Glutathione modulates the formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA100 induced by mineral fibres

Treatment of isolated DNA with crocidolite and man-made vitreous fibre-21 (MMVF-21) significantly increased the concentration of 8-hydroxydeoxyguanosine (8-OHdG) in isolated DNA above background levels and co-treatment with glutathione (GSH) eliminated this effect. Crocidolite, MMVF-21 and chrysotile fibres increased the number of revertants in Salmonella typhimurium TA100 and GSH-deficient strains, TA100/NG-54 and TA100/NG-57, over background levels. This increase was small in TA100 but was greater in the GSH-deficient strains. When these bacterial strains were further depleted of GSH by co-culture with buthionine sulfoximine, all fibres tested caused a significant increase in the number of revertants over the parent strains. Pre-treatment with the GSH precursor N-acetyl-L-cysteine reduced the number of revertants to below that of the parent strain. Previous studies have shown a mechanistic role for iron-catalyzed production of oxygen radicals in the mutagenicity of fibres and this study suggests a protective role for GSH against such oxidative damage possibly by acting as a radical scavenger.     

Genotoxicity of ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine: induction of SCE in human lymphocytes and mutagenicity in Salmonella typhimurium TA 100

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and -10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and +/-2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at -80 degrees C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Shape-dependent effects in a series of aromatic nitro compounds acting as mutagenic agents on S. typhimurium TA98

Freshly isolated hepatocytes of the clawed toad, Xenopus laevis, were cultured for at least 3 days. The viability of the cells was characterized using staining and biochemical methods. In particular, the glucose and glycogen balance was tested. After culture for 16-20 hrs, the cells were subjected to hormonal treatment. Both adrenaline and arginine vasotocin stimulated the release of glucose in a dose dependent manner. 10(-6) M concentrations were strongly effective. The determination of the glycogen balance made it clear that the glucose release is mainly due to glycogenolysis. Using receptor antagonists and agonists, it has been shown that the effect of adrenaline is clearly mediated by beta-type receptors. Arginine vasotocin stimulated glycogenolysis via a type of receptor which is similar to the V2-receptor of mammals. This means that cAMP is involved in the response to both types of hormones which is in contrast to that which is known about the effect of nonapeptides on the liver of mammals.     

Mutagenicity of mixtures of halogenated aliphatic hydrocarbons and polycyclic aromatic hydrocarbons in the Ames test with TA98 and TA100

Within the framework of the assessment of the genotoxic potential of environment samples the Salmonella-microsome-test (Ames-test) is often used as a screening-test. It is one of the most applied biotest systems and possesses a large scientific acceptance. Because most environment samples are mixtures of various substances, possible effects resulting from the combination should be taken into account with regard to the mutagenic potential. In this context we investigated eight polycyclic aromatic hydrocarbons each combined with six halogenated aliphatic hydrocarbons as to their mutagenicity in the Salmonella-microsome-test with TA98 and TA100. For an exogenous metabolizing system, Arochlor 1254 induced rat liver S9-mix was used. Benz-a-pyrene in combination with bromodichloromethane (Ames neg. in TA98 and TA100 +S9) showed an increase in the number of the revertants up to 25% in TA98 and TA100 (+S9). Carbon tetrachloride (Ames neg. in TA98 and TA100 +S9) showed in TA100 (+S9) an increase in the number of the revertants of 18% at most. In the combination 3-methylcholanthrene with dichloromethane the number of revertants in TA98 (+S9) increased by 25% and in TA100 (+S9) by 18%. Hexachloroethane (weakly mutagenic in TA98 +S9) in combination showed in TA98 (+S9) a slightly increased number of revertants with benz-a-pyrene as well with 3-methylcholanthrene. All the other substances tested (chrysene, phenanthrene, anthanthrene, dibenz-a, i-pyrene, triphenylene, fluoranthene) in combination with either tetrachloroethylene or trichloroethene did not cause an increase in mutagenicity.     

Activity of quinolones in the Ames Salmonella TA102 mutagenicity test and other bacterial genotoxicity assays

Eight quinolones were examined for their bacterial mutagenicity in the Ames Salmonella TA102 assay and for their effects in other bacterial genotoxicity assays. In the quantitative Ames plate incorporation assay, all eight quinolones induced His+ deletion reversion in Salmonella tester strain TA102, with maximum reversion observed at about two to eight times the MIC. The quinolones also induced the SOS response. At quinolone concentrations close to the MIC, SOS cell filamentation gene sulA was induced in sulA::lacZ fusion strain Escherichia coli PQ37. RecA-mediated cleavage of lambda repressor in lambda::lacZ fusion strain E. coli BR513 was measurable at about 10 times the MIC, though no induction occurred at 20 micrograms of nalidixic or oxolinic acid per ml. Genotoxicity of quinolones also was observed in the Bacillus subtilis DNA repair assay, in which the mutant strain M45 (recA) was more susceptible to quinolones than its parent strain, H17 (rec+). The results from these analyses indicate that quinolones induce SOS functions and are mutagenic in bacteria; these properties correspond to their antimicrobial activities.     

A plausible mechanism for the mutagenic activity (Salmonella typhimurium TA100) of MX compounds: a formation of CG-CG(+)-CG radical cation by one-electron reduction

Combining our previous QSAR work with recent high-level quantum mechanical calculations, a plausible mechanism for the mutagenic activity of halogenated furanones (so called MX compounds) in Salmonella typhimurium TA100 tester strain is proposed. The mechanism involves one-electron reduction as a key step and it seems reasonable to suggest that the mutagenicity of these direct-acting compounds may be a purely thermodynamic phenomenon, rather than the result of site-specific binding or adduct formation. Overall, the proposed model is consistent with the most experimental findings.     

PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.     

A combination of three mutations, dcd, pyrH, and cdd, establishes thymidine (Deoxyuridine) auxotrophy in thyA+ strains of Salmonella typhimurium

The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C. F. Beck et al., J. Bacteriol. 129:305-316, 1977). In the genetic background used in their selection, the joint acquisition of a dcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy. In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase. The level of UMP kinase activity in the dum mutant was found to be only 30% of that observed for the dum+ strain. Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH+ donor or by complementation with either of two pyrH+-carrying plasmids. Thymidine auxotrophy could be reconstructed in the dum+ derivative (KP1389) by the introduction of a mutant pyrH allele. To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA+ strains, a dcd::Km null mutation was constructed. In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement. A third mutation, cdd (cytidine-deoxycytidine deaminase), was required together with the dcd and pyrH mutations to impart thymidine auxotrophy.     

Comparative assessment of DNA adduct formation, Salmonella mutagenicity, and chromosome aberration assays as short-term tests for DNA damage

DNA adduct formation assay (DAFA) was carried out to compare dose responses with the Ames test and chromosomal aberration test using aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP). In the bacterial mutation test, AFB1 and BaP (0-1 microgram/plate) were all positive in TA97a and TA100 with dose-related revertants. However, the slopes of the dose-response curves were gradual (slope 0.55-3.73, r = .84-.98). In the chromosome aberration test, a significant increase in the percentage of chromosomal aberrations was obtained from male ICR mouse spleen cells treated with AFB1 and BaP, but a dose-related increase was insensitive (slope 0.09-0.23, r = .75-.78). The incidence of chromosomally aberrant spleen cells treated with BaP was significantly increased compared with AFB1. DAFA was performed in vitro with [3H]-AFB1 and [3H]BaP. These two carcinogens were able to induce genotoxicity and showed good dose-related increases in terms of DNA adduct formation (slope 0.78-1.28, r = 1.00). Coefficients of variation (CV) for the slope of each dose-response curve were much lower in DAFA in vitro (CV 15.09- 18.34%) than those in any other test (CV 19.69-99.33%, Ames test; 18.89-44.58%, chromosome aberration test). Furthermore, DAFA in vivo was performed to investigate organotropic DNA adduct formation and persistence in Sprague-Dawley rats ip or orally treated with AFB1 and BaP. DNA adducts were monitored for 48-96 h by enzyme-linked immunosorbent assay (ELISA) using corresponding monoclonal antibodies, 6A10 and 8E11. DAFA in vivo demonstrated that the liver and kidney might be the probable target organs for AFB1 with the highest formation and persistence of DNA adducts and the lung and liver for BaP regardless of the route of administration. The results suggest that DAFA in vitro could be useful for detecting genotoxic compounds, and DAFA in vivo should also be considered as a good alternative method for the screening of organ-specific chemical carcinogens.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9)

Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.     

Identification of a new site for ferrichrome transport by comparison of the FhuA proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans

The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and phi80. All the FhuA proteins contained the region in the gating loops required in E. coli for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by the E. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to phi80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayed S. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.     

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Wheat cDNAs that encode proteins PR-1.1 and PR-1.2 were cloned. Deduced amino acid sequences were homologous to those of pathogen-induced, basic PR-1 proteins from plants. Although expression of PR1.1 and PR1.2 genes was induced upon infection with either compatible or incompatible isolates of the fungal pathogen Erysiphe graminis, these genes did not respond to activators of systemic acquired resistance (SAR), such as salicylic acid (SA), benzothiadiazole (BTH), or isonicotinic acid (INA).     

The location of four fimbrin-encoding genes, agfA, fimA, sefA and sefD, on the Salmonella enteritidis and/or S. typhimurium XbaI-BlnI genomic restriction maps

The recombinant oncotoxin OLX-209 [e23(Fv)PE38KDEL] has been developed to target cancers with erbB-2 expression and is nearing a clinical trial. Important in clinical planning is the selection of patients on the basis of tumor expression of erbB-2. ErbB-2 gene amplification occurs in cancers of the breast, stomach, and ovary. Patients with these diseases and evident overexpression are candidates for OLX-209 therapy. In lung cancer, overexpression of erbB-2 is also frequent, but in most cases, it is not caused by gene amplification. This study demonstrates that OLX-209 has activity on lung cancer cells with varying levels of erbB-2 expression in the presence and absence of gene amplification. In vitro sensitivity of cell lines to OLX-209 is related to erbB-2 expression level. Normal bronchial epithelial cells were not sensitive. Effective treatment of lung cancer cell lines growing as xenografts in nude mice was shown with Calu-3 (a lung adenocarcinoma line with high levels of p185(erbB-2) caused by gene amplification) and three other lung adenocarcinomas (A549, NCI-H1466, and 201T) with lower levels of p185(erbB-2) and no gene amplification. The 201T cell line was isolated recently from a lung tumor with erbB-2 expression in the original tumor. The results of this study indicate that patients with erbB-2-positive, non-small cell lung cancer should be included in clinical trials of OLX-209.     

Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.     

Antibiotic resistance in strains of Salmonella typhimurium and S. enteritidis isolated from poultry in the Czech Republic 1991-1992

Antibiotic resistance has been monitored in 293 strains of S. typhimurium and 260 strains of S. enteritidis isolated from poultry in Czech Republic in the years 1991 and 1992. Ninety per cent of all salmonella isolations examined by disc diffusion method (Bauer et al., 1966) were sensitive to all 8 antimicrobials (chloramphenicol, neomycin, tetracycline, streptomycin, colistin, ampicillin, kanamycin, sulfisoxazol) used for testing. The strains of S. typhimurium were more resistant than S. enteritidis strains, as seen from the percentage of resistant strains, 17.4% and 1.2% respectively. Thirty-two (62.7%) out of 51 resistant strains were multiresistant. The percentage of resistance in S. typhimurium strains was as follows: sulfisoxazol (12.3%), streptomycin (11.3%), tetracycline (4.4%) and chloramphenicol (1.7%).     

Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium

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.     

Role of the C-terminal extension in the interaction of S100A1 with GFAP, tubulin, the S100A1- and S100B-inhibitory peptide, TRTK-12, and a peptide derived from p53, and the S100A1 inhibitory effect on GFAP polymerization

Controversy over the efficacy of many topical wound treatments, particularly growth factors, is common, with many clinical practitioners still confused as to the real value of these agents. A serious lack of knowledge appears to exist concerning the diffusion and distribution of topically applied solutes in wounds. Without this basic understanding there seems little chance of accurately predicting the therapeutic window of drugs targeted at cellular activities, such as division and chemotaxis, and processes, such as collagen lattice deposition and contraction, occurring below the surface of the granulating layer. This study was designed to determine the absorption and distribution of a number of radiolabeled solutes (water, sodium chloride, lidocaine) and growth factors (basic fibroblast growth factor, epidermal growth factor) applied topically to full-thickness excisional wounds in rats during the early (2 d), mid (7 d), and late (12 d) stages of repair. Results showed that water and sodium penetrated deepest into wound sites and that changes in water distribution and retention in the wound paralleled the healing process. Multiple stepwise regression showed that molecular weight and tissue depth, but not day of healing, were significant factors in predicting the concentration of each solute in wound and underlying tissue sites. This finding was consistent with a tissue diffusion model developed in this study. Basic fibroblast growth factor and epidermal growth factor only penetrated slightly into the upper granulating layers of the wound site, and calculation of therapeutic doses, based on the percentage of applied solute reaching the deeper granulating layers, is presented.     

Suppression of the SOS-inducing activity of Trp-P-1 and aflatoxin B1 by meso-dihydroguaiaretic acid from Machilus thunbergii in the Salmonella typhimurium TA1535/pSK1002 umu test

The methanol extract from Machilus thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which requires liver metabolizing enzymes. The methanol extract from M. thunbergii was successively re-extracted with chloroform, butanol and water. A suppressive compound in the chloroform extract fraction was isolated by SiO2 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, and 1H- and 13C-NMR spectroscopy. Meso-dihydroguaiaretic acid inhibited of the SOS-inducing activity of Trp-P-1 in the umu test. Gene expression was suppressed by 62% at less than 0.18 mumol/ml, the ID50 value being 0.08 mumol/ml. Compound 1 was also assayed with aflatoxin B1 (AfB1) and showed a suppressive effect.