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This study was undertaken to investigate the antibacterial and antifungal activities of Rumex tingitanus leaves extracts as well as the identification of bioactive components and their performance in meat preservation. Total phenolics and flavonoids showed the highest content of phenolics and flavonoids in the ethyl acetate fraction (Rt EtOAcF). For antimicrobial efficacy, leaves extract and derived fraction were tested for their capacity to inhibit bacterial and fungal proliferation in vitro and in vivo . The ethyl acetate fraction showed the most potent antibacterial and antifungal activities compared to the others extracts. Thus, the efficacy of this extract to inhibit the proliferation of Listeria monocytogenes in minced beef meat model was examined. This fraction eradicates the L. monocytogenes population in meat in a concentration- and time-dependent manner. A bio-guided purification of the Rt EtOAc fraction resulted in the isolation of the compound responsible for the observed antimicrobial activity. This compound was identified as luteolin by analysis of spectroscopic data. Chemical compounds isolated in this article Luteolin (PubChem CID: 5280445); p -iodonitrotetrazolium chloride (PubChem CID: 64957); Amphotericin B (PubChem CID: 5280965); Gentamicin and (PubChem CID: 6419933); Hexane (PubChem CID: 8058); Methanol (PubChem CID: 887); Ethanol (PubChem CID: 702); Dimethylsulfoxide (PubChem CID: 679); Quercetin (PubChem CID: 5280343); Gallic acid (PubChem CID: 370).

Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes.In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.

Summary Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus . The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a “resurrection” plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.

The flavonoid class of plant secondary metabolites play a multifunctional role in below-ground plant–microbe interactions with their best known function as signals in the nitrogen fixing legume–rhizobia symbiosis. Flavonoids enter rhizosphere soil as a result of root exudation and senescence but little is known about their subsequent fate or impacts on microbial activity. Therefore, the present study examined the sorptive behaviour, biodegradation and impact on dehydrogenase activity (as determined by iodonitrotetrazolium chloride reduction) of the flavonoids naringenin and formononetin in soil. Organic carbon normalised partition coefficients, log Koc, of 3.12 (formononetin) and 3.19 (naringenin) were estimated from sorption isotherms and, after comparison with literature log Koc values for compounds whose soil behaviour is better characterised, the test flavonoids were deemed to be moderately sorbed. Naringenin (spiked at 50 μg g−1) was biodegraded without a detectable lag phase with concentrations reduced to 0.13±0.01 μg g−1 at the end of the 96 h time course. Biodegradation of formononetin proceeded after a lag phase of ∼24 h with concentrations reduced to 4.5±1% of the sterile control after 72 h. Most probable number (MPN) analysis revealed that prior to the addition of flavonoids, the soil contained 5.4×106 MPN g−1 (naringenin) and 7.9×105 MPN g−1 (formononetin) catabolic microbes. Formononetin concentration had no significant (p>0.05) effect on soil dehydrogenase activity, whereas naringenin concentration had an overall but non-systematic impact (p=0.045). These results are discussed with reference to likely total and bioavailable concentrations of flavonoids experienced by microbes in the rhizosphere.

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.

A spectrophotometric assay method for determining succinate dehydrogenase activity is described in which iodonitrotetrazolium chloride is used as a final electron acceptor. The enzyme activity is determined by measuring the formation of formazan due to the tetrazolium salt reduction. The assay is continuous, rapid, simple, and sensitive, and may be used in the determination of enzyme activity either in tissue homogenates or as a marker of the mitochondrial fraction in cell fractionation procedures.

Recent studies have shown that the adminstration of endrn to rodents induced lipid peroxidation in various tissues and decreases glutathione content. These results suggest that endrin produces reactive oxygen species and/or free radicals. We have therefore examined the effects of endrin(4.5 mg/kg) on the production of reactive oxygen species by peritoneal macrophages and hepatic mitochondria and microsomes in rats. The effects of endrin on hepatic mitochondrial and microsomal lipid peroxidation and membrane fluidity as well as the incidence of hepatic nuclear DNA damage were also examined. Twenty-four hours after endrin administration, significant increases in the production of the chemiluminescence by the three tissue fractions were observed. Furthermore, peritoneal macrophages form endrin-animals resulted in 3.0- and 2.8-fold increases in cytochrome c and iodonitrotetrazolium (INT) reduction, indicating enhanced production of superoxide anion. Endrin administration also resulted in significant increases in lipid peroxidation of mitochondrial and microsomal membranes as well as decreases in the fluidity of these two membranous fractions. A significant increase in hepatic nuclear DNA single-strand breaks also occurred in response to endrin administration. The reslts indicate that macrophage, mitochondria, and microsomes produce reactive oxygen species following endrin administration, and these reactive oxygen species may contribute to the toxic manifestations of endrin.

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] and its bioisosteres involves binding to a specific TCDD [Ah] receptor, interaction of this complex with chromatin, and the ultimate production of a pleiotropic response. We have hypothesized that TCDD toxicity also involves the production of oxidative stress [OS], and phagocytic cell activation may be a source of reactive oxygen species [ROS] following TCDD exposure. Evidence is provided for the activation of peritoneal exudate cells [PEC] following exposure of rats to TCDD. Increases of 2.5- and 3-fold in ROS production by PEC were observed 24 hrs following treatment of female Sprague-Dawley rats with a single dose of 50 μg TCDD/kg, utilizing the iodonitrotetrazolium and cytochrome c [cyt-c] reduction assays, respectively. However, no increases in ROS production by isolated PEC were observed following in vitro incubation with 5, 25, 50, 100, 125 and 10,000 ng TCDD/ml of the assay medium using the nitroblue tetrazolium and cyt-c reduction assays. Phorbol myristate acetate was utilized as a positive control and resulted in a 3.5-fold increase in ROS production after a 15 min incubation with 3 × 106 PEC/ml at 37° C. PEC activation may contribute to OS following TCDD exposure, and the process may be receptor mediated.

The factors affecting the measurement of electron transport system (ETS) activity in river biofilm through the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan) have been studied. Methanol extracts INT-formazan more effectively than either propanol or ethanol. A concentration of 0.02% INT was chosen and samples should be incubated for less than 8 h. ETS activity is optimal at a circumneutral pH. ETS stimulators (NADH, NADPH and succinate) added as a check of the assay produced an increase in INT-formazan indicating that ETS activity was being measured. This assay is quick, easy to use and ideal for field studies.

The relative contribution of two major soil microbial groups, fungi and bacteria, to soil respiration was investigated because of their importance in decomposition and nutrient cycling in agroecosystems. The bactericide streptomycin and the fungicide cycloheximide were applied singly and in combination in two glucose-amended (5 g kg−1 soil) and texturally differing agricultural soils, a Valentine sand (Typic Ustipsamment) and a Sharpsburg silty clay loam (Typic Argiudoll). Soils receiving only glucose served as controls. Total and metabolically-active fungal and bacterial microscopic counts were made at 1, 3 and 10 days after amendment. CO2 evolution was measured for 24 h before each sampling for the purpose of relating soil respiration to specific microbial metabolic activity. Intracellular reduction of iodonitrotetrazolium to formazan and accumulation of fluorescein from fluorescein diacetate were used as indicators of metabolically-active bacteria and fungi, respectively. Production of new biomass in controls and fluctuations in CO2 evolution were primarily associated with bacterial activity on the first day, indicating an advantage of bacteria over fungi in initial glucose metabolism. Fungal activity was associated with CO2 production at later sampling times. Reduced respiration, as a result of biocide treatment, occurred only on the first day whereas nontarget or indirect effects of both inhibitors were apparent at later times. Fungal activity rose two to five times above the level of the glucose-amended control on day 3 in streptomycin-treated soils, and bacterial activity was stimulated in the cycloheximide treatment of the Sharpsburg soil on day 10. The effectiveness of streptomycin inhibition on total and active bacterial densities was greater in the Sharpsburg silty clay loam than in the Valentine sand. There was no reduction of total hyphal length due to biocide treatment, and fungal activity with cycloheximide did not differ significantly from that of the control. For our experimental conditions the use of biocides alone did not effectively differentiate between bacterial and fungal activity. Selective staining of metabolically-active microorganisms, however, may offer promise in achieving this goal and appears useful in understanding the importance of bacteria and fungi to nutrient cycling in soil ecosystems.

A thorough investigation on the long-known reactivity of spinach ferredoxin-NADP+ reductase with tetrazolium salts has allowed the setting-up of a new assay, highly specific for this enzyme. By immunological titration it has been determined that more than 92% of the iodonitrotetrazolium chloride reductase activity of spinach leaves' homogenate belongs to ferrodoxin-NADP+ reductase. It has been shown that addition of formazanbinding agents, like Triton X-100, is required for the assay of the partially purified enzyme; furthermore Triton X-100 enhanced considerably the tetrazolium salts reductase activity of all the enzyme preparations tested. The optimal condition of pH, ionic strength and Triton X-100 concentration have been determined. Kinetic experiments yielded a series of parallel lines in the double-reciprocal plots and the kinetic parameters obtained are reported. Moreover it has been shown that at least 75–80% of the activity with tetrazolium salts of ferrodoxin-NADP+ reductase is not mediated by the superoxide radical.

A two stage procedure for the quantitative determination of d -xylose is described. The monosaccharide is first chemically reduced with sodium borohydride to the sugar alditol; the xylitol formed is subsequently quantified enzymically with sheep liver d -glucitol-dehydrogenase. The reduced nicotinamide adenine dinucleotide formed is either determined directly or through formazan production in a linked assay with pig heart diaphorase and iodonitrotetrazolium chloride. The assay is an end point determination and uses reagents that are all available commercially, either singly or in kit form. The potential application of the assay in the determination of other monosaccharides of biological importance (e.g., ribose) is discussed. Data on the estimation of xylose during the breakdown of oat spelt xylan by the rumen fungus Piromonas communis and the formation of extracellular bioppolymer from xyclose by Pseudomonas NCIB 11264 are presented to exemplify applications of the assay procedure.

The source of superoxide anion radical (O2⪰) in aerobic mixtures consisting of NAD[P]H, 5-methylphenazinium methyl sulfate (or its 1-methoxy derivative) and tetrazolium salt was investigated using superoxide dismutase (SOD), Mn(II), ferricytochrome-C, and epinephrine as probes. NAD[P]H + phenazine + O2 was found to reduce nitroblue tetrazolium, iodonitrotetrazolium, and thiazolyl blue in a manner sensitive to agents that dismutate O2⪰, iviz., SOD and Mc(II). It also mediated the reduction of ferricytochrome-C, and augmented the autooxidation of epinephrine to the adrenochrome, without a tetrazolium salt present in the medium. The autooxidation ofepinephrine, but not the reduction of ferricytochrome-C, was found to be sensitive to SOD. Nitroblue tetrazolium, either singly or in combination with SOD, did not stimulate the reduction of ferricytochrome-C. The oxidation of NADH, mediated by a catalytically low concentration of phenazine(+O2) was augmented two-fold by SOD. These observations are consistent with, and lend support to a scheme of redox events (Scheme-3) wherein it is proposed that the source of O2⪰ in the NAD[P]H + phenazine + tetrazolium(+O2) system is the reduced phenazine, that the tetrazoinyl radical (a one-electron reduction product of tetrazolium) may not reduce O2 to O2⪰, that the redox reaction between semiquinone radicals of phenazine and O2 is reversible, and that the disproportionation of semiquinone radicals constitutes an important rate-limiting reaction in the expression of phenazine redox couple.

Homogenates of isolated pancreatic islets contain 40-70 times as much flavin-linked glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) as homogenates of whole pancreas, liver, heart, or skeletal muscle when the activity is assayed with either iodonitrotetrazolium or with dichloroindophenol as an electron acceptor. Intact mitochondria from islets release 3HOH from [2-3H]glycerol phosphate 7 times faster than do skeletal muscle mitochondria. The activity of the cytosolic, NAD-linked, glycerol phosphate dehydrogenase (EC 1.1.1.8) in pancreatic islets is comparable to that of the mitochondrial dehydrogenase so a glycerol phosphate shuttle is possible in pancreatic islets. Diazoxide, an inhibitor of insulin release in vivo and in vitro, inhibits the islet mitochondrial glycerol phosphate dehydrogenase in all three of the assays mentioned above at concentrations that inhibit insulin release and CO2 formation from glucose by isolated pancreatic islets. Diazoxide does not inhibit the dehydrogenase in mitochondria from skeletal muscle, liver, and heart. A slight inhibition in mitochondria from whole pancreas can be accounted for as inhibition of the islet dehydrogenase because no inhibition is observed in mitochondria from pancreas of rats treated with alloxan, an agent that causes diabetes by destroying pancreatic beta cells. The results of this study are compatible with the hypothesis that the mitochondrial glycerol phosphate dehydrogenase has a key role in stimulus-secretion coupling in the pancreatic beta cell during glucose-induced insulin release.

An assay method for glutaminase is described in which the rate of release of glutamate from glutamine is followed in a second step by means of two coupled reactions involving glutamate dehydrogenase and the phenazine methosulfate-catalyzed reduction of iodonitrotetrazolium chloride by NADH. The assay is rapid, sensitive, and reproducible, and may be employed to determine glutaminase activity in crude as well as in purified enzyme preparations.

Eukaryotic cells were extracted from mineral soil by shaking in 50 mM Tris buffer, pH 7.5. This extracted a greater biovolume of cells than shaking in a range of surfactants, blending or sonication. An aliquot of soil suspension was incubated with iodonitrotetrazolium (INT) and then loaded onto a linear density gradient prepared from Percoll and phosphate buffer. After centrifugation. the majority of mineral particles had sedimented but most of the cells were retained in the supernatant. These cells were stained and mounted on a membrane filter for epifluorescence microscopy. Metabolically active protozoa were readily identified as having a well defined cytoplasm and cell wall, stained with acridine orange, a distinct nucleus or nuclei, stained with 4′,6′-diamidino-2-phenylindole, and containing crystals of INT-formazan. This combination of stains was the most reliable for detecting protozoa. Algal cells were easily identified by the red autofluorescence of their chlorophyll. The most-probable-number technique and our proposed method both ranked the protozoan populations from a range of soils in the same order. It is argued that the described technique gives a more accurate measure of active protozoan biomass and a recommended procedure is given.

Electron transport system (ETS) activity was measured in freshwater sediment at 4, 10 and 20°C by following the reduction of 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium (INT-formazan). The rate of INT-formazan formation was dependent upon the incubation temperature and nutrient amendments. Electron transport system activity also correlated very highly with O 2 consumption rates in the same sediment.

Summary Electron transport system (ETS) activity was determined in the estuarine sedimentby measuring the reduction of 2-(-p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan) by sediment enzymes and microorganisms. ETS activity was higher in aerobically incubated sediment than in anaerobically incubated sediment. Oxygen uptake in aerobically incubated sediment correlated highly with sediment ETS activity, whereas CO 2 , evolution and nitrogen fixation (C 2 H 2 reduction) correlated very highly with ETS activity in anaerobically incubated sediment.