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PubMed1997

Regulation of the soxRS oxidative stress regulon. Reversible oxidation of the Fe-S centers of SoxR in vivo.

SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable [2Fe-2S] centers that are presumed to serve as redox sensors. In vitro transcription experiments previously demonstrated that only the oxidized form is active. Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells. Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements. In uninduced cells, 90% of the SoxR was in the reduced form. Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers. Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state. The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters.

Alleles↗
PubMed2001

The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase.

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.

2,4-Dinitrophenol↗
PubMed2001

Ubiquinol:cytochrome c oxidoreductase (complex III). Effect of inhibitors on cytochrome b reduction in submitochondrial particles and the role of ubiquinone in complex III.

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing </=0.06 mol of Q/mol of complex III, and Q-replenished SMP. The results showed that Q was not required for electron transfer from ISP to b, a reaction that was inhibited by antimycin (also by myxothiazol or MOA-stilbene as reported elsewhere). It was also shown that antimycin did not inhibit electron transfer from b (b(H)) to Q, in clear contrast to the assumption of the Q-cycle hypothesis regarding the site of antimycin inhibition.

Animals↗
PubMed2002

Sustained production of H(2)O(2) activates pro-matrix metalloproteinase-2 through receptor tyrosine kinases/phosphatidylinositol 3-kinase/NF-kappa B pathway.

A rate-limiting step of tumor cell metastasis is matrix degradation by active matrix metalloproteinases (MMPs). It is known that reactive oxygen species are involved in tumor metastasis. Sustained production of H(2)O(2) by phenazine methosulfate (PMS) induced activation of pro-MMP-2 through the induction of membrane type 1-MMP (MT1-MMP) expression in HT1080 cells. MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 and -2 levels were changed negligibly by PMS. A one time treatment with H(2)O(2) did not induce activation of MMPs. It was also demonstrated that superoxide anions and hydroxyl radicals were not related to PMS action. PMS-induced pro-MMP-2 activation was regulated by the receptor tyrosine kinases, especially the receptors of platelet-derived growth factor and vascular endothelial growth factor, and downstream on the phosphatidylinositol 3-kinase/NF-kappa B pathway but not Ras, cAMP-dependent protein kinase, protein kinase C, and mitogen-activated protein kinases. PMS did not induce pro-MMP-2 activation in T98G and NIH3T3 cells. This may be related to a low level of MT1-MMP, indicating a threshold level of MT1-MMP is important for pro-MMP-2 activation. Furthermore, PMS increased cell motility and invasion but decreased cell-cell interaction. Cell-matrix interaction was not affected by PMS.

Animals↗
PubMed1982

Lipid metabolism in riboflavin-deficient rats. 2. Mitochondrial fatty acid oxidation and the microsomal desaturation pathway.

1. Oxygen consumption was measured by means of an O2 electrode in mitochondrial suspensions from riboflavin-deficient and pair-fed control rats, using six different substrates. Whereas consumption of O2 by glutamate was only slightly depressed in mitochondria from deficient animals, the consumption of O2 by hexanoate and by palmitoyl-L-carnitine was depressed to approximately half the control value: a highly significant difference. A comparable magnitude of depression was observed for stearoyl-, oleoyl-, and linoleoyl-L-carnitine. There were no major or consistent differences between groups of animals receiving two different types, and two different levels, of fat in their diet. 2. The activity of acyl coenzyme A dehydrogenase (EC 1.3.99.3) in hepatic mitochondrial fragments, measured by cytochrome c reduction with palmitoyl-coenzyme A as substrate, and expressed as maximum velocity (Vmax) with respect to phenazine methosulphate, was also reduced to approximately half the control value in deficient animals. 3. In hepatic microsomes, cytochrome b5 reductase (EC 1.6.2.2) activity was unaffected by riboflavin deficiency, although NADPH-cytochrome c reductase (EC 1.6.2.4) and microsomal flavin content were diminished to approximately half the control values. Acyl CoA (delta 9) desaturase activity (EC 1.14.99.5) was virtually identical in deficient, pair-fed, and ad lib.-fed control groups. 4. It is concluded that the depression of mitochondrial beta-oxidation of fatty acids which is observed in riboflavin-deficient animals is not a secondary result of inanition, and may account for the observed changes in fatty acid profiles of triglycerides and phospholipids. Failure of the microsomal fatty acid desaturation system is less likely to be a major consequence of riboflavin deficiency.

Acyl-CoA Dehydrogenase↗
PubMed1998

Free radical-scavenging properties of lignin.

Lignin is one of the major components of dietary fiber. It is a complex hydrophobic molecule that typically occurs in cell walls with heteroxylans. Our experimental data show that lignin is a free radical scavenger. When the NADH-phenazine methosulfate-nitro blue tetrazolium free radical-producing system is used, an alkali-lignin concentration of 46.29 micrograms/ml that causes 50% inhibition of uric acid production by xanthine oxidase (IC50) is a scavenger of superoxide anion radicals. Spectrophotometric assay has shown that alkali-lignin with an IC50 of 59.08 micrograms/ml inhibits the activity of xanthine oxidase, one of the enzymes related to the production of superoxide anion radicals, and presents a mixed-type noncompetitive inhibition pattern. Using the deoxyribose method, we have found that alkali-lignin is a hydroxyl radical scavenger with an IC50 of 250 micrograms/ml, and using the thiobarbituric acid method, we can see that alkali-lignin inhibits nonenzymatic and enzymatic lipid peroxidation with an IC50 of 72 and 100 micrograms/ml, respectively. Alkali-lignin also hinders the activity of glucose-6-phosphate dehydrogenase, another enzyme related to the generation of superoxide anion radicals, with an IC50 of 123.6 micrograms/ml, and obstructs the growth and viability of cancer (HeLa) cells in a dose-dependent manner. Our experimental results suggest another mechanism whereby the free radical-scavenging activity of lignin in dietary fiber may be involved in the fiber-colon cancer interaction. We also suggest that the ability of dietary fiber to protect against colon cancer may be partly determined by the amount of lignin in dietary fiber as well as the free radical-scavenging ability of lignin.

Animals↗
PubMed1991

Development of monoclonal antibodies reactive with methamphetamine raised against a new antigen.

Monoclonal antibodies (McAbs) specific to methamphetamine (MA) were produced using p-amino MA coupled to bovine serum albumin (BSA) with glutaraldehyde (GA) as an immunogen and with conventional hybridoma techniques. Hybridoma clones secreting the McAbs were selected by an enzyme-linked immunosorbent assay (ELISA) system using both the above conjugate and BSA modified with GA as screening antigens. In the ELISA system were used avidin and biotinyl-alkaline phosphatase which converts nicotinamide adenine dinucleotide phosphate (NADP) into NAD. The final enzyme activity was determined using diformazan of nitroblue tetrazolium formed together with the NAD produced, alcohol dehydrogenase and phenazine methosulfate. The McAbs from 9 clones were characterized by a crossreactivity test using the ELISA. The McAbs recognized MA (100%), methoxyphenamine (8.0%), ephedrine (2.3%), but did not react with metylephedrine, amphetamine, OH-amphetamine, dimethylamphetamine, beta-phenylethylamine, norephedrine, phentermine and ranitidine. An inhibition curve for MA was obtained in the range of 0.75 to 50 ng.

Animals↗
PubMedDate not supplied

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

3T3 Cells↗
PubMed1996

The isolation and some properties of the membrane-bound lactate dehydrogenase of Paracoccus denitrificans.

Membrane-bound lactate dehydrogenase was isolated from the cells of Paracoccus denitrificans by a combination of phase separation and chromatographic methods. It was NAD(+)-independent; phenazine methosulphate and 2,6-dichlorophenolindophenol could act as electron acceptors. It preferred D-lactate over the L-form (K(m) = 0.81 and 4.40 mM, respectively). Relative molecular weight estimations were 54000 +/- 3000 (electrophoresis) and 50000 +/- 5000 (gel chromatography) It was inhibited by thenoyltrifluoroacetone and diethyl pyrocarbonate, SH-reagents were without effect. No other lactate dehydrogenase activity was found in any subcellular fraction of cells grown on D,L-lactate, L-lactate, glucose or succinate.

Acetone↗
PubMed2001

A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

Anthranilate Synthase↗
PubMed2001

Assay of sperm quality in the domestic fowl by MTT reduction.

1. An assay which measures the capacity of spermatozoa from the domestic fowl to reduce the colourless tetrazolium dye MTT to its highly coloured purple formazan pigment has been developed and characterised. 2. The assay has several advantages over a previous tetrazolium-based sperm quality assay: it functions without the unstable phenazine methosulphate and toxic cyanide reagents and, following the reductive reaction, the sperm suspension is solubilised to produce optically clear solutions without centrifugation. 3. For samples of semen from individual males, MTT-reduction is strongly correlated with INT-reduction, which has been previously shown to be a useful predictor of sperm fertilising ability.

Animals↗
PubMed2003

Photochemical degradation of benzotriazole.

Benzotriazole is a commonly used additive in aircraft de-icing fluids. As a result of extensive de-icing activities the compound is detected in the groundwater below de-icing platforms at several international airports. The compound is toxic, and not biodegradable. Laboratory tests have been performed to study if UV irradiation can degrade the compound and reduce the aquatic toxicity. Benzotriazole can be degraded by UV irradiation at pH values below 7. Approximately 65% reduction in the benzotriazole concentration was achieved at a dose of 320 mWs/cm2, and almost 90% reduction was achieved at 1070 mWs/cm2, with an apparent first order relation between the logarithm to the UV dose and the reduction. Benzotriazole is not significantly mineralised by UV irradiation, but transformed into other compounds, of which aniline and phenazine were identified. The metabolites show toxic effects, but they are not as toxic as benzotriazole, resulting in a general decrease in toxicity as a result of UV irradiation.

Aircraft↗
PubMedDate not supplied

Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation.

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.

DNA↗
PubMed1964

CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE.

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.

Animals↗
PubMed1965

Light-induced volume changes in spinach chloroplasts.

A light-dependent mechanism that results in a slow, high-amplitude swelling of spinach chloroplasts in vitro has been discovered. The swelling is readily observed by optical and gravimetric methods, and by the use of an electronic particle counter; all show a 100 per cent increase of chloroplast volume in the light with an approximately 10-minute half-time. The existence of an osmotic mechanism for chloroplast swelling in the dark is confirmed. The volume of illuminated chloroplasts versus NaCl concentration represents the addition of osmotic and light effects. The action of light is enhanced by electron flow cofactors, such as phenazine methosulfate (PMS). However, neither conditions for ATP hydrolysis or synthesis nor NH(4)Cl influence the time course and extent of swelling. Hence, high-amplitude chloroplast swelling is light- (or electron flow), but not energy-dependent. A remarkable inhibitory effect of inorganic phosphate on chloroplast swelling is observed in the light, but not in the dark. Another action of light on chloroplasts is known to result in a shrinkage of chloroplasts which is rapid, reversible, energy-dependent, and requires phosphate. Thus phosphate determines the action of light on chloroplast volume. Since shrinkage is reversible, but swelling is not, it may be that they reflect physiological and deteriorative processes, respectively. Chloroplasts and mitochondria appear to control their volume by similar mechanisms.

Adenosine Triphosphate↗
PubMed1961

Formalin fixation in the cytochemical demonstration of succinic dehydrogenase of mitochondria.

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q(10), and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5 degrees to 10 degrees C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro-BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.

Animals↗
PubMed1962

Variations in the lactic dehydrogenase of vertebrate erythrocytes.

Erythrocytes from representatives of the 5 classes of vertebrates revealed a marked species variation in the number of LDH isozymes, in the distribution of the total LDH activity among these isozymes, and in their electrophoretic mobilities. Starch gel electrophoresis of hemolysates followed by direct histochemical demonstration of LDH activity with nitro blue tetrazolium as dye and phenazine methosulfate as electron transporter showed that closely related species exhibited similar LDH patterns. The rhesus monkey had LDH isozymes of similar pattern to those of human hemolysate but slightly slower in electrophoretic mobility. The goat and sheep each had 1 band of LDH activity in their erythrocytes of identical electrophoretic mobility, whereas the single band in steer hemolysate migrated slightly faster. The 5 bands of chicken hemolysate were quite similar in pattern to the 5 bands of duck hemolysate but migrated slightly faster and exhibited a different distribution of the total LDH activity. The 2 species of snake each had 1 band of LDH activity with identical mobility. Staining occurred with the levorotatory form of the substrate and not with the dextrorotatory form. Examination of more than 380 human hemolysates failed to reveal any differences among individuals in the main LDH bands. The genetic basis for the species differences in erythrocyte lactic dehydrogenases is discussed.

Animals↗
PubMed2006

Neutral red as a probe for confocal laser scanning microscopy studies of plant roots.

BACKGROUND AND AIMS: Neutral red (NR), a lipophilic phenazine dye, has been widely used in various biological systems as a vital stain for bright-field microscopy. In its unprotonated form it penetrates the plasma membrane and tonoplast of viable plant cells, then due to protonation it becomes trapped in acidic compartments. The possible applications of NR for confocal laser scanning microscopy (CLSM) studies were examined in various aspects of plant root biology. METHODS: NR was used as a fluorochrome for living roots of Phaseolus vulgaris, Allium cepa, A. porrum and Arabidopsis thaliana (wild-type and transgenic GFP-carrying lines). The tissues were visualized using CLSM. The effect of NR on the integrity of the cytoskeleton and the growth rate of arabidopsis primary roots was analysed to judge potential toxic effects of the dye. KEY RESULTS: The main advantages of the use of NR are related to the fact that NR rapidly penetrates root tissues, has affinity to suberin and lignin, and accumulates in the vacuoles. It is shown that NR is a suitable probe for visualization of proto- and metaxylem elements, Casparian bands in the endodermis, and vacuoles in cells of living roots. The actin cytoskeleton and the microtubule system of the cells, as well as the dynamics of root growth, remain unchanged after short-term application of NR, indicating a relatively low toxicity of this chemical. It was also found that NR is a useful probe for the observation of the internal structures of root nodules and of fungal hyphae in vesicular-arbuscular mycorrhizas. CONCLUSIONS: Ease, low cost and absence of tissue processing make NR a useful probe for structural, developmental and vacuole-biogenetic studies of plant roots with CLSM.

Microscopy, Confocal↗