J Bacteriol, 1991 Jan, 173(2), 710 - 9 Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein; Zuberi AR et al.; The nucleotide sequence of the Bacillus subtilis fliM gene has been determined . This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium . Expression of this gene in Che+ cells of E . coli and B . subtilis interferes with normal chemotaxis . The nature of the chemotaxis defect is dependent upon the host used . In B . subtilis, overproduction of FliM generates mostly nonmotile cells . Those cells that are motile switch less frequently . Expression of B . subtilis FliM in E . coli also generates nonmotile cells . However, those cells that are motile have a tumble bias . The B . subtilis fliM gene cannot complement an E . coli fliM mutant . A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B . subtilis chromosome . The mutant has a Fla- phenotype . This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B . subtilis . Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed . These loci are regulated by the SigD form of RNA polymerase . We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment . The expression of these proteins is also dependent upon SigD . It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes. Infect Immun, 1991 Jan, 59(1), 398 - 406 Prostaglandin E release from human monocytes treated with lipopolysaccharides isolated from Bacteroides intermedius and Salmonella typhimurium: potentiation by gamma interferon; Nichols FC et al.; The purpose of this investigation was to examine gamma interferon potentiation of lipopolysaccharide (LPS) responses in human monocytes by using phenol-water-extracted (unfractionated) and highly purified LPS preparations isolated from Bacteroides intermedius and Salmonella typhimurium . Phenol-water-extracted LPS preparations from these bacteria were further purified by chromatography over Sepharose-CL-4B . LPS enrichment in pooled column fractions was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation of hydroxy-fatty acid and 2-keto-3-deoxyoctulosonic acid content, protein contamination, and anthrone-reactive material . Monocyte stimulation by LPS, measured as prostaglandin E (PGE) release, was assessed with and without gamma interferon treatment . Cells were either treated simultaneously with gamma interferon and LPS or pretreated with gamma interferon prior to LPS stimulation . PGE release from counterflow-isolated monocytes was quantitated during the 0- to 24-h and 24- to 48-h culture intervals . Contrary to previous results from this laboratory, phenol-water-extracted LPS preparations from B . intermedius and S . typhimurium were similar in their capacities to stimulate PGE release from monocytes . Molecular sieve chromatography was found to remove substantial amounts of high-molecular-weight polysaccharide contaminants only from the B . intermedius LPS but did not significantly alter the potency of either B . intermedius or S . typhimurium LPS . Gamma interferon cotreatment did not potentiate the release of PGE with any of the LPS preparations tested . However, 24-h pretreatment of monocytes with gamma interferon followed by a 24-h exposure to LPS resulted in significant potentiation of PGE release over LPS alone . In addition, B . intermedium preparations were approximately threefold more potent than similarly prepared LPS isolates from S . typhimurium following gamma interferon pretreatment . These results indicate that gamma interferon can selectively potentiate the effects of B . intermedius LPS in human monocyte isolates. Mikrobiyol Bul, 1991 Jan, 25(1), 94 - 107 {The mutagenic effects of sodium nitrite and monosodium glutamate used as food additives demonstrated by the Salmonella microsome test system}; Akin A et al.; In this study, the mutagenicity of sodium nitrite (an antimicrobial agent) and monosodium glutamate (a flavoring agent), which are widely used as food additives, have been evaluated . They were tested for mutagenicity in the Salmonella/microsome test system by employing plate-incorporation test in the absence and presence of rat-liver fraction, S 9 . No mutagenic activity was seen for monosodium glutamate in the Salmonella/microsome test system by using the tester strains Salmonella typhimurium TA98, TA100, TA1535 and TA1538 . Sodium nitrite was found mutagenic on S . typhimurium TA1535, whereas it was found weakly mutagenic on S . typhimurium TA100, in the presence and absence of rat-liver fraction, S 9. Infection, 1991, 19 Suppl 4, S224 - 8 Liposomes and nanoparticles as vehicles for antibiotics; Kreuter J; Colloidal drug carriers such as liposomes and nanoparticles are easily taken up by phagocytic cells and accumulate in the organs of the reticuloendothelial system . Therefore, they hold promise as carriers for the treatment of intracellular infections with antibiotics that would normally not find easy access to intracellular sites . Consequently, in in vitro and in vivo experiments the therapeutic efficacy of substances such as amphotericin B, dihydrostreptomycin, amikacin, ampicillin, stibogluconate against a number of microorganisms including Leishmania donovani, Candida albicans, Staphylococcus aureus, Mycobacterium avium, Listeria monocytogenes, and Salmonella typhimurium was increased significantly by binding to liposomes and nanoparticles. Microbiol Immunol, 1991, 35(3), 187 - 98 The 106-kilobase plasmid of Salmonella braenderup and the 100-kilobase plasmid of Salmonella typhimurium are not necessary for the pathogenicity in experimental models; Horiuchi S et al.; Among 1.041 clinical isolates (77 serovars) of Salmonella which had been derived from cases with acute enterocolitis, 601 (58%) contained one or more plasmids . Large serovar-specific plasmids were seen in 95 of 307 isolates (31%) of Salmonella typhimurium, in 34 of 34 isolates (100%) of Salmonella enteritidis and in 36 of 38 isolates (94.7%) of Salmonella braenderup: the sizes of which were 100, 60 and 106 kilobases (kb), respectively . In order to determine the role of these plasmids in pathogenicity for enterocolitis, the plasmids were eliminated from some strains of S . braenderup and S . typhimurium and the pathogenicity of the plasmid-less strains was compared with that of the parent strains by invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, lesion of mucosal tissue and the Sereny test . The virulence of all the plasmid-less strains was as strong as that of the plasmid-bearing strains in these pathogenicity assay systems . We therefore concluded that the 106-kb plasmid of S . braenderup and the 100-kb plasmid of S . typhimurium are not necessary for their pathogenicity in the experimental models: invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, and Sereny test. Environ Mol Mutagen, 1991, 18(1), 41 - 50 Genotoxicity of nitrated polycyclic aromatic hydrocarbons and related structures on Escherichia coli PQ37 (SOS chromotest); Mersch-Sundermann V et al.; To determine the genotoxicity of nitrated polycyclic aromatic hydrocarbons and related molecules (nPAH) we examined 24 compounds representative of nitroanthracenes, nitrofluorenes, nitronaphthalenes, nitropyrenes, and nitroquinolines for genotoxicity in Escherichia coli PQ37 (SOS-chromotest) . To enhance the sensitivity of the tester strain and optimize metabolic activation we used a modified test protocol and S9-mix composition . All chemicals with the exception of 9-nitroanthracene, 1- and 2-nitronaphthalene, 2-methyl-1-nitronaphthalene, and 5-, 6-, and 8-nitroquinoline induced the SOS system of E . coli PQ37 . As expected from previously referred mutagenicity studies, the highest SOS inducing potencies (SOSIP) were exhibited by the dinitropyrenes (SOSIP = 151-416), 4-nitroquinoline-N-oxide (SOSIP = 62), and 3-nitrofluoranthese (SOSIP = 16) . Except for some nitronaphthalenes, the nPAHs showed their highest genotoxicity in the absence of an exogeneous metabolic activation system . The results were compared to those reported for the bacterial mutagenicity of these substances in Salmonella typhimurium TA98. Environ Mol Mutagen, 1991, 18(1), 35 - 40 Inhibitory effect of lichen constituents on mutagenicity induced by heterocyclic amines; Osawa T et al.; Physodic acid, one of the main constituents of Hypogymnia enteromorpha, inhibited the mutagenicity of indirect mutagens, including benzo{a}pyrene and heterocyclic amines in Salmonella typhimurium TA 98 . In contrast, it was not effective against direct mutagens such as 6-nitropiperonal and adriamycin . Its antimutagenicity was not associated with free-radical scavenging or antioxidative activities . Physodic acid seemed to inhibit the formation of reactive metabolites, such as N-hydroxy-Trp-P-2, by blocking the hepatic microsomal oxidation systems . Another component of H . enteromorpha, physodalic acid, also inhibited mutagenicity of a heterocyclic amine, Trp-P-2, in S . typhimurium TA 98, even though it was reportedly mutagenic in S . typhimurium TA 100. Microb Pathog, 1991 Jan, 10(1), 47 - 59 Expression cloning of Yersinia enterocolitica O:3 rfb gene cluster in Escherichia coli K12; al-Hendy A et al.; The genes of Yersinia enterocolitica serotype O:3 (YeO3) that determine the synthesis of the O-side-chain of the lipopolysaccharide, the rfb region, were cloned into plasmid pBR322 . The O-side-chain of YeO3 was expressed by the clone both in Escherichia coli and Salmonella typhimurium indicating that the entire rfb region was included in the clone . It was shown by restriction mapping, deletion analysis and transposition mutagenesis that about 10.4 kilobase pairs of DNA was essential for the synthesis and expression of the O-side-chain . The correct assembly of the O-side-chain on the cell surface of the clone was confirmed by immunofluorescence microscopy and slide agglutination . Immunoblotting using monoclonal antibody specific for the O-side-chain of YeO3 revealed that the O-side-chain material synthesized by the clone in E . coli was similar to that of YeO3 . The clone did not show the in vitro temperature variation in O-side-chain expression characteristic of YeO3 . Instead analogous O-side-chain was produced both at 25 degrees C and at 37 degrees C . Using transposon Tn2507, which carries a promotorless chloramphenicol acetyltransferase (CAT) gene, transcriptional fusions with the target DNA were generated . When testing the ability of mutated clones to produce CAT, transcription was shown to occur in a uniform direction throughout the whole rfb region . In colony hybridizations, using the cloned insert as a probe, homologous DNA was detected only in pathogenic Y . enterocolitica serotypes. IARC Sci Publ, 1991, (105), 584 - 7 Stability of mutagenic nitrosated products of indole compounds occurring in vegetables; Tiedink HG et al.; Levels of indolylglucosinolates in Brassica vegetables correlated significantly with the amounts of N-nitroso compounds formed in these vegetables after nitrite treatment . Nitrosation of indole-3-carbinol, indole-3-acetonitrile and indole, hydrolysis products of an indolylglucosinolate, resulted in formation of nitrosated products, which were directly mutagenic to Salmonella typhimurium TA100 . The nitrosated products were unstable at pH 2 but stable at pH 8 . Experiments to elucidate the mechanisms behind these differences in stability showed an equilibrium between the nitrosated indole compound and the free compound plus nitrite. IARC Sci Publ, 1991, (105), 558 - 63 Inhibition by fatty acids of direct mutagenicity of N-nitroso compounds; Takeda K et al.; Fatty acids inhibited the direct mutagenicity of N-nitroso compounds in Salmonella typhimurium TA1535, Escherichia coli WP2 and WPhcr-, and E . coli H/r30R (wild) and Hs30R (uvrA) . This inhibitory activity was dependent on the concentration of fatty acids, and fatty acids with longer alkyl chain were more potent . Of the N-nitroso compounds tested, alpha-hydroxy nitrosamines underwent the strongest inhibitory effect . The rate of decomposition was not changed by addition of fatty acids . The partitioning property of the mutagens was altered but not to such a degree as to explain the amount of inhibition . No significant difference in alkylating activity of the N-nitroso compounds was observed in phosphate and acetate buffers . A stronger inhibition of mutagenicity by a butylating mutagen was detected in E . coli WP2 than in WP2hcr- and in E . coli H/r30R than in Hs30R, suggesting that excision repair was a possible mechanism of inhibition . The mutagenicity and cytotoxicity of alpha-hydroxy nitrosamines in Chinese hamster V79 cells were also inhibited by acetate. IARC Sci Publ, 1991, (105), 535 - 7 Modulation of genotoxic activity of tobacco smoke; Balansky RM et al.; Tobacco smoke (TS) caused a three- to nine-fold increase in the frequency of his+ revertants in Salmonella typhimurium TA98 but not in TA97a, TA100 or TA102 . Activation by a post-mitochondrial fraction obtained from the liver of rats pretreated with Aroclor-1254 or methylcholanthrene was required; fractions from phenobarbital-pretreated or untreated rats had no effect . Vitamins A and E, but not ascorbic acid, inhibited the TS-induced mutagenesis by up to 63%, whereas glutathione and cysteine increased it slightly . Na2SeO3, but neither CoCl2 nor caffeine, inhibited the mutagenic effect of TS by 46-56% . In Chinese hamster ovary cells, both Na2SeO3 and caffeine strongly potentiated the number of chromosomal aberrations induced by TS, while theophilline slightly reduced its clastogenic effect . Treatment of mice with TS for 60 min/day increased the frequency of micronuclei in polychromatic erythrocytes in bone marrow and in fetal liver and the number of NCE micronuclei in peripheral blood by four to five fold . Simultaneous treatment of mice with TS and Na2SeO3 reduced the clastogenic effect of TS . Ascorbic acid had no effect on clastogenicity but reduced toxicity as measured by body weight loss . Both Na2SeO3 and ascorbic acid suppressed the induction of TS-induced hyperplastic and metaplastic changes in bronchial mucosa but had no effect on the number of urethane-induced lung adenomas . Vitamins A and E and ascorbic acid may have a protective effect against the toxic and genotoxic activities of TS. IARC Sci Publ, 1991, (105), 404 - 6 Biological and chemical properties of alkanediazotates as active species of N-nitroso compounds; Ukawa S et al.; The mutagenicity and chemical reactivity of (E)- and (Z)-potassium alkanediazotates, as precursors of corresponding alkanediazohydroxides, were investigated . In three microbial strains, Salmonella typhimurium TA1535 and Escherichia coli WP2 and WP2hcr-, the effect of changing the alkyl group on mutagenic potency was similar for (E)- and (Z)-diazotates, N-alkyl-N-nitrosoureas and alpha-hydroxynitrosamines . The capacity to alkylate nicotinamide, measured in an aqueous phosphate buffer, decreased with increasing alkyl chain length . Specific mutagenicity in S . typhimurium TA1535 was linearly related to alkylating activity . These results confirm that alkanediazohydroxides are the active alkylating species of N-nitroso compounds, and that their mutagenicity is determined by their alkylating activity. IARC Sci Publ, 1991, (105), 339 - 42 Mutagenicity, DNA damage and DNA adduct formation by N-nitroso-2-hydroxyalkylamine and corresponding aldehydes; Scherer G et al.; The potent carcinogen N-nitrosodiethanolamine (NDELA) becomes mutagenic to Salmonella typhimurium TA98 and TA100 when activated by alcohol dehydrogenase from yeast or horse liver . Metabolic pathways different from alpha-oxidation might therefore be important for the activation of N-nitroso-2-hydroxyalkylamines such as NDELA . In an in-vitro test system (Namalva cells), neither NDELA nor N-nitrosoethyl-2-hydroxyethylamine was genotoxic, whereas the corresponding metabolites from alcohol dehydrogenase-mediated oxidation, N-nitroso-2-hydroxymorpholine and N-nitrosoethylethanalamine, induced single-strand breaks even at low doses . An immuno-slot-blot assay was used to study the formation of O6-2-hydroxyethyldeoxyguanosine in rat liver after oral administration of different N-nitroso-2-hydroxyalkylamines . When given at equimolar doses (0.375 mmol/kg), DNA hydroxyethylation was considerably lower (6.7 mumol/mol deoxyguanosine) with NDELA than with N-nitrosoethyl-2-hydroxyethylamine (48.7 mumol/mol deoxyguanosine) or N-nitrosomethyl-2-hydroxyethylamine (72.1 mumol/mol deoxyguanosine) . N-Nitroso-2-hydroxymorpholine did not form detectable levels of O6-2-hydroxyethyldeoxyguanosine. IARC Sci Publ, 1991, (105), 244 - 52 Nitrosation of tertiary aromatic amines related to sunscreen ingredients; Loeppky RN et al.; Possible routes to the formation of the sunscreen contaminant, 2-ethylhexyl 4-N-methyl-N-nitrosoaminobenzoate, have been investigated in a study of the nitrosation chemistry of 2-ethylhexyl 4-N,N-dimethylaminobenzoate (Padimate-O) and related tertiary and secondary amines . Padimate-O and the corresponding ethyl ester nitrosate rapidly at 25 degrees C in either N2O3:ether or HNO2:HOAc to produce a mixture of alkyl 4-N-methyl-N-nitrosoaminobenzoate and alkyl 4-N,N-dimethylamino-3-nitrobenzoate, the former of which is the major product . The nitrosative dealkylation of these amines at this low temperature is unusual . Asymmetrical amines exhibit a preference for nitrosative demethylation (methyl versus ethyl or benzyl), but the cleavage ratios in N2O3:ether are time-dependent, suggesting competing mechanisms with different reactant kinetic orders . A radical cation route would explain the unusual reactivity, which may compete with the established nitrosative dealkylation mechanism . 2-Ethylhexyl 4-N-methyl-N-nitrosoaminobenzoate was mutagenic in two strains of Salmonella typhimurium in the Ames assay. J Bacteriol, 1991 Jan, 173(1), 86 - 93 A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein; Pulkkinen WS et al.; The phoP-phoQ-regulated pagC locus is essential for full virulence and survival within macrophages of Salmonella typhimurium . The protein product, DNA sequence, and transcript of pagC were determined . The pagC locus encodes a single 188-amino-acid membrane protein that is similar to the ail-encoded eucaryotic cell invasion protein of Yersinia enterocolitica and the lom-encoded protein of bacteriophage lambda . The similarity of PagC and Ail to Lom leads us to hypothesize that Lom is a virulence protein and that bacteriophage gene transfer and lysogeny could have led to the development of proteins essential to survival within macrophages and eucaryotic cell invasion. Infect Immun, 1991 Jan, 59(1), 449 - 52 Role of ompR-dependent genes in Salmonella typhimurium virulence: mutants deficient in both ompC and ompF are attenuated in vivo; Chatfield SN et al.; A Salmonella typhimurium strain harboring stable mutations in both ompC and ompF was constructed from the mouse-virulent strain S . typhimurium SL1344 . When administered orally to BALB/c mice the strain was attenuated, with the 50% lethal dose (LD50) reduced by approximately 1,000-fold . However, the intravenous LD50 was reduced only by approximately 10-fold . The ompC ompF mutant persisted in murine tissues for several weeks following oral challenge, and mice immunized with this mutant were well protected against challenge with virulent SL1344 . A strain harboring a stable mutation in tppB behaved in a manner similar to that of strain SL1344 in vivo, while a strain harboring mutations in ompC, ompF, and tppB behaved as an ompC ompF mutant in vivo, indicating that the tppB operon is not required for virulence in S . typhimurium. Folia Microbiol (Praha), 1991, 36(3), 240 - 5 Elimination of plasmids pKM 101 and F'lac from Salmonella typhimurium and Escherichia coli by bisammonium salt . The effect of outer membrane pattern; Sykora P et al.; Plasmid-curing activity of N,N'-bis(decyldimethyl)-1,6-hexanediammonium dibromide, BDHD, was tested on six different plasmids in E . coli and plasmid pKM 101 in S . typhimurium . BDHD eliminated the F'lac plasmid from E . coli cells only with a low efficiency . Plasmid pKM 101 was eliminated from S . typhimurium cells significantly and this effect was dependent on an outer membrane pattern . A deep-rough mutant of S . typhimurium is completely resistant to curing activity of BDHD, while part-rough and smooth cells are susceptible to it . In contrast to pKM 101, a cryptic plasmid being present in S . typhimurium cells was not eliminated by BDHD . The curing activity of sodium dodecyl sulfate, acridine orange, crystal violet, and promethazine was also affected by the outer membrane pattern of S . typhimurium cells. Eur J Cancer, 1991, 27(4), 478 - 82 Increased intracellular killing of bacteria in vitro by monocytes of patients with metastatic melanoma before and during treatment with interferon-gamma and interferon-alpha; Osanto S et al.; The effect of recombinant human interferon-gamma (rHuIFN-gamma) and interferon-alpha (rHuIFN-alpha) as in vivo stimuli for the activation of human monocytes was investigated on the basis of the bactericidal activity of peripheral blood monocytes in 11 patients with metastatic melanoma before and during treatment with interferons . Patients received increasing doses of rHuIFN-gamma and a fixed dose of rHuIFN-alpha, both administered subcutaneously three times a week . The rates of intracellular killing of Listeria monocytogenes and Salmonella typhimurium after in vitro phagocytosis by monocytes collected from melanoma patients before interferon treatment were increased (P less than 0.01) by a factor of 1.7 and 1.4, respectively, relative to the rate constants in blood monocytes of healthy donors . During treatment with the interferons, the rates of intracellular killing of the bacteria by patients' monocytes did not further increase . The findings underscore the immunogenicity of malignant melanoma and put into question the macrophage activating activity of IFN-gamma with respect to the bactericidal activity of monocytes. Asia Pac J Public Health, 1991, 5(3), 256 - 61 Observations on the ecology of Salmonella waycross and Salmonella typhimurium on Guam; Haddock RL et al.; A period of high incidence of human Salmonella infections on the island of Guam saw the emergence of S . waycross as the most commonly isolated serotype as well as a concurrent decreasing proportion of isolates due to S . typhimurium . Predation of local rodents by an introduced snake is believed to account for the decreased prevalence of S . typhimurium infections, but reasons for the increased prevalence of S . waycross infections are unknown. IARC Sci Publ, 1991, (115), 255 - 60 Mutagenicity and effects of ochratoxin A on the frequency of sister chromatid exchange after metabolic activation; Hennig A et al.; Primary cultures of hepatocytes derived from untreated rats were incubated in the presence of ochratoxin A for 24 h . Five different strains of histidine auxotroph Salmonella typhimurium were exposed to conditioned cell culture medium before being tested for mutagenicity . A clear hepatocyte-mediated mutagenic response was observed in TA1535, TA1538 and TA100 . In addition, sister chromatid exchange frequency was increased in human peripheral lymphocytes that had been incubated in the presence of conditioned medium derived from ochratoxin A-exposed hepatocytes. J Reprod Fertil Suppl, 1991, 44, 509 - 16 Experimental models of endotoxaemia related to abortion in the mare; Kindahl H et al.; Three different routes of administering Salmonella typhimurium endotoxin to mimic naturally occurring endotoxaemia were tried in the mare . Bolus injection, repeated bolus injections and continuous low-dose infusion were compared with prostaglandin F2 alpha release, leucocyte count and clinical response . A biphasic prostaglandin release and a pronounced leucopenia of almost identical patterns were seen in all models . Repeated bolus injections showed that the second injection initiated only a small prostaglandin release indicating the development of refractoriness to the treatment . A similar refractoriness or desensitization occurred during the low-dose infusion . Flunixin meglumine, a potent inhibitor of prostaglandin biosynthesis, administered in different combinations in association with endotoxin demonstrated that this compound must be used at an early stage to prevent endotoxaemia and its deleterious effects on pregnancy . Taken together, the results show that horses are sensitive to endotoxins such that a short period of challenge (about 30 min) is enough to cause clinical signs and reproductive disorders. Indian J Pathol Microbiol, 1991 Jan, 34(1), 22 - 5 Salmonella typhimurium isolated from bacteraemia; Suresh KP et al.; During a two year period, a total of 15 strains of S . typhimurium were isolated and analysed by phage typing . Of these, 13 were found untypable, while two strains belonged to phage 76 and 22 . All the strains were sensitive to Gentamicin and Cephaloridine . All but one showed multiple drug resistance. Indian J Pathol Microbiol, 1991 Jan, 34(1), 17 - 21 Salmonella phage types in Bombay from 1983 to 1987; Roche RA et al.; A total of 156 strains of Salmonella isolated at T.N . Medical College and B.Y.L . Nair Ch . Hospital, Bombay over a period of 5 years from 1983 to 1987 were subjected to Phage Typing . Out of the 111 Salmonella typhi strains, phage type A was found in highest proportion (45.95%), followed by phage type E1 (15.32%), 0(9.91%), Deg . Vi . (9.91%) and C5(5.41%) . Salmonella paratyphi A had phage type pattern of 1(60.0%), 2(22.86%) and Untypable (14.29%) . Majority of the Salmonella typhimurium isolates (90.0%) were untypable. Arch Microbiol, 1991, 156(4), 307 - 11 Prolonged environmental stress via a two step process selects mutants of Escherichia, Salmonella and Pseudomonas that grow at 54 degrees C; Droffner ML et al.; A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48 degrees C with nalidixic acid selected mutants (T48) able to grow at 48 degrees C . A prolonged incubation at 54 degrees C of the T48 mutants selected mutants (T54) able to grow at 54 degrees C . These mutants were susceptible to the same bacteriophages as the original mesophilic strains . Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48 degrees C or 54 degrees C or on a minimal agar with glucose at 37 degrees C . The T48 alleles mapped in the gyrA region of E . coli or S . typhimurium chromosome . In S . typhimurium the T54 alleles, which permit growth at 54 degrees C, were shown by cotransductional analysis to be linked to gyrA. Arch Microbiol, 1991, 156(4), 281 - 9 Structure and expression of the Chlorobium vibrioforme hemA gene; Majumdar D et al.; The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway . A 1.9-kb clone of genomic DNA from C . vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced . The transforming C . vibrioforme DNA has significant sequence similarity to the E . coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174 . This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E . coli, S . typhimurium, and B . subtilis hemA genes . No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S . typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase . These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway . A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E . coli and B . subtilis hemC genes . This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E . coli and B . subtilis HemC peptides . hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Immunopharmacol, 1991, 13(5), 541 - 8 Effect of the new immunostimulator HAB 439 on cell-mediated immunity against intracellular bacteria; Dickneite G et al.; The isoxazoline derivative HAB 439 was tested for its enzyme inhibiting potency and was found to be an inhibitor of aminopeptidase B (IC50 = 22.5 micrograms/ml) . In further immunopharmacological experiments its efficacy to stimulate cell-mediated immunity was evaluated . HAB 439 was shown to stimulate DTH-reaction against Salmonella typhimurium and Listeria monocytogenes . HAB 439 protected animals against infection by reducing the bacterial load in livers and spleens and by decreasing the mortality rate . Treatment with the antibiotic ampicillin induced a decreased DTH-reaction in mice which was demonstrated to be due to a reduction of the antigen to be presented to the immune system and not to immune suppression . HAB 439 restored the impaired immune response to S . typhimurium and L . monocytogenes in a dose-dependent way . Restoration of DTH was shown to lead to an improvement of protection in ampicillin-treated mice which were challenged with the intracellular bacteria. Nauchnye Doki Vyss Shkoly Biol Nauki, 1991, (7), 58 - 63 {The effect of cyclooxygenase inhibition on the indices of the thrombocyte-vascular link in hemostasis and on the free-radical processes of lipid oxidation in experimental Salmonella intoxication}; Malov VA et al.; Injection of Salmonella typhimurium endotoxin to the laboratory animals (rabbits) in dose of 1 mg/ml (LD84) induces the particular changes in the thrombocyte vessels system of hemostasis: decrease of aggregatory ability of thrombocytes, increase of thromboxane A2 and prostacyclin activation of lipid peroxidation process . Use of indomethacin--the cyclooxygenase inhibitor--leads to less progressive alterations of the studied parameters of the thrombocyte vessels hemostasis and lipid peroxidation processes. Microbios, 1991, 67(272-273), 141 - 9 Physiological effects of the lipopolysaccharide of Vibrio vulnificus on mice and rats; McPherson VL et al.; Vibrio vulnificus is a pathogenic, marine, Gram-negative bacterium which commonly enters and infects humans via open wounds or the ingestion of raw seafood . The lipopolysaccharide (LPS) of V . vulnificus has recently been characterized, but the biological activity of this putative endotoxin is unknown . Three treatment groups were used to test its activity: saline (negative control), the LPS of Salmonella typhimurium (a known endotoxin), and the LPS of V . vulnificus . Whereas, intravenous injections of the S . typhimurium LPS caused mortality in two strains of mice, V . vulnificus LPS was not lethal . However, intraperitoneal injections into rats of both V . vulnificus and S . typhimurium LPS were pyrogenic . Intravenous injections of V . vulnificus LPS in rats caused decreased mean arterial pressure within 10 min which further declined, leading to death in 30 to 60 min . S . typhimurium LPS caused similar responses at the same concentration . The data suggest that the LPS of V . vulnificus may be a factor contributing to the virulence of this organism. Free Radic Biol Med, 1991, 11(6), 545 - 55 Does hydrogen peroxide exist "free" in biological systems? Schubert J, Wilmer JW. Hydrogen peroxide (H2O2) can diffuse far from the site of production to intracellular locations where biological effects may be greater . The diffusion range is extended by H2O2 carriers formed spontaneously by hydrogen bonding with monomeric and polymeric compounds, including amino and dicarboxylic acids, peptides, proteins, nucleic acid bases, and nucleosides . Hydrogen peroxide adducts (HPAs) are readily synthesized, e.g., crystalline histidine (His)-H2O2 adducts . An equilibrium exists between an adduct-forming compound and H2O2 . The detection and relative stabilities of HPAs are measured by the degree of decomposition of H2O2 as influenced by test compounds in buffered solution competing with glucose or fructose for H2O2 . The HPAs delay decomposition of H2O2 up to several hundredfold . The overall charge on an HPA, i.e., its ability to penetrate cell membranes, influences the cytotoxic and clastogenic effects of H2O2 . Growth inhibition of Salmonella typhimurium LT2 by H2O2 is enhanced by neutral HPAs but decreased by anionic HPAs . Addition of catalase 1, 10, or 30 min after inoculation of S . typhimurium LT2 reduces or nearly eliminates partial growth inhibition by H2O2, but a neutral HPA, especially His-H2O2, transported H2O2 into the cells within 1 min, and in about 10 min completely inhibited growth . The stability of HPAs decreases with increasing pH or increasing temperature, while added Fe(II) in the presence and absence of EDTA accelerates H2O2 and HPA decomposition . Calculations indicate H2O2 hydrogen bonds with nucleic acid-base pairs with no apparent bond strain and energy stabilization comparable to normal hydrogen bonding. J Biochem Toxicol, 1991 Winter, 6(4), 277 - 82 Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro; Hajos AK et al.; The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase {EC 1.6.99.2} on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay . NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S . typhimurium TA 98 . In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced . NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98 . The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner . In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98 . NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity . The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP . Such reductive metabolism may lead to increased formation of the penultimate mutagenic species. Int Arch Allergy Appl Immunol, 1991, 96(2), 161 - 7 Delayed-type hypersensitivity antigens detected in culture supernatants of Salmonella typhimurium; Cho N et al.; Protein antigens eliciting delayed type hypersensitivity (DTH) were analyzed and purified from the supernatants of protein-free cultures in which Salmonella typhimurium TV148 organisms had grown . DTH activity was measured by the footpad swelling test in mice immunized with living organisms of S . typhimurium TV148 or Escherichia coli K-12 . DTH activity in the culture supernatant was specific to TV148-immunized mice . This activity was destroyed by pronase . DTH activity was unable to pass through an ultrafilter with an exclusion limit of 10 kD . After condensation of the supernatant and following centrifugation (100,000 g for 1 h), the DTH activities of the sediment and the supernatant were examined, and both showed DTH activity . Further analyses of DTH antigens in the supernatant by HPLC gel filtration separated the activity into three portions . The most active portion was further fractionated by hydroxyapatite HPLC, revealing the presence of two DTH antigens, with molecular weights of 65 and less than 10 kD . These results indicate that the culture supernatant of S . typhimurium TV148 organisms contains a variety of macromolecular protein DTH-eliciting antigens, and one of the antigens is 65 kD, which is dissociated partly by organic solvents into a low molecular weight (less than 10 kD) antigen. Environ Mol Mutagen, 1991, 18(4), 224 - 30 Analysis of Salmonella typhimurium hisD3052 revertants: the use of oligodeoxyribonucleotide colony hybridization, PCR, and direct sequencing in mutational analysis; Kupchella E et al.; A rapid method for determining the DNA sequences of Salmonella typhimurium hisD3052 revertants is presented . DNA colony hybridization was used to analyze revertants previously studied by Isono and Yourno {Proc Natl Acad Sci USA 71:1612-1617, 1974} . Synthetic oligodeoxyribonucleotide probes (18-mers) were able to distinguish sequences that differed by a single base pair . Mutant his sequences not identified by probing analysis were amplified using polymerase chain reaction (PCR) and directly sequenced . The combined use of DNA-colony hybridization and direct sequencing offers a precise and rapid means for the molecular characterization of hisD3052 revertants. Arch Toxicol, 1991, 65(8), 633 - 9 DNA binding, adduct characterisation and metabolic activation of aflatoxin B1 catalysed by isolated rat liver parenchymal, Kupffer and endothelial cells; Schlemper B et al.; In vitro studies with rat liver parenchymal, Kupffer and endothelial cells isolated from male Sprague-Dawley rats were undertaken to investigate cell-specific bioactivation of aflatoxin B1, DNA binding and adduct formation . In the mutagenicity studies, using homogenates of all three separated liver cell populations (co-incubated with NADP+ and glucose-6-phosphate as cofactors for the cytochrome P-450 monooxygenase system) parenchymal, Kupffer and endothelial cells were able to activate aflatoxin B1 to a metabolite mutagenic to Salmonella typhimurium TA 98 . In the case of nonparenchymal cells (i.e . Kupffer and endothelial cells) 10-fold higher concentrations of aflatoxin B1 had to be used to obtain a similar number of revertants to that observed with parenchymal cells . Induction studies with Aroclor 1254 led to a striking decrease in the activation of aflatoxin B1 in parenchymal cells, whereas nonparenchymal cells had a slightly enhanced metabolic activation capacity for aflatoxin B1 . Metabolism studies with microsomes from induced and noninduced cells using testosterone as substrate revealed comparable results: after induction with Aroclor 1254, parenchymal cells showed a 60% decrease in the formation rate of 2 alpha-hydroxytestosterone, whereas the formation rate of this metabolite remained unchanged in nonparenchymal cells; 2 alpha-hydroxytestosterone is specifically formed by cytochrome P-450 IIC11, which also catalyses the activation of aflatoxin B1 to its epoxide . When freshly isolated, intact cells were incubated with tritiated aflatoxin B1, a dose-dependent aflatoxin B1 binding to DNA in parenchymal and nonparenchymal cells was observed . HPLC analysis of DNA acid hydrolysates of all three cell types showed the major adduct to be 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1. Folia Microbiol (Praha), 1991, 36(3), 317 - 8 Colicin E1 export in Salmonella typhimurium wild-type and lipopolysaccharide mutants; Viejo BM et al.; Colicin export was studied in different Salmonella typhimurium strains lacking the O-antigen repeating units (O-) and different strains with different chemotypes for the lipopolysaccharide core, as well as the wild-type strain (O+), to determine the role of lipopolysaccharide length on colicin E1 export . While the lipopolysaccharide length influences the levels of external hemolytic activity in S . typhimurium, no effect was detected on colicin E1 export. Biotech Histochem, 1991, 66(6), 307 - 15 Decontamination of aqueous solutions of biological stains; Lunn G et al.; Aqueous solutions of a number of biological stains were completely decontaminated to the limit of detection using Amberlite resins . Amberlite XAD-16 was the most generally applicable resin but Amberlite XAD-2, Amberlite XAD-4, and Amberlite XAD-7 could be used to decontaminate some solutions . Solutions of acridine orange, alcian blue 8GX, alizarin red S, azure A, azure B, Congo red, cresyl violet acetate, crystal violet, eosin B, erythrosin B, ethidium bromide, Janus green B, methylene blue, neutral red, nigrosin, orcein, propidium iodide, rose Bengal, safranine O, toluidine blue O, and trypan blue could be completely decontaminated to the limit of detection and solutions of eosin Y and Giemsa stain were decontaminated to very low levels (less than 0.02 ppm) using Amberlite XAD-16 . Reaction times varied from 10 min to 18 hr . Up to 500 ml of a 100 micrograms/ml solution could be decontaminated per gram of Amberlite XAD-16 . Fourteen of the 23 stains tested were found to be mutagenic to Salmonella typhimurium . None of the completely decontaminated solutions were found to be mutagenic. Acta Microbiol Pol, 1991, 40(1-2), 65 - 70 Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis; Jankowski S et al.; The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents . The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration . The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum. Lab Delo, 1991, (11), 60 - 3 {DNAase activity, a method of differentiating hospital strains of Salmonella typhimurium}; Lysov PS et al.; Measurements of DNAse activities in Salmonella typhimurium strains of various origins have demonstrated that hospital strains are characterized by the highest DNAse activity . This activity correlates with the presence of R-plasmids in the cells . High DNAse activity of hospital strains is a stable marker, and therefore may be used for intraspecies differentiation of S . typhimurium. J Bacteriol, 1991 Jan, 173(1), 234 - 44 Identification and characterization of dppA, an Escherichia coli gene encoding a periplasmic dipeptide transport protein; Olson ER et al.; We describe the isolation and analysis of an Escherichia coli gene, dppA, and its role in dipeptide transport . dppA maps near min 79 and encodes a protein (DppA) that has regions of amino acid similarity with a peptide-binding protein from Salmonella typhimurium (OppA) . Like OppA, DppA is found in the periplasmic space and thus is most likely a dipeptide-binding protein . Insertional inactivation of dppA results in the inability of a proline auxotroph to utilize Pro-Gly as a proline source . dppA-dependent Pro-Gly utilization does not require any of the three major proline transport systems, demonstrating that DppA is not simply a dipeptidase . An in vivo competition assay was used to show that DppA is probably involved in the transport of dipeptides other than Pro-Gly . Transcription of dppA is repressed by the presence of casamino acids, suggesting that the cell alters its dipeptide transport capabilities in response to an environmental signal. Teratog Carcinog Mutagen, 1991, 11(2), 65 - 76 Analysis of rat lymphocyte activation of benzo{a}pyrene, 2-acetylaminofluorene, and several of their metabolites to mutagenic and DNA-damaging species in vitro; Gao N et al.; Rat lymphocytes are a potentially useful and convenient cell system for monitoring the genotoxic effects of chemicals in vivo, but little is known about the ability of these cells to metabolize promutagens to genotoxic species . In this study, Fischer 344 rat lymphocytes were treated in vitro with benzo{a}pyrene (BaP), 2-acetylaminofluorene (2-AAF), and several of their metabolites, and DNA damage was measured using nucleoid sedimentation analysis . Of the BaP derivatives, BaP 4,5-oxide and BaP 7,8-diol-9, 10-epoxide decreased lymphocyte nucleoid sedimentation, whereas BaP and BaP 7,8-dihydrodiol had little effect . Among the 2-AAF derivatives, N-acetoxy-2-AAF, N-hydroxy-2-AAF, and N-hydroxy-2-aminofluorene damaged rat lymphocyte nucleoids, whereas 2-AAF, 2-aminofluorene, and fluorene produced little detectable damage . The decrease in nucleoid sedimentation produced by N-hydroxy-2-AAF was not inhibited by paraoxon, salicylamide, or 2-aminofluorene, whereas paraoxon inhibited damage produced by N-acetoxy-2-AAF . In co-culture assays, rat lymphocytes increased the mutagenicity of N-hydroxy-2-AAF in Salmonella typhimurium strain TA98, but mediated little or no mutagenic response with BaP, BaP 7,8-dihydrodiol, and 2-AAF . Also, human lymphocytes, but not rat lymphocytes, mediated a positive mutagenic response with BaP 7,8-dihydrodiol in Chinese hamster ovary UV5 cells . Although rat lymphocytes may metabolize certain proximal genotoxic chemicals to DNA-damaging species (e.g., N-hydroxy-2-AAF), these data suggest that in vivo lymphocyte DNA damage is more likely to result from lymphocytes encountering reactive chemical derivatives produced by other cells . It is also clear that differences exist between the ability of human and rat lymphocytes to activate promutagens, and this may impact on the use of the rat model to predict the genotoxicity of chemicals in humans. Teratog Carcinog Mutagen, 1991, 11(2), 103 - 14 DNA strand breaks induced in cultured human and rodent cells by chlorohydroxyfuranones--mutagens isolated from drinking water; Chang LW et al.; Chlorohydroxyfuranones, by-products of chlorine disinfection and drinking water contaminants, are shown to produce DNA strand breaks in human and rodent cells . One chlorohydroxyfuranone, 3-chloro-4-dichloromethyl-5-hydroxy-2{5H}-furanone (MX), a potent bacterial mutagen, induces 232 +/- 89 DNA strand breaks.(cell-microM)-1 in human CCRF-CEM cells over a concentration range of 4.4 to 220 microM . This constitutes a DNA damage potency comparable to dimethylsulfate (DMS) . By comparison, 3,4-dichloro-5-hydroxy-2{5H}-furanone (MA), another chlorohydroxyfuranone which is approximately four orders of magnitude less mutagenic than MX in Salmonella typhimurium strain TA100, is only about tenfold less potent as an inducer of DNA strand breaks in these cells, i.e., 18.2 +/- 3.1 strand breaks.(cell-microM)-1 . The DNA strand-breaking potential of MX is inactivated by prior incubation with a rat liver S9 homogenate . In addition, both chlorohydroxyfuranones are ineffective at producing DNA strand breaks in primary rate hepatocytes (PRH) at concentrations below those which produce cytotoxicity as assessed by release of the cellular enzyme lactate dehydrogenase (LDH) . Prior treatment of the PRH with 750 microM diethyl maleate, a glutathione-depleting agent, did not enhance the cytotoxicity nor the DNA strand-breaking potential of either chlorohydroxyfuranone . This could indicate that glutathione-glutathione-S-transferase is not an important mechanism for the detoxification of these compounds in PRH. Teratog Carcinog Mutagen, 1991, 11(1), 55 - 60 A genotoxicological study of hexachlorobenzene and pentachloroanisole; Siekel P et al.; The potential mutagenic activity of hexachlorobenzene (HCB) and pentachloroanisole (PCA) was investigated . No genotoxicity after application on Salmonella typhimurium (Ames test), Escherichia coli, and human peripheral blood lymphocytes in vitro was observed. Acta Derm Venereol, 1991, 71(1), 77 - 9 Sweet's syndrome associated with Salmonella typhimurium infection; Zillikens D et al.; A 41-year-old woman presented with the typical clinical and pathohistological features of acute febrile neutrophilic dermatosis (AFND) . The disease had been preceded by diarrhoea and vomiting for 2 weeks . Stool cultures proved positive for Salmonella typhimurium infection . Antibiotic therapy and tapering oral steroids led to a complete remission of skin lesions within 2 weeks . To our knowledge, this is the first report of Sweet's syndrome associated with salmonellosis. Boll Ist Sieroter Milan, 1991-92, 70(1-2), 505 - 12 Survival of bacteriophage 1 of the Salmonella typhimurium phage typing scheme in 4 different types of sterile soil; Leonardopoulos J et al.; The survival of bacteriophage 1 of the Salmonella typhimurium phage typing scheme was studied in four different types of sterile soil at 4.20 and 37 degrees C . The longevity of the phage was generally short, not exceeding 36 days and depended on the temperature and the type of soil. Biochemistry, 1990 Dec 25, 29(51), 11280 - 92 Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae; Brian WR et al.; A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains) . A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast . The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes . Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH . The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present . Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz{a}anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz{a}anthracene, 9,10-dihydroxy-9,10-dihydrobenzo{b}fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation . Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine . Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes) . The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family. J Mol Biol, 1990 Dec 20, 216(4), 897 - 910 TonB protein of Salmonella typhimurium . A model for signal transduction between membranes; Hannavy K et al.; The tonB gene product is required for several outer membrane transport processes in bacteria . The tonB gene from Salmonella typhimurium was sequenced and found to be similar to that of Escherichia coli . The TonB protein is highly proline-rich and includes an unusual segment consisting of multiple X-Pro dipeptide repeats . A synthetic peptide corresponding to this segment has been used to raise anti-TonB antibodies . TonB was shown to be associated with the cytoplasmic membrane, apparently anchored via a single hydrophobic N-terminal segment . Protease accessibility studies, and the use of a series of TonB-beta-lactamase fusions, showed that the rest of the TonB protein is periplasmic . Unusually, export of TonB is not accompanied by cleavage of the N-terminal signal peptide . In the accompanying paper, we show that TonB interacts directly with the outer membrane FhuA (TonA) receptor . Thus, TonB must span the periplasm, providing a link between the cytoplasmic membrane and receptors in the outer membrane . On the basis of these data, and those published by other laboratories, we propose a model whereby TonB serves as a "mechanical" linkage that, by transmitting protein conformational changes from the cytoplasmic membrane across the periplasm, acts as a means of coupling energy to outer membrane transport processes . Such a mechanism has general implications for signal transduction within and between proteins. J Mol Biol, 1990 Dec 20, 216(4), 883 - 95 Structure and function of X-Pro dipeptide repeats in the TonB proteins of Salmonella typhimurium and Escherichia coli; Brewer S et al.; The TonB protein is required for several outer membrane transport processes in bacteria . A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy . The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration . This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors . Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein . As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm . These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane. Toxicology, 1990 Dec 17, 65(1-2), 1 - 22 Genotoxicity evaluation of lithium hypochlorite; Weiner ML et al.; Lithium hypochlorite (LiOCl), the pool and spa sanitizer/algicide, was evaluated for genotoxicity in a battery of studies designed to evaluate potential mutagenicity, DNA damage and chromosome aberrations . LiOCl was not mutagenic in the Ames test when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 or in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay in Chinese hamster ovary (CHO) cells without metabolic activation . LiOCl did not induce DNA damage in the unscheduled DNA synthesis assay using rat primary hepatocytes . Effects on metaphase chromosomes were evaluated in vitro in CHO cells at 12 and 18 h exposure without S9 and at 12 and 22 h following a 2 h exposure with S9 . LiOCl induced a statistically significant increase in chromosome aberrations at the high dose only at both harvest times without S9 and at the late harvest time with S9 . There were significant increases in chromosome aberrations at the low dose, low-mid and high doses, but not at the high mid-dose at the early harvest time with S9 . However, LiOCl did not increase chromosome aberrations when tested orally in rats at maximally tolerated doses . Bone marrow cells, collected 6, 24 and 48 h after a single oral dose of LiOCl to rats (100, 500, 1000 mg/kg in males; 50, 250, 500 mg/kg in females) showed no increase in the incidence of aberrations . In general, the weight of the evidence indicates that LiOCl is not genotoxic. J Immunol, 1990 Dec 15, 145(12), 4317 - 21 Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination; Schodel F et al.; Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains . When these live recombinant bacteria were administered i.p . to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies . Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used . With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization . The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p . and oral immunization is consistent with the induction of functional T cell help. Cancer Res, 1990 Dec 15, 50(24), 7789 - 92 Activation of mitomycin C by NADPH:cytochrome P-450 reductase; Bligh HF et al.; Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells . The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase . In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism . In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C . Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed . In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000 . The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions . However, under hypoxic conditions increased sensitivity was not observed . This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells . These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance . P-450 reductase expressed in S . typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway. Eur J Biochem, 1990 Dec 12, 194(2), 655 - 61 Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography; Fischer W; By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids . Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units . Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence . Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues . Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S . typhimurium. Eur J Biochem, 1990 Dec 12, 194(2), 573 - 8 Cloning, nucleotide sequence and regulation of the Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase; Frick MM et al.; The Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase was cloned and sequenced . In total, a sequence of 1286 nucleotide pairs was determined wherein a single open-reading-frame of 1011 bp, encoding a polypeptide of 336 amino acids having 95% similarity with the Escherichia coli pyrD gene product, was identified . A region of hyphenated-dyad symmetry exists within the leader region affording the potential for the formation of a stable secondary structure in the 5' end of the transcript . Mutations from several regulatory mutants were located within the region of dyad symmetry which would impart changes in the transcript within the putative secondary structure, implicating the secondary structure in regulation . Primer extension analysis revealed multiple transcriptional start sites located six to nine nucleotides downstream from the Pribnow box, with the primary initiation site differing in repressing and derepressing growth conditions . The results are discussed in terms of a translational attenuation model for regulation of pyrD expression. Changgeng Yi Xue Za Zhi, 1990 Dec, 13(4), 290 - 5 {Epidemiology and clinical evaluation of Salmonella enteritis}; Suen TY et al.; There was a local epidemic of Salmonella enteritis in southern part of Taiwan during the summer of 1989 . From July through September 1989, a total 162 cases of enteritis were analysed in Chang Gung Memorial Hospital, Kaohsiung . Among them, 46 cases were proved to be Salmonella enteritis by stool and/or blood culture . The identified flora group mainly group B (Salmonella typhimurium, 87%), group C (Salmonella choleraesuis, 6.5%) and group D (Salmonella enteritidis, 6.5%) . The drug resistance of Salmonella enteritis of traditional antibiotics such as ampicillin, chloramphenicol and trimethoprim-Sulfamethoxazole (TMP-SMX) in apparently increasing . We found that 43.5% of cases were uniformly resistant to all 3 antimicrobial agents as mentioned above . Of the 4 infants who developed bacteremia, 2 were less than 3 months old and their blood culture grew out group B . Salmonella, but fortunately no complication were found during hospitalization . The other 2 cases were proved to be caused by group C Salmonella which was reported to have higher incidence of ensuing bacteremia . This study revealed that persistent bacteremia could be present in the absence of fever and toxic signs . Newer, third generation cephalosporins such as cefotaxime of ceftriaxone should be initiated promptly. Int J Food Microbiol, 1990 Dec, 11(3-4), 195 - 204 Use of polymyxin-coated polyester cloth in the enzyme immunoassay of Salmonella lipopolysaccharide antigens; Blais BW et al.; Polyester cloth coated with polymyxin B was used to capture Salmonella typhimurium lipopolysaccharide antigens which were then quantitatively or qualitatively assayed using a specific antibody-peroxidase conjugate . This simple, rapid method can be used to assay a large number of samples by employing a large sheet of the polymyxin-coated cloth onto which multiple samples can be blotted . The method is reproducible and economical, since polymyxin B is relatively inexpensive, stable and available in pure form. J Clin Microbiol, 1990 Dec, 28(12), 2597 - 601 Outbreak of Salmonella typhimurium infection traced to contaminated chocolate and caused by a strain lacking the 60-megadalton virulence plasmid; Kapperud G et al.; We describe an outbreak of Salmonella typhimurium infection, caused by contaminated chocolate produced by one Norwegian company, which occurred in Norway and Finland in 1987 . A total of 349 bacteriologically verified cases were recorded in Norway, and 12 cases were recorded in Finland . There was a predominance of young children among the patients (median age, 6 years), many of whom developed acute hemorrhagic diarrhea . The outbreak strain exhibited a rare phage lysis pattern and a characteristic plasmid profile lacking the 60-MDa virulence-associated plasmid . DNA hybridization failed to demonstrate any DNA sequence homology between the outbreak strain and the virulence plasmid . The outbreak strain was nonlethal for orally infected mice . The finding of only less than or equal to 10 S . typhimurium cells per 100 g of chocolate in about 90% of the positive samples obtained from retail outlets suggested that an inoculum of fewer than 10 organisms may have been sufficient to cause symptomatic disease. Mutat Res, 1990 Dec, 245(4), 251 - 7 N-2 acetylation of 2'-deoxyguanosine by coffee mutagens, methylglyoxal and hydrogen peroxide; Nukaya H et al.; Coffee shows direct-acting mutagenicity in Salmonella typhimurium TA100 and most of this mutagenicity is due to the synergistic effects of methylglyoxal and hydrogen peroxide . The modifications of deoxyribonucleosides by methylglyoxal plus hydrogen peroxide were studied in vitro . When 2'-deoxyguanosine (6.25 mumole) was treated with methylglyoxal (125 mumole) and hydrogen peroxide (125 mumole) in 5 ml of 0.1 M phosphate buffer (pH 7.4) at 37 degrees C for 3 h, N2-acetyl-2'-deoxyguanosine was formed with a yield of 1.1% . Its formation increased time-dependently . By contrast, no appreciable modification of other deoxynucleosides was detected after their incubation with methylglyoxal and hydrogen peroxide under similar conditions . N2-Acetyl-2'-deoxyguanosine was also formed during incubation of 2'-deoxyguanosine with instant coffee. Mol Gen Genet, 1990 Dec, 224(3), 364 - 72 The VANA glycopeptide resistance protein is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes; Dutka-Malen S et al.; Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147 . Vancomycin induced the synthesis of a ca . 40 kDa membrane-associated protein designated VANA . The resistance protein was partially purified and its N-terminal sequence was determined . A 1761 bp DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced . When expressed in E . coli, this fragment encoded a ca . 40 kDa protein that comigrated with VANA from enterococcal membrane fractions . The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing . The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37,400 . VANA was structurally related to the D-alanyl-D-alanine (D-ala-D-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E . coli (28%) . The vanA gene was able to transcomplement an E . coli mutant with thermosensitive D-ala-D-ala ligase activity . Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics . Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target. Carcinogenesis, 1990 Dec, 11(12), 2275 - 9 In vitro inhibition of dihydropyridine oxidation and aflatoxin B1 activation in human liver microsomes by naringenin and other flavonoids; Guengerich FP et al.; Recent in vivo studies in humans have shown a dramatic effect of grapefruit juice in blocking the oxidation of dihydropyridine calcium channel blockers . The flavonoid naringin is the most abundant natural product specific for grapefruit and related citrus--the aglycone naringenin, known to be readily formed from naringin in humans, was found to inhibit the oxidation of the dihydropyridines nifedipine and felodipine in human liver microsomal preparations . These observations were of interest in light of the knowledge that the same human liver cytochrome P450 (IIIA4) appears to be a major catalyst in both nifedipine oxidation and aflatoxin B1 activation . Several flavones inhibited the in vitro activation of aflatoxin B1 in a system employing umuC gene activation due to DNA damage in Salmonella typhimurium TA1535/pSK1002, with naringenin being as effective as any . The high concentration of derivatives of naringenin in certain citrus fruits may be of relevance to cancer chemoprevention involving those carcinogens that are activated by cytochrome P-450IIIA4. Carcinogenesis, 1990 Dec, 11(12), 2171 - 7 Potent genotoxicity of halogen lamps, compared to fluorescent light and sunlight; De Flora S et al.; The light emitted by halogen lamps induced mutations in Salmonella typhimurium and DNA damage in Escherichia coli, as shown by the hypersensitivity of DNA repair-deficient strains . The mutagenicity of halogen lamps was considerably higher than that of fluorescent light and of sunlight, even at much lower illuminance levels . Excision mechanisms and SOS functions were involved in repairing light-induced base-pair substitutions and frameshift errors in bacterial DNA . At variance with solar irradiation, which produces mutagenic effects over a wide UV spectrum, genotoxicity of halogen lamps was almost exclusively due to far-UV wavelengths transmissible through UV-R-250 and UV-R-280 interference filters . The main mutagenic component of fluorescent light (254 nm) were almost 10(4)-fold more mutagenic than near-UV wavelengths (365 nm) . All light sources exhibited some residual mutagenicity even following filtration through various cloths . On the other hand, appropriate glass or plastic covers consistently prevented mutagenic effects . This emphasizes the urgent need for a compulsory shielding of halogen and fluorescent lamps in order to prevent unnecessary exposures to genotoxic and potentially carcinogenic UV radiations. Toxicol Lett, 1990 Dec, 54(2-3), 309 - 15 Prochloraz as potent inhibitor of benzo{a}pyrene metabolism and mutagenic activity in rat liver fractions; Antignac E et al.; We have determined how prochloraz, an imidazole antifungal agent, affects the metabolism of benzo{a}-pyrene by hepatic microsomes from 3-methylcholanthrene treated male rats . The prochloraz-like 7,8-benzoflavone was a potent inhibitor of total benzo{a}pyrene metabolism while miconazole was a weak inhibitor . The proportion of benzo{a}pyrene dihydrodiols formed was decreased whereas phenols were increased by the in vitro addition of prochloraz . Furthermore, a good correlation was obtained between the effects of prochloraz on the microsomal formation of benzo{a}pyrene metabolites and on the mutagenic activity of benzo{a}pyrene in the Salmonella typhimurium test. Mutat Res, 1990 Dec, 242(4), 337 - 43 Mutagenicity of dibenz{a,c}anthracene and its derivatives in Salmonella typhimurium TA100; Kumar S et al.; The mutagenic activities of dibenz{a,c}anthracene (DB{a,c}A), and its 11 derivatives, including 3 diols, 6 phenols and 2 oxepines, were studied in the TA100 strain of Salmonella typhimurium at doses varying from 0 to 20 micrograms/plate in the presence of a rat-liver S9 (9000 x g) preparation . Among the diols of DB{a,c}A tested DB{a,c}A-10,11-diol was the most mutagenic compound . However, it was consistently less mutagenic than the parent hydrocarbon . Oxepine-1 and oxepine-2 which are believed to be the photoisomerized products of DB{a,c}A-1,2 oxide and DB{a,c}A-3,4-oxide, respectively, were also less mutagenic than DB{a,c}A . In contrast to these results, 4-hydroxyDB{a,c}A was almost twice as active as DB{a,c}A, and 2-hydroxy- and 3-hydroxyDB{a,c}A were even more (4-6-fold) mutagenic than DB{a,c}A . The remaining phenols were relatively inactive or weakly active in this mutagenicity assay . These results provide initial evidence that the bay-region theory may not be applicable to the mutagenesis of DB{a,c}A, and that the angular ring substituted phenols of DB{a,c}A may be involved in the metabolic activation of this highly mutagenic hydrocarbon. J Bacteriol, 1990 Dec, 172(12), 7200 - 10 Role of purine biosynthetic intermediates in response to folate stress in Escherichia coli; Rohlman CE et al.; Folic acid plays a central role in anabolic metabolism by supplying single-carbon units at varied levels of oxidation for both nucleotide and amino acid biosyntheses . It has been proposed that 5-amino-4-imidazole carboxamide riboside 5'-triphosphate (ZTP), an intermediate in de novo purine biosynthesis, serves as a signal of cellular folate stress and mediates a physiologically beneficial response to folate stress in Salmonella typhimurium (B . R . Bochner, and B . N . Ames, Cell 29:929-937, 1982) . We examined the physiological response of Escherichia coli to folate stress induced by the drugs psicofuranine, trimethoprim, and sodium sulfathiazole or by p-aminobenzoic acid (pABA) starvation . Analysis of nucleotide pools showed that psicofuranine or trimethoprim treatment of a prototrophic strain or growth of a pABA auxotroph on limiting pABA induced the production of the nucleotide ZTP, as previously observed in S . typhimurium by Bochner and Ames . Accumulation of ZTP and its precursor 5-amino-4-imidazole carboxamide riboside 5'-monophosphate (ZMP) did not correlate well with folate stress in E . coli, as measured by determination of the folate/protein ratios of extracts of treated cells . Treatment of cells with psicofuranine caused a marked accumulation of 5-amino-4-imidazole carboxamide ribonucleotides (Z-ribonucleotides) but a statistically insignificant drop in the folate/protein ratio of cell extracts . Sodium sulfathiazole treatment at a drug concentration that led to a threefold drop in the growth rate and in the folate/protein ratio of treated cells led to little accumulation of Z-ribonucleotides in E . coli A purF his+ strain which produces ZTP and ZMP when treated with trimethoprim was constructed . In this strain, histidine represses the synthesis of both ZMP and ZTP . Treatment of cells of this strain with trimethoprim resulted in a decrease in the folate/protein ratio of cell extracts, but a blockade of Z-ribonucleotide accumulation did not affect the extent of folate depletion seen in treated cells and had only a small effect on the resistance of this strain to growth inhibition by trimethoprim . The patterns of protein expression induced by treatment of this strain with trimethoprim or psicofuranine were examined by two-dimensional electrophoretic resolution of the total cellular proteins . No differences in protein expression were seen when the treatment were performed in media containing or lacking histidine . These studies failed to provide evidence in E . coli for a folate stress regulon controlled by ZTP. J Bacteriol, 1990 Dec, 172(12), 7071 - 84 Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase; Elliott T et al.; Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus . ALA-auxotrophic hemL mutant S . typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway . hemL cells transformed with a plasmid containing the S . typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity . Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid . These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate . In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene . The S . typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide . The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S . typhimurium or E . coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B . japonicum or R . capsulatus, which encode ALA synthase . The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length . Sequence similarity to other aminotransferases was also detected. J Bacteriol, 1990 Dec, 172(12), 7043 - 8 Cloning of the Klebsiella aerogenes nac gene, which encodes a factor required for nitrogen regulation of the histidine utilization (hut) operons in Salmonella typhimurium; Best EA et al.; The nac (nitrogen assimilation control) gene from Klebsiella aerogenes, cloned in a low-copy-number cloning vector, restored the ability of K . aerogenes nac mutants to activate histidase and repress glutamate dehydrogenase formation in response to nitrogen limitation and to limit the maximum expression of the nac promoter . When present in Salmonella typhimurium, the K . aerogenes nac gene allowed the hut genes to be activated during nitrogen-limited growth . Thus, the nac gene encodes a cytoplasmic factor required for activation of hut expression in S . typhimurium during nitrogen-limited growth. J Bacteriol, 1990 Dec, 172(12), 6919 - 29 In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium; Monroe RS et al.; The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers . Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S . typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79 . The S . typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35 . Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2 . In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1 . Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all . An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity . CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter . CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter. J Bacteriol, 1990 Dec, 172(12), 6727 - 35 Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis; Gunasekaran P et al.; The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined . Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E . coli and Z . mobilis consensus sequences . Extracts from E . coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity . However, no transfructosylation activity (exchange reaction or levan formation) could be detected . This sucrase activity was different from that observed with the purified extracellular protein B46 from Z . mobilis . These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity . Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z . mobilis . The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus. J Bacteriol, 1990 Dec, 172(12), 6721 - 6 Overproduction of release factor reduces spontaneous frameshifting and frameshift suppression by mutant elongation factor Tu; Aulin MR et al.; Mutant forms of elongation factor Tu encoded by tufA8 and tufB103 in Salmonella typhimurium cause suppression of some but not all frameshift mutations . All of the suppressed mutations in S . typhimurium have frameshift windows ending in the termination codon UGA . Because both tufA8 and tufB103 are moderately efficient UGA suppressors, we asked whether the efficiency of frameshifting is influenced by the level of misreading at UGA . We introduced plasmids synthesizing either one of the release factors into strains in which the tuf mutations suppress a test frameshift mutation . We found that overproduction of release factor 2 (which catalyzes release at UGA and UAA) reduced frameshifting promoted by the tuf mutations at all sites tested . However, at one of these sites, trpE91, overproduction of release factor 1 also reduced suppression . The spontaneous level of frameshift "leakiness" at three sites in trpE, each terminating in UGA, was reduced in strains carrying the release factor 2 plasmid . We conclude that both spontaneous and suppressor-enhanced reading-frame shifts are influenced by the activity of peptide chain release factors . However, the data suggest that the effect of release factor on frameshifting does not necessarily depend on the presence of the normal triplet termination signal. Infect Immun, 1990 Dec, 58(12), 3899 - 902 Polymorphic expression of defensins in neutrophils from outbred rats; Eisenhauer P et al.; We isolated and characterized a rat neutrophil defensin, RatNP-2, that differs from the previously described defensin RatNP-1 by containing Ser-7 in place of Arg-7 . Although the resulting charge difference rendered RatNP-2 easily distinguishable from RatNP-1 on polyacrylamide gel electrophoresis gels, the two defensins exhibited very similar antimicrobial efficacies against Salmonella typhimurium, Staphylococcus aureus, and Candida albicans . The polymorphonuclear leukocytes of Sprague-Dawley rats obtained from one of two breeders also showed a marked polymorphism for defensin RatNP-4 . This defensin was absent in two of seven animals and present in 1x or 2x relative amounts in the others . These observations indicate that a striking degree of defensin polymorphism exists in the polymorphonuclear leukocytes of outbred rodents. J Infect Dis, 1990 Dec, 162(6), 1397 - 400 Occurrence of secondary attenuating mutations in avirulent Salmonella typhimurium vaccine strains; Lockman HA et al.; The attenuating delta aroA554 mutation in Salmonella typhimurium strain SL3261 was complemented in vitro by selecting for AroA+ recombinant DNA clones . SL3261 containing cloned aroA+ genes did not require exogenous phenylalanine, tryptophan, tryosine, p-aminobenzoic acid, or dihydroxybenzoic acid for growth in defined media . Cloned aroA+ genes did not restore wild-type virulence to SL3261, however, in a murine typhoid model . The delta aroA554 mutation was transduced into S . typhimurium strain SR-11, a mouse-virulent strain recently passaged in mice . The SR-11 delta aroA554 mutant was highly attenuated for mice challenged parenterally . The same cloned aroA+ genes isolated in SL3261 restored the virulence of the SR-11 delta aroA554 mutant to that of wild-type SR-11 . These results suggest that while the delta aroA554 allele remains effective in reducing S . typhimurium virulence, laboratory passage of attenuated vaccine strains may lead to the accumulation of additional attenuating defects. Proc Natl Acad Sci U S A, 1990 Dec, 87(24), 9823 - 7 Method for identifying microbial antigens that stimulate specific lymphocyte responses: application to Salmonella; Warren RL et al.; Vaccine development and understanding of cellular immune stimulatory mechanisms have been impeded by the paucity of data on microbial antigens that stimulate protective immunity . We describe here a general method for identifying and isolating peptide antigens that specifically stimulate sensitized lymphocytes . First, Salmonella typhimurium C5 genomic DNA fragments were subcloned into Escherichia coli by use of the lambda gt11 expression vector . Next, antigens expressed by recombinant phage from this genomic library were tested for their capacity to stimulate proliferative responses in pooled lymphocytes obtained from BALB/c mice infected 14 days earlier with S . typhimurium . Of 2000 recombinant phages tested, 5 stimulated a polypeptide-antigen-specific proliferative response . Physical analyses of these 5 recombinant phages revealed cloned inserts of 0.5-2.4 kilobase pairs representing nonoverlapping regions of the C5 chromosome . Four of the five insert DNAs hybridized at high stringency to both S . typhimurium and Salmonella typhi total chromosomal DNA, suggesting that these pathogens of different host specificity share several antigenic determinants . Use of sensitized primary polyclonal lymphocytes provides a rapid and simple method for screening recombinant DNA libraries for clones that stimulate specific immune responses and avoids the use of cloned lymphocyte cell lines . This approach should be generally applicable to similar studies in different hosts of many other microbial pathogens. Zentralbl Hyg Umweltmed, 1990 Dec, 190(5-6), 536 - 46 {An automated in vitro toxicity test of 25 chemicals}; Jakob R et al.; A computer controlled bench-top analyser was used to establish an automated in vitro-cytotoxicity test . It is based on the continuous monitoring of growth of Salmonella typhimurium TA 98 and TA 100 (strains that are also employed in the classical Ames test (1) for mutagenicity screening) under the influence of the toxic compounds . With this method we can quantify general cytotoxicity and in addition, some questions concerning the mechanism of the toxic influence on the test organisms can be answered . Its simplicity and reliability as well as the good correlation with in vivo data make the new test system suitable for pre-screening the toxicity of a large number of compounds within short time. J Anim Sci, 1990 Dec, 68(12), 4303 - 9 Effect of vitamin E and selenium supplementation on some immune parameters following vaccination against brucellosis in cattle; Nemec M et al.; Twenty-four 7-mo-old beef heifers (Charolais Simmental cross), weighing 213 kg, were used to determine the effect of vitamin E (VitE) and(or) selenium (Se) supplementation on the humoral response to a standard dose of Brucella abortus strain 19 vaccine and on the levels of naturally occurring immunoglobulins (Ig) to several antigens . The treatments were as follows: Group 1, no supplement; Group 2, supplementation with 6 g of elemental Se; Group 3, supplementation with 1,400 IU/d of VitE; and Group 4, Se and VitE supplements combined . There were no significant differences in anti-B . abortus IgG1, IgG2, or IgM antibody levels due to Se, VitE or Se/VitE treatments; the concentrations of IgA antibody were too low to be measured with the ELISA test used . Statistical analysis revealed that the levels of total and IgM natural antibody to Salmonella typhimurium were higher in Group 3 . Perhaps VitE supplementation given in conjunction with B . abortus vaccine enhanced the production of antibody to S . typhimurium in several animals whose humoral system had been activated by previous exposure to this organism. Jpn J Cancer Res, 1990 Dec, 81(12), 1253 - 8 Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines; Degawa M et al.; The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer . The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes . 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido{2,3-b}indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay . By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines. J Med Microbiol, 1990 Dec, 33(4), 235 - 8 Mechanisms of zidovudine resistance in bacteria; Lewin CS et al.; Unlike their parent strains, zidovudine-resistant derivatives of Escherichia coli KL16 and Salmonella typhimurium NCTC 5710 were found to be incapable of incorporating radiolabelled thymidine into their chromosomal DNA . Since incorporation was still prevented in the presence of EDTA, resistance to zidovudine was not associated with a permeability barrier, but appeared to result from the loss of thymidine kinase activity, required for the phosphorylation of zidovudine . Pseudomonas aeruginosa, which is intrinsically zidovudine-resistant, was also shown to be incapable of incorporating thymidine into its DNA, but Staphylococcus epidermidis SK360 and Staph . aureus E3T, which are also intrinsically zidovudine-resistant, possessed thymidine kinase activity . This suggests that two distinct mechanisms of resistance to zidovudine exist in bacteria . Zidovudine resistance did not appear to confer resistance to other antibacterial agents. J Bacteriol, 1990 Dec, 172(12), 6661 - 8 Purification and characterization of the Myxococcus xanthus FrzE protein shows that it has autophosphorylation activity; McCleary WR et al.; Myxococcus xanthus exhibits multicellular interactions during vegetative growth and fruiting body formation . Gliding motility is needed for these interactions . The frizzy (frz) genes are required to control directed motility . FrzE is homologous to both CheA and CheY from Salmonella typhimurium . We used polyclonal antiserum raised against a fusion protein to detect FrzE in M . xanthus extracts by Western immunoblot analysis . FrzE was clearly present during vegetative growth and at much lower levels during development . A recombinant FrzE protein was overproduced in Escherichia coli, purified from inclusion bodies, and renatured . FrzE was autophosphorylated when it was incubated in the presence of {gamma-32P}ATP and MnCl2 . Chemical analyses of the phosphorylated FrzE protein indicated that it contained an acylphosphate; probably phosphoaspartate . FrzE was phosphorylated in an intramolecular reaction . Based on these observations, we propose a model of the mechanism of FrzE phosphorylation in which autophosphorylation initially occurs at a conserved histidine residue within the "CheA" domain and then, via an intramolecular transphosphorylation, is transferred to a conserved aspartate residue within the "CheY" domain. Mol Microbiol, 1990 Dec, 4(12), 2187 - 92 Identification of hypoxanthine and guanine as the co-repressors for the purine regulon genes of Escherichia coli; Meng LM et al.; Addition of purine compounds to the growth medium of Escherichia coli and Salmonella typhimurium causes repressed synthesis of the purine biosynthetic enzymes . The repression is mediated through a regulatory protein, PurR . To identify the co-repressor(s) of PurR, two approaches were used: (i) mutations were introduced into purine salvage genes and the effects of different purines on pur gene expression were determined; (ii) purine compounds which dictate the binding of the PurR protein to its operator DNA were resolved by gel retardation . Both the in vivo and the in vitro data indicated that guanine and hypoxanthine are co-repressors . The toxic purine analogues 6-mercaptopurine and 6-thioguanine also activated the binding of PurR to its operator DNA. Mol Microbiol, 1990 Dec, 4(12), 2111 - 8 Generation of a cytotoxic T-lymphocyte response using a Salmonella antigen-delivery system; Flynn JL et al.; We have constructed a general-use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium . Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S . typhimurium strain . Mice immunized with CS-expressing Salmonella recombinants mount a CS-specific cytotoxic T-lymphocyte (CTL) response . This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice . The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen-delivery system. Poult Sci, 1990 Dec, 69(12), 2248 - 51 Combined halogen disinfectants in poultry processing; Williams DE et al.; Three organic N-halamine compounds (combined halogen disinfectants) were compared with free chlorine (as calcium hypochlorite) as bactericides against Salmonella typhimurium and unidentified normal poultry bacterial flora under controlled conditions of pH, temperature, and halogen demand similar to those encountered in poultry processing . Two of the compounds (3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone) at a concentration of 50 mg/L were found to cause a 99.9999% decline in viable organisms in less than 1 min at 48 C, whereas a third compound (1-bromo-3-chloro-4,4,5,5-tetramethyl-2-imidazolidinone) was found to be less suitable (5.6 min to 99.9999% decline under the same conditions). Poult Sci, 1990 Dec, 69(12), 2244 - 7 Effects of D-mannose on incidence and levels of salmonellae in ceca and carcass samples of market age broilers; Izat AL et al.; Two similar trials were conducted to evaluate the effects of 2.5% d-mannose (DM) in the drinking water of broilers for the first 10 days on incidence and levels of salmonellae in the ceca and on the carcass at market age . Controls received drinking water with no DM . Birds were reared on used litter in floor pens and were inoculated via the drinking water with 10(8) cfu/mL Salmonella typhimurium (ATCC 14028) on Day 3 . At 49 days, 60 birds per treatment were processed and the ceca contents and prechill carcass were evaluated for salmonellae incidence by the most probable number (MPN) method . Results were inconclusive: level of salmonellae in the ceca contents and carcass rinse was significantly lower in control samples than in DM samples in one of the two trials; the reverse was true in the other trial. Poult Sci, 1990 Dec, 69(12), 2128 - 33 Effect of litter condition on microbiological quality of freshly killed and processed broilers; Reiber MA et al.; Two similar trials were conducted to evaluate the effects of litter condition on microbiological quality of freshly killed (feathers intact) and processed broilers . Commercial broilers were reared to 49 days of age on new or previously used litter . Birds in half of the replicate pens were inoculated with Salmonella typhimurium via the drinking water on Days 2, 7, 14, and 21 . Broilers were sampled at the following processing locations: postkill, postpick, prechill, and postchill . Postchill carcasses from birds raised on previously used litter did not have significantly different aerobic plate counts, levels of coliforms, or numbers of salmonellae as compared with carcasses from birds raised on new litter . Live bird inoculation did significantly increase levels of salmonellae on the fully processed carcass. Appl Environ Microbiol, 1990 Dec, 56(12), 3741 - 7 Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact; Bonner CA et al.; The pathway construction for biosynthesis of aromatic amino acids in Escherichia coli is atypical of the phylogenetic subdivision of gram-negative bacteria to which it belongs (R . A . Jensen, Mol . Biol . Evol . 2:92-108, 1985) . Related organisms possess second pathways to phenylalanine and tyrosine which depend upon the expression of a monofunctional chorismate mutase (CM-F) and cyclohexadienyl dehydratase (CDT) . Some enteric bacteria, unlike E . coli, possess either CM-F or CDT . These essentially cryptic remnants of an ancestral pathway can be a latent source of biochemical potential under certain conditions . As one example of advantageous biochemical potential, the presence of CM-F in Salmonella typhimurium increases the capacity for prephenate accumulation in a tyrA auxotroph . We report the finding that a significant fraction of the latter prephenate is transaminated to L-arogenate . The tyrA19 mutant is now the organism of choice for isolation of L-arogenate, uncomplicated by the presence of other cyclohexadienyl products coaccumulated by a Neurospora crassa mutant that had previously served as the prime biological source of L-arogenate . Prephenate aminotransferase activity was not conferred by a discrete enzyme, but rather was found to be synonymous with the combined activities of aspartate aminotransferase (aspC), aromatic aminotransferase (tyrB), and branched-chain aminotransferase (ilvE) . This conclusion was confirmed by results obtained with combinations of aspC-, tyrB-, and ilvE-deficient mutations in E . coli . An example of disadvantageous biochemical potential is the presence of a cryptic CDT in Klebsiella pneumoniae, where a mutant carrying multiple enzyme blocks is the standard organism used for accumulation and isolation of chorismate.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1990 Dec, 4(12), 2119 - 26 Characterization of the type 1 fimbrial subunit gene (fimA) of Serratia marcescens; Nichols WA et al.; The nucleotide sequence of a DNA fragment that contains the fimA gene, encoding the major fimbrial subunit, of Serratia marcescens IA506 and associated flanking sequences has been elucidated . In addition, the origin of transcription has been identified and is located 120 base pairs upstream of the fimA initiation codon . The predicted amino acid sequence of the FimA polypeptide exhibits some degree of sequence homology with the fimbrial subunits encoded by the fimA determinants of Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli and also to Smf2, the major structural component of mannose-resistant (MR) fimbriae of S . marcescens . The Serratia adhesin that facilitates haemagglutination mediated by type 1 fimbriae is less susceptible to inhibition by D-mannose than has been observed to be the case in other type 1 fimbrial adhesins . The molecule conferring this adherence specificity has been shown to be distinct from the fimA gene product and, therefore, is analogous to the fimbrial systems reported in other species. Eur J Immunol, 1990 Dec, 20(12), 2763 - 8 Efficient recognition by rat T cell clones of an epitope of mycobacterial hsp 65 inserted in Escherichia coli outer membrane protein PhoE; Hogervorst EJ et al.; PhoE is a pore-forming protein, abundantly expressed in the Escherichia coli outer membrane . Previous investigations have shown the possibility of inserting antigenic determinants in cell surface-exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein . This method proved to be successful for foot-and-mouth disease virus B cell determinants . We have now shown for the first time that PhoE can also be used as a carrier molecule for T cell epitopes . A well-characterized T cell epitope (180-188) of the 65-kDa heat-shock protein (hsp 65) of Mycobacterium tuberculosis was expressed in PhoE and tested for recognition by specific T cell clones . Specific and efficient T cell proliferation was found after stimulation with this protein construct in vitro . Interestingly, paraformaldehyde fixation of antigen-presenting cells did not abrogate T cell recognition . Thus, in contrast to hsp 65 itself, recognition of epitope 180-188 in the context of PhoE appeared to be independent of antigen-processing events . At the level of polyclonal T cell responses the epitope in the context of PhoE is recognized more efficiently than 180-188 as synthetic peptide or in the context of the hsp 65 molecule itself . These findings indicate that PhoE may serve as attractive vaccine carrier not only for B, but also for T cell epitopes . Furthermore, the possibility for expression of PhoE constructs in attenuated Salmonella typhimurium strains offers the exciting prospect of new types of live oral vaccines expressing selected combinations of B and T cell epitopes. J Bacteriol, 1990 Dec, 172(12), 7151 - 6 Sequence of the pckA gene of Escherichia coli K-12: relevance to genetic and allosteric regulation and homology of E . coli phosphoenolpyruvate carboxykinase with the enzymes from Trypanosoma brucei and Saccharomyces cerevisiae; Medina V et al.; The sequence of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli K-12 and previous molecular weight determinations indicate that this allosteric enzyme is a monomer of Mr 51,316 . The protein is homologous to ATP-dependent phosphoenolpyruvate carboxykinases from Trypanosoma brucei and Saccharomyces cerevisiae . A potential ATP binding site was conserved in all three sequences . A potential binding site for the allosteric activator, calcium, identified in the E . coli enzyme, was only partially conserved in T . brucei and S . cerevisiae, consistent with the observation that the enzymes from the latter organisms were not activated by calcium . The published sequence of the ompR and envZ genes from Salmonella typhimurium is followed by a partial sequence that is highly homologous to pckA from E . coli . The order of these genes and the direction of transcription of the presumptive S . typhimurium pckA gene are the same as those in E . coli . The potential calcium binding site of the E . coli enzyme is conserved in the partial predicted sequence of the S . typhimurium phosphoenolpyruvate carboxykinase, consistent with the observation that calcium activation of the S . typhimurium phosphoenolpyruvate carboxykinase is very similar to that observed for the E . coli enzyme . A pckA mRNA transcript was observed in stationary-phase cells but not in logarithmically growing cells . The mRNA start site was mapped relative to the sequence of the pckA structural gene. Nucleic Acids Res, 1990 Nov 25, 18(22), 6503 - 8 A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes; Sharples GJ et al.; We report the discovery of a novel group of highly conserved DNA sequences located within the intergenic regions of the chromosomes of Escherichia coli, Salmonella typhimurium and other bacteria . These intergenic repeat units (IRUs) are 124-127 nucleotides long and have the potential to form stable stem-loop structures . The location of these sequences within the intergenic regions is variable with respect to known or putative signals for transcription and translation of the flanking genes . Some of the IRU sequences are transcribed, others are probably not . The structure and possible functions of these sequences are discussed in relation to palindromic units and other repeated DNA sequences in bacteria. Biochemistry, 1990 Nov 20, 29(46), 10480 - 7 Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium; Bhatia MB et al.; The chemical mechanism of the phosphoribosyltransferases (PRTases), although largely unknown, may proceed either via a concerted direct-transfer mechanism or with a two-step mechanism involving a carboxonium-like intermediate . To study this question, we have cloned the Salmonella typhimurium pyrE gene, coding for the enzyme orotate phosphoribosyltransferase (EC 2.2.4.10, OPRTase), and developed a bacterial strain that overproduces the enzyme, which we have purified to homogeneity . Initial velocity and product inhibition studies indicated that S . typhimurium OPRTase follows a random sequential kinetic mechanism . This result was further confirmed by equilibrium isotope exchange studies on two substrate-product pairs, PRPP-PPi and OMP-orotate . In addition, the rates of the individual equilibrium isotope exchanges allowed us to conclude that PPi release and PRPP release were the rate-determining steps in the forward and reverse reactions, respectively . Although partial reactions between the two substrate-product pairs, PRPP-PPi and OMP-orotate, were observed, further studies revealed that these exchanges were a result of contaminations . Our results are significant in that S . typhimurium OPRTase, like most PRTases but in contrast to its yeast homologue, follows sequential kinetics . The artifactual partial isotope exchanges found in this work may have implications for similar prior work on the yeast enzyme . In view of the careful isotope effect studies of Parsons and co-workers {Goitein, R.K., Chelsky, D., & Parsons, S.M . (1978) J . Biol . Chem . 253, 2963-2971} and the results obtained by us, we propose that PRTases may involve a direct-transfer mechanism but with low bond order to the leaving pyrophosphate moiety and attacking base. J Biol Chem, 1990 Nov 15, 265(32), 19892 - 7 Purification and characterization of a methionine aminopeptidase from Saccharomyces cerevisiae; Chang YH et al.; Methionine aminopeptidase (MAP), which catalyzes the removal of NH2-terminal methionine from proteins, was isolated from Saccharomyces cerevisiae . The enzyme was purified 472-fold to apparent homogeneity . The Mr of the native enzyme was estimated to be 36,000 +/- 5,000 by gel filtration chromatography, and the Mr of the denatured protein was estimated to be 34,000 +/- 2,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme has a pH optimum near 7.0, and its pI is 7.8 as determined by chromatofocusing on Mono P . The enzyme was inactivated by metalloprotease inhibitors (EDTA, o-phenanthroline and nitrilotriacetic acid), sulfhydryl-modifying reagents (HgCl2 and p-hydroxymercuribenzoic acid), and Zn2+ . Yeast MAP failed to cleave methionine p-nitroanilide . Among 11 Xaa-Ala-Ser analogues (Xaa = Ala, Asp, Gln, Glu, Ile, Leu, Lys, Met, Phe, Pro, and Ser), MAP cleaved only Met-Ala-Ser . MAP also cleaved methionine from other tripeptides whose penultimate amino acid residue is relatively small and/or uncharged (e.g . Pro, Gly, Val, Thr, or Ser) but not when bulky and/or charged (Arg . His, Leu, Met, or Tyr) . Yeast MAP displayed similar substrate specificities compared with those of Escherichia coli (Ben-Bassat, A., Bauer, K., Chang, S.Y., Myambo, K., Boosman, A., and Chang, S . (1987) J . Bacteriol . 169, 751-757) and Salmonella typhimurium MAP (Miller, C., Strauch, K . L., Kukral, A . M., Miller, J . L., Wingfield, P . T., Mazzei, G . J., Werlen, R . C., Garber, P., and Movva, N . R . (1987) Proc . Natl, Acad . Sci . U.S.A . 84, 2718-2722) . In general, the in vitro specificity of yeast MAP is consistent with the specificity observed in previous in vivo studies in yeast (reviewed in Arfin, S . M., and Bradshaw, R . A . (1988) Biochemistry 27, 7979-7984). J Biol Chem, 1990 Nov 15, 265(32), 19535 - 42 The nucleotide-binding site of HisP, a membrane protein of the histidine permease . Identification of amino acid residues photoaffinity labeled by 8-azido-ATP; Mimura CS et al.; The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP . These permeases are energized by ATP . HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport . In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP . HisP was derivatized with 8-azido-ATP (N3ATP) . Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin . The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced . Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively . Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases . These consensus motifs are found in many purine nucleotide-binding proteins . The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed. Biochemistry, 1990 Nov 13, 29(45), 10342 - 50 Comparison of the DNA-alkylating properties and mutagenic responses of a series of S-(2-haloethyl)-substituted cysteine and glutathione derivatives; Humphreys WG et al.; The mutagenicity of 1,2-dibromoethane is highly dependent upon its conjugation to glutathione by the enzyme glutathione S-transferase . The conjugates thus formed can react with DNA and yield almost exclusively N7-guanyl adducts . We have synthesized the S-haloethyl conjugates of cysteine and glutathione, as well as selected methyl ester and N-acetyl derivatives, and compared them for ability to produce N7-guanyl adducts with calf thymus DNA . The cysteine compounds were found to be more reactive toward calf thymus DNA and yielded higher adduct levels than did the glutathione compounds . Adduct levels tended to be suppressed when there was a net charge on the compound and were not affected by substitution of bromine for chlorine, as expected for a mechanism known to involve an intermediate episulfonium ion . Sequence-selective alkylation of fragments of pBR322 DNA was investigated . The compounds produced qualitatively similar patterns of alkylation, with higher levels of alkylation at runs of guanines . The compounds were also tested for their ability to act as direct mutagens in Salmonella typhimurium TA98 and TA100 . None of the compounds caused mutations in the TA98 frameshift mutagenesis assay . In the strain TA100, where mutation of a specific guanine by base-pair substitution produces reversion, all compounds were found to produce mutations, but the levels of mutagenicity did not correlate at all with the levels of DNA al