Yamamoto Itaru

4 (or 5)-diazoimidazole-5 (or 4)-carboxamide and related triazenoimidazoles as antibacterial agents: Their effects on nucleic acid metabolism of Escherichia coli B

Abstract 4(or 5)-Diazoimidazole-5(or 4)-carboxamide (Diazo-ICA) and dialkyl-triazenoimidazoles tested at concentrations of 1 μg/ml and 10 μg/ml, completely inhibit the growth of Escherichia coli B without causing cell lysis. With the latter compounds there is a lag period before growth inhibition. After exposing growing cells to 0.1–0.5 μg/ml of Diazo-ICA for 60 min, the number of viable cells was only about 5 per cent of that before exposure. This compound also affects resting cells. The inhibitory effect of Diazo-ICA can be abolished by the addition of cysteine, but 4(or 5)-aminoimidazole-5(or 4)-carboxamide, hypoxanthine, DNA, RNA, protein and leucine have no affect on the inhibition. At relatively high concentration, 2- azahypoxanthine, which is a cyclized product of Diazo-ICA, also inhibits cell growth, but its effect is abolished by the addition of hypoxanthine. Diazo-ICA, at a concentration of 0.05 μg/ml, inhibits the biosynthesis of DNA in the cells, whereas synthesis of RNA is slightly stimulated and synthesis of protein is not affected. In E. coli B cells incubated with Diazo-ICA (0.01 μg/ml), the incorporation of thymidine-3H into DNA is severely inhibited. However, the incorporation of uridine-3H into RNA is slightly stimulated and that of leucine-3H into protein is not inhibited. These results suggest that the major action of Diazo-ICA in E. coli B is to inhibit DNA synthesis, by interfering with SH-groups in biological systems.

Evidence for the in vivo formation of ascorbic acid 2-O-α-glucoside in guinea pigs and rats

In vivo formation of ascorbic acid 2-O-alpha-glucoside (AA-2G) in guinea pigs and rats given ascorbic acid (AA) orally in combination with maltose was examined. A metabolite of AA which has the same HPLC retention characteristics as authentic AA-2G was detected in the blood, urine and liver of guinea pigs 1-2 hr after their administration. The metabolite was isolated from the urine by chromatographic procedures and identified as AA-2G by its UV spectrum, non-reducibility, susceptibility to alpha-glucosidase hydrolysis, HPLC profile and elementary analysis. The same glucoside was also synthesized by rats and found in the urine, although it could not be determined qualitatively in the blood. AA-2G-forming activities of tissue homogenates from both animals were apparently correlated with their alpha-glucosidase activities and, moreover, both activities were completely inhibited by a specific neutral alpha-glucosidase inhibitor. Thus, we conclude that AA-2G is a possible metabolite produced by enzymatic alpha-glucosidation after a combined administration of AA and maltose to guinea pigs and rats.

Studies on the mechanism of inhibition of xanthine oxidase by 5-diazoimidazole-4-carboxamide and related thioazoimidazole carboxamides

Abstract The mechanism of inhibition of milk xanthine oxidase by 5-diazoimidazole-4-carboxamide (diazo-ICA) and by five related thioazoimidazole carboxamides (thioazo-ICAs) was studied. The extent of inhibition of xanthine oxidase by diazo-ICA and thioazo-ICAs decreased greatly when these compounds were preincubated in a buffer before the addition of substrate and enzyme. In 0.1 M Tris-HCl buffer, pH 7.5, thioazo-ICAs were converted to 2-azahypoxanthine, a cyclized product of diazo-ICA, which inhibits xanthine oxidase slightly. The inhibition of xanthine oxidase by thioazo-ICAs is probably due to this diazo-ICA. With xanthine as a variable substrate, diazo-ICA caused uncompetitive, irreversible inhibition. The inhibition of xanthine oxidase by diazo-ICA was reduced by simultaneous addition of a sulfhydryl compound, such as cysteine, cysteamine or reduced glutathione, but not other amino acids. Diazo-ICA inactivated the enzyme more significantly and rapidly than other sulfhydryl reagents. The inhibitory activity of diazo-ICA was potentiated strongly by Fe 2+ , Mn 2+ , Co 2+ and Cu 2+ , and slightly by Mo 5+ . Treatment of milk xanthine oxidase with diazo-ICA changed the absorption spectrum of the enzyme.

Potent competitive uricase inhibitors--2,8-diazahypoxanthine and related compounds.

Abstract The inhibitory activities of 2,8-diazahypoxanthine and related compounds on uricase of bovine kidney and rat liver homogenates were compared in vitro. 2,8-Diazahypoxanthine was found to be even more inhibitory than 2-azahypoxanthine, 8-azaxanthine, 8-azahypoxanthine or oxonate. From Lineweaver-Burk plots, 2,8-diazahypoxanthine and 8-azaxanthine were shown to be competitive inhibitors of uricase. 2,8-Diazahypoxanthine had no influence on the activity of milk xanthine oxidase or rat erythrocyte hypoxanthine-guanine phosphoribosyltransferase at the concentrations tested. The inhibitory activities of 2,8-diazahypoxanthine and related compounds on rat liver uricase in vivo were compared by measuring their effects on serum urate and allantoin. 2,8-Diazahypoxanthine, administered intraperitoneally, caused the greatest increase in the urate level with a concomitant decrease in the allantoin level. 8-Azaxanthine and 8-azahypoxanthine were both relatively strong inhibitors of uricase in vivo although the latter was not a strong inhibitor in vitro.

Biological activity of diazonium compounds. Studies on the mechanism of action of 4 (or 5)-diazoimidazole-5 (or 4)-carboxamide on 5-hydroxytrypt-amine release from rabbit platelets—II: Comparative studies with N-ethylmaleimide in vitro

Abstract Studies were made on the association of the SH-blocking effect of Diazo-ICA with its activity in releasing 5-HT from isolated rabbit platelets, and the releasing activity was compared with that of NEM. High concentration of NEM, like Diazo-ICA, caused release of 5-HT from platelets, but, unlike Diazo-ICA, its action was independent of the calcium ion concentration in the incubation medium. The calcium-dependent release of 5-HT by Diazo-ICA was completely blocked by a small amount of NEM. The sulfhydryl compounds, cysteine, glutathione and BAL also blocked release of 5-HT by Diazo-ICA. ATP, ADP and inorganic pyrophosphate inhibited release of 5-HT by Diazo-ICA, but AMP and creatine phosphate did not. These findings suggest that the sulfhydryl group and pyrophosphate structures are involved in the mechanism of release of 5-HT from rabbit platelets by Diazo-ICA.

Biological activity of diazonium compounds: Studies on the mechanism of action of 4(or 5)-diazoimldazole-5(or 4)-carboxamide on 5-hydroxytryptam1ne release from rabbit platelets—I: Requirement for calcium ion

Abstract Incubation of 4(or 5)-diazoimidazole-5(or 4)-carboxamide (Diazo-ICA) at 37° with isolated rabbit platelets induces release of 5-hydroxytryptamine (5-HT). This release occurs only in the presence of a trace of calcium ions and the effect is independent of the presence of other plasma components. The release is temperature-dependent, being appreciably lower at room temperature than at 37° and almost negligible at 4°. These results imply that the mechanism of release with Diazo-ICA differs from that with reserpine or bacterial pyrogenic lipopolysaccharide. 4(or 5)-Dialkyltriazenoimidazole-5 or 4)-carboxamides, prepared by coupling Diazo-ICA with corresponding dialkylamines, cause only slight release of 5-HT from platelets, and the release is independent of the presence of calcium ion. In contrast, 4(or 5)-aminoimidazole-5(or 4)-carboxamide (the parent compound of these derivatives), 2-azahypoxanthine (a cyclized product of Diazo-ICA) and S-(5(or 4)-carbamoyl-4(or 5)-imidazolyl azo)cysteine (a coupling product of Diazo-ICA and cysteine) release no 5-HT from platelets. The other diazonium compound tested, diazobenzene sulfonate and diazobenzenesulfonamide, also cause no release.