Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 71 - 8 Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair; Wang G et al.; Nitroxide free radicals have been previously shown to function as superoxide dismutase (SOD) mimics and to protect bacterial and mammalian cells against oxidative damage, particularly from superoxide and hydrogen peroxide . Although nitroxides are generally considered to be non-toxic nor mutagenic, there is no agreement regarding their potential adverse effect . Some toxic effects were observed upon using high concentration of six-membered ring derivatives . Conflicting evidence has also been reported regarding the mutagenic activity of nitroxides toward Salmonella typhimurium . It was also demonstrated that nitroxides exert two opposing effects on exonuclease III deficient cells of Escherichia coli upon exposure to naphthoquinones . The attempts to use nitroxides as contrast agents in nuclear magnetic resonance imaging (MRI) and as a new class of anti-oxidants underscore the need to examine their potential adverse effects . Since nitroxides protected xthA cells from DNA scission caused by H2O2, it was anticipated that they would provide even greater protection for recA DNA repair-deficient cells of E . coli, which are more sensitive to H2O2-induced oxidative stress . The results of the present study showed that: (a) nitroxides exert bactericidal and bacteriostatic effects on recA but not on xthA or wild-type E . coli K12 cells; (b) nitroxides and H2O2 act synergistically on recA cells, both under aerobic and hypoxic conditions; (c) the nitroxide-induced toxicity in recA cells and the synergistic effect with H2O2 were not accompanied by a decrease in the cellular level of reduced glutathione; (d) TEMPAMINE protected against DNA scission induced by H2O2 and 1,10-ortho-phenanthroline chelate of Cu(II) in xthA cells, but potentiated DNA double-strand breakage in recA cells. Mol Gen Genet, 1996 Feb 5, 250(2), 197 - 206 Molecular analysis of the scrA and scrB genes from Klebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme II Scr of the phosphotransferase system and a sucrose-6-phosphate invertase; Titgemeyer F et al.; The Klebsiella pneumoniae genes scrA and scrB are indispensable for sucrose (Scr) utilisation . Gene scrA codes for an Enzyme IIScr (IIScr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), while scrB encodes a sucrose 6-phosphate specific invertase . A 3.7 kbscr AB DNA fragment has been cloned from K . pneumoniae and expressed in Escherichia coli . Its nucleotide sequence was determined and the coding regions for scrA (1371 bp) and scrB (1401 bp) were identified by genetic complementation, enzyme activity test and radiolabelling of the gene products . In addition, the nucleotide sequence of the scrB gene from conjugative plasmid pUR400 isolated from Salmonella typhimurium was also determined and errors in the previously published sequence of the scrA gene of pUR400 were corrected . Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes . Based on the analysis of seven IIScr proteins, a hypothetical model of the secondary structure of IIScr is proposed. J Biol Chem, 1996 Feb 2, 271(5), 2448 - 54 A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin; Matsushita M et al.; Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens . We report here a novel Ca(2+)-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence . When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins . P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains . The latter contained two potential Ca(2+)-binding sites that may be involved in carbohydrate binding . The overall sequence of P35 was highly homologous to porcine ficolins alpha and beta . Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver . P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region . P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides . Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity. Biosci Biotechnol Biochem, 1996 Feb, 60(2), 328 - 9 Antimutagenic activity of caffeic acid and related compounds; Yamada J et al.; Effects of caffeic acid and chlorogenic acid on mutagenicity were studied using the Salmonella typhimurium system . These compounds had inhibitory effects on the mutagenicity of Trp-P-1 and Glu-P-2 . Caffeic acid completely eliminated the mutagenicity induced by activated Glu-P-2 . Some compounds analogous to caffeic acid, such as cinnamic acid, coumaric acid, and ferulic acid, also significantly decreased the mutagenicity of Glu-P-2. Vet Microbiol, 1996 Feb, 48(3-4), 293 - 303 Time course of the serological response to Yersinia enterocolitica O:3 in experimentally infected pigs; Nielsen B et al.; A total of 25 pigs inoculated with Yersinia enterocolitica serovar O:3 and 25 un-inoculated controls were followed weekly by sampling blood and faeces for 70 days post infection (p.i.) . All inoculated pigs were faeces culture positive from day 5 to 21 p.i., whereafter shedding of bacteria declined to < 10% of the pigs at day 49 p.i . and to 0% at day 68 p.i . All control pigs remained Y . enterocolitica O:3 culture negative . When examined in an indirect ELISA using purified LPS from Y . enterocolitica 0:3, sera from all inoculated pigs showed significantly higher optical densities (OD) as compared to the control group . All inoculated pigs had seroconverted at day 19 p.i . and remained seropositive until slaughter at day 70 p.i . The maximum mean anti-LPS response was observed at day 33 p.i . with a positive/negative ratio of 780 . No cross-reactions were observed with sera from 21 pigs, infected with Salmonella typhimurium . At necropsy at day 70 p.i., Y . enterocolitica O:3 was isolated from the tonsils of 20 inoculated pigs, whereas the rest of the gastrointestinal tract and associated lymph nodes were culture negative . The remaining inoculated pigs and all control pigs were culture negative at necropsy at day 70 p.i . The ELISA seems to be a promising alternative to bacteriological culture for detection of Y . enterocolitica O:3 infection in pig herds. Vet Microbiol, 1996 Feb, 48(3-4), 257 - 68 Enzyme-linked immunosorbent assay and immunoblot analysis for detection of antibodies to Borrelia burgdorferi in dogs . The impact of serum absorption with homologous and heterologous bacteriae; Wittenbrink MM et al.; Sera from 665 apparently healthy dogs were examined for antibodies to the Lyme disease spirochete Borrelia (B.) burgdorferi by using an ELISA with a whole cell sonicate of B . burgdorferi sensu stricto reference strain B31 (ATCC 35210) as antigen . To discover false positive reactions due to the unsatisfactory specificity of conventional enzyme-linked immunosorbent assays for B . burgdorferi, sera were absorbed in parallel with both B . burgdorferi and a heterologous sorbent consisting of whole cells of Escherichia coli, Salmonella typhimurium, and eight serovars of Leptospira interrogans . The difference between optical densities obtained in the ELISA after serum absorption with the heterologous sorbent and the B . burgdorferi sorbent was defined as a new value, "ODdiff", for ELISA reactivity specific for B . burgdorferi . ELISA results were confirmed by immunoblot studies . By testing unabsorbed sera, 48 of 665 serum samples (7.2%) were considered ELISA positive . 37 of these 48 sera (77.1%) were apparently false positive: here a similar reduction of ELISA reactivity was obtained after absorption with B . burgdorferi antigen and with the heterologous sorbent (ODdiff approximately equal to 0) . None of these 37 sera gave immunoblot patterns characteristic for canine B . burgdorferi infection. Microbiology, 1996 Feb, 142 ( Pt 2), 217 - 30 Catabolite repression and inducer control in Gram-positive bacteria; Saier MH Jr et al.; Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria . This protein kinase is not found in enteric bacteria such as E . coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control . In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b) . In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc . Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993) . In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated . HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins . These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels . Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor . These two proteins together are believed to determine the intensity of catabolite repression . Their relative importance depends on physiological conditions . Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites . CepA possibly binds sugar metabolites such as FBP as well as HPr(ser-P) . Because HPr(his-P, ser-P) does not bind to CepA, the regulatory cascade is also sensitive to the external PTS sugar concentration . Mutational analyses (unpublished results) suggest that CepA may bind to a site that includes His-15 . Interestingly, both the CepA protein in the Gram-positive bacterium, B . subtilis, and glycerol kinase in the Gram-negative bacterium, E . coli, sense both a PTS protein and a cytoplasmic metabolic intermediate . The same may be true of target permeases and enzymes in both types of organisms, but this possibility has not yet been tested . The parallels between the Gram-negative and Gram-positive bacterial regulatory systems are superficial at the mechanistic level but fundamental at the functional level . Thus, the PTS participates in regulation in both cases, and phosphorylation of its protein constituents plays key roles . However, the stimuli sensed, the transmission mechanisms, the central PTS regulatory proteins that effect allosteric regulation, and some of the target proteins are completely different . It seems clear that these two transmission mechanisms evolved independently . They provide a prime example of functional convergence. Mol Microbiol, 1996 Feb, 19(3), 505 - 9 Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein; Maas WK et al.; Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA . In Escherichia coli, such molecules are encoded by genetic elements called retrons . The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs . Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids . In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E . coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs . We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination . We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein. Mol Microbiol, 1996 Feb, 19(4), 791 - 801 A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8-23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection; Chabalgoity JA et al.; Multiple tandem copies of an immunogenic epitope comprising amino acids 8-23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C-terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains . Expression of the longer fusions was best in strains harbouring a lesion in htrA, a stress protein gene . SL3261, an aroA strain, did not effectively express the longer fusions . Mice immunised with an S . typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC . A non-immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion . In contrast, the S . typhimurium SL3261 aroA vaccine expressing the TetC-tetrameric fusion did not elicit antibody to the peptide . Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro, and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna . We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge . These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV. Mol Microbiol, 1996 Feb, 19(4), 777 - 90 Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria; Jordan A et al.; Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons . Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen . Several factors controlling nrdAB gene transcription have been analysed intensively . Nothing is known about the expression of the nrdEF genes . To study this subject, and after cloning of E . coli nrdEF genes and sequencing of their 5' ends, the promoter of this operon has been identified by primer extension in both bacterial species . The +1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S . typhimurium and E . coli, respectively . Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria . The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system . Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins . Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells . nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA- cells . nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature . nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin . In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions. FEMS Immunol Med Microbiol, 1996 Feb, 13(2), 155 - 60 Purification of a product from Salmonella typhimurium with the ability to inhibit mitogen-induced proliferation of murine splenic T-lymphocytes; Matsui K; We attempted to purify a substance that inhibits mitogen-induced proliferation of murine splenic T-lymphocytes from Salmonella typhimurium . The soluble fraction of a suspension of bacteria disrupted by sonication was chromatographed serially on Mono Q HR, Superdex 200 HR and HiLoad Superdex 75 p.g . columns . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified active substance migrated as a single band corresponding to a molecular mass of 87 kDa . We designated the purified substance S . typhimurium-derived inhibitor of T-cell proliferation (STI), which, at 0.2 microgram/ml and above, inhibited proliferation and augmented CD25 expression of phytohemagglutinin-stimulated murine splenic lymphocytes . These findings suggested that the immunosuppression induced by Salmonella infection may be attributable to STI. Genetika, 1996 Feb, 32(2), 233 - 9 {Genotoxic and mutagenic activity of antineoplastic anthracyclines and their aglycones: study in two test-systems}; Vasil'eva SV et al.; Mutagenic (Ames tests) and genotoxic (SOS chromotest) activities of highly-efficient natural anthracycline monosaccharides possessing antitumor activity-daunorubicin (also known as daunomycin or rubomycin), doxorubicin (adriamycin), and carminomycin-were studied . At the same time, the hypothesis was tested that intercalation of the antibiotic moiety into the helix of cell DNA, which was mediated by the saccharide amino group, played a crucial role in genotoxicity of these anthracyclines . The hydrolysis products of these antibiotics (the corresponding aglycones) and aclacynomycin A (an anthracycline trisaccharide), as well as aclavinone (its derivative aglycone), were studied . All these compounds lacked the saccharide amino group necessary for intercalation . It was found that all anthracycline monosaccharides studied had a strong mutagenic effect on strain TA98 and a moderate effect on strain TA100 of Salmonella typhimurium . Aclacynomycin A was found to have no mutagenic effect on any strain . Lack of the glycoside amino group did not necessarily result in loss of mutagenic activity in the derivative aglycones of anthracycline monosaccharides: they exhibited moderate mutagenic activity in strain TA98 and low but significant activity in strain TA100 . The S9 microsomal fraction did not alter the mutagenic activity of either anthracycline monosaccharides or their aglycones; however, it dramatically increased the mutagenic activity of aclavinone: correspondence between positive responses in Ames tests and the SOS chromotest was found . Apparently, the mutagenic activity of the substances studied in bacterial cells was mediated by inducing the SOS-repair process . If the compound contained the amino glycoside moiety, functional and structural precursors of the SOS response were formed via intercalation of the reagents into the DNA duplex; if the substance did not contain this moiety, the precursors were formed via ionic interaction. J Antimicrob Chemother, 1996 Feb, 37(2), 351 - 6 Mutations of the gyrA gene of clinical isolates of Salmonella typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quinolone drugs; Brown JC et al.; The region of the gyrA gene encoding nucleotides 72 to 557 in ten Salmonella typhimurium clinical isolates received from Vellore, India and six British clinical isolates of a number of Salmonella species has been sequenced . Those exhibiting a decreased susceptibility to 4-quinolone drugs (MICs of ciprofloxacin ranging from 0.128 mg/L to 1.0 mg/L) have been shown to possess a number of different mutations in this region of the gene (Asp-87 to Gly, Asp-87 to Asn and Ser-83 to Phe) . The mutations Asp-87 to Gly and Ser-83 to Phe/Asp-87 to Asn have yet to be reported occurring in S . typhimurium. J Antimicrob Chemother, 1996 Feb, 37(2), 253 - 63 The effect of antibiotics on bacteria under hyperbaric conditions; Hind J et al.; The sensitivity of selected bacteria to a range of antibiotics was tested under hyperbaric conditions used in saturation diving . The effect of hyperbaric helium and oxygen (heliox) on antibiotic stability and on induction of beta-lactamase was also determined . Increased resistance to penicillin (up to 23%) was shown by Staphylococcus aureus and to gentamicin (up to 46%) and rifampicin (up to 18%) by Escherichia coli and Salmonella typhimurium at 36 and 71 bar pressure . Exposure to 71 bar heliox did not affect antibiotic activity but increased the production of beta-lactamase in inducible S . aureus and Bacillus subtilis and production of beta-galactosidase in inducible E . coli . Increased resistance to antibiotics in saturation diving conditions can be attributed in some cases to the influence of hyperbaric pressure on induction mechanisms in bacteria . The experimental system devised for this work is suitable for more detailed examination of the influence of hyperbaric stress on antibiotic resistance and of its effect on induction mechanisms in general. Yeast, 1996 Feb, 12(2), 149 - 67 Analysis of a 36.2 kb DNA sequence including the right telomere of chromosome VI from Saccharomyces cerevisiae; Eki T et al.; The nucleotide sequence of a 36.2-kb distal region containing the right telomere of chromosome VI was determined . Both strands of DNA cloned into cosmid clone 9965 and plasmid clone pEL174P2 were sequenced with an average redundancy of 7.9 per base pair, by both dye primer and dye terminator cycle sequencing methods . The G+C content of the sequence was found to be 37.9% . Eighteen open reading frames (ORFs) longer than 100 amino acids were detected . Four of these ORFs (9965orfR017, 9965orfF016, 9965orfR009 and 9965orfF003) were found to encode previously identified genes (YMR31, PRE4, NIN1 and HXK1, respectively) . Six ORFs (9965orfR013, 9965orfF018, 9965orfF006, 9965orfR014, 9965orfF013 and 9965orfR020) were found to be homologous to hypothetical 121.4-kDa protein in the BCK 5' region, Bacillus subtilis DnaJ protein, hypothetical Trp-Asp repeats containing protein in DBP3-MRPL27, putative mitochondrial carrier YBR291C protein, Salmonella typhimurium nicotinate-nucleotide pyrophosphorylase, and Escherichia coli cystathionine beta-lyase, respectively . The putative proteins encoded by 9965orfF018, 9965orfR014 and 9965orfR020 were found to be, respectively, a new member of the family of DnaJ-like proteins, the mitochondrial carrier protein and cystathionine lyase. Mutat Res, 1996 Feb, 367(2), 83 - 92 Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents; Lee CK et al.; Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher that that of the N-nitrosamines . The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N(1)-nitrosonornicotine (NNN) . These nitrosamines require metabolic activations by cytochrome P-450s for the expression of mutagenicity . Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported, In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9) . All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner . The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine . None of the observed reductions in mutagenicity could be explained by cytotoxicity . These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines. Indian J Exp Biol, 1996 Feb, 34(2), 98 - 102 Antimutagenic effects of polyphenols isolated from Terminalia bellerica myroblan in Salmonella typhimurium; Padam SK et al.; Antimutagenic effect of 2 polyphenolic fractions isolated from T . bellerica in TA98 and TA100 strains of S . typhimurium against 2AF, NPD and 4NQNO has been characterized . Both the fractions were significantly effective against S9-dependent 2AF; less effective against NPD and almost not effective against 4NQNO in TA100 strain . Using 13C-NMR spectral analysis, the TB-3 fraction, which was significantly more effective against 2AF compared to TB-4, was found to be a mixture of 3 tannins while TB-4 was non-tannin fraction . Interaction of polyphenols with S9 proteins may be the probable cause of inhibitory effect of these polyphenols, though the possibility of other mechanisms cannot be ruled out. DNA Cell Biol, 1996 Feb, 15(2), 113 - 23 Structural organization, sequence, and expression of the chicken NRAMP1 gene encoding the natural resistance-associated macrophage protein 1; Hu J et al.; One of the most common causes of food poisoning in humans is salmonellosis, which is frequently caused by ingestion with Salmonella-contaminated poultry products . Several lines of evidence suggest that genetic factors control resistance and susceptibility of chickens to infection with Salmonellae . In the mouse, innate resistance to infection with intracellular pathogens such as Salmonella typhimurium, several species of Mycobacteria, and Leishmania donovani is controlled by the mouse chromosome 1 Nramp1Bcg gene . To investigate the role of NRAMP1 in the differential resistance and susceptibility of chickens to infections with S . typhimurium, we have cloned and characterized cDNA clones corresponding to the chicken NRAMP1 gene . Nucleotide and predicted amino acid sequence analyses indicate that the chicken NRAMP1 polypeptide encodes a 555-amino-acid residue membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif . The peptide sequence identity among chicken, mouse, and human NRAMP1 is 68% . The chicken NRAMP1 gene contains 15 exons and spans 5 kb of genomic DNA . One major and two minor transcription initiation sites were detected using primer extension . Nucleotide sequencing of the promoter region revealed the presence of a classical TATAA element and consensus sequences for binding the myeloid specific PU.1 factor and several lipopolysaccharide (LPS) (NF-IL6 and NF-kappa B) and interferon-gamma (IFN-gamma)-inducible response elements . Similar regulatory elements are found in the promoters of mouse and human NRAMP1 . Northern blot analyses revealed NRAMP1 expression in reticuloendothelial organs (spleen and liver), lung, and thymus . As demonstrated in mice and humans, the macrophage is also a major site of NRAMP1 mRNA expression in chickens . However, the high levels of expression detected in chicken thymus contrast with the absence of expression of the mammalian Nramp1 gene in this tissue. Carcinogenesis, 1996 Feb, 17(2), 297 - 302 A new Salmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5: use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system; Oda Y et al.; The Escherichia coli mu operon was subcloned into a pKK233-2 vector containing rat glutathione S-transferase (GST) 5-5 cDNA and the plasmid thus obtained was introduced into Salmonella typhimurium TA1535 . The newly developed strain S.typhimurium NM5004, was found to have 52-fold greater GST activity than the original umu strain S.typhimurium TA1535/pSK1002 . We compared sensitivities of these two tester strains, NM5004 and TA1535/pSK1002, for induction of umuC gene expression with several dihaloalkanes which are activated or inactivated by GST 5-5 activity . The induction of umuC gene expression by these chemicals was monitored by measuring the cellular beta-galactosidase activity produced by umuC"lacZ fusion gene in these two tester strains . Ethylene dibromide, 1-bromo-2-chloroethane, 1,2-dichloroethane, and methylene dichloride induced umuC gene expression more strongly in the NM5004 strain than the original strain . 4-Nitroquinoline 1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine were found to induce umuC gene expression to similar extents in both strains . In the case of 1-nitropyrene and 2-nitrofluorene, however, NM5004 strain showed weaker umuC gene expression responses than the original TA1535/pSK1002 strain . 1,2-Epoxy-3-(4'-nitrophenoxy)propane, a known substrate for GST 5-5, was found to inhibit umuC induction caused by 1-bromo-2-chloroethane . These results indicate that this new tester NM5004 strain expressing a mammalian GST theta class enzyme may be useful for studies of environmental chemicals proposed to be activated or inactivated by GST activity. Carcinogenesis, 1996 Feb, 17(2), 277 - 82 CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane; Barcelo S et al.; The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats against 9,10-dimethyl-1,2-benz{a}anthracene tumorigenesis, thought to be due, at least in part, to induction of phase II detoxification . We investigated the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible for activation of several carcinogens, including dialkylnitrosamines . Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown to be a potent competitive inhibitor of CYP2E1 with a Ki of 37.0 +/- 4.5 microM . In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA) . Sulforaphane at concentrations of > 0.8 microM inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone . Unscheduled DNA synthesis induced by NDMA (33.5 microM) in mouse hepatocytes was inhibited in a dose-dependent manner by sulforaphane at 0.064-20 microM . Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 micrograms/plate), a direct acting mutagen, in the Salmonella assay . It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of Salmonella employed and cytotoxic only at high concentrations (> or = 0.5 mM) . These findings suggest that inhibition of CYP2E1 by sulforaphane may offer chemoprotection against carcinogenic substrates of this enzyme. Mutat Res, 1996 Feb 1, 349(2), 201 - 8 Efficiency of MucAB and Escherichia coli UmuDC proteins in quinolone and UV mutagenesis in Salmonella typhimurium: effect of MucA and UmuD processing; Clerch B et al.; The role of MucAB and Escherichia coli UmuDC proteins in mutagenesis by 4-quinolone (4-Q) compared to that in UV mutagenesis has been studied in hisG428 Salmonella typhimurium strains . A low-copy plasmid carrying mucAB genes, but not umuDC, promotes reversion of the hisG428 mutation by the 4-Q ciprofloxacin . In contrast, a umuDC plasmid mediates the reversion of hisG428 by UV, although less efficiently than a mucAB one . In addition, a unique copy of mucAB genes is enough to promote UV mutagenesis, whereas, several copies of them are required to detect ciprofloxacin mutagenesis . Therefore, the mutagenic repair of quinolone damage by MucAB proteins is not a very efficient process . The presence of an umuD'C plasmid but not a mucA'B one, slightly increases the reversion of the hisG428 mutation by ciprofloxacin and this finding is further discussed . In contrast, MucA'B are still more active than UmuD'C proteins in UV mutagenesis . These results suggest that the enhanced processing of MucA compared to UmuD would not explain all functional differences between MucAB and UmuDC proteins in the error-prone DNA repair. Arch Microbiol, 1996 Feb, 165(2), 126 - 31 Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB; Van Kuijk BL et al.; Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions . The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa . The enzyme exhibited maximum activity at pH 8.5 and approximately 54 degrees C . The Km values for fumarate and L-malate were 0.25 mM and 2.38 mM, respectively . Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and &beta;-mercaptoethanol under anoxic conditions . EPR spectroscopy of the purified enzyme revealed the presence of a {3Fe-4S} cluster . Under reducing conditions, only a trace amount of a {4Fe-4S} cluster was detected . Addition of fumarate resulted in a significant increase of this {4Fe-4S} signal . The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Appl Environ Microbiol, 1996 Feb, 62(2), 587 - 92 Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples; Yu H et al.; Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water . Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous . Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples . In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E . coli O157 and Salmonella typhimurium in foods and fomites . Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E . coli O157 and S . typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples . An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E . coli O157 and S . typhimurium, respectively . IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay . Some samples, especially fish, gave unexpectedly high background ECL . Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy . Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens. J Bacteriol, 1996 Feb, 178(4), 1187 - 96 Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium; Epelbaum S et al.; The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II . One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI) . The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured . The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter . The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition . This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism . The organization of the pathway thus potentiates the inhibitory effect of SMM . IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate . Valine completely protected strain TV105 from IpOHA at the MIC . A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested . IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration . The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium. J Bacteriol, 1996 Feb, 178(4), 1113 - 9 Effect of the surface composition of motile Escherichia coli and motile Salmonella species on the direction of galvanotaxis; Shi W et al.; We have reported that motile Escherichia coli K-12 placed in an electric field swims toward the anode but that motile Salmonella typhimurium strains swim toward the cathode, a phenomenon called galvanotaxis (J . Adler and W . Shi, Cold Spring Harbor Symp . Quant . Biol . 53:23-25, 1988) . In the present study, we isolated mutants with an altered direction of galvanotaxis . By further analyses of these mutants and by examination of E . coli and Salmonella strains with altered cell surface structure, we have now established a correlation between the direction of galvanotaxis and the surface structure of the cell: motile rough bacteria (that is, those without O polysaccharide; for example, E . coli K-12 and S . typhimurium mutants of classes galE and rfa) swam toward the anode, whereas motile smooth bacteria (that is, those with O polysaccharide; for example, wild-type S . typhimurium LT2) swam toward the cathode . However, smooth bacteria with acidic polysaccharide capsules (K1 in E . coli and Vi in Salmonella typhi) swam toward the anode . Measurements of passive electrophoretic mobility of strains representative of each set were made . We propose that the different directions of galvanotaxis of rough (or capsulate) bacteria and of smooth bacteria are explicable if the negative electrophoretic mobility of flagellar filaments is less than that of rough bodies but greater than that of smooth bodies. J Bacteriol, 1996 Feb, 178(3), 899 - 901 Negative regulation by fliD, fliS, and fliT of the export of the flagellum-specific anti-sigma factor, FlgM, in Salmonella typhimurium; Yokoseki T et al.; The fliD operon of Salmonella typhimurium consists of three flagellar genes, fliD, fliS, and fliT, and is transcribed in this order . It has been shown that an fliD::Tn10 mutation causes an excess export of the flagellum-specific anti-sigma factor, FlgM, resulting in an overexpression of flagellar class 3 operons . In this study, using gene-disruption mutants in the individual genes in the fliD operon, we showed that mutations in any one of the genes in the operon enhanced both FlgM export and the expression of flagellar regulon . This indicates that all three genes in the operon are involved in the negative regulation of FlgM export. J Bacteriol, 1996 Feb, 178(3), 638 - 46 Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product; Choi P et al.; In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in the heme biosynthetic pathway . It has recently been reported that a lac operon fusion to the hemA promoter of E . coli is induced 20-fold after starvation for heme . Induction was dependent on the transcriptional regulator ArcA, with a second transcriptional regulator, FNR, playing a negative role specifically under anaerobic conditions (S . Darie and R . P . Gunsalus, J . Bacteriol . 176:5270-5276, 1994) . We have investigated the generality of this effect by examining the response to heme starvation of a number of lac operon fusions to the hemA promoters of both E . coli and S . typhimurium . We confirmed that such fusions are induced during starvation of a hemA auxotroph, but the level of induction observed was maximally sixfold and for S . typhimurium fusions it was only two- to fourfold . Sequences required for high-level expression of hemA lie within 129 bp upstream of the major (P1) promoter transcriptional start site . Mutants defective in the P1 promoter had greatly reduced hemA-lac expression both in the presence and in the absence of ALA . Mutations in arcA had no effect on hemA-lac expression in E . coli during normal growth, although the increase in expression during starvation for ALA was half that seen in an arcA+ strain . Overexpression of the arcA gene had no effect on hemA-lac expression . Primer extension analysis showed that RNA 5' ends mapping to the hemA P1 and P2 promoters were not expressed at significantly higher levels in induced cultures . These results differ from those previously reported. Infect Immun, 1996 Feb, 64(2), 548 - 56 Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19; Pugh GW Jr et al.; The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins {BCSP}) of Brucella abortus 19 and later challenge exposed with B . abortus 2308 . BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate . Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC) . Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines . Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria . Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic . The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier . However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice . MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D . Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only . The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B . abortus infections. J Biotechnol, 1996 Jan 26, 44(1-3), 161 - 70 Bacterial ghosts: non-living candidate vaccines; Szostak MP et al.; Expression of cloned PhiX174 gene E in bacteria results in lysis of bacteria . It is unique among phage lysis systems as it introduces a transmembrane tunnel structure through the cell envelope complex of Gram-negative bacteria . The resulting bacterial ghosts have intact envelope structures devoid of cytoplasmic contents . E-mediated lysis has been achieved in a variety of Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Klebsiella pneumoniae, and Actinobacillus pleuropneumoniae . Such ghosts, derived from human or animal pathogens, have been proposed as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria . In 'recombinant ghosts', foreign proteins (e.g., viral proteins) are inserted into the inner membrane via specific N-, or C-, or N- and C-terminal anchor sequences prior to lysis . Relevant advantages of (recombinant) bacterial ghosts as immunogens include: (i) inactivation procedures that denature relevant immunogenic determinants are not employed in the production of ghosts used as vaccines or as carriers of relevant antigens; (ii) the recombinant proteins are inserted into a highly immune stimulatory environment; (iii) there is no size limitation of the foreign protein moieties: multiple antigenic determinants can be presented simultaneously; (iv) bacterial ghosts can be produced inexpensively in large quantities; (v) (recombinant) ghosts are stable for long periods of time and do not require the cold chain storage system . Intraperitoneal, subcutaneous or intramuscular applications of recombinant ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and viral components . Initial aerosol vaccinations of swine with ghosts from Actinobacillus pleuropneumoniae showed that protective immunity can be established by this route of application and that the well-preserved surface structures of ghosts obtained by E-mediated lysis are able to target the mucosal immune system. J Biotechnol, 1996 Jan 26, 44(1-3), 91 - 6 Hybrid hepatitis B virus core antigen as a vaccine carrier moiety: I . presentation of foreign epitopes; Schodel F et al.; Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle . Here, we review recent progress in the use of HBcAg as a carrier moiety for heterologous epitopes . To define surface exposed and immunogenic insertion sites for foreign epitopes in HBcAg, peptidic epitopes representing binding sites for virus neutralizing antibodies on the HBV surface antigens were inserted at different positions within HBcAg using genetic engineering in an Escherichia coli expression system (Schodel et al . (1992) J . Virol . 66, 106-114) . While fusion to the N-terminus required a linker to become surface accessible, both fusion to the N-terminus and to the C-terminus was compatible with particle assembly and preserved the native antigenicity and immunogenicity of HBcAg . Fusion to an immunodominant internal site of HBcAg reduced the HBcAg immunogenicity and antigenicity and most drastically enhanced the immunogenicity of the inserted foreign epitope . This internal site of HBcAg was used to express circumsporozoite antigen (CS) repeat epitopes of two rodent malaria parasites and of Plasmodium falciparum (Schodel et al . (1994b) J . Exp . Med . 180, 1037-1046 and Schodel et al . (1995a) 95th ASM General Meeting, Washington DC, Abstr . E61) . When purified from recombinant Salmonella typhimurium, the hybrid HBcAg-CS proteins were particulate and displayed CS antigenicity as well as reduced HBc antigenicity, as compared to native HBcAg . Immunization of several mouse strains with HBcAg-CS hybrid particles resulted in high titered serum anti-CS antibodies representing all murine IgG isotypes . Immunization of mice with HBcAg or HBcAg-CS particles formulated on alum, complete Freunds or incomplete Freunds adjuvant resulted in equivalent anti-CS and anti-HBc serum antibody titres . The possible influence of carrier-specific immunosuppression was examined and pre-existing immunity to HBcAg did not significantly alter the immunogenicity of hybrid HBcAg particles suggesting that they would be useful carrier moieties for repeated immunizations against multiple haptens or in immune subjects after HBV infection . Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site . This indicates that the internal amino acid position in HBcAg is permissive for the inclusion of heterologous functional T helper as well as B cell epitopes . BALB/c mice immunized with HBcAg-CS1 were protected against P . berghei challenge to 90% and 100%, respectively, in two independent experiments. J Mol Biol, 1996 Jan 26, 255(3), 458 - 75 Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images; Katayama E et al.; The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy . In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes of Salmonella typhimurium overproduced in the cytoplasmic membrane of Escherichia coli . The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm . On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible . We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm . It was identified as the C ring, since it was located at the base of the polyhook . Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photogrammetry, and a three-dimensional model of the total basal structure is presented. Proc Natl Acad Sci U S A, 1996 Jan 23, 93(2), 906 - 7 Muller's ratchet decreases fitness of a DNA-based microbe; Andersson DI et al.; Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class {Muller, H.J . (1964) Mutat . Res . 1, 2-9} . The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses . However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high . We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate . Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined . S . typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate . These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations . In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation . This rate is measured for the largest genetic target studied so far, a collection of about 200 genes. Cancer Lett, 1996 Jan 19, 99(1), 15 - 21 All-trans beta-carotene enhances mitogenic responses and ornithine decarboxylase activity of BALB/c 3T3 fibroblast cells induced by tumor promoter and fetal bovine serum but suppresses mutagen-dependent umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002); Okai Y et al.; Although previous epidemiological studies have indicated that beta-carotene is an important agent for the chemical prevention against carcinogenesis, a recent prospective study has strikingly suggested that supplementation with beta-carotene significantly increased the incidence of some types of cancer (The alpha-Tocopherol and beta-Carotene Cancer Prevention Study Group, New Engl . J . Med., 330 (1994) 1031-1035) . To analyze the discrepancy of this problem, the authors analyze the effects of beta-carotene on biochemical and biological events associated with carcinogenesis by in vitro experiments . (1) All-trans beta-carotene enhanced the proliferation and DNA synthesis of BALB/c 3T3 cells induced by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and fetal bovine serum, although beta-carotene itself did not show mitogenic activity . (2) All-trans beta-carotene caused a remarkable stimulation for the early induction of ornithine decarboxylase (ODC) activity after the stimulation of TPA and fetal bovine serum . (3) All-trans beta-carotene exhibited significant antimutagenic activity which suppresses umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by a typical mutagen, 2-aminoanthracene (2-AA) . These experimental results suggest that all-trans beta-carotene might cause beneficial and harmful effects on different phases of carcinogenesis. Mutat Res, 1996 Jan 17, 349(1), 137 - 44 Mutagenicity of nitrophenanthrene derivatives for Salmonella typhimurium: effects of nitroreductase and acetyltransferase; Sera N et al.; To determine the mutagenicity of nitrophenanthrenes, three mononitrophenanthrenes (NPhs), 11 dinitrophenanthrenes (diNPhs) and eight trinitrophenanthrenes (tiNPhs) were synthesized, and their mutagenicity was investigated by using Salmonella typhimurium his- strains TA98, TA100, and TA98NR, nitroreductase-deficient, and TA98/1,8-DNP6, O-acetyltransferase-deficient mutants, and strains YG1021 and YG1026, nitroreductase-overproducing mutants of TA98 and TA100, respectively, and strains YG1024 and YG1029, O-acetyltransferase-overproducing mutants of TA98 and TA100, respectively . 1-, 3- and 9-NPhis induced 329, 620 and 438 revertants per nmol in strain TA100, respectively, and 4,839, 11,309 and 16728 revertants per nmol, respectively, in strain YG1029 . Mutagenicity of 1,6-, 2,6-, 2,9-, 2,10-, 3,5-, 3,6- and 3,10-diNPh was elevated in strains YG1021, YG1024, YG1026 and YG1029 . Among these derivatives, 1,6-, 2,6-, 3,6- and 3,10-diNPhs were more mutagenic in strains YG1024 and YG1029 than YG1021 and YG1026, and they showed a structure-activity relationship between mutagenicity and NO2-substitution . Nitro derivatives substituted at the 3 and 6 positions of their chemical structure strongly mutated both strains YG1024 and YG1029, whereas those substituted at the 9 and 10 positions showed weak mutagenicity . In addition, nitro substituents at positions 4 and 5 were perpendicular while those on positions 2,3,6 and 7 were nearly coplanar to the aromatic ring . Furthermore, 2,6,9-, 3,6,9- and 1,6,9-trinitrophenanthrenes (triNPhs) were mutagenic for strain TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026 . Of the eight triNPhs all except 1,5,10-triNP were mutagenic in TA98 and TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026 . These results suggest that these compounds are mutagens that are activated by O-acetyltransferase esterification following nitroreductase . The nitrated derivatives substituted at the 2(7) and 3(6) positions of the phenanthrene ring were highly mutagenic . The relationship between chemical structure and the mutagenicity of NPh derivatives is discussed. Mutat Res, 1996 Jan 16, 359(1), 17 - 24 DNA strand break by 2,5-dimethyl-4-hydroxy-3(2H)-furanone, a fragrant compound in various foodstuffs; Hiramoto K et al.; 2,5-Dimethyl-4-hydroxy-3(2 H)-furanone (DMHF), produced by Maillard reaction of sugar/amino acid and found in various foodstuffs, showed mutagenicity to Salmonella typhimurium TA100 strain with and without S9 mix, and induced micronucleated mouse peripheral reticulocytes . DNA strand breaking activity of the compound at pH 7.4 increased with the increasing dose of the compound and with the increasing incubation time . The breaking activity was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the incubation mixture . Addition of Fe(III) ion to the incubation mixture enhanced the breaking activity . Incubation of DMHF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristic to DMPO-OH adduct, indicating generation of hydroxyl radical . It was found that DMHF generated hydroxyl radical with an aid of a trace amount of metal ions, and induced DNA strand breaking . Mutagenicity and induction of micronucleated reticulocytes by DMHF may be caused as a result of DNA modification via hydroxyl radical. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 281 - 5 The Salmonella typhimurium flgM gene, which encodes a negative regulator of flagella synthesis and is involved in virulence, is present and functional in other Salmonella species; Schmitt CK et al.; FlgM inhibits the flagella-specific sigma factor FliA and is involved in the mouse-virulence of Salmonella typhimurium . In recent experiments, we observed that: (i) a flgM gene that could function to negatively regulate flagella synthesis was present in a variety of salmonellae; and (ii) the flgM gene derived from Salmonella species that are not normally virulent in mice could complement the S . typhimurium flgM mutant for virulence . Our results suggest that a functional flgM has been retained in most, and perhaps all, Salmonella species, regardless of the motility or virulence phenotype of the strain. FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 161 - 7 Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection; Lacroix FJ et al.; Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously . We have characterized further the LX1054 mutant strain, the most sensitive of them . The chromosomal DNA segment flanking transposon insertion was cloned and sequenced . The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family . LX1054 exhibited a reduced capacity to colonize the intestinal tract . After passages in mice, the mutant strain lost the sensitive phenotype . In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents . Then, the acquired resistant phenotype seemed stable . The data suggested a role of S . typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization . However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S . typhimurium. J Biol Chem, 1996 Jan 12, 271(2), 1232 - 6 Mutants with defective phosphatase activity show no phosphorylation-dependent oligomerization of CheZ . The phosphatase of bacterial chemotaxis; Blat Y et al.; CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis . The mechanism by which the activity of CheZ is regulated is not known . We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al . (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V . M., and Macnab, R . M . (1992) J . Bacteriol . 174, 793-806), for cloning the mutant cheZ genes, overexpressing and purifying their products . We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheY approximately P), and CheY approximately P dependent oligomerization of the mutant CheZ proteins . While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheY approximately P as well as wild-type CheZ . However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheY approximately P . On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheY approximately P dephosphorylation but is not necessary for the binding to CheY approximately P, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheY approximately P, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli. Cell, 1996 Jan 12, 84(1), 165 - 74 Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence; Garcia Vescovi E et al.; Ions are not traditionally thought to act as first messengers in signal transduction cascades . However, while searching for genes regulated by the PhoP/PhoQ virulence regulatory system of Salmonella typhimurium, we recovered two loci whose expression is controlled by the concentration of Mg2+ . To determine whether Mg2+ is the signal modulating the whole PhoP/PhoQ system, we evaluated the gene expression pattern of six PhoP-activated genes . Growth in physiological concentrations of divalent cations repressed transcription of PhoP-activated genes and rendered wild-type Salmonella phenotypically PhoP- . Mg2+ changed the conformation of the periplasmic domain of PhoQ, identifying this protein as a Mg2+ sensor . A mutation in the sensing domain of PhoQ altered the set point for Mg2+ and rendered Salmonella avirulent. Cell Immunol, 1996 Jan 10, 167(1), 8 - 17 Treatment of Candida albicans mannan-specific downregulatory cell populations with divergent concentrations of monophosphoryl lipid A and intact lipopolysaccharide in vitro abrogates their effect on delayed hypersensitivity; Domer JE et al.; We have shown previously that splenocytes from mice injected with Candida albicans mannan (MAN) suppress MAN-specific delayed hypersensitivity (DH) when transferred to immunized recipients and that treatment of donor mice with monophosphoryl lipid A (MLA) derived from Salmonella typhimurium or Salmonella minnesota shortly before transfer abrogated the downregulatory activity . We now show that treatment of splenocytes in vitro at 4 degrees C with 5 ng/ml MLA or 0.05 ng/ml S . typhimurium lipopolysaccharide (LPS) for 30 min before transfer also abrogated downregulatory activity . Higher or lower doses of MLA, 5 micrograms or 5 pg, appeared to increase the suppressor activity slightly (5 micrograms) or had no effect (5 pg) . LPS induced similar effects but the concentrations of LPS required to show the effects were 100-fold less than those of MLA . The effect of MLA appeared to be on cell(s) in the transfer population involved in MAN-specific DH, in that spleen cells from normal mice treated with MLA prior to transfer had no effect on DH . Finally, the population of MLA-responsive cells mediating downregulation could not be concentrated on MLA-coated plates, suggesting that the MAN-specific downregulatory cell(s) either did not bind to MLA or did not bind to MLA with sufficient avidity to remain attached during the washing procedures . The feasibility of abrogating suppression by treatment of lymphoid cells in vitro will allow a more detailed analysis of the mechanism of abrogation. Biochemistry, 1996 Jan 9, 35(1), 65 - 75 Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium . 2 . Cystine disulfides involved in catalysis of peroxide reduction; Poole LB; The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates . This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor . We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms . The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide . Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis . Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC . NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC . Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence . A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle . A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed. Biochemistry, 1996 Jan 9, 35(1), 56 - 64 Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium . 1 . Purification and enzymatic activities of overexpressed AhpF and AhpC proteins; Poole LB et al.; The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties . Recombinant proteins were isolated in high yield (25-33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E . coli cells . We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein . All assays gave identical results whether overexpressed S . typhimurium proteins from E . coli or proteins isolated directly from S . typhimurium were used . Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system . AhpF possesses multiple pyridine nucleotide-dependent activities {5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhydrogenase, and, in the presence of AhpC, peroxide reductase activities} . Although AhpF can use either NADH or NADPH as the electron donor for these activities, NADH is the preferred reductant (Km,app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADPH when analyzed using DTNB reductase assays) . Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities . In contrast, thiol modification of reduced AhpF does not affect transhydrogenase or oxidase activities . These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides within each protein. Biochemistry, 1996 Jan 9, 35(1), 14 - 21 Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase; Tao W et al.; Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate to orotidine 5'-monophosphate (OMP) and pyrophosphate . We have determined kinetic isotope effects on the reaction of OMP with pyrophosphate and with the pyrophosphate analog phosphonoacetic acid . In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosphorylytic reaction . The transition state resembles a classical oxocarbonium ion . Using the recently reported three-dimensional structures of the OPRTase-OMP (Scapin et al., 1994) and the OPRTase-PRPP complexes (Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase . We propose a detailed chemical mechanism which is consistent with these results. Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 279 - 83 The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches; Baumler AJ et al.; We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice . A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice . When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S . typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen . In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine . The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid . Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles . Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S . typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium . These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches. Chem Biol Interact, 1996 Jan 5, 99(1-3), 55 - 72 Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase; Flowers-Geary L et al.; Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test . Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns . PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz{alpha}anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz{alpha}anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo{alpha}pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival . Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals . ortho-Quinones that reduced cell viability produced O2- . and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only . PAH ortho-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104 . They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens . The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104) . These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms . The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals . The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA. Chem Biol Interact, 1996 Jan 5, 99(1-3), 193 - 204 Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia; Lee IS et al.; Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15-oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines . The structures of these compounds were determined by analysis of their spectroscopic data . The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition . The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol . The adduct with beta-mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself . The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677 . The effects of 13-methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells . Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed . The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific . No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15-oxozoapatlin . These data indicate that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression. Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 356 - 9 A novel expression system for Salmonella typhimurium allowing high production levels, product secretion and efficient recovery; Liljeqvist S et al.; A novel expression system for heterologous production in Salmonella typhimurium, taking advantage of the promoter, signal sequence and two IgG-binding domains (ZZ) from staphylococcal protein A, has been investigated . The production of two different fusion proteins, ZZ-M3 and ZZ-M5, was characterized in terms of production levels, product localization (periplasma or culture medium) and product quality after affinity purification . High expression levels and efficient product secretion were obtained, making the system attractive for vaccine development . The potential use of S . typhimurium as host for heterologous production in biotechnology is discussed. Microbios, 1996, 88(357), 199 - 204 Ultrastructural localization of acid phosphatase in some bacteria, after treatment with Lubrol W1; Cherepova N et al.; The ultracytochemical localization of acid phosphatase from some bacteria (Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa) was dependent on the changes in the lipoprotein content of the membranes as a result of the action of the Lubrol W1. Biochimie, 1996, 78(11-12), 1025 - 34 Structure and regulation of the Salmonella typhimurium rnc-era-recO operon; Anderson PE et al.; The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway) . Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA . As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium . Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth . This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli . Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t . and RNase IIIE.c . are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo. Folia Microbiol (Praha), 1996, 41(1), 48 - 52 Euglena gracilis as a supplementary test organism for detecting biologically active compounds; Macor M et al.; The mutagenic activity of more than 120 antimicrobial agents and protective components was investigated . Only Kathon showed a consistent increase in revertant counts in the Ames test on Salmonella typhimurium . The hereditary bleaching test on Euglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate. J Environ Pathol Toxicol Oncol, 1996, 15(1), 21 - 8 Biochemical and genetic interactions of two commercial pesticides with the monooxygenase system and chlorophyllin; Della Croce C et al.; Two commercial preparations of atrazine and zineb were tested on a diploid D7 strain of the yeast Saccharomyces cerevisiae using cells from logarithmic growth phase (with a high level of cytochrome P-450) and from stationary growth phase . The compounds induced marked increases of both gene conversion and point mutation frequencies in the logarithmic phase cells, while in the stationary phase no genotoxic effect was observed . The results obtained employing TA98 and TA100 strains of Salmonella typhimurium (Ames test) confirmed that neither atrazine nor zineb were mutagenic . The interaction between zineb and chlorophyllin, a known antimutagen in several biological systems, has been evaluated in yeast cells from logarithmic growth phase . The results showed that chlorophyllin seems to have a protective role against the genotoxic effects of zineb . The in vivo effects on cytochrome P-450 content (Cyt . P-450) and on monooxygenase activities were examined in hepatic microsomes of induced animals (rat, pig, and rabbit) 24 hrs after acute treatment . The results obtained with atrazine showed that it caused different effects in the three animal species . Preliminary data with zineb indicated that it can act both as an inducer or as an inhibitor of the monooxygenase system, depending on the dose used. Microbios, 1996, 87(351), 89 - 96 Effect of bisquaternary ammonium salts on Salmonella typhimurium; Majtan V et al.; The effect of 4,7-dioxo-3,8-dioxadekan-1, 1{bis(alkyl-dimethyldiammonium dibromides)} on growth, synthesis of macromolecules, respiration and induction of prophage in Salmonella typhimurium cells was studied . Only those compounds with alkyl chains of C10-C12 (octyl, decyl) in their molecules showed antimicrobial efficacy and inhibition incorporation of {14C}-adenine and {14C}-leucine as precursors of macromolecular biosynthesis . The highest inhibition of endogenous respiration was evoked by compounds with a long alkyl chain in the molecule (tetradecyl, hexadecyl) . Endogenous respiration of the cells was more sensitive to a compound with hexadecyl than the respiration of various intermediates of the Krebs cycle . The sub-MICs of compounds with octyl induced a prophage of a lysogenic S . typhimurium strain. Int Arch Occup Environ Health, 1996, 69(1), 33 - 8 Cyto- and genotoxic effects of coordination complexes of platinum, palladium and rhodium in vitro; Bunger J et al.; The growing industrial use of platinum group elements as catalysts, especially in automobile exhaust detoxification (trimetal catalytic converters), is causing increasing occupational and environmental pollution . The cytotoxic and mutagenic properties of industrially used coordination complexes of platinum, palladium and rhodium were investigated using the neutral red cytotoxicity assay on two established cell lines and the Salmonella typhimurium/microsome test system (Ames test) . Cytotoxic effects of the platinum complexes, measured as ED50, occurred at test concentrations of 0.2 mM . The analogous palladium salts tested were 3 times less toxic with ED50 being 0.6 mM, while the rhodium salts proved to be 30 times less toxic (ED50 = 6 mM) . Levels of toxicity of the different complexes of a particular metal did not differ significantly from each other, which indicates that the metal itself is responsible for the toxic effects . In the Ames test, the spontaneous mutation rates increased by factors of 3 to 20 when the four tester strains were exposed to the platinum complexes . The analogous rhodium compounds proved to be considerably less mutagenic, and palladium demonstrated no mutagenic potential . As all of the four tester strains contain different mutations, the mutagenic potential of platinum and rhodium complexes appears to be based on a variety of mechanisms that damage DNA . From these in vitro experiments, it can be concluded that water-soluble complex salts of rhodium are less toxic and have a smaller mutagenic potential than the analogous platinum complexes . For palladium there is no evidence of any mutagenic property . From this point of view, the development of a catalytic converter containing predominantly palladium may be a possible means of minimizing potential health risks from this exhaust detoxification technique. Acta Microbiol Pol, 1996, 45(2), 169 - 80 The mechanism of bactericidal action of normal human serum against Salmonella rods; Mokracka-Latajka G et al.; Forty Salmonella strains sensitive to the bactericidal action of serum were investigated . All these strains were susceptible to complement activated by the classical pathway though in part (60%) of these strains the presence of lysozyme was necessary for killing . S . typhimurium rods were susceptible to only one mechanism of the action of bactericidal factors . On the contrary, S . enteritidis strains were sensitive to three various mechanisms of bactericidal action of serum . Next eight forms of Salmonella typhimurium LT2 differing with respect to the structure of LPS were studied . Original strain was a smooth, form S, and remaining strains were various rough (R) forms such as Ra, Rb1, Rb2, Rc, Rd1, Rd2, Re . The S form was susceptible only to the complete human serum what means that all bactericidal factors of serum were needed for killing . Ra form was susceptible to two independent bactericidal mechanisms: complement (C) activated by the alternative pathway in the presence of lysozyme and C activated simultaneously by both bactericidal C pathways without the participation of lysozyme (al, ac) . The next form, Rb1, besides the mechanism mentioned above was also susceptible to C activated by the classical pathway in the presence of lysozyme (al, ac, cl) . Other forms (Rb2, Rc, Rd1, Rd2, Re) were susceptible to three mechanisms (al, ac, cl) as well as to C activated by the classical pathway without lysozyme (c) . The mechanism (c) was weakly efficient against forms Rb2 and Rc and somewhat more efficient against Rd1 and Rd2 and the most efficient against Re. Acta Pol Pharm, 1996 Jan-Feb, 53(1), 13 - 7 Characteristics of mutagenesis by bleomycin and adriamycin in salmonella typhimurium: action of superoxide dismutase; Ejchart A et al.; The role of reactive oxygen species in adriamycin and bleomycin-induced mutagenicity was investigated in Salmonella typhimurium TA98 and TA102 respectively . Activity of superoxide dismutase (SOD) was inhibited by preincubation of bacteria with diethyldithiocarbamate (DEDTC) . Results of Ames test may suggest the involvement of active oxygen species in bleomycin induced mutagenesis and an absence of their participation in adriamycin induced mutagenesis. Clin Rheumatol, 1996 Jan, 15(1), 83 - 5 Acute anterior uveitis in juvenile Reiter's syndrome; Fischel JD et al.; We describe the case of an acute anterior uveitis (AAU) and reactive arthritis following a gastrointestinal infection of Salmonella typhimurium in a 9-year-old girl . Reiter's syndrome mainly affects men and is unusual in children especially in young females. Clin Rheumatol, 1996 Jan, 15(1), 72 - 4 Recurrent salmonella sepsis with different species in a systemic lupus erythematosus patient; Green L et al.; Infections are a common cause of morbidity and mortality in systemic lupus erythematosus (SLE) patients . The primary disease process and complications of drug management may contribute to this increased susceptibility . A high incidence of salmonella infections have been reported in SLE patients . We report an unusual case of a SLE patient who developed recurrent salmonella sepsis . The first episode with salmonella typhimurium was followed a few months later by an episode of salmonella enteritides sepsis. Chem Res Toxicol, 1996 Jan-Feb, 9(1), 58 - 66 Gastric carcinogenesis: 2-chloro-4-methylthiobutanoic acid, a novel mutagen in salted, pickled Sanma hiraki fish, or similarly treated methionine; Chen W et al.; The customary salting and pickling of fish in high risk gastric cancer regions were modeled to explore the relevant causative chemicals . The fish Sanma hiraki was treated with sodium chloride and sodium nitrite at pH 3 . Previously, it had been found that an extract of the treated fish was mutagenic in Salmonella typhimurium TA 1535 without S9 and also that it induced glandular stomach cancer upon gavage to rats . We now demonstrate that the mutagenicity was enhanced by preincubation of the raw meat for several days before salt-nitrite treatment . HPLC techniques showed that three mutagens were present in the fish extract . One of the mutagens was found to be stable over the pH range of 1.0-9.0 . This mutagen was purified by silica gel solid phase extraction, followed by a series of reverse phase HPLC steps, and was characterized by low and high resolution MS, NMR, and FT-IR . While N-nitroso compounds were generally believed to be associated with gastric carcinogenesis, it was unexpectedly found that the mutagen has the novel structure 2-chloro-4-methylthiobutanoic acid (CMBA) . Based on the structure, it seemed likely that methionine might be the precursor, and this was, indeed, proven . Both salt and nitrite are essential factors for forming this mutagen . The yield of CMBA was linear for chloride concentrations from 0 to 800 mM NaCl . Of 20 amino acids reacted with nitrite and chloride at pH 3, only methionine generated a mutagen for S . typhimurium TA 1535 . Tryptophan gave a product mutagenic in S . typhimurium TA 100 and TA 98, but not TA 1535, and in the case of tyrosine, the mutagen was active only for TA 100 . These results suggest an important role for salt in gastric carcinogenesis and provide new approaches for exploring the formation of mutagens/carcinogens for specific target organs. Chem Res Toxicol, 1996 Jan-Feb, 9(1), 333 - 40 Activation and inactivation of carcinogenic dihaloalkanes and other compounds by glutathione S-transferase 5-5 in Salmonella typhimurium tester strain NM5004; Shimada T et al.; A newly developed tester Salmonella typhimurium NM5004 strain was constructed by introducing a plasmid containing both rat GSH S-transferase (GST) 5-5 cDNA and the umuC"lacZ operon into the host strain Salmonella typhimurium TA1535 and used to examine whether or not GST modified the genotoxic activities of several dihaloalkanes and other compounds . Twenty-nine chemicals that were suggested to be conjugated by GST were compared with regard to their abilities to induce umu gene expression and cause cytotoxicity responses in both the NM5004 strain and the original tester strain (S . typhimurium TA1535/pSK1002, which is devoid of GST activity toward 1,2-epoxy-3-(4'-nitrophenoxy)propane) . Ten chemicals--1,2-dibromoethane,N-(2,3-epoxypropyl)phthalimide, 1,3-dichloroacetone, CH2I2, 1,2-epoxy-3-phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether, 1-bromo-2-chloroethane, 1-bromo-2,3-dichloropropane, CH2BrCl, and CH2Br2--were found to enhance induction of umu gene expression in the NM5004 strain as compared with the TA1535/pSK1002 strain . 1,2-Epoxy-3-(4'-nitrophenoxy)propane and 2,3-dibromo-1-chloropropane were inactivated by GST 5-5 in the NM5004 tester strain, although these chemicals were cytotoxic in both tester strains . Roles of GST 5-5 were also examined for the inactivation of reactive metabolites of several procarcinogens that were formed through oxidation by liver microsomes of polychlorinated biphenyl-treated rats . The results suggest that reactive metabolites (possibly epoxides) of aflatoxin B1, sterigmatocystin, 1,2-dihydro-1,2-dihydroxy-6-aminochrysene, and (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene could be trapped as inactivated GSH conjugates in the NM5004 strain . High-performance liquid chromatographic analysis suggested that exo-aflatoxin B1-8,9-oxide--GSH conjugate was formed during the oxidation of aflatoxin B1 by rat and human liver microsomes in the presence of GSH and several GST enzymes including purified rat theta class GST Yrs-Yrs and rat liver GST (a mixture of alpha and mu class enzymes) . Thus, the present results support the view that the theta class rat GST 5-5 enzyme participates in the activation and inactivation of potential environmental carcinogenic chemicals . This newly developed NM5004 tester strain is of use in the elucidation of roles of GST 5-5 in transformations. Microbiol Immunol, 1996, 40(9), 681 - 4 Purified protein from Salmonella typhimurium inhibits the interleukin-2 response of murine splenic T-lymphocytes activated with anti-CD3 antibody; Matsui K; Previously, we demonstrated that the immunosuppression induced by a purified preparation of Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) can be observed in terms of suppression of the proliferation of murine spleen cells stimulated with a mitogenic lectin . In the present study, I observed that STI inhibited the interleukin-2 (IL-2) response of purified murine splenic T lymphocytes stimulated with anti-CD3 antibody . The flow cytometric analysis of IL-2 receptor (IL-2R) expression on T cells showed that STI specifically suppressed the expression of IL-2R beta and IL-2R gamma . Furthermore, when the IL-2-dependent T-cell line CTLL-2 was incubated with STI, the growth of CTLL-2 cells was significantly inhibited . These results suggest that the target cells for STI are T cells themselves, and that the suppression of T-cell proliferation induced by STI might involve a defect in the IL-2 receptor (IL-2R) function of T cells. Teratog Carcinog Mutagen, 1996, 16(2), 125 - 38 An investigation of some Turkish herbal medicines in Salmonella typhimurium and in the COMET assay in human lymphocytes; Basaran AA et al.; Medicinal plants play a major role in the life of Turkish people and of late medicinal plant usage has increased in many countries . Green plants in general contain mutagenic and carcinogenic substances, but there is little information about the biological activities of herbal medicine . In the present study, therefore, various Turkish medicinal herbs were investigated for their genotoxic potential in the Salmonella typhimurium microsomal activation assay and the alkaline single cell gel electrophoresis (COMET) assay . Extracts from these medicinal herbs and some fractions of these extracts were examined . The species investigated were Arctium minus, Ecballium elatterium, Momordica charantia, Plantago major, Urtica dioica, Viscum album, Salvia triloba, Euphorbia rigida, Stachys lavandulifolia, Acteoside, Abies nordmannia . They are used for various immune disorders and are applied either topically or taken orally as a herbal tea . Of the 19 samples of the extracts and fractions investigated, none produced a positive response in strains TA98 and TA100 with or without metabolic activation, but all produced an increase above negative control values in the COMET assay . Some extracts were investigated further and produced dose-related increases . In the case of Urtica and Euphorbia species, where two fractions from these plants were examined, one fraction produced a greater response than the other . It is suggested that the lesser response of the fractions might be due to less DNA strand-breaking agents in the fractions or they may have antigenotoxic properties . The breaks that are detected in the COMET assay could be alkali-labile AP-sites and intermediates in base- or nucleotide-excision repair and are difficult to interpret in terms of hazard for man . Further studies with additional genotoxicity assays would be required to make such a prediction. Environ Mol Mutagen, 1996, 28(2), 127 - 32 Mutagenicity of cosmetic products containing Kathon; Connor TH et al.; A variety of shampoos, conditioners, skin-care lotions, and other cosmetic products contain the biocide Kathon CG, which is a mixture of two heterocyclic isothiazolinones: methylisothiazolinone and methylchloroisothiazolinone . This mixture and the related biocide, Kathon 886, have been shown to be potent sensitizers and bacterial mutagens . Five cosmetic products that list the components of Kathon on their labels and two that do not were screened for mutagenicity with Salmonella typhimurium TA100 without S-9 . Five of these products and Kathon 886 were further evaluated in TA100 without and with S-9 . Kathon 886, a cosmetic product that contained Kathon, and thin layer chromatography-separated components of Kathon 886 were identified by GC/MS analysis . Three of the five products that listed Kathon were direct acting mutagens with TA100 . The remaining two products were considerably more toxic than the other products and could not be evaluated for mutagenicity . The addition of S-9 reduced toxicity but did not eliminate mutagenicity . The mutagenic evaluation of Kathon 886 resulted in a dose response similar to that seen with some cosmetic products but at a 1,000-fold lower concentration, and activity was also reduced by the addition of S-9 mix . S-9 reduced activity both with and without cofactors present . Thin layer chromatography separation of the components and subsequent identification by GC/MS indicated that methylisothiazolinone was nonmutagenic while methylchloroisothiazolinone was mutagenic . Additionally, a dichlorinated compound was identified which was also mutagenic . In light of these findings and the reported skin sensitization by Kathon CG in various cosmetics, we recommend that additional testing be done to assure the safety of products containing Kathon CG. Clin Rheumatol, 1996 Jan, 15 Suppl 1, 79 - 85 Immunization of HLA-B27 transgenic and non transgenic mice with Salmonella typhimurium results predominantly in the generation of proliferative T cell responses; Ringrose JH et al.; Reactive arthritis (ReA) due to Gram-negative intestinal bacteria or Chlamydia, is associated by an unknown mechanism with HLA-B27 . Like other MHC class I molecules, HLA-B27 presents antigenic peptides derived from intracellular proteins to CD8+ cytotoxic T cells (CTL) . In humans however, CTL specific for ReA associated bacteria have been reported in a limited number of studies . This may be caused by an inefficient in vivo induction of CTL against such micro-organisms . In the present study we addressed the question whether and to what extend mice transgenic for HLA-B27 are able to generate CTL against Salmonella typhimurium after immunization . To this end both HLA-B27 transgenic and non transgenic mice were immunized i.p., i.v . or orally, receiving a secondary challenge four weeks later . One day after infection with Salmonella, bacteria could be cultured from spleen and liver . There was no significant difference in the number of bacteria cultured from these organs between both groups of mice . Spleen cells from all immunized mice proliferated specifically in the presence of heat killed Salmonella but not in the presence of heat killed Yersinia . No proliferation of spleen cells from naive mice was observed in the presence of heat killed Salmonella, excluding the possibility that Salmonella antigens were mitogenic . Only in one out of 6 mice immunized i.v . with Salmonella Salmonella specific CTL could be generated . In order to rule out the possibility that in HLA-B27 transgenic mice the HLA-B27 molecule is not used as a restriction element by murine T cells, CTL were raised against the male minor histocompatibility (mH) antigen H-Y . Both murine class I as well as HLA-B27 restricted CTL could be generated . In conclusion this study demonstrates that MHC class I restricted CTL specific for the Gram-negative bacterium Salmonella typhimurium are difficult to generate in contrast to proliferative responses which can be easily demonstrated . This may comparable in humans where in the majority of studies bacteria specific T cells isolated from ReA patients appear to be CD4+ and class II restricted. Mol Microbiol, 1996 Jan, 19(1), 139 - 44 The specificity of fumarate as a switching factor of the bacterial flagellar motor; Barak R et al.; Fumarate restores to flagella of cytoplasm-free, Che Y-containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another . To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues . Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate . These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate . Aspartate and lactate did not promote switching . Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate) . All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching . Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour. Mol Microbiol, 1996 Jan, 19(1), 1 - 5 Flagellar assembly in Salmonella typhimurium; Aizawa SI; The bacterial flagellum is a motility apparatus in which a long helical filament--the propeller--is driven by a rotary motor embedded in the cell surface . Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure . Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum . The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure . Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins . Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S . typhimurium, is reviewed. Vaccine, 1996 Jan, 14(1), 19 - 24 Evaluation of a phoP/phoQ-deleted, aroA-deleted live oral Salmonella typhi vaccine strain in human volunteers; Hohmann EL et al.; Improved live oral typhoid fever vaccines may be engineered by deletion of Salmonella-specific virulence genes in Salmonella typhi . Ty445, an aroA-deleted S . typhi Ty2 strain also deleted for the phoP/phoQ Salmonella typhimurium virulence regulatory locus, was tested in human volunteers . Volunteers received escalating single doses of the vaccine; subsequently 14 individuals received two doses of 10(10) c.f.u . without significant side-effects . Control vaccinees received four doses of the live oral typhoid vaccine Ty21a . Of controls, 5/8 seroconverted as measured by increases in serum IgG directed against S . typhi O antigen or whole bacterial antigens in ELISAs . Only 2/14 volunteers receiving the experimental vaccine Ty445 seroconverted . Although a delta aroA delta phoP/phoQ S . typhi strain is overattenuated for use as a typhoid fever vaccine, our data demonstrate that the deletion of the phoP/phoQ locus in S . typhi significantly attenuates this human pathogen. Arzneimittelforschung, 1996 Jan, 46(1), 64 - 7 Effects of amphiphilic compounds on metabolic and biological activities of Salmonella typhimurium; Majtanova L et al.; The effect of two quaternary ammonium salts, (1-methyldodecyl)trimethylammonium bromide (ATDBr) and tetramethylammonium bromide (TMABr), as well as that of two amine oxides, (1-methyldodecyl)dimethylamine oxide (ATDNO) and trimethylamine oxid (TMANO), on both metabolic and biological activities of a S . typhimurium strain isolated from nosocomial infection were studied . The compounds with long alkyl chain in the molecule (ATDBr, ATDNO) affected the growth, synthesis of biomacromolecules (DNA, RNA, proteins) and respiration . The compounds lacking the alkyl chain (TMABr, TMANO) were ineffective with exception of the endogenous respiration that they inhibited in a less degree in the highest concentrations tested . The highest inhibition of respiration was evoked by ATDBr and ATDNO in the presence of pyruvate as an exogenous carbon substrate . The sub-MICs of quaternary ammonium salts induced a prophage of the lysogenic S . typhimurium strain, amine oxides were ineffective . The tested compounds decreased at one-fourth of the MICs the permeability reaction . The results showed that both physiologic and metabolic processes were inhibited in dependence on the length of the alkyl chain, however, amine oxides were less effective in biological activities. FEMS Immunol Med Microbiol, 1996 Jan, 13(1), 19 - 28 Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2.- excretion versus H2O2 diffusion; Chateau MT et al.; The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques . Differentiated U937 cells essentially produced extracellular superoxide anion (O2.-), whatever the stimulus used . Monocytes, however, responded to Salmonella typhimurium, phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively . Furthermore, H2O2 but not O2.- was detected in the extracellular oxidative response of monocytes . Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O2.-, providing sufficient concentrations of extracellular horseradish peroxidase were present . However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen by differentiated U937 cells . Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O2.- in the extracellular medium while, within monocytes, O2.- is rapidly dismutated in H2O2 which can eventually diffuse outside the cell . Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells. Rev Argent Microbiol, 1996 Jan-Mar, 28(1), 1 - 8 {Genotoxicity resulting from a complex mixture of environmental origin using 2 bacterial systems}; Lopez L et al.; The genotoxicity of environmental complex mixtures was tested with Ames mutagenicity assay and with Bacillus subtilis rec assay for estimation of primary DNA damage . Soil samples obtained from an area contaminated with petrochemical wastes were sequentially extracted with ethyl ether and methanol . After evaporation to dryness the extracts were resuspended in dimethylsulfoxide for their use in biological tests . No mutagenic response was detected with the Ames test . Ether extract from parcel 1 showed an unusually high toxicity for Salmonella typhimurium TA100 and induced DNA damage in Bacillus subtilis with a clear relationship dose-genotoxic response . The analysis of the results obtained in both assays and the nature of the samples indicate that the genotoxicity detected in the complex mixture is compatible with the one caused by DNA intercalating compounds. Genetics, 1996 Jan, 142(1), 11 - 24 Evolution of coenzyme B12 synthesis among enteric bacteria: evidence for loss and reacquisition of a multigene complex; Lawrence JG et al.; We have examined the distribution of cobalamin (coenzyme B12) synthetic ability and cobalamin-dependent metabolism among enteric bacteria . Most species of enteric bacteria tested synthesize cobalamin under both aerobic and anaerobic conditions and ferment glycerol in a cobalamin-dependent fashion . The group of species including Escherichia coli and Salmonella typhimurium cannot ferment glycerol . E . coli strains cannot synthesize cobalamin de novo, and Salmonella spp . synthesize cobalamin only under anaerobic conditions . In addition, the cobalamin synthetic genes of Salmonella spp . (cob) show a regulatory pattern different from that of other enteric taxa tested . We propose that the cobalamin synthetic genes, as well as genes providing cobalamin-dependent diol dehydratase, were lost by a common ancestor of E . coli and Salmonella spp . and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source . Consistent with this hypothesis, the S . typhimurium cob genes do not hybridize with the genomes of other enteric species . The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes . This process may be an important aspect of bacterial population genetics and evolution. Rocz Panstw Zakl Hig, 1996, 47(1), 77 - 85 {Evaluation of mutagenic activity in water disinfected with chlorine}; Bilyk A et al.; City of Wroclaw is supplied with water from Olawa . The main contaminations of water are high concentration of organic compounds and bacteria count . Raw and drinking water show some mutagenic and carcinogenic properties in Ames tests . To improve the quality of drinking water now technology bored on infiltrated water composed of, coagulation, filtration and disinfection was tested . The goal of investigation and was to examine mutagenic and carcinogenic properties of raw and treated water . Potential carcinogenic activity of volatile disinfection-by-products was estimated by direct analysis of THMs, while for nonvolatile halogenated organic substances Ames test was used . Carcinogenic risk based on THMs concentration could be estimate as 10(-5) for chlorine and 10(-6) for chlorine dioxide . Ozonation and post chlorination did not lowered the risk . Positives results of Ames test obtained for raw water no 2 with Salmonella typhimurium TA100, and for chlorinated treated water with Salmonella typhimurium TA98 . The treatment of water with chlorine transforms same compounds into carcinogenic chlorinated derivatives and does not eliminate its harmful properties . Our results suggest that not all methods of treatment remove harmful to the health components from the water . Consequently in the case of the presence of such compounds in surface water it is necessary to employ appropriate methods and procedures the used Ames test allows rapid determination of the presence of carcinogenic compound in water . In Poland determination of the presence of potential carcinogens in water destined for the supply of urban areas is not obligatory and standard analyses of chemical composition do not give such information . It seems that the mentioned test could be considered for the control of the quality of raw and treated water as an indispensable measure contributing to reducing the health hazard for the population. Jpn J Ophthalmol, 1996, 40(1), 12 - 7 Early expression of intercellular adhesion molecule-1 in the corneal endothelium stimulated by endotoxin: an immuno-scanning electron microscopical study; Yamaguchi K et al.; Three-dimensional localization of the intercellular adhesion molecule-1 (ICAM-1) in the corneal endothelium stimulated by Salmonella typhimurium endotoxin was investigated using immuno-scanning electron microscopy (SEM) . Samonella typhimurium endotoxin, 100 micrograms, was injected in Lewis rats weighing 200 grams . The animals, including the controls, were sacrificed and both eyes enucleated at 0, 3, 12 and 24 hours after injection (n = 3 each time) . After resection, the corneas were immersed in hypothermic University of Wisconsin solution with monoclonal mouse-anti-rat ICAM-1 IgG and then goat-anti-mouse IgG coupled to 15 nm gold particles . Then the corneas were prepared