Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets.
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The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane. (+info)
Chronic inhalation carcinogenicity study of commercial hexane solvent in F-344 rats and B6C3F1 mice.
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The carcinogenic and chronic toxicity potential of commercial hexane solvent was evaluated in F-344 rats and B6C3F1 mice (50/sex/concentration/species) exposed by inhalation for 6 h/day, 5 days/week for 2 years. Target hexane vapor concentrations were 0, 900, 3000, and 9000 ppm. There were no significant differences in survivorship between control and hexane-exposed groups, and clinical observations were generally unremarkable. Small, but statistically significant decreases in body weight gain were seen in rats of both sexes in the mid- and high-exposure groups and in high-expsoure female mice. The only noteworthy histopathological finding in rats was epithelial cell hyperplasia in the nasoturbinates and larynx of exposed groups. This response was judged to be indicative of upper respiratory tract tissue irritation. No significant differences in tumor incidence between control and hexane-exposed rats were found. In mice, uterine tissue from the high-exposure females exhibited a significant decrease in the severity of cystic endometrial hyperplasia compared to controls. An increase in the combined incidence of hepatocellular adenomas and carcinomas was observed in high-exposure female mice. The incidence of liver tumors was not increased in the mid- or low-exposure female mice or in male mice exposed to hexane. An increased incidence of pituitary adenomas was observed in female, but not male mice. This finding was not believed to have been treatment-related because the incidence in the control group was unusually low, and the incidence in exposed groups was not dose-related and was within the historical control range. No other neoplastic changes judged to be treatment-related were observed in tissues from male or female mice. In conclusion, chronic exposure to commercial hexane solvent at concentrations up to 9000 ppm was not carcinogenic to F-344 rats or to male B6C3F1 mice, but did result in an increased incidence of liver tumors in female mice. (+info)
Crystal structure of triosephosphate isomerase from Trypanosoma cruzi in hexane.
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To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2-A resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 A from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design. (+info)
Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.
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Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction. We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC. (+info)
Defining a rob regulon in Escherichia coli by using transposon mutagenesis.
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The Rob protein of Escherichia coli is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the rob gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a rob deletion mutant containing a plasmid that allows for controlled rob gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal rob gene (inaA, marR, aslB, ybaO, mdlA, yfhD, and ybiS). One gene, galT, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of micF is significantly higher in wild-type E. coli than in a rob deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor. (+info)
Anaerobic initial reaction of n-alkanes in a denitrifying bacterium: evidence for (1-methylpentyl)succinate as initial product and for involvement of an organic radical in n-hexane metabolism.
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A novel type of denitrifying bacterium (strain HxN1) with the capacity to oxidize n-alkanes anaerobically with nitrate as the electron acceptor to CO(2) formed (1-methylpentyl)succinate (MPS) during growth on n-hexane as the only organic substrate under strict exclusion of air. Identification of MPS by gas chromatography-mass spectrometry was based on comparison with a synthetic standard. MPS was not formed during anaerobic growth on n-hexanoate. Anaerobic growth with [1-(13)C]n-hexane or d(14)-n-hexane led to a 1-methylpentyl side chain in MPS with one (13)C atom or 13 deuterium atoms, respectively. This indicates that the 1-methylpentyl side chain originates directly from n-hexane. Electron paramagnetic resonance spectroscopy revealed the presence of an organic radical in n-hexane-grown cells but not in n-hexanoate-grown cells. Results point at a mechanistic similarity between the anaerobic initial reaction of n-hexane and that of toluene, even though n-hexane is much less reactive; the described initial reaction of toluene in anaerobic bacteria is an addition to fumarate via a radical mechanism yielding benzylsuccinate. We conclude that n-hexane is activated at its second carbon atom by a radical reaction and presumably added to fumarate as a cosubstrate, yielding MPS as the first stable product. When 2,3-d(2)-fumarate was added to cultures growing on unlabeled n-hexane, 3-d(1)-MPS rather than 2,3-d(2)-MPS was detected, indicating loss of one deuterium atom by an as yet unknown mechanism. (+info)
Interactions of some organic solvents: hydrocarbons and chloroalkene.
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Metabolic and toxicodynamic interactions of some organic solvents in rats repeatedly treated with medium dose levels were examined. It was shown that both n-hexane and ethylbenzene significantly inhibited tetrachloroethylene metabolism during a 2-week period. n-Hexane and tetrachloroethylene enhanced metabolism of ethylbenzene whereas ethylbenzene suppressed n-hexane metabolism only at the end of the experiment. Biochemical changes, especially the drop in the level of non-protein sulfhydryl groups in tissues of rats treated with organic solvent mixtures, were significantly less pronounced than those observed after these chemicals were administered separately. These results demonstrate that metabolic interactions between hydrocarbons and chloroalkene may lead to a modification of the biological response to these compounds. (+info)
Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.
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Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general. (+info)