Infect Immun, 1994 Jun, 62(6), 2229 - 35 Entrance and survival of Salmonella typhimurium and Yersinia enterocolitica within human B- and T-cell lines; Verjans GM et al.; Lymphocytes, located within the Peyer's patches, might be involved in the dissemination of enteropathogenic Salmonella typhimurium and Yersinia enterocolitica bacteria . To test this hypothesis, we have investigated the susceptibility of human B- and T-cell lines to bacterial adhesion and invasion . The two S . typhimurium strains analyzed were highly invasive, while the two Y . enterocolitica (O:8) strains adhered to the B- and T-cell lines but did not enter the cell lines in significant amounts . We hypothesize that the incapability of the Y . enterocolitica (O:8) strains to enter the human B- and T-cell lines is most probably due to the bacterial inability to induce the internalization process upon adhesion to both cell lines . Although immortalized B- and T-cell lines were used in this study, the results presented suggest the possibility that both cell types could play a role in the dissemination of intracellularly residing S . typhimurium in vivo. Biochem Biophys Res Commun, 1994 May 30, 201(1), 16 - 23 Salmonella typhimurium activates human immunodeficiency virus type 1 in chronically infected promonocytic cells by inducing tumor necrosis factor-alpha production; Andreana A et al.; The effect of phagocytosis of Salmonella typhimurium on human immunodeficiency virus type 1 (HIV-1) production was investigated using a chronically infected promonocytic cell line (U1) that contains HIV-1 provirus but produces little or no HIV-1 . The phagocytosis of virulent S . typhimurium by U1 cells resulted in an increased HIV-1 expression as evidenced by significant increase in HIV-1 p24 antigen in culture supernatants . In contrast, heat-killed S . typhimurium failed to induce HIV-1 expression . In addition, phagocytosis of virulent S . typhimurium and not of heat-killed S . typhimurium resulted in a significant induction of tumor necrosis factor-alpha (TNF-alpha) mRNA expression and secretion of TNF-alpha by U1 cells . Furthermore, anti-TNF-alpha monoclonal antibody inhibited S . typhimurium-induced HIV-1 p24 antigen production . These data suggest that S . typhimurium induces HIV-1 expression in U1 cells via production of TNF-alpha. Gene, 1994 May 27, 143(1), 49 - 54 Sequence analysis of the flgA gene and its adjacent region in Salmonella typhimurium, and identification of another flagellar gene, flgN; Kutsukake K et al.; The flagellar genes flgA and flgM are located at the terminus of the region-I flagellar gene cluster on the chromosome of Salmonella typhimurium . The flgA gene is involved in P-ring formation of the flagellar basal body, whereas flgM encodes the anti-sigma factor which acts as a negative regulator of the flagellar regulon . The nucleotide sequence of the DNA fragment containing these flagellar genes and the adjacent region was determined . The flgA gene was found to encode a 219-amino-acid (aa) protein of 23,556 Da . The N-terminal region of FlgA has the characteristics of a typical signal sequence, suggesting that FlgA may function in the periplasmic space where P-ring assembly takes place . The flgM gene was found to constitute an operon together with an ORF which encodes a 140-aa protein of 15,899 Da . A gene disruption mutant was constructed by inserting a cat gene cartridge into the ORF on the chromosome . This mutant showed only weak motility, indicating that the product of the ORF is involved in flagellar formation . Therefore, this ORF was designated as flgN . Electron microscopic observation revealed that most of the flagellar structures produced by the flgN mutant are hook-basal body complexes lacking the filament portions . Based on these results, we concluded that the flgN product is required for the efficient initiation of filament assembly. Gene, 1994 May 27, 143(1), 55 - 9 Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium; Johanson U et al.; We have sequenced fusA, the gene coding for elongation factor G (EF-G), in 18 different mutants of Salmonella typhimurium selected as fusidic acid resistant (FuR) . In addition, we have sequenced two previously described FuR mutants from Escherichia coli . In all cases, the resistance is due to a mutation in one of three separate regions in fusA . The three clusters of mutant sites superimpose on regions that are well conserved, suggesting that they are of a more general functional importance . To further classify the mutants, we have measured the minimal inhibitory concentration (MIC) for Fu and for two other antibiotics which interfere with translocation on the ribosome, kanamycin (Km) and spectinomycin (Sp) . The levels of resistance to Fu for each of the mutants are significantly higher than in the wild type (wt), and vary by about one order of magnitude between the highest and the lowest . Most of the mutants are also more resistant to Km than the wt, although the level of resistance is low and the variation small . In contrast, about half of the mutants are more sensitive to Sp than the wt, with only one being more resistant . Only three of the twenty mutants behave like the wt with respect to the non-selected phenotypes, KmR and SpR. Biochem J, 1994 May 15, 300 ( Pt 1), 271 - 6 Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism; Pemble S et al.; In humans, glutathione-dependent conjugation of halomethanes is polymorphic, with 60% of the population classed as conjugators and 40% as non-conjugators . We report the characterization of the genetic polymorphism causing the phenotypic difference . We have isolated a cDNA that encodes a human class Theta GST (GSTT1) and which shares 82% sequence identity with rat class Theta GST5-5 . From PCR and Southern blot analyses, it is shown that the GSTT1 gene is absent from 38% of the population . The presence or absence of the GSTT1 gene is coincident with the conjugator (GSST1+) and non-conjugator (GSTT1-) phenotypes respectively . The GSTT1+ phenotype can catalyse the glutathione conjugation of dichloromethane, a metabolic pathway which has been shown to be mutagenic in Salmonella typhimurium mutagenicity tester strains and is believed to be responsible for carcinogenicity of dichloromethane in the mouse . In humans, the enzyme is found in the erythrocyte and this may act as a detoxification sink . Characterization of the GSTT1 polymorphism will thus enable a more accurate assessment of human health risk from synthetic halomethanes and other industrial chemicals. Food Chem Toxicol, 1994 May, 32(5), 443 - 59 In vitro effect of vegetable and fruit juices on the mutagenicity of 2-amino-3-methylimidazo{4,5-f}quinoline, 2-amino-3,4-dimethylimidazo{4,5-f}quinoline and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline; Edenharder R et al.; The antimutagenic potencies of the juices of 28 fruits and 34 vegetables commonly consumed in Germany were investigated with respect to the mutagenic activities induced by 2-amino-3-methyl{4,5-f}-quinoline (IQ), and in part by 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) or 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) in Salmonella typhimurium TA98 and TA100 . With IQ, weak to strong antimutagenic activities were found in 68% of the fruits and 73% of the vegetables that were tested . In fruits, strong antimutagenic activities were detected in bananas, blackberries, blueberries, sweet and sour cherries, blackcurrants and redcurrants, pineapple and watermelon . Moderate antimutagenic activities were detected in greengage, kiwi, mangos, honeydew melons and plums . Weak antimutagenic activities were detected in apple, apricot, mirabelle, pears, peaches and strawberries, whereas white and red grapes and raspberries were inactive, and gooseberries and citrus fruits in general possessed marginal or no antimutagenic activities . In vegetables, strong to moderate antimutagenic activities were found for all cruciferous vegetables, except Chinese cabbage, which had only weak antimutagenic activity . Other vegetables with strong antimutagenic activities were beets, chives, horseradish, onions, rhubarb and spinach . Moderate antimutagenic activities were found with green beans and tomatoes, weak activities in eggplant, garden cress, many lettuces, leeks, mangold, cucumber, pumpkin, radish and summer squash . Asparagus, carrots, fennel leaves, parsley, green peppers and radishes were inactive . When fruit and vegetable juices were heated, a considerable reduction of antimutagenic potencies was seen with apple, apricot, kiwi, pineapple, beets, cabbage (Chinese, Savoy, red and white), cauliflower, leafy lettuce, cucumber, onions, radish and rhubarb . Antimutagenic factors in blackberries, blueberries, sweet and sour cherries, honeydew melons, mirabelle, plums, strawberries, Brussels sprouts, chicory greens, eggplant, garden cress, mangold, pumpkin, lamb's lettuce and spinach were, however, remarkably heat stable . Antimutagenic potencies in bananas, blackcurrants and redcurrants, greengages, gooseberries, mangos, watermelon, green beans, kohlrabi, horseradish, tomatoes and chives were partially reduced . Antimutagenic activities in the juices of eight apple cultivars were moderate in two, weak in four, and marginal or absent in two . No major differences, however, were detected in five batches of oranges and three batches each of grapefruits, asparagus, green beans, broccoli, cauliflower, spinach and tomatoes . No (or only minor) differences were seen between IQ, MeIQ and MeIQx and tester strains TA98 and TA100 . Pineapple and celeriac juices inhibited the enzymatic system responsible for the activation of IQ, but had no desmutagenic activity . Peroxidase activity found to be present in broccoli, cauliflower, green beans and tomatoes may contribute to antimutagenic activities in these vegetables. Carcinogenesis, 1994 May, 15(5), 947 - 55 Hepatic and extrahepatic bioactivation and GSH conjugation of aflatoxin B1 in sheep; Larsson P et al.; Whole-body autoradiography of {3H}aflatoxin B1 ({3H}AFB1) in lamb showed a localization of bound labelling, in addition to the liver, in the nasal olfactory and respiratory mucosa, in the mucosa of the nasopharynx, pharynx, oesophagus, larynx, trachea, bronchi and bronchioles and in the palpebral and bulbar conjunctiva . Microautoradiography revealed that the bound material was confined to specific cell types in extrahepatic tissues . Whole-body autoradiography also showed a labelling of pigmented tissues (such as the eye melanin), which can be ascribed to a melanin affinity of AFB1 . In vivo experiments, performed with microsomal preparations of tissues from ewe and lamb showed that several of the extrahepatic tissues were more efficient than the liver in forming DNA-bound AFB1 metabolites . The nasal olfactory mucosa was by far the most effective tissue in this respect . AFB1 induced a high number of gene mutations in Salmonella typhimurium TA100 when incubated with supernatant preparations (9000 g) of the nasal olfactory mucosa, whereas incubations with preparations of the liver resulted in a lower effect . It has been reported that AFB1 can induce nasal tumours in sheep . When microsomal preparations of various tissues were incubated in the presence of reduced glutathione (GSH), but without any addition of cytosolic glutathione-S-transferase (GST), a drastic decrease in the AFB1-DNA binding was seen . Analyses of the water-soluble metabolites formed in the microsomal incubations supplemented with GSH showed fluorescent and ninhydrin-positive metabolites that were not present in the absence of GSH . These results indicate that sheep tissues have intrinsic microsomal GST or cytosolic GSTs associated with the microsomal fraction with a high capacity to catalyse the conjugation of bioactivated AFB1 to GSH . The results of the present study show that several extrahepatic tissues of sheep have a potent capacity to bioactivate AFB1 and also a high capacity to GSH conjugate the bioactivated AFB1. Carcinogenesis, 1994 May, 15(5), 927 - 31 Mutagenic activity of gastric fluid from chewers of tobacco with lime; Niphadkar MP et al.; Although tobacco chewing is strongly associated with a high risk of oral and upper alimentary tract cancers, the nature of mutagenic exposure among users has not been clearly defined . In this study, tobacco-specific and mutagenic exposure of chewers of tobacco with lime was evaluated by analysis of gastric fluid (GF) . The pH, nitrite and cotinine levels of GF samples from chewers and non-chewers were determined and the samples were tested for mutagenicity in the Ames Salmonella/microsome assay using Salmonella typhimurium strains TA98, TA100 and TA102 . Cotinine was not detected in GF from non-chewers while the levels ranged between 0.4-13.64 micrograms/ml in samples from chewers; however, the mean pH values (3.8 +/- 0.4 versus 2.8 +/- 0.3) and nitrite levels (29.40 +/- 1.51 versus 27.39 +/- 0.83 microM) were similar in both groups . While all GF samples from non-chewers were non-mutagenic, samples from chewers were directly mutagenic or upon nitrosation to all the three tester strains and to TA102 strain in the presence of S9 . Experiments using scavengers of reactive oxygen species (ROS) showed that mannitol and benzoate abolished the mutagenic response of TA102, indicating that ROS are principally responsible for oxidative damage . The findings provide specific information regarding the mutagenic exposure among tobacco chewers and suggest that tobacco chewing may be an important risk factor in the development of gastric cancer. Carcinogenesis, 1994 May, 15(5), 917 - 20 Inhibitory effect of vitamin C on the mutagenicity and covalent DNA binding of the electrophilic and carcinogenic metabolite, 6-sulfooxymethylbenzo{a}pyrene; Surh YJ et al.; 6-Sulfooxymethylbenzo{a}pyrene has recently been shown to be a strong hepatocarcinogen in infant male B6C3F1 mice (Y.-J.Surh et al., Biochem . Biophys . Res . Commun., 172, 85-91, 1990) and appears to be an ultimate carcinogenic metabolite of 6-hydroxymethylbenzo{a}pyrene and possibly of benzo{a}pyrene . It produced high levels of aralkyl DNA adducts in the livers of B6C3F1 mice and also exhibited strong direct mutagenicity toward Salmonella typhimurium TA98 without metabolic activation . In the present study we found that ascorbic acid significantly reduced the bacterial mutagenicity and in vitro covalent DNA binding of 6-sulfooxymethylbenzo{a}pyrene . Ascorbic acid forms a mutagenically inactive covalent adduct with 6-sulfooxymethylbenzo{a}pyrene, which appears to account for its novel protective mechanism against this reactive sulfuric acid ester . It seems likely that the formation of this adduct involves aralkylation of an ascorbic acid anion by a presumed carbo cation derived from the electrophilic sulfuric acid ester. Toxicol Appl Pharmacol, 1994 May, 126(1), 39 - 44 Species variation in the genotoxicity of batracylin; Stevens GJ et al.; Batracylin (8-aminoisoindolo{1,2-b}quinazolin-12(10H)-one), an experimental chemotherapeutic agent, is a heterocyclic aryl amine . The presence of the free amino group suggests that this compound may be a carcinogen . Since many carcinogens or their product interact with DNA, the genotoxicity of batracylin was evaluated . The mutagenicity of batracylin was tested in Salmonella typhimurium (TA 98, TA 100, TA 102) with and without Aroclor-induced rat liver S9 . Batracylin induced histidine revertants in all three strains with a higher number of mutants formed in the presence of S9 . The maximum mutant incidence as well as the lowest concentration inducing a positive response was observed with TA 98 which detects frameshift mutations . Genotoxicity was further assessed by the induction of DNA repair in hepatocytes isolated from F-344 rats and two mouse strains differing in N-acetyltransferase activity . In hepatocytes from male F-344 rats, batracylin elicited DNA repair at concentrations of 5 x 10(-8) to 10(-6) M . Maximum repair was observed in both strains of mice at 5 x 10(-5) M batracylin, a concentration that was toxic to rat hepatocytes . Cells isolated from the rapid acetylator strain, C57BL/6J, tended to have higher net grain counts than observed with the slow acetylator (A/J) strain, although the differences were not statistically significant . These results demonstrate that batracylin is genotoxic in bacteria and mammalian cells . The species variation in the genotoxicity of batracylin is consistent with in vivo toxicity studies. J Bacteriol, 1994 May, 176(9), 2619 - 26 The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium; Bergman T et al.; Virulent bacteria of the genus Yersinia secrete a number of virulence determinants called Yops . These proteins lack typical signal sequences and are not posttranslationally processed . Two gene loci have been identified as being involved in the specific Yop secretion system (G . Cornelis, p . 231-265, In C . E . Hormache, C . W . Penn, and C . J . Smythe, ed., Molecular Biology of Bacterial Infection, 1992; S . C . Straley, G . V . Plano, E . Skrzypek, P . L . Haddix, and K . A . Fields, Mol . Microbiol . 8:1005-1010, 1993) . Here, we have shown that the lcrB/virB locus (yscN to yscU) encodes gene products essential for Yop secretion . As in previously described secretion apparatus mutants, expression of the Yop proteins was decreased in the yscN/U mutants . An lcrH yscR double mutant expressed the Yops at an increased level but did not secrete Yops into the culture supernatant . The block in Yop expression of the ysc mutants was also circumvented by overexpression of the activator LcrF in trans . Although the Yops were expressed in elevated amounts, the Yops were still not exported . This analysis showed that the ysc mutants were unable to secrete Yops and that they were also affected in the negative Ca(2+)-regulated loop . The yscN/U genes showed remarkably high homology to the spa genes of Shigella flexneri and Salmonella typhimurium with respect to both individual genes and gene organization . These findings indicate that the genes originated from a common ancestor. J Bacteriol, 1994 May, 176(9), 2596 - 602 Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy; Foster JW et al.; Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3) . It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock) . The genetics of acid resistance was investigated through the use of a new screening medium . The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8 . The medium will lower the internal pH of cells to a lethal level . However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells . Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants . Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC) . Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG) . The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR . An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon . Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance . The atrF mutation mapped within the ent cluster, probably in a fep uptake locus . The atrD locus mapped near metC and may represent an insertion into the S . typhimurium homolog of the Escherichia coli exbB or exbD locus . The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus . The results support the concept of overlapping acid protection systems in S . typhimurium. Infect Immun, 1994 May, 62(5), 1669 - 76 Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination; Schodel F et al.; Avirulent salmonellae expressing foreign genes are attractive for use as oral vaccine carriers . To facilitate the stable expression of heterologous genes without conferring antibiotic resistance, a deletion of the asdA1 gene was introduced into Salmonella typhimurium and S . typhi delta cya delta crp mutant vaccine strains . An asd-complementing plasmid expressing hybrid hepatitis B virus nucleocapsid-pre-S (HBcAg-pre-S) particles was constructed . These hybrid HBcAg-pre-S particle genes were stably expressed in S . typhimurium and S . typhi delta cya delta crp mutant vaccine strains in this balanced, lethal host-vector combination . A single oral immunization of BALB/c mice with a recombinant S . typhimurium delta cya delta crp mutant synthesizing hybrid HBcAg-pre-S elicited potentially virus-neutralizing anti-pre-S serum immunoglobulin G antibodies . In addition, serum immunoglobulin G recognizing S . typhimurium lipopolysaccharide was induced . Distribution in tissue after oral immunization was analyzed in one plasmid-strain combination . The recombinant S . typhimurium colonized the gut-associated lymphoid tissue and the spleen and persisted for over 4 weeks, retaining the HBcAg-pre-S expression plasmid . An isogenic virulence plasmid-cured S . typhimurium delta cya delta crp strain expressing the same HBcAg-pre-S gene had reduced immunogenicity for the carried antigen after oral immunization. Infect Immun, 1994 May, 62(5), 1652 - 7 Systemic and mucosal immune responses in mice orally immunized with avirulent Salmonella typhimurium expressing a cloned Porphyromonas gingivalis hemagglutinin; Dusek DM et al.; Porphyromonas gingivalis produces a variety of virulence factors that may have a function in the periodontal disease process . Determination of the role of these various factors in pathogenesis and identification of a means for protecting the host from the destructive effects of this organism are areas of vigorous investigation . In this study we demonstrate the potential of avirulent Salmonella typhimurium strains to stimulate a specific systemic and mucosal immune response to a cloned P . gingivalis hemagglutinin (HagB) . An avirulent strain of S . typhimurium, chi 4072, expressing the hagB gene of P . gingivalis 381 on the plasmid pDMD1 was intragastrically administered to BALB/c mice . These mice mounted a serum immunoglobulin G (IgG) and IgA primary response against the hagB gene product and a mucosal immune response as measured by evaluation of saliva . IgA antibodies were also detected in bile . These results demonstrate the feasibility of using attenuated S . typhimurium strains as carriers of P . gingivalis virulence factors for subsequent evaluation of the systemic and mucosal immune response against these antigens . This system will provide a means for evaluating the virulence factors of P . gingivalis for their suitability in the construction of potential vaccines. Infect Immun, 1994 May, 62(5), 1551 - 6 Protein phosphorylation in murine peritoneal macrophages induced by infection with Salmonella species; Saito S et al.; Infection of peritoneal macrophages from C3H/HeN and C3H/HeJ mice with Salmonella typhimurium or S . enteritidis induced extensive phosphorylation in a set of proteins with molecular masses of 85, 72, 35, 30, and 23 kDa, which were different from those induced by bacterial lipopolysaccharide . The phosphorylated proteins of 35, 30, and 23 kDa (pp35, pp30, and pp23, respectively) originated from the infecting bacteria, because living bacteria could induce these phosphorylated proteins themselves, and no induction of the proteins occurred in macrophages after phagocytosis of heat-killed or UV-irradiated organisms . When the infected macrophages were disrupted and separated into bacterial and macrophage debris fractions, pp85 and pp72 remained in the macrophage debris fraction, with none in the bacterial fraction . Induction of pp85 and pp72 in infected macrophages was inhibited in the presence of chloramphenicol but not cytochalasin D, suggesting that bacterial growth in the macrophages is necessary for induction of both proteins . Neither of these proteins could be detected in macrophages infected with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Listeria monocytogenes . These results support the view that phosphorylation of the 85- and 72-kDa proteins occurs in the macrophages during the early phases of the interaction between Salmonella organisms and macrophages . The functions of specific proteins remain to be clarified. Am J Epidemiol, 1994 May 1, 139(9), 903 - 9 Protective effect of conventional cooking versus use of microwave ovens in an outbreak of salmonellosis; Gessner BD et al.; The authors conducted an investigation to determine the extent and source of an outbreak of Salmonella typhimurium gastroenteritis that occurred following a community picnic in Juneau, Alaska, in 1992, and to evaluate risk factors for illness . A case-control study among 54 picnic attendees and a retrospective cohort study among 60 members of 17 households who had taken home leftover food from the picnic were conducted . A case was defined as diarrhea with onset 12-72 hours after eating food that had been prepared for the picnic . The case-control study associated illness with eating roast pork from one of two pigs that had been flown in from a Seattle, Washington, restaurant . The roast pork was taken home by persons from at least the 17 households included in the cohort study . The cohort study identified 43 persons who ate roast pork, of whom 21 (49%) became ill . This compared with only one case of illness among 17 cohort members who had not eaten roast pork (relative risk = 8.3, 95% confidence interval 1.2-57.0) . Of 30 persons who ate reheated meat, all 10 who used a microwave oven became ill, compared with none of 20 who used a conventional oven or skillet . The Seattle restaurant had prepared the roast pork by first thawing two frozen pigs for several hours at room temperature and then cooking them in a gas-fired flame broiler . One of the pigs was left unrefrigerated for 17-20 hours after cooking . Compared with conventional methods of reheating, microwave ovens had no protective effect in preventing illness . To prevent outbreaks such as this one, care must be taken to assure that food is both properly cooked and handled and properly reheated. Chem Res Toxicol, 1994 May-Jun, 7(3), 319 - 28 1H NMR characterization of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frameshift hotspot of Salmonella typhimurium hisD3052; Moe JG et al.; The exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was incorporated into 5'-d{ATCGC(PdG)CGGCATG}-3', derived from the hisD3052 gene of Salmonella typhimurium . The modified oligodeoxynucleotide was annealed with the complementary strand 5'-d{CATGCCGCGAT}-3' which contained a CpG deletion . The resulting duplex 5'-d{ATCGC(PdG)CGGCATG}-3'.5'-d{CATGCCGCGAT}-3' required PdG and one adjacent cytosine to be unpaired . Four thymine imino 1H NMR resonances were observed at temperatures below 25 degrees C, which demonstrated formation of a stable duplex with a two-base bulge . PdG was accommodated within the DNA helix, whereas the 3'-neighbor cytosine was poorly stacked and appeared to be extrahelical . The sequential nuclear Overhauser enhancement connectivities between aromatic and H1' protons along the modified strand were interrupted between PdG and the 3'-neighboring unpaired cytosine . On the complementary strand no interruptions were observed . An NOE was observed between the PdG methylene protons H8a,b and the imino proton of the 5'-neighbor base pair . Weaker NOEs were observed between the PdG H8a,b protons and the imino proton from guanine two nucleotides removed in the 3'-direction, and to the amino proton of cytosine located in the complementary strand two nucleotides removed in the 3'-direction . Chemical shift perturbations were also observed for the latter cytosine as compared to the corresponding cytosine in the unmodified fully complementary duplex . These observations provided evidence for a poorly stacked or an extrahelical conformation of this unpaired cytosine . The amino proton resonances of the 3'-neighbor cytosine were not observed, presumably due to increased exchange with solvent . The methylene protons from PdG were shifted upfield relative to the monomer PdG, probably as a result of aromatic ring current shielding, consistent with an intrahelical location . Multimeric derivative oligonucleotides containing the PdG bulge migrated anomalously on nondenaturing polyacrylamide gels, consistent with a structure in which the unpaired nucleotides induced a bend in the DNA. Chem Res Toxicol, 1994 May-Jun, 7(3), 313 - 8 Activation of the Maillard reaction product 5-(hydroxymethyl)furfural to strong mutagens via allylic sulfonation and chlorination; Surh YJ et al.; 5-(Hydroxymethyl)furfural (HMF), one of the major intermediate products in the Maillard reaction, is present in a wide variety of foods . This aldehyde is formed as a decomposition product of glucose and fructose in foodstuffs subject to cooking or heat sterilization . It has been found to possess mutagenic and DNA strand-breaking activity . However, the mechanisms by which HMF exerts its genotoxicity remain unclear . The present study was undertaken to determine if HMF could be metabolically activated via esterification of the allylic hydroxyl group . In support of this concept, the chemically synthesized sulfuric acid ester,5-{(sulfooxy)-methyl}furfural (SMF), exhibited direct mutagenicity at both thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase loci in human lymphoblasts . This reactive ester also induced 8-azaguanine-resistant mutants in Salmonella typhimurium TM677 in a dose-dependent manner . The intrinsic mutagenicity of SMF was enhanced by addition of extra chloride ion to the assay medium . The model allylic derivative, 5-(chloromethyl)furfural, was also mutagenic and cytotoxic in bacteria, but much more active than the sulfuric acid ester in this regard . In contrast to (sulfooxy)methyl and chloromethyl derivatives of HMF,2-{(sulfooxy)-methyl}- and 2-(chloromethyl)furans which lack the aldehyde functionality did not exhibit significant mutagenicity . Rodent hepatic cytosols contained sulfotransferase activity responsible for the formation of the reactive allylic sulfuric acid ester metabolite from HMF. J Toxicol Sci, 1994 May, 19(2), 89 - 96 Mutagenicity tests of polyoxyethylene hydrogenated castor oil 60 (HCO-60); Hirai O et al.; The genotoxicity of polyoxyethylene hydrogenated castor oil 60 (HCO-60) was examined in reverse mutation test in bacteria, chromosome aberration test in vitro and micronucleus test in mice . The in vitro tests were done with and without metabolic activation using rat liver microsomal fraction . HCO-60 did induce neither reverse mutation in Salmonella typhimurium TA100, TA98, TA1535, and TA1537 and in Escherichia coli WP2uvrA, nor chromosome aberrations in Chinese hamster V79 cells . In addition, no increase in micronucleated polychromatic erythrocytes was elicited in the bone marrow of BDF1 male and female mice . It is concluded that HCO-60 was not genotoxic in these in vitro and in vivo assays. Plasmid, 1994 May, 31(3), 288 - 96 Colicin 24, a new plasmid-borne colicin from a uropathogenic strain of Escherichia coli; O'Brien GJ et al.; A new colicin, as defined by cross-immunity tests against the colicin reference set, has been identified from a uropathogenic Escherichia coli isolate (strain 2424), collected from a pyelonephritis patient at Christchurch Hospital . This toxin, colicin 24, is active against E . coli and Salmonella typhimurium, and the genes involved in colicin 24 production, transport, and immunity were found to reside on a 43.54-kb conjugative plasmid, p24-2 . Mutagenesis using mini-Tn10 has identified two consecutive EcoRI fragments on p24-2 that contain all the colicin 24 determinants . A 25.14-kb BamHI-HindIII fragment containing this region was cloned from a mutant with a normal colicin phenotype into pBR322 to form the recombinant plasmid pGOB34 . Subcloning of pGOB34 has resulted in an 8.7-kb fragment containing all the colicin 24 determinants being cloned into pUC18 to form pGOB342. Fundam Appl Toxicol, 1994 May, 22(4), 483 - 93 Does the Delaney Clause of the U.S . Food and Drug laws prevent human cancers? Weisburger JH. The Delaney Clause of the Federal Food, Drug, and Cosmetic Act, enacted in 1958, prohibits the addition to the human food supply of any chemical that had caused cancer in humans or animals . The aim was to prevent cancer in humans . The scientific knowledge on causes of cancer and mechanisms of carcinogenesis in the 1950s can be rationalized to justify enactment of this Clause at that time . Since then, important progress in the fields of mechanism of carcinogenesis and cancer causation, and in analytical chemistry permitting accurate determination of trace amounts of chemicals, suggests that the Clause requires modification based on current knowledge . The documented human carcinogens are DNA reactive or genotoxic . Thus, the Clause should emphasize prohibition of the addition to human foods of proven genotoxins that are likely human cancer risks by contemporary standards . Such genotoxic carcinogens are those reliably positive in a battery of three tests, the Ames test in Salmonella typhimurium, the Williams test with evidence of DNA repair in hepatocytes, and direct documentation of DNA adduct formation in the 32P-postlabeling technique of Randerath. J Clin Microbiol, 1994 May, 32(5), 1322 - 5 Molecular epidemiology of ampicillin-resistant clinical isolates of Salmonella enteritidis; Vatopoulos AC et al.; During the last 6 years in Greece, there has been a significant increase in the number of ampicillin-resistant Salmonella clinical isolates reported . In this study 23 ampicillin-resistant Salmonella strains, consecutively isolated from patients with epidemiologically unrelated cases of food poisoning, were investigated . By serotyping and phage typing, 21 of these strains were identified as Salmonella enteritidis phage type 6a, 1 was identified as Salmonella typhimurium, and 1 was identified as Salmonella saintpaul . By plasmid pattern analysis, the 21 S . enteritidis strains were further differentiated into five groups . Group I consisted of 5 strains (carrying two plasmids of ca . 38 and 34 MDa), group II consisted of 10 strains (three plasmids of ca . 38, 34, and 2.5 MDa), group III consisted of 3 strains (four plasmids of ca . 38, 34, 15, and 2.5 MDa), group IV consisted of 1 strain (five plasmids of ca . 100, 38, 34, 24, and 15 MDa), and group V consisted of 2 strains (three plasmids of ca . 100, 38, and 24 MDa) . Ampicillin resistance was easily transferred to Escherichia coli and was associated with the transfer of the 34-MDa plasmid, classified in the N incompatibility group for all strains of groups I to IV, and with the transfer of the 100-MDa plasmid for the group V strains . EcoRI restriction endonuclease digestions showed an extensive homology among the 34-MDa conjugative R plasmids . Hybridizations of the EcoRI restriction fragments of the 34-MDa plasmids with a TEM-type probe revealed the locus of the beta-lactamase gene to be situated on a ca . 6.6-MDa fragment, common in all plasmids . These results indicate that ampicillin resistance in Greece is due to the spread of a limited number of clones of S . enteritidis phage type 6A, carrying related 34-MDa R plasmids . Work is in progress to obtain a better understanding of ampicillin resistance in S . enteritidis. Poult Sci, 1994 May, 73(5), 640 - 7 Comparison of effects of chicken cecal microorganisms maintained in continuous culture and provision of dietary lactose on cecal colonization by Salmonella typhimurium in turkey poults and broiler chicks; Hollister AG et al.; A mixed bacterial culture derived from the cecal contents of an adult broiler chicken was maintained in continuous-flow culture and tested for effectiveness in Salmonella colonization reduction in broiler chicks and turkey poults . Day-old chicks and poults in two separate experiments were divided into four groups and provided a standard corn-soybean diet with: 1) no culture, no lactose (control); 2) 5% dietary lactose; 3) broth culture by crop gavage; 4) culture by crop gavage and 5% lactose . All groups were challenged orally on Day 3 with 10(4) Salmonella typhimurium . At 10 and 21 d of age the chicks provided culture and lactose had significantly (P < .05) fewer Salmonella per gram of cecal contents than controls . Poults provided culture by gavage and lactose also had significantly (P < .05) fewer Salmonella per gram of cecal contents than control poults, but the number was 100- to 1,000-fold higher than that of the chicks provided the same treatment . The percentage of Salmonella cecal-culture-positive chicks provided culture and lactose was significantly reduced at 10 and 21 d of age in both experiments compared with controls, but the percentage of cecal-culture-positive poults was significantly different from controls only at 21 d in one of the two experiments . Chicks provided culture and lactose had significantly fewer Salmonella colony-forming units per gram and significantly fewer cecal-culture-positive birds than poults provided culture and lactose in both experiments . The results indicate that cultures of cecal bacteria that effectively reduce Salmonella colonization in broiler chicks may not be as effective for reduction of Salmonella colonization in turkey poults. FEMS Microbiol Lett, 1994 May 1, 118(1-2), 159 - 62 Sequence of the fruB gene of Escherichia coli encoding the diphosphoryl transfer protein (DTP) of the phosphoenolpyruvate: sugar phosphotransferase system; Reizer J et al.; The Escherichia coli genome sequencing project in the 47 to 48 centisome region has resulted in the sequencing of the complete fructose operon (fruBKA) . Due to a single base insertion, the presence of the fruB gene went unnoticed . The revised nucleotide sequence of the fruB gene, the deduced amino acid sequence of its protein product, the diphosphoryl transfer protein of the phosphoenolpyruvate: sugar phosphotransferase system, and putative transcriptional regulatory signals of the fru operon of E . coli are here presented and compared with that from Salmonella typhimurium. FEMS Microbiol Rev, 1994 May, 14(1), 3 - 20 Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system proU; Lucht JM et al.; A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Escherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor . Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor . The physiological reaction of the cell to the changing environmental condition is a highly coordinated process . Loss of turgor triggers a rapid influx of K+ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate . The increased intracellular concentration of K(+)-glutamate allows the adaptation of the cell to environments of moderately high osmolarities . At high osmolarity, K(+)-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K+ ions with compounds that are less deleterious for the cell's physiology . These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine . One of the most important compatible solutes for bacteria is glycine betaine . This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline . In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes. Bull Tokyo Dent Coll, 1994 May, 35(2), 79 - 83 B-cell mitogenicity and IL-1 beta production of lipopolysaccharides from various Capnocytophaga strains; Kim SJ et al.; To evaluate the etiological roles of Capnocytophaga species in the pathogenesis of periodontal disease, we examined the immunological activities of lipopolysaccharides (LPSs) from various Capnocytophaga strains . All LPSs from various Capnocytophaga species were mitogenic for BALB/c mouse spleen cells, although the responses were lower than those to reference LPSs from Escherichia coli or Salmonella typhimurium . LPSs of C . sputigena strains had polyclonal B cell activation and adjuvant activity and were comparable to reference LPSs . All LPSs from Capnocytophaga strains activated the interleukin-1 beta production from human peripheral monocytes, although the inducing activities of Capnocytophaga LPSs were lower than those of reference LPSs . It appears that LPSs from various Capnocytophaga strains activate certain immunological responses from lymphocytes and monocytes which may be important in the development and pathogenesis of periodontal disease. Tsitol Genet, 1994 May-Jun, 28(3), 91 - 2, 3 of cover {The mutagenic and modifying properties of new gelatinous substances studied by using the Ames test}; Strizhel'chik NG et al.; Mutagenic activities of new gel-forming substances proposed for the use in pharmacy and food industry were studied by the method of scoring mutations in test microorganisms . Sodium carboxy-methyl starch and sodium carboxy-methyl-cellulose did not induce gene mutations in Salmonella typhimurium TA 98 and TA 100. Tsitol Genet, 1994 May-Jun, 28(3), 37 - 41 {Salmonella typhimurium as a test system for detecting the mutagenic activity of environmental pollutants}; Dugan AM; The mutagenic activity of some dyes, derivatives of anthraquinone and aniline, was studied in tests on Salmonella typhimurium . A total of 16 anthraquinones and 9 anilines were tested . It was detected that 7 anthraquinone dyes did not induce gene mutations in Salmonella . Two substances, 2-chlor-anthraquinone and 1-nitro-anthraquinone, showed a medium-strength effect . Seven dyes (1-methyl-amino-4-bromanthraquinone, leuko-1,4-diamino-anthraquinone, 1,5-diaminoanthraquinone, 1-chlor-anthraquinone, 1,2- and 1,8-dihydroxyanthraquinone, 1-chlor-4-benzil-amino-anthraquinone) were weak mutagens . The metabolic activation system was not effective, i.e . the dyes studied are mutagens with a direct action . A dose dependence was detected . The main mechanism of induction of gene mutations is frameshift . From nine dyes of the aniline series, three compounds showed a weak mutagenic effect: 3-nitro-aniline, anilin-3-sulpho-acid and 4-nitroaniline-2-sulpho-acid . The latter substance is an indirect mutagen . The other two are direct ones . The mechanism of induction of gene mutations is frameshift. J Biochem (Tokyo), 1994 May, 115(5), 965 - 72 Overproduction and characterization of recombinant UDP-glucose pyrophosphorylase from Escherichia coli K-12; Hossain SA et al.; Using oligonucleotide probes synthesized on the basis of partial amino acid sequences, we have cloned and sequenced the gene of Escherichia coli K-12 encoding UDP-glucose pyrophosphorylase . The gene consists of 906 base pairs and encodes a polypeptide of 302 amino acid residues with a calculated molecular weight of 32,941 . Its nucleotide sequence was found to be identical with that recently registered (EMBL, X59940) for a gene coding for an unknown 33-kDa protein, which was later annotated as UDP-glucose pyrophosphorylase on the basis of genetic studies . The UDP-glucose pyrophosphorylase gene, mapped at 27.3 min in the E . coli chromosome, complemented the galU mutation, which renders the bacterium unable to ferment galactose . The recombinant enzyme overproduced in E . coli cells and purified to homogeneity catalyzed the synthesis and pyrophosphorolysis of UDP-glucose by a sequential mechanism . The enzyme required Mg2+ for maximal activity and was inhibited by free UTP and pyrophosphate . The E . coli enzyme shows significant sequence similarities with the enzymes from Acetobacter xylinum and Salmonella typhimurium . However, little or no similarity was found with the eukaryotic enzymes that are involved in the biosynthesis of storage carbohydrates, or with other enzymes acting on similar sugar nucleotides . Thus, UDP-glucose pyrophosphorylases participating in diverse metabolic pathways can be classified structurally into the prokaryotic and eukaryotic groups, even though they have almost identical catalytic properties. Mutagenesis, 1994 May, 9(3), 205 - 24 SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds; Mersch-Sundermann V et al.; To examine the concordance of two microbial genotoxicity short-term assays, 330 experimental results for the SOS chromotest using tester strain Escherichia coli PQ37 were compared with the results of the Salmonella/mammalian microsome mutagenicity assay with Salmonella typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and/or TA1538 . With respect to qualitative features, the concordance between SOS chromotest and Salmonella mutagenicity test results was 86.4% (sensitivity, 78.6%; specificity, 100%; chi 2 = 188.6) . None of the non-mutagens (N = 120) were able to induce the SOS system . Additionally, 45 of the 210 S.typhimurium mutagens (21.5%) did not induce the SOS repair system . On closer examination, the majority of these 45 compounds (84%) were mutagens with activities between 0.001 and 10 rev/nmol . Even though the experimental protocols of both systems were not standardized, the correlation coefficient for the experimental results of the two test systems was 0.7 for the 330 chemicals . Except for aliphatic epoxides (r = 0.47), the mutagenicity/SOS induction correlations for congeneric data sets (polycyclic aromatic hydrocarbons, nitroarenes, nitroarenofurans, mycotins) were even better (r = 0.72-0.95) . Additionally, computer automated structure evaluation (CASE) analyses of the nature of the structural determinants associated with each endpoint indicate extensive homologies . The data can be taken to indicate that the two phenomena reflect common mechanisms of action. Indian J Med Res, 1994 May, 99, 203 - 5 Transferable beta lactam resistance against cephalosporins in Salmonella typhimurium strains in India; Bhatia R et al.; From 14 strains of S . typhimurium which were resistant to three cephalosporins (cephalexin, cefadroxil and sodium cefotaxime) the resistance plasmids were transferred to two different strains (Escherichia coli K12F-Lac-Rifr and S . typhimurium LT2) . The plasmids were autotransferable and the donors as well as transconjugants showed high levels of MIC (80-320 micrograms/ml or more) against these antimicrobial agents . The resistance was demonstrated to be mediated by a 15 kilobase plasmid. East Afr Med J, 1994 May, 71(5), 292 - 6 Antimicrobial susceptibility and presence of extrachromosomal deoxyribonucleic acid in Salmonella and Shigella isolates from patients with AIDS; Kariuki S et al.; The development of multi-drug resistance by enteric bacteria is an increasing problem in the developing countries . There is need to monitor antimicrobial susceptibility of these organisms in order to ensure appropriate treatment and control of infections . Antimicrobial susceptibility patterns, plasmid DNA content and restriction enzyme digests of plasmid deoxyribonucleic acid (DNA) were used to study 175 Salmonella and Shigella species isolated from predominantly HIV-seropositive adult patients in Nairobi, Kenya . All the isolates were sensitive to ciprofloxacin . A significantly higher proportion of Shigella species were resistant to chloramphenicol, cotrimoxazole, streptomycin and tetracycline compared to Salmonella species (p-value < 0.001) . Multi-resistant Salmonella typhimurium isolates had 60, 40 and 5 MDa plasmids, the 5 MDa plasmid was absent in gentamicin sensitive isolates . In addition to 2-10 MDa range of plasmids, multi-resistant Shigella species had a heavy 100-105 MDa plasmid . Restriction enzyme digests were similar for the 60 and 40 MDa plasmid DNA bands from Salmonella typhimurium isolates but did not show any consistency among Shigella spp . Plasmid-encoded multi-drug resistance plays a major role in the spread of resistance among enteric bacteria . It is vital to use drugs rationally in order to control the emergence and spread of multi-drug resistance. Biofactors, 1994 May, 4(3-4), 177 - 80 A thiol-specific antioxidant and sequence homology to various proteins of unknown function; Chae HZ et al.; Yeast and mammalian cells contain a 25 kDa enzyme that protects cellular components against oxidative damage from a system capable of generating reactive sulfur species, but not from a system that generates only reactive oxygen species . Yeast and rat cDNAs corresponding to this thiol-specific antioxidant (TSA) have been cloned and sequenced . Rat TSA is 65.3% identical and 76.2% similar to yeast TSA in amino acid sequence . A search of the GenBank database revealed 12 additional TSA-like proteins, which show sequence identity to rat TSA ranging from 31 to 76% . Except for the AhpC protein identified in Salmonella typhimurium, none of the TSA-like proteins is associated with known cellular functions . AhpC, which exhibits approximately 40% sequence identity to TSA, has been proposed to be a catalytic component of alkyl hydroperoxide reductase . Alignment of rat and yeast TSA with the TSA-like sequences revealed two conserved cysteine residues, one conserved in all 14 sequences and the other in 12 sequences . The most conserved cysteine is located in a well-conserved motif of (hydrophobic residue)6-Pro-non-conserved-residue-Asp-Phe-Thr-Phe-Val-Cys-Pro-Thr-Glu- hydrophobic residue . These results suggest that the TSA-like proteins of previously unknown function may represent a widely distributed family of antioxidants with functions similar to those of TSA and AhpC. Vaccine, 1994 May, 12(6), 513 - 7 Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs; Morona R et al.; Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99 . Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed . These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively . When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens . Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms . Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system. Zhonghua Yu Fang Yi Xue Za Zhi, 1994 May, 28(3), 136 - 9 {Phage typing of Salmonella typhimurium in 20 provinces, autonomous regions and municipalities of China}; He XQ et al.; Thirty strains of Salmonella bacteriophages were isolated from hospital sewage . Eleven strains selected from them, in combination with Felix Salmonella Phage O-I, were used as a phage typing set for serotyping Salmonella . Thirty-one identified types and 20 undefined types were detected in 2348 strains of Salmonella typhimurium collected from 20 provinces, autonomous regions and municipalities in China . The nine types with higher frequencies were 7774 (accounted for 48.0%), 0774 (17.1%), 6774 (7.7%), 4774 (7.3%), 5774 (3.3%) . 3774 (2.2%), 4000(2.1%), 0000 (1.8%) and 7000 (1.7%) . Most outbreaks of nosocomial infection and food poisoning were caused by phage types 7774 and 4774 . Phage types of Salmonella typhimurium in domestic ducks, pigs, rats, snails, and in sewages were analyzed, and their significance in epidemics was discussed. Mol Microbiol, 1994 May, 12(4), 639 - 46 The regulatory RNA gene micF is present in several species of gram-negative bacteria and is phylogenetically conserved; Esterling L et al.; micF RNA post-transcriptionally regulates Escherichia coli outer membrane protein F (OmpF), in response to temperature increase and other environmental stress conditions, by binding to ompF mRNA and destabilizing the message . Southern analyses show that the micF gene is present in related Gram-negative bacteria, including Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa . In addition, Northern analyses indicate that micF RNA and ompF mRNA levels are thermally regulated in several related species in a manner similar to the thermoregulation in Escherichia coli . DNA sequences from Salmonella typhi, Salmonella typhimurium, and Klebsiella pneumoniae show greater than 96% homology in the micF gene when compared to the Escherichia coli micF sequence . Upstream of micF, sequences show considerable variation, although several distinct regions are highly conserved . Some of these conserved regions correspond to known binding sites for the transcription factor OmpR and the DNA-binding protein integration host factor . In addition, E . coli micF RNA incubated with protein extracts from other species forms heterologous ribonucleoproteins (RNPs) . The formation of these heterologous RNPs indicates both the presence of micF RNA-binding protein homologues in other species and a conservation of RNA-protein recognition sites . This work demonstrates that the micF RNA regulatory system is present in other Gram-negative bacterial species and that this system appears to be phylogenetically conserved. Mutat Res, 1994 May 1, 307(1), 83 - 93 Structure-activity relationships within various series of p-phenylenediamine derivatives; Shahin MM; The mutagenicity of 20 p-phenylenediamine derivatives has been investigated in Salmonella typhimurium . Tests were performed in the presence and in the absence of Aroclor 1254-induced liver S9 fractions derived from male Wistar rats . Among five series of compounds tested, nitro-p-phenylenediamines with substituents at the C6 position (4-amino-3-nitro-6-methylaniline; 4-amino-3-nitro-6-methoxyaniline; 4-amino-3-nitro-6-fluoroaniline; 4-amino-3-nitro-6-chloroaniline; and 4-amino-3-nitro-isopropylaniline) were the most mutagenic . In all cases, the compounds were less mutagenic in the absence of S9 than in its presence, but three of the five compounds (the methoxy, fluoro, and chloro derivatives) were still mutagenic without the metabolic activation system . In contrast to the mutagenicity of the C6-substituted compounds, the mutagenicity of analogues with substituents on the C5 position (4-amino-3-nitro-5-beta-hydroxypropylaniline; 4-amino-3-nitro-5-isopropylaniline; 4-amino-3-nitro-5-methylaniline; and 4-amino-3-nitro-5-beta-hydroxyethylaniline) was abolished or reduced . A dramatic reduction in mutagenic activity was also achieved when two methyl groups, instead of one, were added to 4-amino-3-nitroaniline . For example, 4-amino-3-nitro-5,6-dimethylaniline and 4-amino-3-nitro-2,5-dimethylaniline were only weakly mutagenic, and 4-amino-3-nitro-2,6-dimethylaniline as 4-amino-2-6-dimethylaniline; 4-amino-5,6-dimethylaniline; was nonmutagenic . Monocyclic compounds such as 4-amino-2,6-dimethylaniline; 4-amino-5,6-dimethylaniline; 4-amino-2-methoxy-3,5-dimethylaniline, and 4-amino-2,3,5,6-tetramethylaniline were all nonmutagenic in Salmonella typhimurium . The compound 4-amino-2,5-dimethylaniline was weakly mutagenic or nonmutagenic, whereas 4-amino-2,5-dimethoxyaniline was mutagenic . It appears that the mutagenic activity or inactivity of these compounds depends on both the chemical groups present and their positions in the molecule . In this context, it seems that the presence of the NO2 group and the nature of the substituent groups at the C5 and C6 positions on the benzene ring are crucial factors in determining the mutagenicity of these compounds. Mutat Res, 1994 May 1, 307(1), 185 - 92 Characterization of 4-nitro-o-phenylenediamine activation by plant systems; Wilson L et al.; 4-Nitro-o-phenylenediamine (NOP) is a powerful direct-acting mutagen which demonstrates significant enhancement in mutagenicity when exposed to plant enzymatic systems . Evidence implicating the involvement of peroxidactic oxidation in NOP activation has been obtained from plant-cell suspension and isolated enzyme experiments . Using selected cytochrome P450 and peroxidase enzyme inhibitors in conjunction with Salmonella typhimurium strain TA98 and intact plant-cell activating systems as well as isolated horseradish peroxidase enzyme we have further investigated NOP activation by plant systems . The activation of NOP by both plant cells and by horseradish peroxidase was suppressed by the P450 inhibitors methimazole and (+)-catechin and by the peroxidase inhibitors diethyldithiocarbamate and potassium cyanide, but was not suppressed by the P450 inhibitors metyrapone and 7,8-benzoflavone . In addition, peroxidase enzymatic activity was measured and found to be inhibited by methimazole, diethyldithiocarbamate and potassium cyanide but not by (+)-catechin . The data strongly support the involvement of exogenous peroxidase in the plant activation of NOP, but point to a complex metabolic system that requires multistep processing before full mutagenic potential of the plant-activated component of NOP is expressed. Mutat Res, 1994 May 1, 307(1), 157 - 67 Inhibition of the 'spontaneous' mutagenicity in Salmonella typhimurium TA102 and TA104; De Flora S et al.; Thirty-four compounds belonging to various chemical classes were assayed for the ability to modulate the 'spontaneous' mutagenicity in strain TA104 of S . typhimurium, and 17 of them were also assayed in TA102 . All test agents, many of which were already known or suspected to act as inhibitors of induced mutagenicity, had been previously monitored in our laboratory for antimutagenicity towards either 4-nitroquinoline 1-oxide in TA100 and/or cigarette smoke in TA98 with S9 mix . A considerable proportion of test compounds decreased the number of spontaneous revertants in TA104 (44.1%) and/or TA102 (41.2%) to a significant extent, with dose-related and reproducible effects . In almost all cases the antimutagenic effect was genuine and not related to bacterial killing or growth inhibition . The results obtained suggest that the DNA repair background plays a prominent role in the genesis of spontaneous mutations in these strains, containing the hisG428 mutation which is typically reverted by oxidative mutagens . Due to its theoretical and practical implications, the finding that several chemopreventive agents can attenuate the rate of spontaneous reversion deserves attention. Mutat Res, 1994 May, 321(3), 165 - 73 Genotoxic and mutagenic effects of guarana (Paullinia cupana) in prokaryotic organisms; da Fonseca CA et al.; Aqueous extracts of Paullinia cupana (guarana), a species that belongs to the Sapindaceae family, were analyzed for the presence of genotoxic activities in bacterial cells . The extracts of guarana were genotoxic as assessed by lysogenic induction in Escherichia coli and they were also able to induce mutagenesis in Salmonella typhimurium . Addition of S9 microsomal fraction, catalase, superoxide dismutase or thiourea counteracted the genotoxic activity of guarana, suggesting that oxygen reactive species play an essential role in the genotoxicity of aqueous guarana extracts . The genotoxic activity in the extracts was related to the presence of a molecular complex formed by caffeine and a flavonoid (catechin or epicatechin) in the presence of potassium. Mutat Res, 1994 Apr 15, 306(2), 153 - 63 Mechanisms of the DNA breaking activity of mutagenic 5-diazouracil; Hiramoto K et al.; 5-Diazouracil in monohydrated form showed mutagenicity and cytotoxicity on Salmonella typhimurium TA98 and TA100 strains without metabolic activation, and induced mouse micronucleated peripheral reticulocytes . Incubation of a plasmid supercoiled DNA with the compound caused DNA single-strand breaking: the supercoiled form was transformed into an open circular relaxed form and then into a linear form . The breaking was similarly caused in the absence of molecular oxygen . The breaking was not inhibited by superoxide dismutase and catalase, but inhibited by ethanol, butyl hydroxyanisole and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), suggesting the involvement of radical species other than oxygen-derived radical species . Sequencing analysis of the singly 5'-end-labeled DNA fragment showed that the phosphodiester breaking was not site-specific . When Escherichia coli cells were incubated with the compound, the intracellular double-strand DNA was fragmented . The fragmentation was inhibited by ethanol, DMPO, N-tert.-butyl-alpha-phenylnitrone (PBN) and thiol compounds . Generation of the carbon-centered radical was confirmed by the electron spin resonance spin-trapping technique using DMPO and PBN . The mutagenicity and the DNA breaking activity of 5-diazouracil can be ascribed to the carbon-centered radical. Mutat Res, 1994 Apr 15, 306(2), 111 - 7 Mutagenesis by 9-aminoacridine in Salmonella typhimurium: inhibition by glucose and other PTS class A carbon sources; Kopsidas G et al.; Reversion of the hisC3076 frameshift marker of Salmonella typhimurium has been measured following treatment of cells in growth and non-growth media with 9-aminoacridine (9AA) . By varying the carbon source present in a defined medium, it has been shown that mutagenesis is reduced close to the spontaneous level in the presence of glucose whilst significant reductions are also observed with glucosamine, mannose, mannitol, fructose or glucose 6-phosphate . Intermediate mutant yields are observed when lactic acid or glycerol are present, whereas any one of a further group of carbon sources (gluconate, arabinose, ribose, succinate or casein hydrolysate) permit relatively large numbers of mutants to be recovered . Interestingly, when any one of these "high yield" carbon sources is supplemented with glucose the strong inhibitory effect characteristic of glucose is again observed . On the basis of these results, it can be concluded that inhibition of 9AA-induced reversion by a carbon source is not an exclusive property of glucose, although when more than one carbon source is present the inhibitory effect of glucose predominates . Possible explanations for these findings include the active exclusion of 9AA from cells as a direct consequence of glucose transport across the cell membrane . To address this possibility, cells were pre-grown in verapamil, a calcium channel antagonist which is known to increase the mutagenicity of various 9-anilinoacridine derivatives in S . typhimurium . We found that glucose inhibition of 9AA-induced mutagenesis was not relaxed to any significant extent following treatment with verapamil . In a further experiment, two glucose analogues (2'-deoxyglucose and methyl-D-glucoside) known to be actively transported into the cell but not metabolised past the first phosphorylation step were used . These analogues inhibit the transport into the cell of several types of molecules, but since they do not significantly depress 9AA mutagenesis it seems unlikely that blockage of 9AA transport across the cell membrane can be invoked to explain the inhibitory effect of glucose on 9AA mutagenesis . An alternative explanation based on glucose-mediated repression of an error-prone, mutation-generating, DNA-repair process is presented. Biochim Biophys Acta, 1994 Apr 13, 1205(2), 294 - 300 Movement of the F40 domain of flagellin during the morphological transition of bacterial flagella; Seville M et al.; Flagella from Salmonella typhimurium were labeled with various amounts of fluorescein isothiocyanate . The site of labeling was identified as being predominantly in the exterior F40 domain . The fluorescence intensity decreased as the fluorescein density on the flagella increased, indicating self energy transfer between fluoresceins . The fluorescence of modified flagella was measured during the normal-to-curly morphological transition induced by alkaline pH . The morphological transition itself was simultaneously monitored by dark-field microscopy . Concomitant with the transition was a 25% increase in fluorescence for flagella heavily labeled with fluorescein . This was shown to be due to a decrease in the efficiency of energy transfer between fluoresceins on proximal flagellin subunits, implying that the F40 domains undergo relative movement apart during the morphological transition . Closer inspection of the domain movement and morphological transition as a function of pH reveals that the two processes are not exactly concomitant . This indicates the existence of intermediates during the transition . The fluorescence technique, outlined here, provides a means of directly monitoring an organizational 'switch' in the flagellin subunits during the actual morphological transition of flagella. J Appl Bacteriol, 1994 Apr, 76(4), 345 - 9 Survival of Salmonella senftenberg and Salmonella typhimurium in glassy and rubbery states of gelatin; Blissett SJ et al.; Salmonella senftenberg and Salmonella typhimurium survived the production of gelatin sheets containing nutrient broth although there was some evidence of cell damage . Both strains survived but did not grow in glassy states with an a(w) of 0.45-0.28 and rubbery states with an a(w) of 0.93-0.96 for at least 28 d . Survival was less in intermediate states with an a(w) between 0.55 and 0.74 . The results suggest that salmonellas should be excluded from glassy state products in order to prevent salmonellosis. Eur J Biochem, 1994 Apr 1, 221(1), 603 - 9 In vitro synthesis of disialoganglioside (GD1 alpha) from asialo-GM1 using sialyltransferases in rat liver Golgi vesicles; Hidari KI et al.; Two gangliosides were efficiently synthesized from asialo-GM1 (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer) and cytidine 5'-phosphate-N-acetylneuraminic acid (CMP-NeuAc) by using sialyltransferases in rat liver Golgi vesicles in vitro . These gangliosides were rapidly purified by a combination of anion exchange and reverse-phase column chromatographies . The ganglioside structures were determined by TLC analysis, treatment with a sialidase from Salmonella typhimurium LT2, which specifically hydrolyzes alpha 2-3 N-acetylneuraminic acid (NeuAc alpha 2-3) linkages, TLC immunostaining, and 1H-NMR spectroscopy . One of the gangliosides was identified as GD1 alpha {Neu-Ac alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer} . The other ganglioside was determined to be GM1b (NeuAc alpha 2-3Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1-1 Cer), which has been reported in a previous study {Pohlentz, G., Klein, D., Schmitz, D., Schwarzmann, G., Peter-Katalinic, J . & Sandhoff, K . (1988) Biol . Chem . Hoppe-Seyler 369, 55-63} . Finally, GM1b and GD1 alpha were obtained from asialo-GM1 as a starting material in 8.1% and 1.2% overall yields, respectively . This study also suggests that the novel synthetic pathway asialo-GM1-->GM1b-->GD1 alpha may exist in rat liver. Biochem J, 1994 Apr 1, 299 ( Pt 1), 129 - 36 Characterization of the CysB protein of Klebsiella aerogenes: direct evidence that N-acetylserine rather than O-acetylserine serves as the inducer of the cysteine regulon; Lynch AS et al.; The cysB gene of Klebsiella aerogenes has been cloned, sequenced and shown to complement the cysteine auxotrophic phenotype of Escherichia coli cysB mutants . The K . aerogenes cysB gene is predicted to encode a protein of 324 amino acid residues that shares approx . 95% sequence similarity with the Salmonella typhimurium and E . coli CysB proteins . Gel-retardation assays demonstrate that the purified protein binds to DNA fragments containing either the K . aerogenes cysb promoter or the S . typhimurium cysJIH promoter . Acetylserine enhances CysB binding to the cysJIH promoter fragment while diminishing its binding to the cysB promoter fragment . Fluorescence-emission-spectroscopy measurements suggest strongly that N-acetylserine binds to CysB apoprotein but that O-acetylserine does not, and support the notion that N-acetylserine is the physiological inducer of cysteine biosynthesis. J Bacteriol, 1994 Apr, 176(8), 2379 - 85 Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium; Sirisena DM et al.; We report the analysis of three open reading frames of Salmonella typhimurium LT2 which we identified as rfaF, the structural gene for ADP-heptose:LPS heptosyltransferase II; rfaD, the structural gene for ADP-L-glycero-D-manno-heptose-6-epimerase; and part of kbl, the structural gene for 2-amino-3-ketobutyrate CoA ligase . A plasmid carrying rfaF complements an rfaF mutant of S . typhimurium; rfaD and kbl are homologous to and in the same location as the equivalent genes in Escherichia coli K-12 . The RfaF (heptosyl transferase II) protein shares regions of amino acid homology with RfaC (heptosyltransferase I), RfaQ (postulated to be heptosyltransferase III), and KdtA (ketodeoxyoctonate transferase), suggesting that these regions function in heptose binding . E . coli contains a block of DNA of about 1,200 bp between kbl and rfaD which is missing from S . typhimurium . This DNA includes yibB, which is an open reading frame of unknown function, and two promoters upstream of rfaD (P3, a heat-shock promoter, and P2) . Both S . typhimurium and E . coli rfaD genes share a normal consensus promoter (P1) . We postulate that the yibB segment is an insertion into the line leading to E . coli from the common ancestor of the two genera, though it could be a deletion from the line leading to S . typhimurium . The G+C content of the rfaLKZYJI genes of both S . typhimurium LT2 and E . coli K-12 is about 35%, much lower than the average of enteric bacteria; if this low G+C content is due to lateral transfer from a source of low G+C content, it must have occurred prior to evolutionary divergence of the two genera. J Bacteriol, 1994 Apr, 176(8), 2308 - 11 Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium; Raha M et al.; filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli . A spontaneous fliL mutant of S . typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch . However, in-frame deletions of the E . coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis . Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one. J Bacteriol, 1994 Apr, 176(8), 2272 - 81 FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium; Ohnishi K et al.; FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum . We have overproduced and purified FlgD and raised an antibody against it . By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants . In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip . Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook . Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament . However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein . When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously . When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant . Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export. Exp Hematol, 1994 Apr, 22(4), 360 - 5 Ciprofloxacin enhances hematopoiesis and the peritoneal neutrophil function in lethally irradiated, bone marrow-transplanted mice; Kletter Y et al.; We analyzed the effect of in vivo ciprofloxacin and ceftazidime treatment on the development of myeloid progenitors and on the survival of lethally irradiated mice rescued with syngeneic bone marrow transplantation (BMT) . Ciprofloxacin treatment (15 mg/kg per dose three times daily for 5 days) enhanced myeloid progenitor (colony-forming cell {CFU-C}) number in the bone marrow and the survival of mice transplanted with suboptimal doses (1 x 10(5) of s{Ngeneic bone marrow cells (BMC) . Twenty days postirradiation, 50% (38 of 76) of saline-treated mice transplanted with 1 x 10(5) cells died compared with 25% (19 of 76) of ciprofloxacin-treated mice (p < 0.05) . Similarly, ciprofloxacin treatment enhanced survival of mice transplanted with 1 x 10(6) syngeneic bone marrow cells: 50% (38 of 76) of saline-treated mice died within 20 days vs . 15% (12 of 80) of ciprofloxacin-treated mice . In contrast, treatment with ceftazidime did not affect progenitor cell number or survival . On day 8 postirradiation, although lethally irradiated mice transplanted with 1 x 10(5) BMC treated with ciprofloxacin demonstrated similar white blood cell (WBC) and red blood cell (RBC) counts as saline-treated mice, a (1.9 +/- 0.2)-fold increase in the percentage of polymorphonuclear cells (PMN) was observed in the peripheral blood of ciprofloxacin-treated mice . On day 5 postirradiation, ciprofloxacin-treated mice showed a (1.6 +/- 0.2)-fold increase in the number of peritoneal PMN and a 6.5-fold increase in their antibacterial activity towards Salmonella typhimurium in comparison with saline-treated mice. Cancer Res, 1994 Apr 1, 54(7), 1672 - 7 New sublines of Chinese hamster CHL stably expressing human NAT1 or NAT2 N-acetyltransferases or Salmonella typhimurium O-acetyltransferase: comparison of the sensitivities to nitroarenes and aromatic amines using the in vitro micronucleus test; Watanabe M et al.; New sublines of Chinese hamster CHL cells stably expressing human NAT1 or NAT2 N-acetyltransferases or O-acetyltransferase of Salmonella typhimurium were established, and their sensitivities to carcinogenic nitroarenes and aromatic amines were compared using the in vitro micronucleus test . The subline expressing human NAT2 N-acetyltransferase exhibited the highest sensitivity to the clastogenicities of 1,8-dinitropyrene and 2-nitrofluorene . These results raise the possibility that human NAT2 N-acetyltransferase is involved in the metabolic activation of 1,8-dinitro-pyrene and 2-nitrofluorene . Since human NAT2 N-acetyltransferase exhibits a marked genetic polymorphism, the polymorphic status of human N-acetyltransferase could be a genetic predisposing factor to cancers caused by the nitroarenes . In contrast, the subline expressing O-acetyltransferase of S . typhimurium exhibited the highest sensitivity to the clastogenicity of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) when the microsomes prepared from rat liver were present . This suggests that O-acetyltransferase of S . typhimurium has a higher ability to activate IQ than do the human acetyltransferases . Acetyltransferase enzymes of human enteric bacteria might contribute to the metabolic activation of IQ . The sublines could provide a new tool for investigation of the mechanism of metabolic activation and for assessment of cancer risk of nitroarenes and aromatic amines to humans. Infect Immun, 1994 Apr, 62(4), 1381 - 91 Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli; Becker PS et al.; The recent discovery that abundant and immunogenic lipoproteins constitute the integral membrane proteins of Treponema pallidum has prompted efforts to investigate their importance in the physiology and ultrastructure of the organism and in immune responses during infection . Earlier studies identified a 38-kDa lipoprotein of T . pallidum believed to be specific to the pathogen . In the present study, monoclonal antibodies generated against the 38-kDa lipoprotein of T . pallidum reacted with cognate 37-kDa molecules in the nonpathogens Treponema phagedenis, Treponema denticola, and Treponema refringens . Cloning and expression of the 38-kDa-lipoprotein gene of T . pallidum in Escherichia coli revealed that the recombinant product displayed a slightly larger (39-kDa) apparent molecular mass but remained reactive with anti-38-kDa-protein monoclonal antibodies . The recombinant product was processed and acylated in E . coli . DNA and amino acid sequence analyses indicated an open reading frame encoding 403 amino acids, with the first 25 amino acids corresponding to a leader peptide terminated by a signal peptidase II processing site of Val-Val-Gly-Cys . The predicted mature protein is 378 amino acids in length with a deduced molecular weight of 40,422 (excluding acylation) . Southern blotting failed to demonstrate in nonpathogenic treponemes genomic sequences homologous with the 38-kDa-lipoprotein gene of T . pallidum . Computer analysis revealed that the 38-kDa lipoprotein of T . pallidum had 34.2% identity and 58.9% similarity with the glucose/galactose-binding protein (MglB) of E . coli and Salmonella typhimurium . Furthermore, of the 19 amino acids of MglB involved in carbohydrate binding, the 38-kDa lipoprotein had identity with 11 . These studies have allowed the first putative functional assignment (carbohydrate binding) to a T . pallidum integral membrane protein . Recognition of this potential physiological role for the 38-kDa lipoprotein underscores the possibility that the membrane biology of T . pallidum may more closely resemble that of gram-positive organisms, which also utilize lipoproteins as anchored transporters, than that of gram-negative bacteria to which T . pallidum often is analogized. Infect Immun, 1994 Apr, 62(4), 1176 - 84 Induction of early-response genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp); Roach TI et al.; The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage activation for antimicrobial activity against Salmonella typhimurium, Leishmania donovani, and Mycobacterium spp . To determine early events in the activation pathway, the ability of mycobacterial lipoarabinomannan (LAM) to induce early gene (KC and JE) expression in macrophages from susceptible (S) C57BL/10ScSn (Lshs) and congenic resistant (R) B10.L-Lshr mice was investigated . Stimulation with 1.8 microgram of arabinofuranosyl-terminated LAM (AraLAM) per ml resulted in similar kinetics for KC or JE expression in S and R macrophages . However, whereas JE/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA ratios remained equivalent, R macrophages consistently showed enhanced KC/GAPDH ratios within 30 to 40 min of stimulation compared with S macrophages . Significant differences in KC/GAPDH ratios were observed throughout the peak period (0.5 to 6 h) of the KC response and with doses of AraLAM ranging from 0.01 to 2.5 micrograms/ml . Heavily mannosylated LAM from virulent Mycobacterium tuberculosis Erdman, in doses of up to 2.5 micrograms/ml, failed to stimulate KC or JE in S or R macrophages . Gamma interferon alone (25 U/ml) stimulated equivalent JE expression in S and R macrophages and synergized with AraLAM to enhance JE in both . In contrast, AraLAM-induced KC expression was inhibited in the presence of gamma interferon . Agonist/inhibitor studies were undertaken to determine the signal transduction pathways mediating KC expression . The protein kinase C (PKC) inhibitor Calphostin C (200 nM) inhibited AraLAM-induced KC by 34% +/- 4% in S macrophages and 43% +/- 5% in R macrophages; the cyclic AMP-dependent PKA inhibitor KT5720 (2 microM) inhibited AraLAM-induced KC by 33% +/- 4% (S) and 25% +/- 5% (R) . A role for Ca2+ was indicated because ionophore alone stimulated KC expression and synergized with AraLAM to give a dramatically enhanced response . Induction of KC was also inhibited by (i) blocking constitutive nitric oxide (NO) production by preincubation of macrophages with NG-monomethyl-L-arginine (400 microM) (48% +/- 8% {S} and 40% +/- 11% {R}) and (ii) incubation of macrophages with the cyclic GMP-dependent kinase inhibitor KT5823 (4 microM) (65% +/- 4% {S} and 72% +/- 6% {R}) . The manner in which these PKC-, PKA-, and Ca(2+)-dependent, NO-mediated cyclic GMP-dependent kinase signal transduction pathways may relate to function of the candidate Lsh/Ity/Bcg gene Nramp is discussed. J Biochem (Tokyo), 1994 Apr, 115(4), 648 - 54 Purification and characterization of a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex; Watabe S et al.; We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex . Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit . Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme . The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody . Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla . We determined the amino acid sequence of SP-22, which is composed of 195 amino acids . Amino acid 47 was not identified by the sequencer . FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H) . By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation . SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins . Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22. Mol Microbiol, 1994 Apr, 12(2), 277 - 85 Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella; Tominaga A et al.; Flagellin genes (fliC) were detected in two species of the genus Shigella . The fliCSF gene cloned from Shigella flexneri produced normal-type flagella in an Escherichia coli delta fliC strain while the fliCSS genes from two Shigella sonnei strains produced curly-type flagella and their expression is repressible by Salmonella FljA repressor . The fliCSF gene (1650 bp) shared high similarity with the E . coli fliCE gene not only in the 5' and 3' constant sequences but also in the upstream and downstream sequences . The fliCSS genes (1572 bp) shared high similarity with the Salmonella typhimurium fliCS gene in the operator and 3' constant sequences and also shared high similarity with the fliCE gene in the downstream sequence, suggesting that the fliCSS gene has undergone horizontal transfer and recombination . Differences in nucleotide sequences of the central variable regions among the four fliC genes, including fliCE and fliCS, suggest that they started differentiation in each lineage approximately 80 million years ago . Loss of motility in Shigella seems to be evolutionarily a recent event. Vet Microbiol, 1994 Apr, 39(3-4), 245 - 54 Purification and characterization of enterotoxic moiety present in cell-free culture supernatant of Salmonella typhimurium; Rahman H et al.; The enterotoxic moiety present in the cell-free culture supernatants of Salmonella typhiurium strains (p/536 and p/603) was purified to apparent homogeneity by salt precipitation with ammonium sulphate and successive chromatography through Sephadex G-100 and G-200 columns . It was non-dialysable, heat labile at 90 degrees C and active within pH 6-8 . Its activity was completely lost on treatment with trypsin, protease and papain . The enterotoxin appeared to be of high molecular weight (100 kDa) and was highly immunogenic in rabbit . Antigenically, it was not related to cholera toxin, Shiga toxin or the heat labile enterotoxin of Escherichia coli . It did not bind to the GM1 ganglioside . The enterotoxic, delayed permeability and CHO cell elongation activities were attributed to a single protein moiety. Kansenshogaku Zasshi, 1994 Apr, 68(4), 491 - 4 {The mechanism of antigen-presentation of porin from Salmonella typhimurium to T lymphocytes}; Matsui K et al.; Porins from wild type strains of Salmonella typhimurium are known to consist of three species of proteins named 34, 35 and 36K . It is understood that these three species of proteins construct porin trimers and the porin trimers from the wild type strain of S . typhimurium are constructed from homologous species of porin subunits . However, we have demonstrated previously that only porin trimers from wild type strains but not mixtures of homologous porin trimers from mutant strains of S . typhimurium could induce antigen-specific cell-mediated immunity (CMI) . In this study, therefore, a mechanism of antigen-presentation of porin from a wild type strain of S . typhimurium to T cells was studied . Our data showed that porin proteins from the wild type strain received antigen-processing as well as other protein antigens in murine macrophages and were presented with Ia antigen to T cells . These results suggested that porin trimers from wild type strains of S . typhimurium were constructed from heterologous species of porin subunits and only heterologous porin trimers held epitopes for a CMI-eliciting antigen. J Clin Microbiol, 1994 Apr, 32(4), 1035 - 9 Detection of leptospiral DNA by PCR; Kee SH et al.; An EcoRI fragment (1.2 kb) which is highly conserved among Leptospira interrogans isolated in Korea was cloned into pBluescript vector from L . interrogans serovar lai WH20 . The EcoRI fragment was sequenced, and a pair of primers (LP1 and LP2) was designed for PCR assay . PCR amplification of target DNA obtained from cultured L . interrogans showed that 274 bp could be detected when as little as 100 fg of leptospiral genomic DNA was used in the reaction mixture . No amplification of DNA was detected from DNA of Leptospira biflexa serovars patoc and sau paulo, Borrelia burgdorferi, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium . Amplification of 274-bp target DNA could be detected in DNA samples purified from 500 microliters of blood collected from experimentally infected gerbils 2 days after infection, while antibodies to L . interrogans could be detected by the microscopic agglutination test 7 days after infection . The specificity and high sensitivity of the test provided valuable tools for the early diagnosis of leptospirosis. Avian Dis, 1994 Apr-Jun, 38(2), 275 - 81 Effect of mannose on Salmonella typhimurium-mediated loss of mucosal epithelial integrity in cultured chick intestinal segments; Droleskey RE et al.; The effect of incubating Salmonella typhimurium and S . typhimurium cell-free extracts with isolated intestinal segments from 1-day-old chicks was evaluated using scanning electron microscopy . Incubation of segments with intact bacteria or with cell-free extract resulted in the loss of mucosal epithelial integrity after as little as 30 min incubation . Loss of mucosal epithelial integrity was evidenced by the complete shedding of the epithelium . The addition of 2.5% D-mannose to the incubation medium inhibited the loss of epithelial cells, whether intestinal segments were incubated with intact bacteria or with cell-free extract . These results indicate that S . typhimurium exerts a D-mannose-sensitive cytotoxic effect on the mucosal epithelium of isolated intestinal segments and that the cytotoxic effector is present in cell-free extracts of the bacteria. Indian J Med Res, 1994 Apr, 99, 162 - 6 Correlation of phospholipase A & enterotoxin production by Salmonella typhimurium with reference to virulence parameters; Shetty M et al.; Seventy strains of S . typhimurium along with 10 strains each of S . typhi and S . paratyphi B, 3 strains of S . enteritidis and 2 strains of S . senftenberg were examined for phospholipase A (PLA) activity . S . typhimurium positive for PLA activity (17.14%) were subjected to study of other virulence parameters like enterotoxin production, haemolytic activity, surface hydrophobicity and antibiotic sensitivity . A high degree of correlation was observed between PLA activity and enterotoxin production, antibiotic sensitivity and cell surface hydrophobicity respectively. J Infect Dis, 1994 Apr, 169(4), 760 - 5 Salmonella typhimurium induces expression of P glycoprotein (multidrug resistance 1 gene product) in a promonocytic cell line chronically infected with human immunodeficiency virus type 1; Andreana A et al.; This investigation showed that phagocytosis of virulent Salmonella typhimurium by promonocytic leukemia cell line U1, which contains human immunodeficiency virus type 1 (HIV-1) provirus but produces minimal or no virus, and not by uninfected U937 cell line resulted in expression of a functional P glycoprotein . Anti-tumor necrosis factor-alpha (TNF alpha) monoclonal antibody failed to inhibit S . typhimurium-induced P glycoprotein expression . Furthermore, recombinant TNF alpha had no effect on the induction of P glycoprotein expression in U1 cells . These data demonstrate that phagocytosis of virulent S . typhimurium results in an induction of P glycoprotein in association with HIV-1 infection; however, TNF alpha does not appear to play a significant role . Thus, secondary microbial infection in HIV-1-positive persons may play a role in multidrug resistance against antiviral and other antimicrobial agents by an induction of P glycoprotein. Mutat Res, 1994 Apr, 323(4), 167 - 71 Antimutagenicity in Euglena gracilis; Foltinova P et al.; The genotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, alpha-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis . The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100, and TA102, were used . The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102. J Bacteriol, 1994 Apr, 176(8), 2216 - 26 Phenotypic suppression of DNA gyrase deficiencies by a deletion lowering the gene dosage of a major tRNA in Salmonella typhimurium; Blanc-Potard AB et al.; One of the pleiotropic phenotypes of mutations affecting DNA gyrase activity in Salmonella typhimurium is the constitutive deattenuation of the histidine operon . In the present work, we isolated and characterized a suppressor mutation which restores his attenuation in the presence of a defective gyrase . Such a suppressor, initially named sgdA1 (for suppressor gyrase deficiency), was found to correct additional phenotypes associated with defective gyrase function . These include the aberrant nucleoid partitioning of a gyrB mutant and the conditional lethality of a gyrA mutation . Furthermore, the sgdA1 mutation was found to confer low-level resistance to nalidixic acid . The last phenotype permitted isolation of a number of additional sgdA mutants . Genetic analysis established the recessive character of these alleles as well as the position of the sgdA locus at 57 U on the Salmonella genetic map . All of the sgdA mutants result from the same molecular event: a deletion removing three of the four tandemly repeated copies of argV, the gene which specifies tRNA(2Arg), the major arginine isoacceptor tRNA . These findings, combined with the observation of some Sgd-like phenotypes in a tRNA modification mutant (hisT mutant), lead us to propose that protein synthesis contributes, directly or indirectly, to the pathology of gyrase alterations in growing bacteria . We discuss plausible mechanisms which may be responsible for these effects. Mutat Res, 1994 Apr 1, 306(1), 9 - 17 Possible occurrence of new mutagens with the DNA breaking activity in coffee; Kato T et al.; Brewed and instant coffee emitted strong chemiluminescence due to singlet oxygen and excited carbonyls, which may be originated by the Maillard reaction of sugars and amino acids but not by the reaction of polyphenolics . Instant coffee cleaved DNA giving single-strand breaks only after it was purified by high performance liquid chromatography (HPLC) or by gel filtration . Retention times of component(s) with strong DNA breaking activity in HPLC were different from those of chemiluminescence emitters, although they were coeluted on a gel filtration . The major DNA breaking component(s) must be different from chemiluminescence emitters . Active oxygen radicals participated little in DNA breaking because active oxygen radical scavengers had only a marginal effect on DNA breaking of the active gel fraction . DNA breaking by the active gel fraction was inhibited by high concentrations of inorganic salts probably because the salts stabilized the DNA double strands . The active gel fraction was mutagenic to Salmonella typhimurium TA98 without metabolic activation . The number of His+ revertant colonies/g of instant coffee powder was estimated to be 4000. Mutat Res, 1994 Apr, 321(1-2), 81 - 7 Inhibition of the bacterial mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine by ascorbic acid and ascorbyl palmitate; Tyrsina EG et al.; The mechanism of antimutagenic activity of ascorbic acid (AA) and its derivatives was studied using the Salmonella typhimurium TA100 bacterial test system . All substances studied inhibited N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis . Ascorbyl palmitate (AP) markedly decreased the numbers of his+ revertants, behaving as a membrane-active antimutagen . A comparative study of the antioxidative activity of the investigated substances in the methyl oleate (MO) system has demonstrated that AA and its derivatives have pro-oxidant properties within the limits of the concentrations studied . The results obtained do not agree with the common view of the mode of action of these antimutagens, including both inhibition of free radical processes and MNNG reductive inactivation. Mutat Res, 1994 Apr, 321(1-2), 7 - 11 The mutagenicity of nitrite in the Salmonella/microsome test system; Balimandawa M et al.; The bacterial strains of Salmonella typhimurium used for detection of base-pair substitutions (TA1530, TA1535, TA100 and TA102) and various types of frameshift mutations (YG1024, DJ400 and DJ460) were exposed to nitrites in the Salmonella/microsome test system . In agreement with published data, nitrites exhibited mutagenic activity in standard strains TA1530, TA1535 and TA100 . The TA102 strain was insensitive as were the frameshift tester strains despite their genetically manipulated genome increasing their sensitivity. Mutat Res, 1994 Apr, 321(1-2), 65 - 72 Propane 2-nitronate is the major genotoxic form of 2-nitropropane; Kohl C et al.; The mutagenicity of 2-nitropropane in Salmonella typhimurium (strain TA100) was proportional to the pH (range 6.1-9.1) of the medium used for pre-incubation of the agent and for incubation of the agent with the Salmonella . The mutagenicity correlated with an enhanced rate of tautomerase to propane 2-nitronate at relatively high pH as measured by high performance liquid chromatography . Both the mutagenicity in Salmonella typhimurium (strains TA100 and TA102) and the rate of tautomerisation to the nitronate was lower with 2-deutero-2-nitropropane than with non-deuterated 2-nitropropane . Furthermore, 2-deutero-2-nitropropane was less potent in the induction of unscheduled DNA synthesis in rat hepatocytes over a 4-h period . Propane 2-nitronate therefore appears to be pivotal in the causation of the genetic toxicity of 2-nitropropane . The presence of hepatocytes enhanced nitronate production from 2-nitropropane suggesting a contribution from hepatic enzymes in the tautomerisation reaction. Mutat Res, 1994 Apr, 321(1-2), 57 - 63 Absence of unscheduled DNA synthesis in rat hepatocytes treated with mutagenic and cytotoxic chlorinated humic substances; Ubom G et al.; Chlorinated hydrophilic macromolecular humic acids (CHMA) (0.07-3.0 mg per plate) were mutagenic in Salmonella typhimurium strain TA100 . In contrast, the chlorinated derivatives did not induce unscheduled DNA synthesis (nuclear incorporation of {3H}thymidine) in cultured rat hepatocytes even after depletion of intracellular glutathione with buthionine sulphoximine (0.001 mM) and at concentrations of CHMA up to 3.0 mg per plate eliciting cytotoxicity . Glutathione depletion did however potentiate cytotoxicity and hepatocyte glutathione concentrations were lowered by CHMA treatment indicating reactivity of CHMA in the cell. Mutat Res, 1994 Apr, 321(1-2), 27 - 34 DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability; Ferguson LR et al.; Two closely-related aniline monomustards (1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA . The shorter-chain derivative (2) alkylates mainly at guanine N7 sites, while the longer-chain analogue (1) reacts almost exclusively at adenine N1 . The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them . Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains . Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101 . Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid . The adenine-selective compound (1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative (2) . Additionally, only the adenine-selective compound (1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006 . The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion. Mutat Res, 1994 Apr, 321(1-2), 13 - 7 Genotoxicity assessment of the antifungal antibiotic aureofungin in Salmonella typhimurium and Swiss albino mice; Mujumdar AM et al.; The widely used agricultural antifungal agent aureofungin (ARF) was subjected to genotoxicity assessment using the Ames Salmonella assay as well as the in vivo micronucleus test and dominant lethal test in Swiss mice . In the Ames Salmonella spot test, ARF slightly elevated the number of histidine revertants after metabolic activation over a wide dose range (1-1000 micrograms/plate) in TA102 but not in TA97a, TA98 or TA100 . In the preincubation plate incorporation assay with TA102, ARF increased the number of revertants in a dose-dependent manner only after metabolic activation . ARF failed to significantly elevate the frequency of micronucleated polychromatic erythrocytes (PE) in the bone marrow of Swiss mice . It elevated the frequency of dominant lethal mutations in the 7th and 8th weeks at 30 mg/kg body weight, a concentration much higher than the actual concentration used in the field . We conclude that ARF is non-mutagenic in somatic cells in vivo at doses used in the present study, probably mutagenic in stem-cell spermatogonia and may be classified as an equivocal promutagen, possibly acting as a cross-linker. J Biol Chem, 1994 Mar 25, 269(12), 8931 - 6 The 1.9 A x-ray structure of a closed unliganded form of the periplasmic glucose/galactose receptor from Salmonella typhimurium; Flocco MM et al.; The three-dimensional structure of a ligand-free closed form of the glucose/galactose binding protein from Salmonella typhimurium has been determined at a resolution of 1.9 A . The crystallographic R-factor for the refined structure is 17.9% . The model contains all the atoms of the 309 residues of the protein sequence, a calcium ion, and 174 water molecules . The root mean square (r.m.s.) deviations for the whole molecule are: 0.010 A for bond lengths and 2.44 degrees for bond angles, indicating a good stereochemistry for the model . This structure shows that the protein is able to close in the absence of ligand, adopting a conformation similar to the liganded form but slightly more open . Water molecules satisfy the hydrogen bonding ability of the hydrophilic side chains of the binding site in a manner which is reminiscent of the sugars' hydrogen-bonding patterns . Since packing forces are weak, the crystallization event is unlikely to trigger a change from an open to a closed conformation . Instead, the latter must be one of the species in equilibrium in solution which is selected by packing in the crystal lattice. Biochem J, 1994 Mar 15, 298 Pt 3, 711 - 8 The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity . Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins; Qi SY et al.; Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria . An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli . The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined . Folding was critically dependent on the urea and salt concentrations as well as the pH . BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity . In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions . Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity . However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form . A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced . The derivative was functional in the above assays and bound with high affinity to S . typhimurium (apparent Kd = 74 nM) . These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI . The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action. Biochemistry, 1994 Mar 8, 33(9), 2667 - 71 Kinetic mechanism of serine transacetylase from Salmonella typhimurium; Leu LS et al.; Serine transacetylase from Salmonella typhimurium was purified to near homogeneity . A complete initial velocity study in both reaction directions suggests that the enzyme catalyzes the conversion of acetyl CoA and L-serine to O-acetyl-L-serine (OAS) and coenzyme A (CoASH) by a ping pong bi bi kinetic mechanism . Initial velocity patterns in the absence of added inhibitors in both directions are best described by a series of parallel lines . Product inhibition by OAS is competitive with respect to acetyl CoA and noncompetitive with respect to L-serine, while product inhibition by L-serine is competitive against CoASH and noncompetitive against OAS . Glycine and S-methyl-L-cysteine (SMC) were used as dead-end analogs of L-serine and OAS, respectively . Glycine is competitive against L-serine, and uncompetitive against acetyl CoA, while SMC is competitive against OAS and uncompetitive against CoASH . All of the above inhibition patterns are consistent with those predicted for a single site ping pong bi bi kinetic mechanism . The equilibrium constant for the transacetylase reaction is 15 in the direction of serine acetylation . The constant was measured by monitoring the change in CoASH concentration obtained for reactions in which the ratio of acetyl CoA/CoASH was constant and the ratio of OAS/serine was varied . The Keq calculated from the Haldane relationship is in good agreement with the value obtained by direct measurement. J Mol Biol, 1994 Mar 4, 236(4), 1049 - 66 Molecular recognition in proteins . Simulation analysis of substrate binding by a tyrosyl-tRNA synthetase mutant; Lau FT et al.; Alchemical molecular dynamics simulations are performed to determine the difference in the free energy of binding of the tyrosine substrate between the wild type of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and the mutant Tyr169-->Phe . The results are of general interest because the Tyr169 hydroxyl group interacts with the ammonium group of the substrate in a manner corresponding to that found in other amino acid binding proteins (e.g . the Asp receptor of the chemotactic bacterium Salmonella typhimurium and class I major histocompatibility complex molecules) . The calculated free-energy change due to the Tyr169-->Phe mutation is 3.4 kcal/mol (the statistical error is +/- 0.5 kcal/mol) in satisfactory agreement with the experimental value of 3(+/- 0.5) kcal/mol . By use of thermodynamic integration, the contribution of the different terms to the free energy change are estimated . The path dependence of such a decomposition is discussed and it is suggested that the alchemical choice is of primary interest for understanding the interactions involved . There are large protein contributions to the alchemical free energy difference of the bound and free enzyme that cancel in the overall result . Due to this cancellation, the essential interactions contributing to the free-energy change are those between the OH group of Tyr169 and water in the free enzyme and those between the OH group of Tyr169 and the ammonium group of the substrate in the bound system . The results thus support simple models based on a balance of hydrogen bonding interactions. Mutagenesis, 1994 Mar, 9(2), 83 - 92 K-region oxides and imines derived from alkylated benz{a}anthracene congeners: synthesis, stability in aqueous media and mutagenicity; Glatt H et al.; The K-region oxides and imines of benz{a}anthracene, 1-methylbenz{a}anthracene, 7-methylbenz{a}anthracene, 7-ethylbenz{a}anthracene and 7,12-dimethylbenz{a}anthracene were synthesized and characterized (melting point, 1H-NMR and electron impact mass spectra, elemental analysis, IR spectroscopy) . All 10 compounds showed high mutagenic activity in Salmonella typhimurium (reversion of his- strains TA97, TA98, TA100 and TA104) . The arene imines were more potent than the corresponding arene oxides . Alkyl substitutions strongly influenced the activities . Furthermore, all compounds were more active when exposure took place in the absence of inorganic ions than when KCl (125 mM) was present . The influence of the exposure medium was more pronounced with strain TA98 than with strain TA100 . The half-lives of the test compounds were determined from mutagenicity experiments in which the compound was added to the exposure medium at varying times before the bacteria . In dilute sodium phosphate buffer (10 mM, pH 7.4), the half-lives of these chemicals (or their biological activity) varied from 0.5 to 110 min . Addition of KCl (150 mM) did not measurably affect the half-lives of some test compounds and appeared to slightly shorten those of others . Therefore, it is unlikely that the strong effect of KCl on mutagenicity and the dependence of this effect on the bacterial strain used can be explained by influences of KCl on the test compounds . Rather, it appears more likely than an effect of KCl on the bacteria may be an important factor . This study provides further examples of strong influences of unobtrusive media components on mutagenicity . It also demonstrates that small structural changes (alkyl substituents at diverse positions of the aromatic system) may play an important role in chemical reactivity and biological activity. Mutagenesis, 1994 Mar, 9(2), 157 - 62 Characterization of the mutagen 2-amino-3-methylimidazo{4,5-f}quinoline prepared from a 2-methylpyridine/creatinine/acetylformaldehyde model system; Lee H et al.; A mixture of 2-methylpyridine, creatinine and aldehydes was heated in diethylene glycol containing 5% water for 1 h at 140 degrees C . The mutagenic compounds were purified by XAD-2 column chromatography, acid/base partition, blue cotton treatment, thin layer chromatography and HPLC . The active substances purified from each step were monitored by their mutagenicity with Salmonella typhimurium TA98 in the presence of S9 mix . Among the mutagens collected, 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) was isolated from HPLC, and was identified by its UV and mass spectrum using a photodiode array detector and mass spectrometry . Our findings appear to be the first experimental evidence to substantiate the hypothetical pathway for the formation of IQ mutagens from a heated model system consisting of a pyridine or pyrazine derivative, an aldehyde and creatinine or creatine. Mutagenesis, 1994 Mar, 9(2), 107 - 12 Modulation of the mutagenic activity of cigarette smoke, cigarette smoke condensate and benzo{a}pyrene in vitro and in vivo; Balansky R et al.; A series of naturally occurring compounds were tested for the ability to modulate the mutagenicity induced by cigarette smoke (CS), cigarette smoke condensate (CSC) and benzo{a}pyrene (BP) in the