Poult Sci, 1993 Nov, 72(11), 2064 - 8 Location of Salmonella typhimurium during incubation and hatching of inoculated eggs; Cason JA et al.; Location of Salmonella during hatching of broiler chicks was studied in three experiments . Unincubated, fertile hatching eggs heated to 42 C were inoculated by immersion for 15 min in a 16 C physiological saline solution containing approximately 1 x 10(5) cfu/mL of a nalidixic acid-resistant strain of Salmonella typhimurium . All eggs were sanitized externally by wiping with a paper towel wet with 70% ethanol . When incubating eggs were transferred to a hatcher, each was placed in a closed paper bag to minimize cross-contamination . Unhatched eggs were sampled by cutting away the shell over the air cell, sanitizing the inner air cell membrane with 70% ethanol, and removing the contents through the membrane . Shells and membranes were crushed and mixed in 10 mL buffered peptone (BP) . Yolks were dissected out, dipped in 70% ethanol, and mixed with 10 mL BP . Embryos or chicks and all surrounding fluid were rinsed in 100 mL BP . A total of 172 eggs was sampled . Shells and membranes were 100% Salmonella-positive 30 min after inoculation, but only 38% were positive after 17 to 21 days of incubation . No chick rinses were positive before pipping, but 15% were positive after pipping . Yolk samples were 2% positive before pipping versus 8% after pipping . A majority of chicks hatching from positive shells and membranes were Salmonella-negative. Int Immunol, 1993 Nov, 5(11), 1431 - 6 Immunization with live versus killed Salmonella typhimurium leads to the generation of an IFN-gamma-dominant versus an IL-4-dominant immune response; Thatte J et al.; The mechanisms responsible for differential commitment of effector T cells to the production of either the IL-4/5/10 group or to the IL-2/IFN-gamma group of lymphokines during an immune response have not yet been clearly elucidated . We have used Salmonella typhimurium as a model murine bacterial parasite in BALB/c mice for live-cell versus killed-cell immunization and looked at the immune response in terms of delayed type hypersensitivity (DTH), IgG subclass distribution in the serum antibody response, and antigen-specific T cell proliferation and lymphokine secretion . The results indicate that the two forms of immunogen induce qualitatively different immune responses . Intraperitoneal immunization with live bacteria induces an IFN-gamma-dominant immune response associated with a strong DTH reaction and relatively higher levels of specific antibodies belonging to the IFN-gamma-dependent IgG2a isotype rather than the IL-4-dependent IgG1 isotype . Immunization with heat-killed bacteria gives rise to an IL-4-dominated response that shows excellent proliferative capacities in vitro, with lower levels of DTH responses and comparatively high levels of specific antibodies of the IgG1 isotype . IL-2 production in the responses generated by the two modes of immunization, however, is not preferentially associated with IFN-gamma production, unlike the reported profiles of long-lived murine T cell clones in vitro. Fundam Appl Toxicol, 1993 Nov, 21(4), 535 - 45 Comparative effects of immunotoxic chemicals on in vitro proliferative responses of human and rodent lymphocytes; Lang DS et al.; In order to determine the comparability of human and rodent in vitro systems, the direct effects of various therapeutic or environmental chemicals on proliferative responses of lymphocytes of mouse, rat, and human origins were examined and analyzed by a detailed statistical approach . Four compounds of diverse structure and mechanism of action which are known to impair lymphocyte transformation, such as hydroquinone, T-2 toxin, lead nitrate, as well as the widely used immunosuppressive drug cyclosporin A, were chosen as model test substances . T cells were stimulated by phytohaemagglutinin as well as monoclonal antibodies directed at the T cell receptor/CD3 complex, while B cells were activated by the T-independent mitogens, including Staphylococcus aureus cells, Escherichia coli lipopolysaccharide, and Salmonella typhimurium mitogen with specificity for human, mouse, and rat lymphocytes, respectively . In almost all cases the chemicals altered lymphoproliferative responses in a concentration-related manner in all three species . In general, overall similarities in the relative sensitivity of lymphoblastogenesis were obtained when the human dose-response curves were compared to the rodent response curves . Frequent, statistically significant species-dependent discrepancies of the overall response curves between mice and rats were observed . Large, statistically significant differences were observed for inorganic lead, revealing obvious divergences of the effect patterns in all cases, across all species . In this case, rodent species, especially the rat, were very sensitive to immunomodulation by lead, whereas human cells were relatively resistant . It is suggested that direct interspecies comparisons of immunological effects due to chemical treatment in vitro can provide a greater understanding of the relationship between animal and human data, which will improve the confidence of extrapolation from findings in laboratory animals to human health risk. Carcinogenesis, 1993 Nov, 14(11), 2303 - 7 Increased mutagenicity of 1,2-dibromo-3-chloropropane and tris(2,3-dibromopropyl)phosphate in Salmonella TA100 expressing human glutathione S-transferases; Simula TP et al.; We have expressed human glutathione S-transferases GSTA1-1 and GSTP1-1 in Salmonella typhimurium TA100 in order to assess the ability of these enzymes to modulate the mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) and tris(2,3-dibromopropyl)phosphate (Tris-BP) . Both compounds were mutagenic when activated by Aroclor-induced rat liver microsomes . However, when Aroclor-induced rat liver microsomes were used together with the GST-expressing strains the mutagenicity of both DBCP and Tris-BP was markedly potentiated . Neither of the GST-expressing strains potentiated the mutagenicity in the absence of microsomes, indicating that cytochrome P450-mediated metabolism was a prerequisite for GST-mediated potentiation . With DBCP both isozymes had comparable effects on mutagenic frequency, although the highest dose of DBCP was toxic in strains expressing GSTP1-1 . In the case of Tris-BP, GSTP1-1 was much more active in potentiating the mutagenicity . These results indicate that human GSTs can play an important role in the activation of compounds such as DBCP and Tris-BP to mutagenic metabolites. Carcinogenesis, 1993 Nov, 14(11), 2233 - 7 Bacterial and mammalian cell mutagenicity of four optically active bay-region 10,11-diol-8,9-epoxides of the nitrogen heterocycle dibenz{a,h}acridine; Chang RL et al.; The mutagenic activities of the enantiomers of the diastereomeric pair of bay-region 10,11-diol-8,9-epoxides of dibenz{a,h}acridine (DB{a,h}ACR) were evaluated in histidine-dependent strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells . In strains TA98 and TA100 of S.typhimurium, the (-)-{8S,9R,10R,11S} diol-epoxide was the most mutagenic compound, inducing 1200 and 6900 His+ revertants/nmol respectively . The mutagenic activity of each of the remaining three isomers was essentially independent of the bacterial strain used and had 14-72% of the activity of the {S,R,R,S} isomer . However, in Chinese hamster V79 cells, the (+)-{8R,9S,10S,11R} diol-epoxide was the most mutagenic compound (68 8-azaguanine resistant variants/nmol/10(5) cells), inducing from 2 to 11 times as many mutations as the other three isomers . These results are analogous to previous studies with the bay-region diol-epoxides of other polycyclic hydrocarbons in that the isomer with {R,S,S,R} absolute configuration has had variable activity in the bacterial assays, but has generally been the most active in the mammalian cells . Furthermore, this isomer has almost always been highly tumorigenic in the mouse. Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9983 - 7 Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase; Morollo AA et al.; Anthranilate synthase {chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27} catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine . A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-{(1-carboxyethenyl)-oxy}-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC) . Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S . typhimurium . Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan . Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase . ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4 . The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction . The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism. Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9852 - 6 Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase; Crennell SJ et al.; Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases . The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution . The viral enzyme is a tetramer; the bacterial enzyme, a monomer . Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%) . The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+ . Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues . However, an rms fit of 3.1 A between 264 C alpha atoms of the S . typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold . In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes . The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure . Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed. Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10390 - 4 Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism; Jones BD et al.; Invasive Salmonella typhimurium induces dramatic actin rearrangements on the membrane surface of mammalian cells as part of its entry mechanism . These changes, which are best characterized as membranous ruffles, closely resemble the membrane changes that occur when a growth factor binds to its receptor . Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated ruffling and stress-fiber formation, respectively . In addition, actin changes induced by the oncogene ras were also shown to be regulated by rac and rho . Because Salmonella-induced actin rearrangements resemble those caused by growth factors, we investigated whether ras, rho, or rac regulates the membrane ruffling elicited by S . typhimurium . Surprisingly, inhibition of the functions of these GTPases had no effect on the ability of invasive S . typhimurium to induce membrane ruffles on a variety of tissue culture cells including Madin-Darby canine kidney cells, Swiss 3T3 fibroblasts, and Hep-2 cells . These results led us to examine the interactions of S . typhimurium with Henle-407 intestinal cells, which lack epidermal growth factor receptor on their membrane surface . We found no difference in the ability of invasive S . typhimurium to induce membrane ruffling and to enter Henle-407 cells with or without the epidermal growth factor receptor on the membrane surface . We, therefore, conclude that invasive S . typhimurium induces membrane ruffling and its own internalization by a rac-independent, growth factor-receptor-independent signaling pathway. J Cell Biol, 1993 Nov, 123(4), 895 - 907 Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils; McCormick BA et al.; In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane . In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S . typhimurium . We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport . However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued . In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration . Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN . Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells . Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8) . However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration . These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium . Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium. J Bacteriol, 1993 Nov, 175(22), 7290 - 300 dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product; Henrich B et al.; Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli . We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine . Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product . Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA . The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression . The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium . It also displays significant homology to the products of the S . typhimurium opdA and the E . coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae . No potential export signals could be inferred from the amino acid sequence . Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E . coli and used to investigate some of its biochemical and biophysical properties. J Bacteriol, 1993 Nov, 175(22), 7200 - 8 Two global regulatory systems (Crp and Arc) control the cobalamin/propanediol regulon of Salmonella typhimurium; Ailion M et al.; The genes for cobalamin (vitamin B12) biosynthesis (cob) are coregulated with genes for degradation of propanediol (pdu) . Both the cob and pdu operons are induced by propanediol by means of a positive regulatory protein, PocR . This coregulation of a synthetic and a degradative pathway reflects the fact that vitamin B12 is a required cofactor for the first enzyme in propanediol breakdown . The cob/pdu regulon is induced by propanediol under two sets of growth conditions, i.e., during aerobic respiration of a poor carbon source and during anaerobic growth . We provide evidence that, under aerobic conditions, the Crp/cyclic AMP system is needed for all induction of the pocR, cob, and pdu genes . Anaerobically, the Crp/cyclic AMP and ArcA/ArcB systems act additively to support induction of the same three transcription units . The fact that these global control systems affect expression of the gene for the positive regulatory protein (pocR) as well as the pdu and cob operons is consistent with our previous suggestion that these two global controls may act directly only on the pocR gene; their control over the cob and pdu operons may be an indirect consequence of their effect on the level of PocR activator protein . The reported experiments were made possible by the observation that pyruvate supports aerobic growth of all of the mutants tested (cya, crp, arcA, and arcB); pyruvate also supports anaerobic growth of these mutants if the alternative electron acceptor, fumarate, is provided . By using pyruvate as a carbon source, it was possible to grow all of these mutant strains under identical conditions and compare their expression of the cob/pdu regulon . The role of Crp in control of vitamin B12 synthesis suggests that the major role of vitamin B12 in Salmonella spp . is in catabolism of carbon sources; the coregulation of the cob and pdu operons suggests that propanediol is the major vitamin B12-dependent carbon source. J Bacteriol, 1993 Nov, 175(21), 7086 - 91 Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions; Mahan MJ et al.; Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments . We describe here a transductional method using the generalized transducing phage of S . typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes . In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid . Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment . Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient . However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection . In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced . In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid. EMBO J, 1993 Nov, 12(11), 4053 - 62 Molecular genetic analysis of a locus required for resistance to antimicrobial peptides in Salmonella typhimurium; Parra-Lopez C et al.; The innate immunity of vertebrates and invertebrates to microbial infection is mediated in part by small cationic peptides with antimicrobial activity . Successful pathogens have evolved mechanisms to withstand the antibiotic activity of these molecules . We have isolated a set of genes from Salmonella typhimurium which are required for virulence and resistance to the antimicrobial peptides melittin and protamine . Sequence analysis of a 5.7 kb segment from the wild-type plasmid conferring resistance to protamine contained five open reading frames: sapA, sapB, sapC, sapD and sapF, organized in an operon structure and transcribed as a 5.3 kb mRNA . SapD and SapF exhibited similarity with the 'ATP binding cassette' family of transporters including the bacterial Opp and SpoOK, involved in the uptake of oligopeptides; the yeast STE6, necessary for the export of a peptide pheromone; and the mammalian mdr, which mediates resistance to chemotherapeutic agents in cancer cells . SapA showed identity with other periplasmic solute binding proteins involved in peptide transport . The SapABCDF system constitutes a novel transporter for enteric bacteria and the first one harboring a periplasmic component with a role in virulence. Rev Med Chil, 1993 Nov, 121(11), 1245 - 51 {Mutagen extraction from bile of patients with inflammatory biliary pathology: Ames test using blue rayon}; Araya JC et al.; Gallbladder carcinoma is frequent in Chile . The aim of this study was to report the mutagenicity of whole human bile, using the Ames/Salmonella microsome assay with Salmonella typhimurium TA98 . The bile of 19 patients, aged 23 to 64 years old, subjected to cholecystectomy was examined, and mutagen activity was found in 13 (72%) . Mutagens were extracted using blue rayon and three dilutions for the eluted material from blue rayon were used (50, 100 and 200 ul) . The best result was obtained using 200 ul . In some cases, the amount of revertive colonies was very high (over 5 times the control value) . We propose that the bile from these patients possibly contains mutagenic substances with frame shift mutagenic activity and that these substances may be related to gallbladder carcinoma . Our results have addressed the importance of bile studies to elucidate the pathogenesis of gallbladder carcinoma. Mutagenesis, 1993 Nov, 8(6), 577 - 81 Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains; O'Donovan MR et al.; Formaldehyde was examined for bacterial mutagenicity using Escherichia coli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absence of any exogenous source of metabolic activation . Using pre-incubation exposure, clear mutagenicity was seen for TA98, TA100 and TA102, and both E . coli strains . In standard plate-incorporation assays, consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101) . No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538 using either method of exposure . These data confirm the enhanced ability of the pre-incubation method to detect the mutagenicity of formaldehyde both quantitatively, as expressed by numbers of revertant colonies, and qualitatively, in terms of the range of indicator strains reverted . The relatively greater sensitivity of the pre-incubation assay is probably due to better containment of a volatile agent and/or lack of interaction with agar during the initial period of exposure . The findings are consistent with the suggestion that formaldehyde induces lesions in bacteria which are, at least to some extent, excision-repairable, and indicate that the presence of the R-factor plasmid may be required for the expression of its mutagenicity in excision repair-deficient Salmonella. Mutagenesis, 1993 Nov, 8(6), 527 - 32 Genetic differences between the standard Ames tester strains TA100 and TA98; Jurado J et al.; The standard Ames tester strains of Salmonella typhimurium are separated by many steps in their pedigree, some involving mutagen treatments, and contain independently isolated uvrB-bio-gal deletions and rfa mutations . In this work the araD531 mutation was introduced into the Ames tester strains TA100 and TA98 . The responsiveness of the resulting strains (BA15 and BA14) to a number of chemical mutagens was then assessed by monitoring the induction of forward mutations to L-arabinose resistance (Ara test) . Here we have shown that these two strains of the Ames test differ greatly in their responses to mutagens, in ways that are not associated with the mutagenic specificities of the original his mutations . In general, the genetic background of strain TA100 appears to be more sensitive to the killing effects of chemicals than that of TA98 . The greatest differences were found with nifurtimox (NFX) and its analogue, compound 1K . The Ara test responded to the mutagenic effects of these two nitrofurans when carried out in the genetic background of strain TA98 but not in that of TA100 . A higher sensitivity to the lethal effects of NFX and 1K together with the greater nitro-reduction capability of strain TA100 as compared with TA98 might explain the differences . In conclusion, our results indicate that the standard Ames S . typhimurium tester strains are not isogenic and that genetic differences at loci other than his might be significant for mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS) Chem Res Toxicol, 1993 Nov-Dec, 6(6), 895 - 9 The influence of delivery rate on the chemistry and biological effects of nitric oxide; Tamir S et al.; Nitric oxide can be introduced slowly and steadily into aqueous solutions, including cell culture media, over extended periods of time via semipermeable Silastic (a registered trademark of the Dow Corning Corp.) polymer membranes . The rates of introduction are predictable and reproducible and can approach rates of nitric oxide production by stimulated cells, such as macrophages, that express inducible nitric oxide synthases . DNA damage in Chinese hamster ovary cells by membrane-delivered nitric oxide is comparable to that observed in the DNA of stimulated macrophages . Toxicity and mutagenicity of nitric oxide toward Salmonella typhimurium, toxicity of nitric oxide toward Chinese hamster ovary cells, and nitrosation of dimethylmorpholine are all more efficient when nitric oxide is delivered by membrane than when an equivalent amount of gaseous nitric oxide is added by syringe. Chem Res Toxicol, 1993 Nov-Dec, 6(6), 825 - 36 1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisD3052: Hoogsteen base-pairing at pH 5.8; Singh US et al.; The exocyclic DNA adduct 1,N2-propano-2'-deoxyguanosine (PdG) was inserted into the oligodeoxynucleotide 5'-CGC(PdG)CGGCATG-3' and annealed to the complementary oligodeoxynucleotide 5'-CATGCCGCGCG-3' . This sequence is derived from a spontaneous revertant of the hisD3052 gene in a frameshift-sensitive tester strain of Salmonella typhimurium and is a hotspot for two-base pair deletions . The solution structure of the modified duplex was examined by 1H NMR spectroscopy . The exocyclic lesions resulted in loss of Watson-Crick base-pairing capability . Modification resulted in an approximately 24 degrees C decrease in Tm of the duplex . NMR experiments revealed pH-dependent conformational equilibria, which involved the modified base pair and its 3'-neighbor base pair . At pH 5.8, the lesion resulted in a localized perturbation of the B-form helix . PdG was rotated about the glycosyl bond from the anti to the syn conformation, thus placing the propano moiety into the major groove . This resulted in the observation of a strong NOE between the imidazole proton of PdG and the anomeric proton of the attached deoxyribose . Additional NOEs were observed between the methylene protons of the propano moiety and H5 and H6 of the 5'-neighbor cytosine . An imino proton resonance from the cytosine complementary to PdG and protonated at N3, characteristic of a Hoogsteen base pair, was observed at 15 ppm, but was broadened due to exchange with water . The amino protons of the complementary cytosine were shifted downfield from the other cytosine amino protons, characteristic of a Hoogsteen-like conformation at the site of modification . A second equilibrium involved the 3'-neighbor base pair, which alternated between Watson-Crick and Hoogsteen pairing, also via rotation of the guanosine glycosyl bond from the anti to the syn conformer . The conformational exchange of the 3'-neighbor base pair was sufficiently slow on the NMR time scale to allow simultaneous observation of resonances from the Watson-Crick and the Hoogsteen conformers. Mikrobiologiia, 1993 Nov-Dec, 62(6), 1093 - 100 {Antimutagenic effect of culture fluid obtained as a result of propionic acid fermentation}; Vorob'eva LI et al.; It was shown for first time that propionic acid bacteria excrete in the medium substances with antimutagenic activity against mutagenicity of 4-nitroquinoline-N-oxide in Salmonella typhimurium TA100 . The substances act as antimutagens . Antimutagenic action of filtrate of culture liquid increased with time preincubation with mutagen and dose increase . The most activity was found in the culture of logarithmic and early stationary phase (24-48 h) . The substances with antimutagenic activity are formed in the medium with glucose, lactose and lactate . Antimutagenic factor (factors) is dialysable, thermostable and is not presented with peptide. Mol Microbiol, 1993 Nov, 10(3), 685 - 96 Chromosomal domains of supercoiling in Salmonella typhimurium; Pavitt GD et al.; The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains . It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled . We have addressed this question genetically, using a supercoiling-sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome . We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling . These findings have implications for the organization of the bacterial genome. Mol Microbiol, 1993 Nov, 10(3), 655 - 64 The XbaI-BlnI-CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2; Liu SL et al.; We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis . The chromosome of 4600 kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb . Cleavage sites of I-Ceul, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BlnI sites in glt-tRNA are also conserved, but other sites are less conserved . Transposon Tn10, located at 60 different positions in the chromosome of S . typhimurium, was transduced by bacteriophage P22 into S . enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10 . Gene order in S . enteritidis is identical to S . typhimurium LT2 and similar to E . coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2 . Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E . coli K-12 and S . typhimurium LT2 . This inversion resembles the inversion between S . typhimurium and E . coli, but is longer at both ends. Drug Metab Dispos, 1993 Nov-Dec, 21(6), 1048 - 56 Characterization of cytochrome P-450 2B6 in human liver microsomes; Mimura M et al.; A cytochrome P-450 (P-450) enzyme of the CYP2B subfamily was partially purified from human liver microsomes and characterized with respect to immunochemical properties, N-terminal amino acid sequence, and catalytic activities toward typical P-450 substrates . P-450 enzymes were monitored in chromatographic fractions by immunoblotting analysis using antibodies raised against a monkey P-450 2B, as well as several purified human P-450 enzymes . The final P-450 2B preparation thus obtained was contaminated with P-450 3A4, but an N-terminal amino acid sequence matching the sequence predicted from the CYP2B6 cDNA was obtained . The apparent M(r) of this protein was 48 kDa, and the migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the same as that of the P-450 2B6 protein expressed in a human lymphoblast cell line . Immunoblotting analysis of 50 human liver samples revealed that the protein band considered to be P-450 2B6 was detected in only 12 samples, with four of these having relatively high levels . Several activities toward typical P-450 substrates were determined in a reconstituted monooxygenase system containing partially purified P-450 2B6 and compared with those obtained using a highly purified preparation of P-450 3A4 enzyme; we found that most of the activities were similar in these preparations, except that the partially purified P-450 2B6 showed high rates of activation of the mutagens 6-aminochrysene and 3-methoxy-4-aminoazobenzene to genotoxic metabolites in Salmonella typhimurium NM2009 strain.(ABSTRACT TRUNCATED AT 250 WORDS) Acta Trop, 1993 Nov, 55(3), 171 - 80 In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations; Celentano AM et al.; The macrophage function was investigated in mice infected with Trypanosoma cruzi . Two subpopulations of the parasite were utilized, RA and K98 . Strain RA is efficiently internalized by macrophages and is lethal for mice, and clone K98 is poorly phagocytosed by macrophages and is not lethal . Treatment with silica enhanced parasitemia and mortality in mice infected with both parasite subpopulations . Parasitemia kinetics, however, were affected only in mice infected with RA, which suggests that macrophage effector mechanisms may play a more relevant role in this experimental group than in mice infected with K98 . Resistance to Salmonella typhimurium infection and bactericidal activity of macrophages depended upon the T . cruzi subpopulation utilized and the infection period . Infection with K98 induced only a trend towards enhanced resistance to bacterial challenge during both the acute and chronic phases, whereas a significantly enhanced bactericidal activity of spleen and liver phagocytes was observed . Mice acutely infected with RA showed significantly enhanced susceptibility to S . typhimurium infection and lower bactericidal activity . Mice surviving infection with this aggressive strain, however, showed significantly enhanced resistance and bactericidal activities . Mice acutely infected with the RA strain displayed a dissociation between macrophage capacities to control S . typhimurium and T . cruzi . A similar phenomenon was also observed in other parasitoses (schistosomiasis, African trypanosomiasis) . This fact may be due to differences in the lethal mechanisms through which macrophages control these parasites and S . typhimurium. Mutat Res, 1993 Nov, 319(3), 223 - 36 Comparison of three short-term assays: results on seven chemicals . Potential contribution to the control of water genotoxicity; Le Curieux F et al.; Three short-term assays (the SOS chromotest, the Ames fluctuation test and the newt micronucleus test) were used to evaluate the genotoxicity of seven chemicals (4-nitroquinoline 1-oxide, potassium dichromate, formaldehyde, sodium hypochlorite, benzo{a}pyrene, cyclophosphamide and 2-naphthylamine) . In the SOS chromotest, all seven compounds except sodium hypochlorite and cyclophosphamide were found to induce primary DNA damage in E . coli . With the Ames fluctuation test, all seven chemicals except sodium hypochlorite showed mutagenic activity on Salmonella typhimurium TA100, TA102 or TA98 . The newt micronucleus assay detected a clastogenic effect of all seven compounds except formaldehyde on the peripheral blood erythrocytes of Pleurodeles waltl larvae . For each compound, the sensitivity of the tests was compared; (1) the SOS chromotest is the least sensitive assay in every case, (2) the Ames fluctuation test is the most sensitive assay for studied chemicals with direct genotoxic effect and (3) the newt micronucleus assay is the most sensitive test for tested compounds with indirect genotoxic activity . The potential contribution of these three tests in the monitoring of water genotoxicity is discussed. Mutat Res, 1993 Nov, 303(3), 135 - 42 Mutagenicity and antimutagenicity of extracts of three spices and a medicinal plant in Thailand; Higashimoto M et al.; Three kinds of spices (caraway, coriander and black pepper seeds) and a medicinal plant called 'tong tak' in Thai (Baliospermum axillar, a species of the spurge family) were fractionated into hot water, methanol and hexane extracts . These extracts were not mutagenic for Salmonella typhimurium strains TA98 and TA100 by the Ames assay . However, when the extracts were treated with nitrite, samples of the water and methanol extracts were mutagenic for strain TA100 without metabolic activation . The mutagenicity of the nitrite-treated methanol and hot water extracts of black pepper was highest (8380 and 22,200 His+ per 0.1 g of spice powder, respectively), and that of the nitrite-treated hot water extracts of caraway and tong tak was moderate . The hot water extracts were examined for their antimutagenic activity against mutagenicity induced by various carcinogens by the Ames assay, using the preincubation technique . The tested samples (equivalent to 1-2 mg of spice powder) reduced the mutagenicity induced by 2.7 nmole (397 ng) of N-methyl-N'-nitro-N-nitrosoguanidine by more than 84%, and that induced by dimethylnitrosamine (1.48 mg) or ICR-170 (10 ng) by 30-60% . However, they did not inhibit the mutagenic activity of 1-nitropyrene, 3-nitrofluoranthene, AF-2, methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-acetylaminofluorene, benzo{a}pyrene or IQ. Mutat Res, 1993 Nov, 303(3), 127 - 33 Dimethyl sulfoxide (DMSO) is mutagenic for bacterial mutagenicity tester strains; Hakura A et al.; We used the Ames method with the modification of pre-incubation to evaluate the potential mutagenicity of DMSO . We performed the assays using nine different Ames Salmonella strains and Escherichia coli strains WP2 and WP2uvrA . DMSO was found to be mutagenic for Salmonella typhimurium TA1537 and TA2637 (the latter strain being isogenic to TA1537 but carrying plasmid pKM101) and for E . coli WP2uvrA . The mutagenic activity of DMSO observed at a concentration of 33% was about 10 times higher than the background level (65 revertants induced) for TA1537 after 20 min of incubation, where some lethal toxicity was observed . The mutagenicity of DMSO was observed in the presence and absence of rat liver S9 mix. J Bacteriol, 1993 Nov, 175(21), 7006 - 15 Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM; Gillen KL et al.; The flgM gene product has been shown to be a negative regulator of flagellin transcription in Salmonella typhimurium (K . L . Gillen and K . T . Hughes, J . Bacteriol . 173:2301-2310, 6453-6459, 1991; K . Ohnishi, K . Kutsukake, H . Suzuki, and T . Iino, Mol . Microbiol . 6:3149-3157, 1992) . Mud-lac fusions to the flgM gene were isolated and used to characterize the regulation of flgM gene expression . Transcription of the flgM gene was decreased more than 30-fold in strains with the flagellar master regulatory genes, flhC and flhD, deleted . A class 2 flagellar defect caused a slight increase of flgM gene transcription unless a wild-type copy of the flgM gene was present, in which case transcription was decreased threefold . A deletion in the gene for the alternative sigma factor sigma 28 (FliA) caused a fourfold decrease in flgM expression . Insertional inactivation of a gene upstream of the flgM gene (flgA) in a fliA mutant strain caused transcription of the flgM gene to be decreased to a basal level . Northern (RNA) blot analysis confirmed the presence of two transcripts through the flgM gene, one which initiates upstream of the flgM gene and a second which initiates upstream of the flgA gene. Gene, 1993 Oct 29, 133(1), 103 - 8 Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG; Roman SJ et al.; The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation . The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN . During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system . CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown . We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch . We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein . The sequence data revealed a 91.8% amino acid (aa) identity between E . coli and S . typhimurium FliG . Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector . FliG was successfully overproduced and purified to > 98% purity . Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell . Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction. J Biol Chem, 1993 Oct 25, 268(30), 22469 - 79 Membrane topology of a P-type ATPase . The MgtB magnesium transport protein of Salmonella typhimurium; Smith DL et al.; P-type ATPases are a family of cation transport enzymes present in all species from bacteria to mammals whose members mediate membrane flux of all common biologically relevant cations . More than 50 members of this family of transporters have been sequenced; extensive structural data are available, and several members have been analyzed by site-directed mutagenesis . Nonetheless, there is no current consensus regarding their membrane topology . In this work, the Salmonella typhimurium Mg2+ transporting P-type ATPase encoded by the MgtB locus has been used as a model for P-type ATPases . Unlike other prokaryotic P-type ATPases, the MgtB protein is similar in length, amino acid sequence, and hydropathy profile to known eukaryotic P-type ATPases . The membrane topology of MgtB was analyzed by several epitope insertions in MgtB and from the activity of 35 protein fusions between MgtB and the reporter enzymes BlaM (beta-lactamase) and LacZ (beta-galactosidase) . The epitope insertions within MgtB all retained function as assessed by cation uptake assays and were regulated normally by the level of Mg2+ within the growth medium . The epitope insertion and fusion protein data are completely incompatible with the numerous previously proposed models for P-type ATPases predicting 7, 8, 9, or 12 transmembrane segments . Rather, they indicate that MgtB contains 10 transmembrane segments with both amino and carboxyl termini residing within the cytosol . By extension, we suggest that all eukaryotic P-type ATPases contain 10 transmembrane segments with both termini within the cytosol. J Biol Chem, 1993 Oct 25, 268(30), 22269 - 72 A novel intersubunit repair mechanism in the tryptophan synthase alpha 2 beta 2 complex . Critical role of the beta subunit lysine 167 in intersubunit communication; Yang XJ et al.; This study explores intersubunit communication in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium . We find that an engineered mutation in the contact region between the alpha and beta subunits remarkably alters the catalytic and spectroscopic properties of the beta subunit in the alpha 2 beta 2 complex . Ligands that bind to the alpha subunit largely repair the deleterious effects of the beta subunit mutation in the alpha 2 beta 2 complex . The conserved residue chosen for mutation, beta subunit lysine 167, appears to form an ion pair with alpha subunit aspartate 56 in the crystal structure of the wild type alpha 2 beta 2 complex . Although replacement of beta subunit lysine 167 by threonine does not prevent formation of the alpha 2 beta 2 complex, this mutation reduces the rate of synthesis of L-tryptophan from L-serine and indole (beta reaction) 25-fold . Ligands that bind to the alpha subunit (indole-3-glycerol phosphate, indole-3-propanol phosphate, alpha-glycerol 3-phosphate, or potassium phosphate) largely restore the activity of the mutant alpha 2 beta 2 complex in the beta reaction . We conclude that beta subunit lysine 167 plays an important role in intersubunit communication in the alpha 2 beta 2 complex . The striking allosteric effects of alpha subunit ligands on the mutant beta subunit in the alpha 2 beta 2 complex may result from ligand-induced conformational changes in the alpha subunit that are transmitted to the beta subunit and repair the mutational defect in the beta subunit. J Mol Biol, 1993 Oct 20, 233(4), 739 - 52 The 1.7 A refined X-ray structure of the periplasmic glucose/galactose receptor from Salmonella typhimurium; Zou JY et al.; The X-ray structure of the periplasmic glucose/galactose receptor (binding protein) of Salmonella typhimurium (GBP-S) has been refined at 1.7 A resolution with an R-factor of 19.0% . The model contains all 309 residues of the amino acid sequence, 153 water molecules, a calcium ion and beta-D-galactose . The protein consists of two very similar structural domains, each of which is composed a core of parallel beta-sheet flanked on both sides by alpha-helices . Three short stretches of amino acid chain (from symmetrically related portions of the structure) link the domains, and presumably act as a hinge to allow their relative movement in functionally important conformational changes . Galactose is bound between the domains, interacting with a number of side-chains from the loops lining the binding cleft . A combination of hydrogen bonding, hydrophobic and steric effects give rise to tight binding (dissociation constant 0.2 microM) and high specificity . Of nine hydrogen bonding groups, three are aspartate, three asparagine, one histidine (unprotonated), one arginine and one water, contributing 13 hydrogen bonds in total . Additional residues pack against (primarily) non-polar faces of the sugar molecule . The precise arrangement of the hydrogen bonding and hydrophobic residues results in an enclosed binding site with a shape that is a composite of those of the allowed sugar molecules . It is presumed that ligands bind to a more open form of the receptor that then closes by rotation in the hinge . Comparison with the GBP-S structure solved earlier in complex with glucose shows no significant changes, even for the aspartate residue most directly involved with the different sugars . Comparison with the galactose/glucose receptor of Escherichia coli indicates that these two proteins are very similar in overall structure, with the main difference being a 2 to 3 degrees rotation in the hinge . This difference appears to be the result of different crystal packing for the two proteins; it is likely that both conformations are normally found in solution. FEBS Lett, 1993 Oct 18, 332(3), 260 - 2 The effect of sugars on the morphology of the bacterial flagellum; Seville M et al.; Using dark-field microscopy, we have found that certain sugars cause the normal-to-curly helical transition of bacterial flagella . Titration of flagella isolated from Salmonella typhimurium with 16 different carbohydrates showed that: (i) only certain sugars cause the transition . There is no obvious relationship between the simple physico-chemical properties of the sugar and whether the sugar causes the transition or not; (ii) the efficacies of sugars that do cause the transition differ markedly . For these sugars there is a relationship between efficacy and molecular size . These results suggest that the specific, though weak, binding of sugars to sites on flagella cause the morphological transition. Cell Immunol, 1993 Oct 15, 151(2), 336 - 44 Tumor necrosis factor (TNF alpha) regulates intestinal mucus production during salmonellosis; Arnold JW et al.; Mucus production by goblet cells in the gastrointestinal tract following Salmonella typhimurium infection using a ligated ileal loop model in mice was investigated . Assessment of the morphology of the loop tissue after Salmonella challenge revealed generalized tissue inflammation, characterized by edema and an inflammatory cell infiltrate . Villi were shortened and blunted, and crypts contained an increased number of cells with mitotic figures . Production of TNF alpha in the loops followed Salmonella challenge and occurred at the same time as the pathological sequelae . A nearly 50% decrease in the number of goblet cells in infected tissue compared to tissue from noninfected controls was observed at these same times . The sulfation of mucins produced by the goblet cells in infected tissues was increased in the villi but was unchanged in the crypts compared to uninfected tissues . Treatment of mice with antibody to TNF alpha before Salmonella challenge abrogated tissue pathology and returned goblet cell numbers and mucin profiles to those observed in noninfected controls . Our results indicate that TNF alpha may mediate changes in goblet cell expression and mucin sulfation in response to Salmonella challenge. Eur J Biochem, 1993 Oct 15, 217(2), 771 - 9 Comparison of X-ray powder-diffraction data of various bacterial lipopolysaccharide structures with theoretical model conformations; Kastowsky M et al.; X-ray powder-diffraction experiments have been performed on dry samples of lipid A and various rough-mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli . The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples . The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra-LPS . Upon treatment with water-saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections . The increase in bilayer dimensions normally was about 0.3 nm . X-ray intensities were used for the determination of the inner bilayer structure, i.e . for calculation of the one-dimensional electron-density distribution across the bilayer . For lipid A and several Re-LPS, Rd2-LPS, Rd1-LPS and Rc-LPS samples, a striking coincidence of the electron-density distributions in the lipid-A domain was found, suggesting that in all these structures the lipid-A portion is similarly arranged . For Rb1 and Ra-LPS the lipid-A domain could not be resolved due to the limited number of observed reflections . For other Re-mutant lipopolysaccharide samples, quite different X-ray patterns were obtained . Some samples yielded diffraction patterns indicating a very high state of order in the lipid-A domain, whereas, in others, a significantly reduced order in the lipid-A domain was inferred . Comparison of the X-ray data with features of a calculated three-dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure. Biochemistry, 1993 Oct 12, 32(40), 10658 - 65 Inhibition of viral capsid assembly by 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid); Teschke CM et al.; The precursor shells of dsDNA bacteriophages are assembled by the polymerization of competent states of coat and scaffolding subunits . The fluorescent dye 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid) (bisANS) binds to both the coat and scaffolding proteins from the Salmonella typhimurium bacteriophage P22 . It displays little affinity for the polymerized forms of the proteins . The subunits with bound bisANS are incapable of assembling into procapsids . The binding constants of bisANS for both coat and scaffolding protein monomers have been measured and are 7 and 6 microM, respectively . Binding of bisANS to coat protein has little effect on the conformation as determined by circular dichroism and susceptibility to proteolysis . Binding of bisANS to scaffolding protein induces a change in the secondary structure consistent with a loss of alpha-helix, and an altered susceptibility to proteolysis . We suggest that the bisANS is probably binding at sites responsible for intersubunit interactions and thereby inhibiting capsid assembly. Biochim Biophys Acta, 1993 Oct 6, 1202(2), 297 - 304 CheZ mutants with enhanced ability to dephosphorylate CheY, the response regulator in bacterial chemotaxis; Huang C et al.; CheZ is a component of the chemotaxis signal-transduction pathway in Escherichia coli and Salmonella typhimurium . It is responsible for accelerating dephosphorylation of CheY and thereby antagonizing the tumble-promoting activity of CheY . In the absence of functional CheZ, cells are non-chemotactic and tumble constantly . We characterized the effects of two mutations in CheZ, R54C and V166G, that are unusual in that they cause cells to have a smooth swimming bias . These mutations were isolated as second-site suppressors of mutations in the switch complex responsible for regulating the direction of flagellar rotation (Yamaguchi, S., Aizawa, S.-I., Kihara, M . Isomura, M., Jones, C.J . and Macnab, R.M . (1986) J . Bacteriol . 168, 1172-1179) . When produced at low levels in a delta cheZ host strain, CheZ R54C and CheZ V166G supported chemotaxis . However, when moderately overproduced they markedly inhibited chemotactic ability . In vitro studies revealed that these mutations enhanced the ability of CheZ to accelerate dephosphorylation of CheY . These results are discussed in relation to the possible roles and interactions of CheZ in the chemotaxis system. Biochemistry, 1993 Oct 5, 32(39), 10404 - 13 Characterization of the functional role of a flexible loop in the alpha-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis; Brzovic PS et al.; The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies . Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit . Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit {Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E . W . (1987) J . Biol . Chem . 262, 10678-10683} . However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction . Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively . SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme . These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure . Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex . This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities. Toxicology, 1993 Oct 5, 82(1-3), 3 - 20 Heterologous expression of drug-metabolizing enzymes in cellular and whole animal models; Simula AP et al.; In this report we describe the heterologous expression of glutathione S-transferase (GST) and cytochrome P450 reductase (Red) in E . coli and Salmonella typhimurium . The same expression vectors could be applied to both systems and high levels of catalytically active GST and Red were obtained . Interestingly the level of expression was invariably higher in S . typhimurium . The level of the alpha class GST being up to 20% of the total bacterial protein . A further advantage of the salmonella system is that strains were used which can be applied to mutagenicity tests . This system was validated by demonstrating increasing mutation frequency of halogenated hydrocarbons in strains expressing the GST and increased cytotoxicity of mitomycin C in cells expressing P450 reductase. J Nutr, 1993 Oct, 123(10), 1714 - 23 Dietary energy source and density modulate the expression of immunologic stress in chicks; Benson BN et al.; To determine how dietary energy level and source influence feed intake, growth and energy partitioning drug immunologic stress, growing chicks were fed diets based on cornstarch and casein with varying energy densities and injected every other day for 6 d with either saline (control), Salmonella typhimurium lipopolysaccharide or heat-killed Staphylococcus aureus . Salmonella typhimurium lipopolysaccharide decreased growth and feed consumption at low energy densities . When the dietary energy density was increased above 13.4 kJ/g using cornstarch, but not corn oil, the growth depressing effect of immunogens was eliminated . Immunologically stressed chicks had a greater proportion of gain in visceral organs and less in the carcass, regardless of the nutrient density of the diet . Immunologic stress decreased intake of metabolizable energy of chicks fed a diet with low nutrient density and increased it for those fed a diet with high nutrient density . Chicks injected with S . typhimurium lipopolysaccharide lost more energy as heat than controls when differences in metabolizable energy intakes were accounted for and modified their preference between two diets differing in metabolizable energy density and fat content as a result of the challenge . Control chicks selected between the 11.7 and 14.2 kJ/g diets to obtain an energy density of 13.2 kJ/g compared with 12.5 kJ/g in the S . typhimurium lipopolysaccharide-challenged chicks . The S . typhimurium lipopolysaccharide-challenged chicks consumed similar amounts of the low energy diet but decreased intake of the high energy diet. J Bacteriol, 1993 Oct, 175(19), 6368 - 71 Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction; Fong CL et al.; The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined . The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase . H2S production by the E . coli host was, as in S . typhimurium, suppressed by nitrate or glucose in the growth medium . The presence of plasmid-borne phs genes in a S . typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate . These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate. J Bacteriol, 1993 Oct, 175(19), 6328 - 36 cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium; O'Toole GA et al.; The cobA locus of Salmonella typhimurium is involved in the assimilation of nonadenosylated cobinamide, (CN)2CBI, into cobalamin (CBL) under aerobic and anaerobic growth conditions . Aerobically, cobA mutants are unable to assimilate (CN)2CBI into CBL . However, under anaerobic conditions, cobA mutants assimilate (CN)2CBI into CBL as efficiently as cobA+ strains . On the basis of this observation, we postulated the existence of a cobA-independent pathway for the assimilation of (CN)2CBI into CBL that is functional under anaerobic growth conditions (J . C . Escalante-Semerena, S.-J . Suh, and J . R . Roth, J . Bacteriol . 172:273-280, 1990) . In this paper, we report the isolation and initial genetic characterization of derivatives of cobA mutants that are unable to assimilate (CN)2CBI into CBL during anaerobic growth . As demonstrated by complementation analysis, marker rescue, and DNA sequencing data, these mutations are alleles of cobU, a gene involved in the assembly of the nucleotide loop of CBL . We have shown that the block in CBL synthesis in these cobU cobA double mutant strains can be corrected by exogenous adenosyl-CBI . Our data indicate that this new class of cobU mutations blocks CBL biosynthesis but does not destroy the putative kinase-guanylyltransferase activities of the CobU protein . We propose that this new class of cobU mutations may affect an as yet unidentified ATP:corrinoid adenosyltransferase activity of the CobU protein . Alternatively, such mutations may alter the ability of CobU to use nonadenosylated CBI as a substrate. Infect Immun, 1993 Oct, 61(10), 4489 - 92 Role of acid tolerance response genes in Salmonella typhimurium virulence; Garcia-del Portillo F et al.; The atp and fur genes are involved in the acid tolerance response of Salmonella typhimurium . An atp::Tn10 mutant was avirulent in the mouse typhoid model when assayed by oral and intraperitoneal routes . However, a fur mutant was completely virulent by the intraperitoneal route . No relevant differences in intracellular survival or invasion rates were observed for the two mutants in macrophages and epithelial cells . These data indicate that separate acid tolerance response genes may have different roles in S . typhimurium virulence. Epidemiol Infect, 1993 Oct, 111(2), 325 - 35 Bacteriophage as models for virus removal from Pacific oysters (Crassostrea gigas) during re-laying; Humphrey TJ et al.; A study was undertaken to examine the feasibility of using naturally-occurring bacteriophages to assess the impact of re-laying on levels of viral contamination in Crassostrea gigas, the Pacific oyster . Two phages were chosen . One, male-specific (F+), was enumerated using Salmonella typhimurium . The other, a somatic phage, was detected using an, as yet, uncharacterized Escherichia coli . Investigations, using a variety of re-laying sites, demonstrated that numbers of F+ phage in oyster tissue declined more rapidly than those of somatic phage . For example, in oysters placed in commercially-used sea water ponds, F+ phage reached undetectable levels within 2-3 weeks, whereas somatic phage could still be detected 5 weeks after re-laying . The studies suggest that F+ phage may not be a suitable indicator for virus removal and that somatic phage may be better suited to this role. Epidemiol Infect, 1993 Oct, 111(2), 189 - 97 A comparison of multiple drug resistance in salmonellas from humans and food animals in England and Wales, 1981 and 1990; Threlfall EJ et al.; For Salmonella typhimurium from humans in England and Wales, the incidence of multiple resistance more than doubled over the 8-year period 1981-8 and, over the next 2 years, increased by a further 7% . From 1981 to 1988 both resistance and multiple resistance also increased significantly in S . virchow and although multiple resistance did not increase over the next 2 years, the overall incidence of resistance has continued to rise . In 1990 the majority of S . typhimurium from cattle were multiply-resistant and the occurrence of such resistance has quadrupled since 1981 . Multiple resistance has also increased in S . typhimurium from pigs and, to a lesser extent, from poultry . In contrast, multiple resistance has remained uncommon in the poultry-associated serotype S . enteritidis . For S . virchow, multiple resistance was common in a phage type frequently associated with poultry meat imported from France . The continuing use of a range of different antimicrobials in calf husbandry has been an important factor in promoting the emergence of multiply-resistant strains of S . typhimurium in cattle . In contrast, multiple resistance has remained rare in those serotypes associated with poultry, where the use of such antimicrobials has been less intensive . It is hoped that recent recommendations discouraging, in veterinary medicine, the prophylactic use of antibiotics with cross resistance to those used in human medicine will result in a reduction in the occurrence of multiresistant strains in food animals and subsequently in humans. EMBO J, 1993 Oct, 12(10), 3779 - 87 Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri; Groisman EA et al.; The enteric pathogens Salmonella typhimurium and Shigella flexneri differ in most virulence attributes including infectivity, pathology and host range . We have identified a new assemblage of genes responsible for invasion properties of Salmonella which is remarkably similar in order, arrangement and sequence to the gene cluster controlling the presentation of surface antigens (spa) on the virulence plasmid of Shigella . In Salmonella, this chromosomally encoded complex consists of over 12 genes, mutations in which abolish bacterial entry into epithelial cells . Although these genera use distinct invasion antigens, a non-invasive spa mutant of Salmonella could be rescued by the corresponding Shigella homolog . While spa promotes equivalent functions in Shigella and Salmonella, this constellation of genes has been acquired independently by each genus and displays motifs used by diverse antigen export systems including those required for flagellar assembly and protein secretion. Genetika, 1993 Oct, 29(10), 1640 - 5 {Effect of structural features of nitro-derivatives of fluorenone and biphenyl on frameshift mutagenesis in tester strains of Salmonella typhimurium}; Abilev SK et al.; Comparative mutagenic activity of 7 derivatives of biphenyl and fluorenone, 4,4'-dinitrobiphenyl-2,2'-dicarboxylic acid; 4,4',6,6'-tetranitrobiphenyl-2,2'-dicarboxylamide; 2-nitrofluorenone-5-carboxylic acid; 2,7-dinitrofluorenone-5-carboxylic acid; 2,7-dinitrofluorenone-5-carboxylamide; 2-nitrofluorenone-5,7-dicarboxylic acid; 2,4-dinitrofluorenone-5,7-dicarboxylic acid was studied . The highest activity was demonstrated for 2,7-DNF-5-KA and 2,7-DNF-5,7-DK which induced frameshift mutations in the tester strains Salmonella typhimurium TA1537, TA97, TA1538, TA98 . High mutagenicity of these compounds is correlated with the position of nitro-groups and the effects of carboxylic and carboxyamide groups. Eur J Cell Biol, 1993 Oct, 62(1), 152 - 62 Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid; Kaeffer B et al.; Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar . Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo) . The IPI-1 cells were found of fibroblastic lineage . The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells . A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency . Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers . Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells . Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning . These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm . The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line . All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus) . The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig . In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells. Can J Vet Res, 1993 Oct, 57(4), 281 - 7 Virulence of Salmonella enteritidis phagetypes 4, 8 and 13 and other Salmonella spp . for day-old chicks, hens and mice; Poppe C et al.; Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S . enteritidis PT 4 strain from the United Kingdom (UK) . The two PT 4 strains were also compared in orally inoculated adult laying hens . In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids . In orally inoculated one-day old chickens, the UK S . enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain . The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain . The S . enteritidis PT 8 strain and one S . typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice . This strain of S . typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S . typhimurium strain isolated from a pig with septicemic disease. Immunology, 1993 Oct, 80(2), 306 - 12 Influence of the antigen delivery system on immunoglobulin isotype selection and cytokine production in response to influenza A nucleoprotein; Brett SJ et al.; The influence of different antigen delivery systems on antibody isotype and lymphokine profile has been investigated using influenza nucleoprotein as a model antigen system . Mice exposed to live or inactivated influenza virus produced antibody against whole virus or recombinant nucleoprotein (rNP), which was predominantly of the IgG2a isotype . Spleen or lymph node cells from these mice rapidly produced large amounts of interferon-gamma (IFN-gamma), but no detectable interleukin-5 (IL-5) when stimulated in vitro with specific antigen . In contrast, after primary immunization with rNP or p206-229 in different adjuvants (CFA, quil A or alhydrogel), specific antibody was predominantly of the IgG1 isotype and relatively lower amounts of IFN-gamma but no IL-5 were detected following in vitro antigenic stimulation . Secondary immunization, however, resulted in detection of IgG2a antibodies and increased levels of IFN-gamma . IL-5 was only detected after secondary immunization with peptide in adjuvant . Mice infected with aro A- Salmonella typhimurium expressing NP produced antibody of both IgG1 and IgG2a isotypes and large amounts of IFN-gamma and no IL-5, following in vitro antigenic stimulation, and therefore parallelled the pattern seen with whole virus more closely than that seen following primary immunization with protein or peptide in conventional adjuvants . The results suggest that the antigen delivery vehicle influences both quantitative and qualitative differences in the type of immune response elicited, which may be important in determining the potency of protective immunity induced. Antimicrob Agents Chemother, 1993 Oct, 37(10), 2251 - 3 Susceptibilities of oxyR regulon mutants of Escherichia coli and Salmonella typhimurium to isoniazid; Rosner JL; Escherichia coli and Salmonella typhimurium are normally resistant to > 500 micrograms of the antituberculosis drug isonicotinic acid hydrazide (isoniazid; INH) per ml . Susceptibility to INH (< 50 micrograms/ml) has now been found for mutants that are deficient in OxyR, the oxidative stress response regulator . Two OxyR-regulated enzymes, alkyl hydroperoxide reductase and hydroperoxidase I, were identified as playing important roles in INH resistance . OxyR regulon mutants should be useful for identifying other determinants of INH resistance in both E . coli and Mycobacterium tuberculosis and for finding new INH-like drugs. Protein Sci, 1993 Oct, 2(10), 1643 - 7 Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein; He JJ et al.; Electrostatic interactions are among the key factors determining the structure and function of proteins . Here we report experimental results that illuminate the functional importance of local dipoles to these interactions . The refined 1.7-A X-ray structure of the liganded form of the sulfate-binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main-chain peptide NH groups) and van der Waals forces . The sulfate is also closely linked, via one of these peptide units, to a His residue . It is also adjacent to the N-termini of three alpha-helices, of which the two shortest have their C-termini "capped" by Arg residues . Site-directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate-binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues . These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices . The contribution of helix macrodipoles appears insignificant. Appl Environ Microbiol, 1993 Oct, 59(10), 3509 - 12 Comparison of methods for specific depletion of ATP in Salmonella typhimurium; Johnson MS et al.; Three methods of ATP depletion in Salmonella typhimurium were compared . ATP concentrations were lowest after arsenate treatment . Arsenate or alpha-methylglucoside-plus-azide treatment nonspecifically lowered all nucleotide triphosphate levels . Histidine starvation in a hisF mutant was relatively specific for ATP depletion and therefore has potential in distinguishing ATP-dependent processes from processes dependent on other nucleotides. Food Chem Toxicol, 1993 Oct, 31(10), 707 - 15 Bioassay of quinoline, 5-fluoroquinoline, carbazole, 9-methylcarbazole and 9-ethylcarbazole in newborn mice; Weyand EH et al.; Quinoline and carbazole are among the more prevalent aza-arenes present as components of environmental pollutants . Both of these aza-arenes are hepatocarcinogenic to mice when administered in the diet . The hepatocarcinogenic potential of quinoline is consistent with its mutagenic activity in Salmonella typhimurium TA100 and potential to induce unscheduled DNA synthesis (UDS) in rat hepatocytes . Structure-activity studies with fluorinated quinolines indicate that the presence of a fluorine atom at the 5-position of quinoline may inhibit detoxification and result in enhanced genotoxic potency . Quinoline and 5-fluoroquinoline were assayed in newborn CD-1 mice at a total dose of 1.75 mumol to establish their relative tumorigenic activity . Liver tumours developed in 60 and 90% of the male newborn mice treated with quinoline and 5-fluoroquinoline, respectively . The majority of liver tumours observed among the quinoline-treated mice were classified as adenomas . In contrast, liver carcinomas developed in most of the male mice treated with 5-fluoroquinoline . Unlike the well established genotoxic potential of quinoline, there is limited evidence for carbazole having either genotoxic or carcinogenic activity . Whereas carbazole is not mutagenic towards several strains of S . typhimurium, both 9-methylcarbazole and 9-ethylcarbazole are active as mutagens in S . typhimurium TA100 . Carbazole, 9-methylcarbazole and 9-ethylcarbazole were assayed in primary rat hepatocytes to assess their relative potential to induce UDS in rat hepatocytes; only 9-ethylcarbazole did so . These carbazole derivatives were also assayed in newborn CD-1 mice at a total dose of 1.75 mumol . Neither carbazole nor either of these 9-alkylcarbazoles produced a significant tumorigenic response in this bioassay system. Carcinogenesis, 1993 Oct, 14(10), 2039 - 43 DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites; Mentzschel A et al.; 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates . In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA . In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats . In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide . The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling . The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate . Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose . Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes . The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate . Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate . In liver and heart the adducts were detectable only on the first day after completion of the treatment . The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential. J Environ Pathol Toxicol Oncol, 1993 Oct-Dec, 12(4), 185 - 91 The involvement of reactive oxygen species in the direct-acting mutagenicity of 5-nitro-3-thiophenecarboxanilides in Salmonella typhimurium; Hrelia P et al.; The primary basis of 5-nitro-3-thiophenecarboxanilides (NTCAs) direct-acting mutagenicity in Salmonella typhimurium appears to be the reduction of the nitro function to the corresponding hydroxylamine via diamagnetic and free radicals intermediates . In Ames test conditions, mutagenicity of NTCAs may be partly attributed to the action of reactive oxygen species and other radicals generated during the bioreductive process . The nitro anion radical seems to be particularly susceptible to react with oxygen and generate superoxide, as shown by the inhibitory effects exerted by superoxide dismutase on genotoxicity by most NTCAs in the study . On the other hand, hydroxyl radical-trapping agents such as mannitol, the enzyme catalase and other scavengers as 3-tert-butyl-4-hydroxyanisole (BHA), and vitamins C, A, and E showed weak inhibitory potency, and rather increased the mutagenic activity of some NTCAs . Our results contribute to the mechanistic understanding of genotoxic activity of NTCAs. Environ Health Perspect, 1993 Oct, 101 Suppl 3, 53 - 6 Mutagenicity and human chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni; Suttajit M et al.; Leaves of Stevia rebaudiana Bertoni have been popularly used as a sweetener in foods and beverages for diabetics and obese people due to their potent sweetener stevioside . In this report, stevioside and steviol were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 and for chromosomal effects on cultured human lymphocytes . Stevioside was not mutagenic at concentrations up to 25 mg/plate, but showed direct mutagenicity to only TA98 at 50 mg/plate . However, steviol did not exhibit mutagenicity in either TA98 or TA100, with or without metabolic activation . No significant chromosomal effect of stevioside and steviol was observed in cultured blood lymphocytes from healthy donors (n = 5) . This study indicates that stevioside and steviol are neither mutagenic nor clastogenic in vitro at the limited doses; however, in vivo genotoxic tests and long-term effects of stevioside and steviol are yet to be investigated. Environ Health Perspect, 1993 Oct, 101 Suppl 3, 33 - 6 Evaluation of mutagenicity testing with Salmonella typhimurium TA102 in three different laboratories; Muller W et al.; Thirty compounds of various chemical classes were investigated for mutagenicity in a collaborative study (three laboratories) using Salmonella typhimurium TA102 . With five compounds, hydrazine sulfate, phenylhydrazine, hydralazine, glutardialdehyde, and glyoxal, mutagenicity was detected by all laboratories . Formaldehyde was assessed as weakly mutagenic in only one of three laboratories . The remaining 24 agents were uniformly described as non-genotoxic in TA102 . In spite of the overall good qualitative agreement in the mutagenicity results between the three laboratories, some quantitative discrepancies occurred in the dose response of the mutagenic compounds . Varying inter- and intralaboratory differences in the spontaneous rate of revertants were obtained . The usefulness of the tester strain TA102 in routine mutagenicity testing is discussed. Environ Health Perspect, 1993 Oct, 101 Suppl 3, 27 - 31 Metabolism of 2-aminofluorene by human polymorphonuclear leukocytes: more evidence for the association between inflammation and cancer; Isola VJ et al.; Recent investigations have demonstrated the ability of leukocytes to metabolize promutagens or procarcinogens into their genotoxic forms . As a possible explanation for the association between inflammation and cancer, we and others have hypothesized that local accumulations of leukocytes could take up nearby promutagens, metabolize them, and release genotoxic agents that may cause damage in the surrounding tissue . Using a modified, two-step preincubation protocol with Salmonella, we have tested this hypothesis . We have shown that total human peripheral blood leukocytes, cultured in the presence of 2-aminofluorene for 18 hr, can metabolize 2-aminofluorene into agents mutagenic to Salmonella typhimurium strain TA98 . Furthermore, experiments in which polymorphonuclear leukocytes were separated from mononuclear leukocytes demonstrated that the PMNs metabolized 2-aminofluorene to a much greater extent than the MNs. Environ Health Perspect, 1993 Oct, 101 Suppl 3, 21 - 6 Mutagenicity testing of 9-N-substituted adenines and their N-oxidation products; Gorrod JW et al.; Adenine together with certain 9-N-substituted derivatives such as 9-methyl, 9-benzyl, 9-benzhydryl, and 9-trityl were tested against Salmonella typhimurium strains TA97, TA98, and TA100 in the absence and presence of rat hepatic S9 prepared from Aroclor 1254 pretreated rats . All compounds were positive toward TA98 in the presence of the metabolic activating system, whereas they all lacked mutagenic activity in the absence of S9, and toward TA97 and TA100 with or without S9 when tested at 100 ng/plate . A similar pattern was observed for the corresponding 1-N-oxides . 6-Hydroxylaminopurine was not mutagenic toward TA100 at 100 ng/plate, whereas it was toxic toward TA97 and TA98 at this level . When tested at 1 ng/plate, hydroxylaminopurine was still toxic to TA98 but produced twice the spontaneous reversion rate to TA97 without metabolic activation . Surprisingly, 9-methyl-6-hydroxylaminopurine was only active toward TA98 in the presence of S9, whereas 9-benzyl-6-hydroxylaminopurine was highly active toward TA97 and TA100 in the absence of S9 and even more active in the presence of S9 . This compound was inactive toward TA98 in the absence of S9 . The results generally support the concept that nuclear N-oxidation of aminoazaheterocycles is a detoxication process, whereas N-hydroxylation of the exo amino group is a toxication reaction. Avian Dis, 1993 Oct-Dec, 37(4), 1051 - 6 Effectiveness of dietary propionic acid in controlling Salmonella typhimurium colonization in broiler chicks; Hume ME et al.; Newly hatched broiler chicks were provided a corn/soybean meal-based ration treated with propionic acid at 30 mumol/g of feed ration . At 3 days of age, the chicks were challenged orally with 10(4) Salmonella typhimurium . Crop contents from 4-day-old chicks that were provided dietary propionic acid contained significantly higher concentrations of propionic acid (4.0 to 6.8 mumol/g crop contents) than crops from challenged control chicks provided untreated feed (0.9 to 1.5 mumol/g crop contents) . Provision of dietary propionic acid on feed as a dry powder in five trials or a liquid application in three trials had no significant effect on crop or cecal pH . Significant decreases in Salmonella in the crop and ceca were detected in one trial, but the decreases were likely the result of the presence of anti-salmonellae bacteria rather than the dietary propionic acid . Results indicate that propionic acid in the feed was ineffective in reducing Salmonella infection in the crop and ceca. Rev Latinoam Microbiol, 1993 Oct-Dec, 35(4), 345 - 9 Evaluation of the in vitro susceptibility and emergence of mutants resistant to ciprofloxacin among multidrug resistant clinical isolates; Lopes CA et al.; Two hundred and seventy-seven multidrug resistant clinical isolates {K . pneumoniae, (N = 87); E coli, (N = 30); Salmonella typhimurium (N = 100); P . aeruginosa, (N = 30); S . aureus, (N = 30)} from hospitalized patients specimens, were tested in vitro for sensitivity to Ciprofloxacin . Application of the disk diffusion test and determination of the minimal inhibitory concentration by the microdilution method indicated that, almost all isolates were sensitive to the drug . Overall, S . aureus and P . aeruginosa were the less sensitive organisms . Ciprofloxacin-resistant mutants occurred at frequencies of > or = 10(-5)/CFU. Mol Microbiol, 1993 Oct, 10(1), 75 - 86 The influence of DNA topology on the environmental regulation of a pH-regulated locus in Salmonella typhimurium; Karem K et al.; Salmonella typhimurium is exposed to major shifts in H+ concentration both in its natural and pathogenic environments . The organism undergoes extensive changes in gene expression in response to these pH fluctuations . A current question of regulatory biology is how a change in external pH selectively modulates transcription . We have analysed the expression of one such pH-regulated locus, aniG, and found it is controlled by several additional environmental conditions including osmolarity and oxygen . For factors such as osmolarity and anaerobiosis, an environmentally triggered change in DNA supercoiling has been suggested as a means for controlling gene expression . Thus, environmentally induced changes in DNA topology were explored as a possible common means for establishing the multiple controls on aniG . The involvement of DNA supercoiling in the genetic response of S . typhimurium to external pH has not previously been defined . This report establishes that alkaline environments lower the linking number of reporter plasmids when compared to acidic environments . A consistent pattern was then established whereby conditions or mutations leading to either increased or decreased negative supercoiling were associated with altered expression of aniG . A similar relationship was observed for another environmentally regulated locus, proU . The DNA topology effects on aniG expression were dependent on the presence of EarA, the negative regulator of aniG . These data can be explained by a model in which repressor-operator interactions are very sensitive to changes in operator conformation . These environmentally induced topological influences on operator DNA structure contribute to the magnitude of pH control exerted upon aniG. Mol Microbiol, 1993 Oct, 10(1), 113 - 22 Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli; Oppenheim AB et al.; Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes . REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria . These REP sequences may participate in the folding of the bacterial chromosome . In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence . We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically . It is estimated that there are about 70 RIP elements in E . coli . Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome . The possible function of the RIP element is discussed. Mol Microbiol, 1993 Oct, 10(2), 273 - 82 Expression of the gene encoding the major bacterial nucleotide protein H-NS is subject to transcriptional auto-repression; Falconi M et al.; Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E . coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles . CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds . The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase . The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription . The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns-cat fusions . Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation gel analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150 . When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied . Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression. Am J Vet Res, 1993 Oct, 54(10), 1648 - 52 Use of random fragments of chromosomal DNA to highlight restriction site heterogeneity for fingerprinting isolates of Salmonella typhimurium from hospitalized animals; Hansen LM et al.; Random fragments of DNA were obtained from a cosmid library of Salmonella agona genomic DNA . From this library, 2 fragments were chosen and pooled to probe isolates of S typhimurium obtained during an episode of salmonellosis in a veterinary medical teaching hospital . Chromosomal DNA from the Salmonella isolates was digested with restriction endonucleases, and was probed with the random fragments of chromosomal DNA . This procedure resulted in a fingerprint pattern for each isolate . We found that the method permitted discrimination between isolates involved in the disease episode and S typhimurium obtained prior to the episode . We conclude that random fragments of chromosomal DNA are useful for fingerprinting isolates of S typhimurium . Analysis of plasmid DNA obtained from the isolates was not as useful . Some isolates found to be identical by restriction site analysis, had plasmids of different molecular weight . These results indicate that plasmid analysis may not be as useful a fingerprinting tool as previously reported. Mutat Res, 1993 Oct, 303(2), 63 - 70 Identification of heterogenous antimutagenic activities in the extract of edible brown seaweeds, Laminaria japonica (Makonbu) and Undaria pinnatifida (Wakame) by the umu gene expression system in Salmonella typhimurium (TA1535/pSK1002); Okai Y et al.; A significant antimutagenic activity was found in the hot water-soluble extract from a common edible brown alga, Laminaria japonica (Makonbu in Japanese) which showed suppressive effects on umu gene expression of the SOS response against DNA damage in Salmonella typhimurium (TA1535/pSK1002) . The extract showed a drastic antimutagenic activity against 2-acetylaminofluorene (2-AAF)- or 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1)-induced mutagenesis which requires liver-metabolizing enzymes, whereas the same extract exhibited weak but significant inhibitory effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)- or furylfuramide (AF-2)-induced mutagenesis in the absence of liver-metabolizing enzymes . Among these antimutagenic activities, the minor activity was found in the polysaccharide fraction of the extract which showed roughly equal antimutagenic activities against all the mutagens tested . The major activity was detected in the nonpolysaccharide fraction which exhibited a relatively strong antimutagenic activity against 2-AAF- or Trp-P-1-induced mutagenesis but a weak activity against MNNG- or AF-2-induced mutagenesis . The nonpolysaccharide fraction was further separated into high- or low-molecular-weight fractions and the latter fraction showed a much stronger activity than the former fraction . In addition, similar antimutagenic activities were detected in polysaccharide and nonpolysaccharide fractions from the extract of the other edible brown alga, Undaria pinnatifida (Wakame in Japanese) . These experimental results indicate that the hot water-soluble extract of Laminaria japonica or Undaria pinnatifida contains heterogenous antimutagenic activities against typical genotoxic substances . The significance of this finding is discussed from the viewpoint of the protection against genotoxic substances by traditional edible seaweeds in Japan. Mutat Res, 1993 Oct, 303(2), 55 - 61 Mutagenic activity of urban air samples and its modulation by chili extracts; Espinosa-Aguirre JJ et al.; Different samples of ambient particulate organic matter were collected during the summer and winter of 1990 in Mexico City . After dichloromethane extraction, the samples were tested for mutagenicity with derivatives of Salmonella typhimurium possessing high activity of 'classical' nitroreductase (YG1021) or O-acetyltransferase (YG1024), and compared to the mutagenicity of the normal strain YG1020, and to that of a nitroreductase-deficient mutant TA98NR . The two enzyme-overproducing strains were more sensitive to the mutagenic effect of the extracts than the parent and deficient strains . The sensitivity order, i.e., YG1024 > YG1021 > YG1020 > TA98NR, emphasizes the usefulness of the new Salmonella strains in analyzing the mutagenicity of complex mixtures and suggests that some of the direct mutagenic compounds in the urban air samples are nitro-aromatics . Investigations were also conducted to analyze the effect of chili extract on the mutagenicity of an urban air sample . The extract itself showed moderate mutagenic activity and an additive effect was noted when both the chili and air extracts were present . On the other hand, the maximum volume of chili tested produced a decrease in the number of revertants without affecting the background lawn of bacterial growth . The same response was also observed when 1-nitropyrene, 1,6-dinitropyrene or 1,8-dinitropyrene was used as the genotoxic compound, although potentiation instead of addition occurred at low vegetable volumes . At the concentrations found in the chili extract, chlorophyllin and beta-carotene showed an antimutagenic effect against the nitro-aromatic compounds. Mutat Res, 1993 Oct, 292(2), 155 - 63 A new Salmonella tester strain expressing a hamster acetyltransferase shows high sensitivity for arylamines; Ando M et al.; A hamster acetyltransferase, AT-I, has high activities for N-acetylation of arylamines, O-acetylation of N-hydroxyarylamines and N,O-acetyltransfer of N-hydroxyarylacetamides . In the present study, the cDNA was expressed in Salmonella typhimurium TA1538 . The new SAT138 strain expressing high levels of AT-I showed remarkably high sensitivity (> 10,000 fold) for a carcinogenic intermediate, N-hydroxy-2-acetylaminofluorene, in an Ames mutagenesis test as compared to the parental TA1538 strain . SAT138 had 650-1,600-fold higher sensitivities for mutagenesis induced by 2-acetylaminofluorene and benzidine in the presence of S9 mix . Higher sensitivities (32-560-fold) were also observed with N-hydroxy-2-aminofluorene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4-propionylaminobiphenyl and N-hydroxy-phenacetin in the absence of S9 mix . These high sensitivities to arylamines and the related chemicals are accounted for by the efficient expression of AT-I in the cytosol of this bacterium . The unique characteristics of SAT138 having high N-hydroxyarylacetamide N,O-transacetylating activity, which is defective in Salmonella acetyltransferase, provide broadened and high sensitivities for the detection of mutagenic N-substituted chemicals in the Salmonella mutagenesis test. J Biol Chem, 1993 Sep 25, 268(27), 19991 - 7 Purification and characterization of the periplasmic domain of the aspartate chemoreceptor; Milligan DL et al.; In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed . This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis . This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques . The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e . the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate . Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium . At low protein concentrations and in the absence of aspartate, the protein is monomeric . At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric . Two charge variants of the protein have been identified on native polyacrylamide gels . The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm. J Biol Chem, 1993 Sep 25, 268(27), 20299 - 304 Active site lysines in orotate phosphoribosyltransferase; Grubmeyer C et al.; Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes the formation of the nucleotide orotidine-5'-monophosphate from orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP) . The bacterial enzyme, unlike its mammalian homolog, is monofunctional and is a dimer of M(r) 23,000 subunits . The availability of large amounts of highly purified crystalline Salmonella typhimurium OPRTase have enabled us to being structure/function studies on the enzyme . Like other phosphoribosyltransferases, OPRTase binds the highly charged MgPRPP complex, as well as anionic orotate, suggesting an active site containing basic residues . The S . typhimurium sequence (Scapin, G., Sacchettini, J . C., Dessen, A., Bhatia, M., and Grubmeyer, C . (1993) J . Mol . Biol . 230, 1304-1308) contains 12 lysine and 13 arginine residues, of which Lys-26, Arg-99, Lys-100, Lys-103, and Arg-156 are conserved as identities among the sequences of OPRTases from other organisms, with Lys-19 and Arg-161 replaced by the alternative basic residue in one or more sequences . The lysine modifier 2,4,6-trinitrobenzene sulfonate inactivated S . typhimurium OPRTase in a pseudo first-order process, and OMP and PRPP provided good protection against inactivation . Spectral quantitation of trinitrophenyl (TNP) group incorporation showed that inactivation was correlated with incorporation of one TNP group per subunit . Surprisingly, tryptic proteolysis followed by high performance liquid chromatography and amino acid sequence analysis revealed that four peptides, containing three distinct lysines, had been modified . Peptides modified at Lys-26, Lys-100 and Lys-103, as well as a doubly modified peptide containing TNP groups at Lys-100 and Lys-103, were identified . Inactivation kinetics showed that the 3 lysine residues were modified at equal rates . Protection studies demonstrated that Lys-26, and to a lesser extent Lys-100 and Lys-103, were protected against modification by OMP, whereas PRPP protected Lys-26, Lys-100 and Lys-103 . Pyrophosphate protected Lys-100 and Lys-103 . The results suggest active site locations for the sequence-conserved and TNP-modified lysine residues, with Lys-26 interacting with the ribose-5-phosphate moiety of OMP and PRPP, and Lys-100 and Lys-103 interacting with the pyrophosphate moiety of PRPP. Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8576 - 80 Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes; Thier R et al.; Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans . Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity . A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme . The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2 . However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells) . HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations . S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates . This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells . S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-{1-(N2-deoxyguanosinyl)methyl}GSH . Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans. FEMS Microbiol Lett, 1993 Sep 15, 112(3), 251 - 4 Correlation between umuC induction and Salmonella mutagenicity assay for quinolone antimicrobial agents; Power EG et al.; Quinolone antimicrobial agents induce the SOS response in bacteria, including the umuDC genes necessary for error-prone repair . Consequently these drugs may be mutagenic in bacteria with a functional SOS response . Differential killing tests with Escherichia coli WP2 (trp) and its repair-deficient derivative CM871 (trp lexA recA uvrA) indicated that a functional DNA repair system was protective against the action of quinolones, implying that quinolones are causing some form of DNA damage (not necessarily directly) and are therefore genotoxic . Dose-dependent reversion from His- to His+ with quinolones was observed in the Ames test with Salmonella typhimurium TA102 (uvr+) but in no other Salmonella tester strains (all uvr-), suggesting that a functional excision repair system is essential for quinolone-induced bacterial mutagenesis . A significant correlation between SOS inducing potential (SOSIP) and mutagenic potential in the Ames test (r = 0.89) indicated that quinolone-induced mutagenic effects in bacteria are almost entirely due to SOS-processed DNA damage. J Biol Chem, 1993 Sep 5, 268(25), 18633 - 6 Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein; Ishii T et al.; Exposure of mouse peritoneal macrophages to oxidative and sulfhydryl-reactive agents in vitro enhances synthesis of a few cellular proteins that may be important in a self-defense system . A cDNA encoding a novel stress-inducible protein, designated MSP23 (macrophage 23-kDa stress protein), was cloned from a cDNA library of the macrophages by differential screening . A 1.0-kilobase mRNA transcript hybridized with the MSP23 cDNA gradually increased in macrophages upon culture in vitro . Treatment with diethylmaleate or glucose/glucose oxidase, which generates H2O2, markedly enhanced the induction of the transcript after several hours . Cadmium chloride and sodium arsenite also induced the transcript . An antiserum raised against recombinant MSP23 reacted with the 23-kDa stress-inducible protein of the macrophages . The amounts of 23-kDa protein in the cells rapidly increased during culture with diethylmaleate . The mRNA was detected in various tissues, and it was especially high in content in the liver . A search of databases revealed that six proteins of various species from bacteria to the mouse have a sequence homology to MSP23 . One of the proteins is the C22 component of alkyl hydroperoxide reductase, which is induced by hydrogen peroxide in Salmonella typhimurium. J Biol Chem, 1993 Sep 5, 268(25), 18617 - 21 The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium . Characterization of the ATPase activity associated with the purified MalK subunit; Morbach S et al.; The ATPase activity associated with the purified MalK subunit of the maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., Honer zu Bentrup, K., and Schneider, E . (1992) J . Biol . Chem . 267, 8863-8869), was characterized in detail . The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein . Both GTP and CTP also served as substrates . While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher . ATP hydrolysis was strongly dependent on the presence of Mg2+ ions . Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+ . The ATPase activity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%) . ADP and the non-cleavable substrate analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors . The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1 . In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity . This inhibition was blocked in the presence of ATP . These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g . ion-translocating ATPases. Mol Gen Genet, 1993 Sep, 240(3), 360 - 4 The pleiotropic effects of his overexpression in Salmonella typhimurium do not involve AICAR-induced mutagenesis; Flores A et al.; Inhibition of cell division associated with overexpression of hisH and hisF in