Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.9.3.1 (cytochrome oxidase)
8,822 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method is described utilizing the tetrazolium salts neotetrazolium chloride (NTC), triphenyltetrazolium chloride (TTC), C,N-diphenyl-N'-4,5-dimethylthiazol-2-yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of intact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transport chain through Complex I, as evidenced by the fact that the inhibition of MTT reduction was reversible by the addition of malic acid to the culture media. A second site of dark toxicity was found to be Complex IV (cytochrome oxidase). After photoirradiation of the cells, Complex I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the addition of succinic acid, whose electrons enter the transport chain at Complex II. A second and more sensitive site of photoirradiation damage was found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoirradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contrast, TTC reduction was found to be inhibited almost immediately indicating Complex IV is extremely susceptible to photoirradiation damage. Compared to other assays of mitochondrial function requiring subcellular fractionation, the use of tetrazolium salts is simpler to perform and can be done using physiologically relevant conditions.
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PMID:The use of tetrazolium salts to determine sites of damage to the mitochondrial electron transport chain in intact cells following in vitro photodynamic therapy with Photofrin II. 806 21

The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoracetone (TTFA), but inhibition was not observed with rotenone at < or = 2 microM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.
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PMID:Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. 839 Feb 25

The activities of selected enzymes of the respiratory chain system in Onchocerca fasciata (Filarioidea: Onchocercidae) have been investigated histochemically. Thus, the localization and distributions of NADH dehydrogenase (EC 1.6.99.3), succinate dehydrogenase (SDH) (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) were investigated in various tissues of the adult female worm by employing MTT, Nitro BT (dehydrogenases) and DAB (cytochrome oxidase). Different tissues varied considerably in their enzymatic activities. The hypodermis and reproductive tissues showed strong and identical localization of NADH and SDH dehydrogenase activities reflecting high metabolic rates. Little or no dehydrogenase activities were observed in the intestine and cuticle. In contrast to the two dehyrogenases, no activity was observed for cytochrome oxidase in any of the tissues in adult or embryonic stages of the worm. The significance of these results with respect to the energy metabolism of the worm is discussed. It is suggested that O. fasciata lacks a classical, mammalian-type respiratory pathway and that oxidative phosphorylation is of no importance as an energy generating pathway in this essentially anaerobic parasite.
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PMID:Onchocerca fasciata: histochemical demonstration of succinate and NADH dehydrogenase. 896 Jan 99

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
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PMID:Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. 1127 22

Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.
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PMID:Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons. 1176 90

The exact mechanisms of fescue toxicity in animals have yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Gastrointestinal (GI) toxicity of ergovaline (10(-11) to 10(-4) M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1 x 10(-4) M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1 x 10(-4) M ergovaline, in all three assays, treatment means were reduced to approximately 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h exposure, an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced approximately 12% over most concentrations. These findings indicate differentiation state-dependent sensitivity of Caco-2 cells to ergovaline, potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment.
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PMID:Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assays. 1295 76

Oxidative stress has been implicated in neuronal death caused by cerebral ischemia or some neurologic disorders. Chemical hypoxia (term defining the simulation by using respiratory inhibitors) chosen as in vitro ischemic model, was induced in primary cultures of rat cerebellar granule neurons by inhibitors of mitochondrial electron transport such as rotenone or paraquat (complex I), 3-nitropropionic acid (3-NPA, complex II), antimycin A (complex III), or sodium azide (complex IV). All compounds caused neuronal death determined by trypan blue staining and MTT-test. On the other hand, neurotoxicity of rotenone and paraquat but not of 3-NPA, antimycin or azide was significantly abolished by menadione (vitamin K3, 2-methyl-1,4-naphthoquinone). This neuroprotective effect of menadione was associated with a decrease of rotenone-induced free radical production.
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PMID:Menadione reduces rotenone-induced cell death in cerebellar granule neurons. 1537 39

We recently showed that acidosis is protective during hypoxia and detrimental during reoxygenation. We hypothesized that the detrimental effect of acidosis during reoxygenation was due to a negative effect on mitochondrial function. Human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and then reoxygenated for 0, 1, 4, 9, or 21 h. The detrimental effect of acidotic reoxygenation on metabolic activity was evident already after 1 h of reoxygenation, when MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] reduction (percentage of normoxic controls) was significantly higher in cells reoxygenated with neutral compared with acidotic medium both after acidotic hypoxia (83+/-26% versus 67+/-27%, p=0.006) and after neutral hypoxia (51+/-12% versus 41+/-7%, p=0.005). Hypoxanthine, a marker of cellular energy failure, increased more with acidotic compared with neutral reoxygenation both after acidotic hypoxia (after 21 h: 7.7+/-2.7 versus 3.1+/-1.9 microM, p<0.001) and after neutral hypoxia (10.4+/-2.6 versus 7.9+/-2.8 microM, p=0.001). During hypoxia and reoxygenation, there was an earlier reduction in the activity of complex IV compared with complexes II+III, and the ratio between these complexes fell during the first hour of reoxygenation. The reduction in complex IV activity was alleviated with acidotic hypoxia. Acidosis during reoxygenation, however, had no effect on the activity of either complex IV or complexes II+III. We conclude that acidosis during hypoxia increases neuronal survival and preserves complex IV activity. Acidosis during reoxygenation has an early detrimental effect on metabolic activity, but this is not mediated through an effect on the mitochondrial complexes IV or II+III.
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PMID:Acidosis during reoxygenation has an early detrimental effect on neuronal metabolic activity. 1569 2

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).
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PMID:Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease. 1592 94

Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose-dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl-inositol-3-kinase (PI-3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin-stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI-3K- and MEK-dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI-3K rather than MEK supports insulin-mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473-phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI-3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome-c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin-dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.
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PMID:Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis. 1654 48


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