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At least 631 records · Page 35Linked to original sources
PubMed2006

In vitro screening of medicinal plant extracts for macrofilaricidal activity.

Methanolic extracts of 20 medicinal plants were screened at 1-10 mg/ml for in vitro macrofilaricidal activity by worm motility assay against adult Setaria digitata, the cattle filarial worm. Four plant extracts showed macrofilaricidal activity by worm motility at concentrations below 4 mg/ml and an incubation period of 100 min. Complete inhibition of worm motility and subsequent mortality was observed at 3, 2, 1 and 1 mg/ml, respectively, for Centratherum anthelminticum, Cedrus deodara, Sphaeranthus indicus and Ricinus communis. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was carried out at 1 mg ml(-1) and 4-h incubation period, and the results showed that C. deodara, R. communis, S. indicus and C. anthelminticum exhibited 86.56, 72.39, 61.20 and 43.15% inhibition respectively in formazan formation compared to the control.

Animals↗
PubMed1996

Onchocerca fasciata: enzyme histochemistry and tissue distribution of various dehydrogenases in the adult female worm.

Histochemistry studies of key dehydrogenases in the glycolytic pathway and related enzymes and the tricarboxylic acid (TCA)-cycle enzymes were carried out on adult female Onchocerca fasciata. The distribution pattern and enzymatic activities of 6-phosphogluconate dehydrogenase (6-GPDH), lactate dehydrogenase (LDH), mitochondrial glycerol-3-phosphate dehydrogenase (GPDH), nicotinamide adenine dinucleotide (phosphate) [NAD+(P)]-linked isocitrate dehydrogenase (ICDH), and NAD+(P)-linked malate dehydrogenase (MDH) in various tissues of the worm were determined. Moderate to intense enzyme activities were localized in three main areas, namely, the hypodermis, body-wall muscle, and reproductive tissues. Activity of the formazan reaction product was very low, if at all present, in the intestinal epithelium and was completely absent in the cuticle. On the basis of the present results and earlier observations, it is suggested that glycolysis leading to the end product lactate is the main energy-generating pathway in O. fasciata. The presence of significant activity of 6-GPDH indicates that the pentose-phosphate pathway might be operative in O. fasciata. In light of the activity of some of the TCA-cycle enzymes, ICDH and MDH, demonstrable in O. fasciata, it is possible that an additional pathway (pyruvate-succinate) of glucose metabolism via a reverse sequence of the TCA cycle may also be operative in the worm. The possible functional significance of the enzymes detected is discussed with respect to their location.

Alcohol Oxidoreductases↗
PubMed2001

Nitric oxide synthase-1 is enriched in fast-twitch oxidative myofibers.

Nitric oxide synthase-1 (NOS-1) is found in high concentrations in skeletal muscles, where its synthesis product nitric oxide (NO) is reported to be involved in a number of processes, including the modulation of the oxidative metabolism of myofibers. Performing immunoblot analysis and quantification of formazan produced by its specific NADPH diaphorase activity, we found NOS-1 to be enriched in rat skeletal muscles with a high proportion of fast-twitch myofibers. Since these myofibers represent a metabolically heterogeneous subpopulation, we extended our investigation to the level of individual myofibers. Using serial sections we combined myosin heavy chain-based fiber-typing with quantitative succinate dehydrogenase histochemistry to determine three groups of fiber-types, comprising fast-oxidative, fast-glycolytic and slow-oxidative myofibers. Image analysis showed that NOS-1 diaphorase activity is significantly enriched in fast-oxidative myofibers compared with fast-glycolytic and slow-oxidative ones. In order to characterize potential biological effects of the fiber-type-specific enrichment of NOS-1, we performed cytochrome oxidase histochemistry in the presence of the NO donors NOC-9 and SNAP. Both NO donors reduced cytochrome oxidase activity in all myofibers investigated with almost identical semi-maximal inhibition rates, although fast-oxidative and slow-oxidative myofibers contained twice as much basal catalytic activity than fast-glycolytic ones. In summary, we suggest that the NOS-1/NO system of skeletal muscles exerts its biological role especially in fast-oxidative myofibers, since these myofibers express more NOS-1 than fast-glycolytic or slow-oxidative ones and also contain the highest concentrations of cytochrome oxidases as potential target molecules of NO.

Animals↗
PubMed2000

Post-ischemic intraportal adenosine administration protects against reperfusion injury of canine liver.

Although adenosine has been postulated to inhibit ischemia-reperfusion injury in various tissues, its in vivo cytoprotective mechanism is not fully known. The aim of this study was to determine the effect of intraportally infused adenosine on reperfusion injury in the canine liver. Two h ischemia and reperfusion of the liver were induced in beagle dogs by clamping the portal triad. Either adenosine or saline was infused in the portal vein after reperfusion for 60 min. Levels of serum aspartate aminotransferase and alanine aminotransferase and the survival of animals were examined. Hepatic levels of protein carbonyls and glutathione were also measured, as markers of oxidative stress. One h after reperfusion, the liver was perfused with nitroblue tetrazolium and the formation of formazan was observed to evaluate superoxide formation. Twenty-four h after reperfusion, 100% of animals in the adenosine group and 33% of animals in the control group survived. Adenosine significantly decreased the reperfusion-induced increase in serum levels of aspartate aminotransferase and alanine aminotransferase. Adenosine also suppressed the formation of protein carbonyls and the decrease in glutathione levels. Histologically, neutrophil infiltration, superoxide formation, and apoptosis were decreased by adenosine. These results suggest that intraportally infused adenosine attenuates reperfusion injury of the liver, presumably by suppressing the activation of neutrophils and oxidative stress.

Adenosine↗
PubMed2000

A new non-radioactive method for IL-2 bioassay.

An oxidation-reduction (redox) indicator, alamarBlue, was used to measure the bioactivity of interleukin 2 (IL-2). This assay system has several advantages over other bioassays for measuring IL-2. It is a nonradioactive method unlike the conventional tritium-labeled thymidine ([3H]TdR) incorporation assay. The alamarBlue assay is also easier to use than other colorimetric methods, such as the MTT assay, because the alamarBlue assay does not depend on the extraction of insoluble formazan salt, which is time-consuming, error-prone, and cumbersome. Due to its solubility in culture medium and its nontoxicity to cells, alamarBlue provides an easy method to monitor cellular growth using either a fluorescence- or an absorbance-based instrument. The alamarBlue assay is not sample-destructive, unlike the thymidine incorporation and MTT methods. This adds another advantage to the alamarBlue method as the measurement of cellular growth by sample-destructive methods requires as many tubes as time points whereas the alamarBlue method requires only one tube for the entire growth period. In this study, alamarBlue was used to measure the proliferation of the IL-2-dependent cytotoxic T cell line, CTLL-2. The colorimetric change of alamarBlue at 570 nm compared to the reference wavelength, 600 nm, was proportional to the number of viable cells. The sensitivity of the IL-2 assay using alamarBlue was comparable to that of the [3H]thymidine incorporation method. These results demonstrate that the alamarBlue assay is valid for the IL-2 bioassay and that alamarBlue can replace the [3H]thymidine employed in the conventional proliferation assays.

Animals↗
PubMed1990

Myocardial infarct size reduction by the synergistic effect of hyperbaric oxygen and recombinant tissue plasminogen activator.

Fasting mongrel dogs underwent hyperbaric oxygen treatment (HBOT), recombinant tissue plasminogen activator (rt-PA) treatment, and simultaneous HBOT and rt-PA treatment following prior experimental left anterior descending coronary artery occlusion for 2 hours. Thrombosis in and around a copper coil was recorded angiographically at regular intervals, and immediately prior to treatment conclusion. Controls (n = 10) were untreated. Group two animals (n = 10) were treated additionally with 90 minutes of HBOT at 2 atm absolute. Group three animals (n = 8) were treated additionally with 50 mg rt-PA over 90 minutes. Group four animals (n = 10) were treated additionally with simultaneous HBOT and rt-PA over 90 minutes. Myocardial injury was determined by a combination of triphenyltetrazolium chloride histochemical staining and by formazan dye extraction. Damage was measured as a percent of left ventricular cross-sectional area studied. HBOT alone restored 35.9% of oxidative enzyme activity (p greater than 0.001). Treatment with rt-PA alone restored 48.9% of enzyme activity (p greater than 0.001). The combination of HBOT and simultaneous rt-PA resulted in 96.9% restoration of oxidative enzyme activity versus the control group (p greater than 0.001).

Animals↗
PubMed1984

Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase.

The usual method of staining polyacrylamide gel electropherograms for superoxide dismutase activity utilizes a photochemical flux of O2- to reduce nitroblue tetrazolium. Superoxide dismutases intercept O2-, preventing formazan production and thus causing achromatic bands. In the presence of H2O2, catalases also yield achromatic bands during this staining procedure. This is due to local elevation of pO2 by the catalatic decomposition of H2O2. O2, in turn, inhibits the reduction of the tetrazolium by O2-. This phenomenon provides a new activity stain for catalase. A previously described activity stain for catalase has also been reexamined and significantly improved.

3,3'-Diaminobenzidine↗
PubMed1984

Detection of glutathione transferase activity on polyacrylamide gels.

A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues.

Animals↗
PubMed1987

Quantitation of aspartate aminotransferase isoenzymes after electrophoretic separation.

A scheme for the quantitative detection of aspartate aminotransferase isoenzymes and multiple forms after electrophoretic separation is described. Glutamate generated from the aminotransferase reaction is quantitated by using the glutamate dehydrogenase/diaphorase-coupled enzyme system to form a formazan dye. Product inhibition of aspartate aminotransferase by oxaloacetate is prevented by including oxaloacetate decarboxylase in the overlay reagent. Results compare favorably with those of an immunochemical precipitation procedure. The method can also be used to detect quantitatively subforms and atypical forms (genetic variants, immunoglobulin-enzyme complexes) of aspartate aminotransferase.

Animals↗
PubMed1988

An assay for the detection of superoxide dismutase in individual Escherichia coli colonies.

A method for detecting superoxide dismutase activity in individual colonies of Escherichia coli was developed. The assay involves the lysis of individual cells in colonies on filter papers by a series of lysozyme, chloroform, and freeze-thaw treatments. Filters are placed on agar plates to allow diffusion of cellular enzymes into a solid matrix. A nitroblue tetrazolium overlay is applied to detect superoxide dismutase activity. Colonies possessing activity produce achromatic zones against a dark Formazan background. The assay can detect the presence of superoxide dismutase and the relative amount of enzyme as well. This assay provides a method for screening a population of cells for mutants deficient in or overproducing superoxide dismutase.

Bacteriological Techniques↗
PubMed1988

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes.

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.

Animals↗
PubMed1989

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase.

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.

Amino Acid Oxidoreductases↗
PubMed1989

The involvement of superoxide anions in the nitro blue tetrazolium chloride reduction mediated by NADH and phenazine methosulfate.

Oxygen inhibits competitively the reduction of nitro blue tetrazolium chloride (nitro BT) by NADH and phenazine methosulfate (PMS). The oxygen-dependent inhibition is stronger in the presence of superoxide dismutase, whereas cyanide counteracts the oxygen interference. On the other hand, the oxidation of NADH mediated by PMS and dioxygen is affected only marginally by superoxide dismutase and cyanide. Therefore, it is concluded that the involvement of superoxide anions occurs at the level of nitro BT reduction via a nitro blue tetrazolinyl radical, as has been suggested by Picker and Fridovich [1984) Arch. Biochem. Biophys. 228, 155-158) and not at the level of PMS oxidation. The inhibition of the oxygen interference in the nitro BT reduction by cyanide is dependent on the cyanide concentration, whereas in nitrogen cyanide has no effect on the reduction. It is caused by competition between cyanide and oxygen to reduce or oxidize the nitro BT radical to either formazan with concomitant cyanogen production or nitro BT, respectively. For the histochemical localization and analysis of electropherograms of NAD(P)+-dependent dehydrogenase activities, the interference of oxygen can be avoided by anaerobic incubations or by the use of 5 mM nitro BT when incubating aerobically.

Kinetics↗
PubMed1992

Chromogenic redox assay for beta-lactamases yielding water-insoluble products. I. Kinetic behavior and redox chemistry.

We describe a chromogenic detection system for beta-lactamase which yields water-insoluble colored products. The assay is based on kinetic measurement of the appearance of color due to the beta-lactamase-initiated redox reaction. The substrates are C3' thiolate-substituted cephalosporins, which, after enzyme-catalyzed hydrolysis of the beta-lactam ring, undergo elimination of the thiolate ion. This thiolate, in a postenzymatic step, reduces the tetrazolium salts, which are water-soluble colorless compounds, to a colored water-insoluble precipitate of formazan. Our model in this study was a beta-lactamase Enterobacter cloacae P-99-catalyzed reaction of thiolacetate cephalosporin with several tetrazolium salts. We found that the reaction rate is dependent on the concentration of the electron carrier 5-methyl phenazinium methyl sulfate, the pKa of the C3' thiolate substituent of the cephalosporin substrate, and the reduction potential of the tetrazolium salts. A kinetic study of this system yielded a rate law for the reaction. We present a mechanism of the reaction and determination of the kinetic parameters for the process. The sensitivity of this kinetic assay is very high; we detect 3 x 10(-10) M beta-lactamase P-99, which is approximately 30 mIU. The assay times are very short, lasting from 2 to 5 min. The new assay system is particularly suitable for a rapid detection of beta-lactamases in bacterial colonies and in enzyme immunoassays where beta-lactamase may be used as the label.

Cephalosporins↗
PubMed1992

An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide.

An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1992

A quantitative microassay of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on polystyrene plates.

A microadhesion assay that allows the quantitative determination of carbohydrate-mediated cell adhesion to glycoconjugates immobilized on 96-well polystyrene plates has been developed. After dislodging nonadherent cells by centrifugation, specifically bound cells are quantified by colorimetric analysis of a blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide by enzymatic reduction. Carbohydrate specificity of the cell adhesion was demonstrated by inhibition analyses and the general applicability of the assay was proved with indicator cells of three different origins: mouse fibrosarcoma cells, Chang liver cells, and human breast carcinoma cells (MDA-MB 231).

Animals↗
PubMed1995

Superoxide radical and xanthine oxidoreductase activity in the human heart during cardiac operations.

BACKGROUND: The results of clinical trials of xanthine oxidoreductase inhibition in cardiac surgery are encouraging, although studies have failed to localize the enzyme to the human heart and to localize free radical activity to fresh human heart. METHODS: We adapted a histochemical staining technique based on the reduction of nitro blue tetrazolium to formazan by superoxide radical. In six samples of right atrium graded blindly on a scale of 0 through 4, strong staining (median grade, 3) of the microvasculature was seen. This was blocked by allopurinol in paired sections (median grade, 1; p < 0.01). Chemiluminescence can be used as an index of superoxide radical activity. Atrial samples were taken from 13 patients at five time points during coronary bypass grafting and placed in buffered luminol. Then chemiluminescence was measured. RESULTS: A 15-fold rise in chemiluminescence (295.93 +/- 39.47 mV) was demonstrated during reperfusion compared with the control value (19.06 +/- 0.47 mV). Chemiluminescence at 1 minute after release of the cross-clamp was significantly higher (p < 0.05) by analysis of variance versus values obtained before bypass and 1 minute before and 30 minutes after reperfusion. CONCLUSIONS: In this study we have identified superoxide radical activity and a possible generating system (xanthine oxidoreductase) in the human heart.

Aged↗
PubMed1990

Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.

Spectroscopic studies indicated that nitroblue tetrazolium (NBT) could be reduced to blue formazan by several distinct reactions in suspensions of isolated rat cardiac myocytes. Both NADPH- and NADH-linked pathways for reduction of NBT were observed. NADPH-linked NBT reduction showed little activity in the absence of digitonin, but could be stimulated an average of 9.5-fold by digitonin permeabilization of the plasma membrane. NADH-linked NBT reduction occurred in the absence of digitonin, and could be increased an average of 3.5-fold by digitonin treatment. Analysis of the effects of cell viability on the extent of digitonin stimulation with these substrates suggested that the NADPH-linked reaction involved a cytosolic component, while the NADH-linked reaction involved an intracellular membrane enzyme system. With either NADPH or NADH, NBT reduction was completely inhibited by dicoumarol (100 microM). Dicoumarol-insensitive NBT reduction could subsequently be observed following the addition of 2 mM cyanide, a level of cyanide known to inhibit cytosolic superoxide dismutase. Cyanide-stimulated, dicoumarol-insensitive NBT reduction was augmented by the presence of either antimycin or doxorubicin, two agents which enhance superoxide formation by different mechanisms. The results indicate the existence of multiple pathways for both superoxide-independent and superoxide-dependent reduction of NBT. Dicoumarol-insensitive, cyanide-stimulated NBT reduction may be useful as a spectroscopic probe for intracellular superoxide formation.

Animals↗