Bacterial variants produced in culture media containing 2,3,5-triphenyltetrazolium chloride.
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The impact of triazophos, bensulfuron-methyl, chlobenthiazone on soil biochemical characteristics in a paddy soil under controlled moisture (flooded soil) and temperature (25 degrees C) condition was studied. The electron transport system (ETS)/dehydrogenase activity displayed a negative correlation with triazophos, bensulfuron-methyl, chlobenthiazone concentrations, and the activity decreased as the concentration of the pesticides increased. The higher doses, 5 and 10 field rates, of triazophos, bensulfuron-methyl, chlobenthiazone significantly inhibited ETS activity, while lower rates failed to produce any significant reducing effect against the control. The relative toxicity of triazophos, bensulfuron-methyl, chlobenthiazone in decreasing the ETS activity was in the order: triazophos > chlobenthiazone > bensulfuron-methyl, irrespective of the rates of application. Triazophos, bensulfuron-methyl, chlobenthiazone caused an improvement in the soil phenol content and it increased with increasing concentration of triazophos, bensulfuron-methyl, chlobenthiazone. Triazophos, bensulfuron-methyl, chlobenthiazone incorporation did not produce any significant change in soil protein content. The response of biomass phospholipid content was nearly similar to ETS activity. The phospholipid content was decreased with the addition of triazophos, bensulfuron-methyl, chlobenthiazone in the order of triazophos > chlobenthiazone > bensulfuron-methyl; and the toxicity of applied amount was in the order: 10 FR (field rate) > 5 FR > 1.0 FR > 0.5 FR > control.
Express diagnosis using 2,3,5-triphenyltetrasolium chloride as the redox indicator provided in most tests rapid and sufficiently precise determination of the microbial flora sensitivity to antibacterial drugs permitting to start in time the antibiotic therapy of the patients. For rapid response it proved to be useful to incubate beforehand the test material taken from surgical patients within 16 to 18 hours and to increase the indicator concentration up to 2--3 per cent.
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Thiol-containing proteins and low molecular sulfhydryl-containing compounds reduced nitroblue tetrazolium (NBT) in presence of vitamin K3(2-methyl-1,4-naphthoquinone). Cysteine and dithiothreitol (but not glutathione) reduced NBT also in absence of the quinone. Reduction of NBT was accelerated in the weakly alkaline medium and occurred both via O2- -dependent and O2- -independent mechanism. In presence of quinones the thiols appear to affect the manifestation of oxidoreductase reactions estimated by means of NBT-test.
Aliphatic triazenes, such as 1,3-dimethyltriazene, are potent biological alkylating agents because they form alkyldiazonium ions. They are also subject to very rapid proteolytic decomposition, even at physiological pH. The acylated analogues 1,3-dialkyl-3-acyltrizenes are much more stable in aqueous solution, but they also give rise to alkyldiazonium ions. Four acylated 1,3-dimethyltriazenes, where the acyl groups were diethylphosphoryl (DMP), carbethoxy (DMC), acetyl (DMA), and N-methylcarbamoyl (DMM), were studied kinetically. Rate-pH profiles indicated that the acyl group had a profound effect on the mechanism of decomposition. The cytotoxic potential of all four compounds was studied in vitro by using the MTT-tetrazolium assay. The compounds had fair-to-good activity against some cell lines, particularly those deficient in methylation repair. In vivo assays of DMC and DMM against several tumor xenografts in nude mice showed promising activity for some cancers, particularly in the case of DMM. In vitro assays were also carried out on three 1-(2-chloroethyl)-3-methyl-3-acyltriazenes. The acyl groups were carbethoxy (CMC), acetyl (CMA), and N-methylcarbamoyl (CMM). The activity of these compounds largely paralleled that of bis(2-chloroethyl)-N-nitrosourea (BCNU), except for those cell lines which exhibited the Rem phenotype; triazenes were more active in those lines than BCNU. The in vivo activity of CMC, CMA, and CMM was tested in the P388 leukemia assay. All three were active but CMC and CMA proved to be rather toxic. CMM was well tolerated and was examined in several tumor xenografts in nude mice. Significant activity was found against MX-1 mammary carcinoma, against LX-1 small cell lung carcinoma, and particularly against LOX amelanotic melanoma, where complete cures were effected. The antineoplastic activity of the acyltriazenes is well-correlated with their chemical behavior.
The reactions of heteropentalene mesomeric betaine 1 and nitrogen-rich mesoionic tetrazolium-5-amides 4, 11 and 16-18 with electron-deficient unsaturated compounds have been studied. Novel heterocyclic systems, tetrazolo[4,5-a][1,7]benzodiazonine inner salt 2 and 3-oxo-3,7-dihydro-2H-pyrazolo[3,4-b]pyridine 5, have been synthesized by the reactions of dimethyl acetylenedicarboxylate with 1 and tetrazolium-5-anilide 4, respectively, and fully characterized by X-ray crystallography. It has been found that the reactions of other tetrazolium-5-amides are also initiated by the nucleophilic addition of the electron-rich amide nitrogen to the electron-deficient unsaturated compounds.
An in vitro tetrazolium dye (MTT) reduction technique was modified and evaluated for use in the large-scale screening of anticancer compounds by examining the activity of ten clinically used drugs against 16 different human and murine cell populations. Cell populations included colon and mammary adenocarcinomas, melanomas, leukemias, and freshly isolated normal cells. Cell lines were grown in microtiter plates for 18-20 hours prior to a 72-hour continuous exposure to the drugs. Cultures were initiated at cell densities which maximized both the difference in dye reduction and the number of cell doublings between the beginning and end of the drug exposure period. Drug potency, expressed as the 50% inhibitory concentration (IC50), was comparable whether the effect on cell doublings or dye reduction was determined. There was good agreement between this method and the more labor-intensive, conventional method of counting trypan blue dye-excluding cells in a hemacytometer. Implemented as a large-scale, high-capacity system, our adaptation of the MTT technique is a rapid, sensitive, reproducible first-line screening device for detecting anticancer compounds with cytostatic or cytocidal activity.
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Sorbitol dehydrogenases from the cytoplasm of plant, animal, and microbial cells was used to study the effect of superoxide dismutase on histochemical exposure of dehydrogenases after their electrophoretic separation. In some organisms, both enzymes are localized in the same region, which finally leads to the formation of hydrasine tetrazolium, a soluble colourless compound, but not of diformazan, an insoluble stained derivative of tetrazolium nitroblue. The experimental conditions for histochemical exposure of the enzymes in gels are discussed.
We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.
Cercosporin, a toxin produced by members of the fungal genus Cercospora, is a photosensitizing compound which rapidly kills plant cells in the light. We have found that cercosporin, when activated by light in the presence of oxygen, is able to generate both singlet oxygen and superoxide ions. Cercosporin, when illuminated in the presence of O(2), reacted with cholesterol to form the 5alpha-hydroperoxide of cholesterol which is only produced by reaction with singlet oxygen. Cercosporin, in the presence of light, O(2), and a reducing substrate, was also able to reduce p-nitro blue tetrazolium chloride, a compound readily reduced by superoxide. Superoxide dismutase, a scavenger of superoxide, inhibited this reaction. Production of both singlet oxygen and superoxide by cercosporin must be considered when studying the possible mechanisms of resistance to cercosporin.
We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.
Investigations have been undertaken to determine the effect of occupational exposure to organophosphoric pesticides on the behaviour of NBT-dye reduction and spontaneous E rosette formation tests. A significant decrease of the values of the examined cellular parameters of human immunity and of the absolute lymphocyte count in peripheral blood was found in 31 workers, as compared to 30 controls. Moreover, the NBT-dye reduction decrease showed a positive linear correlation with a reduced acetylcholinesterase (AChE) activity. The exposure time showed a negative linear correlation of the phagocytic index of the NBT-dye reduction test. It is concluded that depressed NBT-dye reduction and diminished spontaneous E rosette formation may be regarded as a probable mode of the effect of organophosphoric chemicals on metabolic and membrane damage of human cells. A possible role of endogenous acetylcholine poisoning in the development of these pathologic conditions is also discussed.