S . equi ssp . equi by electrotransformation . The recA gene was cloned from S . suis and sequenced, and this information was used in the construction of a recA mutant of S . suis . Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S . suis and E . coli recA mutants compared with the parental strains . These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S . suis and E . coli . Moreover, cloning of the functional S . suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S . suis but not in E . coli . These results showed that pSET vectors are useful tools for cloning and analyzing S . suis genes in S . suis strains directly .

Prep Biochem Biotechnol, 2001 Feb, 31(1), 13 - 22
Heterologous gene expression in bacterial systems under reduced oxygen tensions . Small-scale optimization precedes industrial fermentation; Taylor-Robinson AW et al.; The expression of heterologous fusion proteins from the anaerobically inducible Escherichia coli nitrite reductase nirB promoter has been described using a number of different industrial regimes, but which have proved impractical for scaling down to suit primary research purposes . This paper describes the novel application of microbiological gas sachets generating anaerobic and microaerophilic environments to evaluate the inducible expression under the influence of nirB of heterologous proteins by attenuated vaccine strains of Salmonella typhimurium . The conditions of reduced oxygen tension model those found in lymphoid organs colonized by Salmonella in vivo and so can be used to optimize the vaccine dose prior to administration . Modeling in vivo promoter inducibility to monitor the stability of a plasmid within attenuated vaccine strains of bacteria offers an attractive alternative to antibiotic resistance, which is not permitted for clinical use in humans . This technological advance may be utilized to optimize heterologous gene expression in any microaerophilic bacterial system as a pilot, prior to production-scale applications.

J Biol Chem, 2001 Jul 6, 276(27), 25411 - 20 Epub 2001 Apr 23.
A newly synthesized, ribosome-bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates; Clark PL et al.; Little is known about the conformations of newly synthesized polypeptide chains as they emerge from the large ribosomal subunit, or how these conformations compare with those populated immediately after dilution of polypeptide chains out of denaturant in vitro . Both in vivo and in vitro, partially folded intermediates of the tailspike protein from Salmonella typhimurium phage P22 can be trapped in the cold . A subset of monoclonal antibodies raised against tailspike recognize partially folded intermediates, whereas other antibodies recognize only later intermediates and/or the native state . We have used a pair of monoclonal antibodies to probe the conformational features of full-length, newly synthesized tailspike chains recovered on ribosomes from phage-infected cells . The antibody that recognizes early intermediates in vitro also recognizes the ribosome-bound intermediates . Surprisingly, the antibody that did not recognize early in vitro intermediates did recognize ribosome-bound tailspike chains translated in vivo . Thus, the newly synthesized, ribosome-bound tailspike chains display structured epitopes not detected upon dilution of tailspike chains from denaturant . As opposed to the random ensemble first populated when polypeptide chains are diluted out of denaturant, folding in vivo from the ribosome may begin with polypeptide conformations already directed toward the productive folding and assembly pathway.

J Am Vet Med Assoc, 2001 Apr 1, 218(7), 1152 - 9, 1100
An outbreak of salmonellosis among horses at a veterinary teaching hospital; Schott HC 2nd et al.; Between May 1996 and February 1997, 27 horses and a veterinary student at a veterinary teaching hospital developed apparent nosocomial Salmonella Typhimurium infection . The source of the multiple-drug resistant Salmonella Typhimurium was a neonatal foal admitted for treatment of septicemia . A high infection rate (approx 13% of hospitalized horses) coupled with a high case fatality rate (44%) for the initial 18 horses affected led to a decision to close the hospital for extensive cleaning and disinfection . Despite this effort and modification of hospital policies for infection control, 9 additional horses developed nosocomial Salmonella Typhimurium infection during the 6 months after the hospital reopened . Polymerase chain reaction testing of environmental samples was useful in identifying a potential reservoir of the organism in drains in the isolation facility . Coupled with clinical data, comparison of antimicrobial resistance patterns of Salmonella Typhimurium isolates provided a rapid initial means to support or refute nosocomial infection . Although minor changes in the genome of these isolates developed over the course of the outbreak, pulsed-field gel electrophoresis testing further supported that salmonellosis was nosocomial in all 27 horses.

J Immunol, 2001 May 1, 166(9), 5741 - 8
Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane; Gallois A et al.; Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages . This survival implies that S . typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes . Recent evidence suggests that S . typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner . To gain insights into the mechanism by which S . typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S . typhimurium . We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion . In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S . typhimurium . Subversion of NADPH oxidase assembly by S . typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S . typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.

FEMS Microbiol Lett, 2001 Apr 13, 197(2), 203 - 8
Characterization of the mutS-proximal region of the Salmonella typhimurium SPI-1 identifies a group of pathogenicity island-associated genes; Pancetti A et al.; The virulence properties of Salmonella enterica are largely encoded within a set of horizontally acquired gene blocks termed pathogenicity islands . One such pathogenicity island, SPI-1, located at centisome 63 of the Salmonella chromosome between the mutS and fhlA genes, encodes a type III protein secretion system and an iron uptake system . We have characterized the mutS-proximal border of this pathogenicity island and have identified two sets of genes, pigAB and pigCD . All four genes have homologs of unknown function in several bacteria that share the ability to establish an intimate association with higher eukaryotic hosts . The expression of at least two of these genes, pigA and pigB, is controlled by SprA, a transcription factor encoded within SPI-1 that controls the expression of genes associated with the type III secretion system of this island . In addition, we found that homologs of the pig genes are also found at different locations of the S . enterica chromosome in association with segments of DNA that exhibit features of pathogenicity islands . The presence of several apparently functional copies of these genes argues for an important role in the biology of this bacterial pathogen . Furthermore, they constitute a valuable tool to identify potential pathogenicity islands.

J Agric Food Chem, 2001 Mar, 49(3), 1426 - 31
Isolation and characterization of antioxidant compounds from Aspergillus candidus broth filtrate; Yen GC et al.; The objectives of this study were to isolate the antioxidative components in the broth filtrate of Aspergillus candidus (CCRC 31543), to characterize their antioxidative properties, and to evaluate their safety . Three major compounds were isolated and identified as 3,3' '-dihydroxyterphenyllin, 3-hydroxyterphenyllin, and candidusin B . In the linoleic acid peroxidation system, the inhibition of peroxidation in these three compounds was greater than 95% and was significantly higher than that of alpha-tocopherol but equal to that of BHA at 12.5-200 microg/mL . As measured using the Rancimat method in lard, 3,3' '-di-OH-terphenyllin exhibited a protection factor value of 7.82, which was substantially higher than those of BHA (5.58) and alpha-tocopherol (4.29) at 200 microg/mL . 3,3' '-di-OH-terphenyllin and 3-OH-terphenyllin also exhibited marked scavenging effects on the alpha,alpha-diphenyl-beta-picrylhydrazyl radicals (94.7 and 96.0%, respectively), which were similar to those of BHA and alpha-tocopherol . Safety studies showed that these three compounds were neither cyto- nor geno-toxic toward human intestine 407 (INT 407) cells, nor mutagenic toward Salmonella typhimurium TA98 and TA100.

Vaccine, 2001 Apr 30, 19(23-24), 3269 - 72
Contribution of MHC class I-dependent immune mechanisms induced by attenuated recombinant Salmonella typhimurium secreting superoxide dismutase to protection against murine listeriosis; Grode L et al.; A recombinant (r)Salmonella typhimurium aroA strain secreting the naturally non-secreted superoxide dismutase (SOD) of Listeria monocytogenes controls murine listeriosis dependent on 'transporter associated with antigen processing' (TAP)-mediated immune mechanisms . TAP1-deficient mice (devoid of most CD8 T cells) vaccinated with this rSalmonella SODs strain succumbed to lethal L . monocytogenes challenge, whereas C57BL/6 mice were protected by this vaccine . Moreover, vaccination of H-2I-Abeta-deficient mice (lacking major histocompatibility class (MHC) II molecules and thus devoid of mature CD4 TCR-alphabeta cells), of TAP1-deficient as well as of beta2microglobulin-deficient mice (devoid of conventional CD8 T cells) with a sublethal dose of L . monocytogenes and subsequent challenge with rSalmonella control or SODs strain revealed contribution of both MHC class I- and MHC class II-dependent immune mechanisms to the control of secondary Salmonella infection . Finally, the clearance of rSalmonella SODs bacteria was achieved in TAP1-deficient animals vaccinated with L . monocytogenes . Our data suggest a role of TAP-dependent mechanisms in priming of protective immunity by rSalmonella micro-organisms secreting SOD.

FEBS Lett, 2001 Apr 13, 494(3), 201 - 7
A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation; Marcus SL et al.; The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium . The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation . Here, we investigated the inositol phospholipid substrate preferences of SigD . Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids . Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates . Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin . Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively . The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain . A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt . Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity . We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

J Wildl Dis, 2001 Apr, 37(2), 399 - 402
Mortality in captive elk from salmonellosis; Foreyt WJ et al.; Salmonella typhimurium DT104 infections of captive elk (Cervus elaphus nelsoni) calves resulted in mortality in eight of 13 affected calves . Salmonellosis in these elk calves was characterized by diarrhea, fever, lethargy, inappetence and depression, and death usually ensued within 72 hr of initial clinical signs . Affected calves did not respond to antibiotic and fluid therapy . The source of the bacteria likely was one or more of the calves when they were captured in the wild at less than 5 days of age . In our captive holding facility, the disease spread quickly and was difficult to control . Phage typing, pulsed field gel electrophoresis, antibiotic sensitivity testing, and plasmid profiles determined that this Salmonella sp . strain was the epidemic strain common to cattle, sheep and humans.

J Leukoc Biol, 2001 Apr, 69(4), 583 - 9
Genetic background of attenuated Salmonella typhimurium has profound influence on infection and cytokine patterns in human dendritic cells; Dreher D et al.; Salmonella typhimurium (ST) can cause infection in man, and attenuated strains are under consideration as live vaccine vectors . However, little is known about the interaction of ST with human dendritic cells (DC) . Here, we compared the consequences of exposure of human, monocyte-derived DC with different attenuated strains of ST . Infection was observed with all four strains tested (wild type, PhoP-, PhoPc, and AroA), but the PhoPc strain was by far the most efficient . Intracellular persistence of wild type and PhoP- was longer than that of PhoPc and AroA, both of which were largely eliminated within 24 h . Most DC survived infection by the attenuated strains, although apoptosis was observed in a fraction of the exposed cells . All strains induced DC maturation, independent from the extent of infection . Although all strains stimulated secretion of TNF-alpha and IL-12 strongly, PhoPc induced significantly less IL-10 than the other three strains and as much as 10 times less IL-10 than heat-killed PhoPc, suggesting that this mutant suppressed the secretion of IL-10 by the DC . These data indicate that infectivity, bacterial elimination, and cytokine secretion in human DC are controlled by the genetic background of ST.

J Food Prot, 2001 Apr, 64(4), 493 - 7
Attachment of bacteria to beef from steam-pasteurized carcasses; Warriner K et al.; The extent to which a bacterial cocktail containing equal numbers of Pseudomonas fragi NCTC 10689, Listeria monocytogenes BL5/2, Salmonella Typhimurium LT2, and Escherichia coli JM 109 attached to loin surface cuts (7 by 5 cm) derived from steam-pasteurized beef carcasses has been evaluated . The extent of attachment was categorized as loosely attached (removed by rinsing), firmly attached (released by stomaching), and irreversibly bound . No significant difference (P > 0.10) in the attachment of bacteria to steam-pasteurized carcasses was found compared with control loin samples that had received no treatment . No significant difference (P > 0.05) was also found in the attachment strength between the different bacterial species tested . Most bacteria inoculated onto the loin cuts were reversibly bound, since they had been removed by rinsing and stomaching . The irreversible attachment of bacteria to loin cuts was found to vary significantly (P < 0.01) among the different carcass sets used but was independent of whether the carcass had undergone steam pasteurization treatment . Use of a bioluminescent strain of E . coli showed that cells bound preferentially to cut edges and convoluted areas on the loin surface and could not be removed by rinsing . The possible mechanisms of bacterial attachment and the suitability of steam pasteurization to remove contamination incurred during slaughter are discussed.

J Food Prot, 2001 Apr, 64(4), 486 - 92
Altering the thermal resistance of foodborne bacterial pathogens with an eggshell membrane waste by-product; Poland AL et al.; Eggshells from egg-breaking operations are a significant waste disposal problem . Thus, the development of value-added by-products from this waste would be welcomed by the industry . The ability of extracted eggshell membranes containing, several bacteriolytic enzymes (i.e., lysozyme and beta-N-acetylglucosaminidase) or other membrane components to alter the thermal resistance of gram-positive and gram-negative bacterial pathogens was evaluated . Mid-log phase cells of Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), Escherichia coli O157:H7 (EC), Listeria monocytogenes Scott A (LM), and Staphylococcus aureus (SA) were suspended in 100 ml of 0.1% peptone water (pH 6.9, 10(7-8) CFU/ml) containing either 0 (control) or 10 g of an eggshell membrane extract and incubated at 37 degrees C for 45 min . Following exposure, membrane-free samples (1.5 ml) were heated in a 56 degrees C (LM, SA), 54 degrees C (SE, ST), or 52 degrees C (EC) water bath from 0 to 14 min in sealed glass reaction vials (12 by 32 mm), and the survivors were recovered on brain heart infusion agar . Population reductions ranging from 27.6% (SA) to 99.8% (LM) (ST, 43.8%; SE, 47.5%; EC, 71.8%) were observed for cells treated for 45 min with extracted membrane, as compared to controls . D-value reductions ranging from 0 (LM) to 87.2% (SE) (SA, 36.7%; EC, 83.3%; ST, 86.3%) were observed when membrane-treated cells were subsequently heat inactivated . The effects of exposure pH, time, temperature, and organic load on membrane activity were also evaluated with Salmonella Typhimurium . Exposure pH (5.0 versus 6.9), time (15 versus 45 min), and temperature (4 degrees C versus 37 degrees C) did not significantly reduce the impact of eggshell membranes on D-values . However, the presence of organic matter (0.1% peptone water versus skim milk) significantly reduced the thermal resistance-reducing capacity of the membranes . These preliminary findings provide information on the potential use of extracted eggshell membranes to alter bacterial heat resistance.

J Food Prot, 2001 Apr, 64(4), 456 - 61
Inhibition and reversal of Salmonella typhimurium attachment to poultry skin using zinc chloride; Nayak R et al.; A skin attachment model was used to determine if ZnCl2 would reverse or inhibit Salmonella attachment to broiler skin . In the reversal experiments, skin samples, treated first with 1 ml of Salmonella Typhimurium suspension (10(8) CFU/ml) for 30 min, were then treated with 25 or 50 mM ZnCl2 for 5 or 15 min . Zinc chloride solutions were applied while the culture was present on the skin . In the inhibition experiments, ZnCl2 solutions were added first; treatment solutions were discarded after 5 or 15 min of application, and then the culture was added . Firmly and loosely attached Salmonella were enumerated on xylose lactose tergitol plates . A duplicate section of skin, subjected concurrently to the above treatments, was observed under a scanning electron microscope to enumerate attached bacteria directly . In the reversal experiments, 25 and 50 mM ZnCl2 reduced (P < 0.01) firmly attached cells by 77 and 89%, respectively, when compared to the control (water) . Micrographs indicated that 25 and 50 mM ZnCl2 reduced (P < 0.1) Salmonella attachment by 69 and 99.9%, respectively, in the reversal experiments . In the inhibition experiments, 25 and 50 mM ZnCl2 reduced (P < 0.01) firmly attached cells by 82 and 91%, respectively . Reduction of Salmonella may be attributed, in part, to the bactericidal activity of ZnCl2 in addition to bacterial cell detachment.

Biochemistry, 2001 Apr 3, 40(13), 3912 - 9
Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains; Reynolds CM et al.; AhpF, the flavoprotein reductase component of the Salmonella typhimurium alkyl hydroperoxide reductase system, catalyzes the reduction of an intersubunit disulfide bond in the peroxidatic active site of the system's other component, AhpC, a member of the peroxiredoxin family . Previous studies have shown that AhpF can be dissected into two functional units, a thioredoxin reductase-like C-terminus (containing FAD and a redox-active disulfide, Cys345-Cys348) and an N-terminal domain containing a second redox-active disulfide center (Cys129-Cys132) . The role of the N-terminal domain as the direct reductant of AhpC, mediating electron transfer from the C-terminal redox centers of AhpF, has been firmly established by several approaches . Not known, however, was whether the transfer of electrons between the C-terminal and N-terminal disulfide centers occurred as an inter- or intrasubunit process in dimeric AhpF . Two heterodimeric AhpF species were therefore created in which one of the two pathways was completely disrupted while the other was left partially intact in each construct . Only the heterodimer containing one monomer of wild type AhpF and a monomer of mutated (and truncated) AhpF exhibited peroxidase activity with AhpC indicating that electron transfer between domains of AhpF is an intrasubunit process.

Biochemistry, 2001 Apr 3, 40(13), 3900 - 11
Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis; Wood ZA et al.; AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes . The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase . In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132) . A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC . Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF . Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E . coli thioredoxin . A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.

Clin Cancer Res, 2001 Mar, 7(3 Suppl), 856s - 864s
Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice; Xiang R et al.; Peripheral T-cell tolerance toward human carcinoembryonic self-antigen (CEA) was broken in CEA-transgenic C57BL/6J mice by an oral CEA-based DNA vaccine . This vaccine, delivered by the live, attenuated AroA- strain of Salmonella typhimurium (SL7207), induced tumor-protective immunity mediated by MHC class I-restricted CD8+ T cells . Activation of these T cells was indicated by increased secretion of proinflammatory cytokines IFN-gamma, interleukin (IL)-12 and granulocyte/macrophage-colony stimulating factor, as well as specific tumor rejection and growth suppression in vaccinated CEA-transgenic mice after a lethal challenge with murine MC38 colon carcinoma cells . These tumor cells were double transfected with CEA and the human epithelial cell adhesion molecule (Ep-CAM)/KSA and consequently served as a docking site for a recombinant antibody-IL2 fusion protein (KS1/4-IL2) recognizing KSA . Importantly, the efficacy of the tumor-protective immune response was markedly increased by boosts with this antibody-IL2 fusion protein, resulting in more effective tumor rejection coupled with increased expression of costimulatory molecules B7.2/B7.2 and intercellular adhesion molecule 1 (ICAM-1) on dendritic cells and intensified release of proinflammatory cytokines IFN-gamma, IL-12, and granulocyte/macrophage-colony stimulating factor from T cells of successfully vaccinated CEA-transgenic C57BL/6J mice . Increased T-cell activation mediated by boosts with KS1/4-IL2 fusion protein after tumor cell challenge was further indicated by expanded expression of T-cell activation markers CD25, CD28, CD69, and LFA-1 . The application of such CEA-based DNA vaccines and its further improved versions may ultimately prove useful in combination therapies directed against human carcinomas expressing CEA self-antigens.

Eur J Biochem, 2001 Apr, 268(8), 2201 - 8
Site-directed mutagenesis of human membrane-associated ganglioside sialidase: identification of amino-acid residues contributing to substrate specificity; Wang Y et al.; Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane-associated sialidase, which hydrolyzes only gangliosides . To cast light on the molecular basis of this narrow substrate preference, predicted active site amino-acid residues of the human membrane sialidase were altered by site-directed mutagenesis . When compared with the active site amino-acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region . Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS-1 cells and characterization of their kinetic properties using various substrates . Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction . The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild-type) with change to a lower Km and a higher Vmax . R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild-type . The inhibition experiments using 2-deoxy-2,3-didehydro-N-acetylneuraminic acid were consistent with the results in the alteration of substrate specificity . The findings suggest that putative active-site residues of the human membrane sialidase contribute to its substrate specificity.

Toxicology, 2001 Mar 28, 161(3), 165 - 77
The metabolism and bioactivation of agaritine and of other mushroom hydrazines by whole mushroom homogenate and by mushroom tyrosinase; Walton K et al.; Whole homogenates of Agaricus bisporus metabolised the mushroom hydrazine agaritine {beta-N-(gamma-L(+)glutamyl)-4-(hydroxymethyl) phenylhydrazine} to generate at least three metabolites . None of these metabolites, however, was the free hydrazine {4-(hydroxymethyl)phenylhydrazine}, the postulated metabolite of agaritine believed to be formed as a result of the loss of the gamma-glutamyl group, the reaction being catalysed by gamma-glutamyltransferase . The three metabolites of agaritine displayed weak mutagenic activity towards Salmonella typhimurium strain TA104 . 4-(Hydroxymethyl)phenylhydrazine, as the N'-acetyl derivative, was metabolised by mushroom tyrosinase to yield a number of metabolites that induced a mutagenic response in S . typhimurium TA104 . Similar to N'-acetyl-4-(hydroxymethyl)phenylhydrazine, agaritine was extensively metabolised by the mushroom tyrosinase but, in contrast, the structurally related N'-acetyl-4-hydrazinobenzoic acid did not serve as substrate of this enzyme, implying a critical role for the hydroxymethyl group at the para-position . In conclusion, the current studies have demonstrated for the first time that: (a) whole mushroom homogenates readily metabolise agaritine but not to the postulated 4-(hydroxymethyl)phenylhydrazine; and (b) mushroom tyrosinase metabolises agaritine and N'-acetyl-4-(hydroxymethyl)phenylhydrazine, in the latter case forming genotoxic metabolites.

Poult Sci, 2001 Apr, 80(4), 411 - 7
Cecal volatile fatty acids and broiler chick susceptibility to Salmonella typhimurium colonization as affected by aflatoxins and T-2 toxin; Kubena LF et al.; Four experiments were conducted using day-of-hatch, mixed-sex broiler chicks to evaluate the effects of aflatoxins and T-2 toxin on cecal volatile fatty acids (VFA) and the susceptibility to Salmonella colonization . All chicks in these experiments were challenged orally with 10(4) cfu of Salmonella typhimurium (ST) on Day 3 . In Experiments 1 and 2, chicks were fed diets containing 0, 2.5, or 7.5 mg aflatoxins/kg of diet and were allowed to develop their microflora naturally . In Experiment 3, all chicks were orally gavaged on the day of hatch with a competitive exclusion (CE) culture (PREEMPT) and were fed diets containing 0, 2.5, or 7.5 mg T-2 toxin/kg . In Experiment 4, the chicks were fed diets containing 0, 7.5, or 15.0 mg T-2 toxin/kg and one-half of the chicks were orally gavaged on the day of hatch with the CE culture . In Experiments 1 and 2, with the exception of increased total VFA at 5 d in chicks fed the 7.5 mg T-2 aflatoxins/kg diet, there were no treatment effects on cecal propionic acid, total VFA, or incidence or severity of ST colonization . In Experiment 3, the only alteration in concentration of cecal propionic acid or total VFA was a significant reduction in total VFA at 5 d in chicks fed the 2.5 mg T-2 toxin/kg diet . No significant treatment differences were observed for numbers of Salmonella cecal culture-positive chicks or for numbers of ST in the cecal contents . In Experiment 4, 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 . In the non-CE-treated chicks, T-2 toxin had no effect on any of the parameters, and 85 to 90% of the chicks were Salmonella cecal culture-positive . In the CE-treated chicks, there was a decrease in propionic acid concentration at 3 and 11 d and an increase in susceptibility to Salmonella colonization of the chicks fed the 15.0 mg T-2 toxin/kg diet . These results indicate that cecal concentrations of VFA can be affected by toxins, such as high concentrations of T-2 toxin, and that resistance to Salmonella colonization may be reduced . Further research is necessary to determine the biological significance of these changes.

EMBO J, 2001 Apr 17, 20(8), 1850 - 62
Type III secretion chaperone-dependent regulation: activation of virulence genes by SicA and InvF in Salmonella typhimurium; Darwin KH et al.; Invasion of the intestinal epithelium by Salmonella sp . requires a type III secretion system (TTSS) common in many bacterial pathogens . TTSS translocate effector proteins from bacteria into eukaryotic cells . These effectors manipulate cellular functions in order to benefit the pathogen . In the human and animal pathogen Salmonella typhimurium, the expression of genes encoding the secreted effector molecules Sip/Ssp ABCD, SigD, SptP and SopE requires both the AraC/XylS-like regulator InvF and the secretion chaperone SICA: In this work, an InvF binding site was identified in the promoter regions of three operons . SicA does not appear to affect InvF stability nor to bind DNA directly . However, SicA could be co-purified with InvF, suggesting that InvF and SicA interact with each other to activate transcription from the effector gene promoters . This is the first demonstration of a contact between a protein cofactor and an AraC/XylS family transcriptional regulator and, moreover, is the first direct evidence of an interaction between a transcriptional regulator and a TTSS chaperone . The regulation of effector genes described here for InvF and SicA may represent a new paradigm for regulation of virulence in a wide variety of pathogens.

Epidemiol Infect, 2001 Feb, 126(1), 3 - 9
Epidemiological studies of human and animal Salmonella typhimurium DT104 and DT104b isolates in Ireland; Murphy TM et al.; A total of 122 human and animal Salmonella Typhimurium DT104 isolates and 6 epidemiologically related DT104b isolates from human and animal products were analysed by pulsed-field gel electrophoresis (PFGE) . Genomic DNA was subjected to macrorestriction with three enzymes, SpeI, SfiI and XbaI . A total of 14 restriction fragment length polymorphism (RFLP) profiles were identified when the PFGE patterns from the three enzymes were combined . The majority of isolates (81.2%) exhibited the same RFLP profile . Six animal DT104 isolates, susceptible to enrofloxacin and resistant to naladixic acid, were identified from the antibiotic susceptibility test . Four of these isolates had a different PFGE profile from the common RFLP . In addition, 4 of the 6 isolates were geographically clustered in one region . It was concluded that there was one predominant strain of S . Typhimurium DT104 in Ireland and that the potential and selection pressures for emergence of fluoroquinolone-resistant isolates were present.

Mutat Res, 2001 Apr 5, 491(1-2), 195 - 209
The effects of 4'-alkyl substituents on the mutagenic activity of 4-amino- and 4-nitrostilbenes in Salmonella typhimurium; Ludolph B et al.; Six derivatives of trans-4-aminostilbene bearing different alkyl groups in the 4'-position and six of the corresponding nitro compounds were synthesized and tested for their mutagenic potency in Salmonella typhimurium strains TA98 and TA100 . Regarding the test series in presence of S9-mix, maximum activity was observed for those trans-4-aminostilbenes and trans-4-nitrostilbenes bearing small alkyl substituents like methyl and ethyl . More bulky substituents reduced the mutagenic potential in the order iso-propyl<sec-butyl<tert-butyl for the aminostilbenes . The corresponding nitrostilbenes showed a similar trend under these conditions although the mutagenic activity of the tert-butyl-substituted compound was unexpectedly high in TA100 . In the series without metabolic activation the nitrostilbenes showed a continuous decrease of mutagenic activity with the size of the substituents (methyl>ethyl>iso-propyl>sec-butyl>tert-butyl) . These trends have been compared with quantitative structure activity relationship (QSAR) model predictions, leading to the conclusion that steric demand is an important factor for mutagenicity of substituted aminostilbenes and nitrostilbenes . The unexpected result for the tert-butyl nitrostilbene tested with metabolic activation may be attributed to a different metabolic pathway.

Mutat Res, 2001 Apr 5, 491(1-2), 183 - 93
Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds; Haack T et al.; Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes . A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds . This effect could be observed in TA98 and TA100 . It was only marginally dependent on the metabolical activation by rat liver S9-mix . Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species . The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions . It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour.The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group . A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix . In contrast, 4'-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position . In addition, the substance shows only little cell toxicity even at higher concentrations . Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.

Mutat Res, 2001 Apr 5, 491(1-2), 119 - 26
The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey; Umbuzeiro GA et al.; Since 1979, the Environmental Agency of Sao Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters . This paper is a compilation of data obtained during the last 20 years from more than a thousand samples . Potencies up to 30,000 revertants/l were observed in 137 positive samples . The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix . A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public . The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively . As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the Sao Paulo State Water Quality Monitoring Program . This assay has proven to be a useful tool in the identification of important pollution sources . Correction and prevention actions in Water Pollution Control Programs were generated as a result.

Mutat Res, 2001 Apr 5, 491(1-2), 45 - 56
Role of reactive oxygen species in cupric 8-quinolinoxide-induced genotoxic effect; You BY et al.; This study demonstrates that cupric 8-quinolinoxide (CuQ) has induced genetic toxicity in bacteria and mammalian cells through a mechanism of reactive oxygen species (ROS) generation . In the Ames test with rat liver S9, CuQ dose-dependently caused a point mutation in Salmonella typhimurium TA100 . The effect of CuQ on DNA damage in HL60 and V79 cells identified in the comet assay is direct and enhanced by the addition of S9 . Meanwhile, the tailing length of comet DNA is related to the increasing dosage of CuQ . The genotoxic effect of CuQ on either gene mutation in bacteria or DNA damage in culture cells can be generally blocked by several antioxidants, e.g . pyrrolidinedithiocarbamate, N-acetylcysteine, Vitamins C and E . Supportive of this observation, ROS generation induced by CuQ can be demonstrated both in vitro and in vivo by using the DCFH-DA fluoroprobe . The CuQ-induced intracellular ROS level is also dramatically inhibited by the above antioxidants . Above results imply that the CuQ-induced genotoxicity could be mediated by ROS generation . The nature of ferrous-dependent and S9-enhancing in CuQ-induced ROS generation hints a Fenton-like reaction or some specific enzymes activation could be involved in this process . Furthermore, a DNA damage- and oxidative stress-dependent protein, P53, could also been induced by CuQ treatments in a time-course and dose-dependent manners . Its expression level is recoverable by antioxidants too . In conclusion, our current study strongly suggests that CuQ induces gene mutation, global DNA damage, and P53 expression through a ROS-dependent mechanism.

Toxicol In Vitro, 2001 Apr, 15(2), 105 - 14
Mini mutagenicity test: a miniaturized version of the Ames test used in a prescreening assay for point mutagenesis assessment; Flamand N et al.; The bacterial reverse mutagenicity test on Salmonella typhimurium, known as the Ames test, is widely used by regulatory agencies, academic institutions and chemical companies to assess the mutagenic potential of raw compounds . Several attempts have been made to miniaturise the Ames test in order to fit the industrial constraint of screening more products at the low quantities available . The major limitation of these miniaturised versions of the Ames test lies in the impossibility to work with all the six strains used in the regular Ames test, especially with those showing a low spontaneous revertant frequency . We describe here a mini version of the regulatory Ames test protocol that allows a significant reduction of the quantity of test substance needed (300 mg) but remains applicable to all Salmonella strains used in the regulatory protocol . In a preliminary study, 10 in-house chemical compounds have been evaluated in the Mini Mutagenicity Test (MMT) together with some positive control substances . A first set of historical data obtained in 1999 as well as the predictivity and the sensitivity of the MMT are presented and compared to those of the regular Ames test.

Biosens Bioelectron, 2000 Jun, 15(3-4), 167 - 72
Operational characteristics of an antibody-immobilized QCM system detecting Salmonella spp; Park IS et al.; A quartz crystal microbalance (QCM) system detecting Salmonella spp . was developed by an anti-Salmonella antibody immobilization onto one gold surface of a piezoelectric quartz crystal surface with sulfosuccinimidyl 6-{3-(2-pyridyldithio)propionamido}hexanoate (sulfo-LC-SPDP) thiolation . The optimum temperature and pH for the antibody-immobilized sensor were 35 degrees C and 7.2, respectively . The frequency shifts obtained were correlated with the Salmonella concentrations in the range 3.2 x 10(6)-4.8 x 10(8) CFU per ml . The system was quite specific to Salmonella spp . and applicable for repetitive use after a regeneration step employing 1.2 M NaOH . A model sample measurement was done for a market milk spiked with Salmonella typhimurium.

J Clin Invest, 2001 Apr, 107(7), 861 - 9
Neutrophil-epithelial crosstalk at the intestinal lumenal surface mediated by reciprocal secretion of adenosine and IL-6; Sitaraman SV et al.; Adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5' AMP . Using intestinal epithelial cell line T84, we studied the effect of adenosine on the secretion of IL-6, a proinflammatory cytokine involved in neutrophil degranulation and lymphocyte differentiation . Stimulation of T84 monolayers with either apical or basolateral adenosine induces A2b receptor-mediated increase in IL-6 secretion, which is polarized to the apical (luminal) compartment . In addition, Salmonella typhimurium, TNF-alpha, and forskolin, known inducers of IL-6 secretion in intestinal epithelial cells, also stimulate IL-6 secretion into the apical compartment . We show that IL6 promoter induction by adenosine occurs through cAMP-mediated activation of nuclear cAMP-responsive element-binding protein (CREB) . We also show that IL-6 released in the luminal (apical) compartment achieves a sufficient concentration to activate neutrophils (from which the adenosine signal originates), since such IL-6 is found to induce an intracellular {Ca(++)} flux in neutrophils . We conclude that adenosine released in the intestinal lumen during active inflammation may induce IL-6 secretion, which is mediated by cAMP/CREB activation and occurs in an apically polarized fashion . This would allow sequential activation of neutrophil degranulation in the lumen -- a flow of events that would, in an epithelium-dependent fashion, enhance microbicidal activity of neutrophils as they arrive in the intestinal lumen.

Arch Soc Esp Oftalmol, 2001 Mar, 76(3), 153 - 7
{Effect of tobramycin with topical diclofenac on arachidonic acid in endotoxin-induced uveitis}; Ruiz Moreno O et al.; PURPOSE: We studied if tobramycin associated to sodium diclofenac modifies the drug effect of the latter on arachidonic acid metabolism in an endotoxin induced uveitis (EIU) model in albino rabbits . METHODS: Experimental uveitis was induced by intravitreal injection of a 10 ng lipopolysaccharide A Salmonella typhimurium endotoxin dissolved in 5 microl saline solution in the right eye of experimental animals . We have used 48 animals (4 groups of 12 animals each) . The control group (G-I) was injected with saline solution (5 microl); the endotoxin group (G-II) was injected with 5 microl of ET; group III and IV were injected with the same amount of ET and treated with topical sodium diclofenac (1%) (Group III) and tobramycin and sodium diclofenac (0.3%) (Group IV) two hours before the administration of endotoxin and then every two hours until 24 hours, when we determined E2 prostaglandin and B4 leukotriene concentration in aqueous humor obtained by paracentesis of the anterior chamber . RESULTS: We observed how the E2 prostaglandin concentration was reduced in the two treatment groups compared with the endotoxin group (p<0.01) . However, there were no differences between groups III and IV (p>0.01) . There was a mild increase of B4 leukotriene in both treatment groups, which was not significant in relationship to the endotoxin group (p>0.01) . No differences were found between the two treatment groups (p>0.01) . CONCLUSION: Using the association of tobramycin does not affect the action of sodium diclofenac in arachidonic acid metabolism in endotoxin induced uveitis.

Vaccine, 2001 Apr 6, 19(20-22), 2945 - 54
Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium; Woo PC et al.; A novel vaccine for hepatitis B virus (HBV) was designed by putting a naked DNA vaccine carrying hepatitis B surface antigen (HBsAg) into live-attenuated Salmonella typhimurium . Mucosal immunization by the oral route in mice showed significantly stronger cytotoxic T lymphocyte (CTL) response than recombinant HBsAg vaccination (P < 0.01 at an effector:target ratio of 100:1), while comparable to intramuscular naked DNA immunization at all effector:target ratios . Contrary to previous reports on naked DNA vaccines given intramuscularly, the IgG antibody response induced by the mucosal DNA vaccine is relatively weak when compared to recombinant HBsAg vaccine (P < 0.001 at day 21) . These findings are supported by a high interferon-gamma but a low interleukin-4 level detected in the supernatant of splenic cell cultures obtained from mucosally immunized mice . As distinct to recombinant HBsAg vaccine which is effective for protection, oral mucosal DNA vaccine should be considered as a candidate for therapeutic immunization in chronic HBV infection, donor immunization before adoptive transfer of HBV-specific CTL to HBsAg positive bone marrow transplant recipients, and immunization of non-responders to recombinant HBsAg vaccine . This strongly cellular and relatively absent humoral response may make this vaccine a better candidate as a therapeutic vaccine for chronic HBV carriers than naked DNA vaccines, as the humoral response is relatively less important for the clearance of HBV from hepatocytes, but its presence may lead to side effects such as serum sickness and immune complex deposition in chronic HBV carriers.

Vaccine, 2001 Apr 6, 19(20-22), 2854 - 61
Nasal vaccination with attenuated Salmonella typhimurium strains expressing the Hepatitis B nucleocapsid: dose response analysis; Nardelli-Haefliger D et al.; Nasal vaccination of mice with recombinant attenuated strains of Salmonella typhimurium is more efficient at inducing antibody responses than oral vaccination . However, mortality was observed when high doses {10(9) colony forming unit (CFU)}, otherwise safe by the oral route, were administered . This observation was counterbalanced by the fact that nasal vaccination was still highly efficient with lower doses (10(6) CFU), which are inefficient by the oral route and this, without any incidents of mortality . Here, we further analyse in mice the effect of nasal vaccination with differently attenuated S . typhimurium strains expressing the Hepatitis B nucleocapsid (HBc) . Surprisingly, as few as 100 CFU were sufficient to induce a maximal HBc specific antibody response, but only if the bacteria were inhaled . Furthermore, we observed no correlation between the inoculum dose and the number of surviving bacteria in cervical lymph nodes and spleen . Examination of lung sections revealed strong inflammation and bronchopneumonia 24 h after nasal vaccination with 10(8) CFU, while only minor signs of inflammation were detected transiently when 10(3) CFU or phosphate buffered saline (PBS) were administered . Our data suggest that the safety issue of nasal vaccination with low doses of the Salmonella vaccine strains should be addressed in humans, as it might be an efficient alternative to oral vaccination.

Biochimie, 2001 Feb, 83(2), 155 - 9
Transcription induces a supercoil domain barrier in bacteriophage Mu; Scheirer KE et al.; In enteric bacteria, chromosomes are partitioned into domains that exhibit restricted supercoil movement . The most common domain barrier detected by gammadelta resolution assays is random with respect to sequence and occurs more frequently in cells growing rapidly in rich medium compared to cells in stationary phase . Transcription generates both positive and negative supercoiling movement . To address the question of whether transcription causes the appearance of new domain boundaries, a transcriptionally active MudI element was substituted for a MudJr-1 element that resides within the cobT gene of Salmonella typhimurium . Mu-specific transcription from the phage early promoter was placed under control of either the wild type (c(+)) or the temperature-sensitive (cts62) repressor . Using a resolution assay with res sites at six chromosomal locations, domain structure was normal in cells carrying the MudAr-1 prophage with a wild type Mu repressor . However, in cells with a MudAr-1 prophage harboring the cts62 repressor, a new domain barrier appeared in > 90% of the cells . Supercoil movement was restricted ahead of but not behind the transcription machinery . We conclude that the strong Mu early promoter induces the appearance of a domain barrier within the limits of a MudAr-1 prophage.

Nat Immunol, 2001 Apr, 2(4), 361 - 7
Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria; Rescigno M et al.; Penetration of the gut mucosa by pathogens expressing invasion genes is believed to occur mainly through specialized epithelial cells, called M cells, that are located in Peyer's patches . However, Salmonella typhimurium that are deficient in invasion genes encoded by Salmonella pathogenicity island 1 (SPI1) are still able to reach the spleen after oral administration . This suggests the existence of an alternative route for bacterial invasion, one that is independent of M cells . We report here a new mechanism for bacterial uptake in the mucosa tissues that is mediated by dendritic cells (DCs) . DCs open the tight junctions between epithelial cells, send dendrites outside the epithelium and directly sample bacteria . In addition, because DCs express tight-junction proteins such as occludin, claudin 1 and zonula occludens 1, the integrity of the epithelial barrier is preserved.

J Mol Biol, 2001 Mar 30, 307(3), 899 - 911
Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins; Raso SW et al.; Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins . The Raman S-H band is a unique and sensitive probe of the local S-H environment . Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein . The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure . The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure . To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine . The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures . Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups . (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein . (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds . (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635 . (iv) Cys290 and Cys496 form weak hydrogen bonds . (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states . The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike . The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway . This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes . The method employed here should be applicable to a wide range of cysteine-containing proteins .

Chemosphere, 2001 Mar, 42(8), 931 - 44
Evaluation of an SOS-Chromotest-based approach for the isolation and detection of sediment-associated genotoxins; Bombardier M et al.; A series of experiments was conducted to evaluate an approach advanced by the St . Lawrence Centre (SLC) of Environment Canada for assessing the genotoxic potential of sediments . The SLC method entails the extraction, isolation and solvent exchange of the organic constituents in sediment, and the testing of these solubilized extracts with the SOS Chromotest (Escherichia coli PQ37) . A total of five sediments, three variously contaminated by organic compounds and two reference materials certified for persistent organic chemicals, were Soxhlet-extracted . Each of the five extracts was then split, with a portion remaining in crude form and another portion fractionated into two molecular-weight classes of organic contaminants, thus yielding a total of 15 extract samples . The ability of the SOS Chromotest to detect genotoxins in the various organic extracts was evaluated and compared with that of the Ames Fluctuation Assay (Salmonella typhimurium, strain TA100) . The intra-laboratory variance associated with the SOS Chromotest was also assessed . Procedural details are presented and results are discussed . The SOS Chromotest results were in good agreement with those of the Ames Fluctuation Assay, especially after metabolic activation . However, the E . coli PQ37 system was slightly more sensitive than the Salmonella assay for detecting genotoxins in the sediment extracts . The SOS Chromotest was also the most discriminating of the two assays, generating SOS-induction factors that were consistent with the organic contamination gradient reported in the sediment samples . The removal of macromolecules from the dichloromethane extracts by size-exclusion chromatography prior to testing enhanced the sensitivity of both test systems . The intra-laboratory variance of the SOS Chromotest ranged from 0.24% to 23.82%, depending on the extract sample . As applied in this study, the SOS Chromotest can serve as a sensitive test for screening the genotoxic potential of uncharacterized sediment extracts . A more sensitive assay would be appropriate, however, as a confirmation for definitive investigations, especially for the detection of direct-acting genotoxins.

Carcinogenesis, 1980 Aug, 1(8), 715 - 9
The influence of molecular size and partition coefficients on the predictability of tumor initiation in mouse skin from mutagenicity in Salmonella typhimurium; Scribner NK et al.; Initiation of tumors in mouse skin is well correlated with mutagenesis in hamster V79 cells (correlation coefficient r = 0.88), but is poorly correlated with bacterial mutagenicity in the Ames assay (r = 0.58) . Efforts to understand the difference in the degrees of correlation led to the observation that if the bacterial mutagenicity data are combined with a molecular size or partition factor, the correlation of initiating activity with this combined parameter approaches that found with the hamster cell mutagenesis . The improvement in correlation (r = 0.84) leads to the suggestion that when a broad size range of compounds is considered a most important factor in accounting for the poor correlation initially observed may be a difference between mammalian and bacterial cells in permeability or target access.

Carcinogenesis, 1980, 1(12), 1049 - 57
Ethylene dibromide and disulfiram: studies in vivo and in vitro on the mechanism of the observed synergistic carcinogenic response; Elliott BM et al.; Two possible mechanisms for the reported carcinogenic synergism between ethylene dibromide (EDB) and disulfiram have been investigated in vivo and in vitro, the first involving increased production of an EDB-derived glutathione mustard and the second increased production of bromoacetaldehyde . Consistent with both of these suggested mechanisms, repeated administrations of disulfiram to rats inreased liver glutathione-S-transferase activity and decreased liver low Km aldehyde dehydrogenase activity . However, when added to a rat liver S-9 fraction in vitro, disulfiram decreased transferase activity and only depressed the dehydrogenase activity after a period of preincubation . Although the mutagenic potency of EDB to Salmonella typhimurium was slightly enhanced in vitro by the addition of a rat liver S-9 fraction, the further addition of disulfiram to the assay medium produced no additional change . Similarly, the addition of a range of S-9 and S-0.5 liver fractions derived from disulfiram-treated rats also failed to enhance significantly its mutagenic potency over the normal S-9 fraction . The general implications of these findings are discussed.

J Food Prot, 2001 Feb, 64(2), 255 - 8
Effect of sodium chlorate on Salmonella typhimurium concentrations in the weaned pig gut; Anderson RC et al.; Salmonella cause economic losses to the swine industry due to disease and compromised food safety . Since the gut is a major reservoir for Salmonella, strategies are sought to reduce their concentration in pigs immediately before processing . Respiratory nitrate reductase activity possessed by Salmonella also catalyzes the intracellular reduction of chlorate (an analog of nitrate) to chlorite, which is lethal to the microbe . Since most gastrointestinal anaerobes lack respiratory nitrate reductase, we conducted a study to determine if chlorate may selectively kill Salmonella within the pig gut . Weaned pigs orally infected with 8 x 10(7) CFU of a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were treated 8 and 16 h later via oral gavage (10 ml) with 0 or 100 mM sodium chlorate . Pigs were euthanized at 8-h intervals after receiving the last treatment . Samples collected by necropsy were cultured qualitatively and quantitatively for Salmonella and for most probable numbers of total culturable anaerobes . A significant (P < 0.05) chlorate treatment effect was observed on cecal concentrations of Salmonella, with the largest reductions occurring 16 h after receiving the last chlorate treatment . An observed treatment by time after treatment interaction suggests the chlorate effect was concentration dependent . Chlorate treatment may provide a means to reduce foodborne pathogens immediately before harvest.

J Food Prot, 2001 Feb, 64(2), 240 - 5
Mutagenicity and identification of mutagenic compounds of fumes obtained from heating peanut oil; Wu SC et al.; Since the fume of cooking oil has been reported to increase the risk of lung cancer, the objectives of this study were to evaluate the mutagenicity and to find the mutagens in the fumes of peanut oil heated to the smoke point . Peanut oil prepared from roasted peanut kernel showed a lower smoke point, less unsaturated fatty acids, more fume formation, and stronger mutagenicity than that from unroasted kernel . Further investigation of mutagenic compounds was performed by the Ames test and gas chromatography/mass spectrometry analysis . Among the 12 compounds identified from the neutral fraction of methanol extract, four compounds at a dose of 10 microg per plate were mutagenic to Salmonella Typhimurium TA98 and TA100 in the order of trans-trans-2,4-decadienal > trans-trans-2,4-nonadienal > trans-2-decenal > trans-2-undecenal . Results report the enal compounds formed as the mutagens in the fumes of peanut oil and indicate that inhaling cooking fumes might cause carcinogenic risk.

Microbiol Immunol, 2001, 45(1), 79 - 83
Effect of constitutively expressed phoP gene on the localization of Salmonella typhimurium within Mac-1 positive phagocytes; Matsui H et al.; Intracellular localization of the wild-type (Spv+), the phoP-constitutively expressed strain (PhoPc), and the spv-deleted strain (Spv-) of Salmonella typhimurium was examined by the use of confocal laser scanning microscopy analysis of immunostained sections of mouse spleens after oral or subcutaneous inoculation . Only 40% of salmonellae of both the PhoPc and the Spv- strains were detected intracellularly within Mac-1 positive cells at day five after oral or day four after subcutaneous inoculation . In contrast, over 85% of salmonellae of the Spv+ strain were detected inside Mac-1 positive cells . In both inoculation trials, the splenic colony-forming unit values for the PhoPc and Spv- strains were significantly lower than the corresponding value for the Spv+ strain . These findings suggest that the constitutively expressed phoP gene of S . typhimurium attenuated virulence by limiting intracellular proliferation within mouse spleen phagocytes, and that the lack of spv genes had the same effect.

Mol Microbiol, 2001 Mar, 39(6), 1595 - 609
The novel sigma54- and sigma28-dependent flagellar gene transcription hierarchy of Vibrio cholerae; Prouty MG et al.; The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum . Flagellar transcriptional regulatory factors have been demonstrated to contribute to V . cholerae virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear . The flagellar genes revealed by the V . cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations . It had previously been demonstrated that the alternative sigma factor sigma54 and the sigma54-dependent activators FlrA and FlrC are necessary for flagellar synthesis . The V . cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor sigma28 . A V . cholerae DeltafliA mutant strain is non-motile, and synthesizes a truncated flagellum . Vibrio cholerae FliA complements both V . cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor . Analysis of lacZ transcriptional fusions of the V . cholerae flagellar promoters in both V . cholerae and S . typhimurium identified sigma28-, sigma54-, FlrA- and FlrC-dependent promoters, as well as promoters that were independent of all these factors . Our results support a model of V . cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes . Class I is composed solely of the gene encoding the sigma54-dependent activator FlrA, which along with the sigma54-holoenzyme form of RNA polymerase activates expression of Class II genes . These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC . FlrC, along with sigma54-holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes . Finally, sigma28-holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes . Thus, this novel V . cholerae flagellar hierarchy has incorporated elements from both the sigma54-dependent Caulobacter crescentus polar flagellar hierarchy and the sigma28-dependent S . typhimurium peritrichous flagellar hierarchy.

Mol Microbiol, 2001 Mar, 39(6), 1585 - 94
An adenosyl-cobalamin (coenzyme-B12)-repressed translational enhancer in the cob mRNA of Salmonella typhimurium; Ravnum S et al.; Expression of the cobalamin (Cbl) biosynthetic cob operon in Salmonella typhimurium is repressed by the end-product . This regulation is conferred mainly at the translational level and involves a cobalamin-induced folding of an RNA hairpin that sequesters the ribosomal binding site (RBS) of the cob mRNA and prevents translation initiation . A combined structural and mutational analysis shows that a cis-acting translational enhancer (TE) element, located 83 nucleotides upstream of the Shine-Dalgarno sequence in the 5'-untranslated region (5'-UTR) of the cob mRNA, is required to unfold the inhibitory RBS hairpin in the absence of cobalamin . The TE element, which consists of 5 nucleotides, is proposed to confer its enhancer function in the absence of cobalamin by interacting with nucleotides in the stem of the RBS hairpin . This interaction destabilizes the RNA hairpin and allows ribosome binding . In the presence of cobalamin, the enhancer function is inhibited . As a result, the RBS hairpin forms and prevents translation initiation . Several additional RNA hairpins in the 5'-UTR were also identified and are suggested to be important for repression . The above data suggest that normal cobalamin repression of the cob operon requires that the 5'-UTR has a defined secondary and tertiary structure.

Mol Microbiol, 2001 Mar, 39(6), 1452 - 63
The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix; Zogaj X et al.; Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus . We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose . The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype . Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined . However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis . The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix . As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.

Biophys J, 2001 Apr, 80(4), 1973 - 85
Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy; Chirico G et al.; O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme . Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation . We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation . We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules . The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm . These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine . Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case . Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity . At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns . The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.

Regul Toxicol Pharmacol, 2001 Feb, 33(1), 2 - 11
Safety evaluation of phosphodiesterase produced from Penicillium citrinum: summary of toxicological data; Kondo M et al.; The toxicity of Enzyme RP-1, an enzyme preparation used to hydrolyze yeast RNA to produce flavor enhancers for use in the food industry, was evaluated in a series of studies . A 5-week dietary toxicity study in Wistar rats was conducted in which animals received Enzyme RP-1 in feed at concentrations of 0, 500, 2000, or 8000 mg/kg body wt/day . A 13-week dietary toxicity study in Sprague-Dawley rats was conducted in which animals received RP-1 concentrate at 0, 0.125, 0.5, or 2% in their diets . At the highest dose levels in both studies, submandibular glands in the oral cavity were enlarged, an effect attributed to protease activity of the enzyme preparation . The no-observed-effect level in rats in the 13-week study was 0.5%, equivalent to 317 mg/kg body wt/day for males and 346 mg/kg body wt/day for females . Based on estimated dietary exposure to the enzyme preparation, the margin of exposure is estimated to be greater than 38,000 . Lack of genotoxic potential was demonstrated by an in vitro reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli strain WP2uvr and by an in vitro chromosome aberration test in CHL/IU cells derived from fibroblasts from the lungs of Chinese hamsters . Finally, the particular strain of Penicillium citrinum, the fungal strain used to prepare Enzyme RP-1, was shown to have low pathogenicity upon a single injection into the tail vein of rats of viable spores at doses up to 2.8x10(5) colony-forming units per animal . The results of these studies demonstrate that the enzyme preparation may be considered safe to workers and consumers when employed in the production of flavor enhancers from yeast .

J Cell Sci, 2001 Apr, 114(Pt 7), 1331 - 41
The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells; Criss AK et al.; The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells . This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria . Members of the Rho family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry . Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two Rho family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S . typhimurium . Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE . Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process . In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry . These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

J Environ Monit, 2000 Apr, 2(2), 161 - 3
Ascorbic acid reduction of active chlorine prior to determining Ames mutagenicity of chlorinated natural organic matter (NOM); Urbansky ET et al.; Many potable water disinfection byproducts (DBPs) that result from the reaction of natural organic matter (NOM) with oxidizing chlorine are known or suspected to be carcinogenic and mutagenic . The Ames assay is routinely used to assess an overall level of mutagenicity for all compounds in samples from potable water supplies or laboratory studies of DBP formation . Reduction of oxidizing disinfectants is required since these compounds can kill the bacteria or react with the agar, producing chlorinated byproducts . When mutagens are collected by passing potable water through adsorbing resins, active chlorine compounds react with the resin, producing undesirable mutagenic artifacts . The bioanalytical and chemoanalytical needs of drinking water DBP studies required a suitable reductant . Many of the candidate compounds failed to meet those needs, including 2,4-hexadienoic (sorbic) acid, 2,4-pentanedione (acetylacetone), 2-butenoic (crotonic) acid, 2-butenedioic (maleic and fumaric) acids and buten-2-ol (crotyl alcohol) . Candidates were rejected if they (1) reacted too slowly with active chlorine, (2) formed mutagenic byproducts, or (3) interfered in the quantitation of known chlorination DBPs . L-Ascorbic acid reacts rapidly and stoichiometrically with active chlorine and has limited interactions with halogenated DBPs . In this work, we found no interference from L-ascorbic acid or its oxidation product (dehydroascorbic acid) in mutagenicity assays of chlorinated NOM using Salmonella typhimurium TA100, with or without metabolic activation (S9) . This was demonstrated for both aqueous solutions of chlorinated NOM and concentrates derived from the involatile, ether-extractable chlorinated byproducts of those solutions.

Gene, 2001 Feb 21, 264(2), 281 - 8
Identification of the cobalamin-dependent methionine synthase gene, metH, in Vibrio fischeri ATCC 7744 by sequencing using genomic DNA as a template; Kasai S et al.; To confirm the presence of cobalamin-dependent methionine synthase (CDMS) in luminous bacteria, which is a prerequisite for the substantiation of our proposals on the physiological function of the lux operon, we identified the CDMS gene (metH) in Vibrio fischeri ATCC 7744 . Two partial metH sequences, one located near the 5'-terminus of the gene and the other near the 3'-terminus, were sequenced by a PCR based method . To design a new set of PCR primers located on the two flanking regions of the gene, the genomic DNA was sequenced by SUGDAT method (sequencing using genomic DNA as a template) upstream or downstream from the respective partial gene sequences . Subsequently a 4.2 kb DNA fragment containing the whole metH was amplified by PCR and sequenced . The number of amino acid residues comprising the protein (1226 amino acids) was comparable to those of known CDMSs . The deduced amino acid sequence showed 85, 74, 55, 31, 30, 52, or 52% identity with that of Vibrio cholerae, Escherichia coli, Deinococcus radiodurans, Synechocystis PCC6803, Mycobacterium tuberculosis, Caenorhabditis elegans or Homo sapiens, respectively . All the predicted amino acid residues for the binding of cobalamin and S-adenosylmethionine were conserved . In the regulatory region of the V . fischeri metH, the binding site of the met repressor, MetJ, was present, although the site is atypically not present in E . coli metH or Salmonella typhimurium metH . It was shown that nucleotide sequences, even long ones, can be determined without a cloning step, if only parts of the DNA fragment to be sequenced are amplified by PCR.

Int J Food Microbiol, 2001 Feb 15, 63(3), 209 - 16
Effect of sodium chloride concentration on the heat resistance and recovery of Salmonella typhimurium; Manas P et al.; The survival of Salmonella typhimurium (ATCC 13311) heated and recovered in media with 0, 1, 2, 3, 4 or 5% (w/w) added sodium chloride was investigated . A protective effect in the heating medium and an inhibitory effect in the recovery medium were observed . The results showed an interaction between the effect on, D(58 degrees C) values, of sodium chloride concentration in both media . Lower concentration in the heating media led to a greater effect of the sodium chloride concentration in the recovery media . When the sodium chloride concentration was the same in both media, the protective effect exerted in the heating media dominated over its inhibitory effect in the recovery media.

Mutat Res, 2001 Mar 1, 474(1-2), 113 - 20
Structure-antimutagenic activity relationships of benzalacetone derivatives against UV-induced mutagenesis in E . coli WP2uvrA and gamma-induced mutagenesis in Salmonella typhimurium TA2638; Motohashi N et al.; The antimutagenic activities of benzalacetone (4-phenyl-3-buten-2-one) and its structurally-related compounds were evaluated through their use as post-treatments for the UV-induced mutagenesis in Escherichia coli WP2s (uvrA) and the gamma-induced mutagenesis in Salmonella typhimurium TA2638, the latter of which is sensitive to oxidants . Structure-activity relationships were studied between IC(50) activity values, i.e . the dose (micromol/ml) at which the mutation frequency is reduced to 50% of the control, and electronic and hydrophobicity properties of the studied molecules . Benzalacetone and benzalacetone analogs, cinnamaldehyde and trans-1,1,1-trifluoro-4-phenyl-3-buten-2-one (TF), inhibited both forms of mutagenesis, but methyl cinnamate, cinnamic acid and cinnamamide did not . The IC(50) values of TF, for UV-induced mutagenesis and gamma-induced mutagenesis, were 0.028 and 0.045 micromol/ml, respectively, and one order of magnitude lower than those of cinnamaldehyde and benzalacetone . The three antimutagenic analogs listed in order of decreasing activity are: TF>>cinnamaldehyde>benzalacetone . This order is proportional to the electron-withdrawing property of the terminal group attached to an alpha,beta-unsaturated carbonyl moiety in the side chain that is known to play an important role in the antimutagenicities of benzalacetone and related compounds . In UV-induced mutagenesis in E . coli WP2s, mono-substituted benzalacetones - the ring-substituents of which have electron-withdrawing properties - showed antimutagenic activity that correlated with their electronic property . In gamma-induced mutagenesis in S . typhimurium TA2638, the antimutagenic activities of mono-substituted benzalacetones were proportional to the substituent hydrophobicities (pi) . The different effects on both the mutation-induced systems is suggested to be related to the relative permeability of the cell membranes and the different sensitivities to mutagens between E . coli WP2s and S . typhimurium TA2638 . In addition, the antimutagenic activity against gamma-induced mutagenesis could be due to the ability of parent compounds or their derivatives to scavenge long-lived organic radicals; the radicals have been described to be generated as a result of the X-irradiation of cells by Koyama et al . {Mutat . Res . 421 (1998) 45}.

Mutat Res, 2001 Mar 1, 474(1-2), 71 - 85
Antimutagenic activity of green tea and black tea extracts studied in a dynamic in vitro gastrointestinal model; Krul C et al.; An in vitro gastrointestinal model, which simulates the conditions in the human digestive tract, was used to determine potential antimutagenic activity of extracts of black tea and green tea . In this paper, results are presented on the availability for absorption of potential antimutagenic compounds present in tea and on the influence of the food matrix on this activity . Between 60 and 180min after the tea was introduced into the model, antimutagenic activity was recovered from the jejunal compartment by means of dialysis: the dialysate appeared to inhibit the mutagenicity of the food mutagen MeIQx in the direct plate assay with Salmonella typhimurium (Ames test) . The maximum inhibition was measured at 2h after the start of the experiment and was comparable for black tea and green tea extract . To determine the influence of food matrices on the antimutagenic activity of tea, the model was loaded with black tea together with milk or a homogenized standard breakfast . The maximum inhibition observed with black tea was reduced by 22, 42 and 78% in the presence of whole milk, semi-skimmed milk, and skimmed milk, respectively . Whole milk and skimmed milk abolished the antimutagenic activity of green tea by more than 90%; for semi-skimmed milk the inhibition was more than 60% . When a homogenized breakfast was added into the model together with the black tea extract, the antimutagenic activity was completely eliminated . When tea and MeIQx were added together into the digestion model, MeIQx mutagenicity was efficiently inhibited, with green tea showing a slightly stronger antimutagenic activity than black tea . In this case, the addition of milk had only a small inhibiting effect on the antimutagenicity.Antioxidant capacity and the concentration of catechins were also measured in the jejunal dialysates . The reduction in antimutagenic activity corresponded with reduction in antioxidant capacity and with a decrease of concentration of three catechins, viz . catechin, epigallocatechin gallate and epigallocatechin . The in vitro gastrointestinal model appears to be a useful tool to study the antimutagenicity of food components.

Microbiology, 2001 Mar, 147(Pt 3), 727 - 33
Salmonella typhimurium thyA mutants fail to grow intracellularly in vitro and are attenuated in mice; Kok M et al.; Salmonella typhimurium ATCC14028 readily multiplies in professional phagocytes in vitro and is highly virulent in mice . Mutants lacking thymidylate synthase activity (thyA) were isolated and shown to be strictly dependent on thymidine monophosphate precursors in the growth medium . The thyA mutants were found to be virtually incapable of intracellular growth and survival in vitro, both in macrophage-like cell line P338D(1) and in the human epithelial cell line Hep-2, and their virulence was impaired in BALB/c mice . Intraperitoneal immunization of mice with two doses of live S . typhimurium thyA provided protection against a challenge with 10(3) times the 50% lethal dose of the virulent parent strain.

Microbiology, 2001 Mar, 147(Pt 3), 701 - 8
Transcription of arcA and rpoS during growth of Salmonella typhimurium under aerobic and microaerobic conditions; Sevcik M et al.; Physiology of the exponential and stationary phase of growth, under both aerobic and microaerobic conditions, of Salmonella typhimurium and its isogenic mutants nuoG::Km, cydA::TnphoA, DeltaarcA and DeltarpoS was studied using luxAB transcriptional fusions with the rpoS and arcA genes . In the wild-type strain, rpoS transcription was greater under aerobic than under microaerobic conditions, whereas transcription of arcA was suppressed by aerobiosis . Under aerobic conditions, no interaction between NuoG, CydA, ArcA and RpoS was detected . Under microaerobic conditions, rpoS was suppressed in the nuoG mutant as compared with the wild-type strain, but it was overexpressed in the cydA and arcA mutants . A deletion in the rpoS gene, on the other hand, resulted in non-restricted, increased arcA expression in stationary-phase cultures under microaerobic conditions . Based on the rpoS transcription in the nuoG mutant the authors propose that the decrease in the NADH:NAD ratio that occurs when carbon sources become limiting serves as a signal for increased rpoS transcription, while active respiration catalysed by CydA and controlled by ArcA downregulates rpoS transcription . When, finally, the RpoS-controlled stationary phase of growth is reached, arcA is suppressed in an RpoS-dependent fashion . Transition into stationary phase under microaerobic conditions is thus controlled by coordinated action of the RpoS and ArcA regulators, depending on subtle changes in the environment.

Biochem Biophys Res Commun, 2001 Mar 9, 281(4), 858 - 65
Anthranilate synthase without an LLES motif from a hyperthermophilic archaeon is inhibited by tryptophan; Tang XF et al.; Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, have been expressed independently in Escherichia coli . The anthranilate synthase complex (Tk-AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (alpha subunit) and Tk-TrpG (beta subunit), at 85 degrees C for 10 min . Further purification of Tk-AS complex was carried out by anion-exchange chromatography followed by gel-filtration . Molecular mass estimations from gel-filtration chromatography indicated that Tk-AS complex was a heterodimer (alphabeta) . The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor . The optimal pH was pH 10.0 and the optimal temperature was 85 degrees C in both cases . Mg2+ was necessary for the anthranilate synthase activity . At 75 degrees C, the K(m) values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 microM, respectively . The K(m) value of Mg2+ was 20.5 microM . The K(m) values of glutamine and NH4Cl were 88 microM and 5.6 mM, respectively . Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by L-tryptophan were not present in Tk-TrpE . Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S . typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE . Nevertheless, significant inhibition by L-tryptophan was observed, with K(i) values of 5.25 and 74 microM for ammonia and glutamine-dependent activities, respectively . The inhibition was competitive with respect to chorismate . The results suggest that the amino acid residues involved in the feedback inhibition by L-tryptophan in the case of Tk-AS complex are distinct from previously reported anthranilate synthases .

J Mol Biol, 2001 Mar 9, 306(5), 1127 - 37
Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS; Renzoni D et al.; The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids . The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein . H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region . We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain . This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration . NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry . Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples . The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs) . Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively . A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor . In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer . The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

J Mol Biol, 2001 Mar 9, 306(5), 915 - 29
A multipartite interaction between Salmonella transcription factor sigma28 and its anti-sigma factor FlgM: implications for sigma28 holoenzyme destabilization through stepwise binding; Chadsey MS et al.; Transcription of the late (Class 3) flagellar promoters in Salmonella typhimurium is dependent upon the flagellar specific sigma factor, sigma28, encoded by the fliA gene . sigma28-dependent transcription is inhibited by an anti-sigma factor, FlgM, through a direct interaction . FlgM can bind both to free sigma28 to prevent it from forming a complex with core RNA polymerase, and to sigma28 holoenzyme to destabilize the complex . A collection of fliA mutants defective for negative regulation by FlgM (fliA* mutants) were isolated . This collection included 27 substitution mutations that conferred insensitivity to FlgM in vivo . The distribution of mutations defined three potential FlgM binding domains in conserved sigma factor regions 2.1, 3.1 and 4 of sigma28 . A subset of mutants from each region was assayed for FlgM binding and transcriptional activity in vitro . The results strongly support a multipartite interaction between sigma28 and FlgM . Region 4 mutations, but not region 2.1 or 3.1 mutations, interfered with the ability of FlgM to destabilize sigma28 from core RNA polymerase . We present refined models for FlgM inhibition of sigma28, and for FlgM destabilization of sigma28 holoenzyme.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Jan-Feb, (1), 40 - 3
{Dependence of immunosuppressive action of lipopolysaccharide on degree of Salmonella pathogenicity}; Borisova EV et al.; Immunosuppressive activity of Salmonella typhimurium extracellular lipopolysaccharide (LPS) was studied . In this study isogenic S . typhimurium strains with different degree of virulence were used . The attenuation of these strains was linked with mutations on their chromosome (altered synthesis of RNA polymerase or gyrase DNA) or their own virulence plasmid (the insertion of transposon Tn-5) . To obtain LPS fraction with different molecular weights, the filtrate of bacterial culture was subjected to gel filtration through a column packed with Sephadex G-200 . The immunosuppressive action of LPS fractions was determined on the model of delayed-type hypersensitivity to nonbacterial antigen in experiments on BALB/c mice . The study revealed that transposon-mediated mutation on plasmid, accompanied by the attenuation of salmonellae, led to the loss of immunosuppressive activity of the high-molecular heat-sensitive component of LPS; only the second heat-resistant component with medium molecular weight retained its activity . The presence of two chromosomal attenuating mutations (rifr nalr) was accompanied by the loss of immunosuppressive activity in both components of LPS.

Water Res, 2001 Mar, 35(4), 913 - 20
The role of indigenous microorganisms in suppression of Salmonella regrowth in composted biosolids; Sidhu J et al.; Composting is commonly used as an effective means of stabilizing wastewater biosolids and reducing pathogens to very low concentrations . However, it has been shown that under certain conditions Salmonella can regrow in previously composted biosolids . Growth of seeded Salmonella typhimurium in composted biosolids ranging from two weeks to two years maturity was monitored . Results from sterile and non-sterile composted biosolids were compared . Seeded S . typhimurium colonized rapidly in sterilized biosolids reaching a maximum population density of more than 10(8) g(-1) . Growth of seeded S . typhimurium was suppressed in non-sterilized compost with a maximum population density of less than 10(3) g(-1) . There was a significant decline in the growth rate of seeded Salmonella in sterilized compost when the compost was stored, suggesting that bio-available nutrients declined with storage . However, in non-sterilized compost this was not the case . This suggests that the indigenous microflora play a significant role in suppression of Salmonella regrowth in composted biosolids . There was a strong negative correlation (-0.85) between the Salmonella inactivation rate and the maturity of compost in non-sterilized compost . The Salmonella inactivation rate was seven times higher in biosolids composting for two weeks as compared to compost stored for two years . This suggests that the antagonistic effect of indigenous microorganisms towards Salmonella declined with compost storage . It was concluded that all composted biosolids had a Salmonella regrowth potential . However, the indigenous microflora significantly reduced this regrowth potential . Long-term storage of compost is not recommended as this may increase the pathogen regrowth potential.

Free Radic Res, 2001 Jan, 34(1), 33 - 44
Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis; Imaeda A et al.; Oxidative stress may be an important factor in the development of diabetic complications . Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes . We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin . Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro . DNA was exposed to either reactive oxygen species or advanced glycation end-products . Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects . Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains . Both pravastatin and simvastatin still lacked protective activity . Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.

Eur J Pharm Sci, 2001 Feb, 12(4), 495 - 504
Studies on the photostability and in vitro phototoxicity of Labetalol; Andrisano V et al.; The purpose of this study was to obtain information on the photochemical and phototoxic properties of Labetalol, a beta-blocker drug . Preliminary information on the drug photoreactivity was achieved using a flow system with a photochemical reactor on-line with a diode array detection system . Photophysical and photochemical investigations on the drug were performed in aqueous solutions at different pH values using spectrophotometric and fluorimetric methods; the photodegradation quantum yield was found to be 2.7 x 10(-3) at pH 5.8 and 1.5 x 10(-2) at pH 11.5 . Forced photodegradation of labetalol solutions under exposure to UVA--UVB radiations (xenon arc lamp) was monitored by reversed-phase liquid chromatography . The main photodegradation products were isolated and characterized by NMR and mass spectrometry; labetalol was found to give 3-amino-1-phenylbutane and salicylamide-4-carboxaldehyde as the main photoproducts . Preliminary phototoxic testings on human keratinocyte cultures were performed evaluating the viability of the cells by the neutral-red uptake assay; mutagenic and photomutagenicity tests were also carried out based on Salmonella typhimurium strains . As a result, labetalol was found to be photolabile,mainly in alkaline medium, but evidences of significant phototoxic and photomutagenic effects by the drug were not observed.

Microbios, 2001, 104(407), 49 - 54
Ultrastructural localization of succinate dehydrogenase in some bacteria, after treatment with Lubrol W1; Cherepova N et al.; The localization of succinate dehydrogenase in some gram-negative and gram-positive bacteria (Salmonella typhimurium, Pseudomonas pseudomallei, Pseudomonas aeruginosa and Listeria monocytogenes) treated with the surface membrane active agent, Lubrol W1, was studied by a cytochemical method combined with electron microscopy.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2735 - 9 Epub 2001 Feb 13.
Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing; Aballay A et al.; Programmed cell death (PCD) in mammals has been implicated in several disease states including cancer, autoimmune disease, and neurodegenerative disease . In Caenorhabditis elegans, PCD is a normal component of development . We find that Salmonella typhimurium colonization of the C . elegans intestine leads to an increased level of cell death in the worm gonad . S . typhimurium-mediated germ-line cell death is not observed in C . elegans ced-3 and ced-4 mutants in which developmentally regulated cell death is blocked, and ced-3 and ced-4 mutants are hypersensitive to S . typhimurium-mediated killing . These results suggest that PCD may be involved in the C . elegans defense response to pathogen attack.

Berl Munch Tierarztl Wochenschr, 2001 Jan-Feb, 114(1-2), 35 - 9
{Meat juice ELISA for determination of Salmonella incidence in slaughter pig herds in Bavaria}; Czerny CP et al.; Meat samples from diaphragm pillars were randomly taken from 3,048 pigs of 52 Bavarian herds after slaughtery . Meat-juice was collected and tested for salmonella antibodies in an indirect ELISA . The number of samples was calculated according to the annual production of slaughter pigs of a farm outlined in the "Leitlinien fur ein Programm zur Reduzierung des Eintrags von Salmonellen durch Schlachtschweine in die Fleischgewinnung" from February 05th, 1998 (< 100 slaughter pigs: 45 samples, 100-200 slaughter pigs: 50 samples, > 200 slaughter pigs: 60 samples per year) . Salmonella antibodies were detected in 48 carcasses (1.6%) of 12 farms (23.1%) . However, 33 (68.8%) of these carcasses were originated from a single farm which had to be classified into category III (prevalence of > 40% in the samples) . No bacteria could be isolated from this farm in a follow up examination . The 51 other farms (98%) were classified into category I (prevalence of < 20% in the samples) . Farms with in/out-management showed a higher degree of reagents (2.1T%) than farms with continuous stabling (0.8T%) . In a pig experimentally immunized with LPS-antigen preparations of Salmonella typhimurium it was shown that antibodies induced were nearly at the same level in all meat samples and even in selected organs (liver, kidney, parotis, mesenteric lymph nodes).

Int J Food Sci Nutr, 2001 Jan, 52(1), 5 - 14
Some microbiological and biochemical studies on the fermentation of 'awaze' and 'datta', traditional Ethiopian condiments; Idris A et al.; The microbial and some biochemical changes during the fermentation of two Ethiopian condiments were studied . The aerobic mesophilic microflora of the ingredients of 'awaze' were dominated by Bacillus species (1.1 x 10(6) cfu/g) and lactic acid bacteria (4.5 x 10(4) cfu/g) . The counts of aerobic mesophilic bacteria declined during the fermentation period . Lactic acid bacteria (LAB) reached the maximum count of 5.9 x 10(9) cfu/g at day 4 and the count remained > 10(8) cfu/g throughout the fermentation . The heterofermentative LAB dominated until day 3; thereafter the homolactics dominated the fermentation . Yeasts appeared at day 6 and increased to 2.5 x 10(6) cfu/g . In 'datta' fermentation, the count of aerobic mesophilic bacteria remained unchanged during the fermentation . LAB initiated the fermentation at a level of 7.1 x 10(4) cfu/g and reached 1.2 x 10(9) cfu/g at day 7 . The homolactic LAB initiated and dominated the fermentation for the first 2 days and the heterolactics took over thereafter . Both fermentations were accompanied by declining pH and increasing titratable acidity . Salmonella typhimurium was inhibited during both fermentations within 48 h . Both 'awaze' and 'datta' had low initial contents of available protein and reducing sugars and these did not show marked differences throughout the fermentation.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 48 - 52
{Protective efficacy of combined administration of the multicomponent vaccine and the immunomodulator myelopid in experimental infections in mice}; Stepanenko RN et al.; The influence of myelopid (MP) on the protective activity of polycomponent vaccine VP-4 prepared from the antigens of opportunistic bacteria was studied on experimental infections of mice, caused by Klebsiella pneumoniae, Salmonella typhimurium and Staphylococcus aureus . In staphylococcal and Klebsiella infections the joint administration of vaccine VP-4 and MP produced more pronounced protective effect than each of these preparations, introduced alone . The protective action of vaccine VP-4 was specially enforced by MP in cases of local staphylococcal infection . Recommendations on the joint use of two or more immunomodulating agents are possible only on the basis of the experimental substantiation of their effect in definite infections.

Microbiol Immunol, 2000, 44(12), 987 - 95
Salmonella typhimurium induces apoptosis in human monocyte-derived macrophages; Zhou X et al.; Salmonella species represent a leading cause of gastroenteritis worldwide . More recently, they have been proposed as putative vaccine delivery vehicles in humans . Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages . In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF . Wild type S . typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30 min post-infection . Three hours later more than 80% of the macrophages in the culture were killed . In contrast, during the same period, macrophages infected with the non-invasive S . typhimurium strain BJ66 remained viable . Chromatin fragmentation is a hallmark of cells undergoing apoptosis . Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells . Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S . typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40% . Our study provides the first evidence that invasive S . typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.

Nat Struct Biol, 2001 Mar, 8(3), 243 - 7
Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium; Morollo AA et al.; We have determined the X-ray crystal structure of the cooperative anthranilate synthase heterotetramer from Salmonella typhimurium at 1.9 A resolution with the allosteric inhibitor l-tryptophan bound to a regulatory site in the TrpE subunit . Tryptophan binding orders a loop that in turn stabilizes the inactive T state of the enzyme by restricting closure of the active site cleft . Comparison with the structure of the unliganded, noncooperative anthranilate synthase heterotetramer from Sulfolobus solfataricus shows that the two homologs have completely different quarternary structures, even though their functional dimer pairs are structurally similar, consistent with differences in the cooperative behavior of the enzymes . The structural model rationalizes mutational and biochemical studies of the enzyme and establishes the structural differences between cooperative and noncooperative anthranilate synthase homologs.

Gene, 2001 Jan 24, 263(1-2), 39 - 48
Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain; Chen L et al.; We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp . PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala) . These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E . coli strain ER2799) . EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure . Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E . coli strain ER2799 . Further characterization of these sites by inserting a full length Ssp DnaE intein identified residue 235 of EPSPS as the optimal position . The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE intein, respectively . Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity . As a control the split EPSPS without the intein-affinity domain did not allow cell growth . The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC) . Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein . As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.

J Antimicrob Chemother, 2001 Mar, 47(3), 315 - 21
Antimicrobial resistance in salmonellae from humans, food and animals in Spain in 1998; Cruchaga S et al.; We studied 1710 Salmonella: spp . isolates from human (1051), food (421) and animal (238) sources . They were tested by the disc diffusion method for susceptibility to 12 different antimicrobial agents . The incidence of resistance and multiple resistance (MR) among the salmonella strains of different origins, the relationship between their most frequent serotypes and phage types (PTs) and their antimicrobial resistance patterns were determined . In general, the incidence of resistance and MR was significantly higher in animal isolates than in human and food isolates (P < 0.05) . Resistance to each individual drug among the human isolates and food isolates was very similar, with resistance to ampicillin, tetracycline, streptomycin and sulphonamides most frequently observed . MR has remained uncommon in Salmonella enteritidis . Nevertheless, 90% of PT6A of the human isolates and 100% of the food isolates were ampicillin resistant and 80 and 60%, respectively, of the PT1 isolates were nalidixic acid resistant . Salmonella typhimurium was the most multiresistant serotype in the three sample populations and ten different patterns of MR were seen . Almost 100% of the Salmonella hadar isolates, from human and food sources, were resistant . We recommend restriction of the use of antibiotics in veterinary medicine in order to reduce the selection and spread of multiresistant strains.

Carcinogenesis, 1980, 1(10), 867 - 70
Mutagenicity in Salmonella typhimurium of N-3-methylbutyl-N-1-methyl-acetonyl-nitrosamine and N-methyl-N-benzylnitrosamine, N-nitrosation products isolated from corn-bread contaminated with commonly occurring moulds in linshien county, a high incidence area for oesophageal cancer in Northern China; Lu SH et al.; Two synthetic N-nitrosamines (N-3-methylbutyl-N-1-methyl acetonylnitrosamine and N-methyl-N-benzylnitrosamine), previously isolated from corn-bread which had been inoculated with moulds occurring in Linshien county, Northern China and subsequent nitrosation by sodium nitrite, were tested in Salmonella typhimurium strains TA1535 and TA100 in the presence of a liver postmitochondrial supernatant from Aroclor-treated rats . A concentration-dependent increase in the number of mutant colonies in both bacterial strains was observed when N-3-methylbutyl-N-1-methylacetonyl-nitrosamine was assayed in liquid suspension and N-methyl-N-benzylnitrosamine in plate incorporation assays . Our finding that mutagenic N-nitrosamines are present in foodstuffs that may be consumed in Linshien county are discussed in relation to the possible etiological role of these compounds in cancer of the oesophagus in that area.

Carcinogenesis, 1980, 1(11), 911 - 23
Some factors affecting mutant numbers in the Salmonella/microsome assay; Booth SC et al.; An examination has been made of some of the parameters which can affect mutant numbers in the Salmonella/microsome assay . The type of minimal media plates used for the assay and the concentration of glucose-6-phosphate, one of the co-factors necessary for mono-oxygenase action, had no effect on mutant numbers . Increases in mutated bacteria resulted from the use of (1) log-phase bacteria, (2) higher NADP concentrations than those normally recommended, and (3) higher phosphate buffer concentrations . Six mutagens, i.e., 2-acetylaminofluorene (AAF), 3,3'-dichlorobenzidine (3,3'-DCB), cyclophosphamide (CY), aflatoxin B1 (AFB1), 3-methylcholanthrene (3MC) and benzo{a}pyrene (BP), all requiring mono-oxygenase activation, were studied with two Salmonella typhimurium strains, TA98 and TA100, and liver preparations from rats given different inducing agent treatments using optimum conditions . Phenobarbitone induction was generally superior to Aroclor-1254 in converting these substrates to mutagens except for the polycyclic hydrocarbon substrates . A comparison of 3-methylcholanthrene, Aroclor-1254, beta-naphthoflavone or phenobarbitone as inducing agents revealed the first three of these to be equally effective in activating BP or 3MC to mutagens, whereas phenobarbitone was less active . Dual administration of 3-methylcholanthrene and phenobarbitone to rats did not result in an additive mutagenic effect using AAF, AFB1 or 3,3'-DCB as substrates, the numbers of mutant bacteria obtained being only equal to that seen with 3-methylcholanthrene alone . These differences were not due to there being different liver protein optima for the various inducing agent treatments . The foregoing results are discussed in relation to attempts to draw up a rigid protocol for mutagenicity testing.

Carcinogenesis, 1980, 1(11), 889 - 92
Effect of methyl substitution on mutagenicity of 2-aminodipyrido; Takeda K et al.; The mutagenicities of 2-aminodipyrido {1,2-a:3',2'-d} imidazole (Glu-P-2, a mutagen obtained from pyrolysate of glutamic acid) and six methyl substituted derivatives of Glu-P-2 were tested with Salmonella typhimurium TA98 and TA100 with S-9 mix . Glu-P-1 (6-Me-Glu-P-2) was the strongest mutagen to TA98 and TA100 . 3-Me-Glu-P-2 was weakest . The presence of a methyl group, and its position were shown to affect the mutagenicity significantly.

Carcinogenesis, 1980 Jul, 1(7), 583 - 7
Mutagenicity and toxicity of chromyl chloride and its vapours; De Flora S et al.; Chromyl chloride (CC), a liquid Cr6+ compound suspected of carcinogenic activity, was assayed for mutagenicity in the Ames reversion test . Due to its high volatility and toxic and corrosive properties, handling of CC required particular precautions . The liquid phase of CC elicited dose-related mutations in Salmonella typhimurium, strain TA100, although it had a limited range of activity because of its toxicity to the bacteria . The mutagenic potency and the toxic activity were of the same order of magnitude as all the water-soluble Cr6+ compounds so far tested . Toxic and mutagenic activities could also be clearly detected by using a variety of modifications of the Ames test in CC vapours which indicates the particular danger of this chromium compound . In analogy with the other Cr6+ compounds previously tested in this laboratory, all the effects observed were decreased in the presence of S-9 mix containing rat liver post-mitochondrial fractions.

Epidemiol Infect, 2000 Dec, 125(3), 473 - 80
Emerging antibiotic resistance in Salmonella typhimurium in Norway; Leegaard TM et al.; The antimicrobial resistance of 809 Salmonella Typhimurium isolates collected from humans in Norway between 1975 and 1998 was studied . The material was subdivided into domestic and foreign isolates according to whether the patient had recently travelled abroad or not . In imported isolates the largest increase in resistance was in 1996 when 35% of the isolates were multi-resistant . The first multi-resistant isolate acquired in Norway appeared in 1994, but already in 1998 23% of the isolates domestically acquired were multi-resistant, and a majority were S . Typhimurium DT104 . We found no ciprofloxacin resistance in domestically acquired isolates . Amplified fragment length polymorphism analysis was performed on selected multi-resistant isolates . The method discriminated well between different multi-resistant isolates, but not between DT104 isolates . Resistant and multi-resistant S . Typhimurium were until 1998 essentially recovered from patients who had travelled abroad, but multi-resistant isolates, mainly DT104, are now also being transmitted within the country.

Oncol Res, 2000, 12(3), 127 - 35
Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector; Zheng LM et al.; Attenuated strains of Salmonella typhimurium, VNP20009 and YS7212, when injected systemically to tumor-bearing mice, accumulated preferentially in tumors at levels at least 200-fold and, more commonly, 1000-fold greater than in other normal tissues . This selectivity occurred in subcutaneously implanted murine tumors, including B16F10 melanoma, M27 lung carcinoma, and colon 38 carcinoma . The preferential accumulation was also manifested in animals bearing human tumor xenografts, including Lox, C8186, DLD1, SW620, HCT116, HTB177, DU145, MDA-MB-231, and Caki . Four to five days after a single IV injection of 1 x 10(6) colony-forming unit (cfu)/mouse, we routinely detected VNP20009 proliferation and accumulation at levels ranging from 1 x 10(8) to 2 x 10(9) cfu/g tumor . The amount of VNP20009 accumulated in the liver ranged from 3 x 10(4) to 2 x 10(6) cfu/g . The distribution of Salmonella in tumors was homogenous; YS7212 could be detected from the periphery to the interior portion of the tumors . Using mice with various immunodeficiencies, we also discovered the same preferential accumulation of Salmonella in tumors implanted in these mice . The use of Salmonella as a protein delivery vector was shown by IV administration of the bacteria expressing either green fluorescent protein (GFP) or cytosine deaminase (CD) into tumor-bearing mice . GFP and CD were detected in tumors, but not in livers, taken from mice inoculated with Salmonella carrying these genes . Bacteria accumulation and CD expression persisted in the tumors for up to 14 days after a single bolus IV administration of bacteria to tumor-bearing mice.

J Environ Pathol Toxicol Oncol, 2001, 20(1), 9 - 14
Antimutagenic potential of extracts isolated from Terminalia arjuna; Kaur S et al.; Terminalia arjuna is an important medicinal plants widely used in the preparation of Ayurvedic formulations used against several ailments . The present investigation was aimed at the fractionation of crude extracts from the bark of T . arjuna in order to isolate and purify the antimutagenic factors present . The antimutagenicity assay was performed to check the modulatory effect of these fractions against NPD, sodium azide, and 2AF, using the Ames Salmonella his+ reversion assay . Most of the phenolic fractions exhibited mutagen specificity against direct-acting mutagens, being effective in suppressing the frameshift mutagen NPD but failing to inhibit sodium azide (base pair substitution)-induced his+ revertants . ET-1 fraction triterpenoid diglycoside showed a marked effect against sodium azide but was ineffective against NPD . In the case of the indirect-acting mutagen 2AF, all the fractions were found to be quite potent in modulating its mutagenicity in both TA98 and TA100 tester strains of Salmonella typhimurium . The results indicate that the bark of T . arjuna harbors constituents with promising antimutagenic/anticarcinogenic potential that should be investigated further.

J Environ Pathol Toxicol Oncol, 2000, 19(4), 333 - 46
Nontoxic, mutagenic, and clastogenic activities of Mate-Chimarrão (Ilex paraguariensis); Fonseca CA et al.; Aqueous extracts of Ilex paraguarariensis (mate-chimarrao), a species that belongs to the Aquifoliaceae family, were analyzed for the presence of genotoxic, mutagenic, and clastogenic activities through bacterial trials based on the induction of the SOS functions, as well as in human lymphocytes in vitro and in mammalian cells in vivo . The extracts of mate-chimarrao were genotoxic, as assessed by lysogenic induction in Escherichia coli, and they also induced mutagenesis in Salmonella typhimurium . They addition of S9 microsomal fraction, catalase, thiourea, or dipyridyl counteracted the genotoxic activity of mate-chimarrao, suggesting that oxygen reactive species play an essential role in the genotoxicity of mate-chimarrao extracts . The extracts were not clastogenic in vivo (bone marrow cells of rats) in our experimental conditions, but we have observed an increased frequency of chromosomal aberrations in mate-chimarrao-treated human peripheral lymphocytes . Our results suggest that a high consumption of mate-chimarrao can potentiate carcinogenesis in the human oropharynx and esophagus.

New Microbiol, 2001 Jan, 24(1), 85 - 9
Salmonella typhimurium-endocarditis secondary to an acquired environmental infection: a case report; Di Bonaventura G et al.; A diabetic, cardiopathic and anemic 44-year-old farmer presented with a seven-day history of remittent fever with evening peaks . Two months before he had undergone amputation of the V-finger of the left hand secondary to a phlegmon caused by an agricultural injury . Prior to amputation, anaerobic culture analysis of phlegmon-pus and selective procedures used to isolate Gram-positive cocci and/or Pseudomonas spp . resulted negative . The diagnosis of endocarditis was supported by isolation of S . typhimurium from blood and by echocardiography showing endocarditic lesions . The source of infection was identified by PCR ribotyping as the same Salmonella typhimurium strain that was present, but not sought, both in the anatomic explanted tissues and from blood samples of the patient . The infection was successfully treated with a combination of gentamicin and ampicillin with consequent improvement in the general clinical picture of the patient . We believe this is the first reported case of S . typhimurium-endocarditis secondary to a phlegmon resulting from an environmental source of infection.

Biol Chem, 2000 Dec, 381(12), 1185 - 93
Mutational scanning of a hairpin loop in the tryptophan synthase beta-subunit implicated in allostery and substrate channeling; Rondard P et al.; The tryptophan synthases from Escherichia coli and Salmonella typhimurium are tetrameric enzymes, with an elongated TrpA.TrpB.TrpB.TrpA structure . Structural studies have identified residues 277-283 of TrpB as a potentially important region for the allosteric communication between the TrpA and TrpB subunits and for the transport of indole between their active sites through a hydrophobic tunnel . To explore the functional role of this region, we analyzed the effects of 19 single and double mutations in TrpB on the tryptophan synthase (TSase) and serine deaminase (SDase) activities of the TrpB2 dimer, either in the presence or in the absence of the TrpA subunit . The mutations of residues 273-283 could be divided into 4 classes . Mutations 1278A, F280G and M282A decreased the SDase and TSase activities of TrpB2 to similar extents . F280A decreased the SDase activity of TrpB2 more than its TSase activity, whereas the reverse was true for Y279L . F280A decreased the activation factor of TrpB2 by TrpA, whereas F280G increased it . The reaction steps and intramolecular contacts that could be affected by the mutations are described . The sequence 278-IYFGM-282, which is present in E . coli and S . typhimurium, is only found in 5 out of 42 organisms, whereas the sequence VLHGX is found in 21 organisms . Our results identified several mutations that could be used as structural probes to analyze precisely the roles of residues 278-282 and their evolution.

Appl Biochem Biotechnol, 2000 Nov-Dec, 89(2-3), 151 - 60
Microbial sensors of ultraviolet radiation based on recA'::lux fusions; Rosen R et al.; Escherichia coli strains containing plasmid-borne fusions of the recA promoter-operator region to the Vibrio fischeri lux genes were previously shown to increase their luminescence in the presence of DNA damage hazards, and thus to be useful for genotoxicant detection . The present study expands previous work by demonstrating and investigating the luminescent response of these strains to ultraviolet radiation . Several genetic variants of the basic recA'::lux design were examined, including a tolC modification of membrane efflux capacity, a chromosomal integration of the recA'::lux fusion, a different lux reporter (Photorhabdus luminescens instead of V . fischeri, allowing the assay to be run at 37 degrees C), and a different host bacterium (Salmonella typhimurium instead of E . coli) . Generally, two modifications provided the fastest responses: the use of the S . typhimurium host or the P . luminescens lux reporter . Highest sensitivity, however, was demonstrated in an E . coli strain in which a single copy of the V . fischeri lux fusion was integrated into the bacterial chromosome.

Mol Cell Biochem, 2000 Dec, 215(1-2), 39 - 46
Role of anaerobiosis in virulence of Salmonella typhimurium; Singh RD et al.; Intestinal pathogens are exposed to various stress conditions during their infectious cycle . Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen . In the present study, a virulent strain of S . typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S . typhimurium . Anaerobically grown S . typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically . Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro . Anaerobic S . typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria . LD50 of anaerobic S . typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria . Cell surface hydrophobicity of anaerobic S . typhimurium was also found to be significantly higher than aerobic bacteria . Thus, it appears that exposure of S . typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.

Environ Pollut, 2001, 111(1), 83 - 8
Toxicity of methyl-tert-butyl ether to freshwater organisms; Werner I et al.; Increased input of the fuel oxygenate methyl-tert-butyl ether (MTBE) into aquatic systems has led to concerns about its effect(s) on aquatic life . As part of a study conducted by University of California scientists for the State of California, the Aquatic Toxicology Laboratory, UC Davis, reviewed existing literature on toxicity of MTBE to freshwater organisms, and new information was generated on chronic, developmental toxicity in fish, and potential toxicity of MTBE to California resident species . Depending on time of exposure and endpoint measured, MTBE is toxic to various aquatic organisms at concentrations of 57-> 1000 mg/l (invertebrates), and 388-2600 mg/l (vertebrates) . Developmental effects in medaka (Oryzias latipes) were not observed at concentrations up to 480 mg/l, and all fish hatched and performed feeding and swimming in a normal manner . Bacterial assays proved most sensitive with toxicity to Salmonella typhimurium measured at 7.4 mg/l within 48 h . In microalgae, decreased growth was observed at 2400 and 4800 mg/l within 5 days . MTBE does not appear to bioaccumulate in fish and is rapidly excreted or metabolized . Collectively, the available data suggests that at environmental MTBE exposure levels found in surface waters (< 0.1 mg/l) this compound is likely not acutely toxic to aquatic life . However, more information is needed on chronic and sublethal effects before we can eliminate the possibility of risk to aquatic communities at currently detected concentrations.

Arch Toxicol, 2000 Dec, 74(10), 638 - 41
Mutagenicity of N-nitrosodiethylamine in the Ames test with S . typhimurium TA1535 is due to volatile metabolites and is not dependent on cytochrome P4502E1 induction; Westphal GA et al.; N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages . No threshold has been detected for these carcinogenic effects . The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages . We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range . Two main modifications were evaluated . Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied . The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture . Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment . For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used . N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535 . NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix . A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535 . In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites . The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites . Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.

J Food Prot, 2001 Jan, 64(1), 58 - 62
Lactic acid sprays reduce bacterial pathogens on cold beef carcass surfaces and in subsequently produced ground beef; Castillo A et al.; Organic acids have been shown to be effective in reducing the presence of pathogenic bacteria on hot beef carcass surfaces; however, application for decontaminating chilled carcasses has not been fully evaluated . In this study, a postchill, 30-s lactic acid spray (500 ml of 4% L-lactic acid, 55 degrees C) was applied onto outside rounds that had been contaminated with Escherichia coli O157:H7 and Salmonella Typhimurium, subsequent to prechill hot carcass treatments consisting of water wash alone or water wash followed by a 15-s lactic acid spray (250 ml of 2% L-lactic acid, 55 degrees C) . The prechill treatments reduced both pathogens by 3.3 to 3.4 log cycles (water wash alone) to 5.2 log cycles (water wash and lactic acid) . In all cases, the postchill acid treatment produced an additional reduction in E . coli O157:H7 of 2.0 to 2.4 log cycles and of 1.6 to 1.9 log cycles for Salmonella Typhimurium . The counts of both pathogens remained significantly lower in ground beef produced from the outside rounds that received prechill and postchill acid spray than from those that received a postchill spray only . These data indicate that organic acid sprays may be successfully applied for pathogen reduction in beef carcass processing after the cooler, especially when combined with prechill treatments.

J Food Prot, 2001 Jan, 64(1), 23 - 9
Survival and growth of Salmonella and Listeria in the chicken breast patties subjected to time and temperature abuse under varying conditions; Murphy RY et al.; Chicken breast patties were inoculated with a mixture of Salmonella Senftenberg, Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Mission, Salmonella Montevideo, Salmonella California, and Listeria innocua . The initial inoculation of bacteria was approximately 10(7) log10 CFU/g . The inoculated patties were processed in a pilot-scale air convection oven at an air temperature of 177 degrees C, an air velocity of 9.9 m3/min, and a low (a wet bulb temperature of 48 degrees C) or high (a wet bulb temperature of 93 degrees C) humidity condition . The patties were processed to a final center temperature of 65 to 75 degrees C . The survivors of Salmonella and Listeria in the processed patties were evaluated . Processing humidity affected the survivors of bacteria . More survivors of Salmonella and Listeria (>2 logs) were obtained for the patties cooked at low humidity than at high humidity . After thermal processing, the patties were stored under air, vacuum, or CO2 at refrigerated (4 degrees C) or thermally abused (8 to 15 degrees C) temperatures . Storage temperature, time, and gas environment affected the bacteria growth . Higher storage temperature and longer storage time correlated to an increased growth of bacteria in the cooked chicken patties . Less Salmonella (2 logs) and Listeria (0.5 to 1 log) cells were obtained in the patties stored under vacuum than in air . Storing the patties in 30% CO2 reduced the growth of Salmonella more than 2 log10 CFU/g . At a CO2 level of 15%, 1 log10 CFU/g of reduction was obtained for Listeria in cooked chicken patties.

J Food Prot, 2001 Jan, 64(1), 17 - 22
Inhibition of in vitro Salmonella Typhimurium colonization in porcine cecal bacteria continuous-flow competitive exclusion culturest; Hume ME et al.; Continuous-flow (CF) chemostate cultures were used as models to determine the potential usefulness of undefined porcine cecal bacteria as competitive exclusion (CE) cultures against colonization by Salmonella Typhimurium . One culture, pCF1, was derived from cecal bacteria of an animal maintained on antibiotic-free feed, while the other culture, pCF4, was derived from cecal bacteria of an animal maintained on feed containing chlortetracycline . The effectiveness against a chlortetracycline-resistant Salmonella Typhimurium was examined in CF cultures maintained in the absence (pCF1 and pCF4) and presence (cpCFl and cpCF4) of chlortetracycline . CF cultures were inoculated with each of 10(2), 10(4), and 10(6) Salmonella Typhimurium CFU/ml . Chemostat inocula of 10(2) Salmonella CFU/ml resulted in no Salmonella Typhimurium being detected at 2 and 3 days postinoculation in pCF1 and pCF4, respectively, and after 2 days in both cpCF1 and cpCF4 . Inoculations of 10(4) Salmonella Typhimurium CFU/ml resulted in clearance from pCF1 and pCF4 within 4 days and within 3 days from cpCF1 and cpCF4 . Following inoculation with 10(6) CFU/ml, no Salmonella Typhimurium were detected in all CF cultures by 6 days postinoculation . The results indicated that in vitro CF cultures of porcine cecal bacteria were able to inhibit the growth of Salmonella Typhimurium . The ability to limit Salmonella Typhimurium growth was not restricted by prior exposure of the cecal bacteria to the feed additive chlortetracycline . The present study demonstrates the potential application of CF cultures as models to aid in the identification of CE cultures against salmonellosis in pigs.

Am J Vet Res, 2001 Jan, 62(1), 72 - 6
Evaluation of polymyxin B in an ex vivo model of endotoxemia in horses; Parviainen AK et al.; OBJECTIVE: To evaluate effects of polymyxin B sulfate (PMB) on response of horses to endotoxin, using an ex vivo model . ANIMALS: 8 healthy horses . PROCEDURE: In a crossover design, 3 doses of PMB (100, 1,000, and 10,000 U/kg of body weight) and physiologic saline solution (control) were evaluated . Prior to and for 24 hours after administration of PMB, blood samples were collected into heparinized tubes for use in 2 assays . For the endotoxin-induced tumor necrosis factor (TNF) assay, blood samples were incubated (37 C for 4 h) with 1 ng of Escherichia coli or Salmonella Typhimurium endotoxin/ml of blood . Plasma was harvested and assayed . For the residual endotoxin activity assay, plasma was collected into sterile endotoxin-free borosilicate tubes, diluted 1:10 with pyrogen-free water, and incubated for 10 minutes at 70 C . Escherichia coli endotoxin (0.1 or 1 ng/ml of plasma) was added to the thawed samples prior to performing the limulus ameobocyte lysate assay . Serum creatinine concentrations were monitored for 1 week . RESULTS: Compared with baseline values, PMB caused a significant dose- and time-dependent decrease in endotoxin-induced TNF activity . Compared with baseline values, residual endotoxin activity was significantly reduced after administration of 10,000 U of PMB/kg . Compared with baseline values, 1,000 and 5,000 U of PMB/kg should inhibit 75% of endotoxin-induced TNF activity for 3 and 12 hours, respectively . Serum creatinine concentrations remained within the reference range . CONCLUSION AND CLINICAL RELEVANCE: Results of the study suggest that PMB is a safe, effective inhibitor of endotoxin-induced inflammation in healthy horses.

Avian Dis, 2000 Oct-Dec, 44(4), 989 - 92
Serologic survey of slaughter-age ostriches (Struthio camelus) for antibodies to selected avian pathogens; Ley EC et al.; Serum samples from 163 slaughter-age ostriches (Struthio camelus) in Ohio and Indiana were tested for antibodies to avian influenza virus (AIV), Newcastle disease virus (NDV), paramyxovirus (PMV) 2, PMV3, PMV7, infectious bursal disease virus (IBDV), Bordetella avium, Mycoplasma synoviae, Mycoplasma gallisepticum, Ornithobacterium rhinotracheale, Salmonella pullorum, Salmonella gallinarum, and Salmonella typhimurium . One ostrich had antibodies to AIV H5N9, 57% of the ostriches had antibodies to NDV, four ostriches had antibodies to both NDV and PMV2, and one ostrich had antibodies to NDV, PMV2, PMV3, and PMV7 . None of the ostriches had antibodies to IBDV, B . avium, M . synoviae, M . gallisepticum, O . rhinotracheale, S . pullorum, S . gallinarum, and S . typhimurium . This is the first report of antibodies to avian influenza and PMV7 in ostriches in the United States.

Avian Dis, 2000 Oct-Dec, 44(4), 968 - 76
Some safety aspects of salmonella vaccines for poultry: distribution and persistence of three Salmonella typhimurium live vaccines; Barbezange C et al.; The purpose of this study was to analyze the safety characteristics of three commercially available live Salmonella vaccine strains (vacT, Zoosaloral, and X3985) in relation to their persistence in individual animals but also within a flock and in the environment . In a first experiment, the digestive and systemic distributions in chickens were followed for 10 days in individually reared chickens that were orally inoculated at 1 day of age . Strain X3985 quickly disappeared from the digestive tract but remained in the liver until the end of this experiment, whereas strains vacT and Zoosaloral colonized the liver as well as the gut for 10 days . In the second trial, behavior of the vaccine strains was studied in groups of 20 chickens during 10 wk after a single oral administration to individual birds . Strain vacT remained in the environment of inoculated animals for 4-5 wk . Six weeks after the inoculation, vacT was not recovered from internal organs such as liver and spleen, and vacT disappeared from the digestive tract between the sixth and the 10th weeks . Comparatively, both Zoosaloral and X3985 vaccine strains persisted longer in the environment (8 wk at least) . Of the vaccine strains, X3985 showed the greatest colonization of both systemic and digestive organs.

Avian Dis, 2000 Oct-Dec, 44(4), 853 - 60
Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures; Himathongkham S et al.; Exponential inactivation was observed for Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure with decimal reduction times ranging from half a day at 37 C to 1-2 wk at 4 C . There was no material difference in inactivation rates between S . typhimurium and E . coli O157:H7 . Inactivation was slower in slurries made by mixing two parts of water with one part of manure; decimal reduction times (time required for 90% destruction) ranged from 1-2 days at 37 C to 6-22 wk at 4 C . Escherichia coli O157:H7 consistently exhibited slightly slower inactivation than S . typhimurium . Log decimal reduction time for both strains was a linear function of storage temperature for manure and slurries . Chemical analysis indicated that accumulation of free ammonia in poultry manure was an important factor in inactivation of the pathogens . This finding was experimentally confirmed for S . typhimurium by adding ammonia directly to peptone water or to bovine manure, which was naturally low in ammonia, and adjusting pH to achieve predetermined levels of free ammonia.

Crit Rev Biotechnol, 2000, 20(4), 265 - 91
Control of foodborne pathogens during sufu fermentation and aging; Shi X et al.; Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated . Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g . After 2 days of fermentation at 30 degrees C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g . After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl . After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels . The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P > 0.05) compared with the count after 1 month . Microorganism in experimental sufu from different aging periods and in commercial sufu were compared . A total of 270 isolates were purified and identified by the BBL Crystal Identification System . From the experimental sufu samples, 49 Bacillus spp . (20.4%), 167 Enterococcus spp . (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous gram-negative bacilli (7.5%) were identified . From commercial sufu samples, 17 Bacillus spp . (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained . Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor . This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.

Cell Microbiol, 2001 Feb, 3(2), 75 - 84
SifA permits survival and replication of Salmonella typhimurium in murine macrophages; Brumell JH et al.; SifA was originally identified as a virulence factor required for formation of Salmonella-induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella-containing vacuole in infected epithelial cells . Here, we demonstrate that deletion mutants of ssaR, a component of the SPI-2 type III secretion system, do not form Sifs in HeLa epithelial cells . This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs . In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP-1-positive compartments . Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures . deltasifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants . Our findings suggest that SifA is an effector of the SPI-2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals . A family of SifA-related proteins and their importance to Salmonella pathogenesis is also discussed.

Cell Microbiol, 2000 Jun, 2(3), 239 - 50
Salmonella typhimurium mutants that downregulate phagocyte nitric oxide production; Eriksson S et al.; To examine the potential and strategies of the facultative intracellular pathogen Salmonella typhimurium to increase its fitness in host cells, we applied a selection that enriches for mutants with increased bacterial growth yields in murine J774-A.1 macrophage-like cells . The selection, which was based on intracellular growth competition, rapidly yielded isolates that out-competed the wild-type strain during intracellular growth . J774-A.1 cells responded to challenge with S . typhimurium by mounting an inducible nitric oxide synthase (iNOS) mRNA and protein expression and a concomitant nitric oxide (NO) production . Inhibition of NO production with the use of the competitive inhibitor N-monomethyl-L-arginine (NMMA) resulted in a 20-fold increase in bacterial growth yield, suggesting that the NO response prevented bacterial intracellular growth . In accordance with this observation, five out of the nine growth advantage mutants isolated inhibited production of NO from J774-A.1 cells, despite an induction of iNOS mRNA and iNOS protein . Accompanying bacterial phenotypes included alterations in lipopolysaccharide structure and in the profiles of proteins secreted by invasion-competent bacteria . The results indicate that S . typhimurium has the ability to mutate in several different ways to increase its host fitness and that inhibition of iNOS activity may be a major adaptation.

Cell Microbiol, 2000 Feb, 2(1), 59 - 68
A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells; Hopkins SA et al.; The attenuated Salmonella typhimurium PhoPc strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines . In this study, the fate of PhoPc bacteria expressing green fluorescent protein was investigated in murine Peyer's patches . The survival of PhoPc was monitored after orogastric inoculation of BALB/c mice . Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen . Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoPc expressing green fluorescent protein at early time points . In addition, live PhoPc were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation . Taken together, these results provide strong evidence that PhoPc is internalized and survives within Peyer's patch dendritic cells . As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S . typhimurium vaccine strains in vivo.

Cell Microbiol, 1999 Jul, 1(1), 33 - 49
Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway; Steele-Mortimer O et al.; In epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella-containing vacuole (SCV) . In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (Igps) without fusing directly with lyosomes . Here, we have investigated early events in SCV biogenesis using immunofluorescence microscopy and epitope-specific flow cytometry . We show that proteins specific to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs . The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when Igp and vATPase are acquired . Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the Igp is acquired, and that these processes occur progressively and not as the result of a single fusion/fission event . These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of Igp . The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways . Our study indicates that intracellular S . typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.

J Immunol, 2001 Mar 1, 166(5), 3440 - 50
Prophylactic tumor vaccination: comparison of effector mechanisms initiated by protein versus DNA vaccination; Zoller M et al.; Clinical success in tumor vaccination frequently does not reach expectation . Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-beta-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms . BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated SALMONELLA: typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the beta-galactosidase protein or mice were vaccinated with both DNA and protein . Although all regimens led to a prolongation of survival time, oral vaccination with transfected S . typhimurium followed by i.v . transfer of protein-loaded DC provided the optimal schedule . In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-beta-gal . As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4(+) and CD8(+) cells and 2) CD8(+) CTL are most strongly activated by vaccination with transformed SALMONELLA:, whereas vaccination with protein-loaded DC is superior for the activation of Th . The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms . The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.

Vet Immunol Immunopathol, 2001 Jan 26, 78(2), 143 - 61
Gamma/delta T cell response of chickens after oral administration of attenuated and non-attenuated Salmonella typhimurium strains; Berndt A et al.; Poultry represents an important source of Salmonella infection in man . Despite intensive research on immunity, little is known about the involvement of T cell sub-populations in the immunological response of chickens against infection with non-host-adapted Salmonella (S.) serovars . In this study, the T cell composition of blood lymphocytes (CD4(+)CD8(+); CD4(+)CD8(-); CD4(-)CD8(+); CD8(+)TcR1(+); CD8(-)TcR1(+), CD8(+)TcR1(-)) after oral administration of the non-attenuated S . typhimurium wild-type strain 421 (infection) or the attenuated vaccine strain Salmonella vac((R)) T (immunization) to day-old chicks was investigated and compared with non-treated chickens by flow cytofluorometry . Additionally, the occurrence of T cell sub-populations (CD4(+); CD8(+); TcR1(+)(gammadelta); TcR2(+)(alphabeta(1))) in ceca, spleen and bursa of Fabricius of the birds was studied immunohistologically . Blood samples and tissues were examined between days 1 and 12 of age.Chicks inoculated with S . typhimurium 421 or Salmonella vac((R)) T showed significantly elevated percentages of CD8(+)TcR1(+) in blood on days 7, 8 and 9, or on day 8 in comparison to control animals . The CD4 to CD8 cell ratio was about 3:1 in infected animals on day 5 of age . In the organs of treated chicks the numbers of CD8(+)(gammadelta) and TcR1(+)(gammadelta) cells had markedly increased on days 4 and 5 in ceca, 8 and 9 in the bursa and 9 and 12 in the spleen . Moreover, infected or vaccinated birds revealed larger quantities of CD4(+) and TcR2(+) T cells in ceca on days 4 and 5 . As shown by double staining, the TcR1(+) cells in the organs of infected animals additionally carried the CD8 antigen.In conclusion, immunization of day-old chicks with the attenuated Salmonella live vaccine strain resulted in the same changes in T cell composition as seen after infection with the non-attenuated Salmonella wild-type strain, but at a lower level . The remarkable increase of CD8(+)TcR1(+)(gammadelta) double positive cells in treated birds indicates an important role of this cell sub-population in the immunological defense of chickens against Salmonella exposure.

J Interferon Cytokine Res, 2001 Jan, 21(1), 53 - 62
The roles of Nramp1 and Tnfa genes in nitric oxide production and their effect on the growth of Salmonella typhimurium in macrophages from Nramp1 congenic and tumor necrosis factor-alpha-/- mice; Ables GP et al.; The macrophages from Nramp1 congenic mice and tumor necrosis factor (TNF)-alpha(-/-) mice were used to examine the functions of Nramp1 and Tnfa genes in nitric oxide (NO) production and Salmonella typhimurium infection . It was confirmed that the level of inducible NO synthase (iNOS)-mediated NO production in Nramp1(r) peritoneal macrophages was generally higher than that of Nramp1(s) macrophages after stimulation by interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone or in combination . Nramp1 mRNA expression in both Nramp1 congenic macrophages was constitutive notwithstanding cytokine stimulation . During infection with S . typhimurium strain 6203, Nramp1(r) macrophages produced a lower amount of NO because of an initial strong reaction and unsustained iNOS gene expression as compared with Nramp1(s) macrophages . An inhibitory effect of the Nramp1(r) gene on bacterial replication was also observed during the early stage of S . typhimurium infection, whereas the effect of TNF-alpha occurred later . NO production and iNOS expression in TNF-alpha(-/-) macrophages were not detected from the start of the bacterial infection or at 24 h after infection . We also observed that S . typhimurium strain 6203 grew more profoundly without TNF-alpha, especially in Nramp1(s) macrophages . These data, therefore, demonstrate that there is cooperation of the Nramp1 and Tnfa genes in NO production and a growth inhibitory effect in response to S . typhimurium infection.

J Agric Food Chem, 2001 Jan, 49(1), 336 - 41
Antimutagenic activity of isoflavone from Pueraria lobata; Miyazawa M et al.; A methanol extract from Pueraria lobata showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) . The methanol extract from P . lobata was re-extracted with hexane, dichloromethane, ethyl acetate, butanol, and water, respectively . A suppressive compound in the dichloromethane and ethyl acetate extract fractions was isolated by SiO(2) column chromatography and identified as tectorigenin (1) by EI-MS and (1)H and (13)C NMR spectroscopy . Compound 1 and its methylated derivative {7,4'-di-O-methyltectorigenin (2)} had the suppressive effects on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against furylfuramide, 4-nitroquinoline-1-oxide, N-methyl-N'-nitrosoguanidine, and activated Trp-P-1, which do not require live metabolic activation by S9 . These compounds also showed suppression of SOS-inducing activity against Trp-P-1 and AfB(1), which requires liver metabolizing enzymes . In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by an Ames test using S . typhimurium TA100.

J Agric Food Chem, 2001 Jan, 49(1), 114 - 7
Influence of processing stages on antimutagenic and antioxidant potentials of rooibos tea; Standley L et al.; The antimutagenic and antioxidant potentials of rooibos (Aspalathus linearis) tea samples, collected from each of its major processing stages, were evaluated according to the Salmonella typhimurium mutagenicity test and the hydrogen donating ability and superoxide anion radical scavenging assays, respectively . Ten random samples were collected before and after fermentation, as well as after sun-drying, sieving, and steam pasteurization . Results indicated that the fermented tea had a significantly (P < 0.05) lower antimutagenic and antioxidant potential than the unfermented tea . Of the different processing stages, the most significant reduction in the antimutagenic and antioxidant property of the tea was found during the "fermentation" step . Sun-drying, sieving, and steam pasteurization also reduced the antimutagenic potential of the tea, although not to the same extent as the first processing step . The hydrogen donating ability was significantly increased after steam pasteurization in comparison to those of fermented and sun-dried tea . Pasteurization did not affect superoxide anion radical scavenging in comparison to fermented tea . Differences seem to exist in the antimutagenicity and antioxidant potencies of the tea sampled at the various stages during processing . A possible role of tea polyphenols in the antimutagenic and antioxdant activities of the tea is suggested as processing caused a significant reduction in the total polyphenolic content.

Eur J Clin Invest, 2001 Feb, 31(2), 138 - 44
Effect of ischemia on intestinal permeability of lipopolysaccharides; Drewe J et al.; The enteral absorption of endotoxin (lipopolysaccharide, LPS) was studied in vitro and in vivo . The absorption of fluorescence (FITC) labelled LPS was investigated by uptake studies with rabbit jejunal brush-border membrane vesicles (BBMV), the human intestinal cell line Caco-2 and in rats . FITC-LPS from Salmonella typhimurium was taken up into BBMV in a dose-dependent manner . Uptake was neither pH- nor Na+-dependent, nor could it be inhibited by unlabelled LPS . 74.5% of vesicle-associated fluorescence was due to adhesion to the vesicular membranes, 25.5% was taken up into an osmotically-sensitive space . Transport of LPS across Caco-2 cell monolayers was dose-dependent . The permeation rate increased significantly under ischemic conditions, i.e . when the cells were incubated under oxygen-depleted conditions . However, no significant differences in transepithelial electrical resistances were observed between oxygen depleted and control cells . The release of lactate dehydrogenase was only marginally different from control cells, indicating cell integrity . In situ, after gut ischemia, a significantly increased uptake of fluorescent LPS was observed by fluorescence laser scanning microscopy . Conclusion LPS is taken up by the intestinal mucosa, predominantly by passive transcellular diffusion . Under ischemic conditions, the permeability of LPS is increased mainly by an enhanced paracellular permeability and epithelial destruction . The findings partly explain the clinically observed development of multiorgan system failure after temporary malperfusion of the gut during major vascular surgery or thromboembolism.

Clin Microbiol Infect, 2000 Nov, 6(11), 593 - 9
Phage types, antibiotic susceptibilities and plasmid profiles of Salmonella typhimurium and Salmonella enteritidis strains isolated in Istanbul, Turkey; Ang-Kucuker M et al.; OBJECTIVE: To examine 13 Salmonella typhimurium and 22 S . enteritidis strains isolated from individual cases of gastroenteritis for their phage types, antibiotic susceptibilities and plasmid profiles . METHODS: The phage typing of S . typhimurium strains was done according to the method of Anderson et al, and the phage typing scheme of Ward et al was used for phage typing of S . enteritidis strains . Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion method . Extended-spectrum beta-lactamase production of the strains was determined by the three-dimensional method . Plasmid profiles of the strains were examined using the method described by Kado and Liu with some modification by Graeber et al . RESULTS: Two S . typhimurium strains were DT 193 and one was DT 22, whereas 10 strains were untypable . PT 4 was the predominant phage type among S . enteritidis strains . Four S . enteritidis strains were DT 6a, three strains were PT 1 and one strain was PT 8, whereas only one strain was untypable . Eleven of 13 S . typhimurium and three of 22 S . enteritidis strains were found to be multiresistant . Ten different resistance patterns among S . typhimurium and four different resistance patterns among S . enteritidis strains were detected . Extended-spectrum beta-lactamase production was detected in 10 of 13 S . typhimurium and in three of 22 S . enteritidis strains . All S . typhimurium strains but one were found to contain at least one plasmid, with molecular masses varying between 4 and 107 MDa, and 11 different plasmid patterns were determined . Plasmid pattern analysis permitted further differentiation of the S . enteritidis strains into nine groups . A serovar-specific virulence plasmid of 36 MDa was detected in 13 of 22 S . enteritidis strains . CONCLUSIONS: The results suggest that the majority of S . typhimurium strains were closely related.

Vaccine, 2001 Feb 8, 19(13-14), 1772 - 82
Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice; Flo J et al.; An attenuated strain of Salmonella typhimurium has been used as a carrier for oral genetic immunization . The eukaryotic expression vector pCMV containing the gene of the glycoprotein D (gD) of the herpes simplex virus 2 was used to transform Salmonellae . The oral immunization with the transformed salmonellae elicited a strong cellular immune response in both, the mucosal and systemic compartments (spleen, ileal lymph nodes and Peyer patches) . The immune response mainly consisted in a dramatic activation of IFN-gamma-secreting cells . Twenty hours following the challenge with five lethal doses of virus, mRNA for IFN-gamma was observed in vaginal tissues from mice immunized with salmonella harboring the plasmid pgD but not in tissues from mice immunized by the intramuscular route with pgD . After an intravaginal challenge all immunized mice survived without developing symptoms . Furthermore, the immunization with Salmonella resulted in a more effective control of viral shedding than intramuscular immunization . We have unequivocally demonstrated by the introduction of an intron in the green fluorescent protein that the expression of the plasmid was due to the transcription of the protein by an eukaryotic nuclear process and not as a result of expression of the protein by the bacteria . Macrophages and dendritic cells were found expressing the protein in systemic and mucosal compartments of the immune system.

Microbes Infect, 2000 Dec, 2(15), 1799 - 806
Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination; Hess J et al.; Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies . Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively . Oral and intravenous (i.v.) vaccination of mice with either construct induced 'transporter associated with antigen processing'-dependent protection against the intracellular bacterial pathogen L . monocytogenes . Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS . typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys(492) strain . In contrast, efficacy of i.v . vaccination with either rSalmonella strain did not significantly differ . Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.

Cancer Lett, 2001 Feb 26, 163(2), 157 - 61
Induction of liver preneoplastic lesions by aminophenylnorharman, formed from norharman and aniline, in male F344 rats; Kawamori T et al.; 9-(4'-Aminophenyl)-9H-pyrido{3,4-b}indole (aminophenylnorharman, APNH), produced by the reaction of norharman with aniline in the presence of S9 mix, is a novel heterocyclic amine (HCA), with mutagenicity to Salmonella typhimurium TA 98 and YG 1024 comparable to that of other HCAs such as 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) . This experiment was designed to investigate its potential to induce glutathione S-transferase placental form (GST-P) positive foci in the liver . Male F344 rats, 7 weeks old, were fed diet containing 0, 10, 20, or 50 ppm APNH for 4 weeks, killed by ether euthanasia and performed complete necropsy . Numbers of GST-P positive foci larger than 0.1 mm in diameter induced by APNH at the dose of 10, 20, and 50 ppm were increased in a dose dependent manner to 0.52, 1.3, and 21 foci/cm2, respectively, with areas of 0.006, 0.01, and 2.3 mm2/cm2 . No such GST-P positive foci were observed in rats fed control diet . These findings suggest that APNH has hepatocarcinogenic potential in male F344 rats.

J Struct Biol, 2000 Nov, 132(2), 142 - 6
A new purification method for overproduced proteins sensitive to endogenous proteases; Saijo-Hamano Y et al.; Proteolysisis a major problem in purification of overproduced proteins for structural studies . We developed a new method to avoid proteolysis of the products even in cases where popular protease inhibitors do not work effectively . When we cloned FlgF, a flagellar rod protein, from Salmonella typhimurium and overproduced it in Escherichia coli, FlgF was highly susceptible to cleavage by endogenous proteases after cell disruption even in the presence of various protease inhibitors . However, FlgF was not digested when the cells were disrupted in the presence of urea, which allowed us to develop the following new purification procedure . After cell disruption in the presence of urea and removal of the cell debris, the supernatant was passed through tandem-connected cation- and anion-exchange columns . Proteases were trapped in the cation-exchange column, and protease-free FlgF was eluted from the disconnected anion-exchange column . This gave a stable full-length product suitable for crystallization trials . The key procedures are cell disruption in the presence of urea and linked ion-exchange chromatography to quickly remove proteases as well as urea . This fast and simple method can be applied to purification of other overproduced proteins that are very sensitive to proteolysis .

Regul Toxicol Pharmacol, 2000 Dec, 32(3), 317 - 25
Mutagenicity studies of FAVOR PAC; Haselbach J et al.; A series of in vitro and in vivo assays have been conducted using FAVOR PAC (CAS Registry No . 9003-04-7), a cross-linked sodium polyacrylate polymer, to test its ability to induce mutations . FAVOR PAC is a member of the FAVOR family of superabsorbent polymers (SAPs) developed by Stockhausen GmbH & Co KG (Krefeld, Germany) . These SAPs are known for their ability to retain large volumes of fluid, even against pressure . The genotoxic potential of FAVOR PAC and its extracts was examined in the following five standard mutagenicity assays: the Salmonella typhimurium and Escherichia coli reverse mutation assay, the mouse lymphoma fluctuation assay, the mouse lymphoma forward mutation assay, the in vivo mouse micronucleus assay, and an in vitro rat DNA synthesis assay . Based on the results of these assays, it was concluded that FAVOR PAC was clearly not genotoxic under any of the conditions of the mutagenicity assays performed .

Microb Pathog, 2001 Feb, 30(2), 101 - 10
Oral immunization of swine with attenuated Salmonella typhimurium aroA SL3261 expressing a recombinant antigen of Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF specific secretory IgA response in the lungs; Fagan PK et al.; Salmonella typhimurium SL3261 (aroA mutant) expressing a recombinant Mycoplasma hyopneumoniae antigen was used to orally immunize swine against porcine enzootic pneumonia . This construct, designated S . typhimurium aro A SL3261 (pKF1), expressed a recombinant protein containing the carboxy-terminal 11 kDa of a 42 kDa M . hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein . Here we demonstrate that this antigen is present in all seven geographically diverse strains of M . hyopneumoniae tested, and is recognized by the swine immune system after experimental infection with the virulent M . hyopneumoniae Beaufort strain . The immune response of swine orally immunized twice with S . typhimurium SL3261 (pKF1) on day 0 and day 14 was evaluated . Oral immunization with S . typhimurium SL3261 (pKF1) primed the immune system to elicit a significant (P<0.05) secretory IgA response against the 15 kDa NrdF antigen in the respiratory tract of swine, post-challenge, compared to control groups . Blood lymphocytes from swine immunized with S . typhimurium SL3261 (pKF1) proliferated significantly (P<0.05) following stimulation with M . hyopneumoniae whole-cell extracts compared to control groups 14 days post-vaccination . Following challenge with virulent M . hyopneumoniae, swine immunized with S . typhimurium SL3261 (pKF1) showed higher average daily weight gains and reduced lung pathology compared to control groups .

Microb Pathog, 2001 Jan, 30(1), 29 - 38
Early responses to Salmonella typhimurium infection in mice occur at focal lesions in infected organs; Khan SA et al.; Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid, with key roles for cytokines and possibly also inducible nitric oxide synthase (iNOS), in mediating host responses to infection . In this paper we demonstrate that iNOS mRNA, protein and enzyme activity is induced within spleens and livers of infected mice as early as 5 h post-infection . Immunohistochemistry and in situ hybridization indicated that iNOS expression occurs predominantly in macrophages in localized, discrete foci in the infected organs . iNOS activity in spleen and liver was not detectable in uninfected control mice . The presence of mRNA encoding pro-inflammatory cytokines (TNFalpha, IL-1beta and IFNgamma) in infected organs was measured using RT-PCR, all three being present from 2 h post-infection onwards, but not before . These data show that there is a very early host response to S . typhimurium infection in mice, limited to foci within the infected organs .

Curr Microbiol, 2000 Jul, 41(1), 39 - 44
Mutations permitting fdhF (formate dehydrogenase H) expression in a selD mutant of Salmonella typhimurium; Chenier I et al.; Translation of fdhF mRNA, encoding formate dehydrogenase H, requires the context-specific insertion of selenocysteine at an opal codon . We have cloned the Tn10 insertion previously shown to block fdhF transcription in Salmonella typhimurium and shown that it lies in selD, which encodes phosphoselenate synthetase . A spontaneous mutant of the selD::Tn10 strain that expresses anfdhF::lacZ protein fusion (where lacZ is fused after the opal codon) was isolated and characterized . Suppression showed the same context requirement as previously reported for SelB insertion of selenocysteine . The suppressing mutation was mapped by P22 co-transduction to the 74-75 min of the Salmonella typhimurium genome.

Mutat Res, 2000 Mar 3, 466(1), 97 - 107
Improved bacterial SOS promoter&Colon;lux fusions for genotoxicity detection; Davidov Y et al.; Escherichia coli strains containing plasmid-borne fusions of Vibrio fischeri lux to the recA promoter-operator region were previously shown to be potentially useful for detecting genotoxicants . In an attempt to improve past performance, the present study examines several modifications and variations of this design, singly or in various combinations: (1) modifying the host cell's toxicant efflux capacity via a tolC mutation; (2) incorporating the lux fusion onto the bacterial chromosome, rather then on a plasmid; (3) changing the reporter element to a different lux system (Photorhabdus luminescens), with a broader temperature range; (4) using Salmonella typhimurium instead of an E . coli host . A broad spectrum of responses to pure chemicals as well as to industrial wastewater samples was observed . Generally, fastest responses were exhibited by Sal94, a S . typhimurium strain harboring a plasmid-borne fusion of V . fischeri lux to the E . coli recA promoter . Highest sensitivity, however, was demonstrated by DPD3063, an E . coli strain in which the same fusion was integrated into the bacterial chromosome, and by DPD2797, a plasmid-bearing tolC mutant . Overall, the two latter strains appeared to perform better and seemed preferable over the others . The sensor strains retained their sensitivity following a 2-month incubation after alginate-embedding, but at the cost of a significantly delayed response.

J Agric Food Chem, 1998 Jan 19, 46(1), 286 - 290
Antifeedant Delphinium Diterpenoid Alkaloids . Structure-Activity Relationships; Gonzalez-Coloma A et al.; The insect antifeedant and toxic activity of the Delphinium diterpene alkaloids 15-acetylcardiopetamine, cardiopetamine along with its amino alcohol, the beta,gamma unsaturated ketone, and the acetylated ketone derivatives were studied in Spodoptera littoralis and Leptinotarsadecemlineata . Cardiopetamine and 15-acetylcardiopetamine strongly inhibited the feeding activity of S . littoralis and L . decemlineata, respectively . Structure-activity studies with S . littoralis showed that the C13 and C15 hydroxy substituents are essential features of the active molecule, while a C13 hydroxy and/or a C15 acetate determined their effect on L.decemlineata . The C11 benzoate group enhanced the biological effect on both insect species . These alkaloids were not toxic to S . littoralis, while their toxicity on L . decemlineata was inversely correlated with their antifeedant effects, the beta,gamma unsaturated ketone derivative being the most toxic . Cardiopetamine showed little antifungal action against several species of plant pathogens and did not have any mutagenic effects on Salmonella typhimurium by means of the Ames test.

J Appl Microbiol, 2001 Jan, 90(1), 131 - 47
Tracing of Salmonella spp . in two pork slaughter and cutting plants using serotyping and macrorestriction genotyping; Giovannacci I et al.; AIMS: The origin of Salmonella contamination of pork products is not well established . In order to further this knowledge, the transmission of Salmonella spp . from live pigs to pork cuts was investigated in two pork slaughter and cutting plants . METHODS AND RESULTS: Salmonella spp . were isolated from both pork (pigs, carcasses, cuts) and the environment before and during slaughterhouse activities . Eight serotypes were identified . XbaI and SpeI macrorestriction distinguished 20 genotypes of Salmonella Typhimurium and 16 genotypes of Salmonella Derby . A major cluster of Salmonella Typhimurium genotypes was common to both plants and all pig-related genotypes, while a predominant pig-related Salmonella Derby genotype was common to both plants . CONCLUSION: None of the Salmonella strains persisted for long periods in the pork-processing environments . SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that contaminated live pigs, because of bacterial spread due to the process and ineffective cleaning procedures, are involved in Salmonella contamination.

Mutat Res, 2001 Jan 25, 490(1), 27 - 34
Mutagenic exposure in the rubber manufacturing industry: an industry wide survey; Vermeulen R et al.; Mutagenic exposure conditions in several rubber manufacturing companies (n=9) in The Netherlands were studied . Mutagenicity of total suspended particulate matter in air (TSPM) and of wipe samples from possible contact surfaces were measured in the Ames mutagenicity assay with Salmonella typhimurium YG1041 in the presence of a metabolic activation system . Large differences in median mutagenicity of TSPM samples were observed between companies (range 49-1056rev/m(3)) and to a lesser extent between production functions (range 129-402rev/m(3)) . The production function curing revealed overall the highest TSPM mutagenicity levels . Forty-one percent of the surface wipe samples revealed mutagenic activity ranging from 26 to 665rev/cm(2) . Mixing had the largest proportion of positive samples resulting in a median surface mutagenic contamination of 39rev/cm(2) . Surface mutagenic contamination, averaged per department/company combination, showed only a weak correlation with TSPM mutagenicity (r=0.28, P=0.05) . Company, production function and total soluble matter (e.g . mass collected upon extraction with organic solvents with different polarity) explained 79 and 81% of the variability in mutagenicity of TSPM and surface contamination levels, respectively . "Company" was identified as the most important exposure determinant for mutagenic activity in TSPM and surface wipe samples . This indicates the importance of company specific determinants like production volume and rubber chemicals used for the encountered mutagenic exposure conditions . Detection of substantial mutagenic activity on possible contact surfaces supports furthermore the potential importance of the dermal route in the uptake of genotoxic compounds of workers in the rubber manufacturing industry.

Mutat Res, 2001 Jan 25, 490(1), 21 - 6
Mut-Test to detect substances suppressing spontaneous mutation due to oxidative damage; Yonezawa Y et al.; Since it has been considered that suppression of spontaneous mutation in cells is related to suppression of spontaneous carcinogenesis, it is significant to detect substances which suppress spontaneous mutation in bacterial cells such as Escherichia coli and Salmonella typhimurium in the environment . However, since the frequency of spontaneous mutation in bacteria is usually very low, generally 10(-8)-10(-10),it is difficult to determine significant suppressive ability of such substances on spontaneous mutation . A new method, Mut-Test, was developed by us, applying Luria & Delbruck fluctuation test, to detect substances which suppress spontaneous mutation using E . coli mutT mutant in which spontaneous mutation frequency due to oxidative damage is enhanced to approximately 500-1000 times of the wild type strain . Suppressive abilities of two hydroxyl radical scavengers: D(-)-mannitol and thiourea, were examined and clear positive results were obtained, suggesting that the radical scavengers are suitable as the positive control for the test . Using Mut-Test, suppressive abilities of four vitamins: L-ascorbic acid, beta-carotene, folic acid and riboflavin; 10 polyphenols: caffeic acid, ellagic acid, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, gallic acid, pyrocatechol, pyrogallol, quercetin and tannic acid which are recognized as antimutagens, were examined . Furthermore, the concentrations for 50% of suppressive abilities of five positive samples, L-ascorbic acid, folic acid, caffeic acid, pyrocatechol and pyrogallol were compared . Negative results were obtained in nine samples, riboflavin, tannic acid, etc . suggesting that their antimutagenic effect on cells may not be related to oxidative damage in cells.

Arch Soc Esp Oftalmol, 2000 Jul, 75(7), 443 - 8
{Study of topical ketorolac effect on arachidonic acid metabolism in experimental anterior uveitis}; Cuevas Andres R et al.; PURPOSE: To study the effect of topical ketorolac-tromethamine in the arachidonic acid metabolism on a model of endotoxin induced uveitis (EIU) in albino rabbits . METHODS: EIU was produced by intravitreal injection in the right eye with 10 ng LPS A Salmonella typhimurium endotoxin in 5ul saline solution . We used 60 animals (5 groups of 12 animals each) . Control group (G-I) was injected with saline (5 ul); endotoxin group (G-II) was injected with 10 ng of ET; group III, IV and V were injected with the same amount of ET and treated with topical ketorolac-tromethamine every 6, 4 and 2 hours respectively . The animals were sacrificed 24 hours after the ET administration and we determined E2 prostaglandin and B4 leukotriene concentration in the aqueous humor . RESULTS: In all treated groups with ketorolac-tromethamine we observed a significant reduction (p<0.05) in aqueous humor concentration of E2 prostaglandin without increase of the B4 leukotriene concentration comparing with endotoxin group . CONCLUSIONS: Ketorolac tromethamine produced an inhibition of ciclooxigenase metabolism of arachidonic acid without increase of lypoxygenase pathway.

Arch Soc Esp Oftalmol, 2000, 75(5), 327 - 32
{Experimental comparative study of the anti-inflammatory effectiveness of pranoprofen}; Andres RC et al.; PURPOSE: To study the antiinflammatory capacity of topical pranoprofen in comparison with other ophthalmological nonsteroidal antiinflammatory drugs (diclofenac and flurbiprofen) in albino rabbits . METHODS: We have produced an endotoxin-induced uveitis by intravitreal injection of 10 ng of Salmonella typhimurium endotoxin . We have used 48 albino rabbits (4 groups of 12 animals each), first group were control group (intravitreal saline solution), which were treated unilaterally every two hours with 2 drops of: Group II, pranoprofen (1 mg/ml) . Group III, diclofenac (0.2 mg/ml) . Group IV, flurbiprofen (0.3 mg/ml) . We determined clinical signs of inflammation and protein concentration in the aqueous humor . The rabbits were sacrificed 24 hours after the endotoxin administration . RESULTS: In all treated groups we observed a significant reduction (p<0.05) in all studied parameter as compared with control group, except for ciliar hyperemia . Different treatment groups did not show differences . CONCLUSION: The antiinflammatory capacity of pranoprofen is comparable to other NSAIDs derived from propionic acid such as flurbiprofen, obtaining a good control of endotoxin-induced uveitis.

Arch Soc Esp Oftalmol, 2000, 75(5), 333 - 8
{Antiinflammatory capacity of topical ketorolac in experimental model of ocular inflammation}; Moreno OR et al.; PURPOSE: We studied the antiinflammatory effect of topical Ketorolac-Tromethamine on a model of endotoxin-induced uveitis in albino rabbits . METHODS: Endotoxin-induced uveitis was produced by intravitreal injection in the right eye of 10 ng lipopolysaccharide (LPS) A Salmonella typhimurium endotoxin in 5 microl saline solution . We have used 60 animals (5 groups of 12 animals each) . Control group (G-I) was injected with saline (5 microl); endotoxin group (G-II) was injected with 10 ng of endotoxin; groups III, IV and V were injected with the same amount of endotoxin and treated with topical ketorolac-tromethamine every 6, 4 and 2 hours respectively . The animals were sacrificed 24 hours after endotoxin administration . We determined the ocular clinical signs and inflammatory cells and protein concentration in the aqueous humor . RESULTS: In all the groups treated with Ketorolac-Tromethamine we observed a significant reduction (p<0.05) in all parameters studied when compared with those of the endotoxin group (G-II) . CONCLUSION: Topical Ketorolac-Tromethamine has demonstrated a significant reduction of endotoxin-induced-uveitis inflammation.

Biochemistry, 2001 Jan 16, 40(2), 361 - 74
Three-dimensional structure of ATP:corrinoid adenosyltransferase from Salmonella typhimurium in its free state, complexed with MgATP, or complexed with hydroxycobalamin and MgATP; Bauer CB et al.; In Salmonella typhimurium, formation of the cobalt-carbon bond in the biosynthetic pathway for adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196 amino acid residues . This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosyl moiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide, its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin . Three X-ray structures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and a ternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 A resolution, respectively . These structures show that the enzyme is a homodimer . In the apo structure, the polypeptide chain extends from Arg(28) to Lys(181) and consists of an alpha/beta structure built from a six-stranded parallel beta-sheet with strand order 324516 . The topology of this fold is very similar to that seen in RecA protein, helicase domain, F(1)ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase where a P-loop is located at the end of the first strand . Strikingly, the nucleotide in the MgATP.CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases . That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate . The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate . In the ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depression at the C-terminal end of the beta-sheet of one subunit; however, the active site is capped by the N-terminal helix from the symmetry-related subunit that now extends from Gln(7) to Ala(24) . The lower ligand of cobalamin is well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit . Interestingly, there are few interactions between the protein and the polar side chains of the corrin ring which accounts for the broad specificity of this enzyme . The corrin ring is oriented such that the cobalt atom is located approximately 6.1 A from C5' of the ribose and is beyond the range of nucleophilic attack . This suggests that a conformational change occurs in the ternary complex when Co(III) is reduced to Co(I).

Virology, 2001 Jan 5, 279(1), 354 - 60
Mucosal vaccination with a recombinant Salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) or HPV16 VLPs purified from insect cells inhibits the growth of HPV16-expressing tumor cells in mice; Revaz V et al.; Human papillomaviruses, mainly type 16 (HPV16), are responsible for cervical intraepithelial neoplasia, which can lead, in association with other factors, to cervical cancer . Both Salmonella recombinant vaccine strains assembling HPV16 virus-like particles (VLPs) and HPV16 VLPs purified from insect cells are able to induce HPV16 neutralizing antibodies in genital secretions of mice after nasal immunization . Anti-HPV16-specific antibodies in cervical secretions of women may prevent genital infection with HPV16, although this cannot be critically evaluated in the absence of an experimental model for genital papillomavirus infection . Induction of HPV16-specific cell-mediated immunity in the genital mucosa could improve the efficacy of a vaccine and a mucosal route of immunization might be necessary to do so . It has been shown that systemic immunization of mice with purified HPV16 VLPs confers protection against an HPV16-expressing tumor cell challenge through the induction of cytotoxic T-lymphocytes . Using the same C3 tumor model, we show that intranasal immunization of mice with purified HPV16 VLPs in a prophylactic setting also induces anti-tumor immunity . More interestingly, mucosal vaccination of mice with a Salmonella recombinant strain stably expressing HPV16 L1 VLPs also induces anti-tumor immunity in prophylactic as well as in therapeutic settings . Our data suggest that attenuated Salmonella strains expressing chimeric VLPs containing nonstructural viral proteins might be a promising candidate vaccine against cervical cancer by inducing both neutralizing antibodies and cell-mediated immunity .

Rocz Panstw Zakl Hig, 2000, 51(3), 299 - 305
{Mutagenicity of organic pollutants adsorbed on airborne particles in the center of Wrocław}; Jadczyk P; With Ames assay there was examined mutagenicity of airborne particles that was sampled in summer and winter in centre of Wroclaw; there were also examined fractions of aliphatic hydrocarbons, aromatic hydrocarbons and polar compounds obtained from suspended matter extracts suspended with column chromatography . The strain of Salmonella typhimurium TA 98 was used with metabolic activation with S9 mix fraction . The samples collected in winter was more mutagenically active than the one sampled in summer . Mutagenicity of suspended matter sampled in summer was determined by compounds that were of more polar character than polycyclic aromatic hydrocarbons soluble in hexane . There was observed a decrease in mutagenic activity of samples in summer due to metabolic activation . There were few polycyclic aromatic hydrocarbons on the dust sampled in summer and they did not display mutagenic activity . Mutagenicity of air particles sampled in winter was determined by polycyclic aromatic hydrocarbons soluble in hexane and polar compounds . There was observed an increase in their mutagenic activity due to metabolic activation . This demonstrates that among them there are present promutagens, which, in mammals, undergo enzymatic transformation to compounds of direct mutagenic activity . Fractions of aliphatic hydrocarbons in the examined range of concentrations did not display mutagenic activity neither in summer nor in winter, both with and without metabolic activity.

Vaccine, 2000 Dec 8, 19(9-10), 1239 - 45
Stabilisation of Salmonella vaccine vectors by the induction of trehalose biosynthesis; Bullifent HL et al.; The growth of an aroA mutant of Salmonella typhimurium (SL3261) in minimal medium containing 0.5 M NaCl resulted in the intracellular accumulation of 2.2 micromol trehalose/mg total protein . The vacuum drying of these bacteria in the presence of trehalose allowed the recovery of 35% of the viable cells that were present before drying . In contrast, bacteria cultured in control medium accumulated 0.4 micromol trehalose/mg total protein and only 5% of the viable cells were recovered after vacuum drying with trehalose . Similar results were obtained when S . typhimurium SL3261, expressing the vaccine antigen (F1-antigen) of Yersinia pestis, was cultured in minimal medium with or without 0.5 M NaCl and dried in the presence of trehalose . Although these results indicate the potential for trehalose stabilisation of vaccine strains of S . typhimiurium, growth in minimal medium containing 0.5 M NaCl resulted in the loss of invasion competence of the bacteria.

Microbes Infect, 2000 Nov, 2(14), 1687 - 92
Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis; Spreng S et al.; In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system . Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E . coli hemolysin and expressed in secreted form by the attenuated S . typhimurium aroA SL7207 . Immunization of MV-susceptible C3H mice revealed that S . typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively . Mice vaccinated orally with a combination of both recombinant S . typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.

Mol Microbiol, 2001 Jan, 39(2), 260 - 71
Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella; Figueroa-Bossi N et al.; Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants . Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process . Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2 . Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence . One such gene, on the Gifsy-2 prophage, codes for the {Cu, Zn} superoxide dismutase SodCI . Other putative pathogenicity determinants were uncovered more recently . These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA) . In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants . A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII . Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1 . Finally, a phage specifically released from strain SL1344 was identified as SopEPhi . Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars . Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

Toxicol Sci, 2001 Jan, 59(1), 59 - 67
Ochratoxin A-induced tumor formation: is there a role of reactive ochratoxin A metabolites?
Zepnik H, Pahler A, Schauer U, Dekant W.
Ochratoxin A is a nephrotoxic and tumorigenic mycotoxin which contaminates a variety of food items, resulting in chronic human exposure . Biotransformation reactions have been implicated in the tumorigenicity of ochratoxin A . The biotransformation of ochratoxin A by cytochromes P450 and other mammalian enzymes was investigated to optimize conditions for bacterial mutagenicity testing . Metabolite formation was assessed by HPLC with UV and fluorescence detection and by LC/MS/MS . When ochratoxin A was incubated with liver microsomes from rats and mice, formation of 4R- and 4S-hydroxyochratoxin A was observed at very low rates . However, oxidation of ochratoxin A was not observed using kidney microsomes from rats and mice . Significantly higher rates of oxidation were seen in liver microsomes from rats pretreated with 3-methylcholanthrene and dexamethasone . Other reported or postulated that ochratoxin A-metabolites were not formed in detectable concentrations . Human cytochromes P450 (3A4, 1A2, and 2C9-1 Supersomes((R))) also showed very low activity with ochratoxin A (<60 fmole/min x pmol P450) . Other enzyme systems used to study possible biotransformation of ochratoxin A were rat and human liver and kidney S-9 fortified with NADPH and glutathione, semipurified glutathione S-transferases, horseradish peroxidase, and soybean lipoxygenase; none of these resulted in detectable biotransformation of ochratoxin A . Using rat liver microsomes with high activity for ochratoxin A oxidation and the other enzyme systems to activate ochratoxin A for mutagenicity testing in the Ames test, mutagenicity was not observed in Salmonella typhimurium TA 100 and TA 2638 . The obtained results suggest that oxidative biotransformation of ochratoxin A occurs at low rates, is catalyzed by cytochromes P450, and is unlikely to form reactive intermediates capable of binding to DNA.

J Clin Invest, 2001 Jan, 107(1), 99 - 109
Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response; Gewirtz AT et al.; This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia . We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present . Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion . PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S . typhimurium supernatants, indicating PIF is of bacterial origin . PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin . In support of this finding, flagellin-deficient S . typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia) . Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia . These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S . typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.

J UOEH, 2000 Dec 1, 22(4), 305 - 15
Enhancing effect of a plastic plasticizer, di-2-ethylhexyl phthalate on umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002); Okai Y et al.; Di-2-ethylhexyl phthalate (DEHP) is the most extensively used phthalate ester as a plasticizer for plastic products made of polyvinyl chloride (PVC), and previous mutagenic and genotoxic studies have shown positive and negative results of DEHP-induced genotoxicity . To elucidate this discrepancy, we reestimated the genotoxicity of DEHP in more detail using the umu C gene expression system in Salmonella typhimurium (TA 1535/pSK 1002) which reflects SOS response against genotoxin-induced DNA damage . Although DEHP itself did not have a significant effect on umu C gene expression in tester bacteria at 0.5 to 4 mM, higher concentrations of DEHP (2 and 4 mM) caused a weak induction of umu C gene expression in the presence of commercially available S-9 mixture . Rat liver S-9 fraction alone also showed a similar weak inducing activity in the absence of substrates for drug-metabolizing enzymes . When DEHP was preincubated with S-9 fraction of various rat organs and applied to the umu C gene expression assay, S-9 fraction of rat pancreas had the strongest inducing activity, and S-9 fractions of liver and intestine homogenates showed weak but significant activities . However, S-9 fractions of lung and kidney homogenates did not exhibit any significant activities . These S-9 fractions have proportional lipase activities comparable with umu C gene expression activities . Furthermore, when DEHP was treated with highly purified lipase from porcine pancreas, a significant umu C gene expression was observed and this expression was enhanced in the presence of 1 or 5 mM bile acids such as choric acid and deoxychoric acid . These results suggest that DEHP itself has no or very low genotoxicity, but enzymatic and non-enzymatic factors in specific tissues induce DEHP-dependent genotoxic activity.

J Biol Chem, 2001 Apr 6, 276(14), 11062 - 71 Epub 2000 Dec 15.
Entropic stabilization of the tryptophan synthase alpha-subunit from a hyperthermophile, Pyrococcus furiosus . X-ray analysis and calorimetry; Yamagata Y et al.; The structure of the tryptophan synthase alpha-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-A resolution, and its stability was examined by differential scanning calorimetry . Although the structure of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the alpha-subunit alone . The alpha-subunit from P . furiosus (Pf-alpha-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S . typhimurium (St-alpha-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix . The structure of the Pf-alpha-subunit was essentially similar to that of the St-alpha-subunit in the alpha(2)beta(2) complex . The differences between both structures were discussed in connection with the higher stability of the Pf-alpha-subunit and the complex formation of the alpha- and beta-subunits . Calorimetric results indicated that the Pf-alpha-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect . On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-alpha-subunit . Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-alpha-subunit.

Microbios, 2000, 103(406), 179 - 96
Comparison of the fermentative alcohol dehydrogenases of Salmonella typhimurium and Escherichia coli; Dailly YP et al.; The adhE gene, encoding the fermentative alcohol dehydrogenase, from Salmonella typhimurium (Genbank accession number U68173) was cloned and sequenced . The Salmonella AdhE protein has 619/878 (70%) amino acid residues identical to the AdhE protein of Escherichia coli . Salmonella AdhE was synthesized only anaerobically . It was present in higher amounts when cells were grown on reduced substrates such as sorbitol, instead of glucose . Growth on glucuronate, which generated no net nicotinamide-adenine dinucleotide reduced (NADH) during metabolism, showed the lowest AdhE levels . Analysis of fermentation products by in vivo nuclear magenetic resonance showed that the proportion of ethanol was highest with sorbitol, intermediate with glucose and negligible with glucuronate . The Salmonella enzyme had a lower Michaelis-Menten constant (Km) for alcohol substrates than AdhE of E . coli although both enzymes displayed a similar Km for nicotinamide-adenine dinucleotide (NAD+) . Although AdhE of E . coli was inactive with alcohols longer than four carbons, the Salmonella enzyme was still active with alcohols up to eight carbons.

Anticancer Res, 2000 Sep- Oct, 20(5B), 3609 - 14
Effects of citrus flavonoids on the mutagenicity of heterocyclic amines and on cytochrome P450 1A2 activity; Bear WL et al.; Heterocyclic amines (HCA's) are promutagens produced by high temperature cooking of meat products and are activated by cytochrome P450 (CYP) lA2 . Using Aroclor 1254 induced rat liver S9 we tested four citrus flavonoids diosmin, naringenin, naringin and rutin for their effects on the mutagenicity of HCA's MeIQx, Glu-P-1*, IQ and PhIP in Salmonella typhimurium TA98 . The effects of the citrus flavonoids on CYPlA2 activity was determined by measuring demethylation of methoxyresorufin (MROD) . MeIQx induced mutagenesis in S . typhimurium was significantly inhibited by all four flavonoids in a concentration dependent manner at 0.25, 0.5 and 1.0 mumole . Glu-P-1 induced mutagenesis was inhibited by rutin and naringenin . IQ induced mutagenesis was significantly inhibited by each flavonoid except diosmin at all three doses . With the exception of diosmin and naringin at 0.25 mumole all four flavonoids at all three doses significantly inhibited PhlP induced mutagenesis . The inhibition of MROD activity by the citrus flavonoids correlated best with the inhibition of MeIQx induced mutagenesis but also correlated with the inhibition of IQ induced mutagenesis except for diosmin and with the inhibition of PhlP induced mutagenesis except for the 0.25 mumole dose of diosmin and naringin . Our data suggest a chemopreventive potential for diosmin, naringin, naringenin and rutin towards CYPlA2 mediated mutagenesis of HCA's.

Chem Res Toxicol, 2000 Dec, 13(12), 1235 - 42
Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine; Plastaras JP et al.; Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido{1, 2-a}purin-10(3H)-one (M(1)G) . M(1)G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood . Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N(epsilon)-oxopropenyllysine . Here we compare the M(1)G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and N(alpha)-acetyl-N(epsilon)-oxopropenyllysine methyl ester . Base propenals are 30-150 times more potent than MDA in M(1)G formation and are 30-60 times more mutagenic than MDA . In addition, the Fe-bleomycin complex, which generates base propenals, induced M(1)G, but gamma-radiation, which generates mostly MDA, did not . M(1)G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions . N(alpha)-Acetyl-N(epsilon)-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA . These differences in potency are consistent with differences in leaving group ability . This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M(1)G adducts.

Mol Microbiol, 2001 Jan, 39(1), 79 - 88
Fis, a DNA nucleoid-associated protein, is involved in Salmonella typhimurium SPI-1 invasion gene expression; Wilson RL et al.; The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co-ordinated expression of many genes located around the Salmonella chromosome . Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells . Salmonella pathogenicity island 1 (SPI-1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host . Regulation of SPI-1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract . As a result of our continued studies of SPI-1 gene regulation, we have discovered that the nucleoid-binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI-1 genes . A S . typhimurium fis mutant demonstrates a two- to threefold reduction in hilA:Tn5lacZY and a 10-fold reduction in invF:Tn5lacZY expression, as well as a 50-fold decreased ability to invade HEp-2 tissue culture cells . This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant . Furthermore, the virulence of a S . typhimurium fis mutant is attenuated 100-fold when administered orally, but has wild-type virulence when administered intraperitoneally . Expression of hilA:Tn5lacZY and invF:Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S . typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC-like regulator of Salmonella invasion genes.

J Immunol, 2001 Jan 1, 166(1), 566 - 73
Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse; Merlin T et al.; LPS(d) mouse strains are characterized by the presence of a defective LPS/tlr4 gene that make them refractory to the biological activity of LPS . One of the mouse strains commonly used to study LPS defects is the C57BL/10ScCr (Cr) strain . However, unlike other LPS(d) strains, the Cr strain also has a heavily impaired IFN-gamma response to micro-organisms . As a consequence, unlike other LPS(d) mouse strains, they do not acquire a partial LPS susceptibility when treated with sensitizing bacteria . Because IL-12 is important for the microbial induction of IFN-gamma, we investigated whether the production or function of IL-12 might be defective in Cr mice . IL-12 mRNA (p35 and p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was inducible in mice injected with live or killed Salmonella typhimurium, and IL-12 (p70) was inducible in macrophages by bacteria . Thus, Cr mice exhibit normal IL-12 responses . In functional tests, splenocytes of untreated or of S . typhimurium-infected mice failed to produce IFN-gamma when stimulated with murine rIL-12 or with a combination of IL-12 and murine rIL-18 or Con A . Furthermore, Cr mice were identical with IL-12p35/p40 and IL-12 receptor beta(1) knockout mice in their impaired in vivo and in vitro IFN-gamma responses to bacteria . Thus, Cr mice carry a second genetic defect unrelated to the Lps/tlr4 mutation that underlies the IL-12 unresponsiveness and contributes to the LPS resistance and impaired innate immune response in this strain.

J Immunol, 2001 Jan 1, 166(1), 473 - 80
Borrelia burgdorferi and other bacterial products induce expression and release of the urokinase receptor (CD87); Coleman JL et al.; The urokinase-type plasminogen activator receptor (uPAR, CD87) is a highly glycosylated 55- to 60-kDa protein anchored to the cell membrane through a glycosylphosphatidylinositol moiety that promotes the acquisition of plasmin on the surface of cells and subsequent cell movement and migration by binding urokinase-type plasminogen activator . uPAR also occurs in a soluble form in body fluids and tumor extracts, and both membrane and soluble uPAR are overexpressed in patients with tumors . uPAR may be a factor in inflammatory disorders as well . We investigated whether Borrelia burgdorferi could stimulate up-regulation of cell membrane uPAR in vitro . B . burgdorferi, purified native outer surface protein A, and a synthetic outer surface protein A hexalipopeptide stimulated human monocytes to up-regulate membrane uPAR as measured by immunofluorescence/FACS and Western blot . The presence of soluble uPAR in culture supernatants, measured by Ag capture ELISA, was also observed . LPS from Salmonella typhimurium and lipotechoic acid from Streptococcus pyogenes also induced the up-regulation of both membrane and soluble uPAR protein by monocytes . Up-regulation of uPAR was induced by conditioned medium from B . burgdorferi/monocyte cocultures . The up-regulation of uPAR by B . burgdorferi was concomitant with an increase in uPAR mRNA, indicating that synthesis was de novo . The expression and release of uPAR in response to B . burgdorferi and other bacterial components suggests a role in the pathogenesis of Lyme disease as well as in other bacterial infections.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14650 - 5
Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors; Niedergang F et al.; Macrophages have long been regarded as the main target encountered by Salmonella typhimurium, a Gram-negative facultative intracellular pathogen that invades the intestinal mucosa . S . typhimurium, however, are first internalized by dendritic cells . To gain new insights into the interactions between Salmonella and the dendritic cells, we compared the fate of wild-type S . typhimurium and the virulence-attenuated PhoP constitutive (PhoP(c)) strain . The PhoP(c) strain is impaired for entry and survival in mammalian cells and is poorly processed by macrophages for antigen presentation on MHC class II molecules . Here, we show that bone marrow-derived dendritic cells can similarly process and present a foreign antigen expressed by the invasive wild-type and the attenuated PhoP(c) S . typhimurium . This property correlates with equivalent entry and survival efficiencies of both strains in dendritic cells . In addition, Salmonella strains mutated in mgtCB, sseC, and orfL genes required for macrophage survival showed no defect in survival in dendritic cells . Together, these results indicate that uptake of Salmonella by dendritic cells and subsequent antigen processing and presentation do not depend on virulence factors important in macrophages.

Vet Microbiol, 2000 Dec 20, 77(3-4), 453 - 63
Further studies on the GS element . A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp . silvaticum but not in Mycobacterium avium subsp . paratuberculosis; Bull TJ et al.; We have recently described the GS element, found in Mycobacterium avium subsp . paratuberculosis (MAP), Mycobacterium avium subsp . silvaticum (MAS) and some isolates of Mycobacterium avium subsp . avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids . Here we describe a further gene of low GC% content (mpa), within the GS element in MAP . mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri . Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB) . In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence . This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E . coli) and IS1415 (R . erythropolis) . Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element . Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster . This cluster has a high GC% content which suggests a 'border' for the GS element . A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element . This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature . Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

J Biol Chem, 2001 Mar 23, 276(12), 9083 - 92 Epub 2000 Dec 06.
A PhoP/PhoQ-induced Lipase (PagL) that catalyzes 3-O-deacylation of lipid A precursors in membranes of Salmonella typhimurium; Trent MS et al.; Pathogenic bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental changes . Some lipid A modifications are important for virulence and resistance to cationic antimicrobial peptides . The two-component system PhoP/PhoQ plays a central role in regulating lipid A modification . We now report the discovery of a PhoP/PhoQ-activated gene (pagL) in Salmonella typhimurium, encoding a deacylase that removes the R-3-hydroxymyristate moiety attached at position 3 of certain lipid A precursors . The deacylase gene (pagL) was identified by assaying for loss of deacylase activity in extracts of 14 random TnphoA::pag insertion mutants . The pagL gene encodes a protein of 185 amino acid residues unique to S . typhimurium and closely related organisms such as Salmonella typhi . Heterologous expression of pagL in Escherichia coli on plasmid pWLP21 results in loss of the R-3-hydroxymyristate moiety at position 3 in approximately 90% of the lipid A molecules but does not inhibit cell growth . PagL is synthesized with a 20-amino acid N-terminal signal peptide and is localized mainly in the outer membrane, as judged by assays of separated S . typhimurium membranes and by SDS-polyacrylamide gel analysis of membranes from E . coli cells that overexpress PagL . The function of PagL is unknown, given that S . typhimurium mutants lacking pagL display no obvious phenotypes, but PagL might nevertheless play a role in pathogenesis if it serves to modulate the cytokine response of an infected animal host.

J Biol Chem, 2001 Mar 23, 276(12), 9066 - 70 Epub 2000 Dec 05.
Evaluation of the mutagenic potential of the principal DNA adduct of acrolein; VanderVeen LA et al.; Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product . Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG) . Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency . Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair . Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E . coli strains . Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG . The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested . In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication . The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

Inhal Toxicol, 2000 Dec, 12(12), 1185 - 204
Trends of polycyclic aromatic hydrocarbon levels and mutagenicity in Santiago's inhalable airborne particles in the period 1992-1996; Gil L et al.; Trends of polycyclic aromatic hydrocarbons (PAHs) for 1992-1996 (cold season) and their mutagenic activity were investigated in organic extracts from the Santiago, Chile, inhalable particles (PM(10)) . The highest PAH concentrations were observed in 1992 and declined dramatically in the following years . During this period, total PAHs decreased 85%, carcinogenic PAHs 82%, and benzo{a}pyrene, the most potent carcinogen, 85% . In spite of this significant decrease, PAH levels in respirable particles were higher than those reported in recent studies in Australia, Europe, and the United States . PAH profiles were analyzed by principal component (PC) analysis and Pearson correlation analysis . PC1 represents 71% of the variance, suggesting that most PAHs might originate predominantly from one main generic source . Higher correlations were obtained for the major carcinogenic PAHs . Most of the samples assayed were highly mutagenic to Salmonella typhimurium both in the presence and in the absence of metabolic activation system (S9), especially in the coarse fraction, but direct mutagenicity did not decline significantly . Incubation of calf thymus DNA with organic extracts from particulate matter and xanthine oxidase allowed the detection of five nitro-PAH-DNA adducts . Thus, nitroarenes might play an important role in the mutagenic activity of inhalable particles in Santiago, representing a high risk for human health.

Inhal Toxicol, 2000 Dec, 12(12), 1173 - 83
Polycyclic aromatic hydrocarbon levels and mutagenicity of inhalable particulate matter in Santiago, Chile; Adonis M et al.; The air in Santiago, Chile, is among the most highly polluted in the world . Due to the high levels of pollutants and the high incidence of respiratory diseases, especially in the most susceptible groups, Santiago has been declared a saturated zone for PM(10), O(3), and CO . The aim of this work was to investigate polycyclic aromatic hydrocarbon levels and mutagenic activity of Santiago s fine and coarse fractions of inhalable particles . The levels of 16 polycyclic aromatic hydrocarbons (PAHs) were determined by high-performance liquid chromatography in organic extracts from respirable particles (OERP) . Respirable particulate matter (fine and coarse) contains high levels of PAHs including six mutagenic ones classified by the IARC as carcinogenic, which represented at least 45% of the total PAH concentration . A seasonal effect was observed, with higher values in months with lower temperatures . Although a significant decline of PAH levels in OERP was observed in the last years, the levels of carcinogenic PAHs are still higher than those reported in cities of the United States, Australia, and Europe . OERP were highly mutagenic and contained direct and indirect mutagens, which produced both frameshift and base substitution mutations in Salmonella typhimurium . In addition, organic extracts from total suspended particles were also highly mutagenic at the tk locus in h1A1v2 human lymphoblasts in culture . In spite of the important decrease in PAHs in the period 1991-1996, direct mutagenic response has not changed significantly, suggesting that the levels of direct mutagenic pollutants (e.g., nitroarenes) have not decreased considerably during the last years . These results suggest a risk for Santiago s inhabitants since pollutants adsorbed in inhalable particles are highly mutagenic and can damage DNA.

Curr Biol, 2000 Nov 30, 10(23), 1539 - 42
Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans; Aballay A et al.; Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions . We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P . aeruginosa and C . elegans genes can be identified that affect this killing, and that most of these P . aeruginosa genes are also important for mammalian pathogenesis . Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S . enteritidis and S . dublin can also kill C . elegans . When C . elegans is placed on a lawn of S . typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days . This killing requires contact with live bacterial cells . The worms die with similar kinetics when placed on a lawn of S . typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E . coli . After the transfer to E . coli, a high titer of S . typhimurium persists in the C . elegans intestinal lumen for the rest of the worms' life . Furthermore, feeding for 5 hours on a 1:1000 mixture of S . typhimurium and E . coli followed by transfer to 100% E . coli, also led to death after several days . This killing correlated with an increase in the titer of S . typhimurium in the C . elegans lumen, which reached 10,000 bacteria per worm . These data indicate that, in contrast to P . aeruginosa, a small inoculum of S . typhimurium can proliferate in the C . elegans intestine and establish a persistent infection . S . typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C . elegans.

Mutat Res, 2000 Dec 20, 472(1-2), 163 - 9
Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029; Sato G et al.; The potential initiation activities of a novel monoamine oxidase type-A (MAO-A) inhibitor E2011, which induced preneoplastic foci in the rat liver, were investigated by comparing the mutagenic activity of E2011, 6-aminobenzothiazole (6-ABT, a structural scaffold of E2011) and its derivatives, which are suggested primary reactive metabolites for E2011-induced hepatotoxicity in the rats in vivo, in the Ames assay system employing two Salmonella tester strains, TA100 and YG1029, a bacterial O-acetyltransferase-overproducing strain of TA100 . E2011, a tertiary amine, showed no mutagenic activity both in the Salmonella typhimurium TA100 and YG1029 with and without S9 mix . On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix, and a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029 . Thus, it appears that primary and secondary amino moieties of benzothiazole derivatives at C(6)-position are the specific structures contributing to their mutagenic activity . In addition, the alkyl group at C(2)-position of E2011, ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C(2)-position in the place of the alkyl group . These results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s).

Mutat Res, 2000 Dec 20, 472(1-2), 139 - 45
Protective effects of hemin and tetrakis(4-benzoic acid)porphyrin on bacterial mutagenesis and mouse skin carcinogenesis induced by 7, 12-dimethylbenz{a}anthracene; Chung WY et al.; Porphyrins which are widespread in nature can interfere with the actions of certain carcinogens and mutagens, and have also been used clinically in photodynamic therapy (PDT) of tumors . Porphyrins such as chlorophyll, chlorophyllin (CHL) and hemin are known to inactivate various mutagens by forming complexes with them . Tetrakis(4-benzoic acid)porphyrin (TBAP) has been developed as a photosensitizer for PDT and its metal complex, MnTBAP has been shown to be efficacious in a variety of in vitro and in vivo oxidative stress models of human diseases . In the present study, we have found that TBAP and hemin exert concentration-related inhibition of his(+) reversion in Salmonella typhimurium TA100 induced by 7, 12-dimethylbenz{a}anthracene (DMBA), and significantly reduced both incidence and multiplicity of skin tumors when topically applied prior to treatment of 12-O-tetradecanoylphorbol-13-acetate in female ICR mice . Covalent DNA binding of DMBA in mouse skin was also significantly inhibited by topical application of TBAP or hemin as well as CHL . These results suggest the chemopreventive potential of compounds containing a porphyrin nucleus.

Mutat Res, 2000 Dec 20, 472(1-2), 129 - 38
Bioactivation of diesel exhaust particle extracts and their major nitrated polycyclic aromatic hydrocarbon components, 1-nitropyrene and dinitropyrenes, by human cytochromes P450 1A1, 1A2, and 1B1; Yamazaki H et al.; The genotoxicities of four samples of diesel exhaust particle (DEP) extracts (DEPE) and nine nitroarenes found in DEPE were investigated after activation catalyzed by human cytochrome P450 (P450) family 1 enzymes co-expressed with NADPH-cytochrome P450 reductase (NPR) in Escherichia coli membranes . The DEPE samples induced umu gene expression in Salmonella typhimurium TA1535/pSK1002 without any P450 system and were further activated by human P450 1B1/NPR membranes . Moderate activation of the DEPE sample by P450 1A2/NPR membranes was also observed, but not by either P450 1A1/NPR or NPR membranes . 1-Nitropyrene (1-NP) was strongly activated by human P450 1B1/NPR membranes . 1,8-Dinitropyrene (1,8-DNP) was most highly activated by P450 1A1 and 1B1 systems for the three DNPs tested . In contrast, 1, 3-DNP was inactivated by P450 1A1/NPR, 1A2/NPR, and 1B1/NPR systems and slightly activated by NPR membranes . 2-Nitrofluoranthene (2-NF) and 3-nitrofluoranthene (3-NF) showed activities similar to 1-NP after bioactivation by P450 1B1/NPR membranes . However, the genotoxicities of 6-nitrochrysene, 7-nitrobenz{a}anthracene, and 6-nitrobenzo{a}pyrene were all weak in the present assay system . Apparent genotoxic activities of DEPE were very low compared with standard nitroarenes in the presence of P450s, possibly because unknown component(s) of DEPE had inhibitory effects on the bioactivation of 1-NP and 1,8-DNP catalyzed by human P450 1B1 . These results suggest that environmental chemicals existing in airborne DEP, in addition to 1-NP, 1,6-DNP, 1,8-DNP, 2-NF, and 3-NF, can be activated by human P450 1B1 . Biological actions of air pollutants such as nitroarenes to human extrahepatic tissues may be of concern in tissues in which P450 1B1 is expressed.

Mutat Res, 2000 Dec 20, 472(1-2), 23 - 36
Soil mutagens are airborne mutagens: variation of mutagenic activities induced in Salmonella typhimurium TA 98 and TA 100 by organic extracts of agricultural and forest soils in dependence on location and season; Edenharder R et al.; As our hypothesis was that soil mutagens are airborne mutagens, possibly modified by soil microorganisms, we checked solvent extracts from agricultural and forest soils collected during late summer in the environment of Mainz, a region highly charged by anthropogenic air pollution, or near Bayreuth, a rural low charged region of Germany, or in a remote region of western Corsica without anthropogenic air pollution for the presence of mutagenicity in Salmonella typhimurium . Levels of mutagenic activities were quantified by calculation of revertants/g from the initial slope of dose-response curves applying tester strains S . typhimurium TA 98 and TA 100 in the absence and presence of an activation system from rat liver (S9) . Three soils from Corsica did not induce mutagenicity under any test condition . However, most soils from Germany exhibited mutagenic activities, though preferentially in strain TA 98, but no statistically significant differences could be detected between 27 soils from the Mainz and nine soils from the Bayreuth regions . On the other hand, no correlation could be detected between the levels of mutagenic activities at any test condition and agricultural practice - rye growing, viniculture, fruit growing, meadow, and fallow - texture of soils - % composition of clay, slit, and sand - or the contents of organic matter . The only significant difference of mutagenicity was, however, found with S . typhimurium TA 98-S9 between forest soils of pH approximately 4.0 as compared with agricultural soils of pH approximately 7.0 . The presence of antimutagens in soil as demonstrated by the course of dose-response curves of the three soils from Corsica may be another possible confounder . Calculation of mean values of mutagenic activities for all soils from Germany gave the following results: S . typhimurium TA 98: 69.7+/-153.2 (-S9); 63.0+/-176.3 (+S9); S . typhimurium TA 100:-144.7+/-399.4 (-S9); 43.3+/-172.0 (+S9) revertants/g of dry soil . In another series of experiments, soil mutagenicity in 10 rye fields near Mainz was monitored for 1 year . It became evident that low levels of mutagenic activities in late summer increased during autumn, reached a peak in late winter, and subsequently, decreased during spring and summer . These results agree with the hypothesis of an airborne origin of soil mutagens, deposition, and an adjacent transformation to non-mutagenic compounds by soil microorganisms.

Mutat Res, 2000 Dec 20, 472(1-2), 1 - 21
The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay; Wesp HF et al.; To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks . Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100) . In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity . No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected . In another series, soils sampled at the roadside and at distances of 10 and 50m of five roads near Mainz expressed 10-20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils . Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated . Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides . The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, approximately 4% to fractions designed polar neutrals, approximately 8% to polar aromatics, approximately 7% to dichloromethane solubles, and approximately 79% to cylohexane solubles, among them approximately 63% acetone soluble compounds . The major part of mutagenicity (55-65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles ( approximately 10% each) summarizing the results obtained with S . typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9 . The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity . With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz{a,h}anthracene (4.18mg/kg soil), benzo{a}pyrene (1.96mg), benzofluoranthenes (0.14mg), and benz{a}anthracene (0 . 18mg) were present in the acetone subfraction of cyclohexane solubles . Concentrations and mutagenic activities, however, did not correlate . Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene . Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5-15cm depth and a subsequent decrease with very little activity remaining deeper than 35cm . In human lymphocyte cultures, the fraction of polar aromatics, 0.01-0 . 3microg/ml, induced 11.27+/-4.76-20.70+/-6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10 . 16+/-4.83 SCE per cell) and 12.77+/-6.53-17.87+/-4.93 SCE per cell in the presence of S9 (solvent control: 8.37+/-3.92 SCE per cell) . However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals . Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000mg/kg p.o . with all fractions.

Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 208 - 12
Use of small fluorescent molecules to monitor channel activity; Jones SE et al.; The Mechanosensitive channel of Large conductance (MscL) allows bacteria to rapidly adapt to changing environmental conditions such as osmolarity . The MscL channel opens in response to increases in membrane tension, which allows for the efflux of cytoplasmic constituents . Here we describe the cloning and expression of Salmonella typhimurium MscL (St-MscL) . The amino acid sequence encoding for this MscL exhibits a high degree of similarity to Escherichia coli MscL (Eco-MscL) . Using a fluorescence efflux assay, we demonstrate that efflux through the MscL channel during hypoosmotic shock can be monitored using endogenously produced fluorophores . These fluorophores are synthesized by a cotransformed gene, cobA . In addition, we observe that thermal stimulation, i.e., heat shock, can induce efflux through MscL .

Arch Androl, 2000 Nov-Dec, 45(3), 169 - 80
Immune responses in rats following oral immunization with attenuated Salmonella typhimurium expressing human sperm antigen; Kuang Y et al.; The HSD-I gene codes a human sperm membrane protein (hSMP-1) and has been assigned the accession number U12978 . The gene is located on human chromosome 9, region p12-p13 . When the 1.7-kb cDNA of HSD-I was digested sequentially with EcoRI, BamHI, and HindIII, a 550-bp cDNA fragment was formed, which codes for the extracellular domain . This fragment was cloned into the asd+ vector pYA3149 to construct pYA3149R . The recombinant plasmid was used to transform an avirulent deltacva, deltacrp, deltaasd vaccine strain of Salmonella typhimurium chi4550 . The hSMP-1 component was localized on the surface of the head of mature rat spermatozoa by an immunofluorescence technique using polyclonal anti-hSMP-1 antibodies . Since rat sperm contain hSMP-1, this rodent can be used to assay the immunogenicity of pYA3149R . Female Wistar rats were immunized by oral administration of the recombinant Salmonella . Anti-hSMP-1 antibodies in blood and vaginal washes of immunized animals were determined . Both body fluids contained significant amounts of the antibodies, showing that the recombinant Salmonella is an effective oral immunogen in rats.

FEMS Microbiol Lett, 2000 Dec 15, 193(2), 187 - 93
Effect of lipoteichoic acid on the uptake of Streptococcus pyogenes by HEp-2 cells; Sela S et al.; Lipoteichoic acid (LTA) is thought to play a role in the interactions between Streptococcus pyogenes and host cells . We have examined the effect of exogenous LTA on the adherence and entry of S . pyogenes JRS4 strain into HEp-2 epithelial cells . LTA markedly inhibited bacterial entry in a concentration-dependent manner, up to 250 microg ml(-1) . In contrast, LTA had only a slight inhibitory effect on adherence . LTA also inhibited the entry but not adherence of Salmonella typhimurium strain into HEp-2 cells . Binding experiments showed a dose-dependent binding of LTA to cells up to 10 microg ml(-1) . Confocal laser microscopy imaging and analysis revealed that LTA was internalized by the epithelial cells and colocalized with F-actin . These results might imply that, following binding, exogenous LTA enters HEp-2 cells and exerts a cytotoxic effect that interferes with bacterial internalization . A possible target for LTA activity might be the actin cytoskeleton, which is known to be essential for bacterial uptake.

Kansenshogaku Zasshi, 2000 Oct, 74(10), 816 - 23
{Drug sensitivity, conjugative R plasmids and plasmid profiles of Salmonella isolated from humans with infectious enteritis}; Tomosawa H et al.; Using 92 Salmonella strains isolated from patients suspected of having infectious diseases of the intestinal tract who visited 13 hospitals in Japan during the six years between 1991 and 1996, we investigated the drug susceptibility, prevalence of conjugative R plasmid, and the plasmid profiles . 1) Of the bacterial isolates tested, 52.2% showed drug-resistance . Regarding the drug-resistance patterns, 70.8% of the isolates were resistant to a single drug, while 29.2% were multi drug-resistant . 2) Dividing the resistance patterns by the serotypes, among Salmonella Enteritidis isolates, single-drug resistance to SM was the most frequent, being detected in 27 isolates . Single-drug resistance to NA and two-drug resistance to SM/TC were the second-most frequent, each being detected in isolates . Among Salmonella Hadar isolates, four isolates showed two-drug resistance to SM/TC, and one isolate showed single-drug resistance to TC . Among Salmonella Typhimurium isolates, one isolate each showed three-drug resistance to ABPC/CER/KM and KM/TC/CP . Among Salmonella Agona isolates, one isolate each showed two-drug resistance to SM/TC and single-drug resistance to SM . Among Salmonella Derby isolates, two isolates showed single-drug resistance to SM . 3) The prevalence of conjugative R plasmid was investigated in 48 drug-resistant isolates, and six isolates (12.5%) contained the plasmid . 4) The prevalence of the plasmid was investigated in 29 drug-resistant S . Enteritidis isolates, and 22 isolates (75.9%) contained the plasmid . These isolated were classified by the plasmid profiles into types H1 to H7 . 5) Regarding the plasmid profiles of the S . Enteritidis isolates, a position corresponding to 60 Kbp was the most frequently detected in 90.5%.

Int Microbiol, 1999 Sep, 2(3), 177 - 84
The virulence plasmids of Salmonella; Rotger R et al.; Certain Salmonella serovars belonging to subspecies I carry a large, low-copy-number plasmid that contains virulence genes . Virulence plasmids are required to trigger systemic disease; their involvement in the enteric stage of the infection is unclear . Salmonella virulence plasmids are heterogeneous in size (50-90 kb), but all share a 7.8 kb region, spv, required for bacterial multiplication in the reticuloendothelial system . Other loci of the plasmid, such as the fimbrial operon pef, the conjugal transfer gene traT and the enigmatic rck and rsk loci, may play a role in other stages of the infection process . The virulence plasmid of Salmonella typhimurium LT2 is self-transmissible; virulence plasmids from other serovars, such as Salmonella enteritidis and Salmonella choleraesuis, carry incomplete tra operons . The presence of virulence plasmids in host-adapted serovars suggests that virulence plasmid acquisition may have expanded the host range of Salmonella.

Med Pediatr Oncol, 2000 Dec, 35(6), 641 - 6
Tyrosine hydroxylase-based DNA-vaccination is effective against murine neuroblastoma; Lode HN et al.; BACKGROUND: The disruption of self-tolerance against neuroblastoma is the ultimate goal of an effective DNA-vaccine . PROCEDURE: Here we demonstrate the induction of a protective immunity against syngeneic murine NXS2 neuroblastoma in A/J mice, following vaccination with tyrosine hydroxylase (TH) derived antigens . Oral gene delivery was accomplished using an attenuated strain of Salmonella typhimurium as a carrier harboring vectors encoding for mTH antigens . RESULTS: Vaccination was effective in protecting animals from a lethal challenge with wild-type NXS2 tumor cells . CONCLUSIONS: These results provide the first evidence of the TH self antigen being recognized by T-cells and demonstrate that a TH-based DNA vaccine is a potentially useful immunotherapeutic strategy for neuroblastoma .

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14097 - 102
The structure of aspartyl dipeptidase reveals a unique fold with a Ser-His-Glu catalytic triad; Hakansson K et al.; The three-dimensional structure of Salmonella typhimurium aspartyl dipeptidase, peptidase E, was solved crystallographically and refined to 1.2-A resolution . The structure of this 25-kDa enzyme consists of two mixed beta-sheets forming a V, flanked by six alpha-helices . The active site contains a Ser-His-Glu catalytic triad and is the first example of a serine peptidase/protease with a glutamate in the catalytic triad . The active site Ser is located on a strand-helix motif reminiscent of that found in alpha/beta-hydrolases, but the polypeptide fold and the organization of the catalytic triad differ from those of the known serine proteases . This enzyme is a member of a family of serine hydrolases and appears to represent a new example of convergent evolution of peptidase activity.

Microbiology, 2000 Dec, 146 Pt 12, 3217 - 26
Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum; Kaiser P et al.; Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts . Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken . Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts . This results in a polymorphonuclear cell influx that contributes to the resulting enteritis . In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S . enteritidis and S . typhimurium, and the host specific serotype S . gallinarum, which rarely causes disease outside its main host, the chicken . The cytokines interleukin (IL)-1ss, IL-2, IL-6 and interferon (IFN)-gamma were measured by quantitative RT-PCR, and production of IL-6 and IFN-gamma was also determined through bioassays . All serotypes were invasive and had little effect on the production of IFN-gamma compared with non-infected cells; S . enteritidis invasion caused a slight down-regulation of IL-2 production . For IL-1ss production, infection with S . typhimurium had little effect, whilst infection with S . gallinarum or S . enteritidis caused a reduction in IL-1ss mRNA levels . Invasion of S . typhimurium and S . enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S . gallinarum caused no increase . These findings correlate with the pathogenesis of Salmonella in poultry . S . typhimurium and S . enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S . gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

J Soc Biol, 2000, 194(2), 105 - 8
{In vitro study of the interference of certain food components with the metabolism of aflatoxin B1}; Ngombo M et al.; Some compounds naturally present in food (quercetin, beta-naphthoflavone), used as food additives (butylated hydroxytoluene, sodium sulfite) or resulting from the way they were cooked (2-aminodipyrido {1,2-a; 3', 2'-d} imidazole, norharmane) can interfere with AFB1 metabolism . These interferences have been studied in vitro by evaluating the production of adducts to glutathione and by the Ames test on Salmonella typhimurium . Whereas all compounds produced a drastic decrease of the mutagenic activity, the first three only (quercetin, beta-naphthoflavone, butylated hydroxytoluene) interfered with the production of the adducts to glutathione.

Arch Toxicol, 2000 Oct, 74(8), 490 - 8
Cytotoxic and mutagenic effects, particle size and concentration analysis of diesel engine emissions using biodiesel and petrol diesel as fuel; Bunger J et al.; Diesel engine exhaust particles (DEP) contribute substantially to ambient air pollution . They cause acute and chronic adverse health effects in humans . Biodiesel (rapeseed oil methyl ester . RME) is used as a "green fuel" in several countries . For a preliminary assessment of environmental and health effects of RME, the particulate-associated emissions from the DEP of RME and common fossil diesel fuel (DF) and their in vitro cytotoxic and mutagenic effects were compared . A test tractor was fuelled with RME and DF and driven in a European standard test cycle (ECE R49) on an engine dynamometer . Particle numbers and size distributions of the exhausts were determined at the load modes "idling" and "rated power" . Filter-sampled particles were extracted and their cytotoxic properties tested using the neutral red assay . Mutagenicity was tested using the Salmonella typhimurium/microsome assay . Despite higher total particle emissions, solid particulate matter (soot) in the emissions from RME was lower than in the emissions from DF . While the size distributions and the numbers of emitted particles at "rated power" were nearly identical for the two fuels, at "idling" DF emitted substantially higher numbers of smaller particles than RME . The RME extracts caused fourfold stronger toxic effects on mouse fibroblasts at "idling" but not at "rated power" than DF extracts . The extracts at both load modes were significantly mutagenic in TA98 and TA100 . However, extracts of DF showed a fourfold higher mutagenic effect in TA98 (and twofold in TA100) than extracts of RME . These results indicate benefits as well as disadvantages for humans and the environment from the use of RME as a fuel for tractors . The lower mutagenic potency of DEP from RME compared to DEP from DF is probably due to lower emissions of polycyclic aromatic compounds . The higher toxicity is probably caused by carbonyl compounds and unburned fuel, and reduces the benefits of the lower emissions of solid particulate matter and mutagens from RME.

J Biol Chem, 2001 Feb 16, 276(7), 4772 - 80 Epub 2000 Nov 22.
Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis; Coppolino MG et al.; Bacterial invasion, like the process of phagocytosis, involves extensive and localized protrusion of the host cell plasma membrane . To examine the molecular mechanisms of the membrane remodeling that accompanies bacterial invasion, soluble NSF attachment protein receptor (SNARE)-mediated membrane traffic was studied in cultured cells during infection by Salmonella typhimurium . A green fluorescent protein-tagged chimera of VAMP3, a SNARE characteristic of recycling endosomes, was found to accumulate at sites of Salmonella invasion . To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a dominant-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion . Inhibition of NSF activity did not affect cellular invasion by S . typhimurium nor the associated membrane remodeling . By contrast, Fcgamma receptor-mediated phagocytosis was greatly reduced in the presence of the mutant NSF . Most important, dominant-negative NSF significantly impaired the fusion of Salmonella-containing vacuoles with endomembranes . These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an NSF-independent mechanism, whereas maturation of Salmonella-containing vacuoles is NSF-dependent.

J Biol Chem, 2001 Mar 2, 276(9), 6779 - 88 Epub 2000 Nov 22.
Structural transformations accompanying the assembly of bacteriophage P22 portal protein rings in vitro; Moore SD et al.; The Salmonella typhimurium bacteriophage P22 assembles an icosahedral capsid precursor called a procapsid . The oligomeric portal protein ring, located at one vertex, comprises the conduit for DNA entry and exit . In conjunction with the DNA packaging enzymes, the portal ring is an integral component of a nanoscale machine that pumps DNA into the phage head . Although the portal vertex is assembled with high fidelity, the mechanism by which a single portal complex is incorporated during procapsid assembly remains unknown . The assembly of bacteriophage P22 portal rings has been characterized in vitro using a recombinant, His-tagged protein . Although the portal protein remained primarily unassembled within the cell, once purified, the highly soluble monomer assembled into rings at room temperature at high concentrations with a half time of approximately 1 h . Circular dichroic analysis of the monomers and rings indicated that the protein gained alpha-helicity upon polymerization . Thermal denaturation studies suggested that the rings contained an ordered domain that was not present in the unassembled monomer . A combination of 4,4'-dianilino-1,1'-binapthyl-5,5'-disulfonic acid (bis-ANS) binding fluorescence studies and limited proteolysis revealed that the N-terminal portion of the unassembled subunit is meta-stable and is susceptible to structural perturbation by bis-ANS . In conjunction with previously obtained data on the behavior of the P22 portal protein, we propose an assembly model for P22 portal rings that involves a meta-stable monomeric subunit.

Shock, 2000 Nov, 14(5), 572 - 7
Complement system is involved in anaphylactoid reaction induced by lipopolysaccharides in muramyldipeptide-treated mice; Kawabata Y et al.; We previously reported that an intravenous injection of specified bacterial lipopolysaccharides (LPS) induced anaphylactoid shock in muramyldipeptide (MDP)-primed mice of various strains, including LPS-resistant C3H/HeJ, accompanied with occasional mortality of mice within 1 h . Prior to shock, rapid accumulation of blood platelets into the lungs and liver followed by degradation of the platelets and tissue destruction were observed . In this report we present the following evidence suggesting that complement activation by LPS is responsible for the anaphylactoid reaction . In C5-deficient DBA/2 mice, the platelet degradation and anaphylactoid reactions did not occur following injection of Prevotella intermedia LPS, although transient platelet accumulation into the lungs and liver was observed . Anti-complement agents K-76 COOH (C5 inhibitor) and cobra venom factor (C5 consumer) protected MDP-primed C3H/HeJ mice from mortality in the anaphylactoid reaction induced by P . intermedia and Salmonella typhimurium LPS, respectively . K-76 COOH also inhibited platelet degradation, but not accumulation, induced by P . intermedia LPS in C3H/HeN mice . LPS specimens carrying mannose-homopolymer (MHP) prepared from wild-type Klebsiella 03 and Escherichia coli 08 and 09 and recombinant E . coli 08 and 09 strains, which have been reported to markedly activate the human complement system probably through the lectin pathway, induced anaphylactoid reactions in MDP-primed C3H/HeJ mice . In contrast, LPS from R-mutant of Klebsiella 03 and the parental strain of the recombinant E . coli strains, which lacked MHP, did not induce anaphylactoid reaction . Based on these findings together with those of our previous studies, we postulated the following mechanism for the anaphylactoid reaction: strong complement activation by specified LPS preparations induced degradation of platelets which have accumulated in the lungs and liver, resulting in acute inflammation accompanied with severe tissue destruction, especially in the lungs, which in turn leads to anaphylactoid reaction . However, the mechanism of platelet accumulation induced by LPS is not yet clear.

Microbiol Immunol, 2000, 44(9), 741 - 8
Lack of evidence of an association between the carriage of virulence plasmid and the bacteremia of Salmonella typhimurium in humans; Chiu CH et al.; The involvement of the virulence plasmid (pSTV) of Salmonella typhimurium in human salmonellosis was examined . Most of the 224 clinical strains isolated from the blood (53) and nonblood samples (171) contained a 90 kb or larger plasmid, most of which were pSTV . The rates of pSTV carriage in the isolates showed no statistically significant difference between those derived from the blood and those from other sources (87% vs . 83%; chi2=0.49, 0.1<P<0.9), suggesting that pSTV may not play a critical role in promoting S . typhimurium bacteremia in humans . Nine strains with representative plasmid profiles were tested for the mouse virulence . The result revealed that these clinical isolates contained all three virulent types known: the avirulent, the highly virulent when a pSTV was present, and the moderately virulent regardless of the presence or absence of pSTV . This indicated that mouse virulence of S . typhimurium did not correlate their virulence in humans . Clinical data showed that most patients with primary bacteremia had underlying immunosuppressive diseases, whereas only a few patients with secondary bacteremia had preexisting diseases (87% vs . 13%; chi2=22.73, P<0.005) . It is suggested that the contribution of pSTV to S . typhimurium bacteremia in humans is likely to be limited, and both the host factor and the microbial virulence determinants on the chromosome are more important than virulence plasmid in predisposing patients to bacteremia.

J Int Med Res, 2000 Sep-Oct, 28(5), 222 - 8
Mutagenicity of Helicobacter pylori in the Ames test using Salmonella typhimurium TA100; Kaneko T et al.; Infection with Helicobacter pylori has been recognized as a risk factor for gastric cancer, although it is still unclear whether strains of this organism behave as carcinogens . We have studied 30 H . pylori clinical isolates from 15 patients with gastric cancer and 15 patients with other gastroduodenal diseases . Bacterial pellet and supernatant fluid of broth cultures were tested for mutagenicity by means of the Ames test using the pre-incubation technique . The average Ames ratio was 1.111 in the bacterial pellet and 1.312 in the supernatant of strains from the gastric-cancer group, and 0.858 and 0.950 in those from the non-gastric-cancer group, respectively . The strains from the gastric-cancer group had a significantly higher ratio than those from the non-gastric-cancer group, although all results were below the cut-off ratio of the Ames test, and were thus regarded as having no mutagenicity . The Ames ratio of the supernatant was higher than that of the bacterial pellet in both groups . The results indicate that all of the H . pylori strains tested revealed no mutagenicity, but the difference in Ames ratio between strains might reflect differences in genotoxicity between the strains.

Food Addit Contam, 2000 Sep, 17(9), 739 - 47
Toxicological studies on Thermomyces lanuginosus xylanase expressed by Fusarium venenatum, intended for use in food; Pedersen PB et al.; The xylanase used in this study was produced by a submerged fermentation of Fusarium venenatum and contained a gene code originating from Thermomyces lanuginosus . The enzyme was subject to a 13-week toxicological test in rats and in vitro tests to document its safety in use . The enzyme is to be applied as a processing aid in the baking industry to improve handling and stability of dough . The enzyme was not found to be mutagenic in the Salmonella typhimurium reverse mutation assay, nor did it cause chromosomal aberrations in cultured human lymphocytes . Oral administration to rats of up to 10.0 ml/kg bw/day (equivalent to a Total Organic Solids dosage of 1.12 g/kg bw/day or a xylanase dosage of 89422 FXU (W)/kg bw/day) for 13 weeks did not cause any adverse effect.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 60 - 8
Identification of amino acid residues of nitrite reductase from Anabaena sp . PCC 7120 involved in ferredoxin binding; Curdt I et al.; The nitrite reductase gene (nirA) from the filamentous, heterocyst-forming cyanobacterium Anabaena sp . PCC 7120 (A . PCC 7120) was expressed in Escherichia coli using the pET-system . Co-expression of the cysG gene encoding siroheme synthase of Salmonella typhimurium increased the amount of soluble, active nitrite reductase four fold . Nitrite reductase was purified to homogeneity . In order to identify amino acid residues involved in ferredoxin (PetF)-nitrite reductase electron transfer in A . PCC 7120, we performed a sequence comparison between ferredoxin-dependent nitrite reductases from various species . The alignment revealed a number of conserved residues possibly involved in ferredoxin nitrite reductase interaction . The position of these residues relative to the {4Fe4S}-cluster as the primary electron acceptor was tentatively localized in a three dimensional structure of the sulfite reductase from E . coli, which is closest related to nitrite reductase among the proteins with known tertiary structure . The exchange of certain positively charged amino acid residues of the nitrite reductase with uncharged residues revealed the influence of these residues on the interaction of nitrite reductase with reduced ferredoxin . We identified at least two separate regions of nitrite reductase that contribute to the binding of ferredoxin.

J Agric Food Chem, 2000 Nov, 48(11), 5440 - 3
Suppression of the furylfuramide-induced SOS response by monoterpenoids with a p-menthane skeleton using the Salmonella typhimurium TA1535/pSK1002 Umu test; Miyazawa M et al.; Suppression of the furylfuramide-induced SOS response by 25 kinds monoterpenoids (hydrocarbons, alcohols, ketones, and aldehydes) with a p-menthane skeleton was studied . Suppression of the SOS-inducing activity by monoterpenoids was determined in the umu test using Salmonella typhimurium TA1535/pSK1002 . The terpene alcohols, ketones, and aldehydes had potent suppressive effects, but the hydrocarbons did not . Especially, (+)-menthol, (+)-pulegone, piperitenone, and cuminaldehyde were shown to have the most potent suppressive effects, and the ID(50) (dose for 50% inhibition) was 0.52 micromol/mL.

Biochemistry, 2000 Nov 21, 39(46), 14183 - 95
Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex; Kreimer DI et al.; The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP . We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex . We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically . The possible basis of this asymmetry is discussed . We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy . Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation . The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.

Mutat Res, 2000 Nov 20, 471(1-2), 157 - 66
An investigation on the antimutagenic properties of South African herbal teas; Marnewick JL et al.; The antimutagenic properties of South African herbal teas were investigated using the Salmonella typhimurium mutagenicity assay . Aqueous extracts of fermented and unfermented rooibos tea (Aspalathus linearis) and honeybush tea (Cyclopia intermedia) both possess antimutagenic activity against 2-acetylaminofluorene (2-AAF) and aflatoxin B(1) (AFB(1))-induced mutagenesis using tester strains TA98 and TA100 in the presence of metabolic activation . A far less inhibitory effect was noticed against the direct acting mutagens, methyl methanesulfonate (MMS), cumolhydroperoxide (CHP), and hydrogen peroxide (H(2)O(2)) using TA102, a strain designed to detect oxidative mutagens and carcinogens . Depending on the mutagen used, the unfermented tea exhibited the highest protective effect . A similar response regarding the protection against mutagenesis was obtained when utilising different variations of the double layer Salmonella assay . The double layer technique proved to be more effective to detect the protective effect of the different tea preparations against the direct acting mutagens . With respect to indirect mutagens, the highest protection was noticed when the carcinogen was metabolically activated in the presence of the tea extract as compared with when the tea extract was incubated in a separate layer with the bacteria . The current data suggest that two mechanisms seem to be involved in the antimutagenicity of the tea extracts towards carcinogens that require metabolic activation: (i) the tea components may interfere with cytochrome P450-mediated metabolism of these mutagens and (ii) the direct interaction between the tea constituents, presumably the polyphenolic compounds, with the promutagens and/or the active mutagenic metabolites . However, the mild and/or lack of protection and in some cases even enhancement of mutagenesis induced by direct acting or oxidative mutagens, provide new perspectives regarding the role of the polyphenolic compounds known to exhibit antioxidant properties, in the protection against mutagenesis in the Salmonella assay . The present study provides the first evidence on the antimutagenic activity of honeybush tea and further evidence on the antimutagenicity of rooibos tea.

Mutat Res, 2000 Nov 20, 471(1-2), 127 - 34
A 50 Hz, 14 mT magnetic field is not mutagenic or co-mutagenic in bacterial mutation assays; Nakasono S et al.; We used bacterial mutation assays to assess the mutagenic and co-mutagenic effects of power frequency magnetic fields (MF) . For the former, we exposed four strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and two strains of Escherichia coli (WP2 uvrA, WP2 uvrA/pKM101) to 50Hz, 14mT circularly polarized MF for 48h . All results were negative . For the latter, we treated S . typhimurium (TA98, TA100) and E . coli (WP2 uvrA, WP2 uvrA/pKM101) cells with eight model mutagens (N-ethyl-N'-nitro-N-nitrosoguanidine, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 4-nitroquinoline-N-oxide, 2-aminoanthracene, N(4)-aminocytidine, t-butyl hydroperoxide, cumen hydroperoxide, and acridine orange) with and without the MF . The MF induced no significant, reproducible enhancement of mutagenicity . We also investigated the effect of MF on mutagenicity and co-mutagenicity of fluorescent light (ca . 900lx for 30min) with and without acridine orange on the most sensitive tester strain, E . coli WP2 uvrA/pKM101 . Again, we observed no significant difference between the mutation rates induced with and without MF . Thus, a 50Hz, 14mT circularly polarized MF had no detectable mutagenic or co-mutagenic potential in bacterial tester strains under our experimental conditions . Nevertheless, some evidence supporting a mutagenic effect for power frequency MFs does exist; we discuss the potential mechanisms of such an effect in light of the present study and studies done by others.

Mutat Res, 2000 Nov 20, 471(1-2), 1 - 6
Mutagenicity of cooked hamburger is controlled delicately by reducing sugar content in ground beef; Kato T et al.; Effect of sugars added to ground beef on the generation of mutagenicity of cooked hamburger was investigated . Mutagenicity of hamburger was assayed by the Ames test using Salmonella typhimurium TA98 strain with metabolic activation after the mutagens were purified by use of blue rayon . Intrinsic reducing sugar content in ground beef was estimated to be 0.07% (w/w) . Mutagenicity of hamburger was sharply or delicately controlled by the amount of a reducing sugar added to ground beef . Mutagenicity was increased more than 2-folds by addition of 0.08% (w/w) glucose, fructose or lactose but decreased to about a half by addition of more than 0.67% (w/w) each of the reducing sugars . Mutagenicity of cooked hamburger was not influenced by addition of sucrose at the ranges between 0.08 and 0.67% (w/w) . When red wine with 0.10% (w/w) equivalent amount of reducing sugars or white wine with 0.13% (w/w) equivalent amount of reducing sugars were added to the ground beef, mutagenicity of cooked hamburger was similarly increased 1.6-1.8-fold . Controlling the reducing sugar content in ground beef would be a simple way to regulate the mutagenicity of cooked hamburgers.

EMBO J, 2000 Nov 15, 19(22), 5951 - 61
Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis; Diederichs K et al.; The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments . Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport . In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain . A dimer of the subunit is attached cytoplasmically to the translocation pore . Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution . The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles . Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore . The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

Am J Physiol Regul Integr Comp Physiol, 2000 Dec, 279(6), R2164 - 72
Bacterial translocation can increase plasma corticosterone and brain catecholamine and indoleamine metabolism; Ando T et al.; The potential contribution of stress-induced bacterial translocation to the activation of the hypothalamo-pituitary-adrenocortical (HPA) axis and brain biogenic amines was assessed . Mice were restrained for various periods, and brain concentrations of tryptophan, catecholamines, serotonin, and their metabolites, plasma corticosterone, and the translocation of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes, spleen, and liver were measured . Restraint induced the translocation of indigenous gram-positive bacteria in only a small proportion of animals, but translocation of gram-negative bacteria did not occur . Restraint induced short-lived increases in plasma corticosterone and brain amine metabolism, whereas bacterial translocation was slower and persisted long after the HPA axis and neurochemical responses had dissipated . When mice were infected with Salmonella typhimurium, spontaneous translocation occurred and plasma corticosterone, interleukin-6 concentrations, and brain catecholamine and indoleamine metabolism were elevated . These findings indicate that the translocation of indigenous gastrointestinal bacteria did not contribute to the HPA axis and neurochemical changes induced by restraint . However, translocation of nonindigenous S . typhimurium with or without restraint did induce HPA and neurochemical responses.

Mutagenesis, 2000 Nov, 15(6), 495 - 502
Comparison of the sensitivities of Salmonella typhimurium strains TA102 and TA2638A to 16 mutagens; Ryden E et al.; The qualitative and quantitative sensitivity of the genetically related, histidine-auxtrophic Salmonella typhimurium strains TA102 and TA2638a to 16 compounds was examined . The compounds were mainly cross-linking and oxidising mutagens, the effects of which were known to be detected by strain TA102 preferentially or by a combination of Escherichia coli WP2 (pkM101) and uvrA/pkM101 . The morphology and number of spontaneous revertants was also compared . Fourteen of the 16 compounds caused reversion in both strains . Bleomycin and streptomycin induced reversion in strain TA102 but not TA2638a . The greater sensitivity of TA102 to these compounds may be associated with the extrachromosomal location of the target genes . The overall quantitative sensitivity of the two strains was similar for the other compounds . The number of compounds that caused reversions at lower doses or produced greater proportional increases were the same in TA102 as in TA2638a . The spontaneous number of revertants, without and with metabolic activation, respectively, was 98 and 130 for TA2638a and 322 and 465 for TA102 . Strain TA2638a formed larger, more uniform colonies than TA102 . The present results together with those of previous studies indicate a high degree of concordance between the sensitivity of strains TA102 and TA2638 for the detection of mutagens . The uniform colony size and lower spontaneous reversion frequency seen with strain TA2638a compared with TA102 would make it more reliable and convenient for routine testing . It is concluded that strain TA2638a should be considered as an alternative to TA102 and included, as well as the two E.coli strains, in the set of bacterial strains used in the standard test battery for mutagenicity testing.

Mutagenesis, 2000 Nov, 15(6), 473 - 7
Oxidative damage and induced mutations in m13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation; Arimoto-Kobayashi S et al.; N:-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes alpha-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations . Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2 . In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA . Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed . Singlet oxygen is likely to account for the formation of 8-oxodGuo . We analyzed the spectrum of mutations in lacZalpha of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA . The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts . A higher level of mutation was observed with the mutM-deficient host than with the -proficient host . Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts . With the mutM-deficient host, we observed an overall increase in the percentage of GC-->TA transversions . In addition we noted that there were fewer GC-->AT transitions than in the mutM-proficient host . With these hosts, different hot spots were observed and a new GC-->TA hot spot was produced . The formation of 8-oxodGuo in DNA, which is known to induce GC-->TA transversion, may contribute to mutagenesis by NPYR plus UVA.

Mutagenesis, 2000 Nov, 15(6), 469 - 71
Assessment of in vitro mutagenicity in Salmonella and in vivo genotoxicity in mice of the mycotoxin fumonisin B(1); Aranda M et al.; Fumonisin B(1) (FB(1)), a mycotoxin produced by Fusarium moniliforme, is a contaminant of cereals with various and complex cellular effects . FB(1) induces liver cancer in rats and has been linked to esophageal cancer in South Africa and China . The mechanisms of FB(1)-induced carcinogenesis are uncertain and the information on FB(1) mutagenic properties is limited and controversial . FB(1) contamination levels in maize and wheat from Chile were found to be similar to those in other countries . FB(1) was devoid of activity in gene mutation assays with Salmonella typhimurium strains TA100, TA102 and TA98 . However, i.p . injection of FB(1) induced an increased frequency of micronuclei in mouse bone marrow polychromatic erythrocytes at 25 and 100 mg/kg . We conclude that FB(1) induces in vivo genotoxicity in the absence of in vitro mutagenicity in Salmonella.

Toxicol Appl Pharmacol, 2000 Nov 15, 169(1), 52 - 8
Metabolism of styrene-7,8-oxide in human liver in vitro: interindividual variation and stereochemistry; Wenker MA et al.; Styrene is an industrial solvent which is mainly oxidized by cytochrome P450 to an electrophilic, chiral epoxide metabolite: styrene-7,8-oxide (SO) . SO has cytotoxic and genotoxic properties; the (R)-enantiomer is more mutagenic to Salmonella typhimurium TA 100 in the Ames test than the (S)-enantiomer . Detoxication proceeds via microsomal epoxide hydrolase (mEH) . Interindividual differences in mEH activity as well as differences in mEH enantioselectivity are important factors for toxic effects of SO . To study the extent of the interindividual variation, microsomal preparations of 20 human livers were incubated with (R)- and (S)-SO separately (1-2000 microM) and Michaelis-Menten kinetics were determined . In addition, samples were genotyped for two genetic polymorphisms of the mEH gene . V(max), K(m) and V(max)/K(m) values of both enantiomers differed three- to fivefold between the livers . No association of the enzyme constants with the genetic polymorphisms of the epoxide hydrolase gene was found . Hydrolysis of the styrene oxide enantiomers proceeded in an enantioselective manner, with the (S)-enantiomer having an approximately six times higher K(m) and five times higher V(max) than the (R)-enantiomer . In vivo, both SO enantiomers are formed; therefore, time course incubations with racemic SO were carried out in vitro to investigate possible interactions between the enantiomers . When racemic SO was used as a substrate, the (R)-enantiomer acted as an inhibitor on the hydrolysis of the (S)-enantiomer . These results indicate that mEH-mediated hydrolysis of SO is subject to appreciable interindividual variation and that hydrolysis of the more toxic enantiomer is favored .

Int J Cancer, 2000 Dec 1, 88(5), 702 - 7
Interactions between N-acetylcysteine and ascorbic acid in modulating mutagenesis and carcinogenesis; D'Agostini F et al.; Both ascorbic acid (AsA, vitamin C) and N-acetylcysteine (NAC), a precursor and analogue of glutathione, possess a broad array of biological properties underlying their protective role in a variety of pathophysiological conditions . However, under certain circumstances, AsA behaves as a pro-oxidant rather than an anti-oxidant and produces adverse effects . This prompted us to evaluate whether NAC could interact with AsA in preventing mutation and cancer . AsA significantly increased spontaneous revertants in the Salmonella typhimurium strains TA102 and TA104, which are sensitive to oxidative mutagens . In contrast, NAC lowered the spontaneous background in TA104 and neutralized the negative effects of AsA . Moreover, NAC and AsA showed additive effects in reducing chromium(VI) and in reverting its mutagenicity . A single i.p . injection of urethane (1 g/kg body weight) to 120 A/J mice resulted, after 4 months, in the formation of a total of 1,532 lung tumors, 425 in the 30 mice treated with the carcinogen only, 404 in those treated with urethane plus AsA, 365 in those treated with urethane plus NAC and 338 in those treated with urethane plus the combination of AsA and NAC (both given daily with drinking water at the dose of 1 g/kg body weight) . Compared to positive controls, tumor multiplicity was poorly affected by AsA, whereas it was significantly decreased by NAC and even more so by its combination with AsA . The overall volumes of lung tumors in the 4 groups were 107.5, 89.3, 61.3 and 49.7 mm(3), respectively . Tumor sizes were slightly but significantly decreased in mice treated with AsA and more so in those treated with NAC and NAC plus AsA, their combination being significantly more effective than each individually . All protective effects elicited by combining the 2 drugs were additive . Therefore, NAC prevents the adverse effects of AsA on spontaneous mutagenicity; at the same time, this thiol behaves in an additive fashion with AsA, inhibiting the mutagenicity of chromium(VI) and the lung tumorigenicity of urethane in mice . These findings suggest that NAC and AsA could conveniently be combined in cancer chemoprevention and other pharmacological interventions .

Biochim Biophys Acta, 2000 Nov 15, 1494(1-2), 54 - 62
A portion of the nucleotide sequence corresponding to the N-terminal coding region of livJ is essential for its transcriptional regulation; Matsubara K et al.; We investigated the regulation of the livJ and livKHMGF operons, which are involved in branched-chain amino acid high-affinity transport in Salmonella typhimurium . When livJ was fused to lacZ at the second codon of livJ to make a livJ-lacZ protein fusion, expression from the livJ promoter was not repressed even under repressing growth conditions; however, expression of an analogous construct of livK-lacZ was repressed . When livJ was fused to lacZ at the twelfth codon of livJ, the expression level under unrepressing growth conditions was elevated, resulting in apparent repressibility of the livJ-lacZ protein fusion . Expression from the livJ-lacZ operon fusion, in which livJ was fused to lacZ 159 bp downstream from the A of the start codon of livJ, was relatively normal under unrepressing growth conditions . Deletion analysis and site-directed base-substitution analysis strongly suggested that cis-acting element for regulation of livJ transcription, 5'-GGCAGGATGTATCG-3', starting at +21 and ending at +34 downstream from the A of the start codon of livJ, was present in the N-terminal coding region of livJ.

Eur J Surg, 2000 Oct, 166(10), 814 - 7
Translocation of Salmonella typhimurium in rats; effect of enteral and parenteral nutrition; Sakamoto K et al.; OBJECTIVE: To study translocation of Salmonella typhimurium from ileal loops in rats fed enterally or parenterally . DESIGN: Laboratory experiment . SETTING: University departments of surgery and microbiology, Japan . SUBJECTS: Male Wistar rats and female BALB/C CrSlc mice . INTERVENTIONS: First experiment: portal venous blood and mesenteric lymph nodes from normally fed rats were cultured under aerobic and anaerobic conditions . Second experiment: various concentrations of S . typhimurium (GIFU 12142) were injected intraperitoneally in mice and their survival was monitored . Third experiment: 7 rats were given total parenteral nutrition for 14 days and 6 were given standard chow and water for the same period . Cultures of S . typhimurium were injected into closed ileal loops and portal and vena caval blood and mesenteric lymph nodes were cultured . MAIN OUTCOME MEASURES: Presence and number of bacteria in all samples, and survival of mice . RESULTS: In the first experiment 3/17 blood samples and 9/17 node samples grew enteric bacteria . In the second experiment all the mice died within 5 days . In the third experiment no sample grew bacteria in the enterally fed group, whereas at least some samples from 5/7 rats in the parenterally fed group grew organisms; the difference was significant (p = 0.02) . CONCLUSION: Total parenteral nutrition encourages the translocation of S . typhimurium from ileal loops.

J Environ Sci Health B, 2000 Nov, 35(6), 751 - 70
Evaluation of the genotoxic and cytochrome P450 monooxygenase-inhibitory potential of Dicuran on procaryotic and eucaryotic test systems; Ramljak S et al.; The effect of the herbicide Dicuran 500 FL (formulated product) on the phenotypical and genotypical changes in procaryotic and eucaryotic organisms was investigated using short-term tests for detecting genotoxins . Since pesticides discharged in the water environment can modulate the mixed-function monooxygenases (MFO) detoxification system of water organisms, the in vivo and in vitro effects of Dicuran on hepatic cytochrome P450 (cyt P450) monooxygenase activities were also examined in juvenile carp (Cyprinus carpio L.) . By measuring the activities of MFO in experimental carp exposed to Dicuran an attempt was made to establish whether Dicuran could be bioactivated by MFO into ultimate mutagens . Our results on the bacterial strains Salmonella typhimurium TA100 and TA98 show that Dicuran does not possess either mutagenic or premutagenic characteristics . The micronucleus test on the erythrocytes of experimental carp did not establish any clastogenic effect either . However, Dicuran significantly inhibited the MFO activity of 7-ethoxyresorufin-O-deethylase (EROD) and benzo{a}pyrene monooxygenase (BaPMO) in the liver of experimental carp in vitro, as well as in in vivo conditions . These findings demonstrate the potentially damaging effect of Dicuran on the xenobiotic metabolizing enzyme systems of fish, and suggest the applicability of described methods for the prediction of the ecotoxicological significance of the presence of pesticides in the water environment.

Lett Appl Microbiol, 2000 Oct, 31(4), 284 - 8
Effects of simulated solar disinfection of water on infectivity of Salmonella typhimurium; Smith RJ et al.; To determine whether cells of Salmonella typhimurium rendered nonculturable by simulated solar disinfection retain infectivity for mice . Bacteria suspended in water were exposed to UVA irradiation for up to 8 h . Culturability, determined by colony forming unit and Most Probable Number counts, fell by six log10 units, while cellular activity determined by the Kogure cell elongation test was retained by approximately 5% of the cells present after 8 h . Intraperitoneal doses of nonculturable cells and active but nonculturable (ABNC) cells exceeding the LD50 of the test organism and BALB/c mouse host, respectively, by 4 and 3 orders of magnitude failed to produce detectable infections . Culturable cells that had been irradiated for 1.5 h were less infective (virulent) than their nonirradiated counterparts . Nonculturable and ABNC cells of Salm . typhimurium produced by UVA irradiation do not retain infectivity for mice . Although ABNC cells could be produced by low cost solar disinfection systems, they do not appear to pose a potential infection hazard.

Microbiology, 2000 Nov, 146 ( Pt 11), 2775 - 83
Susceptibility of calves to challenge with Salmonella typhimurium 4/74 and derivatives harbouring mutations in htrA or purE; Villarreal-Ramos B et al.; Salmonella typhimurium 4/74 is highly virulent for cattle after oral challenge, causing severe diarrhoea, which is sometimes associated with systemic spread of the micro-organism . Although susceptible to oral challenge, groups of cattle were found to be relatively resistant to subcutaneous challenge with this strain . The virulence of S . typhimurium 4/74 harbouring mutations in htrA and purE was also assessed in cattle . Although S . typhimurium 4/74 htrA and purE are attenuated following oral challenge in mice, cattle were highly susceptible to oral challenge with these mutants . As with the parent S . typhimurium 4/74 strain, cattle exhibited greater susceptibility to oral compared to subcutaneous challenge with S . typhimurium htrA and purE mutants . Following subcutaneous challenge with sublethal levels of S . typhimurium 4/74, calves produced significant levels of antibodies to S . typhimurium soluble extract . No correlation was detected between interferon gamma levels in sera and susceptibility to infection by any route . The concentrations of the acute-phase-associated protein haptoglobin were increased in the sera of five of six cattle inoculated subcutaneously, although increases in concentration were smaller in cattle inoculated orally.

Sci Total Environ, 2000 Oct 30, 262(1-2), 159 - 74
The application of reporter gene assays for the determination of the toxic potency of diffuse air pollution; Hamers T et al.; Diffuse air pollution consists of a mixture of numerous compounds . It is emitted by many distributed sources and is omnipresent due to atmospheric transport . Risk assessment of the complex mixture of air pollutants on the basis of the toxicity of the individual compounds is not yet possible because the chemical identity and/or toxicity of the constituencies of a substantial fraction is unknown . In addition, no adequate procedures are available to integrate toxicity data of such complex mixtures, so that an individual risk assessment of the constituents of air pollution disregards possible combination effects . In the present study, an approach has been developed to assess the toxic potency by using in vitro bio-assay techniques . Genotoxicity was assessed in the umu-assay, a reporter gene assay using a strain of Salmonella typhimurium stably transfected with a plasmid (pSK1002) carrying the SOS-gene umuC fused to the reporter gene lacZ . Arylhydrocarbon-receptor activation was assessed in the DR-CALUX-assay, using a stably transfected H4IIE hepatoma cell line containing a plasmid for the luciferase gene under transcriptional control of dioxin-responsive elements . Samples of airborne particulate matter (APM) were collected with a high volume sampler next to a highway and in a natural conservation area . Both assays proved to be applicable to quantify genotoxicity and the presence of polycyclic aromatic hydrocarbons (PAHs) in small extracts from air-filter samples . Results indicate that PAHs from traffic exhausts seem to be largely responsible for an increased genotoxic activity of APM collected down-wind from the highway (western wind) . APM collected at eastern wind directions seems to have a different composition of compounds, with a higher genotoxic activity that is less related to highway-emitted PAH-like compounds . At northern wind directions, APM is relatively less genotoxic and contains less PAHs than at other wind directions . Dioxin-like compounds contribute negligibly to the Ah-receptor agonistic potency of APM . Airborne pollutants with genotoxic and/or PAH-like characteristics form an undesired mutagenic risk, which will be evaluated in further in vivo studies.

Chemosphere, 2000 Dec, 41(11), 1809 - 19
Mutagenic nitrated benzo{a}pyrene derivatives in the reaction product of benzo{a}pyrene in NO2-air in the presence of O3 or under photoirradiation; Ishii S et al.; In order to clarify the contribution of nitrated products to the direct-mutagenic activity of products of the reactions of benzo{a}pyrene in NO2-air under various conditions, heterogeneous reactions of BaP deposited on filter in the air containing 10 ppm of NO2 have been conducted in dark or under photoirradiation . The reaction products have been analyzed by gas chromatography and mutagenicity of the products fractionated by preparative HPLC was assayed for Salmonella typhimurium strains TA98 and YG1024 in the absence of S9 mix . 3,6-dinitrobenzo{a}pyrene and 1,3-dinitrobenzo{a}pyrene, which are strong direct-acting mutagens, largely contributed to the total direct-acting mutagenicity of the dark reaction products in NO2-air . On the other hand, both the dark reaction in the presence of O3 and the photoreaction in NO2-air resulted in the formation of much smaller amounts of nitrobenzo{a}pyrenes than that observed in the dark reaction in the absence of O3 . These results show that the contribution of other direct-acting mutagens to the total direct-acting mutagenicity of the products in these reactions should be considered . Benzo{a}pyrene lactones were identified in a highly mutagenic fraction of the products of the dark reaction in the presence of O3 and photoreaction and a nitrobenzo{a}pyrene lactone was also identified in a highly mutagenic fraction of the dark reaction products in the presence of O3 . Nitrated oxygenated benzo{a}pyrene derivatives such as nitrobenzo{a}pyrene lactone were considered to largely contribute to direct-acting mutagenicity of the products of the dark reaction in the presence of O3 and photoreaction.

Eur J Clin Microbiol Infect Dis, 2000 Sep, 19(9), 679 - 87
Enteric fever and other extraintestinal salmonellosis in University Hospital, Nottingham, UK, between 1980 and 1997; Ispahani P et al.; The clinical spectrum of extraintestinal salmonellosis comprises enteric fever (typhoid and paratyphoid) and invasive infections due to nontyphoidal salmonellae . This study describes the clinical spectrum, management and outcome of all confirmed cases of extraintestinal salmonellosis in patients admitted to University Hospital, Nottingham, UK, between 1980 and 1997 . There were 142 cases (children, 42; adults, 100) of extraintestinal salmonellosis, of which 38 (children, 20; adults, 18) were enteric fever, consisting of 21 cases of typhoid, 12 of paratyphoid A and five of paratyphoid B . All patients with typhoid and paratyphoid A fever were from Indian or Pakistani families and, except for two adults, all were considered to be previously fit . The outcome in patients with enteric fever was excellent, and there were no complications . Of the 104 patients (children, 22; adults, 82) with nontyphoidal salmonellosis, 69 were bacteraemic secondary to gastroenteritis, 10 were bacteraemic without an obvious focus of infection and 25 had focal infections . The three major sites of focal infections were meningitis in five infants, osteomyelitis in two children and three adults, and arterial infections in ten adults . The three most frequently isolated organisms were Salmonella enteritidis (40%), Salmonella typhimurium (25%) and Salmonella virchow (14%) . Sixty-seven percent of these patients had underlying disease(s)/risk factors . In contrast to the outcome of enteric fever, there were 19 deaths (children, 2; adults, 17) in patients with nontyphoidal salmonellosis . Sixteen of the 17 adults who died were over the age of 60 years . Eight (25%) of 32 males over the age of 60 years with nontyphoidal Salmonella bacteraemia had arterial infections . In some patients, the diagnosis of Salmonella arterial infection is likely to be delayed or missed altogether if blood cultures are not obtained . Mortality in patients over the age of 60 years with nontyphoidal Salmonella infections was 28%.

Jpn J Infect Dis, 2000 Aug, 53(4), 164 - 5
An increase in multi-drug-resistant isolates of Salmonella typhimurium from healty carriers in Aichi, Japan; Matsumoto M et al.; To investigate the prevalence of drug-resistant isolates of Salmonella Typhimurium in Aichi, Japan, we performed antimicrobial susceptibility tests for 148 isolates from healthy carriers, and from sporadic and outbreak cases of salmonellosis from 1980 to 1999 . We found an increase in drug-resistant isolates from 56% (37/66) in the 1980s to 74% (61/82) in the 1990s due to increasing examples of four-, five-, and six-drug resistances . Of 98 resistant isolates in 1980-1999, 12 were identified as ampicillin (A)-, chloramphenicol (C)-, streptomycin (S)-, sulfonamide (Su)-, and tetracycline (T)-resistant S . Typhimurium (4 in the 1980s, 8 in the 1990s), whose pattern was identical to that of multi-drug-resistant S . Typhimurium definitive phage type 104 (DT104) which has been recently detected in various developed countries . Six-drug-resistance ACSSuTP (piperacillin), in which P was added to the core pattern of the ACSSuT, was also found in four isolates in the 1980s and seven in the 1990s . Another six-drug-resistant pattern, ACSSuTN (nalidixic acid), appeared in five isolates in the 1990s . These multi-drug-resistant isolates were predominately found in healthy carriers (21/28), suggesting that in Aichi the multi- (five- or six-) drug-resistant isolates of S . Typhimurium have existed in healthy carriers as well as in diarrhea patients in 1980 to 1999.

J Appl Microbiol, 2000 Oct, 89(4), 710 - 8
Efficacy of a commercial polymerase chain reaction-based assay for detection of Salmonella spp . in animal feeds; Maciorowski KG et al.; Salmonellosis is a cyclic problem in the food industry, to which animal feed has been contributory . Current conventional methods of Salmonella spp . detection require 96 h for detection and confirmation . With modern and just-in-time production schedules, a 96-h hold represents a significant expense in storage and decontamination . The commercially available assay, 'BAX for Screening/Salmonella' (BAX), is based on the principle of the polymerase chain reaction and may represent a significant decrease in assay time . Seven fresh feed formulations, two fresh feed ingredients, seven stored feeds and two stored feed ingredients were artificially contaminated with a primary poultry isolate of Salmonella typhimurium and analysed by conventional and BAX methodology . The results of BAX agreed with conventional plating results for 16 of 18 samples spiked with 1200 cfu 10 g(-1) of feed and 13 of 18 samples spiked with 40 cfu 10 g(-1) of feed . Indigenous Salmonella spp . were detected in five of eight samples of poultry diets by conventional methods . With BAX, Salmonella spp . could not be detected in any of the samples after only 7 h of enrichment but could be detected in two dietary samples after 13 h of enrichment and four dietary samples after 24 h of enrichment . Specific sequences of salmonella DNA that were extracted from poultry diets could be detected with BAX.

J Bacteriol, 2000 Nov, 182(22), 6456 - 62
Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis; Dashper SG et al.; Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis . In this paper, we report the characterization of a protein, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by P . gingivalis . IhtB was localized to the cell surface of P . gingivalis by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera . The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose . The growth of heme-limited, but not heme-replete, P . gingivalis cells was inhibited by preincubation with IhtB peptide-specific antisera . The ihtB gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria . Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases . Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity . These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by P . gingivalis.

J Pediatr Surg, 2000 Oct, 35(10), 1494 - 5
Salmonella typhimurium: a rare cause of colonic ulceration and perforation in infancy; Chui CH et al.; A rare case of a healthy infant with colonic ulcers caused by Salmonella typhimurium infection that presented with colonic perforation, hypovolemic, and septicemic shock is discussed . It stresses the importance of considering an infective process such as salmonellosis in the differential diagnosis of colonic ulceration in an infant and illustrates the unique histologic finding of colonic inflammatory changes with sparing of the small intestine.

Vet Res, 2000 Sep-Oct, 31(5), 481 - 90
Development of a complete ELISA using Salmonella lipopolysaccharides of various serogroups allowing to detect all infected pigs; Proux K et al.; Although poultry is recognized as the major source of food-poisoning caused by Salmonella, pork also contributes to human infections . This study was therefore undertaken in order to develop a reliable serological method for the evaluation of the Salmonella status of piglets . A complete ELISA was performed using lipopolysaccharides of Salmonella Typhimurium, Anatum, Hadar and Infantis because these serovars were representative of the serogroups isolated from 30 contaminated fattening farms . S . Enteritidis was also added because of its importance in human infection and to include the O:9 antigen . This method potentially detects 100% of infected pigs . A significant correlation was found between this serological method and the bacteriological data from mesenteric lymph nodes (p = 0.01) . In addition, both sensitivity and specificity were high (97% and 94% respectively) . The ELISA test was therefore used in a cross-sectional study on 4 farms to evaluate when pigs became contaminated: seropositive pigs were only found for the 20 week old finishing pigs . The antibody response to Salmonella in piglets was also investigated: maternal antibodies persisted until 7 weeks of age and post-Salmonella contamination seroconversion was detected from 8 weeks of age onwards.

Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12283 - 8
A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration; Lee CA et al.; In response to Salmonella typhimurium, the intestinal epithelium generates an intense inflammatory response consisting largely of polymorphonuclear leukocytes (neutrophils, PMN) migrating toward and ultimately across the epithelial monolayer into the intestinal lumen . It has been shown that bacterial-epithelial cell interactions elicit the production of inflammatory regulators that promote transepithelial PMN migration . Although S . typhimurium can enter intestinal epithelial cells, bacterial internalization is not required for the signaling mechanisms that induce PMN movement . Here, we sought to determine which S . typhimurium factors and intestinal epithelial signaling pathways elicit the production of PMN chemoattractants by enterocytes . Our results suggest that S . typhimurium activates a protein kinase C-dependent signal transduction pathway that orchestrates transepithelial PMN movement . We show that the type III effector protein, SipA, is not only necessary but is sufficient to induce this proinflammatory response in epithelial cells . Our results force us to reconsider the long-held view that Salmonella effector proteins must be directly delivered into host cells from bacterial cells.

Chem Pharm Bull (Tokyo), 2000 Oct, 48(10), 1500 - 3
Synthesis and properties of novel bifunctional nitrosamines with omega-chloroalkyl groups; Ishikawa S et al.; Novel N-nitroso-N-(acetoxymethyl)-omega-chloroalkylamines were synthesized and their chemical and biological properties were evaluated . The nitrosamines were expected to decompose through omega-chloroalkyldiazohydroxides in aqueous solution, and then to alkylate various cellular macromolecules . N-Nitroso-N-(acetoxymethyl)-2-chloroethylamine rapidly decomposed in aqueous solution, and the reaction rate was apparently independent of the pH of the solution . On the other hand, the rate of decomposition of chloropropyl and chlorobutyl homologs was pH-dependent, and increased in alkaline solution . When mutagenicity was assayed in Salmonella typhimurium TA1535 and TA92 for preliminary evaluation, all three compounds were directly mutagenic . The mutagenicity in Salmonella typhimurium TA1535, which can detect base-pair change mutation, clearly showed that these compounds induced DNA alkylation in vivo . The increase of alkyl chain length in chloroalkyl compounds increased the mutagenic activity, and the activities were stronger than those of the corresponding simple alpha-acetoxy nitrosamines lacking a chloro group, N-nitroso-N-(acetoxymethyl)alkylamines . Furthermore, the positive result in TA92 suggested that chlorinated nitrosamines cross-linked DNA like antitumor chloroethylnitrosoureas and that they are expected to be new lead compounds for antitumor agents.

J Microbiol Methods, 2000 Nov, 42(3), 255 - 63
Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype; Rychlik I et al.; We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry . Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange . In unstained cultures it was possible to distinguish flagellated and non-flagellated cells . nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential . Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains . Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants . Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels . To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts . Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.

Biol Pharm Bull, 2000 Oct, 23(10), 1247 - 9
Antimutagenic activity of 5alpha-cholest-7-en-3beta-ol, a new component from the starfish asterina pectinifera; Han YH et al.; From the butanol fraction of the starfish Asterina pectinifera Muler et Troschel (Asteriidae), we have isolated a new component, 5alpha-cholest-7-en-3beta-ol . Its antigenotoxic and antimutagenic activities were examined by the SOS chromotest with Escherichia coli PQ37 and by Ames test with Salmonella typhimurium TA1538, respectively . 5alpha-Cholest-7-en-3beta-ol showed potent antigenotoxic activity against the mutagens, both MNNG and NQO . For 100% of antigenotoxicity, the concentration of the compound applied against MNNG and NQO were 10 microg and 5 microg per reaction tube, respectively . Its antimutagenic activity with S . typhimurium TA1538 against the mutagen MNNG was very effective . When its concentrations were varied from 1 up to 10 microg dose per plate, the inhibition ratio of revertant CFU of TA1538 per plate was increased accordingly, from 25.2 to 99.2% . These results suggest that 5alpha-cholest-7-en-3beta-ol possesses antigenotoxic and antimutagenic activity and might be useful as a chemopreventive agent.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 101 - 6
Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines; Barbezange C et al.; Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable . Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome . Two out of the three vaccine strains showed genetic instabilities . Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of chi(3985), a genetically modified organism, were variants and showed different macrorestriction profiles.

Food Chem Toxicol, 2000 Oct, 38(10), 893 - 7
Antimutagenicity of ethanol extracts of bee glue against environmental mutagens; Jeng SN et al.; The antimutagenicity of ethanol extracts of bee glue (propolis) (EEBG) was evaluated, using Salmonella typhimurium strain TA98 as a test model, against two direct mutagens, 4-nitro-O-phenylenediamine (4-NO) and 1-nitropyrene (1-NP), and two indirect mutagens, 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and benzo{a}pyrene (B{a}P) with S9 mix . EEBG was shown to suppress the mutagenicity of these compounds in a dose-dependent fashion . To delineate the mechanism of action of the antimutagenic effects of EEBG on the two indirect mutagens IQ and B{a}P, two possible points of blocking were considered: (1) cytochrome P-450 activity (route 1) and (2) interaction with microsome-generated proximate mutagens to generate an inactive complex (route 2) . Our results clearly demonstrated, at a very low dose, remarkable suppression of the mutagenicity of both compounds by inhibiting either route 1 or route 2 pathway . Further studies indicated that EEBG was capable of inhibiting both the activities of hepatic cytochrome P-450 IA1-linked 7-ethoxyresorufin-O-deethylase (EROD) and IA 2-linked 7-ethoxycoumarin-O-deethylase (ECD) in a similar dose-dependent manner . Taken together, we demonstrated that EEBG was a good inhibitor for mutagenicity of direct mutagens, 1-NP and 4-NO, as well as for the indirect mutagens IQ and B{a}P in the presence of S9 mix via inactivation of microsomal enzyme activities (e.g . EROD and ECD) or antagonizing metabolic generation of the proximate mutagens of IQ and B{a}P.

Comp Immunol Microbiol Infect Dis, 2000 Oct, 23(4), 253 - 66
Characterization of Salmonella isolates from beef cattle, broiler chickens and human sources on Prince Edward Island; Abouzeed YM et al.; Non-typhoid Salmonella serovars remain a potential threat to human health, and beef cattle and broiler chickens are possible sources of these organisms on Prince Edward Island (PEI) . In this study, the ceca of beef cattle belonging to fasted and non-fasted groups, and broiler chickens were examined for Salmonella at the time of slaughter . The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the isolates obtained from cases of human salmonellosis on PEI during the study period (1996-97) . The prevalence of Salmonella in beef cattle was 4.6% (11/240) . The rate was significantly higher in fasted cattle (7.46%), than in non-fasted cattle (0.94%) . The prevalence rate in chickens was 32.5% (39/120) . In beef cattle, Salmonella typhimurium phage type (PT) or definitive type (DT) 104 which was resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline, was the most predominant type (64%) . In chickens, S . heidelberg, with resistance to gentamicin, streptomycin and sulfisoxazole, predominated . Of 26 isolates from humans, the most common serovar was S . typhimurium, including a multidrug-resistant strain of DT104 . Examination by PCR revealed presence of the virulence gene invA in all serovars, and the spvC gene in all S . typhimurium isolates, of both beef cattle and human origin . Among the other serovars the latter gene was found in 7 human isolates, but in none of the chicken or beef isolates . All but 3 of the spvC-positive isolates possessed a 90 kilobasepair (kbp) plasmid suggesting that the 3 isolates had the spvC gene on their chromosome . These findings were confirmed by plasmid DNA isolation using 3 different protocols and by sequence analysis of the spvC-PCR product.

Lupus, 2000, 9(7), 515 - 20
Determination of autoantibodies to annexin XI in systemic autoimmune diseases; Jorgensen CS et al.; Annexin XI, a calcyclin-associated protein, has been shown to be identical to a 56,000 Da antigen recognized by antibodies found in sera from patients suffering from systemic autoimmune diseases . In this work hexahistidine-tagged recombinant annexin XI (His6- rAnn XI) was used as antigen in ELISA experiments for determination of autoantibodies to annexin XI in sera of patients with systemic rheumatic autoimmune diseases . Immunoblotting with HeLa cell extract and with His6-rAnn XI as antigen was used for confirmation of positive ELISA results . We found eleven anti-annexin XI positive sera (3.9%) out of 282 sera from patients with systemic rheumatic diseases . The highest number of annexin XI positive sera were found in primary antiphospholipid syndrome (3/17), and in subacute lupus erythematosus (1/6), while lower frequencies of positive sera were found in patients with systemic sclerosis (5/137), rheumatoid arthritis (1/21), and systemic lupus erythematosus (1/58) . Sera from healthy donors and patients with chronic infections were negative, except for one Salmonella typhimurium antibody positive serum . Autoantibodies to annexin XI were found to relate to thrombosis, but not to other clinical or laboratory features . A relation between antibodies to annexins and thrombosis has so far only been known for annexin V.

Mutat Res, 2000 Nov 6, 454(1-2), 45 - 52
Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase; Cooper MT et al.; Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic . The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity . We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S . typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-) . These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction) . Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver . These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine . Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts . These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.

J Biol Chem, 2000 Dec 29, 275(52), 41058 - 63
Structural basis for the impaired channeling and allosteric inter-subunit communication in the beta A169L/beta C170W mutant of tryptophan synthase; Weyand M et al.; We determined the 2.25 A resolution crystal structure of the betaA169L/betaC170W mutant form of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium complexed with the alpha-active site substrate analogue 5-fluoro-indole-propanol-phosphate to identify the structural basis for the changed kinetic properties of the mutant (Anderson, K . S., Kim, A . Y., Quillen, J . M., Sayers, E., Yang, X . J., and Miles, E . W . (1995) J . Biol . Chem . 270, 29936-29944) . Comparison with the wild-type enzyme showed that the betaTrp(170) side chain occludes the tunnel connecting the alpha- and beta-active sites, explaining the accumulation of the intermediate indole during a single enzyme turnover . To prevent a steric clash between betaLeu(169) and betaGly(135), located in the beta-sheet of the COMM (communication) domain (betaGly(102)-betaGly(189)), the latter reorganizes . The changed COMM domain conformation results in a loss of the hydrogen bonding networks between the alpha- and beta-active sites, explaining the poor activation of the alpha-reaction upon formation of the aminoacrylate complex at the beta-active site . The 100-fold reduced affinity for serine seems to result from a movement of betaAsp(305) away from the beta-active site so that it cannot interact with the hydroxyl group of a pyridoxal phosphate-bound serine . The proposed structural dissection of the effects of each single mutation in the betaA169L/betaC170W mutant would explain the very different kinetics of this mutant and betaC170F.

Food Chem Toxicol, 2000 Dec, 38(12), 1113 - 9
Modulatory effects of a tannin fraction isolated from Terminalia arjuna on the genotoxicity of mutagens in Salmonella typhimurium; Kaur SJ et al.; A fraction isolated from Terminalia arjuna was studied for its antimutagenic effect against 4-nitro-o-phenylenediamine (NPD) in TA98, sodium azide in TA100 and 2-aminofluorene (2AF, S9-dependent), a promutagen, in both TA98 and TA 100 tester strains of Salmonella typhimurium using the Ames assay . The fraction inhibited the mutagenicity of 2AF very significantly in both strains while the revertant colonies induced by NPD and sodium azide were reduced moderately . 1H-NMR, 13C-NMR, IR and UV-spectroscopic data of the fraction revealed it to be tannin in nature.

Mol Microbiol, 2000 Oct, 38(1), 31 - 40
Salmonella induces macrophage death by caspase-1-dependent necrosis; Brennan MA et al.; We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis . Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis . In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase-3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP-ribose polymerase (PARP) . Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state . Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase . During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity . Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis . Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis.

Crit Rev Food Sci Nutr, 2000 Sep, 40(5), 399 - 425
Control of foodborne pathogens during sufu fermentation and aging; Shi X et al.; Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated . Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g . After 2 days of fermentation at 30 degrees C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g . After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl . After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels . The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P > 0.05) compared with the count after 1 month . Microorganism in experimental sufu from different aging periods and in commercial sufu were compared . A total of 270 isolates were purified and identified by the BBL Crystal Identification System . From the experimental sufu samples, 49 Bacillus spp . (20.4%), 167 Enterococcus spp . (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous Gram-negative bacilli (7.5%) were identified . From commercial sufu samples, 17 Bacillus spp . (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous Gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained . Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor . This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.

Int J Food Microbiol, 2000 Oct 1, 61(1), 73 - 9
Survival of Salmonella typhimurium and Escherichia coli O157:H7 in cheese brines; Ingham SC et al.; Survival of Salmonella typhimurium and Escherichia coli O157:H7 was studied in model brines and brine from three cheese plants . Three strain mixtures of S . typhimurium and E . coli O157:H7 (10(6) CFU/ml) were inoculated separately into 23% model brine with or without added pasteurized whey (2%) and as a combined inoculum into the commercial brines . The model brines were incubated at 8 and 15 degrees C for 28 days, and the commercial brines at 4 and 13 degrees C for 35 days . Populations of both pathogens in the model brine + whey decreased slowly over 28 days (1.0-2.0 log CFU/ml) with greater survival at 8 degrees C than at 15 degrees C . Corresponding decreases in model brine without whey were 1.9-3.0 log CFU/ml, with greater survival at 8 degrees C than at 15 degrees C . Both S . typhimurium and E . coli O157:H7 survived significantly better (P < 0.05) at 4 degrees C than at 13 degrees C in two of the commercial brines . The survival of each pathogen in the commercial brines at 13 degrees C was significantly influenced by brine pH . Both pathogen populations decreased most rapidly in commercial brines during the first week of storage (2.5-4.0 and 2.3-2.8 log CFU/ml for S . typhimurium and E . coli O157:H7, respectively) with significant recovery (ca . 0.5 log CFU/ml increase) often occurring in the second week of storage . Counts changed little thereafter . Overall, E . coli O157:H7 survived better than S . typhimurium, with differences of 0.1-1.2 log CFU/ml between the two pathogens . Results of this study show that cheese brine could support the survival of contaminating S . typhimurium and E . coli O157:H7 for several weeks under typical brining conditions.

Mutat Res, 2000 Oct 31, 470(2), 141 - 6
Chlorination of harman and norharman with sodium hypochlorite and co-mutagenicity of the chlorinated products; Nakano K et al.; Harman and norharman are widely distributed in the environment and consequently contaminate in domestic waste-water . It has been reported that they have co-mutagenic activity in the presence of non- mutagenic aromatic amines such as aniline and o-toluidine with S9 mix . When these beta-carbolines were treated with sodium hypochiorite under mild conditions, chlorinated derivatives were produced . Among them, 6-chloroharman and 6-chloronorharman showed much more potent co-mutagenic activities than harman and norharman in the presence of o-toluidine toward Salmonella typhimurium TA98 with S9 mix . These results suggest that the chlorination of harman and norharman occurs during disinfection at the sewage plant to produce potent co-mutagens that contaminate river water.

Vaccine, 2000 Oct 15, 19(4-5), 460 - 9
Salmonella typhimurium as a basis for a live oral Echinococcus granulosus vaccine; Chabalgoity JA et al.; A live attenuated Salmonella typhimurium vaccine candidate, LVR01, was constructed by introducing a null deletion into the aroC gene of the parental canine S . typhimurium isolate, P228067 . LVR01 was used to orally deliver to the canine immune system a fatty acid binding protein (FABP) from Echinococcus granulosus (EgDf1), as a fusion protein with fragment C (TetC) of tetanus toxin . Immunization studies demonstrated that live LVR01 is well tolerated by orally vaccinated dogs . There was no detectable shedding of the vaccine strain in the faeces 2 days after immunization . Humoral antibody responses were observed against Salmonella, TetC and EgDf1 . Cellular responses were consistently detected against Salmonella and TetC . A cellular response against EgDf1 was also seen in a proportion of the LVR01 vaccinated dogs . We propose S . typhimurium LVR01 as a carrier for recombinant antigens and a vector for the construction of multivalent oral vaccines for dogs.

FEMS Microbiol Lett, 2000 Oct 15, 191(2), 177 - 82
Co-expression of the Bordetella pertussis leader peptidase I results in enhanced processing and expression of the pertussis toxin S1 subunit in Escherichia coli; Smith AM et al.; Bordetella pertussis is the causative agent of whooping cough . Traditional vaccines against this disease are inherently reactogenic, thus research is currently focussed on the production of less reactive, acellular vaccines . Expression of candidate antigens for these vaccines in Escherichia coli would be preferable, however, several B . pertussis antigens undergo incorrect post-translational processing in E . coli . The leader peptidase gene (lep) of B . pertussis encodes a protein of 294 amino acid residues that shares homology with other prokaryote leader peptidase I sequences . Hydrophilicity analysis based on the predicted amino acid sequence has demonstrated a similar membrane topology to that of E . coli and Salmonella typhimurium leader peptidase I . Co-expression of the B . pertussis lep gene in E . coli strain TOPP2 expressing the pertussis toxin S1 subunit was found to markedly increase the expression and post-translational processing of the S1 protein.

J Microbiol Methods, 2000 Oct, 42(2), 129 - 38
A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells; Guy RL et al.; The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells . Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen . Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g . 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy . Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy . Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches . In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments . The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells . This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.

Int J Food Microbiol, 2000 Sep 25, 60(2-3), 195 - 203
Changes in culturability and virulence of Salmonella typhimurium during long-term starvation under desiccating conditions; Lesn J et al.; The survival of Salmonella typhimurium under desiccation and starvation conditions commonly associated with farm buildings was investigated in a desiccation model system: filtration onto polycarbonate membranes placed in a sealed desiccator with 0.0067 g/m3 absolute humidity . Heterogeneities within bacterial populations in relation to time of desiccation were investigated on a single-cell basis by epifluorescence microscopy coupled with an image analysis system in conjunction with fluorescent dyes Chemchrome V6 and DAPI . Changes in cellular states were compared to the results of plate counts (colony forming units, CFU) on selective (modified semi-solid Rappaport Vassiliadis (MSRV)) and non-selective (nutrient agar (NA) and R2A agar) media, and to the measurements of infectivity and virulence using two animal models (chicks and mice) . During 9 weeks of experimental desiccation, total cell counts (DAPI) of starved S . typhimurium remained stable, as did esterase activity (Chemchrome V6), but DAPI fluorescence intensity decreased slowly . Bacterial cells entered gradually into non-culturable states (decrease of CFU counts on MSRV, NA and R2A agar media) and the total loss of culturability on NA (defined as probability of presence of 1 CFU on the membrane inferior to 10 (-6)) was obtained after 9 weeks . Loss of chick infectivity and mice virulence in animal models occurred more rapidly, within three weeks of experimental desiccation.

Int J Food Microbiol, 2000 Sep 15, 60(1), 33 - 42
Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens; Alpa H et al.; The objective of this study is to determine the interactions between high hydrostatic pressure, pressurization temperature, time and pH during pressurization on death and injury of pressure-resistant and pressure-sensitive strains of four foodborne pathogens: Staphylococcus aureus 485 and 765, Listeria ,monocytogenes CA and OH2, Escherichia coli O157:H7 933 and 931, Salmonella enteritidis FDA and Salmonella typhimurium E21274 . Among these strains S . aureus 485, L . monocytogenes CA, E . coli O157:H7 933 and S . enteritidis FDA were reported to be more pressure-resistant than the respective strain of the same species (Alpas et al., 1999) . In general, viability loss of all pathogens was enhanced significantly as the level of pressure and temperature were increased (P < 0.05) . All the strains except S . aureus 485 demonstrated more than 8 log cycle reduction when pressurized at 345 MPa at 50 degrees C for 5 min . This strain seemed to be the most pressure-resistant strain within the conditions of the study . Pressurization in the presence of either citric or lactic acid increased the viability loss by an additional 1.2-3.9 log cycles at pH 4.5 for both acids at 345 MPa . This study has indicated that high hydrostatic pressure applied in conjunction with mild heat and acidity can be an effective method for inactivating pressure-resistant and pressure-sensitive strains of four foodborne pathogens in organic acid solutions . This combination treatment indicates possible pressure pasteurization applications to liquid foods that have low pH . reserved.

Environ Mol Mutagen, 2000, 36(2), 121 - 6
Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay; Aryal P et al.; The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT) . We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines {Aryal et al., 1999, Mutat Res 442:113-120} . To further enhance the sensitivity of the strain toward HCAs, we developed S . typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S . typhimurium TA1535 . In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S . typhimurium TA1535/1,8-DNP . Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively . We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo{4,5-f}quinol ine(MeIQ), 2-amino-3-methylimidazo{4,5-f}quinoline (IQ),2-amino-3, 8-dimethylimidazo{4,5-f}quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido{1,2-a::3,2'-d}i midazole,3-amino-1, 4-dimethyl-5H-pyrido{4,3-b}indole, and 3-amino-1-methyl-5H-pyrido{4, 3-a}indole . However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain . These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase .

EMBO J, 2000 Oct 2, 19(19), 5071 - 80
Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria; Bishop RE et al.; Regulated covalent modifications of lipid A are implicated in virulence of pathogenic Gram-negative bacteria . The Salmonella typhimurium PhoP/PhoQ-activated gene pagP is required both for biosynthesis of hepta-acylated lipid A species containing palmitate and for resistance to cationic anti-microbial peptides . Palmitoylated lipid A can also function as an endotoxin antagonist . We now show that pagP and its Escherichia coli homolog (crcA) encode an unusual enzyme of lipid A biosynthesis localized in the outer membrane . PagP transfers a palmitate residue from the sn-1 position of a phospholipid to the N-linked hydroxymyristate on the proximal unit of lipid A (or its precursors) . PagP bearing a C-terminal His(6)-tag accumulated in outer membranes during overproduction, was purified with full activity and was shown by cross-linking to behave as a homodimer . PagP is the first example of an outer membrane enzyme involved in lipid A biosynthesis . Additional pagP homologs are encoded in the genomes of YERSINIA: and BORDETELLA: species . PagP may provide an adaptive response toward both Mg(2+) limitation and host innate immune defenses.

Eur J Biochem, 2000 Oct, 267(20), 6126 - 33
AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase; Poole LB et al.; A group of bacterial flavoproteins related to thioredoxin reductase contain an additional approximately 200-amino-acid domain including a redox-active disulfide center at their N-termini . These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine-nucleotide-dependent reduction of cysteine-based peroxidases (e.g . Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides . These enzymes catalyze rapid electron transfer (kcat > 165 s-1) through one tightly bound FAD and two redox-active disulfide centers, with the N-terminal-most disulfide center acting as a redox mediator between the thioredoxin-reductase-like part of these proteins and the peroxiredoxin substrates . A chimeric protein with the first 207 amino acids of S . typhimurium AhpF attached to the N-terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities . Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron-reduced (EH2) and four electron-reduced (EH4) redox states.

Biochemistry, 2000 Oct 3, 39(39), 12069 - 75
Isotope effects in the transient phases of the reaction catalyzed by ethanolamine ammonia-lyase: determination of the number of exchangeable hydrogens in the enzyme-cofactor complex; Bandarian V et al.; Transient phases of the reaction catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been investigated by stopped-flow visible spectrophotometry and deuterium kinetic isotope effects . The cleavage of adenosylcobalamin (coenzyme B(12)) to form cob(II)alamin (B(12r)) with ethanolamine as the substrate occurred within the dead time of the instrument whenever coenzyme B(12) was preincubated with enzyme prior to mixing with substrate . The rate was, however, slowed sufficiently to be measured with perdeutero ethanolamine as the substrate . Optical spectra indicate that, during the steady states of the reactions with ethanolamine and with S-2-aminopropanol as substrates, approximately 90% of the active sites contain B(12r) . Reformation of the carbon-cobalt bond of the cofactor occurs following depletion of substrate in the reaction mixtures, and the rate constant for this process reflects k(cat) of the respective substrates . This late phase of the reaction also exhibits (2)H isotope effects similar to those measured for the overall reaction with (2)H-labeled substrates . With unlabeled substrates, the rate of cofactor reassembly is independent of the number of substrate molecules turned over in the steady-state phase . However, with (2)H-labeled substrates, kinetic isotope effects appear in the reassembly phase, and these isotope effects are maximal after only approximately 2 equiv of substrate/active site are processed . With 5'-deuterated coenzyme B(12) and deuterated substrate, the isotope effect on reassembly is independent of the number of substrate molecules that are turned over . These results indicate that the pool of exchangeable hydrogens in the enzyme-cofactor complex is two-a finding consistent with the hydrogens in the C5' methylene of coenzyme B(12).

Microbes Infect, 2000 Aug, 2(10), 1257 - 63
Interleukin-18 (IL-18) and infectious diseases, with special emphasis on diseases induced by intracellular pathogens; Sugawara I; Interleukin-18 (IL-18) is a novel cytokine mainly produced by activated macrophages . IL-18 was originally called interferon-gamma inducing factor, due to its action in inducing IFN-gamma secretion from Th1 cells, NK cells and NKT cells . It has been reported that IL-18 may play important roles in various diseases including cancer and infectious diseases . This review deals with the roles of IL-18 in infectious diseases, with special emphasis on IL-18 in infectious diseases caused by intracellular pathogens including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes and Salmonella typhimurium.

J Biol Chem, 2000 Dec 22, 275(51), 40316 - 23
The enigma of cobalamin (Vitamin B12) biosynthesis in Porphyromonas gingivalis . Identification and characterization of a functional corrin pathway; Roper JM et al.; The ability of Porphyromonas gingivalis to biosynthesize tetrapyrroles de novo has been investigated . Extracts of the bacterium do not possess activity for 5- aminolevulinic-acid dehydratase or porphobilinogen deaminase, two key enzymes involved in the synthesis of uroporphyrinogen III . Similarly, it was not possible to detect any genetic evidence for these early enzymes with the use of degenerate polymerase chain reaction . However, the bacterium does appear to harbor some of the enzymes for cobalamin biosynthesis since cobyric acid, a pathway intermediate, was converted into cobinamide . Furthermore, degenerate polymerase chain reaction with primers to cbiP, which encodes cobyric-acid synthase, produced a fragment with a high degree of identity to Salmonella typhimurium cbiP . Indeed, the recently released genome sequence data confirmed the presence of cbiP together with 14 other genes of the cobalamin pathway . A number of these genes were cloned and functionally characterized . Although P . gingivalis harbors all the genes necessary to convert precorrin-2 into cobalamin, it is missing the genes for the synthesis of precorrin-2 . Either the organism has a novel pathway for the synthesis of precorrin-2, or more likely, it has lost this early part of the pathway . The remainder of the pathway may be being maintained to act as a salvage route for corrin synthesis.

J Small Anim Pract, 2000 Aug, 41(8), 339 - 41
Outbreak of Salmonella typhimurium in cats and humans associated with infection in wild birds; Tauni MA et al.; An outbreak of Salmonella typhimurium infection in cats and humans in Sweden in 1999, associated with wild birds, is described . In the county of Varmland, 62 sick cats were examined . All were anorectic and lethargic, 57 per cent had vomiting and 31 per cent had diarrhoea . It was considered likely that salmonellosis was transmitted from cats to humans, but there were only a few such cases.

Nature, 2000 Sep 14, 407(6801), 211 - 5
Peroxynitrite reductase activity of bacterial peroxiredoxins; Bryk R et al.; Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants . Superoxide (O2-) arises in all organisms inhabiting aerobic environments . Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref . 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors . The oxidative, inflammatory, mutagenic and cytotoxic potential (ref . 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself . Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production . We considered whether cells might be equipped with enzymes to detoxify peroxynitrite . Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood . Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA . Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori . Thus, peroxynitrite reductase activity may be widespread among bacterial genera.

J Biotechnol, 2000 Sep 29, 83(1-2), 45 - 50
Aromatic-dependent salmonella as anti-bacterial vaccines and as presenters of heterologous antigens or of DNA encoding them; Stocker BA; The development of live bacterial vaccines is reviewed, in particular aromatic-dependent Salmonella, either for protection against the corresponding infections (including typhoid fever) or as carrier-presenter of antigens of unrelated pathogens or of DNA specifying them . Aromatic-dependent Salmonella live vaccines are also compared with BCG and Ty21a and the recent records of exceptional situations are discussed in which aroA (deletion) strains of Salmonella typhimurium cause progressive disease in micePublication Types:

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