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 populati