Oxidation of isonicotinic acid hydrazide (isoniazid) by horseradish peroxidase at the expense of H202 yielded reactive species which were able to reduce nitroblue tetrazolium and bleach p-nitrosodimethylaniline. Nicotinic acid hydrazide oxidation did not cause these effects. At slightly alkaline pH, oxidation of isonicotinic acid hydrazide by horseradish peroxidase proceeded at the expense of molecular 02, and the reaction was oxygen consuming. The addition of H202 abolished 02 consumption. Bovine liver catalase enhanced the rate of nitroblue tetrazolium reduction and decreased the maximal velocity of the reaction proportionately to catalase concentration. During oxidation of isonicotinic acid hydrazide by horseradish peroxidase-H202, splitting of the heme group of horseradish peroxidase took place as shown by the disappearance of the Soret and minor bands in the visible region of the spectrum.Peroxidase activity of Mycobacterium tuberculosis correlates strongly with isonicotinic acid hydrazide (isoniazid) (INH) susceptibility (3, 4). Loss of the enzyme, together with its catalase activity, causes emergence of resistance to the drug (3). Most saprophytes and atypical mycobacteria are relatively resistant to INH (12) and may acquire higher levels of resistance concomitant with the loss of their peroxidase activity and only a partial loss of catalase activity (15). The reason for these differences, as pointed out by Davis and Phillips (3), is that M. tuberculosis has one protein with peroxidase and catalase activity, whereas the saprophytes and atypicals have two proteins, one with peroxidase and catalase activity and the other with catalase activity only. The activities of the former enzyme are INH labile, whereas the activity of the latter is not. Therefore, although peroxidase loss in saprophytes and atypicals heightens the level of resistance, their primary relative resistance seems to be correlated with the presence of the INH-stable catalase. The mechanism by which that catalase confers protection against INH is unknown; however, a plausible explanation is its ability to scavenge H202. This does not necessarily mean that INH toxicity is mediated by H202 but rather that the removal of H202 by catalase inhibits H202-dependent peroxidase-mediated oxidation of INH. The role of peroxidase in mediating cytotoxicity in INH toward mycobacteria has been postulated to involve oxidation of INH (7, 10), with the oxidation products, isonicotinic acid (7), excited triplet-state aldehyde (16), and a yellow pigment precursor (15) being direct or indirect effectors in the mechanism of action of INH.Of interest is the observation that although INH and its meta isomer nicotinic acid hydrazide inhibit the peroxidase enzyme activity equally, the latter has no mycobacteriocidal activity (10). This points out an important role for the pyridine ring in determining the outcome of the metabolism, probably oxidation, of the drug by the microorganism. Thus, although both isomers could bind through their hydrazine group to the enzyme (6...
Crude extracts of Mycobacterium tuberculosis H37Ra, an isonicotinic acid hydrazide (isoniazid) (INH)-susceptible strain which has peroxidase activity, catalyzed the production of catechol from phenol in the presence of INH and H202 as shown by the development of the 444-nm absorption peak of oxidized catechol product. Extracts of the INH-resistant strain of M. tuberculosis H37Ra, which has no peroxidase, did not catalyze the reaction. The rate of development of the 444-nm peak increased proportionately with increased superoxide dismutase concentrations. The hydroxyl radical ( * OH) scavengers dimethylsulfoxide and mannitol inhibited the reaction. Isonicotinamide, isonicotinic acid, and nicotinic acid could not replace INH.The oxidation of isonicotinic acid hydrazide (isoniazid) (INH) by peroxidase seems to contribute to its action (13,18). INH oxidation intermediates appear to include peroxy, carbonyl, and isonicotinyl radicals (9,14). The possibility of interconversion or side reactions of these radicals to yield other radicals, such as * OH and superoxide (02), iS plausible. In a model system, horseradish peroxidase (HRP)-catalyzed oxidation of INH failed to produce * OH (14), although it produced a radical, probably a species equivalent to * OH such as a ferryl ion that could bleach p-nitrosodimethylaniline. Superoxide was produced only in the presence of an electron donor (15). Although HRP is quite similar to the peroxidase of Mycobacterium tuberculosis, they differ in some respects, such as the higher catalase activity and substrate specificity of the mycobacterial peroxidase (5). We have shown previously (14) that low concentrations of catalase accelerated the rate of nitroblue tetrazolium reduction by HRP. Thus, oxidation of INH by the mycobacterial peroxidase could differ in certain respects from HRP-catalyzed oxidation.Hydroxyl radicals, O2, and singlet oxygen (102) are toxic and mutagenic for cells (3,6). Mycobacteria are susceptible to the catalase-H202-halide system (8), which is capable of producing active 02. Also, it was proposed that the carotenoids of mycobacteria function primarily as scavengers of 102, to which mycobacteria are suggested to be susceptible (4). In addition, mycobacteria are susceptible to high 02 concentrations, and the MIC of INH against mycobacteria is lowered by increasing 02 concentration (7). The uptake and metabolism of INH by mycobacteria are aerobic processes, and in the absence of 02, INH has no effect. Since it seems that mycobacteria are susceptible to active 02 and that 02 plays a significant role in the mechanism of INH action, we examined the possibility of an INH-mediated active oxygen production in susceptible mycobacteria through oxidation of INH by endogenous peroxidase. confirmed by the method of the Center for Disease Control (1), and the MICs were found to be less than 1 ,ug for strain 201 and more than 5 ,ug for strain 326. MATERIALSThe catalase activity of each strain was confirmed by the column height technique and by the ability of extracts of each str...
During the course of horseradish peroxidase-mediated oxidation of either o-dianisidine or 2-2'-azino-d1(3-ethyl-benzthlazoline-6sulfonic acid) (ABTS), no 02 consumption took place. When isonicotinic acid hydrazide (isoniazid) (INH) was included in the reaction mixture, 02 The -activity of the mycobacteriocidal drug isonicotinic acid hydrazide (isoniazid) (INH) has been linked to both mycobacterial peroxidase activity (3, 16) and NAD (11,12) in several ways. The peroxidase enzyme of mycobacteria is thought to sensitize these cells to INH, and a strong positive correlation has been found (4). Sensitization of peroxidasecontaining mycobacteria has been postulated to be through the oxidation of the drug with the production of toxic metabolites (8,15
BackgroundHepatitis B is a liver disease primarily caused by hepatitis B virus (HBV) infection. It is distributed worldwide and associated with high mortality and morbidity rates. HBV infections can be avoided by the administration of the currently available vaccine and can be easily diagnosed through commercially available kits. Both the vaccine and the diagnostic kits depend on using the hepatitis B surface antigen (HBsAg) as an antigen. Developing countries such as, Egypt, suffer from the widespread of HBV infections and the limited resources to provide adequate supplies of either the vaccine or the diagnostic kits. Therefore the need for an easy, rapid, low cost method to produce HBsAg is urgently needed within this setting.FindingsTo achieve this goal, the gene encoding the HBsAg(S) protein was cloned and expressed as a fusion protein with a GST tag in Escherichia coli. The recombinant protein was successfully expressed and purified in both good quality and quantity.ConclusionsThe simplified and the relatively low cost of the used protocol make this an attractive alternative to protocols currently used for the purification of HBsAg(S). The exploiting of this achievement for new diagnostics can be directed for application in the developing countries where they are extremely needed.
Washed intact cells of Escherichia coli and Staphylococcus aureus, grown under partial anaerobic conditions in nitrate media, reduced nitrate quantitatively when formate was used as a reducing substrate. Nitrate reductase was applied as an index for bacterial adherence to different target surfaces including uroepithelial cells, HeLa cells and fibrin clots. Nitrate reduction by adhered as well as control cells was determined by quantitative diazotization reaction for nitrite. Variations in the conditions which affect adherence gave rise to corresponding variations in the nitrate reduction index from which bacterial adherence can be conveniently determined under these conditions. This method is simple, reproducible and easy to perform in a short time.
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